Abstract Book of EAVLD2012 - eavld congress 2012

2 nd CONGRESSOFTHEEUROPEAN 

ASSOCIATIONOFVETERINARY 

LABORATORYDIAGNOSTICIANS(EAVLD) 

 

EAVLD 2012 

 

 

14July,2012,KazimierzDolny,Poland 

 

 

 

 

 

 

 

 

 

 

 

organizedbythe 

NationalVeterinaryResearchInstitute,Pulawy

TM marks are owned by IDEXX Laboratories, Inc. 

 

 

BVDV Test 

A new diagnostic solution to detect bovine viral 

diarrhea virus (BVDV) in just 20 minutes. 

EU veterinarians estimate that BVDV costs producers 

over €25 per head, even after vaccination. 1 Test all new 

introductions with the IDEXX SNAP BVDV Test to identify 

persistently infected cattle. 

The IDEXX SNAP BVDV Test: 

 

 

 

To learn more about the 

IDEXX SNAP BVDV Test, 

visit idexx.com/snapbvdv. 

1. Data on fi le at IDEXX Laboratories, Inc. Westbrook, Maine USA.

Welcome 

Dear colleagues, 

Welcome to the 2nd EAVLD congress in Kazimierz Dolny, hosted by the National Veterinary Research 

Institute, Puawy. I am very honoured and proud that our Institute had an opportunity to organize the event. 

Nowadays, a constant demand for a higher quality of diagnostic services, also a competition on the market 

and funding shortages make diagnostic services a hard work. Therefore, the possibility of meeting other 

specialists in the field of veterinary laboratory diagnostics and exchanging of eye-to-eye experience are of 

prime importance, which was also marked by the success of the 1 st EAVLD congress. The sponsors’ 

response to this congress was beyond our expectations and we are pleased that participants will have the 

possibility to get acquainted with the variety of the latest diagnostic developments during the congress. 

Despite the economical situation encountered, by the end of May, 2012 we had over 170 registered 

participants from 26 countries including: Africa, Australia, Europe, North and South America. 

Walking in the footsteps of the first EAVLD congress held in 2010 in Lelystad by the CVI, made our 

preparations easier since we had a tremendous and invaluable back-up and advice from Dr. Willie Loeffen, 

Secretary of the EAVLD and the President of the Organizing Committee of the EAVLD 2010. At the same 

time, we are aware of the responsibility and high expectations from the participants due to the positive 

feedback from 1 st such an event organized in 2010. Despite recent renovations of our Institute and its training 

centre both based in Puawy, we had to find another location due to the space limits. We have chosen 

Kazimierz Dolny, a town situated nearby Puawy, located at the bank of Vistula river. We hope you will 

admire town’s beauty and its flavour when visiting different attractions during the congress free time. 

Myself and the whole Organizing Committee of EAVLD2012 hope that you will find this congress interesting 

and valuable source of the up-to-date knowledge within the field of veterinary laboratory diagnostics. 

We wish you all the best and enjoy your stay in Kazimierz Dolny and in Puawy, both scientifically and 

socially. 

Miroslaw P. Polak 

The Organizing Committee 

of the 2 nd EAVLD Congress 

Welcome to the second EAVLD congress and to the beautiful town of Kazimierz Dolny. I hope that you have 

time to explore the area and are looking forward to the Polish hospitality! 

The congress is being held at a time of great economic uncertainty for many countries and individuals. As 

veterinary diagnosticians we have a duty to continue to make new scientific advances while also working 

more efficiently to help reduced costs to governments and animal owners. We will not be able to do this 

unless we share ideas and learn from each other. I want to thank you for coming and supporting this 

congress and also to all our sponsors for helping to make the congress possible. Please make the most of 

the commercial exhibition – which must be the most concentrated gatherings of companies involved in 

veterinary diagnostics in Europe since our last congress in 2010! 

Please get involved and come to the General meeting on Tuesday at 16.30. If you have ideas for how the 

association may be able to better serve its members please speak up at the General Meeting or discuss with 

an EAVLD board member. 

I wish to especially thank Miroslaw Pawel Polak and the team at National Veterinary Research Institute for all 

their hard work in organising this congress. 

At this meeting I will hand on the presidency of the association to Willie Loeffen who was elected as vice 

president at the last congress in 2010. I want to thank Willie for all the work he has done as secretary over 

the last few years and to wish him all the best for his term of office. He will have my full support and I know 

that he will do an excellent job as your new EAVLD President. 

Andrew Soldan 

EAVLD President

KEYNOTE SPEAKERS 

General Session 

Prof. Dr. Wim H.M. van der Poel 

Prof. Dr. Wim H.M. van der Poel, DVM is senior scientist at the Central Veterinary Institute of 

Wageningen University and Research Centre, The Netherlands. His research activities have 

focussed on veterinary virology, emerging and zoonotic viruses and food borne viruses. He is 

also coordinator of the EPIZONE European Research Group, the network on epizootic 

animal diseases research. Previously, from 1996 to 2004, prof Van der Poel has been 

working on research of viral zoonoses and food borne viruses at the National Institute for 

Public Health and the Environment in Bilthoven, The Netherlands. At this institute, from 

2000-2004, he was also heading the National Reference Laboratory for Microbiological 

Contamination of Bivalve Molluscs. Prof. Van der Poel is a member of a number of scientific 

committees, boards and professional bodies in the field of veterinary virology, and he has 

been involved in many national and international research projects on viruses in animals and 

foodstuffs. Throughout his career he has published a large number scientific papers and 

reviews in this area and he has often served as an ad hoc reviewer for scientific journals and 

granting agencies. In February 2009 he has accepted an honorary visiting chair on Emerging 

and Zoonotic Viruses at the University of Liverpool, United Kingdom. 

Prof. van der Poel graduated in Veterinary Medicine in 1988 and completed his PhD in 

veterinary virology at the Utrecht University in 1995. He is a registered specialist in veterinary 

microbiology within the Netherlands Royal Veterinary Association and a registered research 

worker in medical microbiology within the Netherlands Royal Microbiology Association. 

Prof. Dr. med. vet. Stefan Schwarz 

works in the Institute of Farm Animal Genetics of the Friedrich-Loeffler-Institut (= Federal 

Research Institute for Animal Health) in Neustadt-Mariensee, Germany. He heads the 

research group ‘Molecular Microbiology and Antimicrobial Resistance’ and is involved in both 

surveillance of antimicrobial resistance and analysis of the molecular genetics of 

antimicrobial resistance. He also teaches various academic courses at the University of 

Veterinary Medicine Hannover. Prof. Schwarz is a specialist veterinarian in (a) microbiology, 

(b) epidemiology, and (c) molecular genetics and gene technology. He acts as editor/ 

editorial board member of six international journals. 

Diagnostics at the point of interest 

Andrew Soldan 

qualified as a veterinary surgeon in 1984 from the Royal Veterinary College, University of 

London and then worked in general mixed practice in Yorkshire, UK for 2 years. Following 

the completion of an MSc in Tropical Veterinary Medicine at the University of Edinburgh he 

working in Malawi for 5 years and helped set up an epidemiology unit. During this time he 

specialised in laboratory diagnosis of disease and control methods for tick borne disease in 

local cattle. For the latter work he was awarded a Doctorate of Veterinary Medicine by the 

University of Edinburgh. On return from Africa he worked as a government veterinary 

investigation officer before another 3 year spell in mixed practice. In 1999 he started work 

for the Veterinary Laboratories Agency and was heavily involved in the laboratory response 

to the CSF outbreak in 2000 and the FMD outbreak in 2001. He was head of testing for the 

VLA for 5 years, was Veterinary Director for 1 year and is currently Commercial Director and 

Head of AHVLA Scientific for the Animal Health and Veterinary Laboratories Agency 

following its formation in early 2011. Andrew has been involved in the development of new 

tests and bringing them into routine use in an ISO17025 accredited laboratory. He has also

worked with many diagnostic companies to ensure that new tests developed at the VLA have 

been available to international laboratories and end users. He has taken a special interest in 

tests that can be used outside a specialist laboratory - possibly informed by his time working 

in Africa. Andrew was a founding member of EAVLA and its first president. 

Emerging, re-emerging and wildlife diseases - diagnostic 

possibilities 

Dolores Gavier-Widén 

is a veterinary pathologist, Head of the Research and Development Division, Department of 

Pathology and Wildlife Diseases, National Veterinary Institute (SVA) and Associated 

professor at the Swedish University of Agricultural Sciences, Uppsala, Sweden. She 

obtained her veterinary degree at Buenos Aires University, Argentina, and her MS and PhD 

degrees at the University of California, Davis. She has worked on diagnosis in combination 

with applied research, in particular on infectious diseases of wild animals, for more than 25 

years, most of the time in Sweden and including 40 months of work at the Veterinary 

Laboratory Agency, Weybridge, England. She is the president of the Wildlife Disease 

Association (WDA) (www.wildifedisease.org) and past president of its Section, the European 

WDA. She has participated of several EU FP6, FP7 and LIFE projects, such as the current 

WildTech project, and also of several working groups of the European Food Safety Authority 

(EFSA) and of the OIE ad hoc Group on the validation on diagnostic tests for wildlife, in 

2011. She has contributed to the organization of several WDA, EWDA and other 

conferences, such as Cutting Edge Pathology (European Society of Veterinary Pathology), in 

2011. Dolores believes that surveillance of infectious diseases of wildlife is essential to 

improve and maintain the health of humans, domestic and wild animals. 

New techniques in bacteriology, parasitology and pathology 

Dr. Markus Kostrzewa 

In 1990 Dr. Kostrzewa obtained the Diploma in Biology, University Gießen, Germany, then in 

1993 he obtained the title of Dr. rer. nat, University Gießen, Germany. He joined Bruker in 

1998, where he started as project manager for DNA analysis by MALDI-TOF mass 

spectrometry. Later he became the Head of development of Clincal Proteomics (“ClinProt” 

system, a first mass spectrometry protein profiling system). 

Since 1999 he works in the field of MALDI-TOF MS in microbiology. Dr. Kostrzewa initiated 

and directed the development of microorganism identification by MALDI-TOF mass 

spectrometry (“MALDI Biotyper” system). He is heading development of clinical applications 

for mass spectrometry. Since 2005 he is the Director of Molecular Biology R&D at Bruker 

Daltonics and since 2012 Dr. Kostrzewa is the Vice President of Clinical Mass Spectrometry, 

R&D at Bruker Daltonics.

Sensitive and reliable animal pathogen detection 

QIAGEN veterinary testing solutions 

Subheadline optional 

can run over 1, 2 or 3 lines. 

QuantiFast® Pathogen +IC Kits provide versatile and sensitive detection. 

 

 

 

Simultaneous detection of viral RNA and DNA 

Internal Control for certainty in result interpretation 

Universal protocol for standard and fast cyclers 

Learn more at www.qiagen.com/QuantiFast 

For up-to-date trademarks and disclaimers, see www.qiagen.com. 

Sample & Assay Technologies

Products and services 

for scientists around the world 

The Animal Health and Veterinary Laboratories Agency has developed products and services 

and makes them available to organisations around the world, through AHVLA Scientific. 

For veterinary laboratories these include: 

Specialist laboratory reagents and diagnostic kits 

Extensive range of proficiency testing schemes 

Comprehensive bio-imaging service 

Unique cell characterisation service 

Internationally recognised R & D capability 

Laboratory testing services 

If you require further information on any products and services, 

please contact our Customer Service Team at 

ahvlascientific@ahvla.gsi.gov.uk or visit www.ahvlascientific.com

ScientificProgram 

DAY1 

17:0020:00 

19:0021:00 

DAY2 

08:0010:00 

09:0009:30 

09:3010:15 

10:1510.45 

10:4512:15 

12:3014:00 

14:0014:45 

14:4516.15 

16:1516.45 

 

Sunday,1July2012 

RegistrationdeskinKrolKazimierzHotel(KKH) 

Welcomedrink(patioofKKH) 

 

Monday,2July2012 

Registrationdesk(KKH) 

OpeningCeremony(lecturehallofKKH) 

1 st session[S1.]:GeneralSession 

(allaspectsofveterinarydiagnosis&drugresistanceissues) 

lecturehallofKKH(09:3018:15) 

[S1.]Keynotelecture:TheemergenceofSchmallenbergvirus–Howtorespondto 

newepizooticsinEurope?WimVanderPoel,DVM,PhD,CentralVeterinary 

Institute,WageningenUniversityandResearchCentre,DepartmentofVirology,P.O.Box65,8200 

ABLelystad,Edelhertweg15,8219PHLelystad,TheNetherlands 

Coffeebreak+postersession+sponsors’exhibitions(patioofKKH) 

[S1.]Oralpresentations(6) 

1. Oralfluids–samplematrixforeffectiveherdhealthmonitoring.ChristinaBoss,Life 

Technologies,Germany. 

2. DiagnosticpropertiesofseveralPRRSantibodyELISAsinthecontextofa 

longitudinalstudyofafarmusingdifferentvaccinationstrategies.KatrinStrutzberg 

Minder,IVDGmbH(IVDInnovativeVeterinaryDiagnostics),Germany. 

3. Introductionrateofalowpathogenicavianinfluenzavirusinfectionindifferent 

Dutchpoultrysectors.RuthBouwstra,CentralVeterinaryInstitute(CVILelystad), 

TheNetherlands. 

4. AduplexonesteprealtimeRTPCRfordiagnosisoffootandmouthdisease.Kamila 

Górna,ANSES,France. 

5. ValidationofacompetitiveELISAforthedetectionofantibodiesantip26ofequine 

infectiousanemiavirusinequinesera.RobertoNardini,IZSLAZIOETOSCANA,Italy. 

6. DiagnosticperformanceofacommercialPRRSserumantibodyELISAadaptedtooral 

fluidspecimens:fieldsamples.AndreaBallagi,IDEXXLaboratories,Sweden. 

Lunch(restaurantofKKH)+postersession+sponsors’exhibitions(patioofKKH) 

[S1.]Keynotelecture:Assessingtheantimicrobialsusceptibilityofbacteriaof 

animaloriginsomebasicconsiderations.Prof.Dr.StefanSchwarz,Institutfür 

NutztiergenetikFriedrichLoefflerInstitut(FLI),Höltystr.10,D31535NeustadtMariensee,Germany 


7. SubtypingofswineinfluenzavirusesbymultiplexrealtimePCR.KeesvanMaanen, 

GDAnimalHealthService,TheNetherlands. 

8. Importanceofcontinuousvalidationofmolecularmethodsforroutinediagnosisof 

PRRSVRNAinclinicalsamples.SandraRevillaFernández,AustrianAgencyfor 

HealthandFoodSafety/InstituteforVeterinaryDiseaseControlMödling,Austria. 

9. Viraldiagnosisusingtransmissionelectronmicroscopy.WilliamCooley,AHVLA, 

UnitedKingdom. 

10. Abeadbasedmultipleximmunofluorometricassayforscreeningandconfirmationof 

allmajorpriontypesinsheep.JanLangeveld,CentralVeterinayInstituteof 

WageningenUR,TheNetherlands. 

11. UsingoralfluidfortheserologicalmonitorizationofPRRSVcirculationinagroupof 

infectedgilts.XavierRebordosaTrigueros,HIPRA,Spain. 

12. Enhanceddetectionofbovinerespiratoryvirusesbyinclusionofcohortanimals. 

RonanO’Neill,DAFMLaboratories,CentralVeterinaryResearchLaboratory,Ireland 

Coffeebreak+postersession+sponsors’exhibitions(patioofKKH)

16:4518.15 

20:0023:00 


13. ThefirstyearofobligatoryBVDcontrolinGermany–diagnosticstrategies,results 

andexperiences.HorstSchirrmeier,FriedrichLoefflerInstitut,Instituteof 

DiagnosticVirology,Germany. 

14. EuropeanPRRSv–diagnosticsolutionsforarapidlymutatingvirus.ChristinaBoss, 

LifeTechnologies,Germany. 

15. SerologicalanalysisandmonitoringofIBR.IsitpossibletocontrolIBRgEantibodies 

inabulktankmilk?LourdesPorquetGaranto,HIPRA,Spain. 

16. DevelopmentandvalidationofarealtimePCRzengelmixforthediagnosisand 

quantificationofCoxiellaBurnetii.AleidaVillaEspinosa,EXOPOL,SL.Autovacunasy 

Diagnóstico,Spain. 

17. RingtestevaluationforthedetectionofPRRSVantibodiesinoralfluidspecimens 

usingacommercialPRRSVserumantibodyELISA.AndreaBallagi,IDEXX 

Laboratories,Sweden. 

18. RealtimePCR,MycoplasmaGallisepticum,Mycoplasmasynoviae,Mycoplasma 

meleagridis.AndréFuchs,IDEXXLivestockandPoultryDiagnostics,U.S.A. 

BarbecuepartyinthegardenoftheNationalVeterinaryResearchInstitutein 

Pulawy(transportbybusestoandfromKazimierzDolnywillbeprovidedbythe 

organizer–buseswillleavefromKKHat19:40)

Day3 

09:0009:45 

9:4510:45 

10:4511.15 

Tuesday,3July2012 

2 nd session[S2.]:Diagnosticsatthepointofinterest 

(pensitetests,SNAPtestsandhomemadetestsusedbeyondthelabbencharea) 


[S2.]Keynotelecture:Whendoyouwanttheresult?Howmuchdoyouwanttopay! 

AndrewSoldan,DVM,PhD,HeadofAHVLAScientific,AHVLA–Weybridge,WoodhamLane, 

NewHaw,Addlestone,SurreyKT153NB,UnitedKingdom 


1. DevelopmentofarapidisothermalassaytodetectTaylorellaequigenitalis,the 

causativeagentofcontagiousequinemetritis.SarahNorth,AHVLAWeybridge, 

TechnologyTransferUnit,SpecialistScientificSupport,UnitedKingdom. 

2. Laboratoryvalidationofanimmunochromatographictestfortherapiddetectionofkoi 

herpesvirus(cyhv3)ingillswabs.RobertVrancken,OkapiSciencesN.V.,Belgium. 

3. EvaluationofrapidHRSVstriptestsfordetectionofbovinerespiratorysyncytialvirus. 

WojciechSocha,DepartmentofVirology,NVRIPulawy,Poland. 

4. EvaluationofAlatexagglutinationtestfortheidentificationofClostridiumdifficileof 

porcineorigin.LauraVallsVila,Hipra,DiagnosticCenter,Girona,Spain. 

Coffeebreak+postersession+sponsors’exhibitions(patioofKKH) 

3 rd session[S3.]Emerging,reemergingandwildlifediseasesdiagnosticpossibilities 

(detectiontoolsbasedoncommercialandhomemadetests,theirperformanceandvalidationinthelab) 


11:1512:00 

12:0013:00 

13:0014:30 

14:3016:15 

16:3018:00 

20:0022:00 

[S3.]Keynotelecture:Emergingandreemergingwildlifediseases:pathologyand 

relatedtechniquesfordiagnosticsandforgeneralandtargetedsurveillance. 

DoloresGavierWidén,DVM,MS,PhD,AssociateProf.,DepartmentofPathologyand 

WildlifeDiseases,DeputyHeadofDept.,HeadR&DDivision,NationalVeterinaryInstitute(SVA), 

SE75189Uppsala,Sweden 


1. SchmallenbergvirusoutbreakinTheNetherlands:Routinediagnosticsandtestresults. 

RuthBouwstra,CentralVeterinaryInstitute(CVILelystad),TheNetherlands. 

2. 25yearsofpassivesurveillanceofbatsintheNetherlands.Molecularepidemiology 

andevolutionofEBLV1.BartKooi,CentralVeterinaryInstituteofWageningenUR– 

Lelystad,TheNetherlands. 

3. DiagnosisofQfeverinDairyCattlebyPhasespecificMilkSerology.JensBöttcher, 

BavarianAnimalHealthService,Germany 

4. EQUINEnocardioformplacentitis&abortionoutbreakandfarmbasedriskfactor 

study,20102011.CraigN.Carter,UniversityofKentucky,U.S.A. 

Lunch(restaurantofKKH)+postersession+sponsors’exhibitions(patioofKKH) 


5. AnewdiagnostictoolforbovinetuberculosisIDEXXM.bovisantibodytestkit. 

ChristophEgli,IDEXXLabs,Inc.,U.S.A. 

6. DetectionofSchmallenbergvirusintheUK.AnnaLaRocca,AnimalHealthand 

VeterinaryLaboratories(AHVLA),UnitedKingdom. 

7. DetectionofWNVenzooticcirculationinhorseswithneurologicalsignsandincaptive 

sentinelchickensintheprefectureofThessaloniki,Greece.SerafeimC.Chaintoutis, 

FacultyofVeterinaryMedicine,AristotleUniversityofThessaloniki,Greece. 

8. Schmallenbergvirus:serologicalstudiesinGermanholdings.HorstSchirrmeier, 

FriedrichLoefflerInstitut,InstituteofDiagnosticVirology,Germany. 

9. Differentdiagnostictoolsforabroadrangeofmacavirusesandtheirreservoirand 

susceptiblehosts.ChristineFoerster.Instituteofvirology,Diagnosticlaboratory, 

Germany. 

10. DiagnosticaspectsofSuidherpesvirus1infectioninwildboar.AdolfSteinrigl,AGES 

InstituteforVeterinaryDiseaseControl,Austria. 

11. PreliminaryvalidationoftheIDScreen®SchmallenbergvirusIndirectELISA.Philippe 

Pourquier,IDVet,France. 

GeneralMeetingofEAVLD(EAVLDmembersonly) 

Galadinner(restaurantofKKH)

Day4 

Wednesday,4July2012 

4 th session[S4.]Newtechniquesinbacteriology,parasitologyandpathology 

(newadvancesindiagnostictechniques) 


09:0009:45 [S4.]Keynotelecture:MALDITOFandothernewdiagnostictechniques.Dr.Markus 

Kostrzewa,DirectorofMolecularBiology,R&D,BrukerDaltonikGmbH,Fahrenheitstr.4,D28359 

Bremen,Germany 

09:4511:15 [S4.]Oralpresentations(6) 

1. Suitabilityofrecombinantproteinsforthediagnosisofleptospirosisinpigs.Ulrike 

Ripp,Synlab.vetLeipzig,Germany. 

2. BiologgenerationIII,matrixassistedlaserdesorption/ionisationtimeofflight(MALDI 

TOF)massspectrometryand16srRNAgenesequencingfortheidentificationof 

bacteriaofveterinaryinterest.PeterWragg,AnimalHealthandVeterinary 

LaboratoriesAgency(Weybridge),UnitedKingdom. 

3. Evaluationofthediagnosticperformanceofpeptidecocktailsintheinterferongamma 

assayfordiagnosisoftuberculosisincattle.AlexRaeber,PrionicsAG,Switzerland. 

4. Matrixassistedlaserdesorptionionizationtimeofflightmassspectrometry(MALDI 

TOFMS)inaveterinarydiagnosticlaboratory.AnnetHeuvelink,GDAnimalHealth 

Service,TheNetherlands. 

5. RapididentificationofbovinemastitispathogensusingMALDITOF.GudrunOveresch, 

InstituteofVeterinaryBacteriology,UniversityofBern,Switzerland. 

6. 15minuteELISAusingalowcostcommercialbiosensor.JasonSawyer,AnimalHealth 

andVeterinaryLaboratoriesAgency(Weybridge),UnitedKingdom. 

11:1511:45 Closingceremony,EAVLD2014 

11:4513:00 Lunch(restaurantofKKH)+postersession+sponsors’exhibitions(patioofKKH) 

13:00 EndoftheCongress 

(shuttlebusestoWarsawAirport)

GD Animal Health Service 

Custom made 

animal health solutions 

GD Proficiency testing schemes 

R&D 

Veterinary laboratory 

GD Diagnostics 

Consultancy 

www.gddeventer.com

Oral presentations 

“General Session” 

(1 st session)

S1 - K - 01 

THE EMERGENCE OF SCHMALLENBERG VIRUS – HOW TO RESPOND TO NEW EPIZOOTICS IN 

EUROPE 

Wim H. M. van der Poel 

Central Veterinary Institute of Wageningen University and Research Centre, Lelystad, The Netherlands, Tel. +31320238383, email: 

wim.vanderpoel@wur.nl 

Schmallenberg virus, epizootic, sheep, cattle, goat 

The emergence of Schmallenberg virus 

Schmallenberg virus was discovered in November 2011, 

and named after the village in Germany where it was 

first detected in blood samples from a dairy herd (1). 

The provisionally named “Schmallenberg virus” is an 

enveloped, negative-sense, segmented, single-stranded 

RNA virus. It belongs to the Bunyaviridae family, within 

the Orthobunyavirus genus. Schmallenberg virus is 

related to the Simbu serogroup viruses, which also 

includes ruminant viruses like Shamonda, Akabane, 

Sathuperi, Douglas and Aino virus. Based on what is 

already known about the genetically related Simbu 

serogroup viruses, Schmallenberg virus affects domestic 

ruminants. At its first occurrence in dairy cattle in both 

Germany and The Netherlands Schmallenberg virus 

infections presented with fever and reduced milk yield, in 

the Netherlands also severe diarrhoea (2). In early 

December 2011, congenital malformations were reported 

in new-born lambs in the Netherlands, and 

Schmallenberg virus was detected in and isolated from 

the brain tissue. Thereafter the virus was also detected 

in malformed calves and goat kids. Gross pathology in 

malformed animals and stillbirths (calves, lambs, kids) 

included arthrogryposis, hydrocephaly, brachygnathia 

inferior, ankylosis, torticollis, scoliosis, hydranencephaly, 

hypoplasia of the central nervous system, porencephaly 

and subcutaneous oedema (3). The symptoms can be 

summarised as arthrogryposis and hydranencephaly 

syndrome (AHS). The spatial and temporal distribution 

suggests that the disease is first transmitted by insect 

vectors, in particular culicoides spp and then vertically in 

utero. The detection of SBV in midges (culicoides spp) in 

several countries supports this assumption. 

The emergence of Schmallenberg virus in Europe 

resulted in a rapid increase of malformations in new 

borne lambs and later on calves. In spring 2012 the 

numbers of cases started to decrease, first in sheep and 

then in cattle. By May 2012 Schmallenberg virus had 

affected farms in at least 8 countries in Western Europe, 

and although the epidemic curve seemed to come to an 

end by May 2012 a further spread over Europe still is 

likely (4). Schmallenberg virus clearly causes severe 

disease in ruminants and as a result economic losses 

which may be enhanced by trade restrictions. As the 

family of Bunyaviridae contains several important 

zoonoses, studies were performed to elucidate its 

zoonotic potential. In a rapid risk assessment in Dec 

2011 it was concluded that human infections were 

unlikely but could not be excluded. Therefore both in the 

Netherlands and Germany serosurveys in the human 

population were performed. In the Netherlands 301 

persons exposed to SBV, farmers and veterinarians, 

were tested and in North Rhine-Westphalia 60 cattle and 

sheep farmers were tested. None of the tested 

individuals showed antibody to SBV and it was concluded 

that there is no evidence for zoonotic infection (5). 

How to respond to new epizootics in Europe 

The “Schmallenberg virus experience” again has shown 

that the introduction of a completely new virus on the 

continent can encompass an important threat to animal 

health and public health. Moreover, continued changes in 

human and animal demography, coupled with 

environmental changes and changes within a virus itself 

make it likely that the trend for increased viral disease 

emergence will continue. 

Strategies to improve veterinary and public health 

protection with regard to emerging pathogens have 

focused towards improved surveillance. Improved 

detection of viruses in reservoirs, early disease outbreak 

detection, or broadly based research to clarify important 

factors that favour (re-)emergence. In order to recognize 

and combat viral diseases, it is pivotal to understand the 

epidemiology of these infections. We need to know the 

pathogen, its vertebrate hosts and the methods of 

transmission between these hosts. This should be 

coupled with knowledge of spatio-temporal disease 

patterns together with changes over time. Together, 

these can be used to build a picture of the dynamic 

processes involved in virus transmission that can be 

used to account for observed disease patterns and 

ultimately to forecast spread and establishment into new 

areas. 

Longitudinal veterinary surveillance should include food 

producing animals as well as wildlife and also insect 

vectors should be considered. A main goal of infectious 

disease surveillance is the early detection of new 

emerging pathogens. For this we will primarily be 

dependant of clinicians and laboratories testing field 

samples from potential reservoirs. Case reports will have 

to be generated and combined to early detect new 

emerging pathogens. Electronic systems, preferably 

web-based, could be very helpful to achieve this. In 

addition, improved detection may also be achieved 

through use of syndromic approaches. Syndromic 

surveillance, which collects non-specific syndromes 

before diagnosis, has great advantages in promoting the 

early detection of new emerging diseases before disease 

confirmation. By combining syndromic surveillance with 

case report surveillance in online reporting systems, a 

sensitive early detection system for new emerging 

diseases could be build. 

Novel molecular methods, for example DNA microarrays 

and whole genome approaches offer unprecedented 

opportunities for rapid detection but these require 

significant optimisation and validation before they can be 

deployed broadly. Also due to costs limitations, the rapid 

detection of a new virus will only be feasible by 

employing the different molecular techniques, including 

microarray, (RT)-PCR and whole genome sequencing, in 

a sensible combination. By applying molecular 

approaches, positive detections of a lot of different 

pathogens in a lot of different samples have been 

performed. However, it is much more difficult to proof 

causation. The agent should be present at high 

concentrations and seroconversion should be 

demonstrated. Confidence in a causal relationship 

between a candidate pathogen and a disease is 

enhanced by fulfilment of Kochs’ Postulates (i.e. 

demonstration of the presence of an agent in all cases of 

a disease and not in the absence of disease, replication 

of disease following ex vivo cultivation and introduction 

into a naïve host); however, this will not always be 

feasible. Apart from the fact that this can be extremely 

time-consuming, some viruses cannot be cultured and 

experimental infection can be extremely difficult. 

Prompt detection and instigation of control measures 

such as vaccination are crucial to prevent spread. Cloned

antigens or attenuated vaccines can be modified into the 

appropriate antigenic forms and in the case of 

arboviruses, approaches targeting replication in the 

arthropod vector, or the vectors themselves, may offer 

substantial benefit for control. However, the 

development of a safe and efficacious vaccine including 

its registration for use is very time-consuming. Moreover 

history has learned that it is extremely difficult to swiftly 

design a new reactive vaccination strategy, which means 

that in the first phase of every new epidemic we will 

have to rely on biocontainment and biosecurity 

measurements. 

National and International cooperation can be helpful to 

early identify new pathogens and will be needed to 

develop innovative and rapid control strategies to 

combat emerging diseases. Therefore this should be 

stimulated as much as possible. Knowledge exchange 

and research networking should become a commonplace. 

In addition the One Health approach, involving inclusive 

collaborations between physicians, veterinarians and 

other health and environmental professionals, will be 

more and more important to combat emerging viral 

disease outbreaks. National authorities as well as 

international organisations like WHO, OIE, FAO and also 

ECDC therefore should actively support such cooperative 

initiatives to achieve an optimal response to new 

epizootics in Europe. 

References 

1. Hoffmann B, Scheuch M, Höper D, Jungblut R, Holsteg M, 

Schirrmeier H, Eschbaumer M, Goller KV, Wernike K, Fischer M, 

Breithaupt A, Mettenleiter TC, Beer M (2012) Novel 

orthobunyavirus in Cattle, Europe, 2011. Emerg Infect Dis. 18: 

469-472. 

2. Muskens J, Smolenaars AJ, van der Poel WH, Mars MH, van 

Wuijckhuise L, Holzhauer M, van Weering H, Kock P (2012) 

Diarrhea and loss of production on Dutch dairy farms caused by 

the Schmallenberg virus. Tijdschr Diergeneeskd. 137:112-115. 

3. Van den Brom R, Luttikholt SJ, Lievaart-Peterson K, 

Peperkamp NH, Mars MH, van der Poel WH, Vellema P (2012) 

Epizootic of ovine congenital malformations associated with 

Schmallenberg virus infection. Tijdschr Diergeneeskd. 137:106- 

111. 

4. Armin R.W. Elbers , Willie L.A. Loeffen, Sjaak Quak, Els de 

Boer-Luijtze, Arco N. van der Spek, Ruth Bouwstra, Riks Maas, 

Marcel A.H. Spierenburg, Eric P. de Kluijver, Gerdien van Schaik, 

Wim H.M. van der Poel (2012) Seroprevalence of Schmallenberg 

Virus Antibodies among Dairy Cattle, the Netherlands, Winter 

2011–2012. Emerging Infectious Diseases 18:.. 

5 .Chantal Reusken, Cees van den Wijngaard, Paul van Beek, 

Martin Beer, Gert-Jan Godeke, Leslie Isken, Hans van den 

Kerkhof, Wilfrid van Pelt , Wim van der Poel, Johan Reimerink, 

Peter Schielen, Jonas Schmidt-Chanasit, Piet Vellema, Ankje de 

Vries, Inge Wouters and Marion Koopmans (2012) Public health 

response to an emerging arbovirus: the case of Schmallenberg 

virus (submitted for publication).

S1 - K - 02 

ASSESSING THE ANTIMICROBIAL SUSCEPTIBILITY OF BACTERIA OBTAINED FROM ANIMALS 

Stefan Schwarz 1 , Peter Silley 2,3 , Shabbir Simjee 4 , Neil Woodford 5 , Engeline van Duijkeren 6 , Alan P. Johnson 7 

and Wim Gaastra 8 

1 

Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Neustadt-Mariensee, Germany 

2 

MB Consult Limited, Lymington, UK 

3 

Department of Biomedical Sciences, University of Bradford, Bradford, UK 

4 Elanco Animal Health, Basingstoke, UK 

5 Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency, London, UK 

6 

Centre for Infectious Disease Control Netherlands (CIb), National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands 

7 Department of Healthcare-associated Infections and Antimicrobial Resistance, Centre for Infections, Health Protection Agency, London, UK 

8 

Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands 

Antimicrobial susceptibility testing, interpretive criteria, MIC 50 , MIC 90 , multi-resistance 

Introduction 

In recent years, antimicrobial resistance in bacteria of animal 

origin, including food-producing animals, pet and companion 

animals, fish and other aquatic animals as well as wild animals, 

has gained particular attention. Consequently, an increasing 

number of studies that include antimicrobial susceptibility testing 

have been published. However, an analysis of recently published 

articles revealed a number of frequently occurring shortcomings, 

which may have an impact either directly on the quality of the 

results obtained or on the conclusions drawn. An editorial has 

been published simultaneously in Veterinary Microbiology (1) and 

in the Journal of Antimicrobial Chemotherapy (2) and is intended 

to highlight the major pitfalls and provide guidance for authors, 

and reviewers on the correct performance of antimicrobial 

susceptibility testing as well as the presentation of the obtained 

results and the proper comparison of data from different studies. 

Methodology 

Several methods, like disc diffusion, E-test, agar dilution, broth 

microdilution and broth macrodilution are suitable for in-vitro 

antimicrobial susceptibility testing (AST). Whichever method is 

used, the tests have to be performed in accordance with an 

internationally accepted procedure, such as those published by 

the Clinical and Laboratory Standards Institute (CLSI) among 

others. The documents issued are regularly updated and, since 

the methodologies and interpretative criteria change over time, it 

is important to always follow the latest edition. In contrast to most 

other organizations, the CLSI is unique in that it produces 

separate documents for use in human and veterinary 

microbiology. The CLSI documents are not freely available, but 

must be purchased. 

The status of the various types of documents is clarified below. 

The CLSI for example differentiates between “standards” and 

“guidelines”. A “standard” is a document that clearly identifies 

specific and essential requirements for materials, methods, and 

practices to be used in an unmodified form. A standard may, in 

addition, contain discretionary elements, which are clearly 

identified. In contrast, a “guideline” is a document describing 

criteria for a general operating practice, procedure, or material for 

voluntary use. A guideline can be used as written or modified by 

the user to fit specific needs. 

The current CLSI document for testing antimicrobial 

susceptibilities of bacteria isolated from animals, M31-A3 (3), is 

an approved standard and cannot be used in a modified form. 

Clear and precise instructions on how to perform AST in vitro are 

given. They include, for example, the medium to be used 

(including any supplements required to support the growth of 

specific bacteria), the inoculum density, the incubation time, the 

temperature and test conditions. These instructions are not 

optional, but are strict rules that must be adhered to for good 

laboratory practice. Thus, statements such as “Susceptibility 

testing mainly followed the recommendations given in the CLSI 

document M31-A3” are not acceptable. Any deviation from the 

approved test conditions, such as the use of a different medium 

or extended incubation times for slow-growing bacteria, have to 

be specified and justified by the authors. 

Most AST documents cover testing of numerous different 

bacterial species. However, for several bacterial pathogens 

relevant to the veterinary field, such as Haemophilus parasuis or 

Riemerella anatipestifer, no approved methodology exists. If 

authors adopt a method approved for a phylogenetically closelyrelated 

organism, it must be stated clearly that the method used 

has not been approved for the species tested, but for another 

member the same genus (e.g. if the method for testing 

Haemophilus influenzae is used to test H. parasuis). Whenever 

susceptibility testing is undertaken on bacteria for which there is 

no approved standard available, the methodology chosen has to 

be validated first, as detailed in CLSI document M37-A3 (4). 

Quality controls 

It is essential to test approved AST reference strains in parallel 

with the test strains for quality control (QC) purposes. Lists of 

approved reference strains are included in the documents 

mentioned. They also contain acceptable MIC and zone diameter 

ranges for these reference strains and clearly state the 

methodology (e.g. broth microdilution) and the medium (e.g. 

Mueller-Hinton agar) that the values relate to. The reference 

strains must be relevant to the bacterial species tested, e.g. 

Escherichia coli ATCC 25922 may be used when testing 

Enterobacteriaceae. Furthermore, authors must ensure (a) that 

reference strains are suitable for quality control of the 

antimicrobial agents tested, (b) that the range of concentrations 

(in broth microdilution) tested spans the entire approved QC 

ranges, and (c) that discs (in disc diffusion tests) contain the 

quantity of antimicrobial for which the QC ranges are approved. 

Interpretation of the results 

AST studies seek to categorize bacterial isolates as susceptible, 

intermediate or resistant to each antimicrobial tested, on the 

basis of the MICs or the zone diameters obtained. Such 

classification requires approved interpretive criteria. Currently, 

two different types of interpretive criteria are available, clinical 

breakpoints and epidemiological cut-off values (5,6). The precise 

emphasis of a particular study will dictate which criteria must be 

applied. If data are intended to guide a therapeutic approach (i.e. 

the aim of the study is to determine which antimicrobial agents 

are most likely to lead to therapeutic success), clinical 

breakpoints must be applied. Epidemiological cut-off values 

should be used to describe MIC distributions of bacteria without 

clinical context. Clinical breakpoints and epidemiological cut-off 

values may be very similar or even identical for some 

bacteria/drug combinations; however, authors need to 

understand that epidemiological cut-off values are determined by 

a different approach than clinical breakpoints and do not 

necessarily take into account the results of clinical efficacy 

studies, dosing and route of administration of the antimicrobial 

agents, nor the drug’s pharmacokinetic and pharmacodynamic 

parameters in the respective animal species. The term 

“breakpoint” should be used exclusively for clinical breakpoints 

and “susceptible”, “intermediate” and “resistant” categories 

should also be reserved for classifications made in relation to the 

therapeutic application of antimicrobial agents. When reporting 

data using epidemiological cut-off values, the term ‘resistant’ is 

inappropriate, rather bacteria should be reported as ‘wild type’ if 

the MIC or zone diameter falls within the wild type range, or ‘nonwild 

type’ if the MIC is higher or the zone diameter smaller than 

the wild-type range. 

The CLSI document M31-A3 (3) lists exclusively clinical 

breakpoints and includes the largest collection of approved 

clinical breakpoints for bacteria of animal origin currently 

available, a considerable number of which represent veterinaryspecific 

breakpoints. Many of the latter have been approved for 

specific disease conditions often caused by particular bacterial 

species in defined animal host species. For example, approved 

clinical breakpoints for enrofloxacin in cattle apply exclusively for 

bovine respiratory diseases due to Pasteurella multocida, 

Mannheimia haemolytica and Histophilus somni. The use of 

these breakpoints for other bovine bacteria and different disease

conditions, e.g. Staphylococcus aureus from bovine mastitis, is 

unacceptable. Thus, the scope of application of the veterinaryspecific 

breakpoints is clearly defined and cannot be altered. 

All standards for performance of AST contain interpretive 

criteria which refer specifically to that particular methodology. 

Thus a certain methodology and its associated interpretive 

criteria are an entity, and as such belong together. It is not good 

practice to ‘mix and match’ testing methodologies and interpretive 

criteria issued by different organizations. Authors who perform E- 

tests must refer to the interpretive criteria given by the 

manufacturer of the E-test strips. Since these interpretive criteria 

are not veterinary-specific, but are adopted from human 

medicine, their true value for veterinary pathogens is unkown. 

The same holds for CLSI-approved breakpoints adopted from 

human medicine and listed in CLSI document M31-A3 (3). 

Authors who describe AST of animal isolates, often use incorrect 

or out-dated interpretive criteria derived from their own or others’ 

previous publications. This is also bad practice and often results 

in cumulative errors. Authors must ensure that correct (at the 

time of submission) interpretive criteria are used. In addition, 

there is an onus on reviewers to verify whether the correct 

interpretive criteria were used. 

When comparing rates (percentages) of resistance between 

published studies, authors must make sure that the same 

methodologies and the same interpretive criteria have been used. 

Interpretive criteria often change over time and lowering the 

breakpoint(s) for a specific antimicrobial agent will result in a 

higher percentage of isolates being classified as resistant even if 

the MIC/zone size distribution of the population has not changed. 

As a consequence, an artefactual increase in the percentage of 

resistant strains may be noted (Fig. 1). Publication of the full MIC 

distributions for each species/drug combination reduces the 

potential for this error since the data can be re-analysed by 

others if interpretive criteria change. 

(a) 

Number of Salmonella isolates 

(b) 

Number of Salmonella isolates 

2500 

2006 

2000 

1500 

1000 

500 

0 

2500 

2011 

2000 

1500 

1000 

500 

0 

susceptible: 2 mg/L 

susceptible: 0.06 mg/L 

MARAN 2004 (n = 2195) 

MARAN 2005 (n = 2238) 

resistant: 0.12 mg/L 

56 94 60 13 2 2 

0.06 0.12 0.25 0.5 1 2 4 8 16 

MIC values of ciprofloxacin in mg/L 

resistant: 4 mg/L 

63 79 33 8 5 1 

0.06 0.12 0.25 0.5 1 2 4 8 16 

MIC values of ciprofloxacin in mg/L 

0.27 % 

10.14 % 

Figure 1: Distribution of ciprofloxacin MIC values among 

Salmonella isolates collected in the MARAN studies 2004 and 

2005 and distinctly different percentages of resistance resulting 

from the use of different interpretive criteria in 2004 (a) and in 

2005 (b) 

Such tables or histograms are often large and due to limitations 

on journal space, may need to be provided as supplemental 

material. 

Before performing disc diffusion, authors need to make sure that 

the discs contain the correct quantity of antibiotic for which 

interpretive criteria are available. Unfortunately, for many 

antimicrobial agents a range of discs with varying amounts of the 

antimicrobial agent are commercially available. However, zone 

diameter interpretive criteria are commonly available only for a 

single specific disc strength. For example, discs charged with 10 

µg, 15 µg or 30 µg erythromycin are available, but CLSI 

interpretive criteria and QC ranges for reference strains (3) refer 

only to zone diameters around a 15 µg disc. Since it is not 

possible to adjust the values measured with a 10 µg or a 30 µg 

disc to the values for an approved 15 µg disc, zone sizes 

obtained with the 10 µg or a 30 µg disc can neither be interpreted 

nor validated. 

A standard dilution series for AST consists of doubling antibiotic 

concentrations and includes the reference concentration 1 mg/L 

(e.g. 0.125, 0.25, 0.5, 1, 2, 4, 8 mg/L etc.). E-test strips indicate 

half-log values and so MICs determined by E-test should be 

‘rounded up’ to the next highest value on the standard series. For 

example, if an E-test indicates that growth is inhibited at 0.38 

mg/L (which is not a concentration in the standard series), the 

MIC should be rounded up and reported as 0.5 mg/L. 

MIC 50 and MIC 90 values 

MIC 50 and MIC 90 values as well as the range of values obtained 

are important parameters for reporting results of susceptibility 

testing when multiple isolates of a given species are tested. The 

MIC 50 represents the MIC value at which at least 50% of the 

isolates in a test population are inhibited; it is equivalent to the 

median MIC value. Given n test strains and the values y 1 , y 2 , ..., 

y n representing a graded series of MICs starting with the lowest 

value, the MIC 50 is the value at position n x 0.5 as long as n is an 

even number of test strains. If n is an odd number of test strains, 

the value at position (n+1) x 0.5 represents the MIC 50 value. The 

MIC 90 represents the MIC value at which at least 90% of the 

strains within a test population are inhibited; the 90 th percentile. 

The MIC 90 is calculated accordingly, using n x 0.9. If the resulting 

number is an integer, this number represents the MIC 90 ; if the 

resulting number is not an integer, the next integer following the 

respective value represents the MIC 90 . MIC 50 and MIC 90 values 

should always be presented as concentrations on the standard 

AST dilution series. If using a statistical package to calculate the 

values, never use intermediate values. It should be noted that 

MIC 50 and MIC 90 values are not necessarily suitable parameters 

to describe bimodal or trimodal MIC distributions, although a 

discrepancy of several dilution steps between the MIC 50 and 

MIC 90 values, e.g. MIC 50 at 0.25 mg/L and the MIC 90 at 16 mg/L, 

might point towards the presence of at least two subpopulations 

which differ distinctly in their MICs to a given antimicrobial agent. 

As an example, in a test population of 70 strains, the MIC 50 is the 

value at position 35 and the MIC 90 the one at position 63 in a 

graded series of MICs starting with the lowest MIC value at 

position 1. In a test population of 71 strains, the MIC 50 is the 

value at position 36 and the MIC 90 the one at position 64 in the 

aforementioned graded series of the MICs. 

Although MIC 50 and MIC 90 values can also be calculated for 

small test populations of e.g. 10–30 strains, under such 

conditions few strains with high MICs will have a 

disproportionately high influence on the MIC 50 and MIC 90 values. 

Thus, researchers are encouraged not to overemphasize MIC 50 

and MIC 90 data obtained from small test populations. Since the 

significance of MIC 50 and MIC 90 increases with the numbers of 

strains tested, sufficiently large test populations should be used 

for most meaningful statements on MIC 50 and MIC 90 values. 

Multi-resistance 

The term ‘multi-resistance’ exclusively refers to acquired 

resistance properties. Bacteria may occasionally exhibit intrinsic 

(primary) resistance to certain antimicrobial agents. Intrinsic 

resistance may be based on either the lack or the inaccessibility 

of the antimicrobial target site among the bacteria in question. In 

other cases, intrinsically resistant bacteria produce inactivating 

enzymes, such as species-specific β-lactamases, contain 

multidrug transporters, and/or exhibit permeability barriers. Such 

intrinsic resistances must be excluded when describing multiresistance 

patterns. 

There is no universally accepted definition of ‘multi-resistance’. 

As a consequence, this term is used inconsistently in the 

literature. The following suggestions are intended to provide 

guidance for the most accurate presentation of multi-resistance 

patterns:

(a) If only phenotypic susceptibility testing is performed, 

resistance to three or more classes of antimicrobial agents can 

be referred to as multi-resistance. For example, resistance to 

enrofloxacin, marbofloxacin, difloxacin and orbifloxacin 

represents resistance to one antimicrobial class, since all agents 

are fluoroquinolones and resistance is most likely mediated by 

the same mechanism(s). In the case of fluoroquinolones (and 

some other antimicrobial classes), resistance to a single 

representative of this class of antibiotic agent can reasonably be 

extrapolated to resistance (or reduced susceptibility) to other 

members of that class. However, single class representatives 

cannot always be validly defined, e.g. for -lactams and 

aminoglycosides. In these cases, resistance is not a class effect 

and multiple, diverse resistance mechanisms exist, each of which 

confers resistance to sub-groups of the respective antimicrobial 

class. Resistance to sub-groups should be counted separately 

e.g. resistance to streptomycin and spectinomycin is distinct from 

resistance to gentamicin, kanamycin and/or tobramycin. 

(b) If phenotypic susceptibility testing is supplemented with 

molecular analysis for the resistance genes present, multiresistance 

should be assessed at the molecular level. Bacterial 

isolates exhibiting the presence of three or more resistance 

genes or mutations, all of which are associated with a different 

resistance phenotype (i.e. affecting different antimicrobial classes 

or sub-groups), may be referred to as multi-resistant. Exceptions 

to this rule would include those cases where a single resistance 

gene or a gene complex is associated with resistance to 

structurally and/or functionally different antimicrobial agents, e.g. 

the gene cfr for resistance to phenicols, lincosamides, 

oxazolidinones, pleuromutilins, and streptogramin A antibiotics or 

the erm genes for combined resistance to macrolides, 

lincosamides and streptogramin B antibiotics. 

Bacteria of Animal Origin; A Report. (ISBN Number:1-56238-765-0). CLSI 

document X08-R. Wayne, PA: Clinical and Laboratory Standards Institute. 

Conclusions 

As indicated above, conducting AST and subsequent data 

interpretation is a complex matter. A number of competent 

authorities provide instructions for performing AST and data 

interpretation. Each should be followed precisely. Importantly, 

protocols for AST and data interpretation from different 

authorities cannot be interchanged. AST data intended for the 

recommendation of therapy should be interpreted and reported 

using clinical breakpoints, whereas AST data intended for 

surveillance purposes may be reported using epidemiological cutoff 

values. Moreover, the comparison of data generated in 

different studies requires not only a common methodology, but 

also the preferential presentation of the data as MIC distribution 

which allows for fast and easy re-evaluation of the original data 

even if the interpretive criteria change over time. In addition to the 

two editorials published in 2010 (1,2), CLSI has published in 

2011 a comprehensive report (CLSI document X08-R) 

“Generation, Presentation and Application of Antimicrobial 

Susceptibility Test Data for Bacteria of Animal Origin” (7). This 

report provides additional information on how to avoid mistakes in 

AST methodology, AST data reporting and AST data 

interpretation. 

References 

1. Schwarz, S, Silley, P, Simjee, S, Woodford, N, van Duijkeren, E, 

Johnson, AP, Gaastra, W (2010). Assessing the antimicrobial susceptibility 

of bacteria obtained from animals. Veterinary Microbiology, 141, 1-4. 

2. Schwarz, S, Silley, P, Simjee, S, Woodford, N, van Duijkeren, E, 

Johnson, AP, Gaastra, W (2010). Assessing the antimicrobial susceptibility 

of bacteria obtained from animals. Journal of Antimicrobial Chemotherapy, 

65, 601-604. 

3. Clinical and Laboratory Standards Institute (CLSI), 2008a. Performance 

standards for antimicrobial disk and dilution susceptibility tests for bacteria 

isolated from animals; approved standard – third edition (ISBN Number:1- 

56238-659-X). CLSI document M31-A3. Clinical and Laboratory Standards 

Institute, Wayne, PA, USA. 

4. Clinical and Laboratory Standards Institute (CLSI), 2008b. Development 

of in vitro susceptibility testing criteria and quality control parameters for 

veterinary antimicrobial agents; approved guideline - third edition (ISBN 

Number 1-56238-660-3). CLSI document M 37-A3. Clinical and Laboratory 

Standards Institute, Wayne, PA, USA. 

5. Bywater, R, Silley, P, Simjee, S (2006). Antimicrobial breakpoints – 

definitions and conflicting requirements. Veterinary Microbiology 118, 158- 

159. 

6. Simjee, S, Silley, P, Werling, HO, Bywater, R (2008). Potential confusion 

regarding the term ‘resistance’. Journal of Antimicrobial Chemotherapy 61, 

228-229. 

7. Clinical and Laboratory Standards Institute (CLSI) (2011). – Generation, 

Presentation and Application of Antimicrobial Susceptibility Test Data for

S1 - O - 1 

ORAL FLUIDS – SAMPLE MATRIX FOR EFFECTIVE HERD HEALTH MONITORING 

C.Boss 1 , R. Shah 2 , C. O’Connell 2 

1 

Life Technologies,Darmstadt, Germany 

2 

Life Technologies, Austin, USA 

Oral Fluids, PRRS, PCV2, SIV 

Introduction 

The use of oral fluid as a sample matrix in porcine reproductive 

and respiratory syndrome virus (PRRSV) surveillance has 

increased in many parts of the world over the last few years. 

Advantages of using this matrix are ease of collection, reduced 

stress to the pigs, the low cost of collection and minimum labor 

required. Successful implementation of sample testing through 

the use of this sample matrix has extended to the evaluation of its 

testing for other swine diseases, including porcine circovirus type 

2 (PCV2), swine influenza virus (SIV), and mycoplasma 

hyopneumoniae (M. Hyo). 

Materials & methods 

In a multi-center, collaborative study, cotton ropes were used to 

collect samples of oral fluid from pigs that were experimentally 

infected with PRRSV and PCV2. Additionally, oral fluid samples 

from SIV negative pens were spiked with SIV for evaluation. 

Nucleic acid was extracted from oral fluid samples using a 

MagMAX based magnetic bead extraction and then amplified 

using a TaqMan® based real-time PCR assay for each virus. The 

method is semi automated, involving the use of the MagMAX 

Express-96 Magnetic Particle Processor for the purification of 

nucleic acid. 

For PRRSV testing, in collaboration with Kansas State University, 

oral fluids were collected from pens on the day that the pigs were 

experimentally infected through 40 days post-infection. Serum 

was collected from each pig within the pen on the same day that 

oral fluids were collected from the pen. Serum samples and oral 

fluids were both tested using real-time PCR. 

For PCV2 testing, in collaboration with Iowa State University, 

twenty-four 21-day-old pigs free of PRRSV and SIV were 

assigned to 1 of 4 groups and housed in pens in separate rooms. 

Pen number One was the control (None-Challenge) group. Each 

pig in the other 3 groups received PCV2a strain at 11 weeks of 

age (dpi 0). Six pigs (Pen 3) were re-challenged with PCV2a 

strain at 35, 70, and 105 dpi. Each pig in Pen 4 group 

alternatively received PCV2a (dpi 0 and 70) and PCV2b (dpi 35 

and 105). The two PCV2a strains used were heterologous. Oral 

fluids samples were collected, dpi 0, 2, 4, 6, 8, 10, 12, 14, and 

weekly thereafter until dpi 140. The results are shown in Figure 2 

for the time periods tested using the PCV2 real-time PCR 

reagents (dpi 2-98 

For SIV testing, in collaboration with Iowa State University, a total 

of 180 oral fluid samples were spiked with high, medium and low 

copy numbers of SIV. The extracted nucleic acids were also 

tested with subtyping reagents for the identification of 

hemagglutinin and neuraminidase subtypes. 

of ~20 swine, the proposed method of sample preparation and 

nucleic acid purification efficiently processes multiple samples, 

thereby decreasing screening time. 

A major advantage of this high throughput method, combined 

with the ease of collection of oral fluid, is that large numbers of 

pigs can be tested without increased cost or labor. This provides 

a more efficient and cost effective testing environment and the 

ability to better curb incidences of infection. 

Acknowledgements 

Dr. Bob Rowland, Kansas State University 

Dr. Tanja Opriessnig, Iowa State University 

Dr. Christa Irwin, Iowa State University 

Dr. Jeff Zimmerman, Iowa State University 

Results 

PRRSV nucleic acid was amplified from all oral fluid samples 

derived from the experimentally infected pigs. Lower Cts, 

indicating a higher copy number of viral RNA, were present in the 

serum samples early post-infection; but similar levels were 

detected in serum and oral fluid at later collection points. These 

results indicate a high level of correlation between PRRSV 

detection from serum collected from individual animals and from 

the pen or herd based oral fluid samples. 

Using the SIV screening real-time PCR reagents, SIV nucleic 

acid was detectable in all of the spiked oral fluid samples and at 

each concentration level. Additionally, the results of the subtyping 

study indicate that all positive samples could be sub-typed. 

High titers of PCV2 were detected from day 12 through 28 post 

infection, and virus was detectable throughout the entire testing 

period (dpi 2 – 98). 

Discussion & conclusion 

In summary, oral fluid samples were demonstrated to provide 

sensitive detection of PRRSV, SIV, and PCV2. As these 

samples could be used for the detection of virus in a population

S1 - O - 2 

DIAGNOSTIC PROPERTIES OF SEVERAL PRRS ANTIBODY ELISAS IN THE CONTEXT OF A 

LONGITUDINALSTUDY OF A FARM USING DIFFERENT VACCINATION STRATEGIES 

Strutzberg-Minder K 1 , Seidenspinner M 2 , Schuh H 2 , Böttcher J 3 , Wendt M 4 , Leibold W 5 

1 IVD GmbH, Hannover, Germany, 

2 Dr. Hermann Schuh, Veterinary Practice, Ipsheim, Germany 

3 Bavarian Animal Health Service, Poing, Germany, 

University of Veterinary Medicine, Foundation, 4 Klinik für kleine Klauentiere, 5 Institute of Immunology, Hannover, Germany 

PRRS virus, serology 

Introduction 

Porcine reproductive and respiratory syndrome (PRRS) is one of 

the most important diseases in pig farms almost worldwide (1). 

The PRRS virus (PRRSv) causes severe economic losses due to 

reproductive failures in sows and gilts and respiratory distress in 

piglets and fattening pigs (1). Among the objectives of the study 

was a longitudinal analysis of the antibody response following 

different vaccination strategies by routine diagnostic ELISA. 


The study was performed on an intensive pig farm in Germany 

with about 120 sows, 450 raising sites and 500 fattened pigs in a 

closed system. 8 to 12 piglets of 6 sows examined preliminarily 

were reassembled in 4 different groups with a total of 15 piglets 

in each. Blood samples were taken at 10 weeks (w), 13 w, 16 w, 

21 w, 26 w and 27 w of age. Pigs were vaccinated with Porcilis ® 

PRRS (live European PRRSV strain DV, Intervet International, 

now: MSD Animal Health, USA) and Progressis ® 

(inactivated 

PRRSv European strain P120, Merial, UK) as described in the 

instruction manuals and in the following table. 

Table 1: Vaccination schedule 

Group 1 Group 2 Group 3 Group 4 

Age of pigs 

(red) 

(blue) 

(green) (yellow) 

8 Progressis ® 

11 

Porcilis ® Porcilis ® Porcilis ® 

PRRS 

PRRS 

PRRS 

20 Progressis ® Progressis ® 

25 

Porcilis ® Porcilis ® 

PRRS 

PRRS 

Progressis ® 

Porcilis ® 

PRRS 

Porcilis® 

PRRS 

Serum samples were analysed with the following PRRS antibody 

ELISAs: 

A) HerdChek* PRRS 2XR, IDEXX Laboratories, USA. A 

sample/positive control (S/P) ratio (blanked by normal host 

cells antigen for control) ≥ 0.400 is positive.* 

B) HerdChek* PRRS X3 (now: IDEXX PRRS X3), IDEXX 

Laboratories, USA. An S/P ratio (blanked by negative 

control, NC) ≥ 0.400 is positive.* 

C) INGEZIM PRRS UNIVERSAL, INGENASA, Spain. S/P ratio 

transformed in OD%; an OD% > 15 is positive. 

D) INGEZIM PRRS EUROPA, INGENASA, Spain. S/P ratio 


E) INGEZIM PRRS AMERICA, INGENASA, Spain. S/P ratio 


F) CIVTEST SUIS PRRS E/S (European strains), Laboratories 

HIPRA, Spain. An IRPC (Index Relative to Positive Control) 

value ((sample - NC) / (PC - NC) x 100) > 20 is positive. 

G) CIVTEST SUIS PRRS A/S (American strains), Laboratories 

HIPRA, Spain. IRPC value >20 is positive 

H) INGEZIM DR, INGENASA, Spain. S/P ratio transformed in 

OD%; an OD% > 17.5 is positive. 

I) PRRSv, BioChek, The Netherlands. An OD > 0.500 is 

positive.* 

*Results of ELISA with real numbers were multiplied by 100 to 

yield a positive integer for better comparison of the different 

ELISA results in one diagram (Fig. 1). ELISA H and I were used 

only for group 2. 

Results 

The individual course of antibody development detected by all 

ELISA differed most in group 4. However, the arithmetic means 

smoothed the individual differences (Fig. 1). Results for group 4 

were significantly higher (p ≤ 0.0606) than for group 1 and 2 at 13 

w. Maximum antibody levels were observed between 21 w and 

26 w (10 w to 15 w after priming with Porcilis ® PRRS) for all 

ELISA (Fig. 1). These peaks were more or less independent of 

subsequent boosting with Progressis ® or with Porcilis ® PRRS (s. 

Tab. 1). 

Mean of ELISA results 

3.500 

3.000 

2.500 

2.000 

1.500 

1.000 

0.500 

ELISA B 

0.000 

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

Age of pigs (weeks) 

Group 1 Group 2 Group 3 Group 4 

Figure 1: Pigs' developing antibody response (mean ELISA 

results) with 4 different PRRSv vaccination strategies detectable 

by different ELISAs (here: ELISA B) for PRRSv antibodies 

The earliest positive results after exposition with live PRRSv 

vaccine at 11 w were detected by ELISAs A and B in at least 6 

(group 3) of 15 samples and all means of all groups (Fig. 2) at 13 

w (2 w after priming with Porcilis ® 

PRRS). ELISA H tested 

positive in all but 2 samples at all dates before and after 

vaccination without significant changes in results (Fig. 2). The 

earliest positive results of the other ELISAs C, D, F and I in 

almost all samples and all means of groups 1-3 were at 16 w (5 w 

after priming with Porcilis ® PRRS) (Fig. 2). Only group 4 tested 

positive also by ELISAs C, D and F at 13 w. 

Mean of ELISA results 

450 

Group 2 

400 

350 

300 

250 

200 

150 

100 

50 

0 

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

Age of pigs (weeks) 

ELISA A ELISA B ELISA C ELISA D ELISA F ELISA H ELISA I 

Figure 2: Pigs' developing antibody response with different 

PRRSv vaccination strategies (here: group 2 mean ELISA 

results) detectable by different ELISAs for PRRSv antibodies 


As ELISA H results did not reflect any PRRS vaccination they 

were not useful for understanding antibody development as an 

immunological response to PRRSv. The earliest detection of 

exposing pigs to live PRRSv was possible with ELISA A and B at 

the first sampling (2 w after priming). Pre-immunization with 

Progressis ® (group 4) also made it possible to detect antibodies 

against PRRSv at 13 w by the other positive ELISAs C, D and F, 

but individual serological profiles were very heterogeneous, while 

all other individual serological profiles (groups 1-3) were more 

homogenous and represented appropriately by the group means. 

ELISAs A and B can be used at the latest 2 w after priming with 

PRRSv for early detection, and other ELISAs except E, G and H, 

for monitoring the antibody response. 

References 

1. Zimmermann et al. (2006) in: Straw et al. (eds.), Diseases of Swine, 

9thed., 387-417 

Note: These data are in part results of the thesis of Marc Seidenspinner

S1 - O - 03 

INTRODUCTION RATE OF A LOW PATHOGENIC AVIAN INFLUENZA VIRUS INFECTION IN 

DIFFERENT DUTCH POULTRY SECTORS 

Jose Gonzales 1 , Guus Koch 1 , Ruth Bouwstra 1 , Armin Elbers 1 , J.J. de Wit 2 , Arjan Stegeman 3 

1 Central Veterinary Institute, of Wageningen University and Research Centre (CVI-Lelystad), Lelystad, The Netherlands 

2 Animal Health Service, Deventer, The Netherlands 

3 Utrecht University, Faculty of Veterinary Medicine, Department of Farm Animal Health, Utrecht, The Netherlands 

Avian Influenza, low pathogenic, introduction rate 

Introduction 

Low Pathogenic Avian Influenza (LPAI) viruses of the H5 and H7 

subtype have the potential to evolute to Highly Pathogenic Avian 

Influenza (HPAI) viruses in poultry and therefore infections with 

these subtypes are notifiable . Consequently, member states of 

the European Union have implemented surveillance programmes 

(1). In the Netherlands a syndromic surveillance and serological 

monitoring programme is in place. In the monitoring programme, 

all poultry farms are tested 1-4 times a year. Frequency differs 

between the different poultry types and housing systems (indoor 

and outdoor layer chickens, broilers, ducks, turkeys, etc) based 

on the supposed differences in the risk of introduction of LPAI 

infections. However, quantitative information regarding the 

possible differences in risk between these poultry types is sparse. 

In this study the rate of introduction of LPAI in different poultry 

types was quantified. . 


Data from the Dutch LPAI surveillance programme(2007–2010) 

were analysed using a generalised linear mixed and spatial 

model. All poultry farms should be tested at least once a year. In 

addition, outdoor-layer farms are tested 3 to 4 times per year. 

Farms were identified by their unique farm number (UBN) and 

poultry sector (duck-breeders, duck-meat (meat production), 

turkeys, broilers, indoor-layers, outdoor- layers, pullets and 

breeders). Based on the sampling frequency (time interval 

between samplings), the time at risk of exposure to a LPAI 

infection (“time at risk”) was calculated per poultry sector. For 

poultry sectors sampled once a year or once per production 

cycle, the age of the birds, at the moment of sampling, was taken 

as the time at risk. For poultry sectors sampled more than once 

per production cycle, the average sampling interval was taken as 

the time at risk. Positive cases were defined as: 1) farms with at 

least one seropositive animal – to any LPAI strain – in both, the 

screening test (IDEXX FLockCheck AI MultiS-Screen or agar gel 

precipitation, which is only used for broilers) and the confirmatory 

test (Hemagglutination Inhibition test) or 2) three or more 

positives in the screening test. Furthermore, only primary cases 

were included. 

Results 

The results are summarised in Table 1. Almost all seropositive 

results appeared to be single introductions. Results showed that 

outdoor-layer farms had a 11, turkey 8, duck-breeder 23 and 

meat-duck 13 times higher rate of introduction of LPAI than 

indoor-layer farms. 

Table 2. Relative risk (RR), with accompanying lower (LCI) and 

upper (UCI) 95% confidence intervals, of introduction of a LPAIv 

infection on poultry farms. Indoor - layers were considered as the 

reference category. 

Poultry Type 

RR 

Mean LCI UCL 

breeders 0.3 0.0 2.4 

pullets 0.7 0.1 5.7 

layers indoor 1.0 

layers outdoor 11.0 4.9 24.8 

turkeys 7.6 2.0 29.0 

duck meat 12.8 1.6 102.7 

duck breeders 23.0 6.2 85.7 


Our analysis shows that outdoor-layer farms, duck (breeders and 

meat) farms and turkey farms have a significantly higher risk of 

introduction of a LPAI infection compared to indoor-layer farms. 

Breeder ducks have the highest risk. This could be related to 1) 

their higher susceptibility to infection with LPAI of wild bird origin 

(ducks, geese, swans) than chickens, 2) their long production 

cycle (time of exposure), and 3) their higher exposure to LPAI 

from a contaminated environment and/or contact with wild 

waterfowl. The latter could also be the reason for the higher risk 

observed in outdoor-layer than indoor-layer farms. 

In the Netherlands, turkeys are raised indoors and despite the 

small population of turkey farms, we observed a higher risk of 

introduction of a LPAI infection on turkeys than indoor-layers. 

This higher risk might be partly associated with the apparent 

higher susceptibility of turkeys to LPAI infections than chickens 

(2). We also observed a significant higher risk of introduction in 

meat-duck farms. This was surprising because this poultry type is 

kept indoors and has a short production cycle (6 weeks). The 

above mentioned higher susceptibility of ducks than chickens 

could be one reason for the observed higher risk. Differences in 

the rate of introduction of LPAI could be used to (re)design a 

targeted risk-based surveillance programme. 

References 

1.European Commission, 2007. Commission Decision 2007/268/EC of 13 

April 2007 on the implementation of surveillance programmes for avian 

influenza in poultry and wild birds to be carried out in the Member States 

and amending Decision 2004/450/EC. OJEU ;L 115:3.5.2007, p.2003. 

2. Tumpey, T, M, Kapczynski, D, R, Swayne, D,E 2004. Comparative 

susceptibility of chickens and turkeys to avian influenza A H7N2 virus 

infection and protective efficacy of a commercial avian influenza H7N2 

virus vaccine. Avian Diseases 48:167-176.

S1 - O - 04 

A DUPLEX ONE-STEP REAL TIME RT-PCR FOR DIAGNOSIS OF FOOT AND MOUTH DISEASE 

Kamila Górna, Romey Aurore, Anthony Relmy, Aude Allmandou, Sandra-Blaise Boisseau, Stephan Zientara, 

Labib Bakkali Kassimi 

ANSES, Laboratoire de Santé Animale, UMR1161 ANSES INRA ENVA, Maisons-Alfort 94706 France 

Foot- and –Mouth Disease; Diagnosis; Real Time RT-PCR 

Introduction 

Foot-and-mouth disease (FMD) is a highly contagious transboundary 

disease of wild and domesticated cloven-hooved 

ruminants. It is one of the most economically devastating 

diseases in livestock, due to measures employed to control the 

outbreak and trade loss resulting from embargos on the country. 

The causative agent, the FMD virus (FMDV), is a single stranded 

RNA virus belonging to the Picornaviridae family, genus 

Aphtovirus. There are seven immunologically distinct serotypes 

of the virus, namely types O, A, C, Asia 1, SAT 1, SAT 2 and 

SAT 3. 

Quick diagnosis of FMD is critical for the rapid implementation of 

control measures to limit the spread of the virus and eradicate the 

disease. The recommended real-time RT-PCR method (rtRT- 

PCR) for detection of FMDV is a two-step simplex method 

targeting conserved regions in the internal ribosomal entry site 

within the 5’ untranslated region (IRES) and in the RNA 

polymerase gene (3D)(1). In parallel, a rtRT-PCR targeting a 

cellular gene (endogenous control) is performed to allow 

normalization of results. This method has the disadvantage of 

being performed in two separate steps (RT and PCR) and 

detects only one target at a time. It is therefore time-consuming, 

and since several pipetting steps are required, this method can 

be subject to possible errors and contamination. Multiplex rtRT- 

PCR is a modification of the basic rtRT-PCR method in which 

pairs of primers targeting different targets are used in the same 

reaction. In one-step multiplex rtRT-PCR, the reverse 

transcription and subsequent amplification take place in a single 

tube, thereby reducing the reaction time and number of reaction 

mixtures, and also diminishing the risk of contamination. In this 

study, duplex one-step rtRT-PCRs were established to detect 

and type FMDV. These methods are simple, specific, and 

sensitive for early diagnosis of FMDV infection. 

Material & methods 

Samples: Artificially FMDV spiked samples were prepared by 

making 10 fold serial dilutions of virus stock (strains O1 Manisa, 

A22 Iraq or Asia 1) in FMDV-free bovine tongue epithelial tissue 

suspension. Field bovine epithelium samples (n:19) were 

collected from suspected cases in West Africa. Each sample was 

subjected to virus isolation, Ag-ELISA, Chromatographic test 

(Svanodip) and rtRT-PCR. Virus Isolation: Samples were prediluted 

5 and 10 times in cell culture medium and inoculated into 

goat tongue cell cultures (ZZ-R). Chromatographic test (lateral 

flow device): Svanodip (SVANOVA Biotech AB) test was 

performed according to the manufacturer’s instructions. Antigen 

Capture Elisa was performed as described in the OIE manual 

(1). rtRT-PCR: RNA was extracted from samples by using the 

QIAamp Viral RNA mini-kit (QIAGEN). Two step simplex rtRT- 

PCRs for IRES, 3D and -actin targets were performed using 

TaqMan Reverse Transcription Reagents kit and TaqMan PCR 

Core Reagents kit (Life technologies) following protocols 

described previously (2, 3, 4). One Step duplex rtRT- PCRs were 

performed using the AgPath-IDOne-Step RT-PCR Reagents 

(Life Technologies). The thermal conditions were as follows: 10 

min at 45°C (RT), 10 min at 95°C (denaturation) and 45 cycles of 

15s at 95°C and 1 min at 60°C. The final concentration of primers 

and probes in duplex assay was optimized for IRES, 3D and - 

actin targets and was as previously described for the typing 

protocol (5). Each probe for FMDV specific targets, marked by 

FAM dye, was coupled in the same test tube with β-actin probe 

labelled with VIC dye. 

with two other vesicular disease viruses, swine vesicular disease 

and vesicular stomatitis viruses. Artificially FMDV spiked samples 

containing different amounts of virus were used to compare the 

sensitivity of FMDV diagnostic methods. Both one-step and twostep 

rtRT-PCR methods were 1000 to 10,000 fold more sensitive 

than the antigen capture Elisa and Svanodip test. VI and all rtRT- 

PCRs displayed quite similar sensitivity. The 3D/-actin duplex 

real-time one-step method gave better results than the two-step 

method. The serotype-specific one-step duplex rtRT-PCR was 

slightly less sensitive than the pan-FMDV one-step duplex rtRT- 

PCR method. A better detection of the internal β-actin control 

was observed with the one-step protocol. All rtRT-PCR methods 

were evaluated with 19 field samples. FMDV was isolated from 

all samples. The one-step duplex rtRT-PCR for both IRES and 

3D targets was more sensitive than the two-step simplex rtRT- 

PCR. Moreover, four samples found negative for IRES by a twostep 

method were positive using the one-step method. It was not 

possible to type the 19 field samples by rtRT-PCR, despite the 

presence of FMDV type O and A in these samples, as revealed 

by VP1 sequencing. 


This study reports the development of a one-step duplex rtRT- 

PCR method for detection of FMDV and characterization of the 

three most commonly occurring serotypes worldwide. This 

method is more sensitive than other recommended FMDV 

detection methods tested. Simultaneous detection of FMDV and 

-actin within the same reaction permits the exclusion of false 

negatives that may result from improper extraction or degradation 

of the RNA, and permits normalization of the results. Thus, this 

one-step duplex rtRT-PCR method could be of interest for FMD 

diagnostic laboratories. We were unable to type FMDV from the 

field samples by rtRT-PCR. Analysis of the FMDV VP1 sequence 

from these samples revealed multiple mismatches in the primer 

and probe sequences for type O and type A. This is not 

surprising since the primers and probes were designed from 

sequences of FMDV strains belonging to lineages not present in 

West Africa (5). New primers and probes specific to WA lineages 

will be designed and evaluated. The development of a multiplex 

one-step rtRT-PCR could provide an improved method for rapid 

detection of FMDV, as it should allow detection of more targets in 

the same test and in less time. 


The author thanks Jennifer Richardson for editing the English 

version of the manuscript. 

References 

1-Manuel of Diagnostic Test and Vaccines for Terrestrial Animals, chapter 

2.1.5.1C, 2009, OIE. 

2- M. Reid et al. Detection of all seven serotypes of foot-and-mouth 

disease virus by real-time, fluorogenic reverse transcription polymerase 

chain reaction assay. J. Virol. Methods, 2002, 105 : 67-80 

3- JD Callaham et al. Use of a portable real-time reverse transcriptasepolymerase 

chain reaction assay for rapid detection of foot-and-mouth 

disease virus. YAVMA, 2002, 220, 11:1636-42. 

4- J.F. Toussaint et al. Bluetongue virus detection by two real-time RTqPCR 

targeting two different genomic segments. J. Virol. Methods, 2007, 

140 : 115-128 

5- Reid et al. Detection of FMDV serotypes O, A and Asia1 by real-time 

RT-PCR. EuFMD 2008 Meeting. The global control of FMD – Tools, ideas 

and ideals – Erice, Italy 14-17 October 2008. 

Results 

One step duplex rtRT-PCR methods for pan-FMDV (IRES and 

3D) successfully detected all 7 serotypes of FMDV. The typing 

rtRT-PCRs detected exclusively the specific serotype and did not 

cross react with other serotypes. All tests gave negative results

S1 - O -05 

VALIDATION OF A COMPETITIVE ELISA FOR THE DETECTION OF ANTIBODIES ANTI-p26 OF 

EQUINE INFECTIOUS ANEMIA VIRUS IN EQUINE SERA 

R. Nardini 1 , G.L. Autorino 1 , R.Lorenzetti 1 , P. Cordioli 2 , 1 A. Caprioli, M.T. Scicluna 1 

1 

National Reference Centre for equine infectious diseases, Istituto Zooprofilattico Sperimentale Lazio e Toscana, 00178 Roma Italia. 2 Istituto 

Zooprofilattico Sperimentale Emilia Romagna e Lombardia, Via A. Bianchi 9, 25124 

Keywords: Equine infectious anemia, competitive ELISA, validation 

Introduction 

Equine infection anemia (EIA) is a viral infection of equidae, 

caused by a virus (EIAV) of the Lentivirus genus, Retroviridae 

family. Hematophagous insects belonging to the Tabanidae, 

Hippoboscidae and Muscinae families are involved as passive 

vectors in viral transmission. The infection is usually persistent, 

and can occur as an asymptomatic form or with recurrent febrile 

episodes. Since 2006, Italy has adopted a National Surveillance 

Plan (NSP) (1) consisting in a serological screening of all animals 

except for meat horses. According to the Italian Regulations, the 

official confirmatory test is the Agar Gel Immunodiffusion (AGID) 

test which detects antibodies anti-p26, a highly conserved 

capsidic protein of the virus. Since this technique has a low 

detection limit, development of new and more sensitive tests has 

become necessary. WOAH Manual (2) indicates ELISA, 

immunoblotting and PCR as confirmatory tests for the serological 

diagnosis of EIA. The ELISA, for its characteristics, is the most 

suitable for screening purposes. The aim of this paper is to 

present the results of validation of a competitive ELISA (c-ELISA) 

for the detection of antibodies anti-p26 of EIAV in equine sera. 

Materials and methods 

The procedure of the c-ELISA, object of this validation, is briefly 

described as following. A 96-well microplate is pre-adsorbed 

overnight at 4°C with a monoclonal antibody (Mab) anti-p26. 

Serum samples and a recombinant antigen p26, produced in E. 

coli, are mixed on a separate plate and incubated at 37°C for 75 

minutes. At the end of the incubation, the serum-antigen mix is 

transferred onto the pre-absorbed plate and a second Mab 

conjugated with horseradish peroxidase is added. After another 

incubation at 37°C for 75 minutes, ortophenyl-diamine substrate 

is added and the plate is incubated at room temperature for 15 

minutes in the dark. The reaction is stopped by the addition of 

sulphuric acid and the optical density (OD) is read with a 492nm 

filter. Sera are categorized as positive, negative or equivocal 

according to the percentage inhibition (PI), calculated as the ratio 

between the sample and the internal negative control. 

Validation of the c-Elisa was performed according to WOAH 

Manual guidelines (3) and the aim of this test was for screening 

purposes. 

For its validation the following parameters were evaluated. 

Analytical specificity was estimated at three different levels: 

1. Selectivity, defined as the capability to detect the target 

analyte in the presence of other interferences, was evaluated by 

changing the composition of the wash solution and processing 

positive and negative International Reference Serum (IRS). 

2. Exclusivity, considered as the capacity to discriminate 

target analyte from other crossreactive analytes, was evaluated 

processing ten sera positive for each of the following virus: Feline 

Immunodeficiency Virus, Feline Leukaemia Virus and Visna 

Maedi Virus. Each sample was repeated ten times. 

3. Inclusivity which is how a test can differentiate between 

different serovars. p26 protein is highly conserved in this virus 

and (4) thus evaluation was not necessary. 

Analytical sensitivity is represented by limit of detectability (LOD) 

and in this validation, the LOD of ELISA was compared with the 

AGID LOD, analysing progressive dilutions of a positive IRS in 

both methods. 

Repeatability was evaluated estimating the standard deviation 

(S r ) of the OD values of 30 repetitions of a negative IRS; this 

value was compared with another set of 30 values, processed by 

the same person during the same session. 

Reproducibility was estimated taking into account both qualitative 

and quantitative characteristics of ELISA test. 

a) To analyse qualitative reproducibility a Ring Test was 

performed by eleven laboratories and the statistic K of Cohen 

was calculated. 

b)Quantitative reproducibility was evaluated in seven sessions 

examining 30 replica of negative IRS in each, and calculating S R 

of OD values. 

Diagnostic performances were evaluated from sensitivity (Dse) 

and specificity (Dsp). Minimum number of samples to evaluate a 

sensitivity of 90% and a specificity of 80% with confidence 

interval of 99% and a precision of 5% was calculated. 1095 sera 

from field samples and from European Union Reference 

Laboratory, consisting in 857 positive and 238 negative samples 

were analysed with both ELISA and AGID, using the latter as 

Gold standard (GS). Dse, Dsp, positive and negative predictive 

values, efficacy and test Bias were calculated (5). 

For statistical analysis XL-Stat 2011 ® was used. 

Results 

1.ELISAs performed changing the washing solution did not 

correctly recognise positive and negative sera. 

2.All sera positive for other Lentivirus were classified as negative 

by the ELISA. 

3.LOD of ELISA test is at least 1 Log 10 more sensitive than AGID. 

4.Comparison between two data sets for evaluation of 

repeatability did not show significant differences with test F 

(p=0.42) and S r resulted to be equal to 0.184. 

5.Statistic K value was equal to 0.967. 

6.Comparison between seven data sets for evaluation of 

reproducibility did not show significant differences with test F 

(p=0,092) and S r was equal to 0.129. 

7.Dse and Dsp resulted respectively 100 and 80,3%. 

8.Positive and negative predictive values resulted respectively 

94.8% and 100% 

9.Efficacy of ELISA which is the percentage of samples correctly 

identified (positive and negative together) compared with the GS 

resulted equal to 95.7% 

10.Test Bias which is the ratio between apparent and real 

prevalence resulted 1.05. 

Discussion and conclusions 

The results of the parameters evaluated for the validation of the 

c-ELISA are all satisfactory. In particular, the lower Elisa LOD 

would allow not only the detection of “poor responders” then 

when using the AGID only, but also the possibility of detecting 

earlier, new cases. Although the Dsp is 80%, this value is still 

acceptable for a sreening test compared to the advantages that it 

presents to the AGID, i.e the possibility of its standardization, the 

objectivity of the results reading, the lower amount of reagents 

used and the higher processing of samples per unit time. The use 

of this diagnostic test is an added value, together with other 

measures, in a control programme for AIE. 

ReferenceS 

1.Ordinanza 8 agosto 2010 Piano di sorveglianza nazionale per l'anemia 

infettiva degli equidi. G.U. Serie Generale n. 219 del 18 settembre 2010 

2.Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2010, 

Chapter 2 . 5 . 6 .Equine infectious anaemia 

3.Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 

2010,Chapter 1.1.4/5. Principles of validation of diagnostic assays for 

infectious diseases. 

4.Charles J. Issel, R. Frank Cook (1993)REVIEW ARTICLE A review of 

techniques for the serologic diagnosis of equine infectious anemia J Vet 

Diagn Invest 5: 137-141 

5.Signorelli C. Elementi di metodologia epidemiologica SEU, 2005

S1 - O - 06 

DIAGNOSTIC PERFORMANCE OF A COMMERCIAL PRRS SERUM ANTIBODY ELISA ADAPTED TO 

ORAL FLUID SPECIMENS: FIELD SAMPLES 

Apisit Kittawornrat 1 , John Prickett 1 , Chong Wang 1, 2 , Chris Olsen 1 , Yaowalak Panyasing 1 , Andrea Ballagi 3 , 

Anna Rice 3 , Sergio Lizano 3 , John Johnson 1 , Rodger Main 1 , Chris Rademacher 4 , Marlin Hoogland 4 , 

Jeffrey Zimmerman 1 

1 Department of Veterinary Diagnostic and Production Animal Medicine, 2 Department of Statistics, Iowa State University, Ames, Iowa, United States, 3 IDEXX 

Laboratories, Westbrook, Maine, United States, 4 Murphy-Brown LLC, Ames, Iowa, United States 

Oral fluid, ELISA, Field samples, PRRSV 

Introduction 

Oral fluid samples are of interest because of their ease of 

collection and documented use in surveillance of PRRSV and 

other pathogens (1,2). Previous work showed that a commercial 

PRRS ELISA (HerdChek® PRRS X3 ELISA) could be adapted to 

detect anti-PRRSV IgG in oral fluid specimens (IgG ELISA). The 

objective of the current study was to evaluate the ability of the 

assay to detect anti-PRRSV IgG antibody in pen-based oral fluid 

field samples. 

References 

1. Kittawornrat A, et al. 2010. PRRSV in serum and oral fluid samples from 

individual boars. Virus Res 154:170-176. 

2. Prickett, J et al.: 2011. Prolonged detection of PCV2 and anti-PCV2 

antibody in oral fluids following experimental inoculation. Transbound 

Emerg Dis 58:121-127 

3. Ramirez et al. 2012. Efficient surveillance of pig populations using oral 

fluids. Prev Vet Med (Epub ahead of print). 


Positive samples were derived from a longitudinal field study in 

10 wean-to-finish barns (3). At each site, oral fluid samples were 

collected from the same 6 pens at 2-week intervals (total of 10 

sampling points per barn). Positive oral fluid samples were 

defined as all samples collected from a pen after the first PRRSV 

PCR positive oral fluid sample from that pen (n = 250). Negative 

oral fluid (n = 284) field samples were diagnostic samples 

submitted to the ISU VDL for PRRSV qRT-PCR testing from 

expected-negative herds. 

Results 

The cumulative S/P results for defined positive and negative oral 

fluid samples are given in Figure 1. Of 284 expected-negative 

field samples, all were negative on the IgG ELISA (S/P

S1 - O - 07 

SUBTYPING OF SWINE INFLUENZA VIRUSES BY MULTIPLEX REAL-TIME PCR 

Kees van Maanen 1 , Ingrid Wiggers 1 , Chris Schouten 3 , Tom Duinhof 1 , Paolo Cordioli 2 , Remco Dijkman 1 

1 

GD-Animal Health Service, Diagnostics,Research and Epidemiology,Deventer , the Netherlands 

2 Istituto Zooprofilattico Esperimentale Brescia, the Netherlands 

3 DAP Aadal, Erp, the Netherlands 

Swine Influenza Virus; real-time PCR; subtyping; lung lavage samples; diagnosis 

Introduction 

Swine influenza is an acute respiratory disease of swine caused 

by type A influenza viruses. In Europe, swine influenza is 

considered one of the most important primary pathogens of swine 

respiratory disease and infection is primarily with H1N1, H1N2 

and H3N2 influenza A viruses. Avian-like H1N1 and human-like 

H3N2 swine influenza viruses (SIV) have been considered 

widespread among pigs in Western Europe since the 1980s, and 

a novel H1N2 reassortant with a human-like H1 emerged in the 

mid 1990s. These viruses have remained endemic in European 

pig populations but significant differences in the circulation of 

these strains occur at a regional level across Europe (1,2). 

For diagnosis of swine influenza infections serological and 

virological tests can be used. For detection of subtype-specific 

antibodies HI-tests are commonly used. However, crossreactions 

can occur, and the interpretation can be difficult 

depending on the vaccination and infection history of the herd. 

For diagnosis of recent infections paired serum samples are 

required collected with a 2-3 week interval. 

For rapid detection and discrimination of the different swine 

influenza subtypes several PCRs have been described. However, 

these methods were either conventional PCRs, targetted only the 

Haemagglutinin gene, or the coverage for European SIV strains 

was too low according to our BLAST analysis results. 

Therefore we decided to develop an in-house subtype-specific 

SIV real-time PCR. In the course of the project a prototype realtime 

PCR for detection and differentiation of SIV (Life 

Technologies) became available and was also evaluated. 


In this study two RNA extraction methods (High Pure nucleic acid 

kit, Roche Applied Science, and MagMax AM1836, Life 

Technologies) were compared in combination with either the inhouse 

designed primer/probe sets + AgPath-ID multiplex onestep 

RT-PCR kit (Life Technologies) or a prototype SIV 

Screening qRT-PCR kit (Life Technologies). All tests were run on 

an ABI7500 fast platform. 

Primers and probes were designed in silico using GenBank SIV 

sequences of the last 15 years. Primers and probes should have 

a coverage of at least 95%, and were used in working dilutions of 

10 and 5 µM, respectively. Primers and probes were designed to 

enable all sorts of combinations (singleplex, multiplex H+N, 

multiplex N and multiplex H in parallel). Test results were 

compared with the results of an influenza A real-time PCR 

targetting the M gene that had been validated and implemented 

several years ago. 

Detection limits and efficiency were determined for decimal 

dilution series of H1N1, H3N2, and H1N2 reference viruses, as 

compared with the influenza A matrix real-time PCR. Subtypespecificity 

was evaluated by testing 22 different SIV strains 

provided by the IZSLER institute, Brescia, Italy. Diagnostic 

performance for lung lavage samples was evaluated by testing 

influenza A PCR positive pooled lung lavage samples (n=17) of 

4, 7 and 10 week old piglets. For some pools the lung lavage 

samples that contributed to the pool were also tested individually 

(n=25) in order to compare the reproducibility of subtyping results 

and the presence of dual infections. 

Results 

For the haemagglutinin genes of H1N1, H1N2, and H3N2 viruses 

81, 36, and 61 entries were aligned for the development of 

primer/probe sets. For the neuraminidase genes of the same 

subtypes 67, 28, and 25 entries were aligned for the development 

of primer/probe sets. 

Both for the in-house PCRs and for the prototype SIV Screening 

qRT-PCR kit RNA extraction with the MagMax AM1836 kit 

yielded 10-100 times lower detection limits than the High Pure 

nucleic acid kit. Using the MagMax AM1836 kit for RNA 

extraction, the detection limits of the in-house PCRs and the 

prototype commercial kit were comparable for the neuraminidase 

genes, whereas detection limits showed more variation for the 

haemagglutin genes. Overall, the detection limits of the subtypespecific 

primer/probe sets were equivalent to 10 times higher 

than the detection limit of the influenza A real-time PCR targeting 

the matrix gene. 

Subtype-specificity of the in-house PCR and the commercial kit 

were investigated by blind testing of a panel of Swine influenza 

virus isolates (H1N1 n=10; H1N2 n=4; H3N8 n=8) generously 

provided by the IZSLER institute, Brescia, Italy. The in-house 

PCRs failed to subtype several strains and also one probe 

appeared to be labile. In contrast the prototype commercial kit 

subtyped 21/22 strains correctly (for one sample a dual infection 

was reported, whereas this sample only contained H3N2). All 

strains were detected with the influenza A matrix PCR with Ctvalues 

ranging from 20-33. 

A first diagnostic evaluation of the commercial kit was performed 

by testing influenza A positive (results from another laboratory) 

pooled lung lavage samples (n=17) from piglets of 4-10 weeks of 

age. All pools were also tested in our in-house influenza A PCR 

and results were confirmed. In the subtype-specific PCR 15/17 

pools yielded a single subtype (H1N2: n=9; H1N1: n=5; H3N2: 

n=1), whereas 2 pools yielded 2 or 3 subtypes. Retesting of the 

individual lung lavage samples that contributed to the latter pools 

confirmed the presence of dual infections in the majority of these 

piglets. Retesting of the individual lung lavage samples of three 

pools that were subtyped unequivocally yielded exactly the same 

subtype in all individual samples. 


Influenza viruses are highly variable RNA viruses that show rapid 

evolution of especially the haemagglutinin and neuraminidase 

genes. Therefore, development of reliable PCR protocols for the 

subtyping of these viruses is not easy. Despite the effort we 

spent on selection of primers and probes, the results of our inhouse 

PCR were disappointing and inferior when compared to a 

prototype commercial real-time PCR kit. 

The latter kit showed a good performance when combined with 

RNA extraction on a MagMax AM1836 platform. Detection limits 

were comparable to tenfold higher than the influenza A matrix 

PCR, 95% of well-defined SIV strains were subtyped correctly, 

and the test also demonstrated a good diagnostic performance 

for subtyping of influenza A positive lung lavage samples. 

Although some practices in the Netherlands are very experienced 

in collecting lung lavage samples, the method is cumbersome; 

collecting other types of samples like nasal swabs or chewing 

ropes is much less invasive laborious. However, nasal and oral 

virus excretion is often short-lived. In the next study we will 

evaluate the diagnostic performance of real-time influenza A PCR 

and subtype-specific PCRs for nasal swabs and chewing ropes in 

fattening pigs with acute respiratory disease. 


We thank Life Technologies for generously providing the 

prototype SIV Screening qRT-PCR kit for this study. 

References 

1. Van Reeth, K, Brown, IH, Dürrwald, R, Foni, E, Labarque, G, Lenihan, 

P, Maldonado, J,,Markowska-Daniel, I, Pensaert, M, Pospisil, Z, Koch, G 

(2008). Seroprevalence of H1N1, H3N2 and H1N2 influenza viruses in pigs 

in seven European countries in 2002-2003. Influenza Other Respi Viruses, 

2(3), 99-105. 

2: Brown, IH (2012). History and Epidemiology of Swine Influenza in 

Europe. Curr Top Microbiol Immunol Jan 11. [Epub ahead of print].

S1 - O - 08 

IMPORTANCE OF CONTINUOUS VALIDATION OF MOLECULAR METHODS FOR ROUTINE 

DIAGNOSIS OF PRRSV RNA IN CLINICAL SAMPLES 

Sandra Revilla-Fernández 1 , Adolf Steinrigl 1 , Tatjana Sattler 2 , Friedrich Schmoll 1 

1 

AGES Institute for Veterinary Disease Control, Department for Molecular Biology, Robert Koch Gasse 17, 2340 Mödling, Austria 

2 

Large Animal Clinic for Internal Medicine, University Leipzig, An den Tierkliniken 11, 04103 Leipzig, Germany 

PRRSV, real-time PCR, sensitivity, specificity, validation 

Introduction 

PPRSV is a small, enveloped, positive single-stranded RNA virus 

belonging to the Arteriviridae family, recognised world-wide as an 

important cause of reproductive failure and pneumonia in pigs. 

Since PRRSV was first described at the beginning of the 1990´s, 

wide genomic, antigenic and clinical differences have been 

described in isolates belonging to the European (EU) and North 

American (NA) genotypes. Several molecular-based methods like 

conventional and real-time RT-PCR (RT-qPCR) were 

demonstrated to be fit for detection of PRRSV in clinical samples. 

However, the high mutation rate of PRRSV poses a diagnostic 

challenge (4, 5). Therefore, the purpose of this study was to 

perform an in-house evaluation of recently established PRRSV 

molecular diagnostic methods and to compare them. Six different 

assays were tested with a series of PRRSV EU and NA field 

strains from different subtypes and origin. Diagnostic and 

analytical sensitivity and specificity were extensively studied. This 

is essential to control the disease spread and to guarantee the 

negative status of the herds (1). 


Six different one-step RT-PCR-based assays (Table 1), including 

one published RT-qPCR (3), four commercially available kits and 

one conventional ORF7 duplex RT-nested PCR (2) were tested 

for assay sensitivity and specificity with a series of PRRSV EU 

and NA field strains from Central Europe and Russia, samples 

from two European Ring trials and dilution series of in vitro EU 

and NA transcripts. The target sequences for the commercial RTqPCR 

kits were unknown. All but one test distinguish between 

EU and NA genotypes (Table 1). Runs were performed in two 

different real-time PCR cyclers. The ORF5 region of field 

samples was sequenced and phylogenetically analyzed with 

Bionumerics Software (Applied Maths), using reference strains of 

all PRRSV genotypes and subgroups (4, 5). Quantitative RTqPCR 

data (Cq values) was analysed statistically by 2-sided 

paired t-test. 

Table 1: Summary of PRRSV PCR methods applied for 

validation, references and system descriptions 

conven 

-tional 

RTqPCR 

Assay 

Ref. 

comm. = commercially available 

Differentiation 

NA/EU 

RNA Input 

(µl) 

ORF7 2 yes, one-tube 3.5 

ORF6 3 yes, two-tube 5 

Kit A comm. yes, one-tube 8 

Kit B comm. yes, one-tube 5 

Kit C comm. no 5 

Kit D comm. yes, one-tube 5 

Results 

In general, the commercially available RT-qPCR kits showed 

higher sensitivity than the conventional method. RT-qPCR 

efficiency and sensitivity calculated from serial dilutions of 

standard EU and NA PRRSV strains were optimal. However, 

specificity problems were seen with the published ORF6 based 

assay to detect some regional EU strains and the highly 

pathogenic (HP) NA strains. EU-1 field strains from Austria, 

Slovenia and Russia were equally well detected by all assays 

tested, whereas German strains showed the highest inter-assay 

differences. 

Taking into account different RNA sample inputs, statistical 

analysis of Cq values confirmed relevant differences between 

assays: lowest Cq values, indicating highest analytical sensitivity, 

were consistently observed with kits A, B and C. However, kit A 

did not efficiently detect some EU-2 and EU-unassigned strains, 

whereas kit B showed problems resulting from crosstalk between 

fluorescence channels in one of the real-time PCR cyclers used 

for validation, leading to false positive results. Kit C had 

specificity problems with NA HP strains included in one of the 

ring trials. The conventional RT-PCR method was shown to be 

less sensitive than the commercial real-time PCR Kits. Moreover, 

differentiation of double infection with both genotypes was not 

possible by real-time PCR with kit C and difficult when using the 

RT-nested PCR. 


As shown, in-house validation may be helpful to identify the bestsuited 

methods for PRRSV diagnosis. To overcome these key 

aspects in PRRSV diagnosis, continuous re-validation of applied 

methods is necessary. If this is not possible, diagnostic 

laboratories should closely collaborate with the manufacturers of 

commercial kits, supporting them with new field strains and with 

field observations. 

In many European countries, as in Austria or Germany, both EU 

and NA strains can be detected within the same herd or even 

within the same animal (1, and personal observations). In this 

situation, differentiation of both PRRSV genotypes is directly 

demanded by the clinical veterinarians since it is useful 

information for animal trade and epidemiological studies. 

In conclusion, this validation shows that there is currently no 

single assay or commercial kit with optimal analytic and 

diagnostic sensitivity. This indicates that various laboratory 

technical specifications, including the PCR machines, should be 

also considered for evaluation to avoid false results. The major 

problem to underline is the low specificity of some assays for 

detecting the newly evolved PRRSV strains that are 

underestimated if only PRRSV reference strains are considered 

for validation. Consequently, continuous evaluation and 

adaptation of routinely applied methods to currently circulating 

strains is necessary in each laboratory. 

References 

1. Große Beilage E, Bätza HJ. (2007): PRRSV-Eradikation: Eine Option für 

Schweinebestände in Deutschland? PRRSV-eradication: An option for pig 

herds in Germany? Berl Münch Tierärztl Wochenschr. 120, 11/12,470-479. 

2. Reiner G, Fresen C, Bronnert S, Willems H. (2009): Porcine 

Reproductive and Respiratory Syndrome Virus (PRRSV) infection in wild 

boars. Vet Microbiol. 12;136(3-4):250-8. 

3. Revilla-Fernández S, Wallner B, Truschner K, Benczak A, Brem G, 

Schmoll F, Mueller M, Steinborn R. (2005): The use of endogenous and 

exogenous reference RNAs for qualitative and quantitative detection of 

PRRSV in porcine semen. J Virol Methods. 126:21-30. 

4. Shi M, Lam TT, Hon CC, Hui RK, Faaberg KS, Wennblom T, Murtaugh 

MP, Stadejek T, Leung FC. (2010): Molecular epidemiology of PRRSV: a 

phylogenetic perspective. Virus Res. 154(1-2):7-17. 

5. Stadejek T, Oleksiewicz MB, Scherbakov AV, Timina AM, Krabbe JS, 

Chabros K, Potapchuk D. (2008): Definition of subtypes in the European 

genotype of porcine reproductive and respiratory syndrome virus: 

nucleocapsid characteristics and geographical distribution in Europe. Arch 

Virol. 153(8):1479-88.

S1 - O - 09 

VIRAL DIAGNOSIS USING TRANSMISSION ELECTRON MICROSCOPY 

W.A. Cooley and D. J. Everest 

Animal Health and Veterinary Laboratories Agency-Weybridge, Bio-Imaging Unit, New Haw, Addlestone, Surrey KT15 3NB, UK 

Diagnosis, Electron Microscopy, Virus detection 

Introduction 

The size of most viruses places them well below the resolving 

power of the standard light microscope. However they are quite 

readily viewed and identified by transmission electron microscopy 

(TEM) and therefore TEM has provided contributions to virology 

and in particular to the discovery, detection and diagnosis of 

various viral infections. Virus diagnosis by electron microscopy is 

based on the visualization and morphological identification of 

virus particles. Molecular or immunological methods, such as 

Polymerase Chain Reaction (PCR) and Enzyme Linked Immuno 

Sorbent Assay (ELISA), are very effective in detecting known 

viruses and can be used to scrutinize a large amount of samples. 

However, one of the main advantages of using TEM for viral 

diagnosis is that it does not require organism-specific reagents 

for recognizing the pathogenic agent. Therefore, for new or 

unknown pathogens that may occur in the context of bio-terrorism 

attacks, or as a result of the manifestation of new pathogens, 

electron microscopy remains the only method that can provide a 

quick assessment of all pathogens present in a sample, providing 

an “open view”, which is why electron microscopy is considered a 

“catch all method”. Electron microscopy can also be used to 

investigate viruses isolated in cell cultures, which is useful in 

controlling the results of the virus isolation process, among other 

applications. The AHVLA at Weybridge, UK, provides a rapid 

(diagnosis within 15 minutes of arrival) viral diagnostic service for 

various diseases affecting farmed livestock and wildlife species 

through veterinary surveillance as part of the Great Britain 

Wildlife Disease Surveillance Partnership and also for captive 

and zoological animals. The samples most frequently received for 

viral examination in our diagnostic TEM laboratory are fresh 

unfixed samples such as scabs, warts, gut and faecal samples. 


For viral diagnosis we perform the negative staining procedure. 

This is a simple, rapid and well suited technique for the 

examination of small particulate suspensions. For viewing the 

particulate specimens for organisms such as viruses, a support 

membrane must first be prepared onto our TEM grids. This will 

provide a support platform to hold the small particles. Examples 

of support films we may use include Formvar, Collodion, Butvar, 

and Pioloform. Once the grids are coated with the films, they are 

then stabilized by evaporating a thin carbon coat over them to 

render them conductive and keep them from melting in the 

electron beam followed by plasma glowing, to ensure the grids 

are highly hydrophilic, to obtain maximum sample adsorption The 

received samples are now ground in the presence of a phosphate 

buffer to break up the tissue and release any viral particles which 

may be present. A drop of the ground suspension is then 

adsorbed onto the coated grid for 30 seconds. After wicking dry 

with a piece of filter paper the grid with sample now adsorbed to 

the surface is then floated on a drop of negative stain for about 

one minute. The most common negative stains are 1% (60 mM) 

aqueous uranyl acetate, pH 2-4.5, and 2% (5 mM) 

phosphotungstic acid, pH adjusted to 6.6 with NaOH. After 

approximately 15 seconds on the negative stain any excess stain 

is wicked away and the grid air dried after which it is ready for 

examination by TEM. Structures on the grid are surrounded and 

stabilized by the drying stain, thus they appear as transparent, 

highly detailed negative images within a dark halo of stain. 

Results 

Amongst the most common viruses we diagnose are the 

poxviruses (Fig 1-3). These viruses infect both vertebrate and 

invertebrate animals and are often detected from cattle, sheep 

and goats causing external scabby lesions. The virus group is 

well known as it includes smallpox (variola) an orthopoxvirus. 

Poxviruses are regarded as the major contributor responsible for 

the decline in the UK of the indigenous Red Squirrel (Sciurus 

vulgaris). This is due to its susceptibility to Squirrel pox virus 

(SQPV). This invariable fatal disease was first reported in 1981 

(1) using TEM at the AHVLA. SQPV is approximately the same 

size and shape (ovoid) as the parapoxvirus and is differentiated 

using TEM due to differences in the spiral coat (longitudinal as 

opposed to transverse). TEM is also used to detect ‘enteric 

viruses’ which are an important, but diverse, group of viruses 

found in the intestinal tract of animals (and humans). Examples 

which we commonly detect are rotavirus and adenovirus. 

Adenovirus (Fig.4) is also an emerging problem contributing to 

the decline of the Red Squirrel and was first reported in 1997 (2) 

using TEM at the AHVLA. As mentioned above, the “open view” 

occasionally detects mixed viral infections. A published example 

(3) being both parapox and calicivirus detected in a scab from a 

Grey Seal (Halichoerus grypus). Calicivirus is also the cause of 

Rabbit haemorrhagic disease or viral haemorrhagic disease 

(VHD). This is a highly infectious and often fatal disease that 

affects wild and domestic rabbits and was a notifiable disease in 

the UK for several years during the 1990s. 

Fig. 1. Squirrel poxvirus, 2. Parapoxvirus, 3. Orthopoxvirus, 4. 

Adenovirus. 

Discussion & conclusions 

TEM remains essential for certain diagnostic aspects of Virology 

(and bacteriology). It was and still is necessary for new virus 

characterization and for the initial identification of unknown viral 

agents in particular outbreaks. This especially applies to 

veterinary virology, in which several agents are continuously 

being identified with this technique over the past few years. It is 

also recommended by regulatory agencies for investigations of 

the viral safety of biological products and/or cells. The nature of 

the samples to be analyzed can be tremendously diverse, from 

body fluids or biopsies performed or scabs, warts, gut and faecal 

samples. An additional advantage of electron microscopy is 

through its reassurance in having the picture of the screened 

virus and “a picture is worth a thousand words”. The results by 

TEM are therefore often regarded by many as the 'Gold 

Standard', as viral particles are actually observed. The “open 

view” approach of electron microscopy permits rapid and “catchall” 

detection of viruses, if these are present in a detectable 

concentration, and makes it especially useful as demonstrated 

here for the identification and diagnosis of various animal viruses, 

as well as being used in the initial identification of unknown viral 

agents in particular disease emergencies and outbreaks and/or in 

suspected bioterrorism. 

References 

1. Scott, A. C., Keymer, I. F. and Labram, J. (1981). Parapoxvirus infection 

of the red squirrel (Sciurus vulgaris). Vet. Rec. 109, 202. 

2. Sainsbury, A.W., Adair, B., Graham, D., Gurnell, J., Cunningham, A.A., 

Benko, M. and Papp, T. (2001). Isolation of a novel adenovirus associated 

with splenitis, diarrhoea, and mortality in trans-located red squirrels, 

Sciurus vulgaris. Verhandlungs Bericht über die Erkrankung der Zootiere 

40, 265-270. 

3. Stack M.J., Simpson V.R. and Scott AC (1993). “Mixed poxvirus and 

calicivirus infections of grey seals (Halichoerus grypus) in Cornwall.” Vet. 

Rec. 132: 163-165.

S1 - O - 10 

A BEAD BASED MULTIPLEX IMMUNOFLUOROMETRIC ASSAY FOR SCREENING AND 

CONFIRMATION OF ALL MAJOR PRION TYPES IN SHEEP 

Yue Tang 1 , Jan PM Langeveld 2 , Adriana Gielbert 1 , Jorg G Jacobs 2 , Thierry Baron 3 , Olivier Andreoletti 4 , Takashi 

Yokoyama 5 , Alex Bossers 1 , Maurice J Sauer 2 

1 

Animal Health and Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, UK 

Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands 

Agence Francaise de Sécurité Sanitaire des Aliments Lyon, Lyon, France 

UMR INRA ENVT 1225, 31076 Toulouse, France 

National Institue of Animal Health,Tsukuba, Ibaraki 305-0856, Japan 

Scrapie, discrimination, TSE, BSE, prion, multiplex, assay 

Introduction 

Prion diseases or transmissible spongiform encephalopathies 

(TSEs) in small ruminants are presented in many forms: classical 

scrapie, Nor98/atypical scrapie, CH1641 scrapie and bovine 

spongiform encephalopathy (BSE). The discrimination between 

BSE and CH1641 is especially of interest for food safety reasons, 

but also methods that can directly co recognize atypical 

Nor98/scrapie cases are lacking. 


We previously described a multiplex immunofluorometric assay 

(mIFMA), based on a bead array flow cytometry technology, 

which provided, in a single assay, discrimination between BSE (in 

cattle and sheep) and classical scrapie (Tang et al., 2010). In this 

study, we extended the mlFMA assay to differentiate classical 

scrapie, atypical scrapie, BSE (experimentally infected sheep and 

naturally infected cattle) and CH1641 (both experimental and 

natural CH1641-like infections in sheep). Three prion protein 

(PrP) specific capture antibodies were used, two distinct 

N-terminus specific antibodies 12B2 and 9A2, and core specific 

antibody 94B4 (Fig. 1). Western blotting procedures were as 

described for discriminating bovine BSE types (Langeveld et al., 

2011). 

Figure 1. The principle of discrimination of BSE/CH1641, scrapie 

and atypical scrapie using multiplex IFMA.After PK digestion, 

9A2, 12B2 and 94B4 antibodies can all bind to classical scrapie 

PrP res ; only 9A2 and 94B4 bind substantially to BSE and CH1641 

PrP res ; only 12B2 and 9A2 bind substantially to atypical scrapie 

PrP res 

.The capture antibodies 12B2 for amino acid residues 

93 - 97, 9A2 for ovine PrP N-terminal amino acid residues 102104, 

and 94B4 for amino acid residues 190-197, are coupled to 

distinct bead types. The reporter antibody Sha31 (epitope; 

residues 148-155) is biotinylated. Theoretical idealised profiles of 

MFI readings, associated with 12B2 (black filled bar), 9A2 

(unfilled bar) and 94B4 (grey filled bar) respectively, are indicated 

on the right side of the simplified representation of the PrP res 

protein primary structure. CH1641 may be differentiated from 

BSE by analysis of a minor 14 kDa C-terminal fragment and also, 

the 9A2 region might be different from BSE which would result in 

lower 9A2 values than in BSE. 

suggesting that major PrP Sc cleavage of the N-terminus occurs 

further towards the C terminus in CH1641 than in BSE. The ratios 

of 12B2/94B4 and 9A2/94B4 were similar between experimental 

CH1641 and CH1641-like cases, although two CH1641 like 

subjects displayed slightly elevated ratios of both 12B2/94B4 and 

9A2/94B4. To verify this finding for PrP res , mass spectrometry 

quantification to determine the absolute abundance of the 

peptides associated with all three antibody binding regions 

confirmed that there was a 2.2 fold reduction of peptides 

containing the 9A2 epitope for experimental CH1641 PrP res 

in 

comparison to BSE PrP res . Observation of reduced PrP res 

may 

serve as a new marker for CH1641. Finally, Western blotting 

further confirmed that the CH1641-like cases have to be 

considered as a TSE class different from the experimental 

CH1641 cases. 


This mIFMA may thus provide the basis for simplified TSE 

diagnosis with capability for simultaneous screening and 

differential diagnosis. 


Financial support for this work was provided by the Department 

for Environment, Food and Rural Affairs (Defra, UK; project 

SE1700/ CSA 7335). JPML is partly supported by the Dutch 

Ministry of Agriculture, Nature and Food Quality project number 

WOT-01-002-14 001.01. JPML and OA are partly supported by 

the EU STREP project GoatBSE FOOD15 CT-2006-36353. We 

thank Linda Davis and Sharon Everitt for Western blotting 

analysis. The help of Dr Marion Simmons and the VLA TSE 

Archive team for provision of samples is gratefully acknowledged. 

References 

1. Tang, Y, Thorne, J, Whatling,K, Jacobs, J, Langeveld, J and Sauer, M. 

2010. A single step multiplex immunofluorometric assay for differential 

diagnosis of BSE and scrapie. J. Immunol Meth. 356:29–38. 

2. Langeveld, JPM, Erkens, JHF, Rammel, I, Jacobs, JG, Davidse, A, van 

Zijderveld, F, Bossers, A, and Schildorfer, H. 2011. Four independent 

molecular prion protein parameters for discriminating new cases of C, L, 

and H BSE in cattle. J Clin Microbiol., 49:3026–3028. 

Results 

All three antibodies were shown to bind classical scrapie PrP res 

strongly, whereas in Nor98/atypical scrapie PrP res only 12B2 and 

9A2 binding was observed. PrP res binding of 12B2 was low for 

both BSE and CH1641, as expected. Furthermore, analysis of 

serially diluted samples indicated that the assay provided a 

similar level of sensitivity for atypical scrapie as that found using 

a well established commercial test. Unexpectedly, 9A2 binding to 

CH1641 PrPres was reduced by 2.1 fold both for experimental 

CH1641 and CH1641 like scrapie when compared with BSE,

S1 - O - 11 

USING ORAL FLUID FOR THE SEROLOGICAL MONITORIZATION OF PRRSV CIRCULATION IN A 

GROUP OF INFECTED GILTS 

Martos-Raich, Alba 1 , Coma-Oliva, Ester 1 ; Serra-Martínez, Jordi 2 ; Barrera-Toro, Xavier 3 ; Planasdemunt-Regàs, 

Llorenç 3 ; Maldonado-García, Jaime 1 ; Porquet-Garanto, Lourdes 1 ; Rebordosa-Trigueros, Xavier 1 . 

1 HIPRA 17170 Amer, Girona, Spain. 

2 Biofar Laboratoris, S.L 08261 Cardona, Barcelona, Spain. 

3 AVP Planasdemunt i Associats 17400 Breda, Girona, Spain 

Introduction 

The use of oral fluid-based assays is proving every day that is 

well suited for monitoring of different pathogens in farm animals 1 . 

In the case of PRRSV different studies have shown that pig oral 

fluid (OF) is a suitable sample for monitoring both viremia (PCR) 

and sero-conversion (ELISA) after infection 2 . In this study a new 

methodology to adapt the existing CIVTEST SUIS PRRS E/S and 

A/S ELISA kits (Hipra) to be used with OF matrix instead of 

serum was evaluated under field conditions. The ultimate goal of 

this study was to compare the performance of the ELISA kits 

using individual or pooled serum samples, as well as OF from 

groups of animals. 

PPRS virus, oral fluid, ELISA, alternative for monitoring 


The study included a single group of 10 gilts coming from a 

PRRSV-free farm (as confirmed by PCR and ELISA at the 

beginning of the study). The animals were infected in isolation 

using a field strain of PRRSV Type 1 on day 0. The study lasted 

for 9 weeks (wk) with weekly sampling of blood from each 

individual and OF samples from the group. The presence of 

antibodies to PRRSV Type 1 and 2 was evaluated by using the 

CIVTEST SUIS PRRSV E/S and CIVTEST SUIS PRRS A/S 

ELISA kits respectively (Hipra) and also by Western blot based in 

a European strain. Individual serum samples were tested for the 

presence of viral RNA by Real Time RT-PCR 3 . Serums samples 

were analyzed individually and as a pool of 10 (pool/10). For OF 

serology different conjugates (anti-IgG1,-IgG2, -IgA) for the 

ELISA were evaluated. 

Results 

The incubation conditions for OF were fixed at 4 ºC of a ½ 

dilution of the sample. In all cases an IRPC of 20.0 was used as 

a cut-off (as defined in CIVTEST for individual serum). As seen in 

Table I, results from individual serum showed 80% of positive 

animals at wk3 (100% at wk4). The IRPC values for the pool/10 

and OF samples showed a value above 20 at wk4. Both, the 

mean IRPC value of the group and the IRPC of the pool/10 

increased until the end of the study. By contrast, IRPC values 

obtained for the OF sample showed a maximum at wk4, and then 

dropped down till the end of the trial. No reactivity was observed 

with the A/S kit (data not shown). The use of different conjugates 

for the analysis of OF samples indicated that the most abundant 

type of Ig after infection was IgA (already detectable at wk1 postinfection; 

Figure 1). The kinetics of IgG1 showed sero-conversion 

around 3-4 weeks after infection. IgG2 levels were nondetectable 

(data not shown). The maximum sensitivity (IRPC 

above 20 at wk1) was achieved by using a combination of IgG1 

plus IgA conjugates. 

Figure1. OD450 values obtained with the oral fluid weekly 

samplings using conjugates with different antibody-type 

specificity 


CIVTEST SUIS PRRS E/S has shown a good adaptation to the 

OF matrix. This sample evidenced antibody dynamics similar to 

those observed with the pool/10 serum sample, representing a 

suitable alternative for monitoring PRRSV circulation in pig herds. 

The use of different channels (IgG1 or IgA) for the analysis of the 

OF sample would allow to asses maximum earliness in the 

detection of infection or detection of the fall in post-infection 

immunity. 

References 

1. Prickett, J.R. et al: 2010, Anim Hlth Res. Rev. 11(2): 207-216. 

2. Kittawornrat, A. et al: 2010, Virus Res. Dec;154(1-2):170-176. 

3. Martínez, E. et al: 2008, Res. Vet. Sci. 85: 184-193 

Table I. Serology results obtained with serum and oral fluid using 

the CIVTEST SUIS PRRS E/S at weekly intervals after infection 

(IRPC values above 20.0 appear in shaded boxes) 

PRRS E/S 

% Pos 

(ELISA) 

wk 

0 

wk1 wk2 wk3 wk4 wk5 wk6 wk7 wk8 wk9 

0% 0% 0% 80% 100% 100% 100% 80% 100% 100% 

Mean (IRPC) -1,49 -1,38 8,64 24,32 41,75 46,56 70,47 50,96 72,67 72,00 

Pool/10 

(IRPC) 

-1,80 -2,09 4,19 11,52 23,95 54,09 57,88 48,48 68,97 87,19 

OF (IRPC) -5,20 -4,22 0,13 14,08 46,65 31,48 35,19 29,74 20,52 32,66 

OF/IgG1+IgA 

(IRPC) 

4,33 44,02 60,07 71,66 99,72 96,37 97,72 86,04 80,60 99,95

S1 - O - 12 

ENHANCED DETECTION OF BOVINE RESPIRATORY VIRUSES BY INCLUSION OF COHORT 

ANIMALS 

Ronan O’Neill, Emily Connaghan, Jean Mooney 

CVRL, Virology Division, Backweston, Celbridge, Ireland 

Respiratory virus, bovine, multiple, PCR, diagnostics 

Introduction 

Bovine respiratory disease (BRD) in calves remains a hugely 

significant problem to Irish cattle breeders. Until recently BRD 

diagnostics were quite limited but this has improved due to the 

adoption of molecular techniques, the introduction of a pooled 

swab matrix and a parallel programme of veterinary education. 

These advances have allowed a much more accurate 

representation of the dynamics of BRD in terms of time of year 

and pathogen involved. This study provides support to the 

clinical judgement of vets in making an initial pathogen 

identification and initiating remedial actions at the earliest 

stage. 


The analysis is based on data generated over the three year 

period of 2009 to 2011. The calf age–group dataset consisted 

of 3822 PCR tests from 753 submissions, representing 30% 

(n=12642) and 31% (n=2405) of total live animal respiratory 

submissions over the period. The sample matrix used 

throughout was cotton swabs, without transport media. 

Veterinarians were encouraged to collect up to six swabs from 

the index and cohort animals which were then pooled in the 

lab for testing (same cost) so the analysis includes results 

based on individual swabs and results based for pooled 

swabs. 

The normal respiratory panel consisted of five real-time PCR 

tests for Bovine Herpesvirus 1 (BHV1) (1), Bovine Coronavirus 

(BoCo)(2), Bovine Respiratory Syncytial virus (BRSV)(3), 

Bovine Viral Diarrhoea virus (BVDv)(4) and Parainfluenza 3 

virus (PI3). All swabs were also assessed for nucleic acid yield 

/polymerase inhibition using a B-actin internal control. 

Results 

In calves overall, BHV1 was detected in 8% of swabs tested, 

BoCo in 37%, BRSV in 11%, BVDv in 4% and PI3 in 9%. 

However respiratory samples in this age group are extremely 

seasonal with 46% of samples in months November, 

December and January (Figure 1). In the calf age group, the 

months with the highest rates of detection were December for 

BRSV (44%) and PI3 (26%), November for BoCo (18%) and 

January for BHV1 (15%) and BVDv (15%). 

In calves there were 2622 PCR tests done on individual swabs 

and 1199 PCR tests done on pooled swabs – sufficient 

numbers to allow statistical comparison. On average using 

pooled swabs rather than individual swabs increased the virus 

detection rate for BHV1 by a factor of 2.4 (p

S1 - O - 13 

THE FIRST YEAR OF OBLIGATORY BVD CONTROL IN GERMANY – DIAGNOSTIC STRATEGIES, 

RESULTS AND EXPERIENCES 

Horst Schirrmeier, Günter Strebelow, Martin Beer 

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany 

Pestivirus, disease controlj 

Introduction 

Since November 2004, Bovine viral diarrhea/Mucosal disease 

(BVD/MD) has been a notifiable disease in Germany. An 

obligatory BVD-legislation and control strategy of the Federal 

Government is active since the 1st of January 2011. The 

implemented eradication strategy is focused on the direct 

detection of persistently infected (PI) animals by using antigen 

ELISAs or real-time RT-PCR. 

References 

1. Anonymus (2008). „Verordnung zum Schutz der Rinder vor einer 

Infektion mit dem Bovinen Virusdiarrhoe-Virus (BVDV-Verordnung) v. 

11.12.2008 (BGBl. I S. 2461), neugefasst durch Bek. v. 4.10.2010 I 1320, 

1498,geändert durch Art. 1 V v. 17.12.2010 I 2131, diese geändert durch Art. 1 V 

v. 31.5.2011 I 1002 

2. Schirrmeier, H. Strebelow, G. (2012). BVD-Pflichtbekämpfung – Wie ist 

die epidemiologische Situation in Deutschland?, LBH, 6. Leipziger 

Tierärztekongress – Tagungsband 3: 95-98 


The major goal of the obligatory control program is a fast and 

efficient reduction of the prevalence of PI animals and the 

establishment of so-called “unsuspicious (virus free) animals” and 

farms with a certified status. Therefore, four basic rules were 

defined: 

1. Obligatory investigation of all newborn calves for BVDV 

(antigen/genome) before the age of 6 months 

2. Efficient elimination of all PI animals. 

3. Trade with certified “unsuspicious animals” only 

4. Prevention of reinfections by qualified measures (e.g. 

biosafety and voluntary vaccination) 

Approved methods taking the so-called “diagnostic gap” 

(method-dependent limitation of virus/genome detection due to 

BVDV-specific colostral antibodies) into account, and a system of 

measures for quality control and assurance, like proficiency tests 

or the licensing of diagnostic assays ensure a high standard of 

BVDV diagnostics within the German program. Ear notches are 

the main sample material allowing a reliable and very early 

detection and subsequent elimination of PI animals. All diagnostic 

results are officially documented in a central data base (HIT). 

Additionally, in order to get more information about the molecular 

epidemiology of BVDV in Germany, more than 400 BVDVpositive 

samples or virus isolates from PI animals were collected 

and further analysed by sequencing their 5’ untranslated genome 

region (5´UTR) 

Results 

In the last year, more than 6 million tests for the detection of 

BVDV were conducted in German laboratories. Real-time RT- 

PCR assays and antigen-capture-ELISAs were used in an 

approximately equal amount. 17 410 PI animals had been 

detected and were removed which is a percentage of 0.14% in 

relation to the German cattle population, and of 0.36% in relation 

to all calves born in 2011. The prevalence of PI animals differed 

between the German federal states from 0.02% to 0.58%. In 

6016 cattle holdings at least one PI animal was detected (4.1%). 

Phylogenetic analysis of 445 German BVDV isolates clearly 

showed that only two BVDV subgroups (BVDV-1b and BVDV–1d) 

represented more than 75% of all strains investigated. Apart from 

this dominating subgroups, BVDV-1f (8.8%), BVDV-1e (3.5%), 

BVDV-1h (2.9%), BVDV-1g (0.7%), and BVDV-1k (0.2%) could 

be observed. Regional differences were also noticeable. The 

percentage of BVDV-2 was 5.1%, and the subtype “Germany 2a” 

was the dominating subgroup within this genotype. 


During the first year of the German compulsory BVDV control 

program, more than 17 000 PI animals were detected and 

removed. Furthermore, a reduction of the number of notified PI 

cases was obvious during the last three month due to an efficient 

elimination at the beginning of the year. We therefore conclude, 

that the diagnostic concept, focussed on an as early as possible 

detection and elimination of PI calves is successful. However, 

with the progress in BVDV-eradication, the risk and the economic 

outcome of reinfections have to be taken into consideration, and 

accompanying measures like serological monitoring and 

vaccination have to be implemented into the complex disease 

control system..

S1 - O - 14 

EUROPEAN PRRSV – DIAGNOSTIC SOLUTIONS FOR A RAPIDLY MUTATING VIRUS 

R. Shah 2 , C. Boss 1 , C. O’Connell 2 

1 


2 


Introduction 

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) 

is a highly infectious disease that is endemic in pigs throughout 

Europe. Estimates on its economic impact vary from as little as 

€60 per infected animal to as much as €75 for every animal in 

production on an annualized basis. Detection and removal of 

infected animals followed by surveillance to avoid re-infection is 

the preferred strategy to manage the impact of the disease in 

European Pig Populations. 

The virus is continuously and rapidly evolving, generating “new 

variants” and expanding its diversity. Therefore there is a need to 

update diagnostic solutions at a pace that provides efficacious, 

appropriate and cost competitive solutions to the marketplace. 

In 2010, diagnostic studies showed that a mutation in one of the 

9 open reading frames of the North American PRRS viral 

genome had rendered our Taqman NA/EU PRRSV reagents 

ineffective for detection of certain newly mutated variants. 


Laboratories using the Life Technologies Taqman PRRSV assay 

sent samples to our lab for sequencing analysis. Several of the 

questionable samples were sequenced and the resulting 

sequence information was used to design a set of primers and 

probes that were perfect matches to the new sequence. This new 

set of oligos is refered to as a “patch”. 

The new patch was added to the existing assay and optimized so 

as to increase sensitivity of the North American PRRSV assay 

while not affecting sensitivity of the European PRRSV assay that 

was multiplexed with it. 

Results 

Over 130 samples from different labs that contained the new 

NAPRRSV strain were tested with the patched assay as well as 

the old assay to show that the patch really did pick up the new 

strain that the old assay was missing. No non specific 

interactions were obsereved when the patch was added to the 

old assay reagents. 

The new patched assay enables sensitive and specific detection 

of current and old NAPRRSV strains as well as the ability to 

subtype EUPRRSV strains from NAPRRSV strains. All 

parameters of the old assay such as thermal profile and PCR 

mastermix preparation remain the same. 


Given the rapid mutation rate of PRRSV, the creation and 

maintenance of a diagnostic solution is a moving target. 

Therefore collaborations across veterinary laboratories and other 

key stakeholders is essential for monitoring the degree of 

divergence that is occurring and to aid in building robust 

diagnostic solutions. 

This North American example provides a template, combined 

with newly available technologies such as whole genome 

sequencing, for creating a regionally appropriate assay and 

monitoring program for the detection of rapidly mutating viruses 

like PRRSV. 

References 

Iowa State University, Iowa Select Farms, University of Minnesota 

PRRSV

S1 - O - 15 

SEROLOGICAL ANALYSIS AND MONITORING OF IBR 

IS IT POSSIBLE TO CONTROL IBRgE ANTIBODIES IN A BULK TANK MILK? 

Xavier Rebordosa-Trigueros, Ester Coma Oliva, Santiago Casademunt Garre, Lourdes Porquet-Garanto* 

HIPRA, 17170 Amer (Girona), SPAIN 

IBRgE, Bulk Tank Milk (BTM), lower prevalence detection. 

Introduction 

The detection of antibodies in Bulk Tank Milk (BTM) provides 

an easy and cheap methodology for monitoring the health 

status of the herd 1 . The application of this methodology to the 

detection of IBR-gE antibodies presents some limitations. 

Blocking IBRgE-ELISAs have low sensitivity 2 . So, IBRgE- 

ELISAs are only capable of detecting positive tanks when the 

prevalence in the animals in production is greater than 15- 

20% 3 . Under these conditions, gE detection in the BTM is not 

adequate for monitoring the tank and even less so for the 

classification of the farms. The objective of this work is to 

develop and validate an IgG concentration method to increase 

the sensitivity of the IBR-gE detection systems in BTM 

sample. 


We have developed a simple methodology that in less than 60 

minutes can concentrate up to 30 times the IgGs from a 

sample of 5.0 ml of BTM. To validate this methodology 17 

dairy-farms from the NW of Spain with a known IBR status 

were selected (8 vaccinated and 9 non-vaccinated). From 

each farm we have obtained Individual serum of all the 

animals in production and a sample of each tank. Taking into 

account the IBRgE results in sera the real prevalence of each 

tank was calculated. BTM samples were also analysed for gE 

pure and concentrated. 

Results 

The actual prevalence against gE, calculated from individual 

serum samples in the 17 selected farms was: 0% (5 farms), 

4%, 15%, 17%, 18% 19%, 21%, 24%, 26%, 33%, 43% and 

44% (2 farms). By using pure milk (non-concentrated) only 5 

tanks were consistently detected as positives to gE. These 

tanks corresponded to the farms with the higher prevalence 

values (from 26% to 44%). By contrast, applying to the same 

samples the methodology of concentration of IgGs, all tanks 

were detected positive, even the lowest prevalence one. This 

methodology did not affect the 5 negative tanks that remained 

negative. Fig. 1. 

Figure 1. BTM ELISA IBRgE titres from 17 dairy-farms. 

Prevalence detection from 0 to 44%. Column □ nonconcentrated 

BTM (pure milk) and Column ■ salting-out 

concentrated BTM (IgGs concentration). CIVTEST ® 

BOVIS 

IBRgE interpretation: Higher than 20.0 (%INH) = positive 

sample, Lower than or equal to 20.0 = negative sample. 


These results indicate that the proposed IgG concentration 

system does not affect the specificity of the ELISA but 

increases the sensitivity, having allowed in this study to detect 

tanks with the lower prevalence (4%). 

References 

1.Nylin, B., Strøger, U., Rønsholt, L. “A retrospective 

evaluation of a Bovine Herpesvirus-1 (BHV-1) antibody ELISA 

on bulk-tank milk samples for classification of the BHV-1 

status of Danish dairy herds”. (2000). Preventive Veterinary 

Medicine. 47, 91-105. 

2. Kramps, J.A., Banks, M., Beer, M., Kerkhofs, P., Perrin, M., 

Wellenberg, G.J. and Van Oirschot, J.T. (2004) “Evaluation of 

tests for antibodies against bovine herpesvirus 1 performed in 

national reference laboratories in Europe”. Vet Microbiol., 

8;102(3-4):169-81. 

3. Wellenberg, G.J., Verstraten, E.R.A.M., Mars, M.H., Van 

Oirschot, J.T. (1998) “ELISA detection of antibodies to 

glycoprotein E of bovine herpesvirus 1 in bulk milk samples”. 

Vet Rec. 142(9), 219 – 20.

S1 - O - 16 

DEVELOPMENT AND VALIDATION OF A REAL-TIME PCR ZEN GEL MIX FOR THE DIAGNOSIS AND 

QUANTIFICATION OF COXIELLA BURNETII 

Aleida Villa 1 , José L Arnal 1 , Daniel Serrano 1 , Alfredo A Benito 1 , Rafael Baselga 1 . 

EXOPOL Autovacunas y Diagnóstico. Pol. Río Gállego, 50840 San Mateo de Gállego Zaragoza, España. Tel 976 69 45 25, exopol@exopol.com 

Coxiella burnetii, qPCR, validation, diagnostics 

Introduction 

Coxiella burnetii is the ethological agent of Q Fever a zoonotic 

and emergent disease with implications for public and animal 

health. Reliable diagnosis of C. burnetii is the vertebral point for 

the control of the spread of these bacteria highly infectious. 

Here we describe a novel PCR (qPCR) assay simple, rapid, 

sensitive, specific, robust and well validated for routine diagnosis 

of Coxiella burnetii based in a new type of "ZEN" double 

quencher probe (1) that increases the accuracy, sensitivity and 

significantly decreases the background fluorescence, reducing 

Cq values consistently with a greater precision and signal. In this 

qPCR all the reaction components (master mix, enzymes, 

specific primers, probes, buffer and internal controls) have been 

stabilized within a plate using a technology called gelation (2) 

allowing their use at room temperature and in a "ready to use” 

format. 


To ensure high performance and bio-safety on the DNA process 

extraction, the procedure was performed in an automated Lab 

Turbo robot (Taigen, Bioscience, Taiwan) and compared to an 

established manual DNA extraction method (Mo Bio). The 

samples were run .through the kits according to the 

manufactures` instructions. The qPCR set up protocol were 

single consisting of RNAse/ DNAse free water and 5µl of DNA 

sample Amplification reactions were carried out on a MiniOpticon 

RT System CFB3120 thermal cycler (Bio Rad, software CFX 

manager 2.0). The assay targets a C. burnetii specie specific 

region of the Trans gene was selected, the internal probe trans-p 

was labeled with (FAM/ZEN/Iowa Black). The capacity of the 

ZEN Coxiella burnetii Gel Mix to discriminate between target and 

non target microorganisms was assessed using 1ng/µl of 

genomic DNA from various sources: including 4 reference strain 

and 73 field strain and 1407 clinical cases; from sheep, goat and 

bovine origin: (milk, feces, vaginal exudates, tissue from 

abortions and parturient cows, water and other environmental 

samples) and 40 strains of entities prevalent in ruminants. The 

positives samples obtained by qPCR were used for C. burnetii 

isolation in VERO cell line. The obtained strains were identified 

by immunofluorescence and quantified by qPCR, and then 

confirmed by inoculation in chick embryo yolk sac and genotyping 

in the reference center of Coxiella of Institute Carlos III in Madrid. 

The parameters of analytical and diagnostic sensitivity; analytical 

and diagnostic specificity; precision intra-assay and inter-assay 

were evaluated. 

Results 

ZEN qPCR Coxiella Gel mix demonstrated an excellent ability to 

quantify, defined by its wide dynamic range (at least 7 log units), 

with a minimum detection limit equivalents to 2 genome copies of 

C. burnetii and a maximum value of > 10 10 . The slopes of the 

linear regression curves were calculated within an interval of 7- 

log dilutions and were similar to the theoretical optimum of -3.32 

(-3.475), showing an effective amplification efficiency (E = 0.90), 

and R 2 values close to 1 (0.9988) indicating that the assay was 

extremely linear. Confidence intervals based on the standard 

deviations of the Cq values not overlapped, up to 10 target 

molecules, indicating that it is possible a reliable quantification 

above this limit. The intra-assay CVs (5 replicates and 5 assays) 

ranged between 9.8% and 31%. The inter-assay CVs obtained 

was from 12.6% to 24.5% after 5 independent trials. The 

sensitivity of the real-time quantitative PCR was 2, 6 times higher 

than that isolation in VERO cell line. All isolates (73/194) were 

phase I and highly pathogenic in chick embryos 

Table 1. qPCR positive tissue from abortions Vs Isolation of C. 

burnetii 

Species Positive 

qPCR cases 

C. burnetii 

isolates cases 

% of isolation Vs 

qPCR positive cases 

Bovine 70 41 58,6 

Caprine 31 10 32,3 

Ovine 93 22 23,7 

The results of specificity detection of 40 species of other common 

pathogens agents proved to be negative. The positive rates of 

Coxiella burnetii shedding in abortions were 27.21% (123/452), 

while in post partum cases was very low 5.99% (22/345). The 

excretions of C. burnetii in ruminants herds in productions 

resulted: vaginal swabs 26.81% (74/276); milk tanks 31.58% 

(12/38); pooled milks 22.22% (40/180); bovine feces 2.17(2/90). 

C. burnetii was also detected in ground samples 33.3% (1/3), but 

not on in water (0/10) and cereal (0/3) samples. 


Abortions and/or birth of weak offspring are the most serious 

consequence by C. burnetii infection in animals. Abortions 

usually occur in the second half of gestation (3) . Infected animals 

are usually asymptomatic and is not possible establish any 

relationship between antibody titers and individuals excretion. 

Studies by Rousset et al. (4) 

showed that a non negligible 

proportion of seronegative animals in delivered normally could 

excrete C. burnetii to remain seronegative and spread massive 

amounts of microorganisms during calving. C. burnetii was 

detected in 26.42% of cases of abortions and 61.98% of these 

viable bacteria were recovered in phase I with high pathogenicity 

and 11.36% of them were characterized to belong to genotype III, 

where are included strain from animals, ticks and those that 

cause acute Q fever in humans. These data shows that Coxiella 

infection in rumiants animals have a strong impact on 

environmental contamination and its a potential dangerous 

source for humans infection, even if people have or not contact 

with infected animals. The high number of Coxiella positive cases 

found in this study claim the need for stablish strict health and 

vaccination programs.The qPCR developed and validated for us 

and used in this studies has proved a wonderful diagnostic tool 

and is available commercially. 

References 

1.- CORALVILLE, IA. ZEN double-quenched probes can now be 

combined with a range of fluorescent dyes in: www.idtdna.com 

2.- Biotools. Método de estabilización de Biomoléculas y mezclas 

de reacción complejas mediante la gelificación Patentes 

Internacionales PCT ES2002/000109 PCT ES2004/000024. 

3. Sitio Argentino de Producción Animal. Enfermedad infecciosa 

de los ovinos. S.S.Diab and Uzal F.A. 2007. Disponible en: 

www.produccion-animal.com.ar 

4. Rousset E, Berri M, Durand B, Dufour P, Prigent M, Delcroix T, 

et al. A. Coxiella burnetii shedding routes and antibody response 

after Q fever abortion outbreaks in dairy goat herds.Appl Environ 

Microbiol. 2008, Nov 14. 

5. Guatteo R, Beaudeau F, Joly A and Seegers H. Coxiella 

burnetii shedding by dairy cows. Vet Res 2007 Nov-Dec; 

38(6):849-60.

S1 - O - 17 

RING TEST EVALUATION FOR THE DETECTION OF PRRSV ANTIBODIES IN ORAL FLUID 

SPECIMENS USING A COMMERCIAL PRRSV SERUM ANTIBODY ELISA 

Apisit Kittawornrat 1 , Chong Wang 1 , Gary Anderson 2 , Andrea Ballagi 3 , Andre Broes 4 , Susy Carman 5 , Kent 

Doolittle 6 , Judith Galeota 7 , John Johnson 1 , Sergio Lizano 3 , Eric Nelson 8 , Devi Patnayak 9 , Roman Pogranichniy 10 , 

Anna Rice 3 , Gail Scherba 11 , Jeffrey Zimmerman 1 

1 Iowa State University, 2 Kansas State University, 3 IDEXX Laboratories,Inc., 4 Biovet Inc., 5 University of Guelph, 6 Boehringer Ingelheim Vetmedica Inc., 

7 University of Nebraska, 8 South Dakota State University, 9 University of Minnesota, 10 Purdue University, 11 University of Illinois 

Oral fluid, ELISA, Ring test, PRRSV 

Introduction 

A commercial PRRS serum antibody ELISA (IDEXX 

Laboratories, Inc., Westbrook ME USA) was recently adapted to 

detect anti-PRRSV antibody in oral fluid specimens (1). Based on 

testing of field and experimental samples, diagnostic sensitivity 

and specificity was estimated at 94.7% (95% CI: 92.4, 96.5) and 

100% (95% CI: 99.0, 100.0), respectively, at a sample-to-positive 

(S/P) cutoff of ≥ 0.40. 1 The purpose of this study was to evaluate 

the reproducibility and repeatability of the PRRS oral fluid ELISA 

in a ring test (check test) format. 


A total of 276 oral fluid samples were collected, completely 

randomized, and sent for testing in 12 collaborating diagnostic 

laboratories. In addition to the set of oral fluid samples, each 

laboratory received the materials required for conducting the test: 

ELISA plates (HerdChek® PRRS X3 ELISA, lot #40959-W721), 

reagents, positive and negative controls, pre-diluted conjugate 

antibody, and a copy of the standard operating procedure for the 

PRRS oral fluid IgG ELISA. The laboratories tested the samples 

and returned the results for analysis. Assay results were 

analyzed as S/P ratios, with S/P ratios ≥ 0.40 considered 

positive. 

Results 

Distribution of the results from the 12 laboratories is summarized 

in Figure 1. As is shown in Figure 1, variation in S/P results 

increased as the concentration of antibody in the sample 

increased. Overall, this had little impact on categorical results. 

That is, among the 276 samples tested by the 12 laboratories, 

133 samples tested positive in all laboratories; 136 samples 

tested negative in all laboratories, and 7 samples had discordant 

results (Table 1). With the exception of sample #7, a discordant 

result was reported in each case by only one of the 12 

laboratories. Discordant results for sample #7 were reported at 3 

laboratories, but this may be explained by the fact that all results 

for sample #7 clustered close to the 0.40 cutoff. 

Table 1: Summary of discordant results 

Laboratory 

Sample 1 2 3 4 5 6 7 8 9 10 11 12 

1 0.03 0.00 0.02 0.05 0.00 0.08 0.03 0.51 0.01 0.00 0.06 0.02 

2 0.05 0.01 0.00 0.01 0.01 0.04 0.04 0.03 0.00 0.72 0.00 0.01 

3 0.03 0.01 0.04 0.05 0.00 0.04 0.02 0.01 3.03 0.02 0.02 0.00 

4 1.19 0.03 0.04 0.06 0.00 0.04 0.01 0.08 0.03 0.02 0.04 0.04 

5 0.07 0.04 0.01 0.00 0.02 0.02 0.04 0.00 0.07 0.96 0.00 0.02 

6 0.05 0.03 0.00 0.02 0.02 0.00 0.02 0.03 0.02 0.01 0.04 1.21 

7 0.27 0.26 0.26 0.39 0.45 0.27 0.33 0.41 0.40 0.26 0.26 0.25 


The ring test results showed that the PRRS oral fluid IgG ELISA 

was highly reproducible across laboratories. These results 

support the routine use of this test in laboratories providing 

diagnostic service to pig producers. Thus, herd monitoring based 

on oral fluid sampling could be one part of a PRRSV control 

and/or elimination program. Further, the successful adaptation of 

one assay to the oral fluid matrix suggests that this approach 

could provide the basis for monitoring specific health and welfare 

indicators in commercial swine herds using a "pig friendly" 

approach. 

References 

1. Kittawornrat, A et al.: 2012. Detection of PRRSV antibodies in oral fluid 

specimens using a commercial PRRSV serum antibody ELISA. J Vet 

Diagn Invest 24 (2):262-263 

Figure 1: PRRS oral fluid antibody ELISA results from 237 

samples tested in 12 laboratories

S1 - O - 18 

REAL TIME PCR, MYCOPLASMA GALLISEPTICUM, MYCOPLASMA SYNOVIAE, MYCOPLASMA 

MELEAGRIDIS 

Pablo Lopez 1 , Phyllls I. Tyrrell 2 

1 

IDEXX Laboratories, Livestock and Poultry Diagnostics, Westbrook, Maine, USA 

2 

IDEXX Laboratories, Research and Development, Westbrook, Maine, USA 

Introduction 

Pathogenic Mycoplasma species are widely prevalent, and 

detrimental to poultry health and production. The pathogenic 

Mycoplasma species for poultry include M. gallisepticum (Mg), M. 

synoviae (Ms) and M. meleagridis (Mm). Infection with these 

pathogens can lead to airsaccultis, synovitis, respiratory disease, 

decreased growth and decreased egg production (3). Detection 

has historically been via Hemagglutination Inhibition (HI), Serum 

Plate Agglutination (SPA) and Enzyme Linked Immunosorbent 

Assay (ELISA), with culture isolation as the gold standard of 

testing. The use of Real-time PCR for the detection of pathogenic 

mycoplasmas has emerged as a rapid and highly accurate 

technique for the recognition or confirmation of common 

Mycoplasma species in a flock. The reagent sets use 

hybridization probes to allow for the creation of melting curves 

that provide another level of diagnostic sensitivity and confidence 

in test results. 


All samples were run according to reagent set instructions, using 

IDEXX Mg Detection Reagents (lot #1058), Ms Detection 

Reagents (lot #1060) or Mm Detection Reagents (lot #1062). 

Briefly, reactions consisted of 4 μL Roche Genotyping Master 

Mix, 9 μL nuclease-free PCR grade water, 2 uL of species - 

specific detection reagent and 5 μL DNA extraction sample. 

Cycling conditions were as follows: 

Activation: 95°C, 10 minutes, 1 cycle 

Amplification (45 cycles): 95°C, 20 seconds 

60°C, 20 seconds, single acquisitions 

73°C, 15 seconds 

Melting Curve: 95°C, 1 minute 

45°C, 1 minute 

80°C, 0.14°C/second ramp rate, continuous acquisitions 

All samples were tested on a Roche LC 480 LightCycler at either 

IDEXX Laboratories or field sites, using 96-well reaction plates 

with optical film covers. 

Results 

M. gallisepticum M. synoviae M. meleagridis 

Sample DNA Crossing T m Crossing T m Crossing T m 

Point Point Point 

M. iowae negative negative negative negative negative negative 

M. lipofaciens negative negative negative negative negative negative 

M. cloacale negative negative negative negative negative negative 

M. imitans negative negative negative negative negative negative 

M. gallisnarum negative negative negative negative negative negative 

M. gallinaceum negative negative negative negative negative negative 

M. glycophilum negative negative negative negative negative negative 

M. pullorum negative negative negative negative negative negative 

M. gallopavonis negative negative negative negative negative negative 

10 fg 28.58 63.99 28.92 68.48 28.71 71.48 

1 fg 31.70 63.64 32.22 67.92 32.84 71.42 

nc negative negative negative negative negative negative 

nc negative negative negative negative negative negative 


The use of hybridization probe technology allows for the 

simultaneous generation of crossing point data as well as melting 

curve analysis to confirm positive results. While ELISA 

technology remains the current standard in screening assays for 

avian mycoplasmas, we hope that these standardized reagent 

sets will improve testing confidence and be incorporated into 

laboratory protocols.


“Diagnostics 

at the point of interest” 

(2 nd session)

S2 - K - 01 

WHEN DO YOU WANT THE RESULT? HOW MUCH DO YOU WANT TO PAY! 

Andrew Soldan 

Animal Health and Veterinary Laboratories Agency, AHVLA Weybridge, New Haw, Addlestone, Surrey, KT15 3NB, UK 

The results of some tests are not time critical. For 

surveillance testing of healthy populations to confirm the absence 

of disease the cost of testing and the ability to handle large 

numbers of tests is likely to be more important than the speed to 

result. In many other circumstances a result the day after 

sampling is perfectly adequate. However there are situations 

when a same day result or even a next to patient result would be 

advantageous. In general these tests can justify a higher unit kit 

cost. This paper explores the relationship between cost, 

convenience and speed to result of tests for infectious disease in 

animals. It considers what an ideal rapid patient side test would 

look like. 

In human medicine very large sums are being invested to 

develop point to care testing, especially for molecular agent 

detection. However point of care testing in humans is not the 

equivalent of pen-side testing for animals. It is likely that a spin 

off from the investment in human diagnostics will be significant 

improvements in near patient animal testing. This will be done in 

practice labs, labs set up close to a disease outbreak and in 

regional rather than central labs. The new tests will be rapid, 

accurate, and need little skill or training to run. For veterinary 

applications it is likely that platforms with lower instrument costs 

will find greater acceptance. An issue that both veterinary 

practitioners and regulators will need to address is that owners 

will be able to buy and run many of these tests themselves. 

This paper discusses the major categories of rapid tests for 

serology and agent detection including consideration of some of 

the commercial technologies that are just coming to market. 

Rapid tests, point of care testing, pen-side testing, cost, speed

S2 - O - 01 

DEVELOPMENT OF A RAPID ISOTHERMAL ASSAY TO DETECT TAYLORELLA EQUIGENITALIS, THE 

CAUSATIVE AGENT OF CONTAGIOUS EQUINE METRITIS. 

S. E. North, P. Das, P.R. Wakeley, J. Sawyer 

Technology Transfer Unit, Specialist Scientific Support Department, Animal Health Veterinary Laboratories Agency, Weybridge, United Kingdom. 

Introduction 

Contagious Equine Metritis (CEM) is an infection of the equine 

genital tract, caused by the bacterium Taylorella equigenitalis. 

Identifying T. equigenitalis from equine genital swabs, and 

differentiating it from the closely related Taylorella asinigenitalis, 

using a standard bacteriological culture methodology is laborious, 

slow and relies on the presence of live bacteria. We have 

developed a rapid isothermal assay for the detection of T. 

equigenitalis, the causative agent of CEM, using Recombinase 

Polymerase Amplification (RPA). RPA is a method of amplifying 

DNA or RNA. Like the conventional PCR method of amplifying 

nucleic acid, RPA employs the use of two primers and a probe, 

however, the reaction proceeds at a single temperature (37C) 

and does not cycle through various temperatures as PCR does. 

The reactions can be monitored in real time, based on 

fluorescence readings, or the products of amplification can be 

detected at the endpoint of the reaction on a lateral flow device 

(LFD). 


Equine genital swabs were used to inoculate culture medium 

before a crude DNA extraction was performed, enabling 

comparisons to be made with conventional culture methods. The 

assay was tested, in real time, against 60 equine genital swabs 

(30 positive and 30 negative, as determined by culture). To 

determine the analytical specificity of the assay it was tested 

against a panel of 38 bacterial species, including closely related 

species and equine commensals. The analytical sensitivity of the 

assay was calculated using a dilution series of target DNA which 

was tested in triplicate. A second probe was introduced, specific 

for T. asinigenitalis, which enabled detection and differentiation of 

both T. equigenitalis and T. asinigenitalis. The T. equigenitalis 

assay was adapted, by use of a labelled probe, to enable 

detection of amplification products using a LFD. 

Results 

The assay to identify T. equigenitalis amplified a 121bp fragment 

of the 23S rDNA genes. A crude DNA preparation of a control 

strain of T. equigenitalis could be consistently detected in five 

minutes and the assay will detect as few as 46 gene copies in 12 

minutes. The assay did not cross-hybridise with any of the 38 

different bacterial organisms tested to determine specificity, 

including closely related species and known equine commensal 

bacteria. Fifty-seven of the 60 DNA extracts from genital swabs 

were correctly identified. The introduction of a second probe into 

the assay enabled the simultaneous detection and differentiation 

between T. equigenitalis and T. asinigenitalis. The T. 

equigenitalis assay was successfully adapted to enable detection 

of amplification products using a LFD. 


We have successfully developed a rapid and sensitive isothermal 

assay to identify T. equigenitalis. Additionally, the inclusion of a 

second probe enables the detection of T. asinigenitalis. The 

amplification products can be detected in real time or via LFD. 

This test provides a fast (under 15 minutes) and accurate means 

of identification. The low energy requirements, lack of expensive 

equipment, freeze dried reagents and LFD readout makes this 

test a suitable candidate for rapid diagnosis and treatment in low 

technology laboratories or near to the point of interest. 

Isothermal, amplification, equine, metritis, RPA

S2 - O - 02 

LABORATORY VALIDATION OF AN IMMUNOCHROMATOGRAPHIC TEST FOR THE RAPID 

DETECTION OF KOI HERPESVIRUS (CyHV-3) IN GILL SWABS 

R. Vrancken 1 , N. Goris 1 , T. Leclipteux 2 , F. Lieffrig 3 , S. Wera 1 , J. Neyts 1 and A. Vanderplasschen 4 

1 Okapi Sciences NV, B-3001 Heverlee, Belgium 

2 Coris BioConcept, R&D Department, B-5032 Gembloux, Belgium 

3 CER Groupe, B-6900 Marloie, Belgium 

4 University of Liège, Department of Infectious and Parasitic Diseases (B43b), B-4000 Liège, Belgium 

Immunochromatographic test, Koi herpesvirus, Cyprinid herpesvirus-3 

Introduction 

Carp are one of the most economically valuable fresh-water fish 

in aquaculture. Although the majority of this species is cultivated 

for human consumption (Cypinus carpio carpio), the more 

colourful variant of this species, the koi carp (Cyprinus carpio 

koi), is a popular ornamental fish and may be highly valuable. 

In the late 1990’s, both common carp and koi carp were struck 

with mass mortality, caused by Cyprinid herpesvirus-3 (CyHV-3) 

(1), commonly known as Koi herpesvirus (KHV). Koi herpesvirus 

disease (KHVD) is generally seasonal and may develop rapidly at 

water temperatures between 23 and 25°C inducing mortality up 

to 100% (2). Although clinical signs can be obvious, they are not 

pathognomonic, therefore diagnosis of CyHV-3 relies exclusively 

on laboratory techniques like (q)PCR, virus isolation,… Since 

early detection of KHVD is crucial for disease control, we 

developed an immunochromatographic test for the specific and 

rapid (pond-side) detection of CyHV-3 antigen. 

In this study we evaluated the analytical and diagnostic specificity 

and sensitivity under laboratory conditions. 


Rapid test. Briefly, samples were collected by taking a swab from 

the gills of the animal, which was eluted in 200µl of elution buffer. 

One hundred µl of eluate was subsequently applied to the test 

well of the test cassette and results were read after 15 minutes. 

Analytical specificity. Possible cross-reaction with other 

pathogens affecting carp was evaluated on diseased fish for: 

Ichthyobodo sp., Dactylogyrus sp. and Gyrodactylus sp., 

Ichthyophthirius sp., Trichodina sp., Aeromonas sp., Saprolegnia 

sp., Branchiomyces sp., For Carp Pox virus (CyHV-1) and Spring 

Viremia of Carp virus (SVCV) in vitro cultured virus was used. 

Analytical sensitivity. Serial dilutions of in vitro cultured CyHV-3 

from different geographical regions were used. These included a 

German, an Israeli and two Belgian CyHV-3 field isolates. 

Diagnostic specificity. Animals (n=60) known to be free of CyHV- 

3 were tested using the rapid test as described. 

Diagnostic sensitivity. Two identical animal experiments were set 

up using either the Belgian field strain M1 or M3. Hereto, 120 

common carp (Cyprinus carpio carpio) were distributed over 4 

tanks. Three tanks containing 30 animals were infected by bath 

infection for 1 h at 40 pfu/ml of CyHV-3. One tank of 30 animals 

served as a negative control. Each day, 2 animals per tank were 

sacrificed and tested using the rapid test. All animals found dead 

were analysed as well. DNA of gill tissue was extracted and the 

presence of CyHV-3 was evaluated by PCR (Bercovier TK 

primers) (2). 

Results 

Analytical specificity. No cross-reactivity was observed. Hence, 

the analytical specificity was 100%. 

Analytical sensitivity. All field strains were detected with the rapid 

test with a limit of detection from 1,25 x 10² to 2,4 x 10 4 pfu/ml. 

Diagnostic specificity. No false positive results were observed. 

Hence, the diagnostic specificity was 100%. 

Diagnostic sensitivity. 

Experiment 1: CyHV-3 M1 

The rapid test detected the viral antigen from day 5 post infection 

(p.i.) at a detection rate of 33.3% increasing to 100% on day 9 p.i. 

The detection rate subsequently decreased until the end of the 

observation period (16 days p.i.) (see Figure 1). All animals found 

dead due to KHVD tested positive on the rapid test. Mortality 

started at 7 days p.i. and a relative diagnostic sensitivity of 52.6% 

was estimated when using PCR as golden standard. 

Figure 1. Detection rate in fish infected with CyHV-3-M1. 

Experiment 2: CyHV-3 M3 

The rapid test detected the viral antigen from day 4 p.i. at a 

detection rate of 33.3%, rapidly increasing to 100% on day 7 p.i. 

(see Figure 2). All animals found dead due to KHVD tested 

positive on the rapid test. Mortality was observed from 5 days p.i. 

and a relative diagnostic sensitivity of 71.7% was estimated when 

using PCR as golden standard. 

Figure 2. Detection rate in fish infected with CyHV-3 M3. 


We here present the first rapid immunochromatographic test for 

the specific detection of CyHV-3-antigen. This non-invasive test 

was able to detect the CyHV-3 antigen in a population (aquarium 

or pond) on day 4 or 5 post infection and confirmed the presence 

of CyHV-3 in fish that succumbed to the disease (100% detection 

rate). Although the diagnostic sensitivity of this technique is 

inferior to PCR, this method does have some major advantages 

over PCR and other laboratory techniques. 

The rapid detection of CyHV-3 is crucial in preventing the further 

spread of the disease. With this pond-side test, results are 

available within 15 minutes and carp breeders/koi owners would 

have the opportunity to take the necessary control measures. 

This test does not require trained personnel or expensive 

equipment and samples do not have to be shipped to 

laboratories, thereby saving time. Furthermore, a suspicion of 

KHVD is generally raised in case of mortality and given the 100% 

sensitivity in fish succumbed to KHVD, the detection of an 

emerging infection in the field is highly likely, which makes this 

test fit for purpose. 

In conclusion, we developed the first pond-side test based on 

immunochromatography for the specific detection of CyHV-3 as a 

first line diagnostic tool in the field. 

Acknowledgments 

This study was financed by a project of the Walloon Region (APE 

grant MA-13387-00). 

References 

1. Michel, B., Fournier, G., Lieffrig, F., Costes, B., Vanderplasschen, A. 

(2010). Emerging infectious diseases, 16, 1835-43. 

2. Manual of Diagnostic Tests for Aquatic Animals (OIE, 2009)

S2 - O - 03 

EVALUATION OF RAPID HRSV STRIP TESTS FOR DETECTION OF BOVINE RESPIRATORY 

SYNCYTIAL VIRUS 

Wojciech Socha, Jerzy Rola 

National Veterinary Research Institute, Department of Virology, Pulawy, Poland 

Introduction 

Bovine respiratory syncytial virus (BRSV) is one of the major viral 

pathogens responsible for respiratory tract diseases in cattle 

worldwide. Currently most widely used diagnostic methods for 

direct detection of BRSV are virus isolation test, antigen ELISA 

and RT-PCR (1). Each of them is relatively complicated and 

requires properly equipped laboratory and trained personnel. 

Therefore there is a need for a simple and rapid diagnostic test 

for detection of BRSV in the field conditions. Rapid 

immunochromatographic strip tests detecting viral antigens have 

been developed for closely related to BRSV, Human Respiratory 

Syncytial Virus (HRSV) (2). Due to the high antigenic similarities 

between both viruses it could be theoretically possible to adapt 

this tests for the diagnosis of BRSV. The aim of this study was to 

evaluate this possibility on animals inoculated with BRSV vaccine 

strain. 


Five clinically healthy and serologically negative for BRSV calves 

approximately 6-8 weeks old were selected for experiment. Three 

calves were inoculated intranasally with 2 ml of live vaccine 

Rispoval RS-PI3 (Pfizer). Control calves received 2 ml of sterile 

water. Both groups of calves were housed separately in isolation 

to prevent the spread of the vaccine virus. Nasal swabs were 

taken both from the inoculated and control calves from day -1 to 

28 post inoculation (dpi) and placed in liquid transport medium. In 

the laboratory swabs were shaken, centrifuged and the 

supernatant was stored at -80 o C until examination. 

Three different rapid HRSV strip tests: RSV Respi-strip (Coris), 

TRU RSV (Meridian Bioscience) and BinaxNOW RSV (Inverness 

Medical) were evaluated in the study. All the tests were 

performed according to manufacturer’s instructions. 

The detection limit of all tests was determined by analysing 

a 2-fold dilution series of BT cell cultures infected with BRSV 

strain 375, with each of the strip tests and RT-PCR. Specificity of 

the tests was investigated using two positive reference strains 

(BRSV 375, BRSV A51908) and two strains of other 

paramyxoviruses as a negative control (HRSV A2, BPIV3 SB). 

Another negative control consisting of transport medium from 

UTM-RT Copan system was included. 

Total RNA was extracted from the supernatant of nasal swabs 

using TRI reagent (Sigma) according to the producer’s 

instructions. RT-PCR was performed using Titan One-Tube RT- 

PCR System (Roche) with primers B7 and B8 specific to gene 

encoding glycoprotein G (4). The RT-PCR assay was used as the 

gold standard for this study and samples positive by this method 

were considered true positives. 

Results 

The RT-PCR with primers for glycoprotein G gene was specific 

for BRSV. None of the negative control strains was detected. All 

rapid strip tests reacted positively both with HRSV strain A2 and 

BRSV reference strains and negatively with BPIV-3. The 

detection limit for RT-PCR was 39.1 TCID 50 whereas for each of the 

rapid tests it was around 156 TCID 50 of the virus . 

All three calves vaccinated with the live vaccine Rispoval RS-PI3 

were PCR positive on nasal swabs. In total the vaccine virus was 

found in 12 swabs out of 30 swabs collected from calves 

experimentally vaccinated. Two control calves remained negative 

in PCR throughout the study. Using RSV Respi-strip test virus was 

detected in 7 swabs, four nasal swabs were positive in TRU RSV 

test and 9 in BinaxNOW RSV. All of tests showed negative results for 

control calves. Diagnostic sensitivity, specificity, positive predictive 

value (PPV) and negative predictive value (NPV) were calculated 

for each of the tests (Table 1) 

Table 1. Performance of rapid strip tests for BRSV detection. 

Test 

RSV Respistrip 

Diagnostic 

sensitivity Specificity PPV NPV 

33% 87% 57% 71% 

TRU RSV Test 33% 100% 100% 74% 

BinaxNOW 

RSV 

50% 87% 67% 77% 


Results of our studies confirmed that rapid 

immunochromatographic HRSV tests are able to detected BRSV. 

They also showed high specificity reacting only with HRSV and 

BRSV strains. Positive reaction with HRSV, should not be a 

problem because it is known that this virus has highly restricted 

host range and does not have the ability to infect cattle (3). 

All of the strip tests used in this study were previously evaluated 

in HRSV diagnostics, showing high specificity (97- 98%) with 

sensitivity ranging from 58 to 91%. In our study we made similar 

calculations for the same three strip tests for their performance in 

BRSV detection. Compared with RT-PCR, specificity of strip tests 

was relatively high (87-100%) however their diagnostic sensitivity 

was low (33-50%). This showed that although strip tests reacted 

with similarly high specificity with both BRSV and HRSV, their 

sensitivity was clearly affected when used in detection of bovine 

pathogen. This could be explained by existing differences in 

amino acid sequence of F and N proteins between BRSV and 

HRSV, which can be reason for overall weaker reaction of the 

assays. 

It is also possible that lower diagnostic sensitivity detected in our 

study stemed from different type of samples used for testing. 

Nasopharygneal washes are described as a recommended 

specimen for immunchromatographic tests and previous 

evaluations were done basing on this type of samples. However, 

due to the fact that collection of nasal washes from animals could 

be complicated, we have decided to use nasal swabs as they are 

much easier to get in the field conditions. 

Basing on the evaluation of sensitivity, specificity, PPV and NPV 

of the strip tests, it can be concluded that most reliable was TRU 

RSV. Although its sensitivity was lower compared to BinaxNOW 

RSV it was characterized by very high specificity as well as PPV 

and NPV.That meant that samples positive by TRU RSV were 

true positives. TRU RSV could therefore be used as a supportive 

rapid screen test for BRSV detection at the farm. However, due 

to the limited sensitivity, negative results would have to be 

confirmed by more sensitive tests like RT-PCR. 

References 

1.Larsen, L.E., 2000, Bovine respiratory syncytial virus (BRSV): 

a review. Acta Ve. Scand 41: 1-24. 

2.Selvarangan, R., Abe, D., Hamilton, M., 2008. Comparison of BD 

Directigen EZ RSV and Binax NOW RSV tests for rapid detection of 

respiratory syncytial virus from nasopharyngeal aspirates in a pediatric 

population. Diagn Microbiol Infect Dis 62: 157-161. 

3.Valarcher, J.F., Taylor, G., 2007. Bovine respiratory syncytial virus 

infection. Ve. Res 38: 153-180. 

4.Vilcek, S., Elvander, M., Ballagi-Pordány, A., Belák S., 1994. 

Development of nested PCR assays for detection of bovine respiratory 

syncytial virus in clinical samples. J Clin Microbiol 32: 2225-2231.

S2 - O - 04 

EVALUATION OF A LATEX AGGLUTINATION TEST FOR THE IDENTIFICATION OF CLOSTRIDIUM 

DIFFICILE OF PORCINE ORIGIN 

Introduction 

Clostridium difficile-associated disease (CDAD) in piglets less 

than 1 week old is considered emerging and of great importance 

for its direct effects on piglet performance. Although no direct 

evidence of animal-to-human transmission, isolation and 

accurate identification of CD strains are crucial for both 

diagnostics and epidemiology of CDAD, due to the significant 

overlap between strains implicated in CD infection in humans and 

animals. A definitive diagnostic of CD infection is usually 

achieved by combining toxin testing of stool specimens, along 

with isolation and further toxin production test by CD isolates. 

Jaime Maldonado 1 , Laura Valls 1 

1 

Hipra, Diagnostic Center, Girona, Spain 

Clostridium difficile, latex, agglutination 

In addition to the colony morphology and growth features in 

selective culture media, definitive identification of CD in pure 

cultures is often accomplished by biochemical profiling, and by 

PCR amplification of CD-specific or toxin genes. Commercial 

latex agglutination test (LAT) for presumptive identification of CD 

in stools, broth cultures and solid media are available as an 

alternative to costly and labour-intensive assay. They are rapid 

and simple assays for the early identification of CD. In this study 

we aimed to evaluate the usefulness of a LAT kit for the 

presumptive identification of CD strains of porcine origin. 


The C. difficile Test Kit (Oxoid, Hampshire, UK) was evaluated 

using three groups of bacteria: 1) The test was validated with five 

well identified CD strains of porcine origin from previous studies 

(1), with known toxin and ribotype profiles; 2) Further evaluation 

was carried out with 41 CD field isolates coming from suckling 

piglets suffering from diarrhoea in the 1 st week of age. They came 

from the same number of epidemiologically unrelated pig 

breeding units, and were collected in the period 2008-2011. 

Bacterial culture and identification were performed as previously 

described (1). Definitive identification to species level was carried 

out using the API Rapid ID 32 A kit (BioMerieux, Marcy l'Etoile, 

France); 3) Cross-reactivity in the LAT kit was determined using 

reference and collection strains of swine Escherichia coli (n=4), 

Clostridium perfringens types A, B, C, D and E (n=5), Salmonella 

typhimurium (n=1), Proteus mirabilis (n=1) and Clostridium novyi 

(n=1). The LAT kit was performed as per manufacturer’s 

instructions, with all three groups of bacteria involved in the 

study. Agglutination reactions were scored as negative (0), mild 

(1+), moderate (2+), or strong (3+). 

Results 

When LAT was carried out with well characterized CD antigens of 

porcine origin, it correctly identified all five bacteria with a strong 

(3+) agglutination reaction. Similarly, all 41 field CD strains 

identified by the API system with excellent (24,4%) or good 

(75.6%) identification scores, also tested positive with 1+ 

(14.6%), 2+ (29.3%), or 3+ (56.1%) agglutination reactions. No 

correlation between the two scores was observed. 

Nevertheless, with respect to cross-reactivity, two strains of C. 

perfringens (Types A [3+] and E [1+]), one strain of E. coli (3+), 

and the swine pathogen C. novyi (2+) tested positive in the LAT, 

and hence were incorrectly identified as being CD. 

Figure 1: Latex agglutination test: 1. Swine C. difficile strong 

reaction (3+); 2-4. Cross-reactivity of swine C. novyi (2+), C. 

perfringens type A (3+) and E. coli (3+), respectively; 5. 

Negative control of the test. 6. Positive control of the test. 


Taking together, the results obtained in this study indicates that 

the LAT kit has a good ability to identify CD, since none of the 

collection or field CD strains tested negative. However, the fact 

that three bacterial species that are part of the pig intestinal flora 

have tested positive, indicates that positive results using this kit 

should be interpreted with caution. In fact, the kit manufacturer 

warns of the need to confirm all positive results, which is 

evidenced in this study. 

Considering that a wide variety of CD strains were tested by LAT 

in the present study, it is reasonable to assume that negative 

agglutination test results obtained with the evaluated kit, under 

the described conditions (pure cultures on solid media), may 

correspond to true negative. Consequently, the LAT kit described 

could eventually be used to test porcine bacterial cultures 

suspicious of being CD; positive results must be confirmed by a 

more specific assay, and negative results should be coupled with 

growth features and case history. 

Although CDAD is considered a major threat for the swine 

industry, and despite the obvious similarities between the CD 

strains affecting humans and domestic animals, existing 

diagnostic tools have not been evaluated in depth using clinical 

samples of porcine origin. It would be necessary to do so, with 

those test intended for toxin detection in stools and molecular 

detection of toxin genes, in order to improve the current 

diagnostic approach to CD-related neonatal diarrhoea in swine. 


We thank Carmen Sánchez and Lidia López who collected and 

identified bacterial strains during the study. 

References 

1. Alvarez-Perez S, Blanco JL, Bouza E, Alba P, Gibert X, Maldonado J, 

Garcia. (2009). Prevalence of Clostridium difficile in diarrhoeic and nondiarrhoeic 

piglets. Vet. Microbiol, 137, 302-5.


“Emerging, re-emerging 

and wildlife diseases – 

diagnostic possibilities” 

(3 rd session)

S3 - K - 01 

EMERGING AND RE-EMERGING WILDLIFE DISEASES: PATHOLOGY AND RELATED TECHNIQUES 

FOR DIAGNOSTICS AND FOR GENERAL AND TARGETED SURVEILLANCE 

Dolores-Gavier Widén 

Department of Pathology and Wildlife Diseases, Deputy Head of Department, National Veterinary Institute (SVA), SE‐75189 Uppsala, Sweden 

Wildlife surveillance, emerging diseases, pathology 

The importance of wildlife and wildlife diseases 

Wild animals have high economic, social, ecological and cultural 

value and play fundamental roles in the maintenance of stable 

ecosystems. Wildlife contributes significantly to recreation and 

tourism. Hunting is an important socio-economic activity. Within 

the EU hunting is estimated to be worth €16 billion 1 and there are 

more than 7 million hunters in Europe. 2 

Wildlife trade is a fast growing market and contributes to the 

worldwide spreading of new pathogens and emerging diseases, 

for example giant liver fluke (Fascioloides magna) was introduced 

into Europe in elk from North America. Natural migration of wild 

animals has similar consequences, as exemplified by the global 

spread of highly pathogenic H5N1 avian influenza. Wildlife 

reservoirs maintain infections which may spillover to domestic 

animals, making the control and eradication difficult, for example 

tuberculosis and classical swine fever. Wild animals host 

numerous zoonotic infections. Humans can acquire infections 

through direct contact with wild animals, for instance rabies and 

tularemia, through food consumption, as trichinosis and hepatitis 

E, or from the environment contaminated by wildlife, such as 

hantavirus infections or alveolar echinococcosis. 

Mass mortality in wild animals results in public concern, as seen 

during botulism outbreaks in waterfowl, trichomoniasis epidemics 

in wild finches and morbillivirus infections in seals. Furthermore, 

diseases may have a devastating impact on wildlife populations 

and threaten their conservation, like White Nose Syndrome 

(Geomyces destructans infection) in bats. 

Therefore, the value of maintaining diverse and healthy wildlife 

populations cannot be overestimated. The importance of wildlife 

surveillance and the interest on diagnosing infections in wild 

animals has increased rapidly in Europe and globally. This is 

clearly supported by the Director General of the OIE, who stated 

that “Surveillance of wildlife diseases must be considered equally 

as important as surveillance and control of diseases in domestic 

animals”. 3 

Emerging and re-emerging infectious diseases 

Emerging infectious diseases (EID) include broad categories of 

infections characterized by changes in their epidemiological 

presentation. EID are diseases whose incidence has increased in 

a defined time period and location or threatens to increase. If the 

infection was unknown in the place before, the disease is 

regarded as emerging, but if it had been present at the location in 

the past and was considered controlled or eradicated, the 

infection is considered to be re-emerging. The following situations 

are all examples and mechanisms of EID: new pathogens or 

newly evolved strains of existing pathogens, spread to new 

geographic area (for example through wildlife migration or 

translocation), spread to new populations, increase in 

prevalence, detection of previously unrecognized pathogens and 

old infections reemerging as a result of antimicrobial resistance. 

Emerging infectious diseases (EID) in humans are dominated by 

zoonoses, and constitute 60% of EID events. 4 

A high rate of 

these zoonoses (72%) are caused by pathogens with a wildlife 

origin, and this rate has increased significantly with time 

(controlling for reporting effort). 4 

As a whole, zoonoses from 

wildlife are considered the most significant, growing threat to 

global health of all EID. Vector-borne diseases are frequent 

among EID events (22.8% of events) and these infections have 

also increased over time. 4 Climate change is probably implicated, 

however, it is difficult to demonstrate causal relationships 

between EID events and climate change. Emerging pathogens 

are more likely to be zoonotic or vector-borne with a broad host 

range. 4 Drivers such as human population density, antibiotic drug 

use and agricultural practices are major determinants of the 

distribution of EID events. 4 

Good knowledge and rapid identification of emerging infectious 

diseases occurring in wildlife are essential for the design and 

implementation of preventing, control and mitigating measures to 

protect the health of humans and animals. 

Wildlife health surveillance 

Surveillance, is defined by the OIE) as “the systematic ongoing 

collection, collation, and analysis of information related to animal 

health and the timely dissemination of information to those who 

need to know so that action can be taken”. 5 

General wildlife 

disease surveillance, also named passive surveillance, identifies 

ill or dead wild animals and conducts diagnostic investigations to 

determine the cause of disease or death. The investigations 

include of a broad range of wildlife hosts and pathogens or 

diseases. Targeted surveillance, also named specific or active 

surveillance, identifies a specific infection or disease, 

serologically or demonstrating the pathogen. Surveillance 

informs about occurrence of pathogens, wildlife hosts and 

locations of endemic infections and can also result in the 

detection of new pathogens or diseases. Long term surveillance 

detects changes in disease patterns, which contribute to 

epidemiological analyses and risk assessment. All together, 

surveillance is essential in the control and eradication of 

infectious diseases. 

There is a great variation in the level of wildlife surveillance 

conducted in different countries in Europe. As a whole, more 

than 18,000 wild animals are investigated by general surveillance 

and more than 50,000 by targeted surveillance annually in 

Europe. 6 

The choice of diagnostic tests to be used for surveillance of 

wildlife diseases needs to take into consideration several factors, 

for example the purpose of the testing (such as certifying 

freedom from infection before translocation, estimating 

prevalence, or others), the practicalities and applicability to field 

investigations, the cost of the tests and the known performance 

of the method in the host species to be tested. 

Test validation 

The performance of tests designed and validated for use in 

domestic animals is most often not known when applied to 

wildlife species. Diagnostic testing of wild animals is challenging 

in several ways, for example the samples may be of poor quality 

or difficult to obtain. Likewise, validation of assays for wildlife also 

presents difficulties. In 2011, the OIE established an ad hoc 

group which drafted guidelines on “Principles and methods for 

the validation of diagnostic tests for infectious diseases 

applicable to wildlife”, 7 which take into consideration the intrinsic 

difficulties of validating tests for wildlife. In the guidelines, 

pathways and stages are proposed for validation of tests with two 

different starting points, when a previously validated test in 

domestic animals exists and when it does not. It is proposed that 

when full validation is not possible, the best alternative may be to 

evaluate the fitness of the test in a small number of reference 

samples. These preliminary estimates may give sufficient 

information to obtain provisional acceptance of the test by the 

authorities; this is a new step /category within stage 2 of the 

validation process. 

Pathology 

Pathology is a primary component in general wildlife disease 

surveillance. Rudolph Virchow (1821-1902), a founding father of 

pathology, made seminal advancements in the understanding of 

infectious diseases based on observations at abbatoirs and of 

tissue changes at the cellular level; he was active in the 

implementation of meat inspection. Today, despite the technical

advances, the fundamental tools of pathology are still post 

mortem examination and histopathology, and these are the 

starting point for many diagnostic investigations. Additionally, 

pathology is essential in the early and rapid identification of 

emerging infections and/or changes in disease patterns. 

Pathology techniques 

Histopathology is conducted as a standard on haematoxylin-eosin 

stained sections. Specific stains are used to identify structures 

and substances and to help visualize pathogens, for example 

Congo red for amyloid and Ziehl-Neelsen for acid fast bacteria. 

Enzyme histochemistry can be used to test enzyme activities in 

muscle diseases and in other conditions. Cytology, the study of 

cells spread on slides, is applied on clinical samples, such as 

liquid punctures, or smears of organs. Electron microscopy is 

used to characterize structural changes in cells and tissues and to 

support the morphological changes observed by microscopy. 

Immunohistochemistry is used to reveal specific antigens or 

proteins in tissue sections applying labelled antibodies against the 

particular protein and visualizing the antigen-antibody reaction by 

a marker such as fluorescent dye or enzyme. IHC is a very useful 

technique used to classify tumors and detect bacteria, viruses and 

parasites. Importantly, IHC allows associating the presence of 

pathogens with lesions. IHC can be automated and is widely 

applied to diagnosis. Double-labelling techniques are used to 

identify two or more different proteins, giving information on for 

example cell type and presence of a particular virus in the cell. 

Immuno-electron microscopy uses immunolabelling of ultrathin 

sections, and is visualized with markers as colloidal gold particles. 

Quantifying pathology gives detailed information on lesions 

numbers and size. This results in accurate descriptions, for 

example of degree of advancement and dissemination of lesions, 

and allows comparison of lesion severity between animals, for 

instance, in assessment of disease protection by vaccine 

candidates. Morphometry, the macroscopic or microscopic 

measurement of lesions, or parts of lesions (for example necrosis) 

gives quantitative data. Computerized image analysis allows 

collection of large amounts of data in a short time. Scoring of 

lesions is a semi-quantitative assessment of lesions size and/or 

number, the scoring has to be standardized for each application. 

Imaging techniques developed for clinical application in humans 

allow tridimensional measurements. They can also be used in 

veterinary pathology for measuring lesions in dead animals, 

mostly in experimental settings. Some examples are magnetic 

resonance imaging (MRI) and multidetector computed 

tomography (MDCT), used for example to measure the 

magnitude of lesions in pulmonary tuberculosis. X-rays are used 

frequently in wildlife pathology for forensic investigations to detect 

bullet fragments in carcasses. 

Molecular diagnostics are increasingly used to identify pathogens 

in combination with the diagnostic histopathology. In situ 

hybridization is based on the hybridization of a labelled probe (a 

small specific DNA or RNA sequence); with a complementary 

sequence of for example virus, on a tissue section, the reaction 

can be visualized by chromogens or fluorescein (FISH). PCR and 

the numerous rapidly developing PCR-based techniques are 

widely used to establish an etiological diagnosis. PCR is usually 

performed in unfixed (fresh or frozen) samples, but in many cases 

can be applied to formalin fixed paraffin embedded tissue. The 

target lesion, for example a granuloma, can be micro-dissected 

and tested individually. Microarray technology detects large 

numbers of genomes simultaneously allowing identification of 

multiple infectious agents at the same time in one sample. The 

EU project WildTech, 8 is developing tests for screening wildlife 

populations for infections, including nucleic acid microarrays and 

ELISA technology adapted to microarray format for serology. 

Obtaining the correct sample of target tissues and lesions for 

laboratory testing is of utmost importance, in particular when 

small amounts of tissues are tested. A very sensitive method 

applied to “the wrong sample” can have a small chance of 

detecting the pathogen. 

The importance of networking 

Wildlife disease surveillance is a very extensive field involving 

different diagnostic disciplines and a broad range of wildlife hosts 

and pathogens. It also includes epidemiology and communication 

activities. Networking among disciplines and across national and 

organization boundaries is of great benefit in surveillance 

programs. Organizations such as the European Association of 

Veterinary Laboratory Diagnosticians (EAVLD) 9 and the Wildlife 

Disease Association (WDA) 10 , with its European Section (EWDA) 

are excellent forums. 

Conclusions 

In conclusion, pathology plays a key role in wildlife surveillance 

and in discovering new diseases and is the starting point of 

diagnostic investigations. Importantly, pathology informs about 

the significance, in terms of disease, of the presence of infectious 

agents or particular strains of agents demonstrated by laboratory 

tests. Classical descriptive pathology is today more refined by 

new techniques but cannot be replaced by other disciplines and 

will continue to be a cornerstone in the detection and 

characterization of emerging diseases. Wild animals play an 

increasingly important role in emerging infectious diseases, in 

particular zoonoses. This trend will possibly continue in the 

future. 

Acknowledgement 

WildTech, a project of the European Commission under the Food, 

Agriculture and Fisheries, and Biotechnology Theme of the 7th 

Framework Programme for Research and Technological 

Development, grant agreement no. 222633. 

References 

1. Kenward, R. & Sharp, R. (2008) Use Nationally of Wildlife Resources 

Across Europe, 117-132.: in Manos, P. & Papathanasiou, J. [eds.] (2008) 

GEM-CON-BIO: Governance & Ecosystems Management for the 

Conservation of Biodiversity. Thessaloniki 

2. Federation of Associations for Hunting and Conservation of the EU 

(FACE), at: www.face.eu/aboutus-en.htm 

3. Vallat B. 2008, OIE editorial. Improving wildlife surveillance for its 

protection while protecting us from the diseases it transmits. At : 

www.oie.int/en/for-the -media/editorials/detail/article/improving -wildlifesurveillance-for-its-protection-while-protecting-us-from-the-diseases-ittransmit/ 

4. Global trends in emerging infectious diseases. Kate E. Jones, Nikkita 

G. Patel, Marc A. Levy, Adam Storeygard, Deborah Balk, John L. 

Gittleman, & Peter Daszak. Nature. Vol 451| 21 February 2008| 

doi:10.1038/nature06536 

5. World Organization for Animal Health (OIE) (2009) Terrestrial Animal 

Health Code , 18 th Ed. Glossary. OIE. Paris. Available at: 

www.oie.int/index.php?id=169&L=0&htmfile=glossaire.htm#terme_surveill 

ance). 

6. Establishing a European network for wildlife health surveillance. Kuiken, 

T., Ryser-Degiorgis M.-P., Gavier-Widén, D., Gortázar, C. Rev. Sci. Tech. 

Off. Int. Epiz, 2011, 30 (3), 755-761. 

7. REPORT OF THE MEETING OF THE OIE BIOLOGICAL STANDARDS 

COMMISSION Paris, 14–16 September 2011. At: www.oiw.int/ 

8.WildTech at: www.wildtechproject.com/wildtech/ 

9. European Association of Veterinary Laboratory Diagnosticians 

(EAVLD), at www.eavld.org/ 

10. Wildlife Disease Association (WDA), at : www.wildlifedisease.org

S3 - O - 01 

SCHMALLENBERG VIRUS OUTBREAK IN THE NETHERLANDS: ROUTINE DIAGNOSTICS AND TEST 

RESULTS 

Ruth Bouwstra 1 , Wim van der Poel 1 , Eric de Kluijver 1 , Betty Verstraten 1 , Johan Bongers 1 

1 

Central Veterinary Institute, of Wageningen University and Research Centre (CVI-Lelystad), Department of Virology, Lelystad, The Netherlands. 

Introduction 

At the end of 2011, a new Orthobunya virus named 

Schmallenberg virus (SBV), was discovered in Germany. Soon 

thereafter in the Netherlands, the virus was associated with 

decreased milk production, watery diarrhoea and fever in dairy 

cows, and subsequently also with congenital malformations in 

calves, lambs and goat kids (1,2). By the 20th of December 2011 

in the Netherlands malformations in new-borns of ruminants were 

made notifiable. After a notification by a farmer or veterinarian, a 

maximum of five malformed new-borns per farm were 

necropsied. The diagnosis of Schmallenberg virus disease was 

based on the pathologic findings and RT-PCR test results of 

brain tissue of the malformed new-borns. In addition blood 

samples from mothers of affected new-borns were collected and 

tested for antibodies against SBV using a virus neutralization 

test. 

Schmallenberg virus, routine diagnostics, test results 


Brain samples were tested with the RT-PCR for Schmallenberg 

virus that has been developed by the Friedrich-Loeffler Institute in 

Germany. Serum samples were tested in a virus neutralisation 

test (VNT) against SBV developed by the Central Veterinary 

Institute. A virus isolate from brain tissue of a lamb, 4th passage 

on VERO cells, was used in the test (3). 

Results 

Between 20th of December and March the 8th, in total 1165 brain 

tissue samples were tested in the RT-PCR: 577 originated from 

lambs, 444 from calves and 42 from goat kids. In the VNT 681 

blood samples were tested: 329 originated from ewes, 329 from 

cows and 23 from goats. Results showed that 8% of the tested 

calf brains, 31% of the tested lamb brains and 12% of the tested 

goat kid brains were RT-PCR positive. The number of malformed 

lambs and RT-PCR positive lamb brains decreases over time 

while the number of malformed calves and RT-PCR positive calf 

brains increases (fig 1,2). In the VNT 95% of the ewes, 93% of 

the cows and 23% of the goats tested positive. Combining the 

results of the RT-PCR and the VNT, 20% of all farms tested 

positive in both the RT-PCR (genetic material of SBV in brain 

tissue in malformed new-borns) and the VNT (antibodies against 

SBV in blood from mothers of affected new-borns ). The results 

reported here are based on testing up to the first week of March 

2012. Additional test results which will be available in June will 

be presented also. 

Figure 2: Number of RT-PCR positive (dark grey bars) and RT- 

PCR negative (light grey bars) calf brain tissue samples per 

week. 


In goats the number of seropositives is far lower than in sheep 

and cattle. Less samples from goats were tested and the 

estimated seroprevalence is therefore less precise. Furthermore 

number of RT-PCR positive calves and goat kids is far lower than 

in sheep. Given that goats, in contrast to cattle and sheep, are 

often housed indoors, and the SBV is supposedly transmitted by 

Culicoides vectors, this lower number of test positive results 

would not be surprising. In addition, the difference in pregnancy 

length between cows and sheep might explain why it is more 

difficult to detect genetic material of SBV in brain tissue of calves 

and, assuming that infection of cows and sheep was around the 

same period, it might also explain the difference in number of 

malformations and RT-positives over time. Supposing that all 

malformations notificated are truly caused by the Schmallenberg 

virus, on farm level, diagnostic sensitivity of the RT-PCR is much 

lower in comparison with the VNT. 


We thank our colleagues from the Dispatching Service Unit and 

the Virological Diagnostic Laboratory for execution of the 

diagnostic testing. 

References 

1. Muskens J, Smolenaars AJG, van der Poel WHM, Mars MH, van 

Wuijckhuise L, Holzhauer M, van Weering H, Kock P. Diarrhea and loss of 

production on Dutch dairy farms caused by the Schmallenburg virus. 

Tijdschr Diergeneeskd. 2012; 137: 112-5. 

2. Van den Brom R, Luttikholt SJM, Lievaart-Peteron K, Pperkamp NHMT, 

Mars MH, van der Poel WHM, Vellema P. Epizootic of ovine congenital 

malformations associated with Schmallenberg virus infection. Tijdschr 

Diergeneeskd 2012; 137: 106-11. 

3. Loeffen WLA et al. J Virol Methods 2012 (in preparation) 

Figure 1: Number of RT-PCR positive (dark grey bars) and RT- 

PCR negative (light grey bars) lamb brain tissue samples per 

week.

S3 - O - 02 

25 YEARS OF PASSIVE SURVEILLANCE OF BATS IN THE NETHERLANDS. MOLECULAR 

EPIDEMIOLOGY AND EVOLUTION OF EBLV-1. 

Introduction 

Since 1986 a reactive surveillance program is running in the 

Netherlands to investigate the presence of European Bat 

Lyssavirusses (EBLV-1 and EBLV-2) in bats. In this program all 

contact cases, with human or pets, are send to the laboratory in 

Lelystad and tested for EBLV with a EU prescribed immune 

fluorescence test (IFT). Up to date almost 5000 samples have 

been tested. The main reservoir for EBLV is Eptesicus serotinus, 

334 positive cases so far which is about 22% of the samples 

tested, second species is Myotis dasycneme, with almost 4% 

positive thus far (Table 1). EBLV-2 has not been detected in the 

Netherlands since 1993. 


Bat brains were tested using the prescribed fluorescent antibody 

test (FAT). For conformation a real time PCR test on the N-gene 

was used. On the same nucleic acid isolation used for the realtime 

PCR N and G-gene sequencing was performed on an ABI 

sequencer according to the manufacturers protocol. The species 

of all submissions were determined by a bat expert. 

The MCC phylogenetic tree, estimates of the rate of molecular 

evolution (substitutions per site per year) and the TMRCA for the 

alignments were inferred using a Bayesian MCMC method in the 

BEAST package. 

B. Kooi, A. Vogel and E. Van Weezep 

Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands. 

Lyssavirus, EBLV, bats, surveillance, molecular epidemiology 

Results & Discussion 

We performed a molecular epidemiology study on a collection of 

more then 40 EBLV-1 viruses that currently circulate in the 

Netherlands. Based on the sequences of the coding regions of 

the N and G genes a phylogenetic analysis was performed in 

which the two distinct lineages or subgroups EBLV-1a and EBLV- 

1b were clearly visible (Fig. 1). Surprisingly the larger EBLV-1a 

group could be further subdivided into three phylogenetic groups 

that were consistent with topology. Two groups were 

predominantly found in the northern provinces whereas the third 

group was spread from east to west in the middle of the country. 

A most recent common ancestor analysis was performed which 

confirmed a distinct difference in origin of the two EBLV-1 

subgroups. Whereas the EBLV-1a subgroup migrated into 

Europe via the east-west route, EBLV-1b apparently entered the 

continent from the south, most likely through the Iberian 

Peninsula. 

. 

Table 1: Passive lyssavirus surveillance of bats (1986-2011) 

Species tested postitive 

no determination 10 - 

Myotis mystacinus 22 - 

Myotis nattereri 10 - 

Myotis daubentonii 124 - 

Myotis dasycneme 138 5 

Pipistrellus pipistrellus 2216 - 

Pipistrellus nathusii 358 - 

Nyctalus noctula 77 - 

Nyctalus leisleri 4 - 

Eptesicus serotinus 1491 334 

Vespertilio murinus 13 - 

Plecotus auritus 257 - 

total 4720 339 

Figure 1: Phylogenetic analysis of EBLV-1 based on G-protein 

coding regions of EBLV-1 from bats from the Netherlands (2004- 

2011) 

References 

1. Drummond AJ, Rambaut A. BEAST: Bayesian evolutionary analysis by 

sampling trees. BMC Evol Biol. 2007;7(1):214. 

2. Cliquet, F and Barrat J, 2008. Rabies (chapter 2.1.13). Manual of 

diagnostic tests and vaccines for terrestrial animals, OIE (World 

Organisation for Animal Health), Paris, 1: 304-322

S3 - O - 03 

DIAGNOSIS OF Q FEVER IN DAIRY CATTLE BY PHASE-SPECIFIC MILK-SEROLOGY 

Böttcher J 1 , Frangoulidis D 2 , Schumacher M 1 , Janowetz B 1 , Gangl A 1 , Alex M 1 

1 Bavarian Animal Health Service, Poing, Germany, 

2 

Bundeswehr Institute of Microbiology, Munich, Germany 

Q fever, serology 

Introduction 

Dairy cattle herds are frequently endemically infected by C. 

burnetii the causative pathogen of Q fever. Shedding of C. 

burnetii at calving and in milk is of special concern. Chronic 

infection frequently results in an increased level of milk-shedding. 

The diagnostic value of phase-specific serology was assessed by 

a longitudinal study on milk samples and puerperal swabs in an 

endemically infected dairy cattle herd with about 100 dairy cows. 

PhII titer 

10000 

1000 

100 

10 

PhI-serology 

negative 

positive 


Cows were kept in two groups (MS and RB) with close contact 

within one barn. The same calving boxes are used for both 

groups. Individual milk samples were collected from March 2010 

until December 2011; since August 2010 puerperal swab were 

collected from a proportion of cows within 16 h after calving. 

Samples: 405 milk samples, 59 puerperal, 9 samplings (MS) and 

465 milk samples, 64 puerperal swabs, 10 samplings (RB). 

Phase-specific ELISAs were performed as described (1) with 

minor modifications: Milk samples were diluted 1/5 log 10 and the 

titer was calculated at 20% (OD%) of the positive control (serum 

control diluted 1/400). 

Frequency of positive samples 

1,4 

1,2 

1 

0,8 

0,6 

0,4 

0,2 

0 

swabs 

milk samples 

1003 1004 1007 1008 1009 1010 1011 1012 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 

52 46 98 0 46 57 57 52 0 54 58 51 52 0 56 45 47 0 52 46 

0 0 0 5 11 2 10 9 5 9 8 5 0 7 5 3 6 8 11 6 

Sampling (YYMM) and number of milk-samples and swabs, respectively 

Fig. 1: Frequency of positive qPCR (CI95%) was plotted over 

year/month of sample collection (YYMM, top), the number of milk 

samples (middle) and the number of analysed puerperal swabs (bottom). 

Cut-offs for milk&swabs were ≥10 C.b./ml milk and ≥1 C.b./swab. 

qPCR (C.b. log10/swab and ml milk, resp.) 

10 

8 

6 

4 

2 

0 

1003 

1004 

puerperal swabs 

milk 

1007 

1008 

1009 

1010 

1011 

1012 

1101 

1102 

1103 

1104 

Sampling (YYMM) 

Fig. 2: Amount of C. burnetii-target in qPCR-positive swabs and milk 

samples. qPCR scored positive if the titer exceeded 1 C.b./swab and ml 

milk, respectively. 

Milk samples and puerperal swabs were analysed by quantitative 

(q) PCR (2). Target was quantified by a standard curve derived 

from diluted C. burnetii reference isolate Nine Mile RSA493 

(kindly provided by C. Heydel, Giessen). 

The status of milk-shedding was assessed by two procedures: (1) 

Mean shedding per cow was calculated (C.b./ml) and classified 

as 1000 C.b./ml. (2) Shedding 

pattern was defined as chronic shedding (CS; >3 times ≥10 

C.b./ml) irrespective of negative results in between. Intermittent 

shedding (IS3, IS2, IS1) with 3, 2 and 1 positive result, and 

negative, if all samples gave less than 10 C.b./ml. Classification 

relied on a minimum of three samples except for IS3, this status 

required four samplings. 

1105 

1106 

1107 

1108 

1109 

1110 

1111 

1112 

1 

1003 

1004 

1007 

1009 

1010 

1011 

1012 

1102 

1103 

1104 

Milk sampling (YYMM) 

Fig. 3: PhII-titers in cows (500. Only one multiparous CScow 

remained just below 500. Positive predictive value for PhI 500 

was 40%, whereas it was 25% for PhI 100 and PhII 100 , respectively. 

In contrast to CS-cows, IS1/IS2-cows were characterized by a 

striking instability of PhI- and PhII-results. Even after positive 

puerperal swabs seroconversion in milk was not a regular finding. 

PhI - /PhII + -pattern in young cows indicates the episode of 

puerperal shedding at herd level and PhI 500 is a suitable 

screening for heavy milk shedders. 


The present study was supported financially by the Free State Bavaria and the 

Bavarian Joint Founding Scheme for the Control and Eradication of Contagious 

Livestock Diseases (Bayerische Tierseuchenkasse). 

References 

1. Böttcher, J., Vossen, A., Janowetz, B., Alex, M., Gangl, A., Randt A., and Meier, 

N. (2011): Insights into the dynamics of endemic Coxiella burnetii infection in 

cattle by application of phase-specific ELISAs in an infected dairy herd. 

Veterinrary Microbiology, 151, 291-300. 

2. Böttcher, J., Frangoulidis, D., Schumacher, M., Janowetz, B., Gangl, A., Alex, 

M. (2012): Diagnostic value of Coxiella burnetii phase I and II antibody titers in 

individual milk samples of cows. In preparation. 

1108 

1109 

1111 

1112

S3 – O - 04 

EQUINE NOCARDIOFORM PLACENTITIS & ABORTION OUTBREAK 

AND FARM-BASED RISK FACTOR STUDY, 2010-2011 

Craig N. Carter 1 , Jacki C Cassady 1 , Noah Cohen 2 , Laura A Kennedy 1 , Erdal Erol 1 , Tamara Malm 1 , 

Mike Donahue 1 , Steve Sells 1 , Neil Williams 1 Jacqueline Smith 1 , Roberta Dwyer 3 

1 

Veterinary Diagnostic Laboratory, Department of Veterinary Science, University of Kentucky, Lexington, KY USA 

2 Large Animal Clinic, College of Veterinary Medicine, Texas A&M University, College Station, TX USA 

3 Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY USA 

Equine, horse, placentitis, abortion, epidemiology 

Introduction 

The syndrome now known as equine nocardioform placentitis & 

abortion (NPA) that was first seen in Kentucky in the 1980’s, is 

caused by nocardioform actinomycete bacteria. Crossiella equi, 

characterized and named in 2002 (1), and various species of 

Amycolatopsis, 3 species characterized and named in 2003 (2), 

have been isolated from NPA cases since 1988. Two species of 

Streptomyces (3), both isolated in 1999, have also been 

associated with NPA cases. The infection results in late 

abortions, stillbirths and premature foaling, sometimes leading to 

early neonatal mortality. The epidemiology of transmission is not 

known. Interestingly, nocardioform actinomycetes have rarely 

been isolated from any tissue other than placenta. Most cases 

have occurred in the Bluegrass region of Kentucky (Figure 1) but 

cases have also been diagnosed in Florida (4), Italy (5), and 

South Africa. During the 2010-2011 equine reproductive season, 

the Lexington laboratory identified gram positive branching bacilli 

in 118 cases by culture and PCR. The high incidence seen in 

this season precipitated this farm-level study to identify possible 

risk factors for NPA. 


A 34 question survey instrument was designed to gather 

information on farm size, number of mares, mare density, water 

sources, feeding/grazing practices, pasture management, pre 

and post breeding practices, drugs/supplements used and 

diseases/syndromes seen on the farms. A total of 497 farms 

were contacted with 153 responding. Five of the farms were 

disqualified, leaving 148 for analysis. Data was analysed using 

chi-squared, Wilcoxon rank-sum tests and logistic regression 

analysis using S-PLUS, v8.2. 

Results 

Of the farms surveyed, 98 were affected by NPA during the 2010- 

2011 with 50 farms unaffected. The average number of mares 

affected per farm was 4 (range 1-64). The total number of mares 

at risk on all farms was 8075 with 429 mares diagnosed with NPA 

(5.3% all farms, 6.5% affected farms). The outbreak involved 15 

cities and 27 zip codes. Affected farms tended to have greater 

acreage (p < 0.0001), had more mares bred (p < 0.0002) and 

greater mare density (p = 0.0095). The distribution of the number 

of hours horses were grazed from January-March differed 

significantly for affected farms (median 8 hours) and unaffected 

farms (median 12 hours). The proportion of affected farms using 

progesterone pre-breeding was significantly lower than that of 

unaffected farms (OR = 2.9). The proportion of affected farms 

that administered HCG post-breeding was significantly lower than 

that of unaffected farms (OR = 2.3) Finally, the proportion of 

affected farms that administered NSAIDs pre-breeding was 

significantly lower than that for unaffected farms (OR = 2.8). 


The significant findings of farm size, mare numbers and density 

related to affected farms may be an indicator that simple 

crowding and intensive management of mares is a risk factor for 

NPA. It might also indicate greater opportunities for diagnosis of 

NPA. The finding of longer grazing times January-March on 

unaffected farms may indicate a protective effect, i.e. the barn 

environment may be more risky. Use of progesterone prebreeding, 

HCG post-breeding and NSAIDs pre-breeding might 

indicate practices that reduce risk but further studies are needed 

as the chance for false discovery is high. These factors could be 

better assessed in a case-control study in which the unit of 

analysis is the mare, not the farm. A spatial analysis of the data 

was not performed. 


We owe much gratitude to the farms that participated in the 

survey, the Kentucky Thoroughbred Farm Managers Club, the 

Kentucky Thoroughbred Association (KTA), the Kentucky 

Thoroughbred Owners and Breeders (KTOB), the Kentucky 

Association of Equine Practitioners (KAEP), David Switzer and 

Vickie Garcia of KTA and KTOB, Drs. Ernie Martinez and Stuart 

Brown of the KAEP, and Barbara Dunaway and Susan Neal, 

UKVDL. 

References 

1.Donahue JM., Williams NM., Sells SF and Labeda DP: 2002, Crossiella 

equi sp nov., isolated from equine placentas. International Journal of 

Systemic and Evalutionary Microbiology. 52, 2169-2173. 

2. Labeda DP, Donahue JM, Williams NM, et al.: 2003, Amycolatopsis 

kentuckyensis sp. nov., Amycolatopsis lexingtonensis sp. nov. and 

Amycolatopsis pretoriensis sp. nov., isolated from equine placentas.Int J 

Syst Evol Microbiol. 53(Pt 5):1601-5. 

3. Labeda DP, Price NP, Kroppenstedt RM, et al.: 2009, Streptomyces 

atriruber sp. nov. and Streptomyces silaceus sp. nov., two novel species of 

equine origin. Int J Syst Evol Microbiol. 59(Pt 11):2899-903. 

4. Christiensen BW, Roberts JF, Pozor MA, et al.: 2006, Nocardioform 

placentitis with isolation of Amycolatopsis spp in a Florida-bred mare.J Am 

Vet Med Assoc. 228(8):1234-9. 

5. Cattoli G, Vascellari M, Corrò M, et al.: 2004, First case of equine 

nocardioform placentitis caused by Crossiella equi in Europe. Vet Rec. 

154(23):730-1. 

180 

160 

140 

120 

# of Cases 

100 

80 

60 

40 

20 

0 

90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 06 07 08 09 10 11 

Year 

Figure 1: History of NPA cases diagnosed in Kentucky

S3 - O - 05 

A NEW DIAGNOSTIC TOOL FOR BOVINE TUBERCULOSIS-IDEXX M.BOVIS ANTIBODY TEST KIT 

J. Lawrence 1 , N. Djuranovic 1 , C. Egli 2 

1 - IDEXX Labs, Inc., Livestock and Poultry Diagnostics, Westbrook, Maine, USA 

2 - IDEXX Switzerland,Livestock and Poultry Diagnostics, Bern, Switzerland, USA 

Tuberculosis, ELISA, IFN-g, SICCT, IDEXX 

Introduction 

The IDEXX M. bovis antibody test kit is an ELISA designed to 

detect the presence of antibody to M. bovis in bovine serum and 

plasma samples. This test could improve bTB detection of TB - 

infected animals in TB-infected herds or could be an easy, cost 

effective surveillance tool in TB-negative regions. The M. bovis 

ELISA test is currently in the approval process for the OIE 

registry of tests. Also, all documentation has been submitted to 

USDA for product licensure. 

2. Schiller, I., et al. 2010. Bovine tuberculosis: a review of current 

and emerging diagnostic techniques in view of their relevance for 

disease control and eradication. Transbound. Emerg. Dis. 

57:205–220. 


Characterized serum and plasma samples were obtained from 

worldwide sources and were used to validate the M. bovis ELISA. 

Two temporal series were produced from animals exposed to M. 

bovis and followed over time. Positive samples from 3 different 

countries were obtained from either culture positive animals (n = 

307). Sample sets (n = 100) with varying skin or gamma 

interferon results were evaluated to demonstrate subsets of 

positive animals within TB-infected herds (n = 45) and the power 

of combining tests to increase overall sensitivity. Negative 

samples (n = 1473) were obtained from 4 different countries with 

samples originating from negative herds, states or regions. In 

addition, to understand potential cross-reactivity with other 

Mycobacteria, samples were obtained from animals exposed to 

large doses of M. paratuberculosis, M. avium and M. kansasii or 

from a herd with a high Johne’s antibody levels. 

All samples were evaluated on an M. bovis antibody kit 

manufactured at production scale, according to the standard kit 

protocol. Briefly, samples and kit controls were diluted 1:50 in a 

sample diluent and applied to microtiter plates. After a 60-minute 

incubation, the plates were washed, followed by the addition of 

an anti-bovine HRP conjugate (30-minute incubation). After 

another plate wash, TMB substrate was added. After color 

development of 15 minutes, plates were read on a 

spectrophotometer at 450nm. Sample optical densities were 

compared to those of the kit positive control to derive Sample-to 

Positive (S/P) ratios. Samples with S/P ratios of >/= 0.30 were 

considered positive for M. bovis antibodies. 

Results 

Data from the M. bovis temporal series revealed that animals can 

develop antibody titers within weeks of exposure and that an 

antibody response can be boosted after the application of a skin 

test. The M. bovis ELISA detected 197/307 samples from culture 

positive animals resulting in a sensitivity of 64.2% . Using a single 

test method, between 71.1% and 75.6% of herds would have 

been detected. Combining tests resulted in an increase in herd 

sensitivity to between 86.7% (GIFN and ELISA) and 97.8% 

(GIFN, SICCT and ELISA). On negative sample sets, the M. 

bovis ELISA demonstrated a specificity of 98% with no cross 

reactivity observed on M. paratuberculosis (both experimental 

and field infected) or M. avium samples. Transient, low level 

reactivity was observed with animals inoculated with large doses 

of M. kansasii. 


The strategic supplemental use of the IDEXX M. bovis antibody 

test represents a fast, easy, objective and cost effective option for 

use in bTB programs and can increase overall diagnostic power 

by detectiing subsets of infected animals missed by current 

methods. This test's high specificity allows for an application of 

this test in bTB- negative regions. 

References 

1. de la Rua-Domenech, R., A. T. Goodchild, H. M. Vordermeier, 

R. G. Hewinson, K. H. Christiansen, and R. S. Clifton-Hadley. 

2006. Ante mortem diagnosis of tuberculosis in cattle: a review of 

the tuberculin tests, γ-interferon assay and other ancillary 

diagnostic techniques. Res. Vet. Sci. 81:190-210.

S3 – O - 06 

DETECTION OF SCHMALLENBERG VIRUS IN THE UK 

Julie Peake, Christopher Finnegan, Falko Steinbach, Akbar Dastjerdi and Anna La Rocca 

AHVLA (Animal Health and Veterinary Laboratories), MVIU (Mammalian Virus Investigation Unit), Dept of Virology, Addlestone, UK 

Introduction 

In November 2011, colleagues at the FLI using samples taken in 

Schmallenberg (Germany) detected a new virus. The virus 

identified has subsequently, but tentatively, been named 

Schmallenberg Virus (SBV)(1). This virus belongs to the genus 

Orthobunyavirus within the Bunyaviridae family. Since the end of 

2011, traces of the virus were detected in several European 

countries. In the second half of January 2012, the first PCR 

positive cases were detected in the UK, following reports of 

clinical signs of the disease in aborted lambs from the south-east 

part of the country. 

Materials and Methods 

RNA was extracted using Trizol® (Invitrogen) followed by 

extraction of the acquoeus phase with the QIAamp Viral RNA 

mini Kit (Qiagen) and qPCR carried out with the primers 

described below using the QuantiTect® Virus RT-PCR kit 

(Qiagen). ELISA tests were carried out using the ID Screen® 

Schmallenberg virus Indirect ELISA (ID.VET) 

Results 

The first generation of qPCR assays based on the L segment 

were developed and kindly made available by FLI. In parallel, we 

used the virus sequence information to generate a set of primers 

for an AHVLA-qPCR. Shortly after, a second generation of 

primers and probe for qPCR was available through FLI. 

Subsequently, LSI also marketed a kit. All these qPCRs are 

based on the S genome segment. We are, at present, comparing 

the use of Texas Red as fluorescent dye compared to FAM, in 

the current assays, as this could increase the sensitivity of the 

assay, especially the resolution of results close to the cut-off 

value. 

Using the FLI second generation qPCR we have tested brains of 

sheep and cattle to closely monitor the outbreak across the 

country. We, very interestingly, have observed that the spread of 

the infection has been very quick across south but quite slow 

towards north of the UK. These observations lead us to the 

hypothesis that there could have been several incursions of the 

virus into the UK. 

Discussion and Conclusions 

Since mid-March, the number of positive brains from sheep has 

been declining and this correlates with the advancement of the 

lambing season. However, we have also detected five positive 

cases in April/mid-May in England. This challenges parts of our 

understanding about the transmission of SBV via midges, the 

observation of further cases until mid-May on Channel Islands is 

less astonishing. 

A particular challenging aspect of the SBV diagnosis has been 

the finding that the brains of many submitted suspects with the 

disease case definition were negative in our PCR assay. It is 

considered possible that in some instances the virus has been 

cleared by the developing immune system of the lambs (which 

would be traceable via antibodies to SBV). However, a recent 

study suggested looking also at further tissues besides brain to 

detect SBV (2). 

Ongoing studies in our lab are accordingly looking into the 

comparison of meconium, foetal fluid, and the dissemination of 

SBV in brains. We are also using the recently established 

commercial ELISA to complement PCR results. 

References 

1. Hoffmann B, Scheuch M, Höper D, Jungblut R, Holsteg M, Schirrmeier 

H, et al. Novel orthobunyavirus in cattle, Europe, 2011. Emerg Infect Dis 

2012 Mar;18(3):469-72. 

2. Bilk S, Schulze C, Fischer M, Beer M, Hilnak A, Hoffmann B. Organ 

distribution of Schmallenberg virus RNA in malformed newborns. Vet 

Microbiol 2012 Mar 30 Epub ahead of print. 

Tuberculosis, ELISA, IFN-g, SICCT, IDEXX

S3 - O - 07 

DETECTION OF WNV ENZOOTIC CIRCULATION IN HORSES WITH NEUROLOGICAL SIGNS AND IN 

CAPTIVE SENTINEL CHICKENS IN THE PREFECTURE OF THESSALONIKI, GREECE 

Chrysostomos I. Dovas 1 , Serafeim C. Chaintoutis 1 , Alexandra Chaskopoulou 2 , Nikolaos Diakakis 3 , 

Ilias Bouzalas 1 , Maria Papanastassopoulou 1 , Kostas Danis 4 , Anna Papa 5 , Orestis Papadopoulos 1 

1 

Aristotle University of Thessaloniki, Faculty of Veterinary Medicine, Laboratory of Microbiology and Infectious Diseases, Thessaloniki, Greece 

2 

United States Department of Agriculture-Agricultural Research Service, European Biological Control Laboratory, Thessaloniki, Greece 

3 

Aristotle University of Thessaloniki, Faculty of Veterinary Medicine, Clinic of Companion Animals, Thessaloniki, Greece 

4 

Hellenic Centre for Disease Control and Prevention, Athens, Greece 

5 

Aristotle University of Thessaloniki, Faculty of Medicine, A’ Laboratory of Microbiology, Thessaloniki, Greece 

WNV, detection, characterization, horses, sentinel chickens 

Introduction 

During 2010, a large outbreak of West Nile virus (WNV) 

infections occurred in Greece, with 262 reported human cases. 

Among them, 197 were diagnosed with West Nile neuroinvasive 

disease (WNND) and 35 patients died. The epidemic reoccurred 

in 2011. Laboratory methods, including RT-PCR, ELISA, 

microtiter plaque reduction neutralization test (micro-PRNT), 

indirect immunofluorescence antibody test (IFAT) and virus 

isolation, were used to detect WNV enzootic circulation in horses 

with neurological signs (passive surveillance), as well as in 

captive sentinel chickens (active surveillance), prior to the onset 

of human WNND cases in the prefecture of Thessaloniki, Greece. 


Blood samples were obtained from 17 horses with neurological 

signs, during the epidemic of 2010 (with the first case being 

reported on August 1). Furthermore, blood was collected from 

one horse which showed ataxia during the epidemic of 2011, on 

August 13. Also, in May 2011, 7 coops of 6 chickens each were 

placed at the edges of Thessaloniki City and blood samples were 

collected weekly until October. In conjunction to blood sampling 

from chicken, mosquitoes were sampled using 28 CO 2 -baited 

CDC light traps. 

The sera obtained from the 18 horses were assayed with two IgM 

capture ELISA (MAC-ELISA) kits (IDEXX IgM WNV Ab Test and 

IDVet ID Screen West Nile IgM Capture) for the detection of 

WNV specific IgM antibodies, sign of recent infection, as well for 

specific IgG antibodies, using IFAT. Plasma samples from these 

horses were tested with real-time RT-PCR for WNV (LSI TaqVet 

West Nile Virus – Dual IPC). Chicken sera were assayed by 

competitive ELISA (cELISA) (IDVet ID Screen West Nile 

Competition) for the detection of specific antibodies, and positive 

results were confirmed by micro-PRNT. Plasma samples from the 

seropositive chickens were screened using a WNV specific RT- 

PCR and confirmed using nested RT-PCR, employing specific 

primers for the WNV “Nea Santa-Greece-2010” strain, followed 

by sequencing of the NS3 gene for further molecular 

characterization. Vero cell cultures were inoculated with plasma 

samples from the RT-PCR positive chickens, for virus isolation. 

The collected mosquitoes were being counted and 

morphologically identified. 146 pooled samples, each one 

consisting of 50 mosquitoes of the same species (123 Culex 

pipiens and 23 Culex modestus pools) were examined by three 

different WNV specific real-time RT-PCR protocols, as well as 

with the nested RT-PCR protocol, 

Results 

WNV specific antibodies were detected in all 18 horse cases of 

2010 and 2011, while the IgG IFAT titer was high (>250) in all of 

them. Three horses were found positive with real-time RT-PCR 

(Ct ~39). Regarding chickens, seroconversion was detected in 11 

birds (10 in Western Thessaloniki). The first seroconversion 

occurred on June 29, a month before the onset of human cases 

in the area. The seroconversion rate peaked on August 17 with 4 

seropositive birds and the last seroconversion occurred on 

September 28. WNV was detected in 2/11 seropositive chickens 

with RT-PCR, from samples taken one week prior to 

seroconversion. According to BLAST algorithm, the partial NS3 

sequence presented highest nucleotide sequence identity 

(99,73%) to the strain “Nea Santa-Greece-2010”, responsible for 

the 2010 Greek epidemic. The inferred partial NS3 amino acid 

sequence was 100% identical to that of the “Nea Santa-Greece- 

2010”. The strains maintained the amino acid substitution H 249 P, 

which may be associated with increased virulence. WNV was 

successfully isolated in cell cultures. Chicken serum neutralizing 

antibody titers to WNV ranged between 20 and 40. Culex 

mosquito populations peaked in mid-June, ~10 days prior to the 

first chicken seroconversion, remained high throughout the 

summer and showed a significant drop in September. The most 

prevalent mosquito species in both residential and agricultural 

areas were Culex pipiens, followed by Culex modestus Ficalbi. 

Only one out of the 123 Culex pipiens pools tested was found 

positive for the WNV “Nea Santa-Greece-2010” strain using the 

nested RT-PCR, while the application of all different real-time RT- 

PCR protocols resulted in detection of additional mosquito 

flaviviruses, indicating problems of detection specificity. 

Figure 1. Location of mosquito traps (n=28) and sentinel chicken 

coops for WNV surveillance in Thessaloniki, 2011. Blue bullets 

represent the mosquito traps, while yellow dots represent the 

location of the 7 chicken coops. The 10 seroconverted chickens 

belonged to the coops No. 1, 2 and 3 (Western Thessaloniki), 

while one more chicken belonged to the coop No. 5, in Eastern 

Thessaloniki. 


With passive surveillance, 18 clinical cases of horses were 

detected mainly in suburban areas of Thessaloniki. Unfortunately 

there could not be successfully used as an early warning system, 

given that the first human case in the prefecture of Thessaloniki 

was recorded 15 days prior to the onset of clinical cases in 

horses, both in 2010 and 2011. On the contrary, with the second 

surveillance system, it was shown that the “Nea Santa-Greece- 

2010” strain became endemic in Greece, as it was detected, 

molecularly identified and isolated early in 2011. Most 

importantly, enzootic circulation of WNV was successfully 

detected one month prior to human cases. As a result, health 

authorities were informed in a timely manner and were facilitated 

the successful implementation of preparedness plans to protect 

public health and minimize the impact of the epidemic of 2011. In 

regards to the use of real-time RT-PCR for vector surveillance, 

the methods need further optimization and validation, concerning 

the specificity of WNV detection and results should be further 

confirmed by sequencing. 

References 

Danis, K, Papa, A, Papanikolaou, E, Dougas, G, Terzaki, I, Baka, A, Vrioni, 

G, Kapsimali, V, Tsakris, A, Kansouzidou, A, Tsiodras, S, Vakalis, N, 

Bonovas, S, Kremastinou, J (2011). Ongoing outbreak of West Nile virus 

infection in humans, Greece, July to August 2011. Eurosurveillance, 

16(34), pii=19951.

S3 - O - 08 

SCHMALLENBERGVIRUS: SEROLOGICAL STUDIES IN GERMAN HOLDINGS 

Horst Schirrmeier, Bernd Hoffmann, Martin Beer 

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany 

Introduction 

In August 2011 first indications of a previously unknown disease 

in dairy cattle were observed in North Rhine-Westphalia, 

Germany, and in the Netherlands. Fever, decreased milk 

production and in some cases also diarrhea were the major 

symptoms. In November 2011, the detection of a novel 

Orthobunyavirus in blood samples of acutely diseased dairy cows 

was reported by the Friedrich-Loeffler-Institut, and the the virus 

was named as Schmallenberg virus (SBV). Since December 

2011, congenital malformations were observed, initially in sheep 

and goats, and subsequently also in calves. A real-time RT-PCR 

was developed very early after the first detection, and first 

serological methods were established for serological studies. The 

first findings including investigations about the SBV-prevalence in 

Germany are reported. 


The following methods for the detection of SBV-specific 

antibodies were established and used: 

1. serumneutralization test (SNT) in BHK-21 cells, clone 

CT (L164, CCLV, Insel Riems) using the cell culture 

isolate “Schmallenberg BH80”. 

2. indirect immunofluorescence in a multi-plate format 

with SBV-infected BHK-21 cells, using the SBVinfected 

cell clone BRS5 (L194, CCLV Insel Riems) as 

antigen matrix (Vero cells were used as an alternative 

cell culture). 

3. Indirect ELISA (in house development as well as a 

prototype batch of a commercial ELISA assay). 

Within a nationwide first monitoring study, cattle, sheep and goat 

sera were tested for SBV antibodies. Furthermore, sera from 

malformed calves and pre-colostral sera of newborn ruminants 

were investigated to improve diagnostic options in clinically and 

pathologically suspected, but PCR negative cases. A few sera 

from wild ungulates were also included, and in vitro crossreaction 

studies with other members of the Orthobunyavirus 

family were performed. 

Results & discussion 

SBV-specific antibodies could be detected in serum samples 

from cattle and sheep from all included federal states. The 

observed incidence was within a range of 10% to 60 %, with a 

single outlier of more than 90 %. Positive results were obtained 

most frequently in the epidemiological “core regions” in the 

Northwestern parts of Germany. The number of sero-reactors 

corresponded very well with the frequency of the notified clinical 

cases. However, the in-herd prevalence differed substantially and 

varied between single reactors and up to 80 % positive animals, 

and was generally higher in cattle herds than in sheep flocks. 

Apart from domestic ruminants, antibodies could be also detected 

in samples from bisons, roe deer, red deer and from one camel 

and lama, respectively. Furthermore, SBV-specific antibodies 

could be also detected in samples from PCR-negative animals as 

well as in sera of malformed calves without colostrum uptake. 

Our results will allow better insights into the epidemiolocial 

background of the SBV-infection, will expand the knowledge 

about pathogenetic mechanisms, and are able to substantiate the 

SBV case definition in PCR-negative animals. 

References 

Hoffmann B, Scheuch M, Höper D, Jungblut R, Holsteg M, Schirrmeier H, 

Eschbaumer M, Goller KV, Wernike K, Fischer M, Breithaupt A, 

Mettenleiter TC, Beer M. (2012). Novel orthobunyavirus in cattle, Europe, 

2011, Emerg Infect Dis. 2012 Mar;18(3):469-72 

Orthobunyavirus, serology,

S3 - O - 09 

DIFFERENT DIAGNOSTIC TOOLS FOR A BROAD RANGE OF MACAVIRUSES AND THEIR 

RESERVOIR AND SUSCEPTIBLE HOSTS 

Christine Foerster 1 , Matthias Koenig 1 , Heinz-Juergen Thiel 1 , Jens-Ove Heckel 2 

1 

Institute of Virology, diagnostic laboratory, Justus-Liebig-University Giessen, Germany 

2 

Zoo Landau, Germany 

Macavirus, malignant catarrhal fever, reservoir and susceptible host 

Introduction 

Malignant catarrhal fever (MCF) is a fatal disease of Artiodactyla, 

which is caused by different macaviruses within the subfamily 

Gammaherpesvirinae (GHV) of the family Herpesviridae. Until 

now there are at least 6 distinct macaviruses known to cause 

MCF: Alcelaphine herpesvirus 1 (AlHV-1), ovine herpesvirus 2 

(OvHV-2), caprine herpesvirus 2 (CpHV-2), white tailed deer- 

MCF-virus (MCF-WTD), an unclassified macavirus from ibex and 

a virus resembling alcelaphine herpesvirus 2 (AlHV-2). Other 

closely related viruses are the hippotragine herpesvirus 1 (HiHV- 

1 in roan antelope) and GHV in springbok, impala, oryx, muskox 

and aoudad. 

MCF usually occurs only sporadically. Within the framework of 

differential diagnostics the detection of macaviruses was 

stimulated since the first cases of bluetongue disease were 

noticed in northern Europe. 

Typical reservoir hosts which do not develop clinical disease are 

sheep, goat and wildebeest. Several species of Artiodactyla are 

susceptible to MCF, for example bison, cattle, deer and moose. 

In addition pigs, goats and experimentally infected rabbits show 

typical clinical symptoms like fever, mucosal erosions, nasal and 

lacrimal discharge, corneal opacity and skin lesions. 

Undirected virological methods like virus propagation in cell 

culture and electron microscopy are not promising with regard to 

macaviruses. PCR is the method of choice for laboratory 

diagnosis. Until now most laboratories use the OvHV-2 specific 

PCR (Baxter et al., 1993) for samples of cattle. 

Objective of this work was to compare and improve the 

diagnostic tools for the detection of a broad spectrum of potential 

MCF-viruses. 


The competitive ELISA was clearly superior to SNA with regard 

to time, effort and quality of result. ELISA is recommended for 

routine diagnostic use, especially for reservoir hosts. 

The importance of broadly reactive PCR methods was 

demonstrated by 18 positive results of wildlife samples. All 18 

cases led to negative results in the OvHV-2-specific PCR, but 

reacted positive in the PAN-Macavirus-PCR. Cloning, sequencing 

and phylogenetic analyses of the amplificates led to the detection 

of CpHV-2, CpHV-2-like and BoHV-6-like macaviruses. 

For the first time CpHV-2-induced MCF was detected in two 

Banteng-cattle. Until now, CpHV-2 has been considered as a 

cause of MCF only in deer and moose. This implies that routine 

OvHV-2-specific-diagnostic in cattle is insufficient. 

The establishment of an OvHV-2-CpHV-2-discriminating realtime 

PCR allowed rapid differentiation between the two most common 

causative agents of MCF in Europe. This method is suitable for 

application in routine diagnostics of small ruminants and cattle. 

For the application in wildlife samples the conventional PCR is 

more suited, because primer pairs were selected for a broad 

range of macaviruses and the conventional PCR appeared to be 

more sensitive. 

References 

1. Baxter, S. I., Pow, I., Bridgen, A. and Reid, H. W. (1993). PCR detection 

of the sheep-associated agent of malignant catarrhal fever. Arch Virol 132 

(1-2), 145-59. 

2. Russell, G. C., Stewart, J. P. and Haig, D. M. (2009). Malignant 

catarrhal fever: A review. Vet J 179 (3), 324-35. 


Two new conventional PCR-methods and an OvHV-2-CpHV-2- 

discriminating realtime PCR were established, which allowed to 

detect a broad spectrum of macaviruses. They were compared to 

the commonly used OvHV-2-specific PCR. Amplified fragments 

were cloned, sequenced and used for phylogenetic analyses. 

Furthermore two antibody-test-systems, ELISA and serum 

neutralisation assay (SNA), were compared to each other. 

Samples of 276 wild and zoo animals, comprising 54 different 

genera of the family Bovidae, Cervidae, Camelidae and 

Giraffidae, 230 goats and 58 sheep were examined. 

Results 

39 cases out of 276 wild ruminants (14 %) led to a positive result 

in at least one procedure. In approximately half of the samples 

(19) OvHV-2 was identified, in 18 CpHV-2 or CpHV-2 like virus. 

Samples of one waterbuck contained sequences related to 

BoHV-6. 19 of 39 macavirus-positive animals showed MCF like 

disease. 

CpHV-2 was identified in conjunction with two cases of MCF in 

Banteng cattle. 

5 out of 230 goats (2 %) showed MCF like symptoms and 4 of 

them also pathological and histological signs of MCF. In all 

samples of these animals OvHV-2 could be demonstrated. 

Samples of 92 (42 %) goats obtained positive results in at least 

one PCR method. Interestingly single and also double infection of 

OvHV-2 and CpHV-2 were found. 

In contrast to the goats only OvHV-2 was found in 27 sheep 

samples (47 %). 

Realtime PCR was able to detect OvHV-2 and CpHV-2 in all 

cases of MCF-diseased animals. In latently infected animals high 

cycle treshold values were found. 

In some reservoir hosts antibodies were found but no viral DNA. 

33 of 59 animals (56 %) were found positive via ELISA, but 

negative by SNA, while only one animal (1,6 %) was estimated 

positive by SNA and negative via ELISA.

S3 - O - 10 

DIAGNOSTIC ASPECTS OF SUID HERPESVIRUS 1 INFECTION IN WILD BOAR 

Adolf Steinrigl, Sandra Revilla-Fernández, Eveline Wodak, Zoltán Bagó, Friedrich Schmoll 

AGES Institute for Veterinary Disease Control, Mödling, Austria 

SuHV-1, wild boar, prevalence, molecular characterization 

Introduction 

Many infectious diseases that are strictly controlled in livestock 

may remain unnoticed in wild animals, posing the risk of entering 

domestic animal populations with potentially devastating 

consequences. Suid herpesvirus 1 (SuHV-1), the causative agent 

of Aujeszky’s disease (AD), was eradicated from domestic pig 

populations in many European countries, including Austria. At the 

same time, SuHV-1 infection is common in European wild boar, 

as shown by a number of studies, reporting detection of SuHV-1 

DNA or antibodies in wild boar (1). An additional indicator of 

SuHV-1 presence in wild boar are occasional cases of AD in 

hunting dogs, usually in close connection to hunting events (2). 

Recently, we reported about the first detection of SuHV-1 in 

Austrian wild boar and the molecular characterization of SuHV-1 

field isolates from wild boar and hunting dogs (3). 

In the course of the Wild Animal Survey 2011, a nationwide study 

that was partly financed by the Austrian Federal Ministry of 

Health, additional sera and tissue samples from Austrian wild 

boar were collected and analysed, with the aim to provide a 

representative estimate of the prevalence of both, SuHV-1 DNA 

and antibodies, in Austrian wild boar. This work shows the results 

of this survey and reflects our experiences with detection and 

molecular characterization of SuHV-1 in wild boar. 


Wild boar tissues and sera were sampled according to a survey 

design that was based on recent wild boar hunting bag statistics. 

Anti-SuHV-1 gB ELISA, real-time PCR (qPCR), amplification and 

sequencing of the SuHV-1 glycoprotein C coding region, 

phylogenetic analysis and virus isolation were performed as 

described previously (3). 

Results 

From April 2011 to January 2012, tissues from 298 wild boars 

were sampled, corresponding to 78% of the scheduled sample 

size for the whole country. Wild boar sera of sufficient amount 

and quality were available from 259 animals. Results of 

serological and virological screening are shown in Table 1. All 

qPCR positive wild boars were adult females at 2-4 years of age. 

One of them was also serologically positive, while the other two 

animals were seronegative. 

Table 1: Results of serological (ELISA) and virological (qPCR) 

screening of Austrian wild boars 

Samples Samples Prevalence 

tested positive 

ELISA 259 59 22.8% 

qPCR 298 3 1.0% 

Virus was successfully isolated from one of the qPCR-positive, 

yet seronegative, wild boars: tissue homogenate from a qPCRpositive 

tonsil was inoculated onto PK15 cells, resulting in 

massive cytopathic effect within 24 hours post inoculation. An 

attempt to isolate virus from the other two qPCR positive animals 

was unsuccessful. Phylogenetic analysis of SuHV-1 gC 

sequences amplified from the three qPCR positive wild boars 

sampled in 2011 showed that all three animals hosted virus that 

was identical in gC sequence and belonged to one of the 

previously described lineages of SuHV-1 (3), which are currently 

present in Austria (Figure 1). 

Figure 1: Neighbour Joining tree, based on a 639 bp alignment of 

the SuHV-1 partial gC coding region. Sequences were amplified 

from wild boars (WB), hunting dogs (HD) and from a domestic pig 

(DP). Year of isolation is in brackets. Numbers along the 

branches indicate the percentage of 1000 bootstrap replicates. 


The presented study confirms the presence of SuHV-1 in 

Austrian wild boar. SuHV-1 seroprevalence detected during the 

Wild Animal Survey 2011 was lower than that previously 

described (3), which is probably due to the more representative 

sampling strategy applied for the current study. In contrast to the 

high numbers of wild boars with detectable SuHV-1 antibodies, 

SuHV-1 DNA was detected by qPCR in only a few animals. 

Furthermore, Cq-values of qPCR-positive tissue pools or 

individual organs were relatively high (30 - 38 Cq), indicating a 

low viral load in these tissues. This is also supported by our 

previous observation of weak immunohistochemistry signals in 

qPCR positive tissues (3) and by the fact that virus isolation was 

positive in a single case only. In summary, our results indicate 

that most SuHV-1 infected wild boars harbour virus in a latent 

form, whereas only a small proportion of infected wild boars is 

actually shedding virus and may infect other susceptible animals. 

This is also the most likely explanation for the relatively rare 

cases of AD in hunting dogs, despite rising numbers of wild boars 

in many European countries and the frequent involvement of 

hunting dogs in wild boar hunting. Despite the fact that only few 

potentially virus shedding wild boars were found in this study, 

direct contact between wild boars and domestic pigs could result 

in SuHV-1 transmission. Feeding of wild boar intestines to 

domestic animals, especially pigs, dogs and cats, is another 

reasonable possibility of transmission and must be strictly 

discouraged. 


This study was financially supported in part by the Austrian 

Federal Ministry of Health. The excellent technical assistance of 

the laboratory staff of the Departments for Molecular Biology, 

Virology and Electron Microscopy, and Pathology is 

acknowledged. 

References 

1. Müller T, Hahn EC, Tottewitz F, Kramer M, Klupp BG, Mettenleiter TC, 

Freuling C (2011). Pseudorabies virus in wild swine: a global perspective. 

Arch. Virol., 156, 1691-705. 

2. Cay AB, Letellier C (2009). Isolation of Aujeszky’s disease virus from 

two hunting dogs in Belgium after hunting wild boar. Vlaams 

Diergeneeskundig Tijdschrift, 78, 194-195. 

3. Steinrigl A, Revilla-Fernández S, Kolodziejek J, Wodak E, Bagó Z, 

Nowotny N, Schmoll F, Köfer J (2011). Detection and molecular 

characterization of Suid herpesvirus type 1 in Austrian wild boar and 

hunting dogs. Vet Microbiol., Dec 30. [Epub ahead of print]

S3 - O - 11 

PRELIMINARY VALIDATION OF THE ID SCREEN ® SCHMALLENBERG VIRUS INDIRECT ELISA 

P. Pourquier, 1 , C. Loic, 1 , S. Zientara, 2 , E. Breard, 2 , C. Sailleau, 2 , C. Viarouge, 2 , A.-B.Cay, 3 , N. De Regge, 3 

1 

IDvet, Montpellier, France 

2 ANSES Maisons-Alfort, France 

Introduction 

Schmallenberg virus (SBV) is the name given to a vectortransmitted 

orthobunyavirus related to Shamonda virus, initially 

reported in November 2011 to cause foetal congenital 

malformations and stillbirths in cattle, sheep, and goats. The 

virus has since been detected in Germany, the Netherlands, 

Belgium, France, Luxembourg, Italy, Spain and the United 

Kingdom. 

Serological testing is essential in order to study and control this 

new emerging disease. While SBV specific antibodies can be 

detected by virus neutralization tests and indirect 

immunofluoresence, these techniques are time-consuming, not 

suitable for high-throughput, and do not offer standardized result 


Available as of March 2012, the ID Screen ® 

Schmallenberg 

virus Indirect ELISA is the first ELISA developed for SBV 

diagnosis. The test allows for the detection of SBV antibodies in 

ruminant serum and plasma. It is a rapid, standardized assay 

which is automatable and therefore suited to high throughput 

testing. 

This study presents validation data for this test. Data will be 

added for any second generation tests developed by IDvet 

between March and June 2012. 


- The ID Screen ® Schmallenberg virus Indirect ELISA was 

performed as per manufacturer’s specifications. 

- Specificity was studied using panels of sera collected prior to 

2010. 

- Sensitivity was evaluated on sera tested positive with the virus 

neutralization test (VNT). 

- An experimental infection was performed in April 2012 in order 

to evaluate the seroconversion response in cattle, sheep and 

goats. 

Results & Discussion 

Preliminary validation studies indicate excellent test specificity 

and high correlation with other serological techniques, making the 

the ID Screen ® Schmallenberg virus Indirect ELISA an efficient 

tool for disease surveillance and epidemiological studies. 

3 CODA-CERVA, Department of Viral Diseases, Brussels, Belgium 

Schmallenberg virus, serology, ELISA


“New techniques in bacteriology, 

parasitology and pathology” 

(4 th session)

S4 - K - 01 

MALDI-TOF AND OTHER NEW DIAGNOSTIC 

Markus Kostrzewa 

Bruker Daltonik GmbH, Bioanalytical development, Bremen, Germany 

Microorganism identification, FT-IR spectroscopy, Raman spectroscopy, mass spectrometry, MALDI-TOF MS 

Introduction – the “status quo” 

Since more than one hundred years, identification of 

microorganisms mainly is performed by methods investigating 

their phenotypic characteristics. Gram stain together with 

microscopy in combination gives a first quick hint about identity. 

The screening of series of tests for biochemical capabilities is 

used to generate a pattern which is more or less species specific. 

Although these tests now have been automated for the detection 

of the most prevalent microorganisms, the accuracy of 

biochemical tests is limited, in particular for organisms with few 

characteristic biochemical features like Gram negative nonfermenting 

bacteria of anaerobes. Even worse is the situation for 

rare bacteria, mycobacteria or fungi where mainly labourintensive 

manual methods like API are applied for identification. 

Therefore, there is an urgent need for modern automated 

methods for identification which can be introduced in routine 

laboratories to shorten time-to-result and decrease the overall 

costs of the laboratories. Promising approaches appearing in the 

areas of molecular biology, optical technologies and mass 

spectrometry have appeared through the recent years and are on 

the way to revolutionize microbial analysis. 

Methods based on molecular biology have already been 

introduced in many laboratories, mainly PCR based tests to 

screen for dedicated microorganisms, e.g. food pathogens. While 

having the advantage of high sensitivity and short analysis time 

one drawback of PCR based tests is that they generally need a 

pre-assumption which microorganism is searched for, i.e. a 

dedicated primer design. To apply DNA analysis to broad species 

identification, PCR typically is coupled with subsequent sequence 

determination of the amplicon. Sequencing of the 16S rDNA 

region nowadays is regarded the gold standard for species 

identification. Nevertheless, 16S rDNA sequencing has never 

made it to the routine but is mainly a second- or third-line method 

for cases where other methods have failed. Reasons are the high 

costs, elaborate sample handling, and PCR-related 

contamination risk. Further, in quite a number of cases, 

determination of the 16S ribosomal RNA gene sequence is not 

sufficient for the discrimination of closely related species and 

further gene sequences have to be analysed, increasing time to 

result, costs and labour, significantly. Novel next generation 

sequencing technologies might change the position of 

sequencing in future by their enormous throughput capability, but 

today they are still expensive and labour-intensive research tools. 

The upcoming alternatives 

Vibrational spectroscopy 

Optical technologies have been shown to be powerful for 

microorganism identification as well as for analyses on the 

subspecies level. These techniques are based on a molecular 

fingerprint of the entire biomass, proteins, carbohydrates, lipids, 

nucleic acids. Analyses are not depending on reagent kits, labels 

or drugs, and these technologies are applicable to a broad 

spectrum of organisms. 

Raman spectroscopy is a non-destructive optical technique, 

based on scattering of light by molecules, the so called “Raman 

effect”. The atoms in a molecule move in so called vibrational 

modes. When light, i.e. a photon, and a molecule interact, some 

of the energy of the photon can be transferred to the molecule. 

Thereby, one of the vibrational modes in the molecule is excited. 

The energy of the scattered photon is reduced by the exact 

amount of energy received by the molecule. The energy required 

to excite a molecular vibration is exactly defined and depends on 

the masses of the atoms involved in the vibration, on the type of 

chemical bonds between these atoms, on the structure of the 

molecule and on interactions with its environment and other 

molecules. The spectrum of the scattered light is used as a 

pattern to identify microbes. 

In contrast, Fourier-transformation infrared spectroscopy uses 

absorption of an infrared light spectrum (not a single wavelength) 

to create a fingerprint of the whole cell composition. Mainly 

transmission of the light is used to create the characteristic 

profile. The first deviation of the transmission profile spectrum is 

generated and sophisticated mathematical algorithms are used to 

identify strains by matching against a database or to build 

relational dendrograms for classification. 

Restrictions of the optical technologies is mainly that they require 

a strict standardization of cultivation conditions (time, media, 

temperature) as these are influencing the cell content and 

thereby the optical fingerprint. The speed of the analyses is also 

counteracted by the fact that a pure culture is required (i.e. often 

no analysis from primary culture can be performed). 

Mass spectrometry 

Some technological approaches for identification and further 

analysis of microorganisms based on mass spectrometry may 

have the most impact on microbial characterization in the near 

future. One of these approaches is based on a combination of 

molecular biology and electrospray time-of-flight mass 

spectrometry (ESI-TOF MS). In a first step, several target regions 

of the DNA from the unknown microorganism are amplified with 

generic primer pairs. This is creating a set of amplification 

products with specific molecular masses. Subsequently, the PCR 

products are purified with a magnetic bead based system and 

then injected in liquid phase into the ESI-TOF mass 

spectrometer. During the ionization process, the two DNA strands 

of an amplicon are separated. The mass of both DNA strands is 

measured in the ESI-TOF very accurately. From the combination 

of the accurate masses of the both pairing strands the 

composition of the polynucleotides (i.e. the number of A, C, G, 

and T, respectively) can be determined. The projection of the 

base compositions of the selected PCR products into a database 

is used for the identification of an organism. The more PCR 

products are analysed, the more accurate/discriminative the 

identification can be. One advantage of this technology is that in 

parallel to species identification also other characteristics, e.g. 

resistance genes or virulence factors, can be detected. On the 

other hand, this approach still relies on a relatively complex 

(although in parts automated) workflow, and both the instrument 

as well as consumable costs are significant. 

The second, currently most successful and promising mass 

spectrometry technology is matrix-assisted laserdesorption/ionization 

time-of-flight mass spectrometry (MALDI- 

TOF MS). In contrast to electrospray ionization the MALDI 

process (generating the ions measured in the TOF analyser) is 

performed from a solid sample. For this, the sample is deposited 

on a target plate, overlaid with the so called matrix, generally an 

organic acid related to cinnamic acid, which co-crystalizes with 

the sample. This matrix supports the evaporation of the sample 

and its ionization after absorption of the energy of a pulsed laser 

beam. One advantage of this technology is that the sample can 

be easily prepared offline before measurement and then 

transported or stored until measurement. 

For one molecular biology based approach, MALDI-TOF MS is 

used to detect specific RNA fragments generated by PCR 

followed by transcription of both DNA strands and the cleavage of 

the transcripts by RNAses. The exact masses of the fragments 

can be used as pattern for identification and typing of 

microorganisms. Alternatively, minisequencing/primer extension 

of specific regions after PCR amplification, followed by mass 

determination in a MALDI-TOF MS, has been shown to have 

some potential for typing or resistance detection. 

While none of these DNA based mass spectrometry approaches 

made it to routine yet, MALDI-TOF MS whole cell protein profiling 

has entered many clinical and veterinary microbiology 

laboratories, already. This MALDI-TOF fingerprinting is based on 

the specific pattern of high abundant proteins of microbial cells 

which can be measured quickly and accurately by MALDI-TOF 

mass spectrometry. Sample preparation is very simple, fast and 

cheap: A small amount of a bacterial colony is transferred to a 

position on a MALDI sample target and smeared on it as a thin 

film. More robust cells can be broken by a short extraction with

acid and organic solvent. Then a droplet of matrix solution is 

added. Thereby, the cells are destructed, the protein content gets 

released and is embedded into the matrix crystals which are 

forming after solvent evaporation. During the subsequent MALDI- 

TOF analysis the most abundant proteins, in particular ribosomal 

proteins, are detected as a characteristic molecular profile 

(fingerprint). This pattern then is compared with the profiles 

stored in a database. Identification is done based on highest 

similarity and a minimum identity score. The whole process, from 

sample preparation to identification, can be as fast as ten 

minutes for a single sample/colony, less than one hour for about 

one hundred samples. For difficult to prepare microorganisms like 

actinomycetes, fungi, or mycobacteria special protocols are 

available which allow also their analysis with high success rate. 

Dependance on cultivation conditions is generally low. Thereby, 

this is one of the broadest applicable technologies for microbial 

identification. MALDI-TOF fingerprinting is already used in many 

clinical and veterinary laboratories, frequently as the first-line 

routine identification method. Its accuracy has been reported in 

various publications. Extensive commercial databases are 

available, but also alternative or supplementary laboratoryspecific 

libraries can be established. Recent developments in the 

direction of direct specimen analysis, epidemiology, virulence 

typing, and resistance determination will make the technology 

even more valuable for the clinical microbiology. 

Conclusions 

New technologies for microorganism identification and 

characterization are no more on the horizon, they are on the 

playground. In the near future it can be expected that a 

combination of molecular and spectroscopic/spectrometric 

technologies will substitute most of phenotypic/biochemical 

assays, increasing accuracy, efficiency and speed of the clinical 

microbiology laboratory.

S4 - O - 01 

SUITABILITY OF RECOMBINANT PROTEINS FOR THE DIAGNOSIS OF LEPTOSPIROSIS IN PIGS 

Ripp, Ulrike 1,2 , Truyen, Uwe 2 , Homeier, Timo 2 

1 

Synlab.vet Leipzig, Leipzig, Germany 

2 

Institute of Animal Hygiene and Veterinary Public Health, Faculty of Veterinary Medicine, Leipzig, Germany 

Porcine leptospirosis, LipL32, ELISA 

Introduction 

Leptospirosis is a widespread disease in the world with a high 

zoonotic potential. The momentary gold standard is the 

microscopic agglutination test (MAT). The specificity seems to be 

good, but the sensitivity is controversially discussed. It is lacking 

as a non-objective test procedure. Additionally, the detection of 

antibodies depends on the used serovar panel. Apart from the 

sensitivity, there is a risk of laboratory infection, as the leptospires 

used in the MAT have to be alive. 

Therefore, current research focuses on the development of an 

ELISA for the serological diagnosis of leptospirosis. Whole-cell 

based ELISA did not provide the desired results in sensitivity and 

specificity. Recombinant proteins, especially those that are only 

expressed in pathogenic leptospires, seem to be a more 

promising agent as an antigen. The goal of this study was to find 

a serovar-independent antigen which can be used in a screening 

ELISA in comparison to the MAT. 


Microscopic agglutination test (MAT): 

8 serovars (L. Bratislava, Canicola, Grippotyphosa, Pomona, 

Tarassovi, Sejroe, Copenhageni, Saxkoebing) were used in the 

MAT panel. The highest titer where there were still less than 50% 

of free leptospires to be seen in the dark-field-microscopy, was 

the endtiter. 

Samples with a titer ≥ 1:100 were considered positive. 

Porcine serum samples were screened at dilutions of 1:50, 1:100 

and 1:200. Samples that were still positive at 1:200 were further 

diluted. 

ELISA: 

The recombinant protein LipL32 was used as antigen. It is highly 

conserved among the pathogenic serovars of Leptospira (1) and 

is not expressed in apathogenic leptospires. LipL32 contributes to 

a large amount of the outer membrane proteins of leptospires. It 

has already been tested in an ELISA for human, canine and 

bovine leptospirosis (2,3). 

Flat-bottom microtiter plates were coated with LipL32 and 

incubated over night at 4°C. The optimal antigen concentration 

was determined by checkerboard titration. The range was from 

1,17 g to 0,55 ng / well. 

The plate was blocked the next day with skim milk and again 

incubated over night at 4°C. After washing the plate, serum was 

added at a dilution of 1:100 and incubated for 1 hour at room 

temperature on a shaker. After the next washing step, the 

conjugate (a goat anti-pig antibody that was conjugated to 

horseradish-peroxidase) was added. The plate was incubated for 

1 hour at room temperature on a shaker, washed, and the 

substrate (TMB) was added. The reaction was stopped after 5 

minutes with 0,5M H 2 SO 4 and read at 450/630nm. 

Control sera: 

The positive control serum was gained from a pig that was 

vaccinated 3 times in intervals of 2 weeks with 10 9 CFU (Colony 

forming units) of formalin inactivated Leptospira Pomona. 

The serum of a specific pathogen free pig (that was negative in 

the MAT, too) was used as negative control. 

Results 

Optimum antigen concentration: 

The titration of LipL32 did not show a plateau with additional 

decrease of OD values. Changes of the conjugate and serum 

dilutions as well as different blocking agents and microtiter plates 

did not improve the titration curve. Hence, the antigen dilution 

with the lowest OD for the negative control and still the biggest 

difference between the ODs of the negative and positive control 

was chosen. Weak positive samples were tested to verify that 

they could still be detected. 

It is becoming apparent that MAT negative porcine serum 

samples can be differentiated from MAT positive samples with the 

recombinant protein LipL32. 

OD‐value 

1,600 

1,400 

1,200 

1,000 

0,800 

0,600 

0,400 

0,200 

0,000 

0 1 2 3 

1= positive, 2 = weak positive, 3= negative sera 

Figure 1: 27 porcine sera with different MAT titers tested in the 

LipL32-ELISA 


Leptospirosis is a livestock disease with a serious impact on 

public health. It is important to find a reliable, inexpensive and 

hazardous-free diagnostic test. This study’s target is to test the 

possible utilisation of the recombinant protein LipL32 in an ELISA 

for porcine leptospirosis. 

LipL32 seems to be a potential candidate for an ELISA. The 

serum samples tested until now show different OD values, 

depending on having negative or positive MAT titers. There is a 

correlation of the qualitative results (positive / negative), but there 

seems to be no correlation of the quantitative results (MAT titers / 

OD values). Figure 1 shows that samples with weak positive MAT 

titers (1:100 / 1:200) are in the same range of OD values as 

samples with strong positive MAT titers (≥ 1:800). 

The goal of the ELISA wasn’t to establish a replicate of MAT 

titers, but to give reliable results whether an animal has been 

exposed to leptospires or not. 

The sensitivity and specificity of the ELISA have to be examined 

in further experiments. 


The authors thank Dr. K. Nöckler and E. Luge, Federal Institute 

for Risk Assessment, Berlin, Germany for assistance with the 

MAT. 

References 

1. Haake,D.A., Chao,G. Zuerner, R.L. Barnett, J.K. Barnett, D., Mazel, M., 

Matsunga, J., Levett, P.N., Bolin, C.A., (2000). The leptospiral major outer 

membrane protein LipL32 is a lipoprotein expressed during mammalian 

infection. Infect. Immun. 68, 2276-2285 

2. Dey,S., Madhan Mohan,C. (2004). Recombinant LipL32 antigen-based 

single serum dilution ELISA for detection of canine leptospirosis. Vet. 

Microbiol.103, 99-106. 

3. Bomfim, M.R.Q., Ko, A., Cota Koury, M. (2005). Evaluation of the 

recombinant LipL32 in enzyme-linked immunosorbent assay for the 

serodiagnosis of bovine leptospirosis. Vet. Microbiol. 109, 89-94

S4 - O - 02 

BIOLOG GENERATION III, MATRIX ASSISTED LASER DESORPTION/IONISATION TIME-OF-FLIGHT 

(MALDI-TOF) MASS SPECTROMETRY AND 16S rRNA GENE SEQUENCING FOR THE 

IDENTIFICATION OF BACTERIA OF VETERINARY INTEREST 

Peter Wragg 1 , Luke Randall 2 , Adrian Whatmore 3 

1 Animal Health and Veterinary Laboratories Agency, Laboratory Services Department, Penrith, United Kingdom 

2,3 Animal Health and Veterinary Laboratories Agency, Department of Bacteriology, Weybridge, United Kingdom 

Biolog, Maldi-ToF, 16S rRNA 

Introduction 

The veterinary bacteriologist is currently presented with a wide 

variety of technologies offering potential solutions to problems in 

the identification of bacteria. 

16S rRNA sequencing is perhaps the most familiar of the genomic 

approaches. Nonetheless, genomic methods are not without 

limitations and may suffer similar database validation issues to 

phenotypic systems (1,2). Automated or semi-automated 

metabolic methods have developed rapidly in recent years, with 

multi-analyte systems offering extended databases, although 

frequently less well populated with veterinary taxa. Biolog’s 

Generation III identification system employs a novel method of 

detecting substrate utilisation and an extensive database, 

including veterinary isolates (3). Matrix-assisted laser desorption/ 

ionization time-of-flight mass spectrometry (MALDI-TOF) has 

become widely recognised as one of the most adaptable of the 

chemotaxonomic methods (4). Veterinary strains are wellrepresented 

on manufacturer’s databases. Database 

enhancement initiatives are ongoing for all three technologies in a 

number of research facilities. 

This study was designed to assess the performance of the above 

methods for the identification of a limited but typical range of 

referral isolates of veterinary significance (n=66) by comparison to 

partial 16S rRNA DNA sequencing, with a view to indicating 

potential suitability as a ’front-line’ identification tool. 


The isolates used in this study consisted of field strains referred 

for determinative bacteriology by Regional Laboratories of the 

Animal Health and Veterinary Laboratories Agency (AHVLA), 

although a number of strains from the National Culture Type 

Collection (NCTC, Colindale, London) and the American Type 

Culture Collection (ATCC, Manassas, Virginia) were also included. 

Each field isolate had previously been identified by conventional 

biochemical approaches. Cultures were prepared on 5% sheep 

blood-Columbia agar (Oxoid, Basingstoke, UK), incubated either 

aerobically or in 7.5% carbon dioxide at 37 °C for 18-24h, 

according to cultural characteristics, and tested blind by each of 

the three methods as follows. 

Biolog Generation 3: 

Inocula were prepared according to manufacturer’s instructions 

(Biolog, Hayward, CA). Growth patterns were measured using a 

Microstation reader and analysed using Microlog software. 

Matrix-assisted laser desorption ionization-time of flight mass 

spectrometry (MALDI-TOF): 

Samples were prepared using standard Bruker protocols (Bruker 

Daltonik GmbH, Bremen, Germany) (4) and analysed using a 

Bruker Autoflex II machine. Spectra were analysed with MALDI 

Biotyper software version 2.0.10.0 in comparison with reference 

spectra (MALDI Biotyper reference library version 3). 

16S rRNA gene sequencing: 

Ribosomal RNA sequence identification was based on a minimum 

600bp sequence corresponding to the V2-V4 regions of the gene. 

Sequences were compared with both NCBI and RDP databases 

with criteria of a >99% similarity to the type strain of a 

taxonomically valid species being used as identification criteria for 

species. If matches were below 99% or there were matches >99% 

to multiple taxonomically valid species identification was given to 

the genus level only. 

In the majority of cases, referral for determinative bacteriology 

within AHVLA requires identification to species level. A scoring 

system was devised, using either 16S sequencing or the 

NCTC/ATCC identity as the ‘gold standard’. Where sequencing 

lacked species information for comparison, identification was 

scored at genus level only in all methods. 

Results 

Scores of 3, 1 and 0 were assigned for identifications to species, 

genus and ’ no identification’, respectively. 

Table 1 summarises the performance of each system. 

Table 1: Performance of identification systems 

Numbers of strains identified Biolog MALDI 16S sequencing 

To genus 28 30 29 

To species 27 27 36 

No ID 11 9 1 

Incorrect genus 0 0 0 

Incorrect species 0 0 0 

Overall score 109 111 137 

Scores for MALDI and Biolog may have improved if alternative 

phenotypic data had been considered in determining true identity. 


Scores for MALDI and Biolog were broadly comparable, although 

a number of taxa were identified more specifically by particular 

techniques. A number of isolates proved refractory either by 

MALDI-TOF or Biolog, although local enhancements to databases 

would minimise these occurrences. 

Whilst each methodology offers unique advantages, other factors 

which require consideration are the accessibility of the technology, 

including start-up and running costs, the quality and ease of 

supplementation of the manufacturer’s database and the 

requirement for centralisation of resources and expertise which 

may be implicit in the choice of technique. 


We thank the AHVLA Regional Laboratories at Bury St Edmunds, 

Starcross and Sutton Bonington for provision of field strains, Mark 

Koylass and the Central Sequencing Unit at AHVLA Weybridge. 

References 

1.Janda, J.M., Abbott, S.L. (2007). 16S rRNA gene sequencing for bacterial 

identification in the diagnostic laboratory: pluses, perils and pitfalls. J. Clin. 

Microbiol. 45: 2761-2764. 

2. Woo, P.C., Lau, S.K., Teng, J.L., Tse, H., Yuen, K-Y. (2008) Then and 

now: use of 16S rDNA gene sequencing for bacterial identification and 

discovery of novel bacteria in clinical microbiology laboratories. Clin. 

Microbiol. Infect., 14: 908-934. 

3. Morgan, M.C., Boyette, M., Goforth, C., Sperry, K. V., Greene, S. R., 

(2009). Comparison of the Biolog Omnilog Identification System and 16S 

ribosomal RNA gene sequencing for accuracy in identification of atypical 

bacteria of clinical origin. J. Microbiol. Meth. 79: 336-343. 

4. Eigner, U., Holfelder, M., Oberdorfer, K. et al. (2009) Performance of a 

matrix-assisted laser desorption ionization-time-of-flight mass spectrometry 

system for the identification of bacterial isolates in the clinical routine 

laboratory. Clin. Lab., 55: 289-296.

S4 - O - 03 

EVALUATION OF THE DIAGNOSTIC PERFORMANCE OF PEPTIDE COCKTAILS IN THE INTERFERON 

GAMMA ASSAY FOR DIAGNOSIS OF TUBERCULOSIS IN CATTLE 

Björn Schröder 1 , Roland Hardegger 1 , Marcus G. Doherr 2 , Jean-Louis Moyen 3 , Eamonn Gormley 4 and 

Alex J. Raeber 1 

1 Prionics AG, Schlieren, Switzerland, 2 Vetsuisse Faculty, University of Bern, Switzerland, 3 Laboratoire Départemental d’Analyse et de Recherche, 

Coulounieix-Chamiers, France, 4 University College Dublin, Ireland, 

Tuberculosis, Bovigam, defined antigens, peptide cocktail 

Introduction 

Cattle infected with bovine tuberculosis (bTB) still represent a 

serious regulatory and health concern in a many countries. For 

more than two decades, eradication programs for bTB have 

relied on early detection and removal of infected cattle using the 

intradermal skin test as primary test and the in vitro interferon 

gamma (IFN-) assay as an ancillary assay. The interferon 

gamma assay is generally considered to be more sensitive than 

the skin test while the specificity is slightly lower. To increase the 

specificity of the IFN- assay, defined mycobacterial antigens 

such as ESAT-6 and CFP-10 have been evaluated as alternative 

stimulation antigens in blood cultures and have shown promising 

results although sensitivities were slightly lower than with 

tuberculin stimulation. Combining additional specific 

mycobacterial antigens with ESAT-6 and CFP-10 has been 

proposed to bring the sensitivity of antigen cocktails to the levels 

obtained with tuberculin and at the same time achieving 

specificities comparable to the skin test. We have developed 

peptide cocktails based on ESAT-6 and CFP-10 (1, 2, 3) and 

compared their performance to tuberculins in the IFN- assay for 

diagnosis of bTB in field studies in Ireland and France. 


Lelystad tuberculin PPD antigens (avian and bovine) (Prionics, 

The Netherlands) or a synthetic peptide cocktail were used for 

stimulation of whole blood cultures. Following stimulation, plasma 

was harvested and IFN-γ was measured with the BOVIGAM ® 2G 

(Prionics, Switzerland) sandwich enzyme immunoassay. The 

peptide cocktail Prionics ® PC-HP (Prionics, Switzerland) 

contained overlapping peptides derived from ESAT-6 and CFP- 

10 and in addition peptides derived from Rv3615c and 3 other 

mycobacterial proteins. Diagnostic sensitivity was assessed in 

naturally bTB-exposed animals in Ireland. Diagnostic specificity 

was evaluated in France in animals from 12 herds free of bTB 

according to OIE criteria. Estimates for the test characteristics in 

the absence of a true gold standard were derived by a Bayesian 

model as described by Branscum et al. 2005 (4). 

Results 

Estimates of sensitivity and specificity (median and 95% 

confidence intervals) are shown in Table 1. In France 390 

animals have been included into the specificity trial. Specificity 

estimates for the BOVIGAM ® 

2G with tuberculin PPD from 

Lelystad was calculated with 84% (95%CI: 81% - 87%) whereas 

using the peptide cocktail PC-HP for whole blood stimulation a 

specificity estimate of 94% (95%CI: 92% - 96%) was computed. 

The difference in the specificity estimates of PPD and PC-HP 

was highly significant (p < 0.05; two-tailed exact Fisher test). 

Sensitivity was evaluated from the sample of 201 animals from 

Ireland, with an apparent prevalence (depending on test) 

between 16% and 31%. Sensitivity in this trial was calculated for 

Lelystad PPD with 89% (95%CI: 75% - 97%) whereas with PC- 

HP a comparable sensitivity of 85% (95%CI: 63% - 96%) was 

obtained. 

Table 1: Comparison of diagnostic performance estimates of 

BOVIGAM ® 2G using PPD or antigen peptide cocktail PC-HP for 

stimulation of blood. 

Sensitivity 

Estimates 

95% CI 

Specificity 

Estimates 

95% CI 

PPD 89% 75 - 97% 84% 81 - 87% 

PC-HP 85% 64 - 96% 94% 92 - 96% 


Since 1991 when the BOVIGAM ® IFN- test was approved as an 

official test for bTB in Australia and later on in the European 

Union and the US, numerous field trials were undertaken to 

determine its diagnostic performance. In a review published by 

de la Rua-Domenech et al. (5) the sensitivity of the IFN- test 

varied between 73.0% and 100%, with a median value of 87.6% 

while its median specificity was calculated with 96.6%, with a 

range of 85.0–99.6%. Using a Bayesian modelling approach, we 

have compared the diagnostic performance of the peptide 

cocktail Prionics ® PC-HP with tuberculin PPD for the stimulation 

of blood cultures in the BOVIGAM ® 2G IFN- assay. Our results 

suggest that the sensitivity of the peptide cocktail PC-HP is not 

significantly different from the sensitivity with PPD whereas 

peptide cocktail PC-HP resulted in a specificity that was 

significantly higher than stimulation with PPD. Overall, we 

conclude from these studies that peptide cocktails provide 

superior performance compared to tuberculin PPD in the 

BOVIGAM ® 2G assay which opens up new possibilities for highly 

specific diagnostic tools in the eradication of bTB. 

References 

1. Buddle, BM, Ryan, TJ, Pollock, JM, Andersen, JM, and de Lisle, GW 

(2001). Use of ESAT-6 in the interferon- test for diagnosis of bovine 

tuberculosis following skin testing. Vet Microbiol, 80, 37-46 

2. Sidders, B, Pirson, C, Hogarth, PJ, Hewinson, RG, Stoker, NG, and 

Vordermeier, HM (2008). Screening of highly expressed mycobacterial 

genes identifies Rv3615c as a useful differential diagnostic antigen for the 

Mycobacterium tuberculosis complex. Infect Immun, 76, 3932-3939 

3. Schiller, I, Vordermeier, HM, Waters, WR, Palmer, M, Thacker, T, 

Whelan, A, Hardegger, R, Marg-Haufe, B, Raeber, A and Oesch, B (2009). 

Assessment of Mycobacterium tuberculosis OmpATb as a novel antigen 

for the diagnosis of bovine tuberculosis. Clin Vaccine Immunol, 16,1314-21 

4. Branscum, AJ, Gardner, IA and Johnson WO (2005). Estimation of 

diagnostic-test sensitivity and specificity through Bayesian modeling. Prev 

Vet Med 68, 145-163 

5. De la Rua-Domenech, R, Goodchild, A.T., Vordermeier, H.M., 

Hewinson, R.G., Christiansen, K.H., Clifton-Hadley, R.S. (2006) Ante 

mortem diagnosis of tuberculosis in cattle: a review of the tuberculin tests, 

gamma-interferon assay and other ancillary diagnostic techniques." Res 

Vet Sci 81: 190-210

S4 - O - 04 

MATRIX-ASSISTED LASER DESORPTION IONIZATION-TIME OF FLIGHT MASS SPECTROMETRY 

(MALDI-TOF MS) IN A VETERINARY DIAGNOSTIC LABORATORY 

Annet E. Heuvelink, Refke Peerboom, Erik van Engelen, Abdul A. Hassan 

GD Animal Health Service, Deventer, The Netherlands 

MALDI-TOF, bacterial identification, veterinary laboratory 

Introduction 

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass 

Spectrometry (MALDI-TOF MS) has emerged as a new tool for 

fast and reliable identification of microorganisms (). We evaluated 

the performance of MALDI-TOF MS for the routine identification 

of bacteria in our veterinary diagnostic laboratory. 


Mass spectra were acquired using a Microflex LT mass 

spectrometer and analysed by MALDI Biotyper 3.0 software 

(Bruker Daltonik GmbH, Bremen, Germany). 

First, we determined the accuracy of MALDI-TOF MS 

identification by testing 66 bacterial strains of culture collections, 

in duplicate spots on the MALDI target plate. Second, we 

performed a prospective validation study, including 262 isolates 

collected in the routine laboratory: 156 isolates from post-mortem 

examinations of mammals and 106 mastitis pathogens from 

cow’s milk. The isolates were identified by conventional 

biochemical methods in parallel with MALDI-TOF MS, measured 

in duplicate spots. A selection of Gram-positive cocci, including 

coagulase-negative staphylococci (CNS) and Streptococcus 

uberis isolates, were tested by both direct colony testing and the 

extended direct transfer method described by the manufacturer, 

comprising a rapid partial extraction with 70% aqueous formic 

acid. Finally, the repeatability and the robustness of identification 

of bacteria by MALDI-TOF MS were evaluated. The repeatability 

was determined with five reference strains, by testing 10 cfu from 

one plate, in one run, by one technician. The robustness was 

evaluated by testing the effect of (a) refrigerated storage of fresh 

cultures grown on agar plates (for 24 or 48 h), (b) the use of 

selective solid media, and (c) prolonged incubation (for 48 or 72 

h) either or not followed by a refrigeration step. For each of the 

species included in the robustness test, one reference strain and 

five field isolates were tested. 

some isolates, especially those of S. uberis, more not reliable 

identifications were obtained when performed with cultures stored 

for 48 h in the refrigerator or incubated for 48 h, and especially 

for 72 h prior to identification. 


The present results show that MALDI-TOF MS is a reliable and 

rapid method for identification of most bacterial species 

encountered in our routine veterinary laboratory. No 

misidentifications occurred, only “no reliable identifications”. For 

the majority of the isolates direct colony testing can be performed 

without extraction. For identification of presumptive CNS isolates, 

it is recommended to use the simple partial extraction procedure 

with 70% aqueous formic acid following the instructions of the 

manufacturer. 

One of the additional benefits of MALDI-TOF MS analysis is the 

identification of CNS species. Species level identification of CNS 

will help to elucidate sources, transmission mechanisms and the 

impact of different CNS species on cow health, productivity and 

milk quality, and this knowledge may lead to species-specific 

infection control measures. 

References 

1. Bizzini A, Greub G. Matrix-assisted laser desorption ionization 

time-of-flight mass spectrometry, a revolution in clinical microbial 

identification. Clin. Microbiol. Infect. 2010, 16(11), 1614-1619. 

2. Wieser A, Schneider L, Jung J, Schubert S. MALDI-TOF MS 

in microbiological diagnostics-identification of microorganisms 

and beyond (mini review). Appl. Microbiol. Biotechnol. 2012, 

93(3), 965-974. 

Results 

Of the 66 reference strains tested, 79% were correctly identified 

to species level. The remaining strains were identified to genus 

level (14%) or could not be identified (7%). The latter strains 

belonged to species not included in the BioTyper 3.0 database. 

For 90% of the 156 isolates from post-mortem examinations of 

mammals matching identifications at species level were obtained 

between MALDI-TOF MS and biochemical methods, 8% were 

correctly identified to genus level, and for 2% no reliable 

identifications were achieved. Eighty-nine% of the MALDI-TOF 

MS identifications of 106 mastitis pathogens were concordant 

with standard biochemical identifications. Ten% of the mastitis 

pathogens were identified to species level by MALDI-TOF MS 

(mainly CNS), while standard biochemical identifications of these 

isolates were at genus level only. For one isolate the MALDI-TOF 

MS result diverged from the biochemical identification; MALDI- 

TOF MS identified the isolate as Klebsiella pneumoniae, a 

qualitatively good result at species level, while the API 20E 

(bioMérieux, Marcy l’Etoile, France) result was Klebsiella 

oxytoca, with a doubtful profile. Most bacteria tested in this study 

could be successfully identified at species level using the direct 

colony testing method. However, for 70% of the CNS isolates the 

partial extraction procedure showed to be superior. For S. uberis 

the superiority of the extended direct transfer method appeared 

to be less pronounced, being observed for only 32% of the 

isolates. 

The repeatability of MALDI-TOF MS identification of the 

reference strains of Escherichia coli, Clostridium perfringens, 

Staphylococcus aureus, and Salmonella Typhimurium was 100%. 

For the Streptococcus suis reference strain it was 90%, with 10% 

being not reliably identified. 

In general, MALDI-TOF MS identification results were not altered 

when performed with cultures stored for 24 or 48 h at 

refrigeration temperatures or with cultures tested after 48 or 72 h 

of incubation, nor when selective agars were used. However, for

S4 - O - 05 

RAPID IDENTIFICATION OF BOVINE MASTITIS PATHOGENS USING MALDI TOF 

Gudrun Overesch 1 , Andreas Thomann 1 , Vincent Perreten 1 

1 

Institute of Veterinary Bacteriology, University of Bern, Bern, Switzerland 

Maldi Tof, mastitis, identification, veterinary pathogens 

Introduction 

Correct and rapid identification of pathogens is the main objective 

of bacteriological diagnostic. Phenotypical identification 

procedures using biochemical parameters have been widely used 

in the last decades. Although automatic systems were developed, 

these methods are still time consuming. Therefore molecular 

identification methods, i. e. real time PCR, were established to 

replace biochemical approach when applicable. The exclusive 

use of molecular based diagnostic does not allow performing 

antibiotic susceptibility tests. The recently established matrixassisted 

laser desorption/ionization time-of-flight mass 

spectroscopy (MALDI TOF MS) approach overcomes the 

disadvantages of the methods mentioned above. It is faster than 

PCR and, while working with cultured strains, it is possible to 

perform antimicrobial susceptibility testing. The databases of 

MALDI TOF MS are usually based on spectra generated from 

human isolates [1]. Its use in veterinary bacteriology needs 

validation with livestock derived strains and update of the 

databases with spectra from relevant pathogens of veterinary 

interest. This study presents the establishment of MALDI TOF 

MS technology for the direct identification to the species level of 

relevant bovine mastitis pathogens. 


156 bovine mastitis milk samples from individual cows were 

included. Ten microliter of whole milk was cultured on trypticase 

soy agar plates with 5% sheep blood (Becton Dickinson) at 37 °C 

for 24 hours under aerobic conditions. Colonies were identified 

phenotypically by routine diagnostic procedures [2] [3]. In parallel, 

the same colonies were identified by MALDI TOF MS (Biotyper 

3.0, Bruker) using the direct transfer protocol recommended by 

the manufacturer. Brieflly, material from a single colony was 

taken with a toothpick, smeared on a steel plate in dublicates and 

overlaid with 1 μl of HCCA matrix solution. After air drying, the 

samples were measured using standard settings in the “Flex 

control” software. For calibration and as an internal reference the 

“bacterial test standard” (BTS, Bruker) was applied in triplicate on 

every plate measured. Analysis of spectra with the Biotyper 3.0 

included a comparison against the internal commercial database 

in combination with the institute database. The latter was 

generated using veterinary field strains, which include coagulasenegative 

staphylococci (Table 1), S. pseudintermedius, 

Aerococcus viridans and Streptococcus uberis. All strains were 

identified with VITEK 2 (Biomérieux) and 16SrDNA sequence. 

New profiles were introduced into MALDI TOF MS database. 

Identification was accepted when a score value > 2.000 was 

achieved. All identified strains isolated from mastitis milk included 

in this study are listed in Table 1. 

Results 

After updating the commercial database with veterinary field 

strains (i. e. coagulase-negative staphylococci and. S. uberis) 

155 out of 156 biochemical defined strains were identified from a 

24 hour direct plating culture by MALDI TOF MS analyses (Table 

1). These strains include all highly relevant mastitis pathogens, 

i. e. staphylococci and streptococci species. MALDI TOF MS 

identified all alpha- double- and non-hemolytic Staphylococcus 

aureus. Trueperella (Arcanobacterium) pyogenes, Histophilus 

somni, E. coli and Klebsiella oxytoca were also clearly identified. 

Members of the Streptococcus-, Lactococcus-, Enterococcusgroup, 

which cannot be easily characterized using classical 

methods could also be identified to the species level using 

MALDI TOF MS. Only Corynebacterium bovis could not be 

directly identified by MALDI TOF MS, since this species has not 

yet been introduced in the new database. 


MALDI-TOF MS was shown to represent an excellent method for 

rapid species identification of relevant bovine mastitis pathogens 

by direct analysis of overnight cultures. However, the commercial 

database needed to be expanded with profiles of important 

bovine mastitis pathogens. Further improvement such has 

extraction prior identification should also be engaged to increase 

the identification spectrum. A more detailed identification of 

bacteria present in mastitis milk will also allow to increase the 

knowledge regarding species diversity involved in mastitis. 

Table 1: Biochemical identification and Maldi Tof identification of 

bacterial strains isolated from mastitis milk 

Isolates 

(N) 

Biochemical 

identification 

Maldi Tof identification 

(number) 

39 Streptococcus uberis Streptococcus uberis 

39 

16 

15 

14 

9 

Non haemolytic 

staphylococci 

Double haemolytic 

S. aureus 

Streptococcus-, 

Lactococcus-, 

Enterococcus-group 

Alpha haemolytic 

staphylococci 

Streptococcus 

dysgalactiae subsp. 

dysgalactiae 

8 Enterococcus spp. 

5 

Trueperella 

(Arcanobacterium) 

pyogenes 

S*. xylosus (19) 

S. chromogenes (7) 

S. sciuri (4) 

S. epidermidis (2) 

S. vitulinus (2) 

S. equorum (2) 

S. warneri (1) 

S. devriesei (1) 

S. aureus (1) 

S. aureus (16) 

Lactococcus garvieae (9) 

Aerococcus viridans (3) 

Lactococcus lactis (1) 

Enterococcus devriesei (1) 

Streptococcus uberis (1) 

S. aureus (6) 

S. hemolyticus (5) 

S. xylosus (3) 

Streptococcus dysgalactiae 

subsp. dysgalactiae 

Enterococcus faecalis (4) 

Aerococcus viridans (2) 

Streptococcus equinus (1) 

Enterococcus faecium (1) 

Trueperella 

(Arcanobacterium) pyogenes 

4 Escherichia coli Escherichia coli 

2 

Streptococcus 

agalactiae 

Streptococcus agalactiae 

1 Histophilus somni Histophilus somni 

1 Klebsiella oxytoca Klebsiella oxytoca 

*S. = Staphylococcus 

Acknowledgments 

This study was partially financed by grant 1.11.21 of the Federal 

Veterinary Office (FVO), Switzerland. 

References 

1. Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM, et 

al. Ongoing revolution in bacteriology: routine identification of bacteria by 

matrix-assisted laser desorption ionization time-of-flight mass 

spectrometry. Clin Infect Dis 2009;49(4):543-551. 

2. Guélat-Brechbuehl M, Thomann A, Albini S, Moret-Stalder S, Reist M, 

Bodmer M, et al. Cross-sectional study of Streptococcus species in 

quarter milk samples of dairy cows in the canton of Bern, Switzerland. Vet 

Rec 2010;167(6):211-215. 

3. Moret-Stalder S, Fournier C, Miserez R, Albini S, Doherr MG, Reist M, 

et al. Prevalence study of Staphylococcus aureus in quarter milk samples 

of dairy cows in the Canton of Bern, Switzerland. Prev Vet Med 

2009;88(1):72-76.

S4 - O - 06 

15 MINUTE ELISA USING A LOW COST COMMERCIAL BIOSENSOR 

Jason Sawyer 1 , Jennifer Cork 1 , Rebecca Jones 1 

1 

AHVLA (Weybridge), Technology Transfer Unit, New Haw, Addlestone, Surrey KT15 3NB, UK 

ELISA, IBR, Biosensor, Penside testing, Serology, Vantix 

Introduction 

The use of biosensors for diagnostic testing offers several 

potential advantages, but to date their use for routine diagnostic 

testing has been limited to specific markets (e.g. glucose testing). 

We evaluated the use of a commercially available potentiometric 

biosensor platform - Vantix - to carry out a serological assay 

for antibodies to infectious bovine rhinotracheitis (IBR), caused 

by Bovine Herpes Virus 1 (BoHV-1). 

The Vantix system 1 

comprises a low cost reader unit and 

disposable potentiometric biosensors. The biosensors consist of 

a working and reference electrode coated in a conductive 

polymer (polypyrrole) and covered in a protective plastic film. 

Biochemical or enzymatic activity taking place on the surface of 

the working electrode as a result of immunocomplexes built up on 

the electrode, cause electrochemical changes in the conductive 

polymer layer on and around the working electrode thus 

generating a measurable change in electrical potential (measured 

in millivolts) relative to the reference electrode. The signal 

produced is proportional to the level of analyte under 

investigation. 

IBR is a highly contagious disease of cattle that is endemic in the 

UK and many other parts of the world 2 . ELISA is commonly used 

in the detection and control of this disease and AHVLA uses an 

in-house indirect ELISA. In this assay, BoHV-1 antigens, bound 

to the surface of a microtitre plate, capture BoHV-1 specific 

antibodies present in test samples. Bound antibodies are then 

detected using a horseradish peroxidase (HRP) labelled antibovine 

immunoglobulin and subsequent HRP catalysed oxidation 

of the chromogen substrate 3,3’,5,5’-tetramethylbenzidine (TMB) 

produces a colour change. The in-house ELISA for BoHV-1 used 

at AHVLA has a total assay time of over 3 hours which is typical 

of commercial ELISA tests. 

In this work, the well-characterised and established reagents 

used in the parent ELISA were used to construct an assay on the 

Vantix platform. A panel of serum samples, submitted for 

routine ELISA testing, were then tested with the Vantix assay and 

the results compared to the parent ELISA. 

Table 1: Comparison of methodology of the IBR antibody biosensor 

assay and parent ELISA. 

Biosensor assay 

Indirect ELISA 

Step 

Incubation 

Incubation 

Step 

Time 

Time 

Biosensors and ELISA plates pre coated with BoHV1 antigen 

and stored ready for use 

Incubation with diluted 


5 min 

sample 

sample 

2 h 

8 Washes no soak 0.5 min 

5 x washes 10 second 

soak 

5 min 


Rabbit anti bovine HRP 5 min 


Rabbit anti bovine HRP 1 h 

Conjugate 

Conjugate 

8 Washes no soak 0.5 min 

5 x washes 10 second 

soak 

5 min 

Probes added to TMB 

Plate incubated with 

5 – 20 min 

substrate and read 

TMB substrate. 

4 min 

immediately. Potential 

Addition of Stop 1 min 

difference reading. 

Colorimetric read 1 min 

Total assay time 15 min Total assay time >3.25 h 


Table 1 summarises the steps and timings involved in the 

performing the Biosensor assay compared to the ELISA. 

Biosensor probes were prepared by coating with BoHV-1 antigen, 

and were then stored at +4 o C for up to 12 weeks. To perform the 

assay, the electrodes of the antigen coated biosensors were 

briefly placed in diluted serum, washed, and briefly incubated 

with conjugate. After a final wash step, the sensors were 

inserted into the Vantix reader unit and the working and 

reference electrode placed into TMB substrate. Changes in 

electrochemical potential difference were observed over a period 

of four minutes and recorded using Vantix software. The 

biosensor assay was used to test 194 serum samples, 

comprising 90 positive and 104 negative as designated by the in 

house ELISA. 

% signal relative to weak 

positive control 

250 

200 

150 

100 

50 

0 

IBR Biosensor 

2 x 2 box analysis assay 

Serum Samples Positive Negative Totals 

IBR 

ELISA 

Negative 

Positive 86 4 90 

Negative 2 102 104 

Totals 88 104 194 

Positive 

True positive rate 

(Sensitivity) 

0 

0 25 50 75 100 

False positive rate 

(100 - Specificity) 

Fig.1 Comparison of results obtained when testing serum 

samples using the parent IBR antibody ELISA and the biosensor 

assay. 2x2 table (Top); Box and whisker plot showing biosensor 

results for ELISA negative and positive samples (bottom left) and 

ROC analysis (bottom right). 

Results 

Fig 1 shows the resulting 2X2 Table, Box and Whisker plot and 

ROC curve when the biosensor assay was compared to the 

ELISA. Using the optimal cut-off value (determined by ROC 

analysis and based on a percentage signal relative to the weak 

positive control serum run alongside the test samples) the 

biosensor assay had a sensitivity of 98% and a specificity of 96% 

when compared to the indirect ELISA. Only 6 of 194 samples 

resulting in a different categorisation (positive or negative) 

compared to the ELISA results. Overall, the results demonstrate 

that the biosensor assay produces test results that closely match 

those obtained with the parent ELISA. 


This work has shown that results equivalent to the ELISA can be 

obtained using the Vantix biosensor platform in 15 min. The 

relatively low cost of the biosensors (~ few Euro each) and reader 

(~ few thousand euro) make this an interesting and promising 

technology. The rapid and simple nature of the assays, and the 

fact that established ELISA reagents can readily be converted to 

the platform, suggest Vantix could be useful in a variety of 

diagnostic testing scenarios. These may include rapid testing of 

samples which require fast turnaround or testing in low tech 

laboratories. The generation of an electronic signal offers the 

potential for transmission across mobile networks. Vantix are 

currently developing a hand held device which uses the same 

biosensors but offers the potential for more rapid (

Poster presentations 

“General Session” 

(1 st session)

S1 - P - 01 

LAWSONIA INTRACELLULARIS IN BLUE FOXES IN FINLAND. A CASE REPORT 

Heli Kallio, Heikki Ahola 

Finnish Food Safety Authority Evira, Production Animal and Wildlife Health Research Unit, Seinäjoki, Finland 

Lawsonia intracellularis, blue fox, enteritis, diarrhoea, rectum prolapse 

Introduction 

Lawsonia intracellularis-bacteria is commonly detected in the 

enteritis in domestic pigs with proliferative enteropathy, intestinal 

adenomatosis and ileitis. In addition to pigs, Lawsonia 

intracellularis has been detected in wide variety of domestic and 

wild animals, including wild red foxes, and it is also suggested to 

be found in blue foxes reared in a fur farm. However, 

pathogenesis of Lawsonia intracellularis enteritis in fur farm 

animals is poorly studied. In this study we detected Lawsonia 

intracellularis for the first time in enteritis from blue fox in a 

Finnish fur farm. 

A 1 2 3 4 


A blue fox was submitted to laboratory for diagnostic because of 

occurrence of diarrhoea in a single farm. Symptoms of the foxes 

were, in addition to diarrhoea with loose faeces, decreased 

weight gain, and rectal prolapses. 

Necropsy was performed for the fox. Then intestinal samples 

were prepared for HE- and WS staining. Nested-PCR for the 

epithelium of the intestine was performed as earlier described by 

Jones et al. to detect Lawsonia intracellularis -bacteria. Prior to 

PCR DNA was extracted by boiling procedure according to 

Moeller et al. Intestinal samples were cultured on sheep blood 

agar in aerobic and anaerobic atmosphere, as well as plated onto 

a Campylobacter blood-free selective medium. The Sheather's 

sugar flotation technique was used to detect parasites. 

Results 

In necropsy rectal prolapse, severe hyperemia, oedema and 

thickening of mucosa in the distal ileum, colon and rectum were 

detected. Signs of poor growth and loss of fluid were also seen. 

In histology the fox had proliferative enteritis, colitis and proctitis 

with severe villushyperemia and –necrosis, and hypertrophic 

dilated crypts with vigorously branching structure. By WS-staining 

curved organisms, in the apical cytoplasm of hyperplastic 

epithelium lining intestinal glands, were detected. Using nested- 

PCR, Lawsonia intracellularis was discovered from the intestinal 

samples. No other putative pathogens were identified in 

bacteriological and parasitological studies. 

Figure 2. Amplification of Lawsonia intracellularis after nested- 

PCR analysis from intestine. Negative amplification control (1), 

positive amplification control (2), two separate samples from the 

epithelium of the intestine (3, 4). DNA ladder, 100-1000 bp (A) 


The results obtained by this study show the Lawsonia 

intracellularis caused diarrhoea for the first time in Finnish blue 

fox farm. The fox examined showed typical changes in necropsy 

and histology. Lawsonia intracellularis was detected by PCR and 

WS-staining. Eriksen et al. reported earlier adenomatosis in blue 

foxes reared in a fur farm, most likely caused by Lawsonia 

intracellularis. Lawsonia intracellularis has been detected also 

from faeces of wild red foxes. However, according to our 

knowledge, other reports about Lawsonia intracellularis in foxes 

have not been published. Significance of Lawsonia intracellularis 

for fox farming is still poorly understood and further studies are 

required. 

References 

1. Tomanova,K Literak,I, Klimes,J Pavlacik,L, Mrlik,V and Smola,J ( 

2003). Lawsonia intracellularis in Wild Mammals in the Slovak 

Carpathians. Journal of Wildlife Diseases, 39(2), 407-411 

2. Eriksen,K, Landsverk,K.T and Bratberg,B. (1990). Morphology and 

immunoperoxidase studies of intestinal adenomatosis in the blue fox, 

Alopex lagopus. Journal of Comparative Pathology, 102, 265-278 

3. Jones,GF, Ward,GE, Murtaugh,MP, Lin G, Gebhart,CJ (1993) 

Enhanced detection of intracellular organism of swine proliferative enteritis, 

ileal symbiont intracellularis, in feces by polymerase chain reaction. J Clin 

Microbiol, 31(10), 2611-5 

4. Møller,K, Jensen,TK, Jorsal,SE, Leser,TD, Carstensen,B (1998) 

Detection of Lawsonia intracellularis, Serpulina hyodysenteriae, weakly 

beta-haemolytic intestinal spirochaetes, Salmonella enterica, and 

haemolytic Escherichia coli from swine herds with and without diarrhoea 

among growing pigs. Vet Microbiol, 30;62(1), 59-72 

Figure 1. Curved organisms in the apical cytoplasm of 

hyperplastic epithelium lining intestinal glands stain positively in 

WS-staining.

S1 - P - 02 

DIAGNOSTIC PERFORMANCE OF A COMMERCIAL PRRS SERUM ANTIBODY ELISA ADAPTED TO 

ORAL FLUID: LONGITUDINAL RESPONSE IN EXPERIMENTALLY-INOCULATED POPULATIONS 

Apisit Kittawornrat 1 , John Prickett 1 , Chong Wang 1, 2 , Chris Olsen 1 , Yaowalak Panyasing 1 , Andrea Ballagi 3 , 

Anna Rice 3 , Sergio Lizano 3 , Raymond Rowland 4 , Jeffrey Zimmerman 1 

1 Department of Veterinary Diagnostic and Production Animal Medicine, 2 Department of Statistics, Iowa State University, Ames, Iowa, United States, 3 IDEXX 

Laboratories, Westbrook, Maine, United States, 4 Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, Kansas 

Introduction 

Previous work showed that a commercial PRRS ELISA 

(HerdChek® PRRS X3 ELISA) could be adapted to detect anti- 

PRRSV IgM, IgA, and IgG in oral fluid specimens. Further, the 

protocol for the IgG ELISA for oral fluid samples was readily 

amenable to the routine performance of the assay in highthroughput 

diagnostic laboratories. This suggested the possibility 

of a cost-effective method to routinely monitor commercial swine 

populations for maternal antibody, vaccination compliance, and 

herd immune parameters using oral fluid sampling. The purpose 

of the present study was to evaluate the ability of the PRRS oral 

fluid IgG ELISA to detect anti-PRRSV IgG antibody in pen-based 

oral fluid samples from experimentally inoculated pigs over time. 


In nine trials, ~200 pigs per trials were intramuscularly (IM) 

inoculated with PRRSV isolate NVSL 97-7895. Oral fluid 

samples were collected on 0, 5, 7, 9, 11, 14, 17, and 21 days 

post inoculation (DPI). All oral fluid samples were randomized 

and tested for anti-PRRSV antibodies using the PRRS ELISA 

protocol for oral fluids: 1:2 oral fluid sample dilution, 250µl sample 

volume, 16 hour incubation at 4°C, and detection of reaction 

using anti-pig IgM, IgA, and IgG FC (1). 

Results 

Anti-PRRSV antibody isotype (IgM, IgA, IgG FC ) kinetics in 

experimental samples are shown in Figure 1 for oral fluid 

samples collected on DPI 0, 5, 7, 9, 11, 14, 17, and 21. Mean 

IgM, IgA, and IgG S/P ratios on DPI 7 were 1.14 (95% confidence 

interval (CI) 1.04, 1.24), 0.09 (95% CI 0.06, 0.12), and 0.59 (95% 

CI 0.50, 0.68), respectively. The mean IgM S/P ratio peaked at 

DPI 9 (1.88, 95% CI 1.78, 1.97) and declined rapidly to a mean 

S/P of 0.10 (95% CI 0.09, 0.13) on DPI 21. Mean IgA and IgG 

S/P ratios peaked at DPI 11 (1.41, 95% CI 1.29, 1.53) and DPI 

14 (3.69, 95% CI 3.52, 3.85), respectively. Mean IgA and IgG 

S/P ratios remained relatively constant until the end of study at 

DPI 21. 

Oral fluid, ELISA, Experimental samples, PRRSV, Antibody 

Figure 1: Kinetics of anti-PRRSV antibody isotypes (IgM, IgA, 

IgG FC ) in experimental samples 


These results indicated that anti-PRRSV antibodies in oral fluid 

are amenable to rapid detection post-infection. Testing based on 

oral fluids could provide an efficient, cost-effective approach to 

PRRSV monitoring in commercial herds and surveillance in 

elimination programs. 

References 

1. Kittawomrat, A et al.: 2012. Detection of PRRSV antibodies in oral fluid 


Diagn Invest 24 (2):262-269

S1 - P - 03 

DETECTION OF PRRSV ANTIBODY IN ORAL FLUID SPECIMENS FROM INDIVIDUAL BOARS USING A 

COMMERCIAL PRRSV SERUM ANTIBODY ELISA 

Apisit Kittawornrat 1 , Mark Engle 3 , John Johnson 1 , John Prickett 1 , Chris Olsen 1 , Trevor Schwartz 1 , Daniel 

Whitney 1 , Kent Schwartz 1 , Anna Rice 4 , Andrea Ballagi 4 , Sergio Lizano 4 , Chong Wang 1,2 , Jeffrey Zimmerman 1 

1 Department of Veterinary Diagnostic and Production Animal Medicine, 2 Department of Statistics, Iowa State University, 3 PIC North America, 

Hendersonville, Tennessee, USA, 4 IDEXX Laboratories, Westbrook, Maine, USA 

Oral fluid, Individual boar, ELISA, PRRSV 

Introduction 

Oral fluid specimens are used in human medicine for the 

detection of a variety of infectious agents, hormones, and drugs. 

Oral fluid samples are of interest in swine medicine because they 

are easily collected, yet highly efficacious for the surveillance of 

PRRSV and other pathogens using PCR-based assays (1,3). 

Recent work showed that a commercial PRRS serum antibody 

ELISA (IDEXX Laboratories, Inc., Westbrook, ME USA) could be 

adapted to detect PRRSV antibody in oral fluid specimens (2). In 

the current study we describe the kinetics of the ELISAdetectable 

anti-PRRSV IgG response in oral fluids collected from 

individually-housed boars. 


The study was conducted in 72 boars ranging from 6 months to 

3.6 years in age. Boars were under the ownership of PIC North 

America (Hendersonville, TN USA) and housing, study 

procedures, and protocols were approved and supervised by the 

PIC USA Health Assurance and Welfare department. 

Boars were assigned to three trials (I, II, III). Boars (n = 24) in 

Trial I were intramuscularly (IM) inoculated with 2 ml of a 

modified live virus (MLV) vaccine (RespPRRS®, Boehringer 

Ingelheim Vetmedica, Inc., St. Joseph, MO USA). Boars (n = 22) 

in Trial II were IM inoculated with 2 ml of a Type 1 PRRSV field 

isolate. Boars (n = 24) in Trial III were IM inoculated with 2 ml of a 

PRRSV Type 2 isolate (MN-184). 

Boars were monitored for 21 days post inoculation (DPI). Oral 

fluid samples were collected daily using 5/8" 3-strand 100% 

cotton rope. Serum samples were collected from all boars on DPI 

-7, 0, 7, 14, 21 and from 4 randomly selected boars on DPIs 3, 5, 

10, and 17. Thereafter, serum and oral fluid were assayed for 

PRRSV antibody using the ELISA protocol appropriate for each 

sample type (serum or oral fluid). 

Results 

The ELISA S/P response in oral fluid and serum samples 

followed similar patterns over time (Figure 1). Individual boar oral 

fluid samples were ELISA positive from DPI 8 to DPI 21 (Table 

1). Overall, 96% of the results were in agreement, i.e., 145 oral 

fluid samples and 150 serum samples were ELISA positive. 

Table 1: Detection of PRRSV antibody in oral fluid samples from 

individually housed boars 

PRRSV 

ELISA-positive boars (%) by day post inoculation 

0 8 9 10 11 12 13 14 17 & 21 

MLV 0 0 38 71 88 96 100 100 100 

Type 1 0 17 42 59 82 82 81 85 100 

Type 2 0 17 65 83 96 96 100 100 100 

Total 0 11 48 71 89 91 91 93 100 


The ring test results showed that the PRRS oral fluid IgG ELISA 

was highly reproducible across laboratories. These results 

support the routine use of this test in laboratories providing 

diagnostic service to pig producers. Thus, herd monitoring based 

on oral fluid sampling could be one part of a PRRSV control 

and/or elimination program. Further, the successful adaptation of 

one assay to the oral fluid matrix suggests that this approach 

could provide the basis for monitoring specific health and welfare 

indicators in commercial swine herds using a "pig friendly" 

approach. 

References 

1. Kittawornrat A, et al. 2010. PRRSV in serum and oral fluid samples from 

individual boars. Virus Res 154:170-176. 

2. Kittawornrat A, et al. 2012. Detection of PRRSV antibodies in oral fluid 


Diagn Invest 24(2):262-269. 

3. Ramirez et al. 2012. Efficient surveillance of pig populations using oral 

fluids. Prev Vet Med (Epub ahead of print). 

Figure 1: Mean PRRS antibody ELISA kinetics over time: ora 

fluid vs. serum samples

S1 - P - 04 

DIAGNOSIS OF MAIN HAEMOPARASITIC DISEASES OF CATTLE BY REAL-TIME PCR. 

Villa Aleida 1 ; Benito AA 1 ; Arnal JL 1 ; Serrano JD 1 and Baselga R 1 . 

1 EXOPOL Autovacunas y Diagnóstico. Pol. Río Gállego, 50840 San Mateo de Gállego Zaragoza, España. Tel 976 69 45 25, exopol@exopol.com 

Piroplasmosis, Anaplasmosis, Diagnostic, qPCR, cattle 

Introduction 

Piroplasmosis and Anaplasmosis are the most important blood 

parasitic diseases in cattle and responsible for significant 

economical losses and mortality in livestock from many countries. 

Although these diseases occur mainly in tropical and subtropical 

areas, they are also becoming an increasing and serious problem 

in Europe ( 1 ). Clinical signs in haemoparasitic diseases are quite 

similar and include anemia, pyrexia, weakness, weight loss and 

drop in production levels. Bovine Piroplasmosis is caused by 

pathogenic Babesia and Theileria species. Th. parva and Th. 

annulata are the most important pathogens in this genus, 

although only Th. Annulata has been described in Europe ( 2 ). 

There are several pathogenic Babesia species affecting cattle (B. 

major, B. bovis, B. divergens and B.bigemina), but B. bigemina 

and B. bovis are the most frequently described ( 3 ). Bovine 

Anaplasmosis is caused by rickettsia of genus Anaplasma. A. 

marginale is considered the most pathogenic specie while A. 

centrale has been implicated in mild or less severe cases of this 

disease ( 4 ) . 

Laboratory diagnosis of these pathogens included 

their microscopical identification in blood smears; however, this 

method requires highly qualified personnel and is not reliable for 

detecting pre-symptomatic or carrier animals. Serological tests 

can also be used; although problems of cross-reactivity between 

pathogens of same genus have been reported. Recently, some 

techniques of polymerase chain reaction (PCR) have been 

developed and allowed a specific and sensitive detection of these 

agents ( 5 ) . 

The main objective of this study was evaluate the 

feasibility of a Real-time PCR (qPCR) assay for the diagnosis of 

Th. annulata, Th. parva, B. bigemina, B. bovis, A. marginale, A. 

centrale and A. phagocitophylum, in a multiparametric test for a 

specific identification and also quantification of these main tickborne 

pathogens of cattle. 


Samples: A total of 122 cases (330 blood, 58 serum and 9 tissue 

samples) submitted to our laboratory with suspect of 

haemoparasitic disease were evaluated. Some of these cases 

(10% aprox) had macroscopical findings and histopathological 

lesions of haemolytic syndrome. Most of cases 93% (113/122) 

came from 21 provinces in Spain and the rest from Portugal. 

Whole blood, serum and tissue samples were subjected to DNA 

extraction individually or pooled up to five per case. 

DNA extraction: A commercial kit “Genomic mini Kit LGD 500 

LabTurbo” and an automated DNA/RNA extraction System 

“LabTurbo 36 compact system C3620” (Taigen Bioscience 

Corporation, Taiwan) were used for DNA extraction following the 

manufacturer's instructions. DNA purity (260/280) and DNA yields 

were determined using a micro-volume spectrophotometer 

(Quawell UV Q5000, software 4.0). 

Quantitative Real-Time PCR (Genesig Ltd): The qPCR assays 

used in this study identified the following pathogen-specific 

targets: Tams1 gene (Merozoite-piroplasm surface antigen) for 

Th. annulata; p104 gene (Microneme-rhoptry antigen) for Th. 

Parva; 18S gene (18S ribosomal RNA) for B. bigemina; 18S 

gene (18S ribosomal RNA) for B. bovis; msp4 gene (Major 

surface protein 4) for A. marginale; gen; msp2 gene (Major 

surface protein 2) for A. centrale and msp4 gene (Major surface 

protein 4) for A. phagocytophylum. A unique qPCR protocol were 

performed for all these pathogens according manufacturer's 

instructions, briefly: 10µl of MasterMix, 1µl of pathogen-specific 

primer/probe (FAM labeled, BHQ quenched), 5µl of RNAse/ 

DNAse free water and 5µl of DNA sample (> 5ng) or control 

template conformed the PCR reaction in a final volume of 20 µl. 

Amplification reactions were carried out on a MiniOpticon RT 

System CFB3120 thermal cycler (Bio Rad, software CFX 

manager 2.0) using a unique thermal profile for all qPCRs 

consisting of an initial enzyme activation step of 10 min a 95º C, 

followed by 50 cycles of: a denaturing step of 10 sec at 95º C, 

and an annealing/data collection step of 60 second at 60º C. 

Positive cut-off value was established in ≤ 45 cycles. Detection 

limits and quantification were performed by 10 fold dilutions (10 6 

a 10 0 ) of specific DNA plasmids for each pathogen. Negative 

samples were evaluated for DNA integrity by amplification of 

Beta-actina house-keeping gene. 

Results 

This multi-parametric qPCR assay was robust, specific and 

showed an analytical sensitivity for at less 10 2 copies of the target 

DNA. The Cq values for positive controls ranged from 25 to 35 

and the qPCR efficiencies were around 95% with R 2 

values 

above 0.990. A total of 122 cases were evaluated in this study 

and 49% (59/122) of them were positive by qPCR for at less one 

of these pathogens. Haemoparasites were detected in blood 

samples 52% (171/330), but also in tissue 22% (2/9) and serum 

38% (22/58) samples. Nearly 10% of cases had macroscopical 

and/or histopathological lesions compatible with haemolytic 

syndrome. Microscopical identification of Piroplasmids and 

Anaplasma were found in blood and/or tissue samples from 8 of 

these cases and all of them had one or more positive result for 

haemoparasites by qPCR. From the overall cases Th. annulata 

27% (33/122) and A. marginale 27% (33/122) were the more 

frequent detected pathogens, followed by B. bigemina 16% 

(20/122), B. bovis 2% (3/122) and A. phaghocytophylum 2% 

(2/122). No positive cases were recorded for Th. Parva (0/122) 

and A. centrale (0/122). Concurrent infections with Th. annulata, 

A. marginale and B. bigemina were detected in 15% (9/57) of the 

positive cases. 


Specific identification of haemoparasites infecting cattle is a 

crucial step for control of these diseases; because measures and 

treatment depend of the implicated agent. Although microscopical 

identification is possible it is not always reliable even for most 

expertice technicians. Clinical diagnosis is also difficult, by the 

similar symptomatology in these diseases. Our results clearly 

demonstrate the value of these qPCRs as a specifics and 

sensitive diagnostic tools for the rapid detection of main tickborne 

disease of cattle in blood and tissue sample from infected 

animals. The use of this qPCR multi-parametric assay allow a 

complete diagnosis of the main haemoparasitic diseases in a 

rapid way and reducing cost by pooling samples, up to five, per 

case. It also has the advantge for no competition of multiple 

primers by the target DNA, such ocurrs in a qPCR multiplex 

where a DNA depletion could happen mainly in animals with 

mixed infections. Our results showed a high percentage of 

haemoparasitic infections, mainly Th annulata, A. marginale and 

B. bigemina, in the evaluated cases from the Iberian peninsula 

and justified the use of specific and sensitive diagnostic test, as 

this qPCR, to study the real epidemiological situation of these 

disease in the cattle population from these countries. 

References 

1. Paul Heyman, Christel Cochez, Agnetha Hofhuis, Joke van der Giessen, 

Hein Sprong, Sarah Rebecca Porter, et al. A clear and present danger: 

Tick-borne diseases in Europe. Expert Rev Anti Infect Ther. 2010; 8(1):33- 

50. 

2. R. Cassini, F. Marcer, F. di Regalbono, G. Cancrini, S. Gabrielli, A. 

Moretti, et al. New insights into the epidemiology of bovine piroplasmoses 

in Italy. Vet Parasit 2012(184):77-82.2. R. Cassini, F. Marcer, F. di 

Regalbono, G. Cancrini, S. Gabrielli, A. Moretti, et al. New insights into the 

epidemiology of bovine piroplasmoses in Italy. Vet Parasit 2012(184):77- 

82. 

3. S. Almeria, J. Castellà, D. Ferrer, A. Ortuño, A. Estrada-Peña, J.F. 

Gutiérrez. Bovine piroplasms in Minorca (Balearic Islands, Spain): a 

comparison of PCR-based and light microscopy detection. Vet Parasitol 

2001(99):249–259. 

4. L. Ceci, N. Decaro, E. Lorusso, P. Paradies, G. Elia, V. Martella, et al. 

First report of bovine anaplasmosis by Anaplasma centrale in Europe, 

molecular identification and phylogenetic analysis. Vet Res commun 

2008(32):S236-S266. 

5. Oficina Internacional de Epizootias (OIE). Manual de las Pruebas de 

Diagnóstico y de las Vacunas para los Animales Terrestres. 2008.

S1 - P - 05 

DEVELOPMENT AND EVALUATION OF A NEW AND ORIGINAL EXTRACTION PROTOCOL TO 

DETECT MYCOBACTERIUM AVIUM SUBSP PARATUBERCULOSIS IN BOVINE FECES BY REAL TIME 

PCR 

Blanchard Béatrice. 1 , Versmisse Yann. 1 , Rouillard Tony. 2 

1 ADIAGENE, 38 rue de Paris 22000 Saint Brieuc, FRANCE 

2 

AES CHEMUNEX, Rue Maryse Bastié, 35172 Bruz, FRANCE 

Mycobacterium paratuberculosis (Map), real-time PCR, diagnosis, low shedder. 

Introduction 

Mycobacterium avium ssp paratuberculosis (MAP) is the 

etiological agent of Johne’s disease, a granulomatous enteritis 

that can affect cattle, sheep and goats and other non ruminant 

wildlife species. Numerous countries have set up programmes to 

control the disease. It requires the development of highthroughput 

sensitive diagnostic methods for the direct detection 

of infected animals. The performance of the PCR methods was 

often hampered by low sample input and inhibition. To increase 

the sensitivity of diagnostic test, we developed an original 

protocol to concentrate Map from 6g of faecal samples and 

compared the real time PCR results obtained with classical DNA 

extraction from 1g. 


A negative faeces collected from a paratuberculosis-free herd 

was spiked with serial dilutions of a MAP titrated suspension to 

4670, 2333, 1167, 233 and 117 bacteria per g. DNA extraction of 

each sample spiked was repeat four times using the new protocol 

(NP) developed by Adiagene. Briefly, 6g +/- 0.2 g of faeces were 

placed in a bottle with 40 ml of sterile distilled water, mixed 15 

seconds and allowed to settle 10 to 20 minutes. 10 ml of the 

obtained supernatant were transferred on an ADIAFILTER and 

centrifuged 5 minutes at 3 000 g. The obtained pellet was 

resupended then disrupted 10 minutes at 30 Hz with a mixer mill 

and 300 mg of glass beads and centrifuge 5 minutes at 15 000 g. 

Then DNA was extracted from 200 μl of the supernatant. 

5µL of each DNA solution was analyzed with the ADIAVET ® 

PARATB REALTIME PCR kit (Adiagene). 

The detection limit of the diagnostic method is the smallest 

amount of MAP per sample generating 100% positive results. 

Evaluation of the new protocol on field samples 

329 faecal samples were collected from different veterinary 

diagnostic laboratories. The common point among these samples 

is that they were sent to the laboratory for MAP analyses; they 

came from different area, from different herds with or without 

history of paratuberculosis. 

All the samples were analysed in parallel with two DNA extraction 

protocols. 

This first was performed using Adiapure ® 

(Adiagene) DNA 

extraction protocol (SP). The analyses were carried out from 1g 

of faecal sample as recommended by the manufacturer. 

The second DNA extraction was realised using the new protocol 

(NP). It was achieved with 6g +/- 0.2 g of faeces as previously 

described. 

5 µl of the each DNA solution obtained from the 2 different 

protocols was analysed with ADIAVET ® PARATB REALTIME kit. 

difficult to obtain a good sensitivity of the test due to the presence 

of inhibitor and the biology of the bacteria. 

Foremost, it was important to increase the quantity of treated 

faecal sample. Most of the works done for MAP detection like 

culture or PCR treat up to 1g of faeces. Even the new technique 

combining magnetic beads separation and PCR uses a minimum 

amount of samples. 

The originality of our protocol is to be able to treat up to 10g of 

faeces in order to concentrate the MAP present in the sample. In 

the standard protocol (SP) the DNA of 1g of faeces are treated. 

After homogenization and DNA purification, 0.15mg are used for 

PCR analyse; With the new protocol (NP), 6g are extracted and 

15mg of feces are used for PCR analyse. So, we are able to 

concentrate 100 times the sample and increase the sensitivity of 

the whole method. Prior study have suggested that high 

shedders yield > 50CFU/tube and low shedders

S1 - P - 06 

VALIDATION OF REAL-TIME PCR TEST FOR THE DETECTION AND QUANTIFICATION OF COXIELLA 

BURNETII FOR ABORTION CONTROL PROGRAM 

Leborgne Maëlle 1 , Blanchard Béatrice 1 , Gracieux Patrice 1 , Rouillard Tony 2 

1 

ADIAGENE, 38 rue de Paris 22000 Saint Brieuc, FRANCE 

2 

AES CHEMUNEX, Rue Maryse Bastié, 35172 Bruz, FRANCE 

Coxiella burnetii, Quantitative PCR, Diagnosis, Zoonosis 

Introduction 

Q fever, a zoonosis caused by the obligate intracellular bacterium 

Coxiella burnetii, is endemic throughout the world and infects 

arthropods, birds, pets, domestic and wild mammals, and 

humans. 

The recent developments in the EU, especially the increase in 

confirmed human cases of Q fever in the Netherlands, call for 

special consideration as regards the risks posed by Q fever for 

humans and animals. The European Commission requested 

further scientific advices and human risk assessment, as regards 

Q fever in animals (1). For EU it’s important to assess the 

significance of the occurrence of Q fever in the EU Member 

States for a better understanding of the scale and distribution of 

the disease and infection. In this context, French authorities 

organise a national program of Q fever surveillance. The control 

measures in infected herds consist first in preventing 

contamination of susceptible animals in reducing Coxiella 

shedding in infected ruminants. So, it is necessary to detect and 

quantify the amount of bacteria in infected samples in order to 

eliminate or separate the heaviest shedders from the herds. 

To reach this goal it is important to improve and standardize the 

diagnostic method in laboratories. We have developed a 

quantitative real time PCR test according to the guidelines for the 

development and validation of veterinary PCR (AFNOR, XP U47- 

600-2) (2). The new ADIAVET ® kit was validated by the French 

national reference laboratory for Q fever (ANSES, Sophia- 

Antipolis, France). 


One-well quantitative real time PCR was developed to detect 

simultaneously the C. burnetii IS1111 and a housekeeping gene 

GAPDH as an endogenous internal control of extraction and 

amplification. 

The specificity of the PCR test was tested against a panel of 

bacteria and virus commonly found in bovine, caprine or ovine 

samples. 

The detection limit of the PCR (LD PCR ) generating a positive result 

in 95% of cases and the quantification limit of the PCR (LQ PCR ) 

were calculated with a standard genomic DNA solution provided 

by the French national reference laboratory for Q fever (ANSES- 

Sophia Antipolis). 

The national control program recommended to collect vaginal 

swabs or placenta swabs after abortion. 

Four PCR methods, including DNA extraction from sample and 

PCR, were evaluated. The detection limit and the field of 

quantification of each method were calculated. 

Table 1: Determination of the detection limit and of the field of 

quantification for each method. The results are expressed in 

C.burnetii/ml. 

Vaginal 

swab 

Placenta 

swab 

QIAamp ® DNA Mini Kit 

Detection limit 

of method 

Field of 

quantification 

NucleoSpin ® Tissue 

Detection limit 

of method 

Field of 

quantification 

300 500 à 1.10 6 300 500 à 1.10 6 

300 500 à 1.10 6 300 500 à 1.10 6 


To assess the sanitary status of the herds, the animals are 

considered as clinically infected if the swab contains more than 

10 4 C. burnetii/ml. Thus, the performances of the real-time PCR 

developed match with the requirements for the management of 

C. burnetii infection. 

This quantitative real time PCR test was validated by the French 

national reference laboratory for Q fever. Currently, it’s used in 

France for a national program of Q fever surveillance. In addition, 

this kit may be also used for the epidemiologic diagnostic of C. 

Burnetii in faeces, milk, tissues and foetal liquid. 

References 

1. EFSA (European Food Safety Authority) (2010). Panel on Animal 

Health and Welfare (AHAW): More, S., Stegeman, al. Scientific Opinion on 

Q Fever. EFSA Journal, 8(5):1595. [114 pp.]. 

doi:10.2903/j.efsa.2010.1595. May. 

2. AFNOR XP U47-600-2 (juin 2011), Méthodes d’analyse en santé 

animale – PCR (réaction de polymérisation en chaîne) - Partie 2 : 

exigences et recommandations pour le développement et la validation de 

la PC 

Results 

This PCR test was assessed against a panel of 130 samples 

issues from organisms preferencially found in the same ecologic 

niche and/or close phylogeneticaly, among which Legionella. No 

cross reaction was observed. 

The analysis Probit showed that LD PCR was to 1.5 Genome 

Equivalent/reaction (5µL). LQ PCR was calculated to 2 Genome 

Equivalent/reaction (5µL). 

The results of the evaluation of different methods are showed in 

the following table.

S1 - P - 07 

ANALYTICAL EVALUATION OF A SWINE INFLUENZA VIRUS PCR ON AVIAN SAMPLES 

J. C. Pedersen 1 , C. Boss 2 , A. Burrell 3 , C. O’Connell 3 

1 

National Veterinary Services Laboratory (NVSL), Ames, Iowa. USA 

2 


3 


Introduction 

Swine influenza is a highly contagious viral infection of pigs that 

has a significant economic impact on affected herds. The disease 

is caused by Swine Influenza Virus (SIV) which is a group of 

specified viruses that are members of the family 

Orthomyxoviridae and placed in the genus influenzavirus A. 

Pigs play a unique role in the ecology of Influenza Type A, in that 

they have receptors in their respiratory tract that will bind swine, 

human, and avian influenza viruses. Consequently, pigs have 

been called the ‘mixing vessels’ for the development of new 

influenza viruses when swine, avian, and/or human influenza 

viruses undergo genetic reassortment in pigs. Subtypes of SIV 

that are most frequently identified in pigs include classical and 

avian H1N1, human (hu) H1N1 and H1N2, reassortant (r) H3N2, 

and rH1N2. 

In theory, a PCR based test which detects RNA coding for the 

matrix and nucleoprotein genes of SIV, an Influenzavirus A, 

should be able to detect similar viral RNA sequences in avian 

samples. The USDA licensed Swine Influenza Virus RNA Test Kit 

(VetMAX ® -Gold SIV Detection Kit) was used to test RNA 

extracted from a panel of clinical Avian Influenza Virus samples 

representing all 16 HA subtypes in order to assess the 

performance of the test in species other than swine. 


A panel of 34 cultured isolates (stock virus) representing all 16 

HA Influenzavirus A subtypes was assembled at the NVSL. RNA 

was extracted from the samples using the MagMAX ® Viral RNA 

Isolation Kit following manufactures’ instructions. Undiluted RNA 

extracted from the stock viruses was tested using the VetMAX ® - 

Gold SIV Detection Kit real time PCR from Life Technologies 

following the product insert instructions. The test detects RNA 

representing two different targets on the SIV matrix gene as well 

as an additional target on the nucleoprotein gene. 

Results 

Strong amplification signals were obtained (Ct range: 13.2-20.9) 

for each of the panel members tested. The quality standards 

including positive and negative extraction conotrols, positive kit 

control and no template controls performed as expected. 


This preliminary study gives an indication that further validation 

studies are warranted to support additional claims for use of this 

product for the detection of Influenzavirus A in avian samples. 

On a global level, the use diagnostic assays for the detection of 

certain reportable diseases is becoming more highly regulated in 

order to ensure that clinical decisions are being made from high 

quality laboratory results. In light of this, the advantages listed 

above must be understood along with the limitations of these 

reported data. 

Limitations: The VetMAX ® -Gold SIV Detection Kit is a USDA 

licensed product for the detection of Swine Influenza Virus (SIV) 

RNA isolated from nasal swab samples. Off-label uses of the 

product in the United States are not supported by the 

manufacturer and any additional claims for performance of the kit 

must be supported by rigorous studies which must be submitted 

and approved by the USDA before any additional claims can be 

made in the US. Usage claims in other countries outside the US 

are subject to the regulatory requirement(s) within each country 

of use. 

References 

OIE Terrestrial Manual 2010, Chapter 2.8.8. Swine Influenza, accessed 30 

Mar 2012 

SIV, PCR, Avian Samples

S1 - P - 08 

QUALITY CONTROL OF PRRSV VACCINATION BY PARAMETERS OF IMMUNITY 

Böttcher J 1 , Janowetz B 1 , Alex M 1 , Seidenspinner M 2 , Schuh H 2 , Strutzberg-Minder K 3 , Wendt M 4 , Leibold W 5 

1 

Bavarian Animal Health Service, Poing, Germany, 

2 

Dr. Hermann Schuh, Veterinary Practice, Ipsheim, Germany 

3 

IVD GmbH, Hannover, Germany, 

University of Veterinary Medicine, Foundation, 4 Klinik für kleine Klauentiere, 5 Institute of Immunology, Hannover, Germany 

PRRS, vaccination, immunity 

Introduction 

Control of porcine reproductive and respiratory syndrome (PRRS) 

mainly relies on vaccination. Quality control of vaccination should 

focus on diagnostic parameters which are associated with 

immunity, i.e. neutralizing antibodies (nab) and Interferon-gamma 

(IFN-γ) response. The level of maternally derived nabs (MDNab) 

has to be included as an additional variable. 


Six sows were tested for nabs 2 weeks (w) post partum. Piglets 

were classified by MDNab in “0.05, i.e. the lowest 

value of the IFN-γ-standard titration, or OD SC = 4 

< 4 

1 2 3 4 

vaccination group 

NT_US 

0 

Fig.2: Genotype-specificity of NT (n=58) and IFN-γ (n=29) in w27. 

Interferon-gamma (ng/ml) 

1000 

100 

10 

IFN_EU 

Vaccination group 2 

IFN_US 

Fig.3: PRRSV-EU-IFN-γresponse. 

Data for 12, 4, 1 

and 8 piglets are shown for 

groups 1, 2, 3 and 4, 

respectively. Groups 2 and 

3 showed marked IFN-γresponse 

against Marc145 

(NC). The effect of MDNab 

in group 1 was significant 

(p=0.036, Mann-Whitneytest, 

two-tailed probability). 


This study was financially supported by the Free State of Bavaria and the 

Bavarian Joint Founding Scheme for the Control and Eradication of contagious 

Livestock Diseases. This study was performed as a part of the thesis of Mr. Marc 

Seidenspinner. 

References 

1. Böttcher, J., M. Ritzmann, A. Gangl (2006): Felduntersuchungen mit einem 

„Porcine Reproductive and Respiratory Syndrome Virus“ (PRRSV) 

Neutralisationstest. Tierärztl. Umschau, 61, 550 – 559. 

Neutralization titer (PRRSV EU) 

Neutralization titer (PRRSV EU) 

12 

10 

8 

6 

4 

2 

0 

12 

10 

8 

6 

4 

2 

0 

Nab-titer of sows 

>= 4 

= 4 

< 4 

2 7 10 13 16 21 25 26 27 

weeks of life

S1 - P - 09 

PTS FOR BVDV ANTIGEN DETECTION BY GD - ANIMAL HEALTH SERVICE 

Rianne Buter 1 , Jet Mars 1 , Wim Swart 1 , Huub van de Sande 1 , Kees van Maanen 1 

1 

GD-Animal Health Service, Diagnostics,Research and Epidemiology,Deventer , the Netherlands 

Proficiency tests, nucleic acid amplification techniques, ISO/IEC 17043:2010, BVDV, PCR 

Introduction 

Quality control should be an integral element in the 

implementation of nucleic acid amplification techniques (NATs) 

e.g. PCR in diagnostic laboratories. Dealing with test 

characteristics like specificity and sensitivity, variations in 

qualitative and quantitative results, contamination and inhibition 

control mechanisms remains an on-going challenge. External 

quality controls are a useful tool to improve a laboratories quality 

system. 

At this moment ,different proficiency tests for nucleic amplification 

techniques (NAT’s) e.g.PCR are available at GD, such as for 

Bovine Viral Diarrhoea Virus (BVDV), mastitis pathogens, 

Porcine Circovirus type 2 (PCV-2), Porcine Reproductive and 

Respiratory Syndrome Virus (PRRSV) and Brachyspira 

hyodysenteriae. 

In general, the purpose of proficiency testing is to determine the 

performances of individual laboratories for specific tests. All PTS 

organized by GD are performed according to ISO/IEC 

17043:2010 guidelines. This abstract presents the (anonimized) 

results of the BVDV antigen and genome detection PTS that was 

organized in 2011. 


The BVDV antigen detection PTS consisted of 11 inactivated, 

freeze-dried sera and 1 inactivated and freeze dried tissue 

sample, which were sent to each of the participants with the 

request to test the samples for BVDV antigen/genome using all 

PCR and antigen ELISA techniques in operation at the time. The 

samples were identified by sample numbers (#1 - #12) only. 

Before shipment homogeneity and stability of the samples were 

checked. The samples were sent by TNT courier to participants 

all over the world but mainly in Europe and the deadline for 

testing and reporting was 6 weeks. The participants were asked 

to analyse the samples in duplicate in two different test runs. 

Results 

Statistical calculations were performed when there were at least 

6 laboratories participating within one test system. The boxplot 

represents the measurements for each sample of all laboratories 

participating in a commercial BVD ag ELISA (figure 1) and the 

XY-diagram represents the average z-scores of all laboratories 

using the commercial ELISA. (figure 2) 

Figure 1 BVDV ELISA boxplot 

For the BVDV real time PCR only the within lab reproducibility 

could be calculated (table 1), since there were too many 

differences in RNA isolation methods, PCR mixtures, PCR 

cyclers e.g. 

Table 1: BVDV Real-time PCR within-lab-reproducibility 

Lab 

Mean value 

Within Lab 

reproducibility sd 

A 28.20 0.10 

B 36.15 2.39 

C 28.85 1.54 

D 30.66 1.53 

E 30.96 0.19 

F 29.78 0.24 

G* 37.25 . 

H 30.54 0.39 

I 32.18 0.24 

J 28.53 0.37 

K 29.29 0.95 

M 30.27 2.29 

N* 29.03 . 

O 29.84 0.41 

P 30.11 1.48 

Q 28.36 0.44 

R 29.23 0.87 

S 30.94 0.60 

Labs marked with * reported only single results. 


In this PTS specificity did not seem to be a problem for both 

ELISA and PCR, since almost all labs scored a BVDV negative 

sample and a serum sample spiked with a heterologous RNA 

virus correctly negative. 

Detection limits of the ELISAs used in this PTS, as measured by 

TCID 50 /mL, were relatively high with all spiked samples up to a 

titre of 10 4,05 TCID 50 /mL being scored negative by all participants. 

Also (maternal) antibodies can interfere with virus detection by 

ELISA, as demonstrated by the results for samples #6 and #10. 

Since such interference does not occur with PCR, critical 

evaluation of discrepant test results is always needed when PCR 

and ELISA are both used in the context of BVDV diagnosis and 

compulsory or voluntary control and eradication programmes. 

Most of the laboratories that participated in this PTS used realtime 

PCR for BVDV detection. The detection limit of real-time and 

conventional PCRs as compared with the antigen detection 

ELISAs used in this PTS is much lower as demonstrated by the 

fact that allmost all serum samples spiked with BVDV-1 virus with 

a titre of 10 1,9 TCID50/mL or higher scored positive. The overall 

results of the laboratories that participated in this first GD BVDV 

antigen and genome PTS were reasonably good. Most labs have 

reliable assays in place for detection of BVDV PI-animals. 


We thank our product-sales manager Annemiek Slothouber for 

her support and enthusiasm. 

We thank all participants for their enthusiasm, their quick 

response and their valuable comments. 

Figure 2 BVDV ELISA scatterplot

S1 - P - 10 

PRESENTATION OF THE RESULTS OF THE ANNUAL PROFICIENCY TEST FOR THE SEROLOGICAL 

DIAGNOSIS OF EQUINE INFECTIOUS ANEMIA CONDUCTED IN ITALY BETWEEN 2006-2010. 

Ricci I 1 , Gasperetti L. 1 , D'Alonzo A. 1 , Nardini R. 1 , Giusti C. 1 , Caprioli A. 1 , Forletta R. 1 . 

1 

National reference centre for equine infectious Anemia, Istituto Zooprofilattico Sperimentale Lazio e Toscana 

Keywords: Equine infectious anemia, agar gel immunodiffusion, proficiency test 

Introduction 

Equine infection anemia (EIA) is a viral infection of equidae, 

caused by a virus (EIAV) of the Lentivirus genus, Retroviridae 

family. Since 2006, Italy has adopted a national surveillance plan 

based on the serological screening of all animals older than 6 

months except for meat horses. According to the Italian 

Regulations, the official confirmatory test is the agar gel 

immunodiffusion (AGID), which detects antibodies against the 

p26, a highly conserved and primary immunogenic structural 

protein of the virus (1). The serological analysis of the samples, 

collected for the surveillance plan, can only be performed by the 

network of the national official laboratories, belonging to the 

Isitituti Zooprofilattici Sperimentali, mostly operating according to 

the ISO IEC 17025 and annually participating to proficiency tests. 

In this context, since 2002, the national reference centre for 

equine infectious anemia (CRAIE) has conducted inter-laboratory 

agid proficiency tests (2) with an increase in the number of 

participating laboratories. 

The aim of this paper is to present and discuss the results of the 

proficiency tests conducted in Italy during the years 2006-2010 

for the diagnosis of EIA using the AGID test. 


Over the period 2006-2010, 65 laboratories participated to the 

annual proficiency test, performing both methods prescribed for 

AGID for AIE: “the Coggins test” which uses two layers of agar 

(1.5% and 0.7% respectively and wells of 7mm in diameter and 3 

mm apart) and the OIE prescribed by the OIE (3). The panel 

distributed to each laboratory was each year made up of 10 sera, 

3 negatives and 7 positives, with different level of antibodies 

against the P26 antigen (high to weak positives). Only in 2008, 

the panel was constituted of 2 negative and 8 positive sera. 

Before dispatching the samples to each laboratory, they were 

subjected to homogeneity and stability tests for their reactivity (2), 

so as to correctly evaluate reproducibility and repeatability 

parameters. Statistical analysis was carried out using K Cohen 

statistic, which was calculated for each laboratory and for all 

laboratories gathered together (4). 

Results 

Of the 65 participating laboratories, 27 constantly had a K value 

equal to 1. The results of the different laboratories per year are 

reported in table 1. 

Table 1: K value results per year. 

Results of the 65 laboratories per year 

2006 2007 2008 2009 2010 

N. of laboratories 

0.21-0.40 8 1 2 1 0 

0.42-0.60 5 0 0 0 1 

0.61-0.80 7 2 0 2 1 

0.81-1 45 62 63 62 63 

K Value 

On the other hand when considering the samples not correctly 

identified (K < 1) in both methods for all laboratories, those 

mostly misclassified were weak positive, 7.6%, against the 1.4% 

of the strong positive sera. 

When taking into account the concordance of samples for the two 

different methods, the percentages of error for different AGID 

methods were respectively 5.2 for the Coggins test and 3.8 for 

the OIE method. 

The K analysis conducted to evaluate the concordance among 

the different laboratories for each year is reported in table 2. The 

results using the two different AGID methods are not significantly 

different. 

Table 2: K value results for all raters per year using both AGID 

methods. 

K value results among the raters per year 

YEAR 2006 2007 2008 2009 2010 

AGID Coggins 0.78 0.94 1 0.94 0.95 

AGID OIE 0.77 0.98 1 0.97 0.98 


The constant increase of participating laboratories to the 

proficiency tests during the years is an important element for 

evaluating the efficiency of the diagnostic system at national level 

for detecting EIA infection. These results stress the importance of 

performing such trials to achieve efficient diagnostic standards for 

each official laboratory as well as for the national diagnostic 

network. 

Furthermore, the increase in the number of laboratories during 

the years, having a K value > 0.8, demonstrates an improvement 

of the diagnostic performances, as also the achievement of good 

quality standards, with very satisfactory reproducibility and 

repeatability parameters for both AGID tests, even if better for the 

AGID OIE. It is important to underline that some difficulties 

occurred in the interpretation of positive samples with lower 

levels of antibody against the p26 antigen (weak positive sera). 

This represents a problem especially because this error during 

routine performance is expected to be higher in consideration of 

an expected higher attention in reading a proficiency test. This 

may constitute a potential bias of the results obtained, generating 

the calculation of a better proficiency than the effective. 

In this respect it our opinion that a more sensible technique such 

as the ELISA should be introduced to improve the efficacy and 

efficiency of the surveillance system. The introduction of such a 

screening test, with its characteristic of high sensitivity, rapidity 

and objectiveness is surely a more useful instrument for the 

laboratories performing EIA diagnosis, especially within an official 

surveillance and control programme. Another important issue 

emerging from our results is that the laboratories having lower 

level of concordance were those testing fewer samples in their 

diagnostic routine. This further remarks the importance of the 

experience in the execution and interpretation of a test such as 

the AGID. 

References 

1. Ordinanza 8 agosto 2010 Piano di sorveglianza nazionale per l'anemia 

infettiva degli equidi. G.U. Serie Generale n. 219 del 18 settembre 2010 

2. Guidelines OIE 1998. Guidelines of the Office International des 

Epizooties for laboratory quality evaluation, for international reference 

standards for antibody assays and for laboratory proficiency testing. Rev. 

Sci. Tech. Off. Int. Epiz., 17 (2), 600-609 

3. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2010, 

Chapter 2 . 5 . 6 .Equine infectious anaemia 

4. Langton S.D., Chevennement R., Nagelkerke N., Lombard B., 2002. 

Analysing collaborative trials for qualitative microbiological methods: 

accordance and concordance. International Journal of Food Microbiology 

79: pp. 175-181 

5. Charles J. Issel, R. Frank Cook (1993)REVIEW ARTICLE A review of 

techniques for the serologic diagnosis of equine infectious anemia J Vet 

Diagn Invest 5: 137-141

S1 - P - 11 

ANTIMICROBIAL SUSCEPTIBILITY OF AVIBACTERIUM (HAEMOPHILUS) PARAGALLINARUM 

ISOLATES RECOVERED IN THE RUSSIAN TERRITORY 

A.V. Chernyshov 1 , O.I. Ruchnova 1 , O.P. B’yadovskaya 1 

1 FGBI ARRIAH, Vladimir, Russian Federation 

Avibacterium paragallinarum, Haemophilus paragallinarum, Infectious Coryza, antimicrobial susceptibility 

Introduction 

One of the most widely spread respiratory infectious diseases 

is infectious coryza. The disease is caused by the Gramnegative 

bacterium of Avibacterium paragallinarum species 

previously known as Haemophilus paragallinarum (1, 2, 5). 

The disease belongs to highly contagious diseases. It is 

manifested by catarrhal inflammation of nasal mucosa and air 

cells, as well as by the swollen-head-like syndrome and in rare 

cases by pneumonia. Chicks and chickens are susceptible to 

infectious coryza. Economic losses are caused by the stunting 

of chicks (up to (10-40%), drop in egg production (up to 41%) 

and mortality (up to 10%). (1, 5) 

Avian infectious coryza is reported from many countries of the 

world (2, 5). 

Currently, different antibacterial preparations are used in 

industrial poultry production but the violation of requirements to 

their use can lead to the emergence of resistant bacterial 

strains. Therefore, study of susceptibility of microorganisms to 

antibiotics has a vital significance for the bacterial infection 

therapy. 

The present work was focused on the study of antimicrobial 

susceptibility pattern A. paragallinarum field isolates from 

poultry from the RF farms. 


Pieces of lungs, tracheal and infraorbital sinus washings 

received from poultry of different age between 2009-2011 were 

tested for the presence of infectious coryza agent. Thirty-five 

A. paragallinarum field isolates were used in the given study. 

A. paragallinarum was identified based on the results of 

studying morphological, cultural, biochemical and genetic 

properties of the isolates (1, 2, 5). 

Susceptibility to antimicrobial preparations was determined 

using Mueller-Hinton agar supplemented with 5% of sheep red 

blood cells by Kirby-Bauer disk diffusion test (4) using a kit of 

disks impregnated with different antibiotics (Research Center 

of Pharmacotherapy, St. Petersburg). The degree of 

resistance of the isolates was determined according to the 

instruction to the kit. 

Results 

During 2009-2011 224 samples of pathological materials from 

broiler chicks and chickens were tested for avian infectious 

coryza. Total 35 isolates of A. paragallinarum were isolated 

from chickens and broiler chicks including 57.1% of isolates 

from infraorbital sinus swabs, 34.3% - from lung and 8.6% - 

from tracheal swabs. 

The recovered bacteria possessed tinctorial, morphologic, 

cultural and biochemical properties typical of A. paragallinarum 

and consistent with Bergey’s manual of systematic 

bacteriology and works by P.J. Blackall [et al.] (1, 2). 

Results of the study of A. paragallinarum susceptibility to 

antibacterial preparations are presented in Table 1. 

It was established that most isolates of A. paragallinarum were 

characterized by a wide range of resistance to antibacterial 

preparations. In particular, to lincosamide group (clindamycin) 

(94.5%), tetracycline (tetracycline (82.9%), doxycycline 

(87.9%)), co-trimoxazole (trimethoprim) (82.9%), natural 

penicillins (benzylpenicillin) (82.9%), third generation 

cephalosporins (cefixime) (71.4 %), second generation 

fluoroquinolones (ofloxacin (60 %), ciprofloxacin (intermediate 

susceptibility 42.9 % of isolates), norfloxacin (51.4 %)) and 

macrolide group (erythromycin) (62.9 %). 

A. paragallinarum isolates had high susceptibility mainly to 

inhibitor-protected penicillins (amoxicillin 

clavulanate/amoxiclav) – 77.1% of strains, first and second 

generation aminoglycosides (gentamicin, amikacin 

respectively) and nitrofuran group (furadonin) – more than 60 

%. 

Table 2. Antimicrobial susceptibility of A, paragallinarum 

isolates. Number of isolates in % of total number. R – 

resistant, S – susceptible, I – intermediate susceptibility. 

No. Antibiotic name S I R 

1 Ofloxacin 34,3 5,7 60,0 

2 Ciprofloxacin 31,4 42,9 25,7 

3 Norfloxacin 34,3 14,3 51,4 

4 Levofloxacin 34,3 22,9 42,9 

5 Tetracycline 0,0 17,1 82,9 

6 Doxycycline 12,1 0,0 87,9 

7 Clindamycin 0,0 5,7 94,3 

8 Trimethoprim 17,1 0,0 82,9 

9 Benzylpenicillin 17,1 0,0 82,9 

10 Amoxiclav 77,1 5,7 17,1 

11 Ampicillin 28,6 25,7 45,7 

12 Laevomycetin 62,9 17,1 20,0 

13 Erythromycin 5,7 31,4 62,9 

14 Gentamicin 60,0 22,9 17,1 

15 Amikacin 65,7 22,9 11,4 

16 Furadonin 57,1 25,7 17,1 

17 Cefuroxime 48,6 17,1 34,3 

18 Cefoperazone 48,6 22,9 28,6 

19 Сefixime 11,4 17,1 71,4 


A high rate of resistance to erythromycin (62,9%) in our 

research according with Yuan-Man Hsu [et. al.] (1) results. He 

also marked the resistance of the majority of tested field strains 

to this preparation. Data on tetracycline resistance are similar to 

P. J. Blackall (4) investigations. 

Our investigation, on the contrary, demonstrated high 

percentage of isolates resistant to aminopenicillin, erythromycin 

and benzylpenicillin in contrast to the results presented by E.S. 

Vargas and H.R. Terzolo (10). 

In conclusion, A. paragallinarum isolates have wide resistance 

range to large spectrum of antimicrobials, in particular 

tetracyclines (82.9 %), lincosamides (94.3 %), co-trimoxazole 

(82.9 %), natural penicillines (82.9 %). The majority of bacteria 

were susceptible to aminoglycosides (57.1 %), laevomycetin 

(62.9 %), inhibitor-protected penicillins (77.1 %) and nitrofurans 

(57.1 %). 

These data on antibiotic resistance should be helpful in 

planning strategies for the control of A. paragallinarum infection 

in poultry. 

References 

1. Blackall, P.J. (1989) The Avian Haemophili. Clin. Microbiology 

Reviews. Vol.2(3). P. 270-277. 

2. Blackall, P.J. (1999) Infectious Coryza: Overview of the disease and 

new diagnostic options. Clin. Microbiology Reviews. Vol. 12, №4. P. 627- 

632. 

3. Hsu, Y.-M., Shieh, H.K., Chen, W.-H. [et al.] (2007) Antimicrobial 

susceptibility, plasmid profiles and haemocin activities of Avibacterium 

paragallinarum strains. Vet. Microbiology. Vol.124. P. 209–218. 

4. Jorgensen, J.H., Turnidge, J.D. (2007) Susceptibility test methods: 

dilution and disk diffusion methods. In P. R. Murray, E. J. Baron, J. H. 

Jorgensen, M. L. Landry, and M. A. Pfaller (ed.), Manual of clinical 

microbiology, 9th ed. ASM Press, Washington, D.C. P. 1152–1172. 

5. Vargas, E.S., Terzolo, H.R. (2004) Haemophilus paragallinarum: 

Etiology of infectious coryza. Vet. Mex. Vol. 35, №3. P. 245–259.

S1 - P - 12 

COMPARISON BETWEEN ANALYTICAL SENSITIVITY OF RABIES VIRUS ISOLATION IN 

NEUROBLASTOMA CELL CULTURE WITH ANALYTICAL SENSITIVITY OF MOUSE INOCULATION 

TEST 

E.V. Chernyshova 1 , N.A. Nazarov 1 , A.E. Metlin 1 

1 FGBI "Federal Centre for Animal Health"(FGBI "ARRIAH"), Vladimir, Russian FederationRabies, 

diagnosis, virus isolation in neuroblastoma cell culture, mouse inoculation test, analytical sensitivity 

Introduction 

Rabies is an acute viral zoonotic infection characterized by 

lesions of the central nervous system impairment which almost 

always results in death. 

The main scheme of rabies laboratory diagnosis in animals 

adopted in the Russian Federation (RF) (State Standard 26075- 

84) consists of pathological material testing (brain tissue) using 

fluorescent antibody test (FAT). When FAT is negative mouse 

inoculation test (MIT) should be carried out (4). 

In laboratories with appropriate conditions and staff competence 

the OIE recommends using virus isolation in neuroblastoma cell 

culture instead of MIT (2). The reasons for such a 

recommendation are the following: 

Both methods are of high diagnostic sensitivity and specificity; 

Minimum and maximum time for diagnosis using virus isolation in 

neuroblastoma cell culture is from 4 to 12 days respectively and 

MIT performance takes from 12 to 30 days in average; 

It is necessary to confirm the results of MIT using FAT in addition; 

Virus isolation in neuroblastoma cell culture is more humane and 

less cost-consuming. 

In the EU countries when FAT is negative the virus isolation in 

cell culture is performed only in case of animal-human contact 

(France, Germany). In some countries (Lithuania) in case of 

absence of such contacts pathological materials are just 

passaged once in cell culture. 

Currently, many regional veterinary laboratories of the RF are 

capable of running diagnostic tests using cultural methods. 

Introduction the “Methodical instructions on rabies virus isolation 

in mouse neuroblastoma cell culture” into veterinary laboratory 

practice in the RF is of great importance. 

The study is aimed at the comparison of analytical sensitivity 

between methods of rabies virus isolation in cell culture and 

rabies virus isolation in white mice (1, 3). 

neuroblastoma cell culture. As for our experiments, the difference 

was 1 lg10. 

Thus, our results provide one more basis to replace a 

MIT the method of rabies virus isolation in mouse neuroblastoma 

cell culture in laboratories and research institutions dealing with 

rabies diagnosis. 

References 

1. Anthony R. Fooks. New Diagnostic Tools for Rabies in Animals (2009) 

http://www.oie.int/eng/A_RABIES/presentations_rage/S21%20NewDiagno 

sticTools_ProfFooks.pdf 40 p. 

2. OIE. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 

Vol. 1. – 6nd ed. - Paris, (2008). - Chap. 2.1.13. - p. 304-323. 

3. Rabies General Aspects &Laboratory DiagnosticTechniques (2007) 

http://www.whoindia.org/LinkFiles/Communicable_Diseases_Rabies_- 

General_Aspects_&_Laboratory_Diagnostic_Techniques.pdf 72 p. 

4. State Standard 26075-84 Methods of laboratory diagnosis for rabies 

(1984), 9 p. 


The following materials were used for the study: mouse 

neuroblastoma N-2a cell line; white outbred mice (6-8 g); mouse 

brain-adapted rabies virus (CVS strain; 5.66 lg TCID 50 /ml); antirabies 

fluorescent immunoglobulin produced by the FGBI 

“ARRIAH”. 

Analytical sensitivities of the methods were determined by 

titration of rabies virus CVS strain in mouse neuroblastoma N-2a 

cell line and in white mice. 

The titre was expressed as the highest virus dilution at 

which specific fluorescence in cell culture or deaths of mice were 

observed. 

Specific deaths of mice were confirmed by FAT. 

Results 

A 10% suspension of original mouse-brain-adapted CVS strain of 

rabies virus was prepared in RPMI medium for inoculation. 

Rabies virus was preliminary titrated using a series of tenfold 

dilutions of the prepared suspension. The highest dilution of the 

virus suspension with the specific fluorescence corresponded to 

1.66 lg TCID50/ml titer. 

In order to determine sensitivity more accurately five twofold 

dilutions of the virus suspension with 1.66 lg TCID50/ml titer were 

prepared. Test results demonstrated that the last dilution with the 

specific fluorescence corresponded to 1.358 TCID50/ml titer. 

Seven twofold dilutions of the virus suspension with 2.66 

TCID50/ml titer were prepared to determine sensitivity of the MIT. 

Test results demonstrated that the last dilution accompanied by 

specific deaths in mice corresponded to 2.358 TCID50/ml titer. 


A conclusion was made after comparison of the obtained results 

that the MIT had a lower analytical sensitivity in comparison to 

analytical sensitivity of rabies virus isolation in mouse

S1 - P - 13 

IDENTIFICATION OF GLOBICATELLA SANGUINIS ISOLATED FROM PNEUMONIA IN A GOAT 

M. Cocchi , S. Deotto, T. Di Giusto, M. Bregoli 

Istituto Zooprofilattico Sperimentale delle Venezie, Sezione territoriale di Udine, Basaldella di Campoformido (UD), Italy 

Globicatella sanguinis, pneumonia, goat 

Introduction 

Globicatella (G.) sanguinis was described in 1992. This 

bacterium is a catalase-negative, facultative anaerobic, gram 

positive coccus. It has been associated with various diseases in 

humans, such as bacteraemia, meningitis and infections of the 

urinary tract. In veterinary medicine, G. sanguinis was described 

in meningoencephalitis in lambs (1, 3). The aim of this study was 

to describe an episode of pneumonia in a goat produced by G. 

sanguinis. 


Two 1-year-old Tibetan goats out of a total of four goats 

belonging to a farm located in Friuli Venezia Giulia region (North 

east of Italy) showed unspecific clinical signs such as anorexia, 

depression, and permanent decubitus. These goats died after 

four days by the onset of the symptoms, and one of them was 

submitted for a necropsy. Bacteriological examinations were 

performed from lungs, brain and liver as follows: swabs were 

streaked onto blood agar base (BAB) and eosin-methylene blue 

agar (EMB). Brain Heart Infusion broth (BHI) was inoculated, too. 

Plates and tubes were incubated at 371°C for 48 hrs, in 

aerobiosis. BAB was incubated in anaerobic condition, too. From 

intestine a Perfringens agar base (PAB) other than EMB plate 

was used, incubated in anaerobiosis. It was obtained by using 

ANAEROGEN System, with an anaerobic indicator (Oxoid). 

Depending on morphology of the colonies, Gram staining, 

catalase and oxidase tests were performed. Miniaturized 

commercial tests were also used for identification. Agar plates 

and tubes were supplied by the service for media of the Istituto 

Zooprofilattico Sperimentale delle Venezie. Antimicrobial test was 

performed with disk diffusion method, in accordance with Clinical 

and Laboratory Standards Institute (CLSI) guidelines (2). Tested 

antimicrobials were: penicillin (10 g, Becton Dickinson), 

ampicillin (10 g, Becton Dickinson), oxacillin (10 g, Becton 

Dickinson), tetracycline (30 g, Oxoid), sulfamethoxazoletrimethropim 

(23,75-1,25 g –Oxoid-) and cephalothin (30 g – 

Oxoid-). Reading was done using the established diameter for 

Streptococcus (S.) spp other than S. pneumoniae. 

Results 

Necropsy. Macroscopic lesions recovered from lungs referred to 

acute bronchopneumonia and tracheal and laryngeal oedema. 

Catarrhal enteritis and hepatomegaly were observed, too. 

Bacteriological tests. Cultures from lungs and liver revealed pure 

culture of -haemolytic, pin-pointed (2-3 mm of diameter), circular 

colonies. Subsequently, analyses revealed a gram positive, 

catalase-negative, coccus-shaped organism. 

Figure 1 shows the morphology of the bacterium after 24 hrs of 

incubation, on BAB. Biochemical identification was performed 

with a commercial kit, API rapid ID 32 Strep (Biomerieux). Both 

the gram positive strains showed identical biochemical profiles 

(22274063110), with a percentage of identification of 99.9%, 

T=0.92. Only fermentation of ribose resulted as an atypical test 

(negative in our reaction). Cultures from the intestine showed E. 

coli and Clostridium perfringens. 

Antimicrobial test. G. sanguinis revealed resistance to oxacillin, 

and to tetracycline, while it was sensitive to other tested drugs. 


Pneumonia is one of the most common respiratory problems in 

small ruminants throughout the world. In goat herds, pneumonia 

increases production costs associated with expensive treatments. 

Infectious and non-infectious agents can cause inflammation of 

the lungs. In goats the most frequent causes of respiratory 

infection and death are Pasteurella multocida and Mannheimia 

haemolytica. They are commonly found in the upper respiratory 

tract of healthy goats, too. Transportation stress, viral infections, 

parasites, poor management conditions, increase goats' 

susceptibility to these agents of pneumonia. In our case, the 

identification of G. sanguinis in pure culture from diseased lungs 

could be indicative of the pathological role of this bacterium. 

The performed analysis on other organs revealed the presence of 

G. sanguinis in liver, too. Differences are in reference to the 

growth in different atmospheres: culture from lungs showed G. 

sanguinis both in aerobic and in anaerobic conditions. Cultures 

from liver revealed the presence of the bacterium only in aerobic 

incubation. The fact that all the goats in the farm showed identical 

clinical signs is strongly indicative that this bacterium was also 

responsible for the pathology in the other goats. 

In veterinary medicine, G. sanguinis was isolated in a case of 

suppurative meningoencephalitis in lambs (1). In our case 

macroscopic analysis revealed an acute bronchopneumonia. Any 

lesion was recovered from the brain. 

Regarding identification, in this paper we named the bacterium in 

accordance with the profile given by a miniaturized system. The 

phenotypic findings are consistent with those described for this 

bacterium species, except for pyrrolidonil arylamidase test. It is 

pertinent to note that G. sanguinis belongs to a broad range of 

Gram-positive, catalase-negative new taxa recently founded in 

human and animal clinical samples. Some authors revealed that 

misidentification can occur between Aerococcus viridans and G. 

sanguinis, because of very similar biochemical profiles (1,5). 

Moreover, looking into literature only a few number of isolates 

were tested, with some differences in the results (1,3). For these 

reasons, subsequent investigations using different methods, such 

as molecular genetic tools should accompany the identification of 

this bacterium. 

The improvement of the identification systems of these unusual 

gram-positive, catalase-negative bacteria may contribute to 

making progress in current epidemiological knowledge on host 

distribution and range of clinical conditions. 

Data about antimicrobial susceptibility test showed resistance 

varied significantly among the tested strains (5). In our report 

resistance to tetracycline does not agree with the data of 

Seegmuller et al (4). 

To the author’s knowledge this is the first report about G. 

sanguinis in an episode of pneumonia in a goat. 

References 

1. Vela, A.I., Fernandez E., Las Heras A., Lawson, P.A., Dominguez L., 

Collins M.D., and Fernandez-Garayzabal J. (2000). Meningoencephalitis 

associated with Globicatella sanguinis infection in lambs. J Clin Microbiol 

vol 38, 4254-4255. 

2. Clinical and laboratory standards institute (CLSI). M31-A3. Performance 

standards for antimicrobial disk and dilution susceptibility tests for bacteria 

isolated from animals. Vol 28. n 8. 

3. Collins, M.D., Aguirre, M., Faclam, R.R., Shallcross J., and Williams 

A.M. (1992) Globicatella sanguis gen.nov., sp. Nov., a gram-positive 

catalase-negative bacterium fropm human sources. J Appl. Bacteriol. 

73:433-437. 

4. Seegmuller I., Van der Linden M., Heeg C., and Reinert R.R. (2007). 

Globicatella sanguinis is ana etiological agent of ventriculoperitoneal 

shunt-associated meningitis. J Clinical Microbiol. Febb. 666-667. 

5. Shewmaker, P.L., Steigerwalt, A.G., Shealey, L., Weyant, R., and 

Facklam, R. (2001). DNA relatedness, phenotypic characteristics, and 

antimicrobial susceptibilities of Globicatella sanguinis strains. J Clinic 

Microbiol. Nov 4052-4057. 

Figure 1: G. sanguinis after 24h incubation on blood agar base, in 

aerobic atmosphere

S1 - P - 14 

KAIZEN PROCESS APPLIED AT THE DIAGNOSTIC SERVICE OF FACULTÉ DE MÉDICINE 

VÉTÉRINAIRE, UNIVERSITÉ DE MONTREAL 

Estela Cornaglia, Véronique Boyer, Jean-François Brodeur, Guy Fontaine 

Université de Montréal, Faculté de médicine vétérinaire, Diagnostic Service, Saint-Hyacinthe, Canada 

Continuous improvement, Kaizen 

Introduction 

The diagnostic service (SD) at the Faculté de medicine 

vétérinaire de l’Université de Montréal, include 20 laboratories or 

services unities. First line laboratories as bacteriology, virology, 

parasitology, clinical pathology, pathology, molecular diagnosis, 

serology; and specialized unites as ichtyopathology, food safety, 

genomics, wildlife, OIE reference EcL laboratory, Actinobacillus 

pleuropneumoniae serology and serotyping. 

Our staff is around 90 people, 26 veterinarians and 14 

professionals, among them 19 PhD and 13 graduated ACVP. 

Continuous improvement and accreditations are a priority for us 

to assure the quality of our service. We’ve chosen Kaizen as a 

principal tool to realize them. 

Kaizen means "continuous improvement" or "change for the 

better". It refers to philosophy or practices that focus upon 

continuous improvement of processes in manufacturing, 

engineering, supporting business processes, and management. 

It comes from Japan. 

Improvement can be broken down between innovation and 

Kaizen. Innovation involves a drastic improvement in the 

existing process and requires large investments. Kaizen 

signifies small improvements as a result of coordinated 

continuous efforts by all employees. Management works 

continuously towards revising the current standards, once they 

have been mastered, and establishing higher ones. Kaizen 

methodology includes making changes and monitoring results, 

then adjusting. Large-scale pre-planning and extensive project 

scheduling are replaced by smaller experiments, which can be 

rapidly adapted as new improvements are suggested. 

Kaizen is a process-oriented thinking versus a result-oriented 

thinking. Kaizen concentrates on improving the process rather 

than on achieving certain results. 


The following Kaizen’s methodologies and concepts were 

applied: 

- 5S activity. 5S is the name of a methodology that uses a list 

of 5 Japanese words which are seiri, seiton, seiso, seiketsu 

and shitsuke. In English: sorting, straightening, systematic 

cleaning, standardizing, and sustaining 

- Activities based on teamwork. It involved technicians, support 

staff, quality assurance manager, professionals and directors 

- This small multidisciplinary group worked in improving their 

own work environment and productivity. 

- This group was guided through the kaizen process by an 

external consultant. 

- This group prepared a Value Stream Mapping to select 

specific areas of improvement that would be addressed 

through Kaizen events and “just-do-it” activities. 

- The consultant facilitated an ideation/brainstorming processes 

to identify improvement options and obtained participant 

feedback. 

- The group reported Kaizen results to all employees and 

celebrated success 

Figure 1: Workplace before Kaizen 

Figure 1: Workplace after Kaizen 

Results 

Kaizen increased employee satisfaction by added-value activities 

and improvement of the work environment). 

By improving standardized activities and processes, kaizen 

eliminated waste (lean manufacturing). 

Kaizen process identified expected measurable improvements. 

It prepared an action plan with a list of activities required to 

realize those improvements. 


Kaizen process allowed improving work environment and working 

distribution. It helped to eliminate waste and to recover time for 

value added activities. 

References 

1. Imai, M (2007). Gemba Kaizen, L’art de manager avec bon sens.

S1 - P - 15 

ACCURACY OF PARATUBERCULOSIS ANTIBODY ELISA AT PREDICTING FECAL SHEDDING OF 

MYCOBACTERIUM AVIUM SUBSP PARATUBERCULOSIS IN CATTLE 

Rakel T. Fernández 1 , Carmen Eiras 2 , Carmen Calvo 2 , Ignacio Arnaiz 2 , Fco. Javier Diéguez 1,3 

1 

Santiago de Compostela University (Veterinary Faculty), Department of Anatomy and Animal Production, Lugo, Spain 

2 

Animal Health and Production Laboratory, Lugo, Spain 

3 

Santiago de Compostela University (Veterinary Faculty), Institute of Food Analysis and Research, Lugo, Spain 

Paratuberculosis, bovine, ELISA, fecal culture 

Introduction 

Paratuberculosis, also called Johne's disease, is chronic 

granulomatous enteritis caused by Mycobacterium avium subsp. 

paratuberculosis (MAP). This infectious disease has a worldwide 

distribution affecting mainly ruminants, both domestic and wild, 

but has also been isolated from other animal species. It causes 

great economic losses in cattle farming 1 . 

ELISA is an essential tool in paratuberculosis control programs, 

although bacterial culture remains the reference confirmatory 

test. In this line, the aim of this study was to determine the 

accuracy of the ELISA test as a predictor of the MAP fecal 

elimination "status" (determined by means of fecal culture) of 

individual animals. 


The study was carried out in Galicia (north-west Spain). Galicia is 

the major cattle-farming region of Spain. It was responsible for 

35% of the milk and 12% of the beef produced in Spain, 

constituting approximately 1.7% of the milk and 1.3% of the beef 

produced in the European Union. 

Bacteriological culture was performed in fecal samples from 239 

animals that were collected during the period 2007-2010. 181 

animals were negative to fecal culture and 58 were culture 

positive animals. Serological analysis (ELISA) was also 

performed in serum samples from the same animals. 

Bacterial culture was performed as described by the World 

Organization for Animal Health (OIE) (2008) 2 . Briefly, 1 g of feces 

was added to 20 mL of sterile distilled water, and tubes were 

shaken for 30 min and then allowed to stand undisturbed for 30 

min. Five millilitres of the supernatant were added to 0.75% 

hexadecylpyridinium chloride (HPC) (Sigma). Tubes were 

inverted several times and allowed to stand undisturbed for 18 h 

at room temperature for decontamination. Triplicate Herrold’s egg 

yolk medium (HEYM) culture slopes containing amphotericin B, 

vancomycin and nalidixic acid were inoculated with 0.1 mL of the 

undisturbed sediment, incubated at 37º C and observed at 2- 

week intervals for 16 weeks. Suspect colonies were evaluated for 

mycobactin dependence along with morphology and acid-fast 

staining. Positive-staining colonies were confirmed by PCR. For 

every nine fecal samples a positive control (a positive field 

sample) was included. 

The ELISA used was “PARATUBERCULOSIS ANTIBODY 

SCREENING” (Institute Pourquier, France). False-positive results 

were reduced by pre-absorbing the samples with sonicates of the 

environmental mycobacterium Mycobacterium phlei. Samples 

were considered positive at a % sample:positive ratio of 55% or 

more. 

Receiver operating characteristic (ROC) procedure was used to 

evaluate the overall diagnostic accuracy to estimate the antibody 

titer that was the best cut-off point in terms of sensitivity and 

specificity as a predictor of the bacteriological "status" of the 

animals (culture positive/negative). 

Results 

The ROC curve -as measure of the accuracy of ELISA antibody 

titers to predict the bacteriological “status”- indicated a good 

diagnostic discrimination (Figure 1). The area under the curve 

was 0.878. The best overall diagnostic precision was in the cutoff 

point 155.7 with sensitivity of 84.5% and specificity of 78.9% 

(Table 1). 

Figure 1: ROC curve for overall accuracy of antibody titers as a 

predictor of MAP bacteriological “status” 

Table 1: Sensitivities and specificities obtained from ROC 

procedure 

Cut off point Sensitivity 1-Specificity 

71.4 1.00 0.81 

133.1 0.91 0.34 

147.2 0.88 0.26 

155.7 0.84 0.22 

160.1 0.83 0.20 

164.2 0.81 0.18 

167.7 0.78 0.17 

309.2 0.00 0.01 


Traditionally the interpretation of ELISA is dichotomous (positive 

or negative) based on the cut-off designed to optimize the 

sensitivity and specificity to detect animals with antibodies. The 

use of antibody titers on a continuous scale can improve the 

amount of diagnostic information obtained by this technique. The 

ELISA evaluated in this study, as is the case of previous studies 

with different ELISAs 3 could allow a more accurate estimate of 

the situation of the animal, in this case related to the 

bacteriological “status”. 

References 

1. Kennedy, DJ, Benedictus, G (2001). Control of Mycobacterium avium 

subsp. paratuberculosis infection in agricultural species. Scientific and 

Technical Reviews, 20, 151-79. 

2. World Organization for Animal Health (OIE) (2008). Manual of diagnostic 

test & vaccines for terrestrial animals, Chapter 2.1.11. 

3. Collins, MT (2002). Interpretation of a commercial bovine 

paratuberculosis enzyme-linked immunosorbent assay by using likelihood 

ratios. Clinical and Diagnostic Laboratory Immunology, 9, 1367-71.

S1 - P - 16 

EVALUATION OF AN ELISA ASSAY AS AN ANCILLARY METHOD IN A HERD WITH A NATURAL 

TUBERCULOSIS INFECTION 

I. Arnaiz 1 , C. Eiras 1 , M.L. Luaces 2 , J. Fraga 3 , M. Rodríguez 3 , C. Calvo 1 

1 Laboratorio de Sanidade e Produción Animal de Galicia, Consellería do Medio Rural e do Mar, Xunta de Galicia, Lugo, España 

2 

Área Veterinaria de Lugo, Servicio Provincial de Ganadería de Lugo, Consellería do Medio Rural e do Mar, Xunta de Galicia, Lugo, España 

3 

Área Veterinaria de Vilalba, Servicio Provincial de Ganadería de Lugo, Consellería do Medio Rural e do Mar, Xunta de Galicia, Lugo, España 

Keyword: Tuberculosis, ELISA, IFN-g, SIT, isolation 

Introduction 

The single intradermal tuberculin (SIT) test and the Interferongamma 

assay (IFN-g) are the tuberculosis diagnostic tools in 

the tuberculosis eradication program currently used in Spain. 

These tests based on cell-mediated response can detect 

animals in the early stages of infection (1), although not all 

affected animals are detected with these tests. Humoral 

immune response is found in cattle with natural infection and 

this response could be detected by the use of an ELISA assay. 

ELISA testing is not routinely used in bovine tuberculosis 

control programs mainly due to a reduced sensitivity (2) 

although it has been suggested that it might be used as a 

complement to the tuberculin test, especially for the detection 

of anergic tuberculous cattle (3). 

The aim of this study is the evaluation of the use of SIT test 

and the IFN-g assay together with an ELISA assay in order to 

obtain the most complete detection of tuberculosis infected 

animals in a herd previously confirmed to have tuberculosis. 


314 animals from a tuberculosis infected beef herd from 

Galicia, in north-west Spain, were tested using the SIT test. 

The IFN-g detection assay (BovigamTM, Prionics AG, 

Switzerland) was used in parallel with the SIT test. The 16 

positive SIT animals (of which 15 animals were also detected 

by IFN-g assay) and 17 cattle that were only detected by IFN-g 

assay were slaughtered and their tissues were collected for 

Mycobacterium tuberculosis complex (MTC) isolation. At the 

same time, 20 cows older than 10 years were slaughtered too. 

Tissues from the 53 slaughtered animals were inoculated into 

the selective media (4). The MTC was recovered and 

confirmed by real-time PCR (5) in 18 cattle. 

The plasma collected for the IFN-g assay was also used on 

the ELISA assay for M. bovis antibody (IDEXX Laboratories). 

18 cattle were detected with the ELISA assay. 

The agreement and kappa statistic measurements were 

calculated by comparing the MTC isolation with SIT test, IFN-g 

and ELISA assays respectively. 

References 

1. Rua-Domenech R, Goodchild AT, Vordermeier HM, Hewinson RG, 

Christiansen KH, Clifton-Hadley RS: Ante mortem diagnosis of 

tuberculosis in cattle: a review of the tuberculin tests, gamma-interferon 

assay and other ancillary diagnostic techniques. Res Vet Sci 2006, 

81:190-210. 

2.Ritacco V, Lopez B, Barrera L, Nader A, Fliess E, de Kantor: Further 

evaluation of an indirect enzyme-linked immunosorbent assay for the 

diagnosis of bovine tuberculosis. Zentralbl Veterinarmed B 1990, 37:19- 

27. 

3. Plackett P, Ripper J, Corner LA, Small K, de Witte K, Melville L, 

Hides S, Wood PR: An ELISA for the detection of anergic tuberculous 

cattle. Aus Vet J 1989, 66:15-19. 

4. Idigoras P., Beristain X., Iturzaeta A., Vicente D., Pérez-Trallero E. 

2000. Comparison of the automated nonradiometric BACTEC MGIT 

960 system with Löwestein-Jensen, Coletsos, and Middlebrook 7H11 

solid media for recovery of mycobacteria. European Journal of Clinical 

Microbiology Infections Diseases 19: 350-354. 

5. Huard R.C., Oliveira-Lazzarini L.C., Butler W.R., van Soolingen D., 

Ho J.L. 2003. PCR-Basaed method to differentiate the subspecies of 

the Mycobacterium tuberculosis complex on the basis of genomic 

deletions. Journal of Clinical Microbiology, vol 41, n 4: 1637-1650. 

Results 

10 bovine with MTC recovered were SIT test positive. 16 were 

positive in the IFN-g assay and the ELISA detected 13 of the 

18 cattle with MTC recovered (one of them was negative in 

SIT test and IFN-g assay). One bovine with MTC recovered 

was negative in the three tests; this animal was slaughtered 

because it was more than10 years old. 

The kappa statistic shows a lower agreement for the SIT test 

(0.39) and the IFN-g assay (0.36) with the MTC isolation than 

the ELISA assay (0.58). 


The results suggest that the use of IFN-g and ELISA assays 

as ancillary techniques might help to detect a larger number of 

tuberculosis infected animals in a TB-confirmed herd. These 

assays complement each other because they may detect 

animals in different stages of tuberculosis. 


We thanks IDEXX Laboratories, Inc., for providing the ELISA 

test for detection of M. bovis antibodies. We also would like to 

thank the Official Veterinary Services from Vilalba for assisting 

with abattoir sampling, the Regional Animal Health Veterinary 

Services for their support and the Laboratory of Animal Health 

of Galicia.

S1 - P - 17 

VIROLOGICAL SURVEILLANCE AND CHARACTERIZATION OF AVIAN-LIKE REASSORTANT H1N2 

SWINE INFLUENZA VIRUSES IN SWEDEN 

Giorgi Metreveli , Eva Emmoth, Lena Renström 

National Veterinary Institute, Department of Virology, Immunobiology and Parasitology, Uppsala, Sweden 

Introduction 

The influenza A virus subtypes H1N1, H1N2 and H3N2 are 

prevalent in pig populations worldwide. In 2009 and 2010 there 

was the first reported isolations and demonstrations of natural 

reassortants of H1N2 viruses in pigs in Sweden (1,2). 

Characterized Swedish isolates possessed avian-like SIV H1N1 

HA and European H3N2 SIV-like NA. They were compared 

regarding their molecular composition and biological 

characteristics. 


Virus was isolated from the clinical material by infecting Madin 

Darby Canine Kidney (MDCK) (ATCC CCL-34) cells, following 

standard cell culture procedures. Viruses were analyzed by endpoint 

titration through cpe using 96-well plates containing MDCK 

cells, in 10-fold dilutions assaying eight replicates of 50 µl per 

dilution, and the virus titres after 6-8 days were calculated 

according to Kärber (3). NA enzyme activity and drug inhibition 

assays were performed with methylumbelliferone 

nacetylneuraminic acid (MUNANA) as the substrate. Genetic 

analyses were conducted on the clinical material and on the 

MDCK cell isolates. Total RNA was prepared from virus-infected 

MDCK cells by the QiagenRNeasy Mini kit, according to the 

manufacturer’s instructions (Qiagen, Hilden Germany). 

Sequencing templates were generated by amplification of each 

gene segment using one-step RT-PCR (QIAGEN OneStep RT- 

PCR Kit). The PCR products were purified using the ‘Purification 

Kit from Promega’ and sequenced using the fluorescent dye 

terminator method with an ABI PRISM® Big Dye Terminator 

Cycle Sequencing v3.1 Ready Reaction kit (Perkin Elmer, 

Waltham, MA, USA) on an ABI PRISM® 310 genetic analyzer 

according to the manufacturer’s recommendations (Applied 

Biosystems).Sequence data analyses, multiple alignments of the 

DNA sequences of each gene, were performed using CLC, Main 

Workbench 5.0.2 (CLC bio A/S, Aarhus, Denmark). 

Results 

The most remarkable result was a truncated coding region for 

PB1-F2 in the earlier isolates and a full length coding region in 

the more recent isolates. Concerning biological properties, these 

viruses reached lower titre and showed reduced 

cytopathogenicity in MDCK cells compared with an avian-like 

H1N1 isolate A/swine/Lidkoping/1193/2002 belonging to the 

same lineage as the 2009 and 2010 isolates. 

Figure 1: Always put legend below the figure 


Recent serological investigations in Swedish pig population has 

also shown that this uncommon avian-like reassortant H1N2 SIV 

variant appears to be gaining a stronger foothold among Swedish 

pig populations and producing more clinical disease. The 

molecular genomic differences found here indicate that the virus 

population is steadily evolving. In order to determine the effect on 

the swine industry and influenza ecology, further surveillance 

investigations and detailed analyses are needed. 

References 

1 Bálint, A., et al., The first Swedish H1N2 swine influenza virus isolate represents 

an uncommon reassortant. Virology Journal, 2009. 6 Oct 28(180). 

2. Metreveli, G., et al. Comparison of two H1N2 swine influenza A viruses from 

disease outbreaks in pigs in Sweden during 2009 and 2010.Virus Genes, 2011. 

Apr;42(2):236-44 

3. Kärber, G., Beitrag zur kollektiven Behandlung pharmakologischer 

Reihenversuche. Archiv für experimentelle Pathologie und Pharmacologie, 1931. 

162: p. 480-483. 

Influenza A, swine, genetic characterization

S1 - P - 18 

A TOOL TO ASSESS ANTIMICROBIAL RESISTANCE PATTERNS IN DAIRY FARMS 

Gerolimetto E. 1 , Cibin V. 1 , Marafin E. 1 , Zavagnin P. 1 , Longo A. 1 , Ramon E. 1 , Ruffa M. 1 , Menin R. 2 , Carraro A. 2 , 

Pozza G. 1 , Lettini A.A. 1 ,Ricci A. 1 

1 

Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), Italy 

2 ASL 15 Alta Padovana, Cittadella (PD), ITALY 

Antimicrobial resistance, E. coli, Enterococcus spp., dairy farms 

Introduction 

Resistance to antimicrobials is of major public health concern. 

The inappropriate use of antimicrobials may contribute to the 

emergence of antibiotic resistant commensal bacteria, with the 

subsequent risk of widespread dissemination of antibiotic 

resistance determinants (1). To our knowledge few studies 

describing antimicrobials resistance patterns of indicator bacteria 

in dairy farms have been published (2). Aim of this study is to 

evaluate a tool to identify antimicrobial resistance patterns of 

commensal bacteria at dairy herd-level highlighting differences 

among animal categories. 


30 dairy farms in the north-eastern part of Italy were sampled 

between September 2010 and January 2011. Individual faecal 

samples were collected from 4 groups of animals taking into 

account the intra-herd animal population distribution: 15, 5, 5 and 

up to 5 samples from lactating cows (LA), dry cows (DC), heifers 

S1 - P - 19 

CHARACTERIZATION OF EXTENDED-SPECTRUM ß-LACTAMASE (ESBL)-PRODUCING 

ESCHERICHIA COLI BOVINE ISOLATES IN NORTHWEST SPAIN. 

V. Gómez 1,2 , A. Mora 2 , A. Méndez 1 , R. Mamani 2 , C. López 2 , A. Lamas 1 , C. Eiras 1 , J. Blanco 2 

1 Laboratorio de Sanidade e Produción Animal de Galicia, Consellería do Medio Rural e do Mar, Xunta de Galicia, Lugo, España 

2 

Departamento de Microbioloxía e Parasitoloxía. Laboratorio de Referencia de Escherichia coli. Facultade de Veterinaria. USC, Lugo, España 

Keyword: Escherichia coli, ESBLs, bovine, CTX-M 

Introduction 

Extended-spectrum ß-lactamases (ESBLs) confer bacterial 

resistance to all beta-lactams except carbapenems and 

cephamycins. In 1989, a new family of ESBLs called CTX-M 

was characterized with a potent hydrolytic activity against 

cephalosporins such as cefuroxime, cefotaxime and cefepime. 

At least 65 ß-lactamases CTX-M are currently known and 

spread all over the world (1). 

The emergence and wide dissemination of ESBLs among 

clinical Escherichia coli (E. coli) isolates in hospitals, 

community patients, food-producing animals and household 

pets in recent years are of great concern and represent a 

problem for the treatment of infectious diseases (2). 

The aim of this study was the genotypic and phenotypic 

characterization of E. coli strains isolated from bovine. 

3. Locatelli C, Caronte I, Scaccabarozzi L, Migliavacca R, Pagani L, 

Moroni P. 2009. Extended-spectrum β-lactamase production in E. coli 

strains isolated from clinical bovine mastitis. Vet. Res. Commun. 33: 

141-144. 

4. López-Cerero L, Egea P, Serrano L, Navarro D, Mora A, Blanco J, 

Doi Y, Paterson DL, Rodríguez-Baño J, Pascual A. Characterisation of 

clinical and food animal Escherichia coli isolates producing CTX-M-15 

extended-spectrum β-lactamase belonging to ST410 phylogroup A. Int J 

Antimicrob Agents. 2011 Apr;37(4):365-7. Epub 2011 Feb 16. PubMed 

PMID: 21330111. 

5. Cortés P, Blanc V, Mora A, Dahbi G, Blanco JE, Blanco M, López C, 

Andreu A, Navarro F, Alonso MP, Bou G, Blanco J, Llagostera M. 

Isolation and characterization of potentially pathogenic antimicrobialresistant 

Escherichia coli strains from chicken and pig farms in Spain. 

Appl Environ Microbiol. 2010 May;76(9):2799-805. Epub 2010 Mar 12. 

PubMed PMID: 20228098; PubMed Central PMCID: PMC2863447. 

Material and Methods 

From 2003 to 2009, 38 strains of ESBL-producing E. coli were 

isolated from clinical cases of bovine mastitis and diarrhoea 

from dairy farms in Galicia, northwest Spain. 

The phenotypic characterization was determined by BD 

PhoenixTM Automated Microbiology System and double-disk 

synergy test. 

Genotypic ESBL confirmation, virulence gene, phylogenetic 

groups, and amplification of fragment blaCTX-M were carried 

out by PCR. Finally, the strains were serotyped, and molecular 

characterized by multilocus sequence typing (MLST) and 

pulsed-field gel electrophoresis (PFGE). 

Results 

Analysis of the 38 isolates showed that 74% (28 strains) and 

26% (10 strains) produced CTX-M-14 and CTX-M-32 

enzymes, respectively. A statistical significant association was 

found between CTX-M-14 and resistance to gentamicin, and 

CTX-M-32 and resistance to aztreonam and ceftazidime. Of 

the 38 E. coli isolates, 30 (79%) carried at least two virulence 

gene typical of extraintestinal pathogenic E. coli strains 

(ExPEC). 

The determination of phylogenetic groups showed that the E. 

coli isolates belonged to A (50%), B1 (31.5%) and D (18.4%) 

phylogroups. None of the bovine isolates belonged to the 

phylogenetic group B2 associated with human virulence in 

ExPEC. 

In accordance with the high diversity identified by serotyping, 

PFGE molecular analysis revealed highly heterogeneous 

profiles. Only one successful clonal group, the O20:H8/HNT- 

B1-ST448 CTX-M-14-producing, was detected. 


Our survey demonstrates that Galician bovine is a reservoir of 

CTX-M-14 and CTX-M-32-producing E. coli strains, with 

important associated resistances to aminoglycoside, 

tetracycline, sulphonamide, fluoroquinolone and 

chloramphenicol. 

Although person-to person spread is recognised as the main 

vehicle of spread of ESBL-producing E. coli in hospital and 

the community, the primary reservoirs of such organisms are 

still in discussion. ESBL-producing E. coli have been isolated 

from food animals in many countries, particularly poultry and 

cattle (3, 4, 5), so farm animals are now recognised as 

important carriers of BLEEs. Further studies are required to 

determine the true zoonotic potential of these ESBL-producing 

strains. 

References 

1. Cantón R, Coque TM. 2006. The CTX-M beta-lactamase pandemic. 

Curr. Opin. Microbiol. 9:466-475. 

2. Carattoli A. 2008. Animal reservoirs for extended-spectrum ß- 

lactamase producers. Clin. Microbiol. Infect. 14:117-123.

S1 - P - 20 

OVINE ABORTION CAUSED BY YERSINIA PSEUDOTUBERCULOSIS – A CASE REPORT 

Varpu Hirvelä-Koski, Minna Nylund 

Finnish Food Safety Authority, Research Department, Oulu, Finland 

Ovine abortion, pseudotuberculosis 

Introduction 

Yersinia pseudotuberculosis is a gram-negative organism that 

causes various syndromes including mesenteric lymphadenitis 

and septicaemia in wild and domestic animals as well as in 

humans. Wild birds and rodents are considered to be 

reservoirs of the bacterium. Sporadic abortions due to 

placentitis have been reported in sheep, goats and cows (1,2). 

We describe here a case of ovine abortion caused by Yersinia 

pseudotuberculosis. 


The samples were obtained from an organic farm keeping 

about 300 ewes in central Finland. The ewes were traditional 

Finnish homebred sheep. Two of the ewes aborted before the 

lambing season with an interval of two weeks, approximately 

one month before the estimated lambing. Samples from the 

second abortion were sent to the laboratory, unfortunately 

frozen. 

Two fetuses and the afterbirth were obtained to the laboratory. 

The samples were examined using gross pathology and 

histopathology (HE stain). Lung, liver and contents of 

abomasum as well as the afterbirth were cultivated on the 

following media: trypticase soy agar incorporated with 5 % 

bovine blood (aerobic incubation), fastidious anaerobe agar 

(FAA) (microaerophilic), FAA (anaerobic), selective FAA for the 

detection of Campylobacter fetus (microaerophilic), Sabouraud 

agar (aerobic), salmonella enrichment media (pre-enrichment 

broth, modified semi-solid Rappaport Vassiliadis medium). 

STAMP stain was used for the detection of brucellosis using 

material from lung, liver and afterbirth. All plates were 

incubated at 37⁰+ 1⁰C. The identification of Yersinia 

pseudotuberculosis was based on colony morphology, gramstain, 

oxidase reaction, production of acid from glucose, typical 

growth on MacConkey agar and API 20 E profile. 

Wild rodents are considered to be one reservoir of Yersinia 

pseudotuberculosis. The population densities of wild voles has 

been high in Finland in recent years. It is possible that this 

phenomenon has increased the infection pressure caused by 

Yersinia pseudotuberculosis. 

References 

1. Karbe, E, Erickson, ED (1984). Ovine abortion and stillbirth due to 

purulent placentitis caused by Yersinia pseudotuberculosis. Vet. Pathol. 

21, 601-606 

2. Juste, RA, Minguijón, E, Arranz, J, Fuertes, M, Beltrán de Heredia, I 

(2009). Lamb mortality in an outbreak of Yersinia pseudotuberculosis 

mastitis, as a collateral effect of colostrum feeding for Lentivirus-control. 

Small Ruminant Res. 86, 46-53. 

3. Hallanvuo, S (2009). Foodborne Yersinia. Identification and 

molecular epidemiology of isolates from human infections. Acad. Diss., 

University of Helsinki, Finland. National Institute for Health and Welfare, 

Research series no 11, Helsinki University Print. 

Results 

The fetuses, a male and a female weighed 347,5 and 600,8 g, 

the fetal length being 21 and 26 cm, respectively. In gross 

pathology no specific lesions could be found. Both fetuses and 

the afterbirth had quite intense autolytic changes. 

Yersinia pseudotuberculosis was isolated as a pure growth 

from the afterbirth. Growth was observed on all non-selective 

media at all atmospheres. The bacterium was detected also 

from the inner organs of both fetuses. The growth was most 

abundant in cultivations from abomasum. Other pathogenic 

organisms were not detected. 

The bacterium was a gram-negative small rod, oxidase 

negative, acid but not gas was produced from glucose. There 

were small brown colonies on MacConkey. API 20 E profile 

suggested identification to Yersinia pseudotuberculosis. 

Histopathological examination revealed suppurative placentitis 

and non-suppurative conjunctivitis. In the villi of the cotyledons 

there were multiple necrotic foci with neutrophilic and 

mononuclear infiltrates. There were masses of coccobacilli and 

infiltrations of mononuclear and plasma cells with a few 

granulocytes in the lamina propria of the conjunctiva of both 

fetuses. In other internal organs the autolytic changes were 

quite intense and any specific lesions could not be found. 


Yersinia pseudotuberculosis is often observed as the cause of 

septicemia in wild animals in Finland, e,g, hare. The bacterium 

has also caused several large outbreaks of food-borne 

illnesses in humans in Finland in the last decade. Yersinia 

pseudotuberculosis has been reported as a rare causative 

agent of sporadic abortion of sheep and goat. However, as far 

as we know, this is the first time that it was isolated from ovine 

abortion samples in Finland.

S1- P - 21 

ANALYSIS OF THE EFFICIENCY OF THE MCMASTER METHOD IN RAYNAUD’S MODIFICATION IN 

DETECTION OF TOXOCARA SP. AND TRICHURIS SP. EGGS IN CARNIVORES FAECES 

Maciej Kochanowski, Joanna Dąbrowska, Jacek Karamon, Tomasz Cencek 

National Veterinary Research Institute, Departament of Parasitology and Invasive Disease, Pulawy, Poland 

McMaster method, efficiency, carnivores, Toxocara, Trichuris 

Introduction 

Carnivores animals are the hosts of the important zoonotic 

intestinal parasites. Faeces of these animals, containing eggs of 

dangerous parasites are a potential source of infection for 

humans. The particular importance in the spread of dangerous 

invasive parasitic forms may have a common use of this faeces 

as natural organic fertilizers. The aim of study was to determine 

the efficiency of McMaster method in Raynaud’s modification 

(Raynaund, 1970) in the detection of parasite eggs of the genera 

Toxocara and Trichuris present in the faeces of carnivores, using 

faeces samples enriched with known numbers of eggs. 


Four variants of this McMaster method (counting in one grid, two 

grids, the whole McMaster chamber and flotation slide) were 

used to examine dog faeces samples enriched with Toxocara sp., 

Trichuris sp. and Ascaris suum eggs. A. suum eggs were the 

control of influence of the parasite eggs properties on the results. 

One hundred and sixty enriched samples were prepared from 

dog faeces (20 repetitions for each eggs level) containing 15, 25, 

50, 100, 150, 200, 250 and 300 eggs of the three types of eggs in 

1 g of faeces. Moreover, to compare the influence of kind of 

faeces on the efficiency of the method 160 pig faecal samples 

were prepared and enriched with A. suum eggs.in the same way 

Results 

The highest (worst) limit of detection (Table 1) in all McMaster 

variants were obtained with eggs of Toxocara sp. (from 25 to 250 

eggs / g faeces, depending on the variant) - about twice higher 

than for Trichuris sp. and A. suum eggs (from 15 to 100 eggs / g 

feces). The highest (worst) limit of detection for flotation in the 

tube were obtained for Trichuris sp. eggs (100 eggs / g) - it was 

4-times higher than the results for other types of eggs (25 eggs / 

g). The best results of the limit of detection, sensitivity, the lowest 

coefficients of variation (providing about repeatability) were 

obtained with the use of whole McMaster chamber variant. There 

was no significant impact of faeces properties (dog and pig 

faeces) on the obtained results. Multiplication factors (to estimate 

the real number of eggs in 1 g of faeces) were calculated on the 

basis of the transformed equation of the trend line illustrating the 

relationship between number of eggs found by the examination 

and the number of eggs added to the sample. Multiplication 

factors for eggs of Toxocara sp. and Trichuris sp. were higher 

than planned by Raynaud, and for A. suum were comparable with 

them. The multiplication factor estimated for flotation in the tube 

(flotation slide variant) for all types of examined parasite eggs 

were significantly higher than that given by Raynaud (Table 2). 

Table 1: Limit of detection for all variants of the McMaster method 

in Raynaund’s modification 

Variants 

Dog faeces 

Pig faeces 

of the method Toxocara Trichuris Ascaris Ascaris 

one grid 250 100 100 50 

two grids 100 50 25 25 

whole chamber 25 15 15 15 

flotation slide 25 100 25 25 


Most of the publications about detection of parasite eggs are 

based on material from natural or experimental infection. For that 

reason the real number of eggs in faeces can not be properly 

assessed (Vadlejch et al, 2011; Mes et al, 2007). Therefore, in 

order to obtain the most reliable results in our experiment the 

material enriched with a known number of parasite eggs was 

used. The results of experiments indicate that detection of 

parasite eggs of carnivores by McMaster method in Raynaud’s 

modification differs from the basic parameters of this 

modification. There were significant differences in the efficiency 

of the method according to the type of parasite eggs. In the case 

of Toxocara sp. eggs examined in the McMaster chamber there 

were observed significantly lower numbers of detected eggs in 1 

g of faeces than obtained for Trichuris sp. and A. suum eggs. 

Most likely reason of the lowest detection Toxocara sp. eggs in 

the McMaster chamber is the specific shell structure of these 

eggs – namely, they have strong adhesive properties 

(Overgaauw, Knapen, 2008). It can result in higher losses of 

parasite eggs during performing of the method. The variant in the 

tube (flotaion slide) was characterized by the lowest number of 

detected eggs in the case of Trichuris sp. It is probably related to 

the high specific gravity of the Trichuris sp. eggs affects to less 

effective flotation process in tube. According to our study the 

detection of Toxocara sp. eggs in McMaster chamber is twice 

lower than planned by Raynaud. In the case of Trichuris sp. eggs 

it is slightly lower. However, A. suum eggs in pig and dog feces, 

were detected in number close to the assumptions of McMaster 

method in Raynaud’s modification. Detection of all three types 

parasite eggs as a result of the tube flotation (flotation slide 

variant) is several times lower than the assumptions of Raynaud's 

modification (which is at the level of detection obtained in the 

variant of two grids of McMaster chamber). Our results allowed 

us to assess the real efficiency of the McMaster method in 

Raynaud’s modification in carnivore feces samples enriched with 

a known number of parasite eggs. The investigation carried out 

with selected (important to the diagnostic and the zoonotic point 

of view) two types of nematodes (Toxocara sp. and Trichuris sp.) 

showed a significant effect of parasite genus on the detection 

efficiency. However, it is desirable to extend calibration of the 

method for other kinds of parasites whose eggs differ significantly 

in the morphology - in particular with regard to the eggs of 

tapeworms. 

References 

1. Mes T., Eysker M., Ploeger H., 2007. A simple, robust and semiautomated 

parasite egg isolation protocol. Nature Protocols. 

2. Overgaauw P., Knapen F., 2008. Toxocarosis, an important zoonosis. 

European Journal of Companion Animal Practice Vol. 18 Issue 3 (2008). 

3. Raynaud J., 1970. Etude de l’efficacite d’une technique de coproscopie 

quantitative pour le diagnostic de routine et le controle des infestations 

parasitaires des bovins, ovins, equines et porcins. Annales de 

Parasitologie (Paris). 45 pp. 321-342 

4. Vadlejch J., Petrtyl M., Zaichenko I., Cadkova Z., Jankovska I., 

Langrova I., Moravec M., 2011. Which McMaster egg counting technique is 

the most reliable? Parasitology Research 109(5): 1387-1394 (2011) 

Table 2: Multiplication factors for estimation of the number of 

eggs in 1g of faeces, for all variants of the McMaster method in 

Raynaund’s modification 

Variants 

Dog faeces 

Pig faeces 

of the method Toxocara Trichuris Ascaris Ascaris 

one grid 244 145 108 111 

two grids 120 78 54 63 

whole chamber 40 26 22 22 

flotation slide 49 62 35 43

S1 - P - 22 

MILK AMYLOID A AND ITS USEFULNESS IN THE LABORATORY DIAGNOSIS OF MASTITIS 

Gabriel Kováč 1 , Csilla Tóthová 1 , Oskar Nagy 1 , Martin Kováč 2 

1 University of Veterinary Medicine and Pharmacy, Clinic for Ruminants, Košice, Slovak Republic 

2 Regional Veterinary and Food Administration, Košice, Slovak Republic 

Dairy cows, mastitis, milk amyloid A, haptoglobin, serum amyloid A 

Introduction 

Mastitis is recognized as one of the most important diseases 

affecting dairy cattle. Raised levels of major acute phase proteins 

in cattle, haptoglobin and serum amyloid A, have previously been 

shown in milk from cows with clinical mastitis as a result of the 

leakage of these proteins from the blood to the milk (Eckersall et 

al., 2001). Further investigations showed an extrahepatic 

synthesis of specific isoform of amyloid A directly in mammary 

epithelial cells, the so called milk amyloid A (MAA) (McDonald et 

al., 2001). 

Therefore, this work was aimed at the evaluation of the influence 

of clinical and sub-clinical mastitis in dairy cows on the 

concentrations of MAA in milk samples, and selected acute 

phase proteins in blood serum. 


Into the evaluation we included 41 dairy cows of a low-land black 

spotted breed and its crossbreeds with various clinical findings on 

the mammary gland. These cows were in the 3rd – 4th lactation, 

but not in the period shortly after parturition. Clinical examination 

of the mammary gland was performed by visual inspection and 

palpation. Clinical mastitis was diagnosed by the presence of 

observable signs of inflammation in the infected quarter such as 

swelling, heat, pain or redness, and by the presence of clots and 

flakes in the milk, or by its abnormal color or consistency. To 

detect sub-clinical mastitis the Californian Mastitis Test (CMT) 

was performed. According to the results of the clinical 

examination of the udder and to the results of CMT the animals 

were divided into 4 groups: Group I – cows without clinical 

changes on the mammary gland and with negative CMT (n = 7), 

Group II – cows without clinical changes on the mammary gland 

and with weakly positive CMT (n = 12), Group III – cows without 

clinical changes on the mammary gland and with strongly positive 

CMT (n = 13), Group IV – cows with clinical changes on the 

mammary gland and changes in milk appearance (n = 9). 

Milk samples were collected into plastic tubes by hand-stripping. 

Blood samples were collected by direct puncture of v. jugularis. 

Milk samples were used to assess the concentrations of milk 

amyloid A (M-SAA, ng/ml), and haptoglobin (Hp, mg/ml) and 

serum amyloid A (SAA, μg/ml) were assessed in blood samples. 

Haptoglobin was assessed using commercial colorimetric kits 

(Tridelta Development, Ireland) in microplates. SAA and M-SAA 

were analysed by commercial ELISA kits (Tridelta Development, 

Ireland). The optical densities were read on automatic microplate 

reader Opsys MR (Dynex Technologies, USA) at an optical 

density of 630 nm for Hp, and 450 nm using 630 as reference for 

SAA and M-SAA. 

The analysis of the significance of differences in measured 

values between cows with various clinical findings on the 

mammary gland was performed by Kruskal-Wallis nonparametric 

ANOVA test and Dunn's Multiple Comparisons Test. 

Results 

M-SAA concentrations in milk samples differed significantly 

between the groups (P < 0.001), with concentrations in samples 

from cows with clinical mastitis (group IV) being significantly 

higher than in samples from groups I and II (P < 0.001 and P < 

0.05, respectively, Table 1). The concentrations of M-SAA in milk 

samples increased with increasing CMT score. 

The serum concentrations of Hp showed also tendency of 

gradual significant increase with increasing CMT score and 

clinical changes on the mammary gland (P < 0.05, Table 1). The 

highest mean Hp concentration we found in cows with clinically 

manifested signs of mastitis. Similarly, SAA concentrations 

differed significantly between the evaluated groups of cows (P < 

0.05), with the highest mean concentration in animals with clinical 

signs of mastitis. However, the differences in the obtained results 

of Hp and SAA concentrations between the evaluated groups of 

cows were less significant (P < 0.05) compared with MAA 

concentrations. The mean SAA concentrations found in cows 

from group I and group II were roughly uniform. 

Table 1: Concentrations of evaluated variables in dairy cows 

Variable I II III IV P < 

M- x 325.7 A,B 1433.1 a 3910.4 A 6073.8 B,a 

SAA ±sd 173.8 949.2 2145.8 4414.0 

0.001 

Hp 

x 0.046 0.122 0.299 0.329 

±sd 0.053 0.263 0.314 0.339 

0.05 

SAA 

x 29.7 27.6 a 48.2 71.5 a 

±sd 27.6 28.0 42.5 31.5 

0.05 

The same indexes in lines mean significance of differences in values 

between the groups: a – P < 0.05; A, B – P < 0.01 


The results of our study indicate that inflammatory diseases of 

the mammary gland lead to an increase in the concentrations of 

M-SAA. Compared to Hp and SAA, M-SAA is synthesized directly 

in the mammary epithelia of the udder in response to infection 

(Jacobsen et al. 2005). Therefore, M-SAA is believed to be a 

more sensitive indicator of mastitis; it accumulates in milk only 

during mammary inflammation. Petersen et al. (2005) reported 

that M-SAA concentrations, similarly to our results, were higher in 

quarters with mastitis compared to healthy quarters. 

Moreover, mastitis may activate systemic inflammatory reactions, 

including the induction of the synthesis of acute phase proteins 

by the liver. The results of our study showed that the 

concentrations of Hp and SAA were higher in serum from cows 

with clinical mastitis, and increased with increasing CMT score. 

These results are in line with previous studies (Eckersall et al. 

2001). It appears that localized severe inflammation of the udder 

is sufficiently intense to induce a measurable systemic acute 

phase response. The concentrations of measured acute phase 

proteins had a tendency to be higher in the serum from the cows 

with local signs of mastitis and also in cows without clinical 

changes on the mammary gland, but with positive CMT. 

However, the finding that the differences in Hp and SAA 

concentrations observed between the groups of cows were less 

significant than the differences in M-SAA concentrations means 

that the measuring of serum concentrations of some acute phase 

proteins would be less useful to the evaluation of the severity of 

mastitis than the measuring of the concentrations of M-SAA 

directly in milk samples. 

Generated data suggest the usefulness of M-SAA for diagnosis 

of bovine sub-clinical mastitis, as well as in the determination of 

the severity of mastitis, suggesting that its assessment as a part 

of the laboratory diagnosis would be a valuable supplementation 

to the proper clinical diagnosis and determination of other blood 

laboratory parameters. 


This work was supported by VEGA Scientific Grant No 1/0592/12 

from the Ministry of Education, and by Slovak Research and 

Development Agency under contract No. APVV-0475-10. 

References 

1. Eckersall, PD, Young, FJ, McComb, C, Hogarth, CJ, Safi, S, Weber, A, 

McDonald, T, Nolan, AM, Fitzpatrick, JL (2011). Acute phase proteins in 

serum and milk from dairy cows with clinical mastitis. Vet Rec, 148, 35-41 

2. Jacobsen, S, Niewold, TA, Kornalijnslijper, E, Toussaint, MJM, Gruys, E 

(2005). Kinetics of local and systemic isoforms of serum amyloid A in 

bovine mastitic milk. Vet Immunol Immunopathol, 104, 21-31 

3. McDonald, TL, Larson, MA, Mack, DR, Weber, A (2001). Elevated 

extrahepatic expression and secretion of mammary-associated serum 

amyloid A3 (M-SAA3) into colostrums. Vet Immunol Immunopathol, 83, 

203-211 

4. Petersen, HH, Gardner, IA, Rossitto, P, Larsen, HD, Heegaard, PMH 

(2005). Accuracy of milk amyloid A (MAA) concentration and somatic cell 

count for diagnosing bovine mastitis. Proceedings of the 5 th International 

Colloquim on Animal Acute Phase Protein, Dublin, Ireland, March 14-15, 

2005, 43-44

S1 - P - 23 

RELIABLE AND EARLY DETECTION OF SALMONELLA IN PIGS 

Tilman Kühn 1 , Patrik Buholzer 2 

1 

Synlab.vet Leipzig, Leipzig, Germany 

2 

Prionics AG, Schlieren, Switzerland 

Introduction 

Salmonellosis is one of the most important zoonotic diseases and 

can cause serious clinical symptoms in humans. Pigs have been 

recognized as an important source of Salmonella infections. The 

existence of this disease presents great risks for human health. 

Salmonella infections of animals intended for the food industry 

play an important role in public health as these animals are 

considered to be the major source of human Salmonella 

infections. The PrioCHECK Salmonella Ab porcine 2.0 was 

developed for large-scale screening of pigs and for application in 

Salmonella control programs. Here we show that the test clearly 

discriminates positive and negative samples through its large 

dynamic range. As the test also allows earlier detection of a 

Salmonella infection in pigs, than competitor tests, it is the 

diagnostic tool of choice for Salmonella control programs. 


Two separate experiments were performed. In the first 

experiment serum samples of ten pigs (4 negative and 6 

Salmonella positive samples) were tested with both the 

PrioCHECK Salmonella Ab porcine 2.0 and a competitor test. 

Additionally, the Salmonella status was determined in ten meat 

juice samples with both tests. 

In the second experiment, 40 pigs were experimentally inoculated 

with live Salmonella vaccine. Serum samples were taken at the 

day of inoculation, 3, 6 and 9 weeks post inoculation and were 

tested with the PrioCHECK Salmonella Ab porcine 2.0 and two 

competitor tests both validated for use in Germany. The results of 

the three tests were compared. 

Results 

In the first experiment, both tests identified all positive and 

negative samples correctly, but the positive samples gave a 

much higher values with the PrioCHECK Salmonella Ab porcine 

2.0 and therefore results were more clear-cut than those obtained 

with the competitor test. The average percentage positivity (PP) 

of positive samples of the PrioCHECK Salmonella Ab porcine 2.0 

exceeded that of the competitor test by over 70 %. However, the 

PP values of negative samples were equal to, or lower than those 

of the competitor test. This means that the test does not generally 

generate high values, but is specific for positive samples. 

The PrioCHECK Salmonella Ab porcine 2.0 also earlier identifies 

animals experimentally infected with Salmonella. In serum 

samples of experimentally infected pigs, the PrioCHECK 

Salmonella Ab porcine 2.0 identified 25 samples as positive at 3 

weeks post inoculation. 17 out of these 25 samples were missed 

by at least one competitor test (13 samples) at three weeks post 

inoculation, or only detected by the other tests at a later time post 

inoculation (4 samples). 


The data show that the results obtained with PrioCHECK 

Salmonella Ab porcine 2.0 showed a large dynamic range and 

therefore allow reliable discrimination between positive and 

negative samples. This dynamic range means that the cut-off of 

40% PP -- implemented in most countries -- could easily be 

lowered to 20% without loss of specificity. 

The application of the PrioCHECK Salmonella Ab porcine 2.0 

reliably helps control Salmonella infections even early after 

infection. 

References 

1. B. Nielsen, D. Baggesen, F. Bager, J. Haugegaard, P. Lind Veterinary 

Microbiology 47 (1995) 205-218 

2. H.M.F. van der Heijden. First International Ring Trial of ELISA’s for 

Salmonella- antibody. Berl. Münch. Tierärztl Wschr. (2001) 389-392. 

3. PrioCHECK® Salmonella Ab porcine 2.0, Prionics AG 

http://www.prionics.com/diseases-solutions/salmonellosis/ 

Food safety, diagnostic test, Salmonellosis

S1 - P - 24 

DETECTION OF BVD VIRUS ON PERSISTENTLY INFECTED CALVES BY REAL TIME REVERSE 

TRANSCRIPTION PCR ON EAR NOTCHES 

Damien MAGNEE 1 , Julie CHARROT 1 , Stéphane DALY 1 , Sandrine MOINE 1 , Eric SELLAL 1 

1 

LSI Laboratoire Service International, LISSIEU, France 

PCR, BVDV, Ear Notches, PI Calves, Magnetic Beads 

Introduction 

Bovine Viral Diarrhea Virus is a pestivirus of the Flaviviridae 

family. The virus is transmitted by direct contact between 

animals. Vertical transmission plays an important role in the 

epidemiology and infection pathogenesis. In cattle, fetus infection 

can cause abortions, stillbirths, teratogenic effect or persistent 

infection of newborn calf (PI). It is therefore in the interest of 

farmer to detect PI animals earliest after birth and then prevent 

virus spreading in the herd. The BVD diagnosis is performed on 

blood and serum, but there is a doubt about the possibility of 

virus detection in the presence of calf colostral antibodies, in the 

first day of life. 

In 2011, the French National Federation “Groupe de Défense 

Sanitaire” decided to study the feasibility of detecting the 

Persistently Infected (PI) calves by direct diagnosis on ear 

notches samples picked up while animals are tagged. In the 

frame of BVDV eradication, this system could become universally 

used if its sensitivity is equivalent to the detection on blood by RT 

rtPCR 


A study was conducted in Eastern France on 19 calves (3 days to 

4 weeks old) with confirmed PI status. Bloods and ear notches 

were collected for each animal. 

For each sample, nucleic acids were extracted with silica 

columns methods and with a magnetic beads method developed 

by LSI. Real-Time PCR was performed using the TaqVet 

BVDV Screening kit. 

The results obtained for bloods and ear notches were compared 

for each sample, to validate the using of skin samples for the 

BVDV diagnosis. 

The second part of the study consists to validate the using of pool 

of 10 samples of ear notches. Each positive eluate of skin sample 

for PI animals was pooled with 9 negative BVDV eluates, to 

obtain a pool of 10 samples. After RNA purification by column or 

magnetic beads, Real-Time PCR was performed with LSI kit. 

The last part consists of a study of the storage conditions of ear 

notches, from the sampling to the analysis. The samples were 

stored 7 days at 3 different conditions: -20°C, +4°C and room 

temperature. The RNA purification was performed with silica 

columns methods. 

Results 

For the comparison between blood and ear notches, the results 

showed a correlation of 100% (Figure 1). The mean difference 

between the both matrixes is about 0.7 Ct. Ear notch matrix 

shows an equivalent sensitivity than blood for PI calves detection. 

30 

25 

Blood 

Ear Notch 

Concerning the use of pool of 10 samples, the results show a 

good correlation between individual and pool of 10 results, with a 

Ct value difference of 3.2 (figure 2). This difference corresponds 

to the ear notches dilution factor of 10 (log [10] = 3.3Ct). Ear 

notch is then a reliable sample for PI detection in individual or in 

pool of 10 samples analysis. 

Figure 2: Ear Notch Analysis in pool of 10 

Concerning the last part of the study, for the storage conditions of 

ear notches, no significative difference was observed on the PCR 

sensitivity at -20°C or at +4°C (∆Ct of 0.6, between D0 and D7). 

The storage at room T°C shows a significative variability on Ct 

values. So the room temperature is not adapted for the storage of 

skin samples (∆Ct of 2.7, between D0 and D7). (Figure 3) 

Ct value 

40 

35 

30 

25 

20 

15 

10 

5 

0 

D0 

D7 at RT° 

D7 at +4°C 

D7 at ‐20°C 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18 19 

Calves 

Figure 4: Stability of Ear notch samples 


Our results show that (i) ear notch matrix offers the same 

sensitivity for PI calves detection than blood, (ii) the detection of 

one PI calf amoung a pool of 10 samples can be performed on 

both matrixes, and (iii) the ear notch is a stable matrix (at +4°C 

and -20°C) for the diagnosis by PCR. 

The complete analysis method developed on ear notches 

(TaqVet BVDV Screening and MagVet Universal Isolation 

kit) is sensitive enough to detect a PI calf in pool of 10 samples or 

individual analysis. Then it could be used in large scale to control 

the disease and decrease the incidence of the PI in bovine herds. 

Ct value 

20 

15 


A special thank to the LVD55-Segilab laboratory to their active 

participation in this project. 

10 

5 

0 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18 19 

Calves 

Figure 1: Comparison on blood and ear notches matrixes 

The results obtained with columns or magnetic beads are similar 

for BVD and IPC detection, with a mean difference of 1 Ct 

between both methods. Extraction with beads shows equivalent 

results on PCR sensitivity than columns.

S1 - P - 25 

GENOTYPIC VARIABILITY DETERMINATION OF PATHOGENIC PROTOTHECA 

Sara Marques 1 , Eliane Silva 1 , Gertrude Thompson 1 , Volker Huss 2 

1 

University of Porto, ICBAS – CIBIO, Porto, Portugal 

2 

University of Erlangen-Nuremberg, Biology Department – MPP, Erlangen, Germany 

Prototheca spp.; bovine mastitis; internal transcribed spacer (ITS); 16S rDNA; intergenic spacer (IGS) 

Introduction 

Members of the genus Prototheca are ubiquitous nonphotosynthetic 

green algae that can be associated with 

pathologies in humans and animals (1). The known pathogenic 

species in the genus are P. zopfii, P. wickerhamii and P. 

blaschkeae (2). Both P. zopfii and P. blaschkeae have been 

associated with bovine mastitis (1, 2). Presently this pathology is 

recognized as endemic worldwide and is considered a public 

health problem (3). Prototheca identification is generally achieved 

by phenotypic and molecular characterization, the latter being 

used to easily identify Prototheca species. Phylogenetic 

relationships of Prototheca can be inferred from 18S and 28S 

rDNA sequence comparisons (1, 2, 4). The ribosomal internal 

transcribed spacer (ITS) regions and plastid ribosomal RNA 

operon are less conserved than 18S and 28S rRNA genes and 

provide greater interspecific differences that are useful for the 

differentiation of strains (5). Thus, the aim of the present study 

was to determine the genotypic variability within P. zopfii and P. 

blaschkeae strains by amplification and sequencing of these 

regions. 


Prototheca isolates used in this study belong to a major collection 

of milk pathogens obtained from different dairy herds from the 

Northwest of Portugal. Thirty seven isolates were previously 

identified as P. zopfii genotype 2 and five as P. blaschkeae (1). 

These isolates were spread and grown on Sabouraud Dextrose 

Agar medium (Merck Laboratories, Darmstadt, Germany). In this 

study PCR amplification of the nuclear ITS region and plastid 16S 

rDNA, intergenic spacer (IGS) and partial 23S rDNA were 

performed. Briefly, genomic DNA preparation was performed as 

previously described (1). For amplification of the ITS region 

several sets of primers were applied and some additives were 

used to improve the reaction. The amplification products were 

analysed on a 0.8% (wt/vol) agarose gel after staining with 

ethidium bromide. Since some of the amplifications produced two 

bands, these were purified with the QIAquick ® PCR purification kit 

(Qiagen, Crawley, UK) and cloned into Topo ® XL PCR Cloning 

Kit (Invitrogen, Carlsbad, USA). As some sequences displayed 

similarities to the Prototheca plastid IGS region between 16S and 

23S rDNA, several other primers for the ITS region were tested 

and amplification of the 16S rDNA, IGS and part of the 23S rDNA 

were also performed. 

Results 

ITS amplification of Prototheca isolates retrieved bands of the 

expected size only in few cases (Fig. 1). Sequencing of the 

amplified products retrieved nuclear ITS of only 3 P. zopfii strains 

with some differences between them. No ITS sequences were 

obtained for P. blaschkeae. 

zopfii isolates had identical sequences, and the 5 P. blaschkeae 

isolates sequences could be assigned to 2 groups which differed 

in two positions within the 16S rRNA gene and one position in the 

tRNA-alanine gene. 

Figure 2: 16S rDNA amplification of some P. zopfii (Pz1-2) and P. 

blaschkeae (Pb1-2) isolates with 16S universal primers. All PCR 

fragments have a size close to the expected size of about 1,500 

bp. Lane EcoRI + HindIII – molecular weight standard, -C, 

negative control without DNA. 


The variability of nuclear ITS and plastid rDNA sequences 

between and within P. zopfii and P. blaschkeae strains is shown 

for the first time. Within the ITS sequences of the P. zopfii 

isolates, similarities between 92 and 96% were found. The plastid 

sequences obtained for P. zopfii and P. blaschkeae isolates 

showed only 82.7% similarity between each other. Similarity of 

the complete 16S rDNA alone was 85.3%, which was lower than 

that of the respective 18S rDNA (98.0%) (1). Solving the 

amplification problem and sequencing of the ITS region together 

with the plastid rRNA operon from all isolates will be of great 

value for Prototheca population genetics and epidemiology. 


This work was supported by Fundação para a Ciência e 

Tecnologia, Portugal, grant SFRH/BD/28892/2006. 

References 

1. Marques, S, Silva, E, Kraft, C, Carvalheira, J, Videira, A, Huss, V, 

Thompson, G (2008). Bovine mastitis associated with Prototheca 

blaschkeae. J Clin Microbiol; 46, 1941-1945. 

2. Janosi, S, Ratz, F, Szigeti, G, Kulcsar, M, Kerenyi, J, Lauko, T, Katona, 

F, Huszenicza, G (2001). Review of the microbiological, pathological, and 

clinical aspects of bovine mastitis caused by the alga Prototheca zopfii. Vet 

Q, 23, 58-61. 

3. Malinowski, E, Lassa, H, Klossowska, A (2002). Isolation of Prototheca 

zopfii from inflamed secretion of udders. Bull Vet Inst Pulawy, 46, 295-299. 

4. Moller, A, Truyen, U, Roesler, U (2007). Prototheca zopfii genotype 2: 

the causative agent of bovine protothecal mastitis? Vet Microbiol, 120, 

370-374. 

5. Pröschold, T, Darienko, T, Silva, PC, Reisser, W, Krienitz, L (2011). The 

systematics of Zoochlorella revisited employing an integrative approach. 

Environ Microbiol, 13, 350-364. 

Figure 1: ITS amplification of some P. zopfii (Pz1-6) and P. 

blaschkeae (Pb) isolates with ITS primers. All PCR fragments 

should have a size of approximately 1,300 bp, however different 

sizes were found and in some isolates two bands were amplified. 

Lane ClaI – molecular weight standard, -C, negative control 

without DNA. 

The complete plastid 16S rDNA of most of the isolates was 

amplified (Fig. 2). Almost complete sequences of the plastid 

rRNA operon were obtained for 8 Prototheca isolates. Three P.

S1 - P - 26 

DETECTION OF SAXITOXINE (PSP MARINE BIOTOXIN) IN MOLLUSCS BY ELISA 

Miroslaw M. Michalski, Katarzyna Grądziel-Krukowska 

National Veterinary Research Institute, Department of Hygiene of Food of Animal Origin, Puławy, Poland 

Head of the Department: prof. Jacek Osek 

Marine biotoxins, saxitoxine, Elisa, intoxication 

Introduction 

Marine biotoxins are a group of natural toxins that sometimes 

accumulate in shellfish. Many biotoxins are produced by marine 

algae (phytoplankton, including diatoms and dinoflagellates) and 

can accumulate in fish or shellfish if they ingest these algae. 

There are several types of illnesses, caused by marine biotoxins, 

that are connected with the consumption of contaminated 

shellfish. They include Paralytic Shellfish Poisoning (PSP), and 

Diarrhetic Shellfish Poisoning (DSP), Amnesic Shellfish 

Poisoning (ASP). Saxitoxin is a naturally produced marine 

biotoxin by several gonyaulacoid or gymnodinioid dinoflagellates, 

including Alexandrium, Gymnodinium, Pyrodinium, and has also 

been found in freshwater cyanobacterial strains such as 

Cylindrospermopsis raciborskii 

The toxins responsible for PSP are heterocyclic guanidines 

(saxitoxins) and there are over 21 known congeners. Substitution 

at R4 results in substantial changes in toxicity. Saxitoxin (STX) 

binds with a high affinity to site 1 on the voltage dependent 

sodium channel, inhibiting channel opening (1, 3) 

Table 1 

Type of shellfish 

1.Mule 

Mytilus edulis 

2.Oysters 

Crassostrea gigas 

3.Vongole 

Tapes 

semidecussatus 

4.Scallops 

Pecten spp. 

Number of 

samples 

Number of samples: 

bld 1 /pos 2 /exc 800 

µg/ 3 

24 5/19/0 

13 3/10/0 

Country of 

origin 

Holland, 

Norway, 

France 

Holland, 

France 

12 2/10/0 Italia 

10 2/7/0 Holland 

Total numbers of 

sam-samples 

59 

1 - bellow limit of detection 

2 - range 50-800 µg/kg 

3 – concentartion in molluscs meat more than 800 µg/kg 

PSP toxic syndrome is due primarily to the consumption of 

molluscs contaminated by PSP toxins as a result of filter-feeding 

by toxic dinoflagellates. After intoxication of PSP, the effects are 

predominantly neurological and include tingling, burning, 

numbness, drowsiness, incoherent speech, and respiratory 

paralysis. Symptoms of the disease develop fairly rapidly, within 

0.5 to 2 hours after ingestion of the shellfish, depending on the 

amount of toxin consumed. In severe cases respiratory paralysis 

is common, and death may occur if respiratory support is not 

provided (1, 2, 3). In the EU law a limit for PSP toxins in bivalve 

molluscs is laid down at 80 g STX eq/100 g of shellfish meat (4). 


The RIDACSREEN ® FAST PSP SC test was used for the 

determination of PSP toxins in shellfish. This assay is a direct 

competitive Elisa for PSP and related algae toxins in mussels. All 

reagents are contained in the test kit. Detection limit of test is 50 

µg/kg of shellfish meat. A total of four different types of mussels 

were used for analysis. Live shellfish samples used in this study 

were collected from warehouses and markets. A total number of 

59 samples were investigated. Preparation of samples and Elisa 

test was performed according to the test producer instruction. 


Fifty nine samples of shellfish meat were analysed by the Elisa 

test. In 46 samples PSP biotoxins were found, expressed as 

saxitoxin-HCl, in range 50-800 µg/kg. In 12 samples the 

concentration of PSP toxins was bellow the limit of the detection 

of the test and no sample with more than 800 µg/kg of PSP toxins 

in mussels. The present experiment clearly showed that the 

shellfish available on Polish marked are, as a food, safe for 

consumers. 

References 

1. EFSA. 2009. Marine biotoxins in shellfish – Saxitoxin group. 

Scientific Opinion of the Panel on Contaminants in the Food 

Chain. The EFSA Journal, 2009, 1019, 1-76 

2. Michalski Miroslaw. Biotoksyny morskie - występowanie i metody 

analizy. Żywność.Nauka.Technologia.Jakość. 2006, 3(48), 16-22 

3. Michalski Mirosław: Paralityczne toksyny morskie jako zagrożenie dla 

zdrowia konsumenta. Medycyna Weterynaryjna 2007, 63(12), 1530-1533 

4. Regulation (EC) No 853/2004 of The European Parliament and of the 

Council of 29 April 2004 laying down specific hygiene rules for food of 

animal origin. 

Results 

The content of saxitoxine determined by the Elisa test in the four 

species of shellfish is shown in table 1.

S1 - P - 27 

PROFICIENCY TESTING (PT) FOR CHEMICAL LABORATORIES PERFORMING ANALYSIS OF MEAT 

AND MEAT PRODUCTS ORGANISED BY NVRI IN PULAWY 

Michalski Miroslaw 

National Veterinary Research Institute, Department of Hygiene of Food of Animal Origin, Puławy, Poland 

Head of the Department: prof. Jacek Osek 

Proficiency testing, z-score, chemical analysis 

Introduction 

The aim of this study is the qualification of laboratories with help 

of interlaboratory proficiency testing (PT) in range of analysed 

substances. Results of analyses should be credible with 

previously determined standard uncertainty of measurement. 

The harmonization of correct results of chemical analysis is 

secure by the validation of analytic methods, standard uncertainty 

for individual measurement or for the method of analysis, using 

certified reference materials of and participation in proficiency 

testing (3). Information on the precision and accuracy of the 

results are to be taken into consideration in the design of the 

assay (3,4,5). In case of lack of reference materials or reference 

certified materials, participation in PT is the only method for 

confirming the technical competences of the laboratory (1,5). 


Pasteurized meat product was used for analysis. Every 

participant received sample together with instruction of 

transportation as well as a document which allows to introduce 

the sample in a quality system of laboratory. In this sample 

laboratory personnel should analyse at least one of parameters 

as sodium nitrate, salt (NaCl) by Mohr method, water, fat, 

phosphorus, proteins, ash, hydroxyproline, and starch. Deadline 

of realisation of analyses was appointed by organizer. The 

homogeneity of material was estimated on the basis investigation 

of eight random choosen samples from designed to analysis, 

applying the criterion of the homogeneity Ss0,3 . 

Statistical calculations and homogeneity assessments have been 

conducted in accordance with the principles stipulated in ISO 

13528:2005 “Statistical methods for use in proficiency testing by 

inter-laboratory comparisons”. 

In the proficiency test 25 laboratories participated (official 

veterinary labs as well as private) and 31 analysts. Laboratory 

code numbers were known only for laboratory and organizer. The 

participants of PT applied the standardized method or such, 

which they use during normal analysis, after their validation. 

Reference values for each analysed parameter were calculated 

with a help of algorithm A, according to Standard PN-ISO 57255: 

2000, based on results from participating laboratories. 

The calculated reference values as well as standard deviations 

made it possible to calculate z-score accoprding to the formula: 

z 

* 

x 

X 

* 

s 

where, 

s*- standard deviation for proficiency assessment 

X*- assigned value 

x - participant result 

For the purposes of performance assessment for a single round, 

z scores are interpreted as follows: 

z≤ 2.00 satisfactory result 

2.00 < zand < 3.00 questionable result 

z≥ 3.00 unsatisfactory result 

Table1: Results of statistical calculation for tested samples 

NaNO 3 

[mg/kg] 

NaCl 

[g/100g] 

Nitrogen 

[g/100g] 

Water 

[g/100] 

Fat 

[g/100g] 

Phosphoru 

s [mg/kg] 

Ash 

[g/100g] 

Starch 

[g/100g] 

Hydroxypr 

oline 

[g/100g] 

Reference 

value 18,39 2,22 2,84 76,07 1,09 4308,3 2,91 1,78 0,040 

Std 3,39 0,05 0,03 0,12 0,12 100,92 0,03 0,16 0,005 

Coefficient 

of variation 

cv (%) 

18,43 2,25 1,06 0,16 11,01 2,34 1,03 8,99 12,50 

Table 2: The number of results for respective parameters, which 

z score carried out |z| > 2 

Number of 

results 

2

S1 - P - 28 

SPECIFIED RISK MATERIAL REMOVAL PRACTICES: 

CAN WE REDUCE THE BSE HAZARD TO HUMAN HEALTH? 

D. Pitardi 1 , D. Meloni 1 , C. Maurella 1 , D. Di Vietro 1 , L. Nocilla 1 , A. Piscopo 2 , E. Pavoletti 3 , M. Negro 4 , M. Caramelli 1 , 

E. Bozzetta 1 . 

1 Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta, Via Bologna 148, 10154, Torino, Italy. 

2 Azienda Sanitaria Provinciale 1AG, Viale della Vittoria 321, 92100, Agrigento, Italy. 

3 Azienda Sanitaria Locale VC, Via Benadir 35, 13100, Vercelli, Italy. 

4 Azienda Sanitaria Locale CN1, Corso Francia 12, 12100, Cuneo, Italy. 

BSE; vCJD; Spinal cord; SRM Contamination; Alternative slaughter practices. 

Introduction 

Following the bovine spongiform encephalopathy (BSE) 

epidemics across Europe in the early 1990s, the removal of 

designated BSE specified risk material (SRM) became 

mandatory to minimize the risk to consumers of exposure to the 

infectious agent. Despite this preventive measure, crosscontamination 

of edible meat with SRM can occur during 

conventional slaughter (1-4). 

Currently, there are no markers that can identify the presence of 

SRM in meat as a whole. Nevertheless, some assays are able to 

detect traces of CNS, hence this parameter is officially taken as 

indicative of SRM contamination. 

In this two-stage study, we carried out a survey to estimate the 

prevalence of carcass contamination at two slaughterhouses, one 

large and the other medium-sized; we then compared three 

different methods (conventional vs. suction vs. water-jet) for 

spinal cord removal employed at the large slaughterhouse to 

assess their performance in preventing the spread of CNT over 

the carcass. 


Prevalence of CNS contamination 

The prevalence of CNS contamination by the conventional 

technique was estimated from a total of 216 carcasses from a 

large slaughterhouse and 196 from a medium-sized one. In both 

abattoirs the carcasses were split with a hand-guided belt-type 

saw and the spinal cord cut along its length was removed from 

each side of the carcass. Sampling was performed immediately 

after spinal cord removal. 

Alternative techniques 

Two alternative spinal cord removal techniques were compared 

to the conventional method. In both techniques, the SRM is 

extracted before the carcass is split (by suction or by water-jet). 

Comparative study 

The estimated sample size (50 carcasses) was sampled for each 

SRM removal method: conventional; suction; and water-jet. The 

specimens were withdrawn immediately after carcass splitting. 

Sample collection and preparation 

Bovine older than 12 months were included in the study. Samples 

were collected from a defined area on the medial surface of each 

half of the split carcass. The area was selected and marked off 

on the paravertebral muscles. 

Testing activity 

A commercially available ELISA kit (Ridascreen® Risk Material 

10/5, R-Biopharm), which detects GFAP as a marker for CNS, 

was used to analyze the samples. In order to facilitate application 

of the test for screening purposes, in previous studies we 

validated its qualitative use by plotting an ROC curve to set a 

useful cut-off value (2). 

Results 

Using a qualitative approach, samples were defined as positive if 

CNS tissue was detected at a concentration ≥0.049% (2). 

Samples tested positive in 130/216 carcasses (60.2%, 95% CI 

53.3-66.8) from the large slaughterhouse and in 152/196 (77.6%, 

95% CI, 71-83.2) of those from the medium-sized one (Table I). 

The conventional slaughter technique was associated with an 

overall prevalence of CNS contamination of 68.4% (95% CI, 53 - 

83). 

The comparative study showed a CNS contamination of 62% 

(95% CI, 47.2-75.3) associated with the conventional technique, 

60% (95% CI, 45.2-73.6) with the suction technique, and 36% 

(95% CI, 22.9-50.8) with the water-jet system (Table II). The 

difference among the three methods appeared to be significant 

(P=0.0047). 

Table I: Conventional method - positive samples stratified 

according to contamination level and type of abattoir. 

Large abattoir Medium abattoir 

CNS contamination positive samples positive samples 

(%) 

(%) 

Low ( ≥ 0.049 and

S1 - P - 29 

LOW PATHOGENIC AVIAN INFLUENZA VIRUS INFECTIONS ON POULTRY FARMS IN THE 

NETHERLANDS 

Sylvia Pritz-verschuren 1 , Jeanet van der Goot 1 , Johan Bongers 1 , Guus Koch 1 

1 

Central Veterinary Institute of Wageningen UR,Lelystad,The Netherlands 

Avian Influenza Virus, low pathogenic, poultry 

Introduction 

Outbreaks of highly pathogenic avian influenza (HPAI) have a 

devastating economic impact on the poultry industry and also 

causes social distress. In countries free of HPAI, epidemics start 

after introduction of low pathogenic AI virus which may evolves to 

HPAI viruses. Early detection of AI infection is crucial for the 

control of outbreaks. Infections of LPAI viruses in laying birds is 

subclinical or causes mild symptoms such as a drop in egg 

production. 

Early detection of AI virus infection is crucial to prevent HPAI 

outbreaks. Therefore, in the Netherlands LPAI infections are 

detected by early warning and compulsory serological monitoring 

programmes of all poultry farms. 

In the period of 2006-2011, an increase in the number of LPAI 

virus infections in poultry was observed. A number of possible 

causes is discussed. 


Serological samples were screened in an competitive NP 

antibody ELISA and the positive samples were confirmed and 

typed by hemagglutination inhibition and neuraminidase inhibition 

test. 

For detection of the virus an in house developed PCR with the M- 

gene as target is used. Positive samples were tested in an H5 

and H7 specific PCR described by the community EU reference 

lab (AHVLA, Weybridge). H5 and H7 negative samples were 

subtyped using a generic, hemagglutinin and neuraminidasespecific 

RT-PCR (1) followed by sequencing the PCR product. 

Results 

The number LPAI introductions either detected by PCR and/or 

antibodies increased in the period 2006-2011, especially since 

2009 (Table 1). Although there is a marked increase in the 

number of detections, the period is too short to speak about a 

tendency. 

Table 1. Number of poultry farms where LPAI viruses and/or 

antibodies were detected in period 2006-2011 

Year 

Number of LPAI Number of primary 

detected farms infections 

2006 4 2 

2007 13 8 

2008 10 9 

2009 11 10 

2010 20 18 

2011 33 23 

Total 91 70 

A number of possible causes for the increase of LPAI detections 

are: 

More intensive search for LPAI viruses and introduction of the 

early warning. 

The early warning program was started in 2008/2009. However 

most farms (57/70) are detected by serological monitoring which 

was in place since 2004. So, this can’t be an explanation for the 

increase. 

Higher sensitivity of the serological test used for the monitoring. 

Sera are tested in an ELISA by the Dutch Animal Health Service 

(AHS). In January 2009 the AHS has changed to another test. 

However, the validation tests revealed no differences in 

sensitivity between the old and new test. 

of the Erasmus Medical Centre in Rotterdam there is no 

sufficient evidence to assume such an increased prevalence of 

LPAI viruses in wild birds. 

An increase of the number wild birds in the Netherlands. 

Reports of Dutch Bird research group (SOVON) shows that the 

number of wild birds was more or less constant in the period 

2004-2009. Thus this also can’t explain the increase of the 

number of detection. 

More contact between poultry and wild birds due to the increased 

number of outdoor-layer farms. 

Data of the Product Boards for Livestock, Meat and Eggs (PVE) 

shows that the total number of outdoor-layer farms was lower in 

2008 and 2009 than in the years before and gradually increased 

again in 2010 and 2011. 

Different subtypes are found on the poultry farms (Table 2). In 

poultry H5, H7 and H8 viruses and/or antibodies are the most 

common in the period 2006-2011. For serology the 

neuraminidase inhibition test is used for typing, however the test 

is not suitable to test large numbers of sera samples and 

therefore the neuraminidase type is not yet determined for most 

farms, because 57/70 farms were detected in the serological 

monitoring programme. Therefore the most common N-type is 

not known. 

Table 2. Avian Influenza virus subtypes detected in poultry farms 

in the Netherlands in period 2006-2011 

HA N1 N2 N3 N4 N5 N6 N7 N8 N9 N?* Totaal 

H1 2 1 3 6 

H2 2 1 3 

H3 2 2 

H4 

H5 1 10 11 

H6 1 1 3 5 

H7 1 1 2 4 4 12 

H8 3 9 12 

H9 3 1 4 

H10 2 1 3 

H11 

H12 1 1 

H13 

H14 1 1 

H16 

H?* 1 13 14 

Totaal 4 5 3 5 2 6 49 74 

* Typing of the haemagglutinine and/or neuraminidase was not 

possible. 


Every year in the Netherlands more infections with LPAI viruses 

are detected in poultry farms . Up to now we did not identify an 

obvious explanation for the increase of LPAI virus introduction in 

poultry. However, different subtypes are found. The most 

common H-types are H5, H7 and H8. 

References 

1. Gall,A, Hoffmann, B, Harder, Timm, Grund, C, Ehricht, R, Beer, M 

(2009). Rapid an Highly Sensitive Neuramidase Subtyping of Avian 

Influenza Viruses by Use of a Diagnostic DNA Microarray. J. of Clin. 

Microbiol., 47, 2985-2988 

A increased prevalence of LPAI viruses in wild birds. 

The prevalence varies per season, year, wild bird and location, 

but from a wild bird surveillance studies performed by the group

S1 - P - 30 

DETECTION OF BRACHYSPIRA HYODYSENTERIAE AND BRACHYSPIRA PILOSICOLI IN SWISS PIGS 

USING A COMBINATION OF CULTURE AND PCR 

Sarah Prohaska 1 , Helen Huber 1 , Titus Sydler 2 , Max M. Wittenbrink 1 

1 Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Zurich, Switzerland 

2 

Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Switzerland 

Introduction 

Swine dysentery and porcine intestinal spirochaetosis caused by 

pathogenic Brachyspira (B.) spp. (B. hyodysenteriae and 

B. pilosicoli) lead to considerable economic loss and are 

therefore of increasing importance to the pig industry. 

B. hyodysenteriae colonise the epithelium of the large intestine 

and subsequently induce severe mucohaemorrhagic colitis 

showing the typical signs of diarrhoea containing blood and plugs 

of mucus (1). Infections with B. pilosicoli however are 

characterised by moderate growth loss and mild diarrhoea (2). 

The goal of this study was to evaluate the presence of 

B. hyodysenteriae and B. pilosicoli in Swiss pig herds by a 

combination of culture and PCR. 

Swine, Brachyspira spp., culture, PCR 


Over a time period of three years (2009-2011) ligated colon 

sections (n=140) from dissected pigs as well as faecal swabs 

(n= 661) collected on 228 Swiss pig farms were subjected to a 

cultural assay and subsequent PCR for the detection of 

B. hyodysenteriae and B. pilosicoli. Faecal swabs were taken 

from pigs suffering from acute diarrhoea. 

For culture, the selective BJ-Agar (Trypticase soy agar 

supplemented with 5% cattle blood and Colistin, Vancomycin, 

Spectinomycin, Spriramycin and Rifampicin) was used (3). Plates 

were incubated under anaerobic conditions at 42°C for 4-6 days. 

To evaluate the presence of Brachyspira spp., native 

preparations of colony material were investigated by darkfield 

microscopy. If spirochaetes were found, DNA was extracted from 

suspicious colonies. Subsequently, PCR using two primer pairs 

for the detection of B. hyodysenteriae and B. pilosicoli was 

performed (4). 

Results 

In two of the 140 (1.4%) tested colon sections B. hyodysenteriae 

could be detected, whereas B. pilosicoli was not found in these 

samples (Figure 1). 

In terms of faecal swabs, cultivation and subsequent confirmation 

by PCR yielded B. hyodysenteriae in 122 (18.5%) samples and 

B. pilosicoli in 33 samples (5.0%). However, analysis of seven 

(1.0%) swabs revealed a co-infection of B. hyodysenteriae and 

B. pilosicoli (Figure 2). 

Figure 2: Results of faecal swabs 


The combination of culture and PCR for diagnosis of Swine 

dysentery and intestinal spirochaetosis proofed to be a 

successful tool. 

The results of this study show a remarkable presence of 

pathogenic Brachyspira spp. in Switzerland. B. hyodysenteriae 

was detected in samples taken from pigs from 12 of 26 cantons 

in Switzerland. Due to this situation, the Swiss pig health service 

implemented a monitoring system of pathogenic Brachyspira spp. 

starting 2012. 

References 

1. Hampson D.J., Atyeo R.F., Combs B.G., 1997. Swine Dysentery 175- 

209. In: Intestinal Spirochaetes in Domestic Animals and Humans 

(Hampson D.J. and Stanton T.B.), CAB International, Wallingford, UK. 

2. Taylor D.J., Simmons J.R., Laird H.M., 1980. Production of diarrhoea 

and dysentery in pigs by feeding pure cultures of a spirochaete differing 

from Treponema hyodysenteriae. Vet. Rec. 106, 326-332. 

3. Dünser M., Schweighardt H., Pangerl R., Awad-Masalmeh M., Schuh 

M., 1997. Schweinedysenterie und Sprirochaetendiarrhoe – vergleichende 

Untersuchungen serpulinenbedingter Enteritiden. Wien. Tierärztl. Mschr. 

84, 151-161. 

4. La T., Philips N.D., Hampson D.J. 2003. Development of a Duplex PCR 

Assay for Detection of Brachyspira hyodysenteriae and Brachyspira 

pilosicoli in Pig Feces. J.Clin. Microbiol. 41, 3372-3375. 

Figure 1: Results of ligated colon samples

S1 - P - 31 

PRRSV DIAGNOSTICS BY RT-PCR AND DIFFERENT ELISA SYSTEMS IN SERUM AND ORAL FLUID 

OF PRRSV VACCINATED PIGS 

Tatjana Sattler 1 , Sandra Revilla-Fernández 2 , Adolf Steinrigl 2 , Manfred Berger 2 , Friedrich Schmoll 2 

1 

University Leipzig, Large Animal Clinic for Internal Medicine, Leipzig, Germany 

2 

AGES, Institute for Veterinary Disease Control, Mödling, Austria 

PRRSV, ELISA, PCR, oral fluid, pig 

Introduction 

PRRSV infection is widely distributed all over the world. PRRSV 

eradication programs have been developed and PRRSV negative 

herds and farms have been established. PRRSV diagnostic tools 

with high sensitivity and specificity are essential to control the 

disease and to guarantee the negative status of the herds (1). 

Furthermore, specimens like oral fluid are advantageous in terms 

of costs, working time and animal welfare, and therefore have 

been recently proposed for disease monitoring (2). The objective 

of this study was therefore to evaluate PRRSV RNA and antibody 

detection in pig oral fluid by RT-qPCR and commercial ELISA 

test systems, respectively. Furthermore, the performance of 

different ELISA systems was compared both, in blood serum and 

oral fluid of pigs. 


Ten pigs (from 8 to 20 weeks of age) from a PRRSV negative 

farm were injected with an EU-type PRRSV life vaccine (Porcilis ® 

PRRS, Intervet, Unterschleißheim, Germany). Blood and oral 

fluid samples were taken individually from each pig before, and 4, 

7, 14 and 21 days after vaccination. After automated RNA 

extraction, serum and oral fluid samples were analysed with an 

in-house PRRSV RT-qPCR assay (3). 

PRRSV antibody detection in serum and oral fluid was performed 

with three different ELISA systems: a) IDEXX PRRS X3 Ab-Test 

(IDEXX GmbH, Ludwigsburg, Germany), b) INGEZIM PRRS 

UNIVERSAL, c) INGEZIM PRRS DR (both Ingenasa, Madrid, 

Spain). Serum samples were diluted according to manufacturer’s 

instructions, whereas oral fluid samples were tested without 

dilution. 

Results 

In serum samples (table 1), antibodies were detected in seven 

out of ten pigs by day 14 after vaccination. One pig 

seroconverted at day 21. Two pigs did not seroconvert at all 

throughout the study period and also remained RT-qPCR 

negative throughout the whole study. Three out of ten pigs were 

RT-qPCR positive in serum as early as day 4 after vaccination, 

while most pigs (7/10) became positive by day 7. One pig 

became RT-qPCR positive as late as day 14, while other pigs 

became negative in RT-qPCR at this time-point. This animal was 

also the last to seroconvert during the study period. 

Table 1: Number of serum samples positive by PRRSV RT-qPCR 

and by three different PRRSV antibody ELISA testkits (a, b, c – 

see material and methods) 

day 0 (no 

vaccination) 

day 4 after 

vaccination 

day 

7 

day 

14 

day 

21 

PCR 0/10 3/10 7/10 5/10 3/10 

ELISA 

a) 

ELISA 

b) 

ELISA 

c) 

0/10 0/10 0/10 7/10 8/10 

0/10 0/10 0/10 7/10 7/10 

0/10 0/10 3/10 7/10 8/10 

In oral fluid (table 2), both RT-qPCR and ELISA positive results 

(in two of the tested ELISA kits) were observed, although 

sensitivity was significantly reduced in comparison to serum. The 

INGEZIM PRRS DR ELISA (Kit c) did not show positive results at 

all in oral fluid. 

Table 2: Number of oral fluid samples positive by PRRSV RTqPCR 

and by three different PRRSV antibody ELISA testkits (a, 

b, c – see material and methods) 

day 0 (no 

vaccination) 

day 4 after 

vaccination 

day 

7 

day 

14 

day 

21 

PCR 0/10 0/10 2/10 2/10 0/10 

ELISA 

a) 

ELISA 

b) 

ELISA 

c) 

0/10 0/10 0/10 2/10 3/10 

0/10 0/10 0/10 2/10 4/10 

0/10 0/10 0/10 0/10 0/10 


PRRSV-antibodies could be detected in serum of some pigs as 

early as day 7 after vaccination with the INGEZIM PRRS DR 

ELISA (Kit c), which was the most sensitive of the three ELISA 

systems. Results of the other two ELISA systems did not differ 

substantially from each other, although one pig remained 

antibody negative in serum at day 21 with the INGEZIM PRRS 

UNIVERSAL (Kit b), while it was found positive in both other 

ELISA systems. A seroconversion in this pig, tested with 

INGEZIM PRRS UNIVERSAL ELISA, could be expected later 

than day 21 after vaccination. 

Interestingly, some animals can remain seronegative despite 

vaccination, as was the case in this study in two out of ten pigs. 

The most likely reason was failure of the vaccine virus to 

replicate in these animals, as indicated by negative RT-qPCR 

results at all time-points tested. A similar finding was described in 

a previous vaccination study, where one out of 20 vaccinated 

gilts remained seronegative (4). 

Comparison of RT-qPCR and ELISA results obtained from 

matched oral fluid and serum samples suggest that testing of.oral 

fluid is much less sensitive. Lower amounts of both PRRSV RNA 

and antibodies and/or the presence of inhibitory factors can be 

expected in oral fluid (5). No positive results in oral fluid samples 

were found with the INGEZIM PRRS DR ELISA (kit c). 

It can be concluded that all tested ELISA systems were similarly 

able to detect PRRSV antibodies after PRRSV life virus 

vaccination, whereas INGEZIM PRRS DR ELISA seems to be 

the most sensitive. Reliable PRRSV antibody detection in oral 

fluid will require validation of test kits for this medium. RT-qPCR 

results confirmed the antibody detection results in both, serum 

and oral fluid. 

References 

1. große Beilage, E, Bätza, HJ. (2007): PRRSV-eradication: An option for 

pig herds in Germany? Berl Münch Tierärztl Wochenschr. 120, 11/12, 470- 

479. 

2. Kittawornrat, A, Prickett, J, Chittick, W, Wang, C, Engle, M, Johnson, J, 

Patnayak, D, Schwartz, T, Whitney, D, Olsen, C, Schwartz, K, 

Zimmerman, J. (2010): Porcine reproductive and respiratory syndrome 

virus (PRRSV) in serum and oral fluid samples from individual boars: will 

oral fluid replace serum for PRRSV surveillance Virus Res. 154, 1-2,170-6. 

3. Revilla-Fernández S, Wallner B, Truschner K, Benczak A, Brem G, 

Schmoll F, Mueller M, Steinborn R.(2005): The use of endogenous and 

exogenous reference RNAs for qualitative and quantitative detection of 

PRRSV in porcine semen. J Virol Methods. 126, 1-2, 21-30. 

4. Sipos, W, Indik, S, Irgang, P, Klein, D, Schuh, M, Schmoll, F (2002): 

Transfer of EU-ML-PRRSV (Porcilis PRRS) from vaccinated to nonvaccinated 

gilts. Proc 17th IPVS Congress, Ames, Iowa, USA, 418 

5. Chittick, WA, Stensland, WR, Prickett, JR, Strait, EL, Harmon, K, Yoon, 

KJ, Wang, C, Zimmerman, JJ (2010): Comparison of RNA extraction and 

real-time reverse transcription polymerase chain reaction methods for the 

detection of Porcine reproductive and respiratory syndrome virus in 

porcine oral fluid specimens. J Vet Diagn Invest, 23, 248-53

S1 - P - 32 

RELIABLE DETECTION AND TYPING OF PRRSV USING MULTIPLEX REAL-TIME RT-PCR 

Christine Gaunitz, Carsten Schroeder, Marco Labitzke, Eva V. Knoop, Jörg Gabert 

Labor Diagnostik GmbH Leipzig, Leipzig, Germany 

PRRSV, High Pathogenic, real-time RT-PCR, simultaneous detection 

Introduction 

Infections with the Porcine Reproductive and Respiratory 

Syndrome Virus (PRRSV) in swine are very prevalent and 

economically most important for the swine industry. Clinical signs 

are respiratory disease in piglets and reproductive failure in 

pregnant sows. 

PRRSV is a single-stranded RNA virus of the family Arteriviridae, 

order Nidovirales, and classified into the European (EU/I) and 

North American (NA/II) genotype. These genotypes show only 

55-70 % homology of nucleotides in the different genes. In 2006 

in China a highly pathogenic (HP) NA-strain emerged, which 

shows a deletion of around 30 amino acids in the non-structural 

protein 2 (Nsp2). Infection with the HP-strain is characterized by 

high fever and high mortality in pigs of all ages. In the last years 

there have been detected also new isolates from Eastern Europe. 

Purpose of this study was to develop a real-time PCR which 

allows reliable detection and differentiation of PRRSV strains in 

one test run. 


To evaluate analytical sensitivity of VIROTYPE ® PRRSV, titration 

studies with in-vitro RNA were performed. 

Analytical specificity was proven by testing 12 PRRSV reference 

strains from cell culture. 

Diagnostic sensitivity was proven by testing the EPIZONE ring 

trial panel 2011. 

Serum, tissue and saliva samples were tested in comparison to 

other PCR assays. Two commercial PCR kits and VIROTYPE ® 

PRRSV (Labor Diagnostik GmbH Leipzig, Germany) were used 

according to the manufacturer´s recommendations. 

Results 

The high analytical sensitivity of the VIROTYPE ® PRRSV kit was 

proven by a titration series of in vitro RNA (PRRSV 

EU1/EU2/NA/HP [10 8 copies/well to 10 copies/well]), performed 

in triplicates of ten-fold dilutions. A high correlation between 

number of copies and amount of amplification product was 

demonstrated in the range of 10 8 to 10 3 and 10 8 to 10 2 copies, 

respectively (Fig. 1). 

PRRSV EU I 

PRRSV NA 

Figure 1: Evaluation of analytical sensitivity. 

PRRSV EU II 

PRRSV HP 

The analytical specificity was proven by testing a RNA-panel of 

12 PRRSV reference strains comparatively with VIROTYPE ® 

PRRSV and two commercial available tests (Test I and Test II). 

All reference strains were detected well with the VIROTYPE ® 

PRRSV. In comparison to Test II, the VIROTYPE ® 

PRRSV kit 

detects the EU strains around two Ct-values and the NA strains 

around five Ct-values more sensitive. The results of commercial 

test I and VIROTYPE ® PRRSV are comparable (Tab. 1). 

Table 1: Evaluation of analytical specificity. 

VIROTYPE ® PRRSV commercial Test I commercial Test II 

Strain 

EU NA HP IC PRRSV EU NA IC EU NA IC 

Ct 

FAM 

Ct 

TEX 

Ct 

Cy5 

Ct 

HEX 

Ct 

FAM 

Ct 

FAM 

Ct 

FAM 

Ct HEX 

Ct 

FAM 

Ct 

Cy5 

Ct 

HEX 

EU Stendal V953 25.22 - - 34.61 24.62 24.08 - 29.89 24.95 - 35.66 

Intervet 22.02 - - 33.22 20.41 19.41 - 28.85 24.65 - - 

Cobbelsdorf 23.86 - - 32.65 22.91 22.20 - 29.32 26.02 - - 

Stendal V852 23.24 - - 39.96 23.50 21.86 29.68 26.16 - 36.98 

Stendal V1904 24.30 - - 39.06 24.48 23.47 29.30 27.63 - 37.41 

Stendal V1952/97 26.29 - - 35.41 26.32 24.51 31.32 28.12 - 36.06 

Stendal V1445/99 - 21.39 - 34.85 23.80 - 21.17 30.78 - 24.92 34.10 

USA VD28775/2 - 21.35 - 29.14 23.94 - 21.61 30.40 - 24.07 39.08 

USA VD29949/17 - 22.41 - 28.48 27.72 - 22.18 29.65 - 26.89 - 

USA 2 - 19.83 - 30.64 23.07 - 20.44 30.81 - 23.64 - 

USA 18 - 19.06 - 31.48 20.47 - 19.50 30.39 - 26.54 - 

China - 20.87 22.44 28.36 22.82 - 20.54 29.89 - 25.84 - 

Mean Value EU 24.16 23.71 22.59 26.26 

Mean Value NA 20.82 23.64 20.91 25.32 

In comparison to an in-house PCR (Kleiboeker mod.) performed 

at Friedrich-Loeffler-Institute, VIROTYPE ® 

PRRSV detects all 

samples of the Epizone Ring Trial 2011 more sensitive. 

VIROTYPE ® 

PRRSV reliably detects the samples of the EU-2, 

EU-3, EU-4 and the atypical EU genotype 1 (Tab. 2). 

Table 2: Results from the EPIZONE ring trial panel 2011. 

VIROTYPE® PRRSV Kleiboeker (mod.) 

PRRSV-strain Genotyp RNA Dil. EU NA HP IC EU NA 

EU Stendal V954 EU-1 10 -2 24.96 - - 26.88 28.24 - 

EU Stendal V954 EU-1 10 -3 28.41 - - 27.06 31.53 - 

EU Stendal V954 EU-1 10 -4 31.61 - - 26.91 34.12 - 

EU Stendal V954 EU-1 10 -5 34.63 - - 27.14 37.61 - 

EU Stendal V954 EU-1 10 -6 - - - 28.11 - - 

EU Stendal V1952/97 EU-1 10 -1 23.08 - - 27.23 25.82 - 

EU Stendal V1952/97 EU-1 10 -2 26.57 - - 27.66 29.17 - 

EU Stendal V1952/97 EU-1 10 -3 29.54 - - 26.99 32.19 - 

EU Stendal V1952/97 EU-1 10 -4 32.50 - - 26.86 35.25 - 

EU Stendal V1952/97 EU-1 10 -5 36.92 - - 27.87 40.31 - 

Cobbelsdorf EU-1 1:500 23.33 - - 27.26 26.13 - 

Stendal V852 EU-1 1:500 23.55 - - 27.54 25.78 - 

Stendal V1904 EU-1 1:500 24.37 - - 27.37 26.72 - 

Aus_LT3 EU-2 01:10 24.80 - - 27.59 25.93 - 

BY_Bor/59 EU (atypical) 01:10 32.81 - - 27.79 33.63 - 

BY_231/SZA EU-3 01:10 21.01 - - 27.74 23.78 - 

BY_8380/OKT_2p EU-4 01:10 25.70 - - 27.59 28.36 - 

China NA-HP 10 -2 - 20.01 22.14 26.22 - 21.83 

China NA-HP 10 -3 - 23.93 25.79 26.89 - 26.06 

China NA-HP 10 -4 - 27.61 29.25 27.56 - 28.43 

China NA-HP 10 -5 - 29.93 31.71 26.96 - 32.34 

China NA-HP 10 -6 - 37.1 34.96 27.50 - 35.20 

Stendal V1445/99 NA 1:500 - 21.63 - 27.22 - 23.17 

USA VD29949/17 NA 1:500 - 22.74 - 26.75 - 24.44 

USA 2 NA 1:500 - 21.12 - 27.51 - 22.43 

Mean Value 27.12 25.51 29.62 26.74 

Standard Deviation 4.19 5.77 4.17 4.88 


To evaluate sensitivity and specificity of VIROTYPE ® 

PRRSV, 

titration studies with in-vitro RNA were performed. Serum, tissue 

and saliva samples were tested in comparison to other 

commercial PCR assays. Testing the EPIZONE PRRSV ring trial 

panel, all strains could be detected reliably. East European EU 

strains and the atypical EU strain scored positive. 

VIROTYPE ® 

PRRSV allows the reliable and simultaneously 

detection of PRRSV EU and NA genotype and the HP strain from 

porcine blood, serum, tissue, bronchial swabs, bronchial lavage, 

saliva, semen and also cell culture samples. The assay internal 

control guarantees the control of extraction as well as 

amplification. Due to the high sensitivity of VIROTYPE ® PRRSV 

pools of five samples can be tested. VIROTYPE ® PRRSV is easy 

to use with only one reaction-mix. 


The EPIZONE ring trial panel and the panel of 12 PRRSV 

reference strains were kindly provided by K. Wernike and B. 

Hoffmann, Federal Institute for Animal Health, Germany. 

References 

1. Wernike et al., Porcine reproductive and respiratory syndrome virus: 

inter-laboratory ring trial to evaluate real-time RT-PCR detection methods, 

submitted – under review

S1 - P - 33 

APPLICATION OF A HERD PROGRAMME BASED ON CONTROL MEASURES AND LABORATORY 

MONITORING TO ERADICATE A LEPTOSPIRA OUTBREAK IN A LARGE DAIRY CATTLE HERD 

M.T. Scicluna, G. Manna, F. Rosone, A. Caprioli, R. Frontoso, , G.L. Autorino 

Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, 00178 Roma Italia 

Leptospira serovar hardjo, management, laboratory diagnosis 

Introduction 

In Italy, the application of severe sanitary measures, including 

animal movement restriction are mandatory on the diagnosis of 

Leptospira infection and are lifted only after the removal of all 

seropositive animals. In the attempt of reducing economic losses 

deriving from these actions, a herd control programme was 

applied in a dairy intensive farm, in which the infection had been 

diagnosed, based on the combination of preventive, treatment 

and biosecurity measures, aiming at the sterilization of carriers 

and prevention of new cases of infection (1,2,3,4). The primary 

objective was to accelerate the control/eradication of the infection 

which was evaluated by monitoring the absence of its circulation 

with the aid of serological and molecular techniques and not as 

requested by the Italian Regulation, through the sole removal of 

the seropositive animals. Following is the description of the 

supplementary measures adopted and the actions applied for the 

assessment of the efficacy of the control protocol. 


Following the notification of leptospira diagnosis, an extensive 

serological investigation was undertaken in the farm of 600 

producing cows, to verify if the infection was actively circulating: 

the random sampling scheme (Table 1) adopted, allowed the 

estimation of a prevalence of at least 50%, with a standard error 

of 10% and a confidence interval of 95%. The serological method 

used was the microagglutination test (MAT) described in the OIE 

manual, employing the serovar hardjo. A sample was considered 

positive when presenting a titre > to 1/100 (6). 

The supplementary control plan applied on the farm was based 

on the following points: 

- preventive measures, based on vaccination to limit new cases 

contributing to the further spread of leptospirosis. The vaccine 

employed, produced by the Istituto Zooprofillatico Sperimentale 

of Umbria and Marche, contained serovar hardjo and was 

administered as prescribed to all animals. 

- antibiotic treatment was adopted to sterilize carrier animals and 

eliminate primary source of infection. Two different antibiotics 

were chosen for economical and management reasons. (1,6) The 

more economical, Penistrep ®, 25mg/kg b.w., was for 3 

consecutive days used in animals managed individually. To avoid 

any impact of treatment on the sale of milk, antibiotics were 

administered when the producing animals were dried off. 

Treatment was conducted so as to ensure that all animals of the 

same group received it all together, to avoid infection of 

seronegative subjects, after the disappearance of the effect of 

treatment. These animals received a single dose of 20 mg/kg 

b.w., of Duphaciclina 300 LA®. 

- Biosecurity measures aimed at further limiting the presence of 

leptospira in the environment, consisted in: isolation of the 

different units and their drinking systems in relation to whether 

these had undergone antibiotic treatment and complete 

vaccination; exclusive use of artificial insemination; systematic 

draining of paddocks to avoid water and urine stagnation; 

confinement, feeding and milking last of untreated animals. 

On conclusion of the control plan, actions were set up to verify its 

efficacy. These consisted in the microbiological examination of 

272 urine samples of the animals in the different productive units, 

using the same sampling scheme described for the 

serosurveillance. These were examined in a SYBR Green Real 

Time PCR, specific for pathogenic Leptospira (7). Advantage of 

this test is that it can identify the carrier state even in a 

serologically positive animal and compared to the microbiological 

isolation, is fast and highly specific. Although reaction inhibition 

could occur due to the type of sample, using the test at herd 

level, increases the sensitivity of the method. 

In addition, a serological control was carried out on 50 animals 

born after the conclusion of the programme, used also as 

seronegative sentinels, placed in the productive units for the 

detection of an active circulation. The number of sentinel animals 

examined aimed to reveal an infection prevalence of 5% (IC 

95%). These animals were retested at the end of their in-contact 

period, of two months, ensuring exposure, incubation period and 

also the production of a detectable serological response. 

Results & discussion 

The results of the serosurvey (table 1) demonstrate that 

leptospira infection was actively circulating in the farm. The 

supplementary measures adopted were considered as successful 

on the basis of the interruption of the leptospira infection in the 

different production units, as indicated by the negative results 

obtained for the urine samples and the seronegativity of the 

sentinels after their in-contact period with serological positive 

animals. Further evidence of the efficiency of the control 

programme was also the seronegativity of all animals born after 

the conclusion of the adoption of these measures. 

The adoption of such a herd control programme would be of 

benefit for both the farmers as well as the Veterinary Authorities, 

in that they would have more instruments for the control of this 

infection. 

Table 1. Sample scheme for serological survey. 

Productive Consistency Examined Positive N° of 

Unit 

(positive) 

animals 

(Titres) 

Heifers 60 34 (5) 5 1(1/100) 

1(1/200) 

3(1/400) 

Pregnant 

Heifers 

160 54 (38) 38 27 (1/100) 

3(1/400) 

8(1/200) 

Dry cows 140 51 (37) 37 29(1/100) 73 

8(1/200) 

Primipare 120 48 (26) 26 26 (1/100) 54 

Pluripare 160 60 (35) 35 34(1/100) 58 

1(1/200) 

Fresh 50 33 (12) 12 10(1/100) 36 

Lactating 

cows 

2(1/200) 

Problem 80 44 (25) 25 25 (1/100) 57 

animals 

Sick bay 20 17 (7) 7 7 (1/100) 41 

References 

1. Bolin CA, Alt DP. 2001. Am J Vet Res. 62(7):995-1000 

2. Cortese VS, et al., 2007. Vet Ther. 8(3):201-8 

3. Little TW, et al., 1992. Vet Rec. 1;131(5):90-2 

4. Little TW, et al., 1992. Vet Rec. 24;131(17):383-6 

5. Levett PN Clin. Microbiol. Rev. 2001 Apr.,: 296–32 

6. Alt DP, et al.,2001.J Am Vet Med Assoc.1;219(5):636-9 

7. Ahmed A, et. al. 2009. PLoS One 4: Vol 4, Issue 9:1-8 

Estimted 

prevalence % 

15 

70

S1 - P - 34 

THE SERUM PROTEIN ELECTROPHORETIC PATTERN IN CALVES WITH CHRONIC RESPIRATORY 

DISEASES 

Csilla Tóthová 1 , Oskar Nagy 1 , Gabriel Kováč 1 

1 University of Veterinary Medicine and Pharmacy, Clinic for Ruminants, Košice, Slovak Republic 

Calves, electrophoresis, respiratory diseases, protein fractions 

Introduction 

Together with diarrhoea, respiratory tract diseases constitute the 

main health problem and overall the most common cause of 

morbidity and mortality in calves and young cattle. In case of 

dairy calf pneumonia, diagnosis and treatment are mainly based 

on the observation of clinical symptoms. However, in many 

cases, the infected calves show only mild clinical symptoms that 

could be easily missed in a group of calves on a farm (Gänheim 

et al., 2007). Therefore, there is a need for objective parameters 

that are suitable as indicators of health or disease in calf herds 

applicable in the laboratory diagnosis of diseases. Analytical 

methods like serum protein electrophoresis could be useful for 

identifying diseased animals. Although it provides useful 

information on the pathological conditions associated with 

disorders of the protein profile, the serum protein electrophoresis 

in cattle with various organ diseases is a rarely used laboratory 

method. Therefore, the objective of this study was to determine 

whether chronic respiratory diseases in calves cause changes in 

the serum protein electrophoretic pattern. 


Twenty-five calves with clinical signs of chronic respiratory 

diseases of various degrees were included into this study. The 

calves were of a Slovak spotted breed at the age of 4-6 months. 

Into the evaluation we included the calves with clinical signs of 

the disease manifesting for more than 2 weeks. To compare the 

evaluated variables between sick and healthy animals, 29 

clinically healthy calves of the same age and breed in good 

general health were used as a group of controls. 

Blood samples were collected by a direct puncture of v. jugularis. 

The separated serum was analyzed for the total serum protein 

concentrations and identification of serum protein fractions. 

Total protein (TP, g/l) concentrations in blood serum were 

assayed in the automated biochemical analyser Alize (Lisabio, 

France) by the biuret method using commercial diagnostic kits 

(Randox). Serum protein fractions were separated by zone 

electrophoresis on the buffered agarose gel at pH 8.8 on an 

automated electrophoresis system Hydrasys (Sebia Corporate, 

France) using commercial diagnostic kits Hydragel 7 Proteine 

(Sebia Corporate, France) according to the procedure described 

by the manufacturer. The electrophoretic migration was 

performed for 15 minutes at 20 °C constantly at 10 W, 40 mA, 

and 240 V. After migration, the gels were stained in amidoblack 

staining solution, and then destained by acidic solutions and 

dried completely. The electrophoretic gels were scanned, and the 

serum protein fractions were visualized and displayed on the 

densitometry system Epson Perfection V700 (Epson America 

Inc., USA). Protein fractions were identified and quantified by 

computer software Phoresis 5.50 (Sebia Corporate, France). 

Serum proteins were separated into the following fractions in the 

order from fastest to slowest mobilities: albumin, alpha 1 (α 1 )-, 

alpha 2 (α 2 )-, beta 1 (β 1 )-, beta 2 (β 2 )-, and gamma (γ)-globulins. 

Albumin: globulin ratios (A/G) were computed from the 

electrophoretic scan. 

Results 

The total serum protein concentrations obtained in the calves 

suffering from chronic respiratory diseases were significantly 

higher than the values recorded in healthy animals (P

S1 - P - 35 

DEVELOPMENT OF A BROADLY DETECTING ASSAY FOR FLAVIVIRUS USING A NOVEL CONCEPT 

Frederik Widén 1 , Mikael Leijon 1 , Alia Yacoub 1 

1 

SVA, VIP, Uppsala, Sweden 

2 

Affiliation Institute name, Department name, City, Country 

Introduction 

The flavivirus genus is a large group containing enveloped single 

stranded RNA viruses. At least 70 different species of flaviviruses 

have been identified, several cause disease in animals or man 

and many can be transmitted from animals to man by vectors. 

Climate change and globalisation may cause the virus to appear 

in new areas. This can be illustrated with the detection in Europe 

of Usutuvirus, WNV lineage 2 and Bagazavirus which were 

previously present only in Africa or very rarely in Europe. 

Because of a changing situation it is appropriate to test 

suspected cases with broadly detecting assays. Previously 

developed assay for broad detection of flavivirus have poor 

sensitivity and show strong variation in sensitivity depending on 

virus species. This is very unsatisfactory and therefore a PCR 

assay based on a novel approach was developed and tested on 

a panel of different flaviviruses. 


The following viruses were included in the test panel: WNV 1 and 

2, Usutuvirus, TBEV (3 strains, Yellow fever virus, Dengue virus 

1-4 Japanese Encephalitis virus (two strains) and unrelated virus 

(pestivirus and hepacivirus). Several other viruses were used to 

test the specificity. Amplification primers were designed using 

Jalview etc. The assay was run on a Corbett Rotorgene 3000 

real-time thermocycler. 

Results 

All tested flaviviruses were detected while the unrelated virus was 

not detected. The sensitivity of the assay was similar for all tested 

viruses. For WNV the sensitivity was similar to the specific WNV- 

PCR. 


Our results clearly demonstrate that it is possible to detect many 

different flaviviruses with very good sensitivity. It is likely that this 

assay will also detect emerging variants of previously unknown 

flaviviruses. This assay is an important diagnostic tool for 

surveillance of flavivirus infections. 


Thanks are due to the Swedish institute of infectious disease 

control, to Cecile Baronti at the European Virus Archive project 

and to Tamas Bakonyi and Norbert Novotny for providing virus 

strains. 

References 

1. Moreau et al.(2007) A Real-Time RT-PCR Method for the Universal 

Detection and Identification of Flaviviruses Vector borne and zoonotic 

diseases.7,467-477 

Flavivirus, PCR, WNV, detection

S1 - P - 36 

EXTENDING THE SHELF LIFE OF COMMERCIAL ELISA KITS 

Michael O’Connor, Goretti McDonagh and Ronan G. O’Neill 

Virology Division, Department of Agriculture, Food & the Marine Laboratories, Backweston, Celbridge, Co. Kildare, Ireland 

Kits, ELISA, shelf-life, longevity 

Introduction 

In 2012, veterinary laboratories must use an ever expanding 

range of ELISAs to detect antibodies to, and antigens of, a large 

number of viruses which cause endemic, exotic and emerging 

diseases. The demand for such non-routine testing is impossible 

to anticipate, frequently resulting in kits or portions of kits 

exceeding their stated expiry dates. Veterinary virology has 

developed through in-house and non-commercial ELISAs, 

microtitre cell culture neutralisation tests and virus isolation 

techniques. Reagents and controls for these tests were produced 

in-house or received as gifts. They are in limited supply, valuable 

and often unique. The precedent has been that such reagents 

were stored under optimal conditions and used for as long as 

they worked. COULD THE SAME APPLY TO COMMERCIAL 

ELISAS? 


Competitive and indirect commercial ELISA kits for the antibodies 

to, or the antigens of, classical and African swine fever viruses, 

foot and mouth disease virus, swine vesicular disease virus, 

African horse sickness virus, bluetongue virus, porcine 

reproductive and respiratory syndrome virus, the ruminant 

pestiviruses, ovine herpesvirus 2 and Coxiella burnetii were 

evaluated. Kits stored beyond their stated expiry dates were 

compared at intervals with in-date kits. Control sera remaining 

from used kits and proficiency test sera were retested. Results 

obtained with the same samples using within-date and postexpiry 

date kits were compared. Anticipated shelf life on storage 

was assessed by accelerated degradation at 37 o C. 

Results 

Kits stored at 4 o C for up to 5 years beyond their expiry date 

satisfied all manufacturers’ validity criteria, detected all control 

samples and performed satisfactorily in EU and National 

Reference Laboratory proficiency tests. Positive control serum 

from a blocking ELISA for antibodies to classical swine fever 

virus, stored for 98 months (8 years) at 4 o C, reacted similarly to 

the control serum supplied with the current kit. 

Table 1: Effect of storage for 5 years on the performance of a 

blocking ELISA kit for antibodies to porcine reproductive and 

respiratory syndrome virus 

9/02/05 

Dates tested to 27/09/06 3/10/07 23/03/09 16/04/10 

18/05/05 

Time beyond 

kit expiry date - 9 to - 6 + 10 + 23 + 40 + 53 

in months 

Optical density 

of negative 1.5 1.4 1.7 1.3 1.4 

control serum 

Percentage 

inhibition of 

positive 

control serum 

95 89 89 87 88 

Accelerated degradation at 37 o C for a simulated time of 27 

months had no effect on kits for African horse sickness virus 

antigen and African swine fever virus antibody, although a 

simulated time of 12 months significantly impaired the 

performance of an African horse sickness virus antibody kit. 

Table 2: Percentage inhibition (PI) of NRL proficiency test sera 

examined for antibodies to FMD serotype O using post-expiry 

date blocking ELISA and in-date kit. PI ≥ 50 is positive 

Serum 

ID 

Kit 10 months 

post expiry date 

Same kit 21 

months post expiry 

date 

New in-date kit 

B107 16 22 19 

B108 100 100 100 

B109 95 95 94 

B110 18 28 23 

Ready-to-use substrate/chromogen was sometimes found to 

have developed colour on prolonged storage at 4 o C. This was 

easily recognised and readily replaced by spare reagent from 

newer kits/different manufacturers or from commercial sources. 

Table 3: Time beyond expiry date (in months) of 5 different 

commercial ELISAs for antibodies to bluetongue virus, which all 

gave identical, correct results in NRL proficiency test 

ELISA Kit 1 Kit 2 Kit 3 Kit 4 

Kit 

5 

Indirect (I) or competitive (C) I C C C I 

Months beyond kit expiry date - 6 + 23 + 3 + 18 + 9 

Throughout this study, the only less than satisfactory ELISA kit 

detected was a recently purchased within-date one, which 

yielded atypical and much reduced, although still valid, control 

readings coupled with poor specificity. This kit was replaced free 

of charge by the manufacturer. 


The demonstration of a shelf life of up to 5 years beyond the 

manufacturers’ expiry date means that ELISA kits from 

previous/occasional studies can be safely used. These could 

rapidly identify or exclude novel, rare, exotic or emerging 

infections, rather than waiting for fresh kits from laboratory 

suppliers. It can also reduce wastage, especially where kit usage 

has unexpectedly declined or kits have been stockpiled. Kit 

suppliers may also expand their diagnostic range without the 

need to continuously produce all items in their catalogues. 

Four primary antibodies to cell markers were found by 221 

laboratories to be effective beyond their expiry dates (1). Sixtyfive 

polyclonal and monoclonal antibodies, up to 131 months (11 

years) beyond their expiry date, showed no significant 

deterioration (2). 

The overall performance of the ELISA, rather than its expiry date, 

should be the absolute determinant of whether it may be used. 

Performance monitoring should include not only the meeting of all 

validity criteria but maximum and minimum ODs, sensitivity, 

specificity, trends, time for chromogen development, room 

temperature etc. Critical evaluation of the results by the 

laboratory worker will enable early detection of problems and 

rapid identification of significant results, enhancing the value of 

performing ELISA tests. 

ELISA kits should be stored at the correct temperature, usually 

4 o C, throughout their life. Care should be exercised in 

withdrawing reagent aliquots (aseptically or in aliquots) and 

removing plate strips/sections. Unused materials/components 

should not be left at room temperature while awaiting test 

completion. The substrate/chromogen must be examined before 

use by decanting into a clear container. SOPs and Quality 

Manuals must take account of the likely greatly extended shelf 

life of ELISA kits. 

Every laboratory worker using an ELISA plate or strip has the 

immediate reassurance of seeing that validity criteria and control 

parameters have been met as each test is performed and 

therefore can have appropriate and logical confidence in the 

subsequent results. 

References 

1. Tubbs, RR, Nagle R, Leslie K, Pettigrew NM, Said JM, Corwin DJ, 

Rickert RR & Roche PC. Extension of useful reagent shelf life beyond 

manufacturers’ recommendations. Arch Pathol Lab Med 1998, 122, 1051– 

1052. 

2. Balaton AJ, Drachenberg CB, Rucker C, Vaury P & Papadimitriou JC. 

Satisfactory performance of primary antibodies beyond manufacturers’ 

recommended expiration dates. Appl Immunohistochem Mol Morphol 

1999, 7, 221–225.

S1 - P - 37 

MONITORING OF BOVINE VIRAL DIARRHEA VIRUS (BVDV) IN DAIRY CATTLE HERDS IN POLAND 

Aleksandra Kuta, Mirosław P. Polak, Magdalena Larska, Jan F. Żmudziński 

National Veterinary Research Institute, Department of Virology, Puławy, Poland 

Bovine viral diarrhea, bulk tank milk, Ab ELISA, RT-PCR 

Introduction 

Bovine viral diarrhea virus (BVDV), a Pestivirus of the family 

Flaviviridae is an important pathogen of dairy cattle. BVDV 

infection can lead to: reduced milk production, diarrhea, 

respiratory disorders, interfility, abortion, stillbirth, birth of 

persistently infected (PI) calves. Controlling BVDV is very 

complicated and time-consuming. PI animals may be missed if 

laboratory monitoring is not implemented at herd level. These 

animals shed BVD virus throughout their entire life and they 

are the main source of infection in a herd. This is the reason 

why we should constantly test cattle for the presence of BVDV 

infection. Farmers’ expectation is that this should be done at 

the lowest cost. The aim of the study was to assess the status 

of BVDV infection in selected dairy herds based on bulk tank 

milk serology and RT-PCR. 


The material for the study consisted of 162 samples of bulk 

tank milk collected in 60 dairy herds in Poland. The size of 

these herds was between 15 and 565 cattle. Only one herd 

was vaccinated against BVDV and in another one herd clinical 

problems were observed. 

The SVANOVIR BVDV-Ab ELISA was used to detect 

antibodies in bulk tank milk and 35 samples with high 

Corrected Optical Density (COD) values were additionally 

tested by reverse transcriptase – polymerase chain reaction 

(RT-PCR). Viral RNA was extracted from the milk somatic cells 

using TRI Reagent (SIGMA-ALDRICH, USA), according to the 

manufacturer’s instructions. RT–PCR was carried out using 

a commercial kit (Titan One Tube RT – PCR System, Roche, 

Germany), following the manufacturer’s protocol. Amplification 

was done using primers specific for 5’UTR region, sequences 

of which were published by Vilcek et al. (2). RT-PCR products 

were visualized by electrophoresis in 1.5% agarose gel. 

The results of antibody ELISA were interpreted according to 

Swedish system of classification. Herds were allocated into 4 

different classes based on corrected optical density (COD) 

value. Class: 0 – (COD:

S1 - P - 38 

DETECTION OF BOVINE LEUKAEMIA VIRUS PROVIRAL DNA IN DENDRITIC CELLS BY IN SITU PCR 

Maria Szczotka, Jacek Kuźmak, Ewelina Iwan 

National Veterinary Research Institute, Biochemistry Department, Pulawy, Poland 

Bovine leukemia, dendritic cells, PCR in situ 

Introduction 

Bovine leukaemia virus (BLV), like human and simian T- 

lymphotropic viruses (HTLV-I/II, STLV-I/II, STLV-L), belongs to 

the Deltaretrovirus genus of Retroviridae family and causes 

enzootic bovine leukosis (EBL), a disease of the 

lymphoreticular system. 

Dendritic cells (DCs) are most potent antigen presenting cells 

(APCs) with unique ability take up and process antigens in the 

peripheral blood and tissues. They subsequently migrate to 

draining lymph nodes, where they present antigen to resting 

lymphocytes. They express higher levels of MHC class II and 

accesory molecules on their surface than other professional 

APCs. DCs are generally considered as relatives of monocytes 

and macrophages and to be of myeloid origin. They appear to 

derive from progenitor also capable of forming granulocytes 

and macrophages, although more recently a committed DC 

progenitor, possibly a downstream precursor, has been 

identified. The myeloid mature DCs emphasize by the direct 

development of a form of DCs from blood monocytes. All these 

lines of evidence for a myeloid origin of DC derive from culture 

studies using GM-CSF. The aim of presented study was to 

develop in situ PCR for the detection of proviral DNA of BLV in 

DCs of naturally infected cattle. The results of provirus 

detection by in situ PCR were compared with those obtained 

using conventional PCR i.e. solution phase PCR 


Animals. Investigations were performed on the group of 9 

naturally infected with BLV leukaemic and 5 healthy cows. 

Samples. Blood samples, bone marrow, spleen and lymph 

nodes were examined in this experiment. Blood was collected, 

centrifuged in Histopaque gradient, density 1.077. Cells from 

interphase were collected and monocytes were separated with 

the use of magnetic microbeads labelled with MoAb CD14. 

Samples of lymphoid organs were cut, digested with 

collagenase-DNase, centrifuged and incubated with 

immunomagnetic microbeads coated with monoclonal 

antibody. Cells were passed through the magnetic sorter, the 

cells retendent on the magnet were eluted, washed and placed 

in cell culture for 2 weeks in RPMI in the presence of IL-4 and 

GM-CSF. Cells were grown in special labteks in incubator at 

37 o C in 5% of CO 2 . After that cells were washed, fixed and 

PCR in situ was performed in termocycler. Detection was 

performed with the use of anti-DIG peroxidase conjugate and 

AEC or alkaline phosphatase substrates. 

Immunofluorescence test (IF) and flow cytometry were 

performed with the use of MoAbs anti gp51 –BLV glycoprotein 

for determination of gp51 expression. Flow cytometry was 

used for determination of DCs immunophenotype in healthy 

and naturally infected with BLV cattle. Expression of cytokines: 

IL-6, IL-10, IL-12p40 and IL-12p70 was measured with 

commercial ELISA sets. 

Control. As positive control the foetal lamb kidney 

permanently infected with BLV - (FLK-BLV) cell line was used 

Results 

Generated from the blood and inner organs dendritic cells had 

typical shape, veils and processes. The expression of gp51 

glycoprotein was determined by flow cytometry. 

In dendritic cells infected with BLV we observed very high 

percentage of determinants: CD11a, CD11b, CD11c and 

MHC-II class and very high expression of IL-6, IL-10. IL-12p40 

and IL-12p70 in culture fluids. Leukemic DCs exhibited DCs 

morphology and had a phenotype of mature DCs. The 

expression of gp51 glycoprotein of BLV on leukaemic DCs was 

detected in flow cytometry and immunofluorescence. The cells 

of samples with positive reaction in in situ PCR were dark 

brown stained. The negative controls were unstained. In situ 

PCR results agreed with those of provirus detection using 

conventional PCR and localization of proviral material in cells 

was visible. 

The results of PCR in situ: 

DCs-blood DCs - spleen DCs bone marrow 

DCs – blood 

T 

The results of IF: 

DCs - spleen 


In the present study, DCs generated from peripheral blood and 

different lymphoid tissues of cattle naturally infected with BLV, 

were examined for the presence of proviral DNA. In the first 

approach, called also direct in situ PCR, dNTP’s, labelled with 

digoxygenin, biotin or other markers, are incorporated into 

amplification products; the second approach involves two 

stage process which combines in situ PCR plus in situ 

hybridization with DNA probe. In our study we have used the 

first strategy and digoxigenin labelled dNTP’s. The presence of 

gp51 expression was determined as well in IF as in flow 

cytometry. We obtained agreement in results of in situ PCR 

with results of other test. The DCs infected with BLV had more 

delicate morphology with many vacuoles in cytoplasm. 

Infection with BLV caused an increase in cytokines expression 

and changes in the percentage of surface molecules on 

dendritic cells. With the use of in situ PCR the localization of 

proviral load could be detected. 

Application of in situ PCR would be recommended as a 

method to confirmation of infection with BLV. 


The research was supported by the Polish State Committee for 

Scientific Research, grant No N N 308 622 138. The authors 

thank the Polish State Committee for Scientific Research for 

financial support. 

References 

1.Mirsky M.L., Olmstead C.A., Da Y., Lewin H.A. 1996: The prevalence 

of proviral bovine leukemia virus in peripheral blood mononuclear cells 

at two subclinical stages of infection. J Virol, 70, 2178-2183. 

2. Naif H.M., Brandon R.B., Daniel R.C.W., Lavin M.F. 1998: Bovine 

leukaemia proviral DNA detection in cattle using the polymerase chain 

reaction. Vet Microbiol, 1, 25, 117-129. 

3. Steinman R.M. 1991. The dendritic cell system and its role in 

immunogenicity. Annu Rev Immunol. 9, 271-296. 

4. Ballagi-Pordany A., Klintevall K., Merza M., Klingeborn B., Belak S. 

1992 : Direct detection of bovine leukemia virus infection: Practical 

applicability of a double polymerase chain reaction. J Vet Med B, 39, 

69-77.

S1 - P - 39 

BOVINE BLOOD DENDRITIC CELLS IN CATTLE INFECTED WITH BOVINE LEUKAEMIA VIRUS (BLV) 

Maria Szczotka, Jacek Kuźmak 

National Veterinary Research Institute, Biochemistry Department, Pulawy, Poland 

Bovine leukemia, dendritic cells, immunophenotype 

Introduction 

Dendritic cells (DCs) play an important role in the immune 

system. These cells are specialized in the presentation of 

antigens. DCs are leukocytes derived from bone marrow and 

they are the only cells able to present antigen to naïve T cells. 

They initiate primary immune response, exist in all lymphoid and 

most non-lymphoid tissues and express both MHC class I and 

class II antigens. They not only have an enhancing effect on the 

acquired immunity development, but can induce tolerance and 

can be manipulated to prevent and treat allergic and autoimmune 

diseases. These cells are present in most tissues and comprise a 

small fraction of the leukocytes through the body, representing 

only about 1% of peripheral blood mononuclear cells. DCs are 

heterogenous cells that can be divided into the few functionalldistinct 

subsets on the basis of their phenotype, possibility of 

migration and ability for cytokine production. They modulate both 

innate and adaptive immune response. 

The aim of the study was the isolation of monocytes from bovine 

blood with the use of immunomagnetic beads coated with 

monoclonal antibody, generation of DCs and determination of 

immunophenotype and cytokine expression. 


Isolation of monocytes. 

Investigations were performed on a group of 9 cows naturally 

infected with BLV and 4 healthy, BLV-negative animals in ELISA 

and PCR that served as negative control. Peripheral blood 

mononuclear cells were isolated from whole blood treated with 

EDTA-K 2, by standard density centrifugation in Histopaque 

(density 1.077). The cells from interphase were collected, 

washed twice by centrifugation and incubated with 

immunomagnetic Microbeads (Miltenyi Biotec, Germany), coated 

with monoclonal mouse anti-human CD14 antibody. After 

incubation, cells were centrifuged, cell pellet was resuspended in 

AutoMacs Rinsing Solution and placed on a magnetic column 

MACS LS (Miltenyi Biotec). The magnetically labelled CD14+ 

cells were retained on the column and eluted after removal of the 

magnetic column from the magnet. The viability of the cells was 

assessed by trypan blue exclusion. The purity of the monocyte 

fraction was higher than 90%, determined by FACS analysis. 

Monocyte culture. 

Isolated monocytes were cultured in RPMI 1640 medium 

(containing FCS, glutamax and antibiotics) at 37 0 C in humidified 

5%CO 2 atmosphere, in the presence of granulocyte-macrophage 

colony stimulating factor (GM-CSF) and recombinant bovine 

interleukin- 4. The cells were fed every 3 day and cultivated for 7- 

10 days.during this period, the monocytes transformed into DCs. 

Cytology of DCs 

After 7-10 days, the cultured cells were dislodged by gentle 

pipetting, washed, cytocentrifuged onto slides, air-dried at room 

temperature and stained with Giemsa’s stain. Morphological 

observations were performed under the light microscope 

Olympus, equipped with computer system Lucia (Nikon). 

Ultrastructure of DCs was examined using scanning electron 

microscopy (SEM). Briefly, the cells were fixed in 2,5% 

glutaralaldehyde in 0.1M cacodylate buffer, dehydrated in serially 

graded alcohols (70%-100%), and embedded. Ultrathin sections 

were cut, counterstained, and then observed under SEM. 

Immunophenotype of DCs in both groups of cows was 

determined in flow cytometer with the use of specific monoclonal 

antibodies for CD markers and fluorescent conjugates. 

Results 

In normal bovine blood, the number of monocytes was very low: 

1%-4%. After magnetic separation we obtained a fraction of 

positively enriched (95%-97%) labelled cells. The CD14 positive 

cells had typical monocyte morphology with great nucleus and 

small cytoplasm. These cells cultured 24 h in the presence of 

GM-CSF and IL-4 in a growth medium transformed to the 

dendritic cells. Some of them were elongated and had dendritelike 

processes or edges. After 72 h, almost all cells had typical 

dendrites: some of them were divided on the end. DCs of infected 

cows cultured in vitro had very strong expression of CD11a, 

CD11b, CD11c, MHC-I and MHC-II. Expression of gp51 

glycoprotein was determined in BLV infected cows.by flow 

cytometry 

Tab.1. The immunophenotype of dendritic cells in leukaemic and 

control cattle (% cells – mean values) 

BLV(+) 

CD 

14 

CD 

11a 

CD 

11b 

CD 

11c 

MHC 

I 

MHC 

II 

gp5 

1 

1 week 83,6 42,5 89,5 58,4 84,6 17,0 5,6 

2 weeks 87,9 53,6 84,9 30,0 79,5 16,4 4,1 

3 weeks 92,7 63,2 95,7 31,6 77,5 20,5 5,4 

BLV (-) 

CD 

14 

CD 

11a 

CD 

11b 

CD 

11c 

MHC 

I 

MHC 

II 

1 week 49,4 38,8 67,3 26,5 80,4 20,5 - 

2 weeks 41,6 27,3 75,7 26,5 51,3 9,2 - 

3 weeks 88,4 23,4 72,2 11,2 28,9 10,5 - 

4 weeks 12,7 10,4 41,3 2,1 1,4 30,7 - 

gp5 

1 


The great amount of work performed in the recent years has led 

to the definition of DCs as cells identified by peculiar morphologic 

characteristics and specific functions, such as the ability to 

activate naive T cells. Very little is known about their origin and 

pathways of differentiation. Developing the immunogenic 

potential of DCs for vaccines – and what is very important for 

cancer therapy in humans – requires better access to this cell 

type. This report demonstrates that DCs can be derived from 

positively-selected monocytes in cattle. Large amounts of DCs 

can be obtained after the in vitro exposure of peripheral blood 

monocytes to the effects of cytokine combinations, mainly 

interleukin IL-4 and GM-CSF, but the ability of monocytes to 

convert to DCs in vivo has not been so far established. Although 

their origins are still controversial or remain unknown, myeloidderived 

and lymphoid-derived DCs lineages have been 

described. Through their ability to determine the differentiation of 

either Th1 or Th2 cells, distinct DCs subsets may present 

antigens in vivo in an immunogenic or tolerogenic fashion. It is 

important that the immunostimulatory properties of DCs are 

dependent on their maturity state: immature DCs have been 

shown to be capable of inducing antigen-specific inhibition of in 

vivo T-cell function in humans. Moreover, DCs are potent to 

improve vaccine efficacy and are still being investigated as an 

important tool in cancer therapy. The availability of sufficient 

numbers of efficient antigen-presenting DCs facilitates effective 

vaccine development and the study of T-cell-mediated responses 

to tumour-associated antigens and cancer therapy. 

References 

1.Banchereau, J, Steiman, R.1998, Dendritic cells and the control of 

immunity. Nature, 392, 245-252 

2. Dubois, B, Fayette, J.1999. Dendritic cells directly modulate B cell 

growth and differentiation. J Leukoc Biol, 66, 224-230. 

3. Pinchuk LM, Boyd BL, Kruger EF. 2003. Bovine dendritic cells 

generated from monocytes and bone marrow progenitors regulate 

immunoglobulin production in peripheral blood B cells. Comp Immunol 

Microbiol Infect Dis, 26, 233-249.

S1 - P - 40 

PORCINE CIRCOVIRUS TYPE 2 IN POSTWEANING PIGS WITH ANTIBIOTIC NON-RESPONSIVE 

DIARRHEA 

Anna Szczotka 1 , Jacek Żmudzki 1 , Tomasz Stadejek 2 , Zygmunt Pejsak 1 

1 

National Veterinary Research Institute, Department of Swine Diseases, Pulawy, Poland 

2 

Warsaw University of Life Sciences - SGGW, Faculty of Veterinary Medicine, Department of Pathology and Veterinary Diagnostics Warsaw, Poland 

Pigs, PCV2, diarrhea 

Introduction 

Porcine circovirus type 2 (PCV2) is a causative agent 

of postweaning multisystemic wasting syndrome (PMWS). The 

diseases affects 5 – 15 weeks old pigs and is characterized by 

wasting, dyspnea, enlarged lymph nodes, pallor and jaundice. 

Sometimes diarrhoea could also be observed and this clinical 

manifestation should be differentiated from other swine enteric 

diseases, like proliferative enteropathy (PE ) and swine dysentery 

(SD). The etiological agents of these diseases are, respectively: 

Lawsonia intracellularis (L. Intracellularis) and Brachyspira 

hyodysenteriae (B. hyodysenteriae). 

The aim of the study was to analyze the presence of porcine 

circovirus type 2 (PCV2) in cases of antibiotic non-responsive 

diarrhoea and to evaluate the possible role of the virus in 

development of enteritis in pigs. For differential diagnosis 

identification of L. Intracellularis and B. hyodysenteriae was 

performed. 


Internal organs and feces were collected from 76 pigs, 5 – 19 

weeks old, from 50 farrow-to-finish farms, PMWS-positive or 

PMWS-suspected. Sections of lymph nodes and intestines 

(ileum, caecum and colon) were analyzed for presence of PCV2 

DNA by in situ hybridization test (ISH) (2). They were also 

hematoxilin-eosin (HE) stained for standard histopathological 

examination. Addtionally, fecal samples were tested for presence 

of B. hyodysenteriae and L. intracellularis by PCR (3). 

Results 

In samples from 37 pigs, 10 – 17 weeks old, large amounts of 

PCV2 DNA, typical for PMWS, were detected in lymph nodes by 

ISH. In this group, in samples from 19 pigs, PCV2 was also found 

in abundant amount in samples of ileum. The remaining 18 pigs, 

PCV2-positive in lymph nodes, were negative in ileum. In 

samples from only 1 animal lymph nodes were negative for PCV2 

in ISH, but virus was detected in considerble amount in ileum. In 

HE stained sections of lymph nodes histopathological lesions 

characteristic for PMWS were identified. Similar lesions were 

observed in PCV2-positive samples of ileum. Seventy samples of 

feces were negative in PCR for B. hyodysenteriae and 

L. intracellularis. DNA of L. intracellularis was found in feces from 

3 pigs and mixed infection caused by both bacteria was detected 

in 3 animals 

Figure 2: Presence of multinucleated giant cells (arrows) in 

mesenteric lymph node (left); multiple intracytoplasmic inclusion 

bodies in ileum (right). Hematoxylin and eosin (HE), 200x. 


According to the obtained results in PMWS-affected pigs similar 

lesions could be observed both in lymph nodes and in ileum and 

they correlate with clinical outcome of disease. Also, it was found 

that presence of PCV2 in ileum could be correlated with 

diarrhoea in PMWS- free animal, which confirms that the 

emergence of this virus as an intestinal pathogen may represent 

a new phenomenon (1). In the animals negative for 

B. hyodysenteriae and L. intracellularis and PCV2 other 

causative agents of diarrhoea should be considered. 


This work was supported by the research grant N N308 075634 

from Polish Ministry of Science and Higher Education 

References 

1. Jensen TK, Vigre H, Svensmark B, Bille-Hansen V (2006) Distinction 

between porcine circovirus type 2 enteritis and porcine proliferative 

enteropathy caused by Lawsonia intracellularis. J Comp Pathol 135: 176– 

182. 

2. Stadejek T., Podgórska K., Kołodziejczyk P., Bogusz R., Kozaczyński 

W., Pejsak Z. (2006) Pierwszy przypadek poodsadzeniowego 

wielonarządowego zespołu wyniszczającego świń w Polsce. Medycyna 

Wet., 62 (3), 297-301. [First report of postweaning multisystemic wasting 

syndrome in pigs in Poland]. 

3. Żmudzki J, Osek J, Stankevicius A, Pejsak Z (2004) Rapid detection of 

Brachyspira hyodysenteriae and Lawsonia intracellularis in swine faecal 

and mucosal specimens by multiplex PCR. Bull Vet Inst Pulawy 48: 207- 

214. 

Figure 1: Large amount of PCV2 antigen in inguinal superficial 

lymph node (left) and in ileum (right). In situ hybridization (ISH), 

200x.


“Diagnostics 

at the point of interest” 

(2 nd session)

S2 - P - 01 

EVALUATION OF FIELD BASED METHODS FOR DETECTION OF CLASSICAL SWINE FEVER BY PCR 

Ann-Sophie Olofson 1 , Frederik Widén 1 , Neil Leblanc 1 

1 

SVA, VIP, Uppsala, Sweden 

Introduction 

Classical Swine Fever is a disease of high economical 

importance. Outbreaks in pigs can be extremely costly due to 

mortality, production losses and trade restrictions. In Europe this 

disease is circulating among wild boars. This virus reservoir 

constitutes an important risk for spill over to domestic pigs and 

therefore it is important to detect outbreaks among wild boars as 

early as possible. Field based PCR detection of CSFV offers the 

possibility of very early detection and is therefore an important 

tool for restricting and combating the outbreak. 


Several different PCR instruments, like Cepheid Smartcycler, 

Corbett Rotorgene and Tetracore T4 etc. were compared for 

detection of CSFV using wet kit, dry kit and conventional 

reagents for PCR and several different filter papers, handheld 

magnetic comb and conventional methods for RNA extraction. 

Results 

Using the field based methods for extraction of RNA and field 

based PCR it was possible to detect CSFV with good sensitivity. 

Specific data will be presented. 


The results demonstrates that a truly field based detection of 

CSFV can be accomplished using a battery driven PCR 

instrument an filter paper based extraction of RNA. 


Thanks are due to Tetracore Inc, Rockville, USA for providing 

PCR instruments and reagents 

References 

1. ,Wakeley PR,Errington, J, Squirrell D.Use of a field-enabled extraction and 

PCR to detect BVDV. Veterinary Record 2010;166:238-239 

doi:10.1136/vr.b4783 

PCR, field based, penside, CSFV, Swine Fever

S2 - P - 02 

NEW IMMUNOASSAYS FOR DIAGNOSIS OF ASFV BASED ON VP72: CAPTURE ELISA FOR 

DETECTION OF IGM SPECIFIC ANTIBODIES AND PEN-SIDE TEST FOR BLOOD SAMPLES. 

Teresa Perez 2 , Carmina Gallardo, 1 Angel Venteo 2 , Ana Ranz 2 , Raquel Nieto 1 , Ana Simon 1 , Paloma Rueda 2 , 

Patricia Sastre, P 2 M Luisa Arias 1 , Antonio Sanz 2 

1 CISA-INIA, MADRID, SPAIN; 

2 

INGENASA, MADRID, SPAIN 

ASFV, PEN-SIDE TEST, diagnostic, IgM 

Introduction 

African swine fever (ASF) is a complex and lethal disease of 

swine caused by a large double-stranded DNA virus belonging 

to the Asfaviridae family. The acute forms of the disease are 

devastating and result in very high mortality that may reach 

100% producing a major negative effect on national, regional 

and international trade. ASF it has been endemic in most of 

sub-Saharan African countries and in Sardinia (Italy) until 

2007. In April 2007 its remarkable potential for trans-boundary 

spread was amply demonstrated appearing in the Republic of 

Georgia with subsequent outbreaks to Armenia, Azerbaijan 

and Russia. Currently ASF is considered as being established 

in the Russia Federation threatens to other regions of Europe, 

central Asia and even China, which has the largest pig 

population in the world. Since there is no vaccine available, 

rapid and specific diagnostic procedures are an essential 

component in affected countries. The early appearance and 

subsequent long-term persistence of antibodies make antibody 

detection techniques a key factor in the control of the disease. 

To this end, INGENASA in collaboration with the European 

Union Reference Laboratory for ASF (CISA-INIA), have been 

working in the development an initial standardization of a 

Capture ELISA for detection of specific IgM. In addition has 

been improved a rapid, one-step immunochromatographic strip 

(pen-side test) INGEZIM PPA CROM for its applicability in the 

field to detect specific anti-ASF antibodies directly in blood 

specimens besides serum. 


Although more studies are required for further validation, these 

preliminary results indicate that the p72 based IgM-capture- 

ELISA could be very useful tool for early detection of ASFV 

infection. 

In addition, the analyses of blood samples using the 

immunochromatographic assay INGEZIM PPA CROM allow us 

to detect specific antibodies against ASFV with appropriate 

values of sensitivity and specificity showing the capability of 

the assay for rapid diagnosis of ASF in the field and where 

laboratory support and skilled personnel are limited. 

References 

1. Sánchez-Vizcaíno et al. Scientific report submitted to EFSA on 

African Swine Fever. (2009), 1-141. 

2. J. M. Sánchez-Vizcaíno et al. African Swine Fever: An 

Epidemiological Update. Transboundary and Emerging Diseases, 2012 

(59), S1: 27–35. 

These studies have been made in collaboration with CISA-INIA 

(ASFRISK Project) 


Three different ASFV proteins were used as antigen for the 

IgM-ELISAs which comprised the fused p30-p15 protein, the 

p72 protein and the uncharacterized protein of 33kDa encodes 

by the K205R ORF. For initial standardization and validation of 

the IgM-ELISAs, a panel of experimental serum samples was 

used. The samples were obtained from four independent 

experimental infections using the Kenyan ASF viruses 

belonging to genotype IX and X, the current East Europe 

circulating virus of genotype II and the attenuated and non 

haemadsorbing Portugal ASFV strain NH/P68 (NHV) 

belonging to p72 genotype I. 

To test the applicability of the immunochromatographic assay 

INGEZIM PPA CROM to detect antibodies in blood samples, in 

this study were included a wide panel of paired experimental 

and field porcine serum and blood samples available at CISA- 

INIA. 

The results were compared to those obtained using the OIE 

serological prescribed assays (OIE 2008) as gold standards. 

Results 

The results obtained in the initial standardization and validation 

of the IgM-ELISAs, showed that the new developed IgM 

capture ELISA, using p72 as antigen, detected specific IgM to 

ASFV at early times post infection. These positive samples 

remained negative using HT and K205R based IgM-ELISAs, 

as well as using the OIE prescribed serological assays. 

Results obtained with experimental infections using the 

attenuated and non haemadsorbing Portugal ASFV strain 

indicated that specific antibody response was detected on 

blood samples since day 6 p.i. using the 

immunochromatographic assay INGEZIM PPA CROM until the 

end of the experiment. Same result was obtained using the 

OIE (Indirect ELISA) and INGENASA (Competition ELISA) 

conventional assays.

S2 - P - 03 

PORTABLE PLATFORMS FOR THE DETECTION OF AFRICAN SWINE FEVER VIRUS TESTED IN 

FIELD CONDITIONS IN NORTHERN UGANDA 

Neil LeBlanc 1 , Edward Okoth 2 , Mikhayil Hakhverdyan 1 , Charles Masembe 3 , Richard Bishop 2 , 

Sándor Belák 1 , Karl Ståhl 1 

1 National Veterinary Institute, Uppsala, Sweden 

2 International Livestock Research Institute, Nairobi, Kenya 

3 Makerere Univeristy, Kampala, Uganda 

African Swine Fever, Penside, Real-time PCR 

Introduction 

African swine fever virus (ASFV) is a serious transboundary 

animal disease in Suidae, causing high rates of morbidity and 

mortality. The objective of this study was to produce a portable 

molecular platform lab that is suitable for use in the field or 

modestly equipped laboratories. 

In Southern and Eastern Africa, the presence of the sylvatic 

cycle involving wild Suidae and soft ticks within the Ornithodoros 

family means that the risk of introduction of ASFV into domestic 

swine is always present. However, ASFV can become 

established and maintained in domestic and wild pig populations 

for prolonged periods. In fact, due to low biosecurity, pig to pig 

transmission and contamination are considered the main factors 

in maintaining circulation of the virus. In Uganda, small pig 

holders form a key part of the food supply. The ability to detect 

ASFV at penside can be a key aspect in control of the disease 

and education of the pig owners to create an awareness of 

biosecurity. 


The T-COR 4 portable real-time PCR thermocycler was used for 

amplification and detection (Figure 1). In addition, materials for 

three methods of DNA extraction from whole blood were brought 

in for testing. They included a magnetic bead extraction method 

(Nordiag AB), FTA elute indicating cards, and Tego sample 

cards. 

The real-time PCR assay (ASFV UPL) employed was a new, 

recently published assay based on the universal probe library 1 

(Roche).The reagents used for the assay included the TaqMan 

Fast Advanced Master Mix, as this provided a robust mix that 

was thermostable to deal with field conditions. The primary field 

location was in an area near Koch Goma, Gulu District, Uganda; 

which had no electricity or running water. The laboratory was run 

three times over an eight day period, twice in Koch Goma and 

once in Kampala. 

Results 

The assay was tested in ASFV ring trials from two European 

projects (Epizone and ASFRISK) as well as another panel of 

isolates representing all 22 genotypes of ASFV. They were also 

tested with the 2011 clinical ring trial samples prepared by the 

EURL. The assay performed well and is designed for ease of use 

in the field. The ASFV UPL assay was pre-mixed for use in the 

field, so assay preparation consisted of simply pipetting the mix 

into reaction tubes. The results indicate that platforms now can 

be established at pen-side or in field laboratories that perform at 

a level comparable to sophisticated molecular laboratories. 

Discussions & Conclusions 

The field trial revealed that these methods have potential for 

application in research and monitoring. 

Figure 1. Real-time PCR results from field site in Koch Goma, 

Uganda 

Extraction 

The magnetic bead extraction method appears to be robust and 

the most efficient. However, it involves a good deal of pipetting 

and various reagents. The Tego extraction is extremely simple 

and requires little time but sensitivity is lower. The FTA method 

also works well but there is significant drying time a 30 minute 

heating step, which means that although the method is simple, it 

does take some time. The sensitivity appears in between the two 

former methods. 

Assay 

The ASFV UPL assay worked was sensitive, reliable and robust. 

It is simple to use because it can be pre-mixed before going in 

the field. 

Instrument 

The TCOR 4 instruments functioned very well and managed to 

do several runs in a genuine field setting without fail. Re-charging 

the instruments by plugging into a vehicle outlet using a 12V 

universal adapter was effective in keeping the field lab functional 

while off the grid for several days. 

Future 

Further efforts are now ongoing in collaboration with the 

International Livestock Research Institute in Kenya and Makerere 

University in Uganda. This includes another trial to facilitate the 

use of a portable ASFV platform in a major field study. 

References 

1. Fernández-Pinero, J, Gallardo, C, Elizalde, A et al. (2012). Molecular 

Diagnosis of African Swine Fever by a New Real-Time PCR Using 

Universal Probe Library. Transbound. and Emerg. Dis., 

DOI: 10.1111/j.1865-1682.2012.01317.x


“Emerging, re-emerging 

and wildlife diseases – 

diagnostic possibilities” 

(3 rd session)

S3 - P - 01 

BEAD-BASED SUSPENSION ARRAY FOR THE SIMULTANIOUS DETECTION OF ANTIBODIES 

AGAINS THE RIFT VALLEY FEVER VIRUS NUCLEOCAPSID AND GN GLYCOPROTEIN 

René Achterberg 1 , Fimme Jan van der Wal 1 , Matthijn de Boer 2 , Hani Boshra 3 , Alejandro Brun 3 , Kitty Maassen 1 , 

Jeroen Kortekaas 1 

1 Central Veterinary Institute of Wageningen UR, Department of Bacteriology, Lelystad, The Netherlands 

2 Department of Infectious Diseases and Immunology, Virology Division, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands 

3 Centro de Investigación en Sanidad Animal (CISA-INIA), Carretera de Valdeolmos-El Casar s/n, Valdeolmos, Madrid, Spain 

Introduction 

Rift Valley fever virus (RVFV) is a mosquito borne virus that was 

first isolated in 1930 in Kenya, and has since caused devastating 

outbreaks throughout Africa. 

The glycoprotein Gn and the nucleocapsid protein N were used 

to develop a Luminex assay for the simultaneous detection of 

antibodies against these proteins. 

The resulting assay was evaluated using a well characterized 

panel of sera. The assay correctly identifies seropositive serum 

samples. In addition, results show that the assay may be used to 

differentiate infected from vaccinated animals (DIVA) when new 

vaccines based on RVFV glycoproteins are were used. 


Recombinant Gn and N proteins were coupled to Luminex 

paramagnetic MagPlex beads using standard coupling chemistry. 

The assay was evaluated using ring trial sera (1), field sera, and 

experimental sera from lambs vaccinated with a recombinant 

NDV virus expressing the RVFV Gn and Gc proteins (2). 

The sets include sera from sheep, cattle and humans and were 

tested using anti-species IgG and anti-species IgM. 

Results 

Ring trial (1) sera (57 sheep, 13 cattle) were defined ‘positive’ if 

at least one of the ELISAs in the ring trial gave a positive result at 

all participating laboratories. Most of the sera that scored positive 

in the ring trial scored positive in the Gn/N IgG Luminex assay. 

Sera that scored positive in the ring trial, but negative in the IgG 

Luminex assay, were found positive in the IgM Luminex assay. 

The results obtained from the field sera (11 ovine, 5 bovine and 

16 human) using the Gn/N IgG Luminex assay corresponded with 

data (unpublished) from an IgG ELISA. 

Animals, vaccinated with a Gn vaccine that lacks N, over time 

developed an IgG response exclusively against Gn, as 

demonstrated with the Gn/N IgG Luminex assay. 


The RVFV Gn/N Luminex assay can be used for the 

simultaneous detection of antibodies against the RVFV Gn and N 

proteins in a single sample. The assay is capable of detecting 

IgG and IgM antibodies in multiple species and performs 

comparable to existing ELISAs. The results further show that the 

assay may be used as DIVA test to accompany glycoprotein 

based RVFV vaccines. 


The authors would like to thank Catherine Cêtre-Sossah, Philippe 

Marianneau, Michel Pépin, Martin Eiden, Francesc Xavier Abad 

Morejón de Girón, Christiaan A. Potgieter and Shirley J. Smith for 

sera, Jan Wichers and Rob Moormann for helpful discussions. 

This research was funded by the Dutch Ministry of Economic 

Affairs, Agriculture and Innovation. 

References 

1. Kortekaas et al., European ring trial to evaluate Rift Valley fever virus 

ELISAs, submitted 

2. Kortekaas, J., de Boer, S.M., Kant, J., Vloet, R.P., Antonis, A.F., 

Moormann, R.J., 2010. Rift Valley fever virus immunity provided by a 

paramyxovirus vaccine vector. Vaccine 28, 4394-401 

RVFV, serology, Luminex, DIVA, suspension array

S3 - P - 02 

LIPOPOLYSACCHARIDE-CAPTURE ELISA FOR THE DETECTION OF LEPTOSPIRA 

BORGPETERSENII SEROVAR HARDJO IN SHEEP 

Zbigniew Arent, Colm Gilmore, William Ellis 

OIE Leptospirosis Reference Laboratory, Veterinary Sciences Division, AFBI, Belfast, Northern Ireland, UK 

Leptospira, leptospirosis, sheep, ELISA 

Introduction 

Endemic infection of sheep with Leptospira borgpetersenii 

serovar Hardjo (type Hardjo Bovis) is now recognised in many 

countries. Many authors demonstrated that infection in sheep is 

economically and clinically significant, particularly with regards to 

reproductive problems (1).This type of infection is often attributed 

to abortion, stillbirth, agalactia and death of weak newborn lambs. 

In addition, Hardjo infection is an occupational zoonosis of those 

who work with sheep. All the evidence points to sheep being an 

alternative maintenance host for this serovar. The efficacy of 

epidemiological or diagnostic studies of leptospirosis in sheep 

relies mainly on the correct identification of animals which have 

been exposed to infection. However, seroprevalence studies in 

sheep are difficult to interpret because the microscopic 

agglutination test (MAT) - standard serological test, detects only 

short-live immunological responses and many previously 

exposed and/or infected animals are seronegative. . Therefore 

there is a need for more sensitive test than the MAT as an 

indicator of past exposure. 

Leptospiral lipopolysaccharide (LPS) has been identified as an 

immunodominant antigen, both in response to infection and to 

vaccination. It has proved useful for diagnosing previous 

exposure of cattle to Hardjo infection as it can be used to detect 

specific IgG antibodies to be suitable for diagnostic purposes as it 

is more specific and it may give some indication of the immune 

status of an animal. 

The aim of this study was the development of antibody-capture 

ELISA using a monoclonal antibody directly against LPS epitope 

and its optimisation for use in sheep 


Plates were coated with monoclonal antibody against Leptospira 

borgpetersenii serovar Hardjo lipopolysaccharide produced and 

characterized earlier in our laboratory (2). 

Carbohydrate (CH) antigen was extracted from culture of 

Leptospira borgpetersenii serovar Hardjo type Bovis strains 0294 

using the hot-phenol–water method. 

Mouse monoclonal anti-ovine IgG antibodies prepared earlier in 

our laboratory were used to prepared conjugate. The monoclonal 

Ig’s were purified on a Protein G column and covalently attached 

to horseradish peroxidase (HRP) using periodate oxidation 

method. TMB substrate was used as colorimetric indicator. 

After reading the plate at 405 nm results were normalized by 

expressing the corrected optical density (Serum OD – Negative 

Control OD) as the percentage of positivity (%P) of the positive 

control serum. The optimal cut-off value for the ELISA was 

determined by receiver operating characteristic (ROC) analysis. 

Correlation between MAT and ELISA was evaluated using 560 

field sheep serum samples 

Results 

A cut-off point was calculated using 80 serum samples obtained 

from flocks with no history of Leptospirosis and which had no 

serological evidence of exposure to serovar Hardjo and 60 

Hardjo MAT-positive sheep sera. The ROC analysis estimated 

the optimized ELISA cut-off as %P ≥ 4.5, with corresponding 

diagnostic sensitivity at 95% (95% Cl: 86.1 - 99.0) and specificity 

at 98.75% (95% Cl: 93.2 - 100.0) (Fig1). 

Sensitivity (%) 

100 

80 

60 

40 

20 

Fig 1 ROC curve analysis 

0 

cut-off value: >4.5 

0 20 40 60 80 100 

100-Specificity (%) 

The percentage of positive results obtained by ELISA test and 

MAT applied to field sera are presented in Tab. 1. In the MAT, 

reactions to serovar Hardjo were with 28 ovine sera (5.0%) 

reacting at 1/100 or greater. When using ELISA, 82 (14.6%) of 

the sera were positive. Only 5 (0.9%) sera were positive in MAT 

and negative in ELISA test. 

Tab. 1 Comparison of ELISA and MAT results 

ELISA 

Positive 

ELISA 

Negative 

MAT 

≥ 1:100 

MAT 

Negative 

TOTAL 

for ELISA 

14.6% 85.4% 

TOTAL 

for MAT 

4.6% 0.4% 5.0% 

10.0% 85.0% 95.0% 


The results of the study demonstrated that the ELISA was more 

sensitive than MAT. The differences in correlation between the 

MAT and ELISA tests was to be expected since the tests 

measure different classes of antibody. The MAT detects both IgG 

and IgM antibodies whereas the ELISA used in this study detects 

only IgG. This shows that our ELISA has some limitation 

especially as an individual animal test, but despite this, the higher 

sensitivity, when compared with the MAT indicates obvious 

advantages in use of the ELISA as a flock test, where the aim is 

to estimate exposure and immunity levels in the flock. The 

ELISA seems to be a valuable complement to serological 

diagnosis of leptospirosis but cannot replace MAT, which 

represents the gold standard. 

Advantages of LPS-captured ELISA test: 

- Leptospira Hardjo specific 

- Better indicator of past exposure than MAT 

- Safe and easy to use 

- Suitable for screening large numbers of sera 

References 

1. Ellis et al. (1983) Possible involvement of leptospires in abortion, 

stillbirths and neonatal deaths in sheep. Vet Rec 26, 291-293. 

2. Yan K.-T. et al. 1999. Development of an ELISA to detect antibodies to 

a protective lipopolysaccharide fraction of Leptospira borgpetersenii 

serovar hardjo in cattle. Vet Microbiol 69, 173-187.

S3 - P - 03 

EVALUATION OF DIRECT BLOOD POLYMERASE CHAIN REACTION FOR RAPID DETECTION OF 

AFRICAN SWINE FEVER VIRUS 

Claudia Gabriel 1 , Marianna Khachatryan 1 , Immanuel Leifer 1,2 , Sandra Blome 1 , Johanna Zemke 1 , 

Bernd Hoffmann 1 , Martin Beer 1 

1 

Friedrich-Loeffler-Institut, Institute of Diagnostic Virology, Greifswald – Insel Riems, Germany 

2 

Present address: Institute of Virology and Immunoprophylaxis, Mittelhaeusern, Switzerland 

Introduction 

Most polymerase chain reaction (PCR) techniques for the 

detection of African swine fever virus (ASFV) require labourintensive 

and expensive DNA extraction steps. Nowadays, PCR 

kits are available that allow PCR directly from blood samples. 

Here, we report on the evaluation of such kits in combination with 

routine primers. 


In the initial phase of the study, two direct PCR kits were 

assessed using positive EDTA blood samples obtained from 

animals experimentally infected with a highly virulent ASFV 

isolate from Armenia: A) the Phusion® Blood Direct PCR Kit 

(Finnzymes Oy, Vantaa, Finland; now Thermo Fisher Scientific, 

Waltham, USA) and B) the KAPA Blood PCR Master Mix 

(PEQLAB Biotechnologie GmbH, Erlangen, Germany). For 

specific detection of ASFV, primers published by King et al. 

(2003) were used. 

Both test kits were optimised for gel-based PCR regarding 

sample volume and temperature profile referring to the 

manufacturer’s recommendations. Subsequently, the direct PCR 

Kits were tested with negative samples, positive samples from 

animal trials, and dilution series of positive samples in 

comparison with the routinely used real-time PCR assay. 

Results 

After preliminary optimisation, ASFV was reliably detected in 

blood samples from experimentally infected pigs. Both with these 

experimental samples and dilution series of positive samples, the 

direct PCR proved to be as sensitive as established real-time 

PCR protocols. 


Concluding, this assay is favourable in both economical and 

technological terms and allows the analysis of very small sample 

volumes. Moreover, direct blood PCR could be advantageous for 

pen-side diagnostic applications. 

References 

1. King DP, Reid SM, Hutchings GH, Grierson SS, Wilkinson PJ, Dixon LK, 

Bastos AD, Drew TW. (2003). Development of a TaqMan PCR assay with 

internal amplification control for the detection of African swine fever virus. J 

Virol Methods, 107(1):53-61 

African swine fever virus, Diagnosis, Direct PCR

S3 - P - 04 

DEVELOPMENT OF A SCHMALLENBERG VIRUS ANTIBODY ELISA 

Christian Schelp 1 , Yann Senechal 1 , San Pun 1 , Daniel Schumacher 1 , Dragos Gradinaru 2 , Christoph Egli 1 , Jean- 

Luc Troch 1 , John Lawrence 3 , Serge Leterme 3 

Introduction 

Schmallenberg virus was first identified in Germany in late 2011 

as a new Orthobunyavirus genetically highly similar to viruses of 

the Simbu serogroup. The virus infects ruminants such as cattle, 

sheep and goats. Schmallenberg virus infections have been 

recently confirmed in several European countries including 

Germany 1 , The Netherlands, Belgium, France, United Kingdom, 

Luxembourg, Italy and Spain. Clinical signs include reduced milk 

yields, fever, diarrhea, abortions and malformed newborns. Virus 

detection by real time RT-PCR or virus cultivation is not the 

method of choice for infection monitoring due to the very short 

viremic period. Therefore, there is an urgent need for an antibody 

ELISA to identify infected animals. 


Hundreds of samples originating from clinically affected herds, 

from virus positive animals and from areas with no 

Orthobunyavirus presence are being collected. Samples will be 

further characterized by using an inhouse immunofluorescence 

assay, PCR and epidemiological and clinical data. 

An antibody ELISA will be described. Microtiter plates will be 

coated with inactivated Schmallenberg-virus antigen. Binding of 

Schmallenbergvirus antibodies will be visualized by colour 

change in the wells of the microtiter plate. The diagnostic 

relevance of the result will be assessed by comparing the optical 

density (OD) of the samples with the OD of the positive control. 

Results 

Preliminary data will be presented. Specificity and sensitivity data 

will be calculated using characterized samples. The data will 

allow for setting a cut-off to identify Schmallenbergvirus antibody 

positive and negative samples. 


The data will be discussed and conclusions will be proposed 

according to obtained antibody test performance. 


We kindly acknowledge the collaboration with the FLI (Friedrich- 

Loeffler-Institut), Insel Riems, Germany. 

We also would like to thank for the assistance by various 

institutes and veterinarians across Europe in helping to collect 

samples and additional data. 

References 

1. Hoffmann, B, Scheuch, M, Höper, D, Jungblut, R, Holsteg, M, 

Schirrmeier, H, Eschbaumer, M, Goller, K.V., Wernike, K, Fischer, M, 

Breithaupt, A, Mettenleiter, T.C., Beer, M (2012). Novel Orthobunyavirus in 

Cattle, Europe, 2011. Emerging Infectious Diseases, vol. 18, 469-472 

1 

IDEXX Switzerland AG, Liebefeld, Switzerland 

2 IDEXX Montpellier SA, Montpellier, France 

IDEXX Laboratories, Westbrook, USA 3 

Schmallenberg virus, antibody ELISA, Orthobunyavirus

S3 - P - 05 

DETECTION OF ECHINOCOCCUS MULTILOCULARIS IN FOXES’ FAECES BY PCR WITH THE USE OF 

DILUTED DNA SAMPLES – COMPARISON OF DIFFERENT METHODS OF DNA ISOLATION 

Jacek Karamon, Jacek Sroka, Tomasz Cencek 

National Veterinary Research Institute, Department of Parasitology and Invasive Disease, Pulawy, Poland 

Key words: Echinococcus multiloculais, PCR, copro-DNA, inhibitors, foxes 

Introduction 

Echinococcus multioloculais infection is one of the most 

dangerous zoonoses and still remains a significant public health 

problem. Definitive hosts of this tapeworms are carnivores 

(especially foxes, but also dogs) which disperse the invasive 

eggs with faeces. A lot of prevalence study are conducted in 

populations of foxes and dogs, in order to estimate the infection 

risk for people. In wild carnivires post mortem examinations of 

intestines are recommended - among them the SCT – regarded 

as the “gold standard”. But there are many situations when 

investigation must be carried out in vivo (for example examination 

of dogs populations). Therefore there are some techniques for 

detection of Echinococcus infection in definitive hosts by feaces 

examination and among them PCR for copro-DNA detection is 

one of the most usefull and sensitive. However, there are many 

problems connected with PCR inhibiting factors contained in 

faeces. The aim of this investigation was to compare the 

effectiveness of different methods of E. multilocularis DNA 

isolation from faeces for PCR performed with different DNA 

dillutions - to choose the variant minimizing the inhibiting influent 

of faeces. 


Intestines (small and large) used in investigation were obtained 

from 35 foxes. Small intestines were examined by sedimentation 

and counting technique (SCT). Samples of faeces (about 2 g) 

collected from the end of rectum were frozen (-20 o C) for 

molecular examination., 

DNA from samples were isolated and purified with the use of 3 

different methods: 

(L)– with the use of QIAamp DNA Stool Mini Kit (Qiagen) 

according to protocol for larger volumes of stool (1g samples 

were used) 

(S)– with the use of QIAamp DNA Stool Mini Kit (Qiagen) 

according to standard protocol for (200 mg samples were used) 

(T)- with the use of QIAamp DNA Mini Kit (Qiagen) according to 

standard producer protocol (20 mg samples were used) 

DNA isolated in each method were tested by PCR in 6 variants:. 

one not diluted (1/1) and 5 diluted (1/2.5, 1/5, 1/10. 1/20, 1/40) 

A nested PCR method was used, as described by Dinkel et al. 

(1998) with some modifications concerning reaction mixture and 

time conditions of amplification (Karamon et al 2012). The 

sequence for amplification was part of the E. multilocularis 

mitochondrial 12S rRNA gene. In a second stage of PCR the 

fragment specific for E. multilocularis (250 bp) was amplified. 

In case of 25 foxes additional faecal samples were collected and 

examinmed at once for detection od Taenidae eggs by McMaster 

flotatuion technique. 

Fig. 1. Percentage of E. multilocularis positive samples detected 

by SCT and PCR (3 variants of DNA isolation) and Taenidae 

positive samples found by flotation. 

. 

% of positive samples 

60,0 

50,0 

40,0 

30,0 

20,0 

10,0 

0,0 

51,4 

45,7 

40,0 

34,3 

20,0 

SCT PCR L PCR S PCR T Flotation 

(Taenidae 

eggs) 

Results 

E. multilocularis tapeworms were found in 18 from 35 (51,4%) 

intestines examined by SCT. The intensity was ranged from 2 to 

1055 worms per intestine. 

The highest number of positive results in PCR (16) were obtained 

in isolation variant (L) (isolation with the use of larger stool 

volume), and then 14 and 12 respectively for (S) and (T) variants 

of isolation. In one case (sample No. 19) sample negative in 

SCT was estimated as positive in PCR (L). 

In some samples positive results in PCR were obtained only in 

diluted DNA. From 25 faeces examined by flotation only in 5 

(20%) Taenidae eggs were detected 

Table 1. Selected results obtained by PCR for detection of E. 

multilocularis copro-DNA (in different DNA dilution and with the 

use of 3 different variants of DNA isolation) in comparison to 

microscopic sedimentation and counting technique (SCT). 


Blocking of PCR by inhibitors contained in faeces is the known 

problem in molecular diagnostics. When the concentration of 

inhibiting substances in the sample is high, the elimination of 

them during DNA purification (also by using dedicated methods) 

may be insufficient In that cases significant amount of them leave 

in the DNA sample and can block the PCR - causing possibility of 

false negative results. Some authors demonstrated that samples 

which block the PCR may be identified by repeated examination 

of all negative ones with adding to each one additional positive 

control (Dinkel et al 1998). But in such method all identified 

inhibiting samples had to be eliminated from further analysis at 

all, because there were still no answer which ones were really 

positive or negative. 

However, our investigation showed that in E. multilocularis PCR 

additional using diluted DNA (besides non diluted) could avoid 

false negative results causing by PCR inhibition. In the (L) 

method of purification using additionally 1/10 dilution of DNA 

seems to be enough. But, when DNA purification was done by 

the method not dedicated for faeces, DNA samples required 

more and higher dilutions (because of higher inhibitors 

concentration). 

References 

1. Dinkel, A. et al Echinococcus multilocularis in the definite host, 

coprodiagnosis by PCR is an alternative to necropsy. J. Clin. Microbiol. 36, 

1871–1876 (1998) 

2. Karamon et al. The first detection of Echinococcus multilocularis in 

slaughtered pigs in Poland. Vet Parasitol 185 327– 329 (2012)

S3 - P - 06 

COMPARISON OF CULTURE AND PCR FOR DETECTION OF MYCOBACTERIUM AVIUM SSP. 

PARATUBERCULOSIS IN BOVINE FAECES 

Selina Keller 1 , Nicole Cernela 2 , Roger Stephan 2 , Max M. Wittenbrink 1 

1 

Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Zurich, Switzerland 

2 

Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, Switzerland 

Introduction 

Paratuberculosis is an incurable granulomatous enteropathy 

caused by Mycobacterium avium ssp. paratuberculosis (MAP) 

affecting primarily wild and domestic ruminants with animals up to 

six months of age being most receptive (1). Common route of 

infection is faecal-oral with faeces or milk and incubation time 

ranges from months to years. Together with the late onset of 

symptoms, this makes diagnosis of paratuberculosis very 

challenging. The aim of this study was to compare a direct real 

time PCR with a cultural approach for screening faecal samples 

of healthy animals from different farms with a history of 

paratuberculosis. 


This study comprised faecal samples of 1266 animals from 13 

dairy cattle and 10 sucking cow herds. In these herds at least one 

animal had been diagnosed with paratuberculosis in the last 5 

years. Herd sizes ranged from 11 to 130 animals. 

Paratuberculosis, culture, PCR, cattle 

Culture and PCR were performed from three pooled faecal 

samples at a time (2). For culture, a decontamination with 4% 

NaOH and 5% oxalic acid was used to reduce the accompanying 

bacterial flora before the 422 faecal pools were transferred onto 

selective media containing Mycobactin J, namely Löwenstein- 

Jensen (LJ) and Herrold’s Egg Yolk agar (HEYA) (3). Suspicious 

colonies were inoculated into Middlebrook 7H9 broth enriched 

with ADC and Mycobactin J (4). After one week of multiplication, 

Ziehl-Neelsen staining was performed for detection of acid fast 

bacilli. For confirmation of MAP, real time PCR based on the F57 

element was used (5). Moreover, for direct detection of the 

pathogen in pooled faecal samples, the same real time PCR 

protocol was performed. 

Results 

After incubation for 16 weeks, in 62 of the 422 (14.7%) faecal 

pools cultivation of MAP succeeded (Figure 1), of which 48 

(77.4%) grew on HEYA, 2 (3.2%) on LJ and 12 (19.6%) grew 

both on LJ and HEYA (Figure 2). However, only 9 of 422 (2.1%) 

faecal pools tested positive by direct real time PCR. In general, 

HEYA revealed positive cultures already after 9 weeks of 

cultivation, whereas 11 weeks were necessary for visible growth 

on LJ. 

For identification of the MAP spreading animals, samples (n=186) 

of the 62 positive tested pools were individually subjected to 

culture and PCR. Cultivation of MAP succeeded in 59 of the 186 

(31.6%) faecal samples, whereas only 12 of 186 (6.4%) tested 

positive by direct real time PCR. 

Figure 2: Growth of MAP on HEYA and LJ 


The results of this study show that for MAP detection in faecal 

samples from animals without clinical signs, the cultural assay is 

a more sensitive method compared to F57 real time PCR. 

Nevertheless, considering the long cultivation time and the workintensive 

procedure, the applicability in view of eradication 

systems remains restricted. The limited performance of PCR, 

however, may be due to the fact that in the cultural assay 1244- 

times more faecal material is used. Results from this study also 

highlight the impact of the medium chosen for cultivation since 

HEYA revealed a much higher prevalence of MAP compared to 

LJ. 

References 

1. Bögli-Stuber, K., et al. (2005): Detection of Mycobacterium avium 

subspecies paratuberculosis in Swiss dairy cattle by real-time PCR and 

culture: a comparison of the two assays, 

J Appl. Microbiol. 99:587–597. 

2. Kalis, C.H.J., et al. (2000): Culture of strategically pooled bovine fecal 

samples as a method to screen herds for paratuberculosis, Vet. Diagn. 

Invest. 12:547–551. 

3. Beerwerth, W. (1967): Die Züchtung von Mykobakterien aus dem Kot 

der Haustiere und ihre Bedeutung für die Epidemiologie und Bekämpfung 

der Tuberkulose, Prax. Pneumol. 21:189–202. 

4. Glanemann, B., et al. (2004): Detection of Mycobacterium avium 

subspecies paratuberculosis in Swiss dairy cattle by culture and serology, 

Schweiz. Arch. Tierheilk., 146:409-415. 

5. Bosshard, C., et al. (2006): Application of an F57 sequence-based realtime 

PCR assay for Mycobacterium paratuberculosis detection in bulk tank 

raw milk and slaughtered healthy dairy cows, J Food Prot. 69:1662-7. 

Figure 1: MAP detection in faecal pools by PCR and culture.

S3 - P - 07 

HEPATITIS E VIRUS IN DOMESTIC SWINE AND WILD BOAR FROM GERMANY 

Oliveira-Filho, E.F, Bank-Wolf, B.R., Thiel, H-J., König, M. 

Institute of Virology, Justus-Liebig University Gießen, Schubertstr. 81, 35392 Gießen 

Introduction 

Hepatitis E is an emerging zoonotic disease distributed 

worldwide. The causative agent Hepatitis E virus (HEV) 

belongs to the genus Hepevirus of the virus family 

Hepeviridae. Another member of the family not assigned to a 

genus so far is the avian HEV. 

HEV is non-enveloped with a single stranded RNA genome of 

positive polarity. The genome encompassing approximately 

7.2 kb encodes three open reading frames. Members of HEV 

can be subdivided into at least 4 genotypes (1-4). Virus 

isolates from rats and rabbits may belong to additional new 

genotypes. 

HEV may cause acute hepatitis in humans but infection often 

remains subclinical. Besides man HEV was also found in 

domestic animals such as swine as well as in wildlife species 

like wild boar, deer, rabbit and rat. So far, no clinical disease 

has been associated with HEV infection in these animals. 

In this study we intended to investigate the presence of HEV in 

wild boar and domestic swine in Germany. Phylogenetic 

analyses were performed to form the basis for molecular 

epidemiology and the improvement of diagnostic tests. 


A total of 105 porcine faecal samples and 124 sera from wild 

boar were tested by conventional RT-PCR using different 

primer combinations. In addition a quantitative real time PCR 

(qPCR) test for HEV was set up. 

PCR amlicons were cloned and nucleic acid sequences 

obtained and compared to sequence data from GenBank. 

Phylogenetic analyses using partial as well as complete 

sequences of the HEV capsid gene were performed. 

Results 

A fragment of 241 nucleotides was detected in 13.7% of the 

sera from wild boar and in a single faecal sample from a 

domestic swine (0.95%). All viruses could be assigned to 

genotype 3 of HEV. Sequence diversity was significant with 

differences of up to 21.6% among the wild boar samples. 

The complete capsid protein gene was cloned and sequenced 

from three wild boar samples and the single positive domestic 

swine. Based on this sequence data the isolate from domestic 

swine was assigned to HEV subtype 3a and the three wild 

boar viruses to subtypes 3a and 3i. 

Additional analyses indicated recombination between 

members of HEV subtypes as one reason for sequence 

diversity. 


Our study indicates that HEV is circulating among wild boar in 

Germany and may be transferred to domestic swine. These 

data support observations by others (1, 2). 

The close relationship of the animal and human HEV 

sequences implies the risk for zoonotic transmission of the 

virus. Phylogenetic analyses should be based on complete 

capsid gene sequences to allow a significant separation of 

subtypes. 


Samples from wild boar were kindly provided by Landesbetrieb 

Hessisches Landeslabor Giessen. 

References 

1. Schielke, A, Sachs, K, Lierz, M, Appel, B, Jansen, A, Johne, R 

(2009). Detection of hepatitis E virus in wild boars of rural and urban 

regions in Germany and whole genome characterization of an endemic 

strain. Virol J. 14;6:58. 

2. Wenzel JJ, Preiss J, Schemmerer M, Huber B, Plentz A, Jilg W. 

(2011). Detection of hepatitis E virus (HEV) from porcine livers in 

Southeastern Germany and high sequence homology to human HEV 

isolates. J Clin Virol. 52(1):50-4. 

HEV, zoonosis, wild boar, phylogenetic analysis

S3 - P - 08 

EMERGENCE OF SCHMALLENBERG VIRUS: 

DEVELOPMENT AND VALIDATION OF A REAL-TIME PCR DETECTION KIT 

Damien MAGNEE 1 , Stéphane DALY 1 , Sandrine MOINE 1 , Eric SELLAL 1 

1 

LSI Laboratoire Service International, LISSIEU, France 

Schmallenberg, Real-Time PCR, Outbreak, 

Introduction 

Between August and December 2011, outbreaks of disease in 

adult cattle, abortions and births of malformed animals in sheep, 

cattle and goats were reported in the Netherlands, Germany and 

Belgium. A new virus was identified as a cause of these problems 

and was named “Schmallenberg virus” after the place where it 

was first identified. 

This virus belongs to the Bunyaviridae family, genus 

orthobunyaviridae and is closely related to Akabane, Aino and 

Shamonda viruses. Preliminary studies have suggested that this 

virus affects mainly ruminants and must be transmitted by a 

vector (biting midges of the genus Culicoides, mosquitoes...). 

We present here the development of new real-time PCR kits for 

identification of Schmallenberg virus, by targeting the S segment, 

and the different validation steps leading to final authorisation for 

SBV diagnosis by the French National Reference Laboratory 

(ANSES Maisons-Alfort) and by the Friedrich-Loeffler-Insitut in 

Germany. 


Systems for specific detection of Schmallenberg Virus were 

designed on the basis of the sequence deposited on Genbank. A 

first system was designed on the L segment. Then, according to 

the FLI recommendations, we realised a new design, based on 

the detection of the S segment. The LSI SBVS kit allows the 

simultaneous detection of SBV target and an endogenous IPC. 

Detectability of the both kits was compared with the FLI design 

using serial dilution of SBV RNAs provided by the Friedrich- 

Loeffler Institut (Germany). Analytical specificity and sensitivity of 

L and S segment kit were assessed using several field samples 

(brains), coming from France and Belgium. 

Specificity of prototype kits was evaluated on a panel of ruminant 

pathogens and other orthobunyaviridae. 

Both prototype kits were sent to the Friedrich Loeffler Institut and 

the S segment prototype kit was sent to the French NRL 

(ANSES-Maisons Alfort) for evaluation of their specificity, 

sensitivity, detectability and repeatability on field samples, in 

order to receive official validation for diagnosis in France and 

Europe. 

SBV virus detection is preferably performed on brains of aborted 

fetus, but the virus can also be located in blood, serum and 

spleen. For brain and spleen samples, a grinding step is 

necessary. 

Results 

The both systems showed good specificity, with detection of all 

positive samples and no detection of other ruminant pathogens. 

These kits have equivalent detectability with the FLI designs, with 

better sensitivity for the S segment relative to the L segment (for 

FLI and LSI design). 

On 34 field samples (brains coming from stillbirths with 

malformations), we have better detectability with LSI systems, 

with 29 positives samples with LSI S segment PCR and 25 

positives with FLI PCR. 

Characteristics of the both kits obtained at LSI were confirmed at 

the French NRL and the Friedrich Loeffler Institut. Kits showed 

good sensitivity, specificity and detectability (Table 1). 

Table 1: Official evaluation of LSI SBV Segment S kit 

Sensitivity 

and 

specificity 

Detectability 

ANSES (France) 

37 samples with 

100% correlation 

2 negatives samples 

with FLI system are 

positive with LSI kit 

Gain of 1 log 

FLI (Germany) 

40 field samples 

with 100% 

correlation 

100% correlation 

with FLI system 

The SBV-S segment kit commercialised by LSI received official 

authorisation for utilisation in the French network for diagnosis of 

Schmallenberg virus and was also validated by the FLI, for use in 

European market. 


We showed here all the steps leading to official validation of the 

SBV LSI kit in Real-Time rt-PCR, by the French National 

Reference Laboratory and the Friedrich Loeffler Institut. 

The first step was to develop a PCR kit, targeting the L segment, 

with exogenous IPC. After recommendation by the FLI to target 

preferably the S segment and request of the French NRL to use 

an endogenous IPC, we develop a second kit with segment S 

detection, with endogenous IPC. 

The initial validation at LSI shows good results in sensitivity, 

specificity and detectability. A panel of 34 field samples show 

good results, for SBV target and for IPC (figure 1). 

Figure 1: Results on field samples for SBV-S LSI kit 

All characteristics obtained at LSI were confirmed at the French 

NRL and at the FLI, giving to this kit an official authorisation for 

use in SBV diagnosis in France and Europe. 


A special thank to the French NRL (ANSES Maisons-Alfort) and 

Bernd Hoffmann (FLI) for their scientific expertise and their help, 

in this particular context. 

References 

1. Hoffmann, B, Scheuch, M (2012). Novel Orthobunyavirus in Cattle, 

Europe, 2011. Center for Disease Control and Prevention, Vol 18 N° 3, p1- 

6

S3 - P - 09 

PRELIMINARY VALIDATION OF A SOLID- PHASE COMPETITIVE ELISA FOR THE DETECTION OF 

ANTIBODIES AGAINST WEST NILE DISEASE VIRUS IN HORSE SERA 

G. L. Autorino 1 , A. Caprioli 1 , F. De Simone 2 , R. Frontoso 1 , D. Lelli 2 , R. Nardini 1 , F. Rosone 1 , M. T. Scicluna 1 . 

1 

National Reference Centre for Equine Infectious Diseases, Istituto Zooprofilattico Sperimentale Lazio e Toscana, 00178 Roma Italia. 2 Istituto 

Zooprofilattico Sperimentale Emilia Romagna e Lombardia, Via A. Bianchi 9, 25124 Brescia Italia 

Keywords: West Nile Disease, serological diagnosis, competitive elisa, validation 

Introduction 

In 1998, Italy reported the first equine outbreaks of West Nile 

Disease (WND). From 2001, a national veterinary surveillance 

system was adopted in areas considered at risk of WNV 

introduction, based on the passive surveillance of avian mortality 

and on the repeated serological testing of sentinel horses first in 

screening ELISA tests available commercially, either in a 

competitive format or based on the detection of IgG and IgM. 

Positive samples are then examined in the confirmatory test 

represented by the plaque neutralization reduction test. A solidphase 

competitive ELISA was developed and a preliminarily 

validation was carried by an interlaboratory trial. The data of the 

this preliminary validation is presented and discussed. 


The procedure for the competitive ELISA (C-ELISA), object of 

this validation, is briefly described as follows. 96 well microplate 

are pre-adsorbed with a monoclonal antibody (Mab), recognising 

the domain III (Ed III) of WND virus (WNDV), are prepared ready 

for use. The reaction is started by the addition of the antigen, a 

cell-culture cryolysate of inactivated virus, on to the plate and its 

incubation for 90 minutes at 37°C. In parallel to the first 

incubation, serum samples and controls are diluted 1:5 and 1:10 

on a separate plate. The samples and controls are then 

transferred on the adsorbed plate containing the antigen and 

incubated for 60 minutes at 37°C. After washing the plate, the 

same Mab used for adsorption is added conjugated with 

horseradish peroxidase. Following another incubation period of 

90 minutes at 37°C, ortophenyl-diamine substrate is added and 

the plate is incubated at room temperature for 15 minutes in the 

dark. Sulphuric acid is used to stop the enzymatic reaction, which 

is read using an optical density (OD) of 492nm. Sera are 

categorized as positive or negative according to percentage 

inhibition (PI), calculated as the ratio between sample and 

reaction control. 

Validation was performed according to WOAH Manual guidelines. 

The aim for this ELISA test is for screening purposes. 

Nine laboratories were involved in this validation. Pre-adsorbed 

plates, reagents (Mab and antigen) and a panel of sera were sent 

to each of them. The latter was made up of 20 sera, each 

replicated twice, and numbered from 1 to 40. The panel derived 

from the sera of five horses, two experimentally infected, two 

repeatedly vaccinated and one negative. The order of the sera 

was different for each laboratory. Each participant was requested 

to carry out the test three times, each time carried out by a 

different operator and on different days. The OD of each test was 

registered and returned in an Excel file. Validation criteria for the 

ELISA were the following: mean OD of control reaction higher 

than 1.0; OD of negative control < 50% of OD of control reaction 

for both dilutions; OD of positive control > 50% of OD of control 

reaction in both dilutions. Runs that did not comply with these 

criteria were discarded. 

In analysing all data the following parameters were estimated: 

Qualitative accuracy, estimated by: 

Sensitivity (Se) and specificity (Sp), Cohen K value for each 

laboratory, Weighted Cohen K value for each laboratory, 

Cohen K value for all laboratories gathered together. 

K values were calculated by comparing the expected results, 

positive and negative, with those obtained, for Cohen K value 

and negative, weak, medium and strong positive for Cohen K 

value and weighted Cohen K value. 

In considering both qualitative and semi-quantitative 

characteristics of the ELISA test, repeatability and reproducibility 

were estimated using the following parameters: 

Coefficient of variation (CV), Accordance, Concordance, 

Concordance Odds Ratio (COR), K value of all laboratories 

results gathered together. 

For details regarding the calculation of these parameters, articles 

by Langton et al., Quatto and Soliani et al. can be consulted 

(1,2,3,4). 

Results 

1.Both sensitivity and specificity resulted 100%. 

2.K values are shown in figure 1. K for all laboratories resulted 

equal to 0.76. 

K VALUE 

1,2 

1 

0,8 

0,6 

0,4 

0,2 

0 

K (2 cat.) WEIGHTED K (4 cat.) K(4 cat.) 

LAB 1 LAB 2 LAB 3 LAB 4 LAB 5 LAB 6 LAB 7 LAB 8 LAB 9 

Figure 1: K values for each laboratory: not weighted with 2 and 4 

categories, and weighted with two categories. 

3.Values of accordance, concordance and COR are showed in 

table 1. 

Table 1: Values of Accordance, concordance and COR for each 

serum and all laboratories together 

Serum N° Accordance Concordance COR Serum N° Accordance Concordance COR 

1 86,71 81,75 1,46 11 62,14 45,11 2,00 

2 95,29 95,24 1,01 12 75,43 74,87 1,03 

3 92,43 90,48 1,29 13 64,00 41,27 2,53 

4 100 100 1 14 66,86 52,78 1,80 

5 76,29 56,88 2,44 15 100 100 1 

6 95,29 95,24 1,01 16 100 100 1 

7 65,86 49,74 1,95 17 100 100 1 

8 86,71 81,75 1,46 18 100 100 1 

9 100 100 1 19 100 100 1 

10 100 100 1 20 100 100 1 

K value of all laboratories together not compared with expected 

results resulted equal to 0.72. 


From the results of the evaluated parameters the test is suitable 

for screening purposes. Accuracy is highly satisfactory since 

sensitivity and specificity resulted equal to 100%. Furthermore, K 

values indicate a degree of concordance almost perfect 

according to the classification of Landis et al. (5) Qualitative 

repeatability and reproducibility are also satisfactory since CV 

values are all less than 20%, value set as acceptable limit; 

accordance and concordance are also close to 100% in more 

than the half of sera. COR resulted very close to 1 for all sera 

except for two of them, however with a maximum value of 2.53. 

An additional advantage of this c-ELISA is that it can be extended 

to the testing of other species for which it has to be validated. To 

fulfil the remaining criteria set by WOAH, a second phase of the 

validation procedure is necessary in which further parameters 

need to be evaluated, among which are the performances of this 

test on field samples. 

References 

1.S.D. Langton et al ” Analysing collaborative trials for qualitative 

microbiological methods: accordance and concordance”, International 

Journal of Food Microbiology 79 (2002) 175-181 

2.http:/www.dsa.unipr.it/soliani/soliani.html 

3.J. Richard Landis e Gary G. Koch del 1977 “The measurement of 

observer agreement for categorial data” Biometrics, Vol. 33, pp.159-174. 

4.P. Quatto (2004). “Un test di concordanza tra più esaminatori”. In: 

Statistica, vol. 64, n. 1, pp. 145-151 

5.J. Richard Landis e Gary G. Koch del 1977 “The measurement of 

observer agreement for categorical data” Biometrics, Vol. 33, pp.159-174.

S3 - P - 10 

SIMULTANEOUS DETECTION AND DIFFERENTIATION OF INFLUENZA A VIRUS 

AND NEWCASTLE DISEASE VIRUS BY ONE STEP RT-PCR. 

D. Nidzworski 1 , L. Rabalski 1 , B. Szewczyk 1 , K. Śmietanka 2 , Z. Minta 2 

1 

Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk, Department of Molecular Virology, Gdansk, Poland 

2 

National Veterinary Research Institute in Pulawy, Department of Poultry Diseases, Pulawy, Poland 

Influenza virus, Newcastle disease virus, diagnosis, RT-PCR, 

Introduction 

Newcastle disease Virus (NDV), a member of the 

Paramyxoviridae family and Influenza A virus (IAV), from the 

Orthomyxoviridae family, are two main avian pathogens causing 

serious economic problems in poultry farming (3,4). NDV strains 

are classified into three major pathotypes: velogenic, mesogenic 

and lentogenic (2). Avian Influenza (AI) viruses are also divided 

into low pathogenic (LPAI) and highly pathogenic (HPAI) strains 

(1).Both viruses are enveloped, single stranded, negative-sense 

RNA viruses which give similar symptoms ranging from subclinical 

infections to severe disease, including loss in egg 

production, acute respiratory syndrome and high mortality, 

depending on their level of pathogenicity. This hinders the 

diagnosis based on clinical and post mortem examination only. 

Most of the currently available molecular detection methods are 

also pathogen-specific and require to perform more than one RT- 

PCR to confirm or exclude the presence of both pathogens. 

To overcome this disadvantage, we have applied One Step 

Duplex RT-PCR method to distinguish between those two 

pathogens. The main objective of the project was to develop new, 

fast and non-expensive method, which could be used in any 

veterinary laboratory. 


Thirty six viruses: sixteen ND Polish pigeon strains, four ND 

reference strains, four Influenza and twelve other (negative 

control strains) viruses were isolated from allantoic fluids of SPF 

embryonated eggs. The 4-week-old SPF chickens were also 

infected with both viruses (NDV – LaSota; IV – H7N1) Swabs 

from cloaca and trachea were collected and examined (Tab.1). 

Table 1: The experimental design 

Actions 

Day of experiment 

Inoculation of birds (4 week old) with 

1 

LaSota and H7N1 strains 

Collection of cloacal and oral swabs 4 

Collection of cloacal and oral swabs 5 

RNA was extracted using an RNeasy Nini Kit (Qiagen, Valencia, 

CA, USA). After RNA isolation, One Step RT-PCR were carried 

out. 

Two sets of primers based on conserve fragments of the M gens 

of NDV and IV were used in this study: 

NDV-F-4011: 5’ GTCCCAAATACCGGAGACCT 3’ 

NDV-R-4142: 5’ TTGTTTGCCACAACCCTACAG 3’ 

IV-F-VII/25: 5’ AGATGAGTCTTCTAACCGAGGTCG 3’ 

IV-R-VII/124: 5’ TGCAAAAACATCTTCAAGTCTCTG 3’ 

The reaction was carried out according to the Transcriptor One- 

Step RT-PCR Kit protocol (Roche Diagnostics, Mannheim, 

Germany). The reaction mixture contained: viral RNA, both sets 

of primers (0,3µM each), buffer (1x), Transcriptor Enzyme Mix 

and water. Condition of reaction presents Tab. 2 

Table 2. Conditions of One Step RT-PCR reaction. 

Step Temp. Time Cycles 

Reverse 

transcription 

50ºC 30 min 1 

Initial 

denaturation 

94ºC 7 min 1 

Denaturation 94ºC 10 sec 

Annealing 53 ºC 30 sec 10 

Elongation 68 ºC 105 sec 

PCR profile 

Denaturation 94 ºC 10 sec 

Annealing 60 ºC 30 sec 25 

Elongation 68 ºC 105 sec 

Final 

Elongation 

68 ºC 7 min 1 

Two products were expected: one for NDV – 152 base pair 

fragment contained conserve M gene fragment; and one for IV – 

102 base pair fragment also contained conserve M gene 

fragment. The results were visualized in 2,5% agarose gel. 

Results 

All NDV and Influenza virus strains isolated from eggs and from 

oral or cloacal swabs were detected by One Step RT-PCR 

method. The results of analysis are presented in Figure 1 and 

Tab. 3. 

NDV IV NDV+IV ntc M 

152 bp 

102 bp 

Figure. 1. Analysis of One Step RT-PCR reaction. 2,5% Agarose 

gel with EtBr. ntc – no template control, M – Mass marker (Hyper 

Ladder II, BioLine) 

Table 3. Results of analysis of infected birds and comparison with 

other previously described methods. 

Chicken no 41 Chicken no 31 

AIV NDV AIV NDV 

A OS B OS A OS B OS 

4 dpi O 20.48 + n.d. n.d. 20.71 + 29.82 + 

4 dpi C 22.76 + 29.74 + 24.80 + >35 - 

5 dpi O 20.68 + 27.34 + 21.69 + 33.69 + 

5 dpi C 22.07 + n.d. n.d. 23.40 + >35 - 

Chicken no 42 Chicken no 10 

AIV NDV AIV NDV 

A OS A OS A OS B OS 

4 dpi O 21.68 + n.d. n.d. 21.98 + 35.00 + 

4 dpi C 23.94 + n.d. n.d. 19.55 + 33.39 + 

5 dpi O 21.86 + 34.55 + 23.34 + >35 - 

5 dpi C 25.01 + 34.97 + 20.72 + n.d. n.d 

AIV – Avian Influenza Virus; NDV – Newacastle Disease Virus; dpi – day 

post infection; O – Oral swab; C – Cloacal swab; n.d. – not detected; A – 

method of detection based on Spackman et al., 2002 (6); B – method of 

detection based on Mia Kim et al., 2008 (5); OS – One Step RT-PCR 

Method; “+” – positive; “-“ – negative. 


Birds infected by Newcastle Disease or Influenza virus give very 

similar symptoms, hard to distinguish by veterinarians even 

during post-mortem examination. 

For this reason, the new One Step RT-PCR method for direct 

detection and differentiation of NDV and IV was developed. One 

Step RT-PCR assay can be applied by veterinary diagnosticians 

for preliminary differentiation between NDV and Influenza virus. 

The ability to detect both major avian pathogens in a very easy 

procedure makes this method very attractive for application in 

veterinary laboratories. 

References 

1. Alexander, D.J. – Orthomyxovirus infections. in: Viral Infections of Birds, 

red: J.B. McFerran, M.S. McNulty – Elsevier Science, London 1993, 287- 

316 

2. Beard, C, Hanson, R (1984). Newcastle Disease. In: Hofstad, M, 

Barnes, H, Calnek, B, Reid, W, Yoder, H. (Eds.) Disease of Poultry, 8 th ed. 

Iowa State University Press, Ames, 452-470. 

3. Capua, I., Alexander, D.J. - Avian influenza and human health - Acta 

Tropica 83 (2002), 1–6 

4. Lamb, R, Collins,P, Kolakofsky, D, Melero J, Nagai,Y, Oldstone, M, 

Pringle,C, Rima, B. Family paramyxoviridae. In: Fauquet, C, Mayo M, 

Maniloff J, Desselberger, U, Ball, L. (Eds.) Virus Taxonomy, VIII th Report of 

the International Committee on Taxonomy of Viruses. Elsevier Academic 

Press, San Diego, 655-668. 

5. Mia Kim, L., Suarez, D.L., Afonso, C.L., 2008. Detection of a broad 

range of class I and II Newcastle disease viruses using a multiplex real 

time reverse transcription polymerase chain reaction assay. J. Vet. Diagn. 

Invest. 20, 414-425. 

6. Spackman, E, Senne, DA, Myers, TJ, Bulaga, LL, Garber, LP, Perdue, 

ML, Lohman, K, Daum, LT, Suarez, DL , 2002. Development of a real-time 

reverse transcriptase PCR assay for type A influenza virus and the avian 

H5 and H7 hemagglutinin subtypes.J. Clin Microbiol., 40, 3256

S3 - P - 11 

DOLPHIN MORBILLIVIRUS INFECTION IN A CAPTIVE HARBOR SEAL (PHOCA VITULINA) 

Sandro Mazzariol 1 , Francesco Grande 2 , Cristina Pilenga 2 , Paola Modesto 3 , Cristina Biolatti 3 , Giovanni Di 

Guardo 4 , Alessandra Mondin 5 , Simone Peletto 3 , Pier Luigi Acutis 3 

1 Department of Comparative Biomedicine and Nutrition (BCA), University of Padua, Legnaro, Italy 

2 Zoomarine Italia, Rome, Italy 

3 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Turin, Italy 

4 Department of Comparative Biomedical Sciences, University of Teramo, Teramo, Italy 

5 

Department of Animal Medicine and Health, University of Padua, Legnaro, Italy 

morbillivirus, DMV, harbor seal, transmission, phylogeny 

Introduction 

During the last 25 years morbilliviral infections have caused 

dramatic mortalities among several aquatic mammal species and 

populations worldwide (1). In particular, Dolphin Morbillivirus 

(DMV) represents a major biological threat for free-ranging 

cetaceans. In the Mediterranean Sea, DMV has been responsible 

for two epidemics, the first of which caused a dramatic mortality 

among striped dolphins (Stenella coeruleoalba) between 1990 

and 1992, while the second one was firstly reported in the 

western Mediterranean in 2006-2008, moving thereafter eastward 

along the French and the Italian coastlines. In June 2011, one 

bottlenose dolphin (Tursiops truncatus) and one striped dolphin 

stranded along the Tyrrhenian coastline, close to Rome. Both 

animals were found molecularly (RT-PCR) positive to 

Morbillivirus. Personnel from a marine park helped the stranding 

network with the bottlenose dolphin, which stranded alive and 

died after one day of rehabilitation. Standard quarantine protocols 

were applied, in order to avoid any contact with cetaceans kept in 

the park. On July 25 th , 2011, an 8-years-old male harbor seal 

(Phoca vitulina) hosted inside the aforementioned park was 

submitted for post-mortem examination after a short history of 

anorexia, tremors, abdominal contractions, and polyuria with 

hypothermia and vomit in the last phases. 


A complete post-mortem investigation was carried out the day 

after death. Samples from main organs (brain, lungs, heart, liver, 

kidneys, GI tract, spleen, lymph nodes) were fixed in 10% neutral 

buffered formalin, paraffin-embedded and routinely processed for 

histopathology. Samples from intestine and kidneys were 

submitted for routine microbiological exams while cerebral, 

hepatic, cardiac, splenic, pulmonary and lymph node tissues 

were preserved frozen for molecular analyses. 

Molecular investigations were carried out to look for the presence 

of Canine Distemper Virus (CDV), Phocine Distemper Virus 

(PDV) and Cetacean Morbillivirus (CeMV) nucleic acids. Multiple 

sequence alignment was performed with the software BioEdit 

using CLUSTAL W. An identity matrix was created to compare 

sequenced products with morbillivirus sequences deposited in 

GenBank. Phylogenetic analysis was performed using MEGA 5.0 

software. The tree topology was inferred by the maximum 

likelihood method with Kimura 2-parameter model. The reliability 

of the topology was tested by bootstrapping 1,000 replicates. 

Results 

Necropsy revealed a mild gastro-enteritis and splenitis due to 

Aeromonas hydrophyla, along with hepatic and renal necrosis 

presumably due to endotoxemia. Besides these pathological 

findings, a generalized and diffuse lymph node enlargement was 

observed, along with a severe meningeal hyperemia and 

choroidal edema. Histologically, a severe and diffuse reactive 

lymphadenopathy was apparent, with lesions being characterized 

by lymphoid cell depletion, hyalinosis, and several multinucleate 

syncytia. A mild, multifocal, chronic choriomeningitis with white 

matter spongiosis and demyelination were also apparent, with 

eosinophilic inclusion bodies being also found in the cytoplasm of 

brain neurons and glial cells. Furthermore, the lung showed a 

mild, chronic bronchointerstitial pneumonia, with simultaneous 

evidence of bronchiolar epithelial cell hyperplasia/hypertrophy. 

Pathological findings suggested a morbilliviral infection, which 

was confirmed by RT-PCR, with CeMV-specific genome 

sequences being subsequently amplified from the brain of this 

seal. CDV and PDV molecular analyses were negative. Similarity 

was 97.6-99.5% with known DMV sequences and 85.9-89.6% 

with other CeMV members (Porpoise Morbillivirus and the 

tentatively named Pilot Whale Morbillivirus). Phylogenetic 

analysis confirmed clustering of the harbor seal CeMV sequence 

with previously reported DMV sequences from cetacean species 

(Figure 1). 

53 

75 

DMV AJ608288 

95 DMV AY586536 

DMV Z36978 

DMV AJ224705 

94 

DMV Sc/2007 HQ829973 

75 DMV Gme/2007 HQ829972 

Harbor seal 

PWMV 20E FJ842382 

90 

PMV IRL88 FJ648457 

PMV 2990 AY586537 

MeV UK140H94 U29285 

69 

RPV LA96 JN234010 

PPRV ICV89 EU267273 

PDV AF479277 

CDV EU716337 

SeV X56131 

0.2 

Figure 1: Maximum Likelihood dendrogram, based on the Kimura 

2-parameter model, illustrating haemagglutinin gene (H) 

nucleotide sequence relationships of morbilliviruses. 


During the last epidemic outbreak, morbilliviral infection was 

reported in several species besides striped dolphins, namely pilot 

whales (Globicephala melas), bottlenose dolphins, Risso’s 

dolphins (Grampus griseus), and fin whales (Balaenoptera 

physalus). Although the susceptibility of these cetacean species 

was already known, no reports other than the present one have 

ever described DMV transmission to harbor seals. Indeed, 

infection of pinniped species with morbilliviruses of cetacean 

origin was previously speculated on the basis of serological 

cross-reactivity and sequence homology (2). On a global scale, 

mortality events in both this and in other pinniped species such 

as grey seals (Halichoerus grypus), Bajkal seals (Pusa siberica), 

and Caspian seals (P. caspica) have been respectively linked to 

PDV and to CDV, with the former agent being more closely 

related to CDV than to any other known morbillivirus, while DMV 

appears to be more closely related to Rinderpest Virus (RPV). 

Apart from their diverging phylogeny and their phylogenetic 

distances, DMV and CDV could share similar epidemiologic 

features. This hypothesis is strongly supported by the 

simultaneous evidence of morbilliviral infection in the two freeranging 

dolphins found beached ashore near Rome before this 

captive seal died. Therefore, a possible route of virus (DMV) 

entry into the facility could have been represented by either the 

personnel and/or the instruments used for the rehabilitation of the 

Morbillivirus-infected bottlenose dolphin that was found stranded 

alive one month before. In conclusion, this report emphasizes 

that, apart from cetaceans, also pinniped species such as harbor 

seals are susceptible to DMV infection and related mortality. 

Therefore, strict quarantine procedures should be enforced also 

in structures where pinnipeds are kept when injured or stranded 

cetaceans are hospitalized within these facilities. 

References 

1. Di Guardo G, Mazzariol S, Fernández A. (2011). Biologically threatened 

dolphins and whales. Environmental Microbiology, 13(11), 2833-4. 

2. Van de Bildt MWG, Martina BEE, Vedder EJ et al. (2000). Identification 

of morbilliviruses of probable cetacean origin in carcases of Mediterranean 

monk seals (Monachus monachus). Veterinary Record, 10, 691-4.

S3 - P - 12 

PRELIMINARY VALIDATION OF THE ID SCREEN AFRICAN SWINE FEVER INDIRECT ELISA BASED 

ON THREE RECOMBINANT ASF PROTEINS 

Pourquier, P. 1 , Loic, C. 1, 

1 


African Swine Fever, diagnostics, serology, ELISA 

Introduction 

African swine fever (ASF) is a highly contagious, viral 

haemorrhagic disease of pigs, warthogs, European wild boar and 

American wild pigs caused by the African Swine Fever Virus 

(ASFV). There is neither treatment nor vaccine for ASF. To 

prevent introduction and spread of the disease, countries 

implement strict surveillance, control and eradication programs 

which require accurate and reliable diagnostic tests. 

In this context, IDvet has launched the ID Screen ® African Swine 

Fever Indirect ELISA. This test detects anti-ASFV antibodies in 

domestic or wild pigs, in serum, plasma or blood filter paper 

samples. 

Unique features of the ID Screen ® African Swine Fever Indirect 

ELISA include the coating of three recombinant ASFV antigens 

(P32, P62, and P72), and the ability to use the test with blood 

filter paper samples as well as serum and plasma. 

This study presents the preliminary validation data obtained for 

this ELISA kit on both serum and filter paper samples. 

References 

1. Barderas MG et al, 2000. Serodiagnosis of African Swine Fever using 

the recombinant protein p30 expresses in insect larvae. Journal of 

virological methods, 89:129-136. 

2. Gallardo C et al, 2009. Recombinant antigen targets for serodiagnosis of 

African Swine Fever. 16 :1012-1020. 

3. Gallardo C et al, 2006. Antigenic properties and diagnostic potential of 

African Swine Fever virus protein p62 expressed in insect cells. Journal of 

clinical microbiology, 44 : 950-956. 

4. Hutching GH et al, 2006. Indirect sandwich ELISA for antigen detection 

of African Swine Fever virus : comparison of polyclonal and monoclonal 

antibodies. Journal of virological methods, 131:213-217. 

5. OIE, 2008. Manuel terrestre de l’OIE : African Swine Fever. Chapitre 

2.8.1. 

6. Sanz et al, 1985. Monoclonal antibodies specific for African Swine Fever 

Virus proteins. Journal of virology, 54:199-206. 


The ID Screen ® African Swine Fever Indirect is based on three 

recombinant ASFV antigens (P32, P62, and P72) and an antiswine-HRP 

conjugate. Analyses were performed as per 

manufacturer’s specifications. 

Results 

Specificity 

- 556 disease-free sera from domestic pigs (France and Norway), 

wild boars (France), and Iberian pigs (Spain) were tested. All 

samples were found negative. 

- 90 negative animals were tested by both the serum and filter 

paper protocols. All sera were found negative by both protocols. 

Sensitivity 

- 3 sera were tested from pigs vaccinated on day 0 and day 24 

with the ASF strain Ourt88/3, challenged on day 42 with a mild 

ASF strain (ANSES, Ploufragan, France), and bled on day 62 or 

63. After challenge, all three animals gave strong positive results 

with the ID Screen ® African Swine Fever Indirect ELISA. 

- 8 reference sera provided by the Community Reference 

Laboratory (CRL), CISA-INIA, Spain were analysed. The ID 

Screen® African Swine Fever Indirect ELISA accurately identified 

all sera. 

- 92 sera from infected herds (Sassari, Sardinia, Italy) were 

tested in parallel by the ID Screen ® ELISA and the CRL ELISA 

reagents (CISA-INIA, Spain). Test correlation was found to be 

95.7%. The relative sensitivity and specificity were 97.67% and 

97.83%, respectively. 

- 3 sera were titrated and tested by both the serum and filter 

paper protocols. The measured analytical sensitivity was similar 

regardless of the sample type tested (serum, Whatman 1 or 

Whatman 3 filter paper). 


The ID Screen ® African Swine Fever Indirect ELISA is an efficient 

and reliable tool for the diagnosis of ASF in wild and domestic 

species. 

It is the only commercial ELISA based on 3 recombinant 

antigens. Both the serum and filter paper test protocols show 

excellent sensitivity and specificity 

Collecting blood using filter paper facilitates sample collection in 

the field. With the ID Screen protocol, filter paper samples may 

be tested in a 96-well deep-well plate, making sample processing 

faster and less prone to mix-ups.

S3 - P - 13 

ONE STEP RT-PCR FOLLOWED BY MSSCP AS A NOVEL METHOD FOR A FAST AND RELIABLE 

DETECTION AND DIFFERENTIATION OF MIXED INFECTION WITH NEWCASTLE DISEASE VIRUS OF 

DIFFERENT ORIGIN. 

L. Rabalski 1 , D. Nidzworski 1 , B. Szewczyk 1 , K. Śmietanka 2 , Z. Minta 2 

1 

Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk, Department of Molecular Virology, Gdansk, Poland 

2 

National Veterinary Research Institute in Pulawy, Department of Poultry Diseases, Pulawy, Poland 

MSSCP, differentiation of strands, RT-PCR, Newcastle disease 

Introduction 

Newcastle disease virus (NDV) is an avian enveloped virus 

containing linear non-segmented single-stranded RNA (Lamb et 

al., 2005). It belongs to the Mononegavirales order, 

Paramyxoviridae family and Avulavirus genus. NDV strains are 

classified into three major pathotypes: velogenic, mesogenic and 

lentogenic (Beard and Hanson, 1984). Vaccination (with live 

virus) does not protect against the spread of virulent form of the 

virus. Detection of the field virus in immunized population of birds 

by means of conventional diagnostic methods is costly and time 

consuming. To overcome this disadvantage, we have applied 

One Step RT-PCR and Multitemperature Single-Strand 

Conformational Polymorphism (MSSCP) methods in our 

laboratories to distinguish between pathogenic and vaccine 

strains of the NDV. Classical single-strand conformational 

polymorphism (SSCP) analysis is based on the observation that 

single stranded DNA fragments attain a number of 

conformational forms which may be separated by native 

polyacrylamide gel electrophoresis giving a characteristic pattern 

of electrophoretic bands. Point mutations, other minor changes in 

nucleotide sequence, as well as physico-chemical conditions like 

ionic strength, pH and temperature may have significant effect on 

electrophoretic pattern of single stranded DNA (Orita et al., 

1989). By sequential changes of separation temperature it is 

possible to increase the resolution of single-strand DNA bands; 

this technique was named MSSCP (where M stands for 

“multitemperature”) (Kaczanowski et al., 2001). 


SPF chickens housed in isolation were infected oculonasally with 

three different strains of NDV (LaSota – lentogenic, Roakin – 

mesogenic and Italy – velogenic). Swabs from cloaca and 

oropharynx were collected at different time-points post infection 

(Tab.1). After RNA isolation and One Step RT-PCR, 123bp DNA 

of viral fusion (“F”) gene fragments were obtained (Fig. 1). PCR 

products were denatured (3’ in 97C) and subjected to MSSCP 

electrophoresis where, after silver staining, they gave specific 

ssDNA band patterns. 

Table 1: The experimental design 

Actions 

Inoculation of birds with 

LaSota and Roakin NDV 

strains 

Inoculation of birds with Italy 

NDV strain 

Collection of cloacal and 

oropharyngeal swabs 

Day of experiment 

1 

3 

3, 4 ,5 ,6 ,7, 10 

Results 

Three embryo-grown NDV strains (separately and mixed 

together) were tested for the level of detection. We found it 

possible to determine the presence of the minor variant of the 

virus, even when its contribution was less than 0.1% of total 

sample. After MSSCP analysis, characteristic ssDNA band 

patterns were obtained for each NDV strain. Both artificially 

mixed and swab samples showed a combined pattern of bands 

making it easy to identify the composition of samples (Fig. 2). 

Figure 1: 123bp PCR product at 7 days after infection (digit - 

identification of the chicken, G - oropharyngeal swabs, 

K – cloacal swabs, M - HyperLadder IV - 100bp-1000bp) 

Figure 2: MSSCP patterns of 123bp PCR product (M - 

HyperLadder IV - 100bp-1000bp, L – LaSota, R – Roakin, 

I – Italy, number – level of matrix concentration) 


MSSCP combined with One Step RT-PCR can be applied for the 

detection and preliminary characterization of NDV without the 

need for nucleotide sequencing. The ability to diagnose multistrain 

infections and to detect the minor viral variant are the main 

advantages of the proposed method. Additionally, we confirm the 

observation that vaccination does not protect against virus 

replication and shedding in chickens. 

The developed method can be especially helpful in the detection 

of field infection in flocks immunized with live vaccines provided 

that we know the MSSCP pattern of an NDV strain used for 

vaccination. In such cases an RT-PCR method alone yields 

postitive results irrespective of the number of strains present in 

the tested sample while the sequencing shows the overlapping 

peaks of fluorsecence. If the MSSCP patterns of NDV present in 

the sample are not known, there is a possibility to cut the bands 

from the gel and sequence them separately. 

References 

1. Orita, M, Iwahana, H, Kanazawa, H, Hayashi, K, Sekiya, T (1989). 

Detection of polymorphisms of human DNA by gel electrophoresis as 

single-strand conformation polymorphisms. Proc. Natl. Acad. Sci.USA, 86, 

2766-70. 

2. Kaczanowski, R, Trzeciak, L, Kucharczyk, K (2001). Multitemperature 

single-strand conformation polymorphism. Electrophoresis, 22, 3539-45. 

3. Lamb, R, Collins,P, Kolakofsk, D, Melero J, Nagai,Y, Oldstone, M, 

Pringle,C, Rima, B. Family paramyxoviridae. In: Fauquet, C, Mayo M, 

Maniloff J, Desselberger, U, Ball, L. (Eds.) Virus Taxonomy, VIII th Report of 

the International Committee on Taxonomy of Viruses. Elsevier Academic 

Press, San Diego, 655-668. 

4. Beard, C, Hanson, R (1984). Newcastle Disease. In: Hofstad, M, 

Barnes, H, Calnek, B, Reid, W, Yoder, H. (Eds.) Disease of Poultry, 8 th ed. 

Iowa State University Press, Ames, 452-470.

S3 - P - 14 

DEVELOPMENT OF MULTISPECIES SEROLOGICAL ASSAYS TO DETECT ANTIBODIES SPECIFIC 

FOR Mycobacterium bovis IN SERUM SAMPLES 

Lisset López 1 , Ana Ranz 1 , Angel Venteo 1 , Teresa Pérez 1 , Tamara Ruiz 1 , L DeJuan 2 , Beatriz Romero 2 , Mariana 

Boadella 3 , Christian Gortázar 3 , Beatrice Boniotti 4 , Paolo Pasquali 5 , Paloma Rueda 1 

1 1 INGENASA, Madrid Spain; 2 VISAVET, Madrid, Spain; 3 IREC, Barcelona, Spain; 4 I.Z.S. della Lombardia e dell'Emilia, Brescia, Italy ; 5 Istituto Superiore di 

Sanità di Roma, Roma, Italy 

Tuberculosis, PEN-SIDE TEST, DIAGNOSTIC, ELISA DR 

Introduction 

Bovine tuberculosis (TB) is a chronic bacterial disease of 

animals and humans caused by M. bovis. In a large number of 

countries bovine tuberculosis is a major infectious disease 

among cattle, other domesticated animals, and certain wildlife 

populations. Transmission to humans constitutes a public health 

problem. For years eradication programmes have been carried 

out. Traditional mycobacterium culture remains the gold 

standard method for routine confirmation of infection but 

Delayed Hypersensitivity test and gamma-interferon test are the 

ones used in these programmes. Detection of antibodies 

specific could be a useful alternative for TB diagnosis. Different 

antibodies detection assays have been described although all of 

them present sensitivity problems 

The aim of this study has been to develop and validate 

multispecies assays based on Double Recognition ELISA 

(ELISA-DR) and Immunochromatography (IC) techniques for 

detection of antibodies specific of M. bovis. using the 

recombinant protein MPB83, expressed in insect cells. 


Recombinant proteins ESAT6, CFP10, MPB64, MPB70 y MPB 

83 were obtained, expressed and purified. Finally, MPB83 was 

selected as the antigen for serological assays (ELISA and IC 

assays) 

To validate these assays, sera from different species previously 

catalogued by gamma interferon assay (72 bovine sera), by 

culture (46 buck, 205 wild boar) or by necropsy (205 deer and 

138 wild boar experimental sera) were used. Moreover, cross 

reactivity with M. avium subsp. paratuberculosis was 

determined. For this propose sera and plasma of 9 cows and 9 

goats positive to Paratuberculosis and negative to TB were 

used. 

Samples from different wild species such as alpaca, elephant 

and American bison are going to be tested. In addition sera 

collected from wild boar and deer are being evaluated. 

Table 2. 

Ref: ELISA DR 

SPECIES N Accuracy 

CATTLE 13 77% 

WILD BOAR 200 90% 

BUCK 46 89% 

DEER 22 77% 


INGENASA has developed two multispecies assays based on 

the use of the recombinant protein MPB83 expressed in insect 

cells. More than 300 samples from different species have been 

analyzed giving high percentages of sensitivity and specificity. 

Although higher number of samples is necessary, preliminary 

results suggest that both diagnostic approaches are very useful 

methods for control and surveillance of TB infection. 

The immunochromatographic assay can become a useful tool in 

situations where laboratory support and skilled personnel are 

limited. 

References 

1. Schiller et al. Transboundary and Emerging Diseases. (2010), 

57:205-220 

2. Boadella et al. Preventive Veterinary Medicine (2012), 104:160-164 

3. Waters et al. Clinical and Vaccine Immunology (2011), 18:1882-1888 

(KBBE-2007-1-3-04) Strategies for the eradication of bovine tuberculosis 

(TB-STEP) 

Results. 

Preliminary results show that both assays detect antibodies 

specific of TB. None of them showed cross-reaction with 

antibodies to PTB. Depending on the species analyzed, 

sensitivity and specificity of ELISA-DR range between 70-100% 

and 92-97%, respectively (Table I). Concerning to IC, accuracy 

with respect to ELISA DR ranged between 77 and 90% 

depending on the species studied (TABLE II). 

Table 1 

ELISA-DR 

SPECIES N Sensitivity Specificity REFERENCE 

CATTLE 72 88% 96% Bovigam® 

WILD BOAR 138 100% 97% Necropsy 

WILD BOAR 200 82% 92% Culture 

BUCK 46 70% 94% Culture 

DEER 205 93% 92% Necropsy

S3 - P - 15 

FIRST REPORTED LOW PATHOGENCITY AVIAN INFLEUNZA VIRUS SUBTYPE H9 INFECTION OF 

DOMESTIC FOWL IN ENGLAND 

C. Daniel Parker 1 , Scott M. Reid 2 , Allan Ball 1 , William J. Cox 2 , Steve C. Essen 2 , Amanda Hanna 2 , 

Marek J. Slomka 2 , Richard M. Irvine 2 , Ian H. Brown 2 

1 Slate Hall Veterinary Practice Ltd, Unit 7 Highgate Farm, Over Road, Willingham, Cambridge, CB24 5EU 

2 Animal Health and Veterinary Laboratories Agency-Weybridge, Woodham Lane, New Haw, Addlestone, Surrey, KT15 3NB 

Avian influenza virus, H9 real-time RT-PCR, broiler breeders, UK poultry, Egg drop in poultry 

Introduction 

Poultry outbreaks, caused by avian influenza (AI) viruses of 

H9N2 subtype, have been reported in many parts of Asia and the 

Middle East since the 1990s where they may be considered as 

being endemic (1, 2). Although H9 virus infections are typically 

characterised as being of low pathogenicity (LP) and are not 

classified as notifiable disease (3), LPAI H9N2 poultry outbreaks 

in these regions continue to be characterised clinically by 

respiratory disease and a reduction in egg production (4). 

However, since the more recent emergence of a significant H9N2 

poultry problem in Asia and the Middle East, the present paper 

presents the first detailed epidemiological investigation of a H9 

poultry outbreak in the UK and indeed in Europe. This occurred in 

the East Anglia region in December 2010 in a broiler breeder 

flock, and an important aspect of the outbreak investigation was 

the successful use of molecular methods to identify and 

characterise the AIV subtype. 


Disease suspicion was based on acute drops in egg production in 

two of four sheds, poor egg shell quality and evidence of 

diarrhoea. Cloacal and oropharyngeal swabs from 60 chickens 

from each of two affected houses were pooled (n=48) into groups 

of five swabs obtained from the same anatomical site. All swab 

pools were inoculated into 10-day-old embryonated SPF fowls’ 

eggs, and allantoic fluids were harvested and tested for the 

presence of any haemagglutinating agent (5). Four AI real-time 

reverse transcription polymerase chain reaction (RRT-PCR) 

assays were used to test the RNA extracts: (i) the Matrix (M)- 

gene assay for generic AI detection; (ii) H5 and H7 AI virus RRT- 

PCR assays to test for notifiable avian influenza (NAI); (iii) an H9 

RRT-PCR; (iv) selected RNA extracts were tested by an N1 RRT- 

PCR. The RNA extracts were simultaneously screened for 

Newcastle disease virus (NDV) using primers and probes 

targeting the L-gene of NDV. RNA extracted from the positive 

cloacal swab specimen A/chicken/England/1415-51184/10 was 

used for nucleotide sequencing. The HA and neuraminidase (NA) 

genes were amplified by conventional reverse transcription (RT)- 

PCR, sequenced and analysed phylogenetically. 

poultry for 40 years vindicates the need for continued vigilance 

and surveillance of AI viruses in poultry populations. 


The authors thank Andrew Gibson, Mark Spelman and Simon 

Rednall from the farm management team, Sue Graham of Slate 

Hall Veterinary Practice Ltd for her laboratory assistance and Dr 

Dennis Alexander of AHVLA for technical assistance. This study 

was supported by Defra contract ED1300: Scanning Surveillance 

for Disease in Poultry and Game Birds in England, Scotland and 

Wales. 

References 

1.Alexander, DJ (2007). An overview of the epidemiology of avian 

influenza. Vaccine 25:5637–5644. 

2.Capua, I, Alexander, DJ (2007) Avian influenza infections in birds – a 

moving target. Influenza and Other Respiratory Viruses 1:11–18. 

3.OIE (World Organisation for Animal Health). (2008). World Health 

Organisation for Animal Health, Terrestrial Animal Health Code, chapter 

10.4 “Avian influenza”. OIE: Paris. Available at: 

http://www.oie.int/eng/normes/mcode/en_chapitre_1.10.4.pdf 

4.Swayne, DE, Halvorson, DA (2003). Influenza. In ”Diseases of Poultry”, 

pp135-160. Eds: YM Saif, HJ Barnes, JR Glisom, AM Fadly, LR 

McDougald and DE Swayne. Ames, Iowa: Iowa State University Press. 

5.OIE (World Organisation for Animal Health). (2010). Manual of 

Diagnostic Tests and Vaccines for Terrestrial Animals, chapter 2.3.4. 

“Avian influenza”. Paris: OIE. Available at: 

http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.03.04_AI. 

p 

Results 

Attempted virus isolation in embryonated SPF fowls’ eggs was 

unsuccessful. H9N1 LPAI virus infection was confirmed by RRT- 

PCR. Sequencing of the haemagglutinin and neuraminidase 

genes revealed high nucleotide identity of 93.6% and 97.9% with 

contemporary North American H9 and Eurasian N1 genes, 

respectively. Epidemiological investigations were conducted to 

identify the source of infection and any onward spread. 


This is the first known report of an H9 subtype avian influenza 

infection in poultry in England. The infecting subtype was H9N1 

and phylogenetic analysis of the HA and NA genes indicated 

North American and Eurasian origins for these two genes, which 

suggested that this AIV may be a reassortment that consists of 

genetic segments of diverse geographic origin. The source of the 

influenza virus infection could not clearly be established. 

Following the infection, epidemiological investigations failed to 

identify any human, inter- or intra-company contacts that could 

have introduced the virus onto the farm. No other breeder or 

poultry farms in the area were known to be infected. Infection 

followed a period of extremely cold weather and snow which 

could have compromised disease security. Wild bird activity 

around the farm buildings was significantly higher during this cold 

weather period as birds foraged for feed. In the present episode, 

clinical signs were relatively mild in the poultry with no mortality. 

However, this first reported detection of H9 LPAI virus in UK

S3 - P - 16 

DETECTION OF IBV QX IN COMMERCIAL POULTRY FLOCKS IN THE UNITED KINGDOM 

Richard M. Irvine 1 , Rebecca M. Jones 1 , Richard J. Ellis 1 , Jennifer L. Cork 1 , 

Jane Errington 2 , William J. Cox 1 , Vanessa Ceeraz 1 , Alisdair M. Wood 3 , Scott M. Reid 1 , 

Ian H. Brown 1 

1 Animal Health and Veterinary Laboratories Agency – Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK; 

2 Animal Health and Veterinary Laboratories Agency – Penrith, Merrythought, Calthwaite, Penrith, Cumbria CA11 9RR, UK; 

3 Animal Health and Veterinary Laboratories Agency – Lasswade, International Research Centre, Pentland Science Park, Bush Loan, Penicuik, Midlothian 

EH26 0PZ, UK. 

Infectious bronchitis virus, IBV QX strain, IBV real-time RT-PCR, IBV S1 genotyping, UK commercial poultry flocks 

Introduction 

The QX strain of infectious bronchitis virus (IBV) was first 

detection in China between 1995 and 2004, associated with 

nephritis, mortality and egg production problems in IBVvaccinated 

flocks (1). Strains of IBV showing a close relationship 

with the so-called Chinese QX strain of IBV (IBV QX) have 

recently been detected in the UK. Following the initial isolation of 

a QX-like variant strain serologically unrelated to the major UK 

contemporary IBV strains from a small backyard flock during 

2007, European IBV QX-like strains have been detected in 

backyard and hobby flocks. Since 2009, such strains have also 

been detected in samples submitted for diagnostic investigation 

from commercial poultry flocks (2). 


Diagnostic samples testing positive by IBV real-time reverse 

transcription polymerase chain reaction were subject to partial 

sequencing of the S1 gene (3). Primers and probes were 

optimised for sequencing a fragment of the S1 gene (c. 450 bp). 

For an initial genotyping test, a 140bp region was compared to a 

library of IBV S1 gene sequences. 

Results 

Comparison of the entire 450bp region for the QX strains allowed 

separate sub-lineages to be identified. Whilst the European IBV 

QX-like genotypes detected from commercial poultry flocks in 

Great Britain (GB) and Northern Ireland are closely related, 

differences are apparent in the S1 sequence, revealing separate 

sub-lineages (Figure 1). The greatest differences occur between 

European IBV QX-like strains detected from backyard poultry and 

commercial flocks. Furthermore, strains from some commercial 

poultry flocks in GB share a higher degree of S1 sequence 

homology with each other and a strain detected from poultry in 

The Netherlands. 

SA12- 008180 

SA12- 013762 

SA12- 007589 

SA12- 009984 

SA12- 009987 SA12-013717 

SA12-013719 

SA12-013721 

SA12-013722 

SA12-011754 

SA12-011755 

SA12-006209 

SA12-006211 

SA12- 010211 

SA12- 010573 

SA12- 010575 

SA12- 010825 

SA12- 010827 

SA12-005257 

SA12-010215 

SA12-004866 

SA12-006034 

Jun- 164-S3 

AVP - 09- 023587 

AVP - 09- 023588 

SA12- 011173 

Poulvac-QX -Vaccine 

QX AV2150/07 

SA12-014043 

AV418-trachea 

AVI-08- 035383 

AVI-08-022254 

SA12-011670 


Together, these data revealed differences by sector, suggesting 

that separate routes of introduction may have occurred. 

Emergence of a novel variant such as IBV QX might threaten 

commercial poultry production as currently-used vaccines might 

not be fully protective and the severity of disease caused by 

novel strains may differ from that caused by extant strains. This 

may result in substantial losses. Experimental studies have 

demonstrated that protection may be afforded against infection 

with IBV QX-like strains in young chickens by existing vaccines 

and vaccine combinations (4). However, further work is required 

to fully understand the epidemiology of IBV QX in UK poultry 

populations. 


This study was supported by Defra contract ED1300: Scanning 

Surveillance for Disease in Poultry and Game Birds in England, 

Scotland and Wales. 

References 

1. Liu, SW, Zhang, QX, Chen, JD, Han, ZX, Liu, X, Feng, L, Shao, YH, 

Rong, JG, Kong, XG, Tong, GZ (2006). Genetic diversity of avian 

infectious bronchitis coronavirus strains isolated in China between 1995 

and 2004. Archives of Virology 151: 1133-1148. 

2. Irvine, RM, Cox, WJ, Ceeraz, V, Reid, SM, Ellis, RJ, Jones, RM, 

Errington, J, Wood, AM, McVicar, C, Clark, MI (2010). Detection of IBV QX 

in commercial broiler flocks in the UK. Veterinary Record 167:877-879. 

http://veterinaryrecord.bmj.com/content/167/22/877.2.full.pdf 

3. Jones, RM, Ellis, RJ, Cox, WJ, Errington, J, Fuller, C, Irvine, RM, 

Wakeley, PR (2011). Development and Validation of RT-PCR Tests for the 

Detection and S1 Genotyping of Infectious Bronchitis Virus and other 

Closely Related Gammacoronaviruses Within Clinical Samples. 

Transboundary and Emerging Diseases 58 (5): 411–420. 

http://onlinelibrary.wiley.com/doi/10.1111/j.1865- 

1682.2011.01222.x/abstract. 

4 Terregino, C, Toffan, A, Beato, MS, Nardi, R, Vascellari, M, Meini, A, 

Ortali, G, Mancin, M, Capua, I (2008). Pathogenicity of a QX strain of 

infectious bronchitis virus in specific pathogen free and commercial broiler 

chickens, and evaluation of protection induced by a vaccination 

programme based on the Ma5 and 4/91 serotypes. Avian Pathology 37 

(5) : 487-493. 

0.01 

Broilers; Broiler breeders; Layer; Backyard 

Figure 1: Evolutionary relationships

S3 - P - 17 

APPLICATION OF REVERSE TRANSCRIPTION REAL-TIME PCR TO DETECT THE 

SCHMALLENBERG VIRUS GENOME 

Nikolai Salnikov, Helena Nikitina, Sodnom Tsybanov, Denis Kolbasov 

National research institute for veterinary virology and microbiology of Russia, Pokrov, Russia 

Schmallenberg disease, virus, PCR 

Introduction 

The disease Schmallenberg was registered among cattle, sheep 

and goats in Germany, the Netherlands, Belgium, Britain, France, 

Italy, Luxembourg and Spain (3,4). The Schmallenberg virus is 

a member of the genus Orthobunyavirus family Bunyaviridae. 

Established the pathogenicity of the Schmallenberg virus for 

sheep, goats and cattle. Clinical signs of the disease in 

cattle include fever, drop in milk yield (50%), loss of appetite and 

sometimes diarrhea. If an adult ewe or heifer was infected in the 

early stages of pregnancy, the fetal infection can occur, 

leading to serious consequences: abortion, birth of premature or 

dead fetuses and lambs, goats and calves with 

various malformations. At detection in animals the characteristic 

clinical signs of the Schmallenberg disease carry out 

activities for virus isolation in cell cultures, detection of the 

virus genome by PCR and antibodies to it in the neutralization 

test (1,2). The aim of this work was the development the test - 

system to detect the Schmallenberg virus genome by real-time 

quantitative reverse transcription PCR (RT-qPCR). 


In our work the referent RNA samples of the Schmallenberg virus 

were used. Also we used the different strains of such viruses, as 

Nairobi sheep disease, Akabane, Rift Valley fever and 

bluetongue. Extraction of viral RNA was performed using "Trizol'' 

(Trizol Reagent, Life Technology, USA). RT-qPCR was carried 

out on the thermocycler Rotor Gene 6000 (Corbett Research, 

Australia). Cloning of the amplified Schmallenberg virus 

genome's fragment was performed using the "Ins TAclone PCR 

cloning kit"(Fermentas, Latvia). The synthesis of RNA from 

cloned DNA inserts was performed using the kit "RiboMAX Large 

Scale RNA Production System T7" (Promega, USA). 

preparations of heterologous viruses and RNA extracted from the 

intact samples has not been received positive results. This 

proves the specificity of the primers and the probe included in 

the test - system. The analytical sensitivity of the developed test - 

system is 7,178*10^5 RNA copies / mkl. 


We thank for the referent RNA samples of the Schmallenberg 

virus Dr. Martin Beer and Dr. Bernd Hoffman, Institute of 

Diagnostic Virology, Friedrich Loeffler Institute, Germany. 

References 

1. Gibbens, N. Schmallenberg virus: a novel viral disease in northern 

Europe. 2012. Vet. Rec.17: 58. 


H, et al. Novel orthobunyavirus in cattle, Europe, 2011. Emerg. Infect. 

Dis. 2012 Mar. 

3. Latest posts on Pro Med-mail. International society for infectious 

diseases, 2011-2012. http://www.promedmail.org. 

4. Weekly disease information/WAHID Interface/OIE, 2011-2012.- 

http://www.oie.int. 

Results 

With order to select primers the available in Gen Bank nucleotide 

sequences of Schmallenberg virus Genome were analyzed using 

the programs Bio Edit 7.0 and Oligo 6.0. As a result a pair of the 

primers flanking the fragment of 130 bp within the gene N of 

Schmallenberg virus was designed. For detection of amplification 

products in real-time the probe Taq-man, which contains at the 

5'-end the fluorophores HEX and at the 3'-end – quencher BHQ2 

was designed. Analytical sensitivity of the method was 

determined using in vitro transcripts synthesized on the template 

of the recombinant plasmid with the Schmallenberg virus 

genome fragment's insertion. Ten-fold serial dilutions of in 

vitro transcribed RNA (of known concentration) were used to 

determine analytical sensitivity of RT-q PCR. The limit of 

sensitivity considered the maximum dilution at which a positive 

result was registered. 

The calculated value of analytical sensitivity of RT-PCR in realtime 

was 7.178*10^5 RNA copies /mkl, which corresponds 

to 3,698*10^6 copies of RNA per reaction. To evaluate 

the specificity of the test - system the RNA samples of Nairobi 

sheep disease, Akabane, Rift Valley fever and bluetongue 

viruses were examined. We also examined the RNA samples 

extracted from the intact cultures of kidney cells of antelope, 

BHK-21/13, CV-1, blood samples and organs from intact animals 

(goats, sheep, cattle, mice). With any of the examined RNA 

samples has not been a received positive result in RT-qPCR. By 

using the developed method over 500 blood samples and 15 

samples of placenta and meconium from clinically 

healthy pregnant heifers imported from Germany and the 

Netherlands in 2011-2012 in the farms of Nizhny Novgorod, 

Moscow and Kaluga regions were examined. This also was 

not obtained positive results. 


The designed test -system is suitable to detect the 

Schmallenberg virus genome in blood samples and 

organs of sheep and cattle. In the investigation of RNA

S3 - P - 18 

PREVALENCE OF COXIELLA BURNETII ANTIBODIES IN SHEEP AND GOATS IN FINLAND 

Teresa Skrzypczak 1 , Tiina Autio 2 , Jaana Seppänen 1 , Sinikka Pelkonen 1,2 

1 

Finnish Food Safety Authority, Evira, Research and Laboratory Department, Veterinary Bacteriology, Helsinki, Finland 

2 

Finnish Food Safety Authority, Evira, Research and Laboratory Department, Veterinary Bacteriology, Kuopio, Finland 

Coxiella burnetii, Q-fever, sheep, goat, prevalence 

Introduction 

Coxiella burnetii is an intracellular pathogen which causes a 

highly contagious zoonotic disease, Q fever. The infection is 

endemic worldwide and has been detected in several European 

countries in goat, sheep and cattle populations (2, 4). The 

transmission of the infection from goats to humans has caused 

severe zoonotic epidemic in the Netherlands in the last few years 

(3, 5). In Finland, C. burnetii infection was detected for the first 

time in 2008 in a dairy herd. Seropositive animals were found in 

ELISA tests made for export control. A succeeding PCR test of 

the bulk tank milk sample was positive for C. burnetii. No clinical 

signs of Q fever were observed in the herd. A later performed 

nationwide prevalence study of dairy herds in 2009 indicated that 

the prevalence of C. burnetii infection in cattle population was 

extremely low. Only two out of 1238 tested herds were 

seropositive (1). In this study we assessed the prevalence of C. 

burnetii antibodies in sheep and goat populations in Finland. 

References 

1. Autio, T., Skrzypczak, T., Pohjanvirta, T., Pelkonen, S. 2010. 

Low prevalence of Coxiella burnetii (Q-fever) antibodies in dairy herds in 

Finland. 25th World Buiatrcis Congress, Chile. 

2. Guatteo, R., Seegers, H., Taurel, A.-F., Joly, A., Beaudeau, F. 2011. 

Prevalence of Coxiella burnetii infection in domestic ruminants: A critical 

review. Vet. Microbiol. 149:1-16. 

3. Roest, H.I.J., Tilburg, J.J.H.C., van Der Hoek, W., Vellema, P., van Zijderveld, 

F.G., Klaassen, C.H.W., Raoult, D. The Q fever epidemic in The Netherlands: 

history, onset, response and reflection. 2011. Epidemiol. Infect. 139: 1–12. 

4. Ryan, E, Kirby, M, Clegg, T, Collins, D,M. 2011. Seroprevalence of 

Coxiella burnetii antibodies in sheep and goats in the Republic of Ireland. 

Vet. Record, 169, 280b 

5. Schimmer,B, Luttikholt, S, Hautvast, J.L.A, Graat, E.A.M, Vellema, P, 

van Duynhoven, Y.T.H.P.2011. Seroprevalence and risk factors of Q fever 

in goats on commercial dairy goat farms in the Netherlands, 2009-2010. 

BMC Vet. Res. 2011,7:81 


We used serum samples collected for sheep Meadia-Visna-virus 

(MV) and goat Caprine arthritis-encephalitis-virus (CAE) 

surveillance study in year 2010. According to the legislation, all 

herds having 20 eves or more must be tested for MV and CEA. 

The smaller herds remained out of this study. 

In total, we obtained 8545 ovine sera from 246 farms and 819 

caprine sera from 24 farms. From the herds with less than 50 

animals, all animals were included in the study. From herds with 

more than 50 animals, 50 sera were chosen randomly. The 

samples of individual herds were combined into pools of ten 

samples. Altogether 740 pooled samples from sheep were 

tested. Caprine samples were mainly studied individually, except 

from one herd with more than 200 animals. In case of a positive 

test result, all samples from a positive pool were tested 

separately. 

The samples were tested using CHEKIT Q-fever antibody ELISAtest 

(IDEXX, Liebefeld-Bern, Switzerland). 

Results 

All the tested samples were negative for C. burnetii antibodies. 

The negative results indicate that the seroprevalence of C. 

burnetii in the ovine population was less than 0.0004 % and 

caprine population less than 0.0048 %, with 95 % confidence. 


This was the first time that Q fever antibodies were analyzed in 

sheep and goats in Finland. All tested farms and animals were 

negative. There were altogether 129 091 sheep on 1349 farms 

and 4 890 goat on 165 farms at the time of sampling in 2010. The 

sampling covered 6.6 % of sheep and 16.7 % of goat populations 

in the country, and represented 18.2% of ovine and 14.5% of 

caprine farms situated in different parts of the country. The study 

shows extremely low prevalence of C. burnetii infection in sheep 

and goats in Finland. The finding is in line with the detected low 

prevalence of the infection in dairy cattle herds in 2009 (1). The 

Nordic climate, small herd size of sheep and goat farms and 

small density of these farms may have accounted for the 

favorable situation. We conclude that C. burnetii infection is very 

rare in domestic ruminants in Finland. 


We thank Riikka Mäkinen for excellent technical assistance.

S3 - P - 19 

DETECTION OF SCHMALLENBERG VIRUS IN BOVINE SEMEN BY ONE-STEP REAL-TIME RT-PCR 

Remco Dijkman 1 , Jet Mars 1 , Gerard Wellenberg 1 

1 

Animal Health Service, Deventer, The Netherlands 

2 

Affiliation Institute name, Department name, City, Country 

Schmallenberg virus, semen, real-time PCR 

Introduction 

For the detection of the Schmallenberg virus (SBV) in serum, 

tissues and body fluids, a real-time PCR, based on the detection 

of specific regions of the L-segment of the Schmallenberg virus, 

was developed (1). Recently, two real-time PCR methods, based 

on the detection of S-segment sequences, were developed too. 

However, these PCR methods have not been validated for 

semen samples. 

In this study, we have examined whether the L-PCR, S3-PCR 

and an “in-house” S-PCR were suitable for the detection of SBV 

in bovine semen. 


Besides the L1 and S3 PCR, which were developed by FLI 

(Germany), we also used an “in-house” pan-orthobunyavirus S- 

PCR (OBV-S PCR) real-time PCR targetting the orthobunyavirus 

S-segment for the detection of SBV in bovine semen samples. 

RNA was extracted with the AM1836 extraction kit (Life 

Technologies) using the MagMAX express 96 system (Life 

Technologies). The AgPath-ID one-step RT-PCR kit (Life 

Technologies) was used for amplification. PCR amplification was 

performed on a ABI7500 sequence detection system from 

Applied Biosystems (Life Technologies). 

The validation of the three PCR tests was based on the 

determination of the detection limit (lowest detectable amount of 

SBV per mL semen) for undiluted (fresh semen) and diluted 

semen (straw) samples. 

Therefore, ten-fold dilutions from a Schmallenberg virus stock 

solution with a known titer (10 4.9 TCID50/mL) were used for 

spiking semen samples and subsequently used for analyses. 

This study was also focused on spiking experiments to determine 

the negative influences of inhibitory compounds in semen 

samples as they may lead to false-negative results. 

As it is unknown whether SBV is excreted by semen, and natural 

SBV infected semen samples were not available, spiking 

experiments have been performed with undiluted (fresh semen) 

and diluted semen (straw) samples. Undiluted semen from 8 

different bulls (bulls A t / H) and diluted semen from 14 bulls 

(bulls I t / m V) were used. The recovery of the SBV in undiluted 

and diluted semen samples was determined by PCR and 

compared with the results of control samples. 

Results 

In silico analyses showed that the primers and probes of the L- 

and S3-PCR are specific for SBV, and that the selected primers 

and probe of the OBV S-PCR recognize conserved regions within 

the S-segment of SBV, also present in other orthobunyaviruses. 

The results of the detection limits of the three real-time PCR 

methods are reported in Table 1. 

straws were comparable or to a maximum of 4 Ct-values higher 

than the Ct-value of the Positive Reference sample (PBS matrix). 

The semen samples without spiking were negative in all three 

PCR methods (Figure 1). 

Figure 1: Ct-values of 1:3 diluted (fresh) semen and diluted 

semen (straws)spiked with SBV as obtained by L-, S3- and the 

OBV-S PCR. 


This validation study showed that the L-, the S3-, and the OBV-S 

PCR are able to detect SBV in 1:3 diluted fresh semen and 

diluted (straw) semen samples. The detection limits of the S3- 

and the OBV-S PCR are lower compared to the detection limit of 

the L-PCR. 

In addition, the spiking experiments, to examine the influence of 

inhibitory compounds in bovine semen samples, showed that no 

negative influences were recorded in 1:3 diluted semen and in 

diluted semen (straw) samples. Therefore, fresh semen needs to 

be diluted prior to the RNA-extraction and semen straws can be 

examined by PCR without further dilution. 

The use of an internal control, to check for inhibitory effects of the 

matrix, was not examined in this study, but can be the scope for 

further validation. 

As fresh semen can be diluted 1:3, and semen in straws are 

diluted approximately between 1:20 and 1:30, the screening of 

fresh semen is more sensitive than straws. 

It might be useful to use the OBV-S PCR as a general screening 

PCR, and the S3-PCR can be used as a screening and/or 

confirmatory PCR. 

References 


H, et al. Novel orthobunyavirus in cattle, Europe, 2011. Emerg Infect Dis 

(epub). 

Table 1: Detection limits of the three real-time SBV PCR methods 

Test method 

Detection limit (TCID50/ml) 

Undiluted semen Diluted semen 

Straws 

L-PCR 10 1.9 10 0.9 

S3-PCR 10 -0.1 10 -1.1 

OBV-S PCR 10 -0.1 10 -0.1 

Results showed that inhibitory effects were recorded for undiluted 

(fresh) semen, while no inhibitory effects were recorded by PCR 

when these semen samples were 1:3 pre-diluted. Ct-values of 

the 1:3 diluted fresh semen samples and the diluted semen

S3 - P - 20 

INTERFERON-GAMMA ASSAY TO DIAGNOSE Mycobacterium bovis INFECTION IN PIGS 

Michele Pesciaroli 1 , Piera Mazzone 2 , Cinzia Marianelli 1 , Sara Corneli 2 , Miriam Russo 3 , Vincenzo Aronica 3 , Michele 

Fiasconaro 3 , Massimo Biagetti 2 , Marcella Ciullo 2 , Monica Cagiola 2 , Paolo Pasquali 1 and Vincenzo Di Marco 3 

1 Istituto Superiore di Sanità, Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare, Italy, 

2 Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Italy, 3 Istituto Zooprofilattico Sperimentale della Sicilia,Italy 

Mycobacterium bovis, pig, γ-IFN test 

Introduction 

Different species of animals are susceptible to Mycobacterium 

bovis (MB), the causative agent of bovine tuberculosis (bTB) and 

may represent both "reservoir" of infection or "spillover host". 

Swine, until a few years ago, was considered only a spillover host 

(1). In areas where MB is still present in cattle which share the 

pasture with the pig population, the marginal role attributed to the 

pig in the maintenance of infection of MB is strongly questioned. 

When high rates of infection are observed also in pigs, this 

species may play the role of true "reservoir" (2). The diagnostic 

procedures used to identify in vivo MB infected cattle, including 

skin test (IDT) and γ-interferon (γ-IFN) test, are also adopted in 

other animal species (3,4) but their application in pigs has not 

been completely evaluated. The aim of this study was to 

calculate the sensitivity of the γ-IFN test in pigs infected with MB, 

compared with the results of post-mortem diagnostic tests 

(pathological examination and culture). 


Animals: The Black Sicilian Pig is a traditional Italian breed native 

of the Nebrodi Park (Sicily). These pigs are usually reared in freerange 

farms and share the pasture with cattle. In this area the 

prevalence of TB in cattle ranges from 5.36% to 8.58% (data 

2010 Italian Ministry of Health). For the study, 100 Blacks Sicilian 

Pigs, coming from farms with bTB positive cases (2), aging from 

12 to 24 months, were randomly sampled at the abattoir of Mirto 

(Sicily). In addition, 15 healthy pigs from tuberculosis-free herds 

were sampled at the same abattoir and used to set up the 

method and as negative controls. 

Protocol and dose response curves of IFN-γ: Samples of whole 

blood of the 15 healthy pigs were stimulated with 5μl/ml of 

Pokeweed mitogen (PWM) (Sigma-Aldrich, MS, USA) and 5μl/ml 

of "Phosphate Buffered Saline" (0.01M PBS, pH 7.2). The 

production of γ-IFN by lymphocytes was evaluated using a 

sandwich ELISA (Porcine IFN-gamma Quantikine ELISA Kit, 

R&D Systems, Mn, USA) on the supernatant collected after 24 

hours of incubation at 37 °C at 5% CO 2 . The stimulation with PBS 

did not induce any production of IFN-γ; on the contrary, the blood 

samples stimulated with PWM, showed an average production of 

γ-IFN of 2633 µg/ml, with a standard deviation of 688.2 µg/ml. 

The samples stimulated with PWM, with a value below 1250 

µg/ml (calculated as mean - 2 SD), were excluded from the 

analysis because considered unresponsive. 

γ-IFN test: Heparinized blood samples, taken at the 

slaughterhouse, were dispensed in aliquots of 1.5 ml in 24-well 

plates (Costar, Corning Incorporated, Corning, NY, USA). The 

lymphocyte stimulation was performed in duplicate: PBS, for the 

evaluation of the basal value of γ-IFN (N); PWM (5 μl/ml); bovine 

PPD (10μg/ml); avian PPD (10μg/ml). The PPDs (purified protein 

derivatives), used in the experiment, were both produced by the 

Istituto Zooprofilattico dell’Umbria e delle Marche (IZSUM), 

according to the official protocols (EC Regulation N o 1226/2002). 

The values obtained from each sample, expressed in units of 

Optical Density (OD), were interpreted as reported below. 

Table 1. Possible outcomes following lymphocytes stimulation 

with avian and bovine PPDs (OD 450 nm ) 

AOD & BOD < 2N 

Negative 

AOD or BOD > 2N 

AOD 2 N Positive for M. avium 

BOD 2 N Positive for M. bovis 

BOD/AOD 0,9 Positive for M. avium 

AOD & BOD > 2N BOD/AOD 1,1 Positive for M. bovis 

0.9 BOD/AOD 1,1 Doubt 

N = baseline value (value obtained with PBS only) 

AOD = value obtained after stimulation with avian PPD 

BOD = value obtained after stimulation with bovine PPD 

Pathological examination (EAP): The macroscopic examination 

was conducted at the slaughterhouse in accordance with 

conventional inspection methods. Samples of lymph nodes (LN) 

from the head (mandibular, retropharyngeal, parotid LN), the 

thorax (bronchial LN), and the abdomen (hepatic, gastric, 

intestinal LN) were also collected from all the pigs. 

Microbiology: Bacterial culture for MB was performed according 

to the Italian Ministerial Decree (DM 592/1995) on each lymph 

node. Suspect colonies, testing positive for acid fast staining, 

were further identified through bio-molecular techniques (3). 

Results 

Of the 100 pigs involved in the study, 2 subjects did not respond 

to stimulation with PWM; these animals were excluded from the 

final evaluation. EAP and culture identified respectively 26 

(26,5% ) and 19 (19,4%) positive pigs . γ–IFN assay identified 22 

positive pigs among the 26 with tubercular lesions and 15 among 

the 19 with positive bacterial culture (Table 2). The sensitivity of 

the test, assuming as gold standard the traditional tests, resulted 

84,6% (95% CI 52.6-100%) and 78,9% (95% CI 44.8-100%) 

respectively. 

Table 2: Comparison between the results obtained in γ-IFN test, 

EAP and culture for M. bovis. P: Positive N: Negative. 

Lesion 

Culture for M. bovis 

P N P N 

P 22 4 15 11 

γ- IFN 

N 4 68 4 68 

Total 26 72 19 79 


Our results demonstrate the circulation, in the Nebrodi Park, of 

MB also in the swine population that inhabits this area (2), 

supporting the hypothesis of an epidemiological role of the Black 

Sicilian pig as a significant reservoir of infection. Therefore, the 

implementation of future control strategies also in this species is 

needed. In this perspective, γ-IFN test could represent a good 

tool for intra vitam diagnosis of bTB in pigs. The study results 

show, in general, an acceptable level of agreement between 

traditional diagnostic tests and γ-IFN test. In particular, as shown 

in Table 2, there is a better agreement with the presence of 

tubercular lesions rather than with the MB isolation. This feature 

can be explained with a generally low sensitivity of the cultural 

test (4). Animals with positive reaction at γ-IFN test showing 

neither macroscopic lesions nor MB infections could be assumed 

as false positive. However, they could also be ascribed to the 

capacity of this immunoassay to identify recently infected 

subjects with still unapparent lesions at least during the routine 

post-mortem inspection. These preliminary results encourage us 

to plan future research to optimize γ-IFN test performance in 

swine. This study was founded by the Research Project of the 

Seventh Framework Program “Strategies for the eradication of 

bovine tuberculosis” (Contract Number 212414 – Acronyms TB- 

STEP). 

References 

1. O’Reilly L. M., Daborn C. J., (1995) The epidemiology of Mycobacterium 

bovis infections in animals and man: a review. Tubercle and lung disease. 

The official journal of the International Union against Tuberculosis and 

Lung Disease, 76 Suppl. 1: 1-46. 

2. V. Di Marco, P. Mazzone, MT. Capucchio, MB. Boniotti, V. Aronica, M. 

Russo, M. Fiasconaro, N. Cifani, S. Corneli, E. Biasibetti, M. Biagetti, ML. 

Pacciarini, M. Cagiola, P. Pasquali, and C. Marianelli. “Epidemiological 

Significance of the Domestic Black Pig (Sus scrofa) in Maintenance of 

Bovine Tuberculosis in Sicily” J Clin Microbiol. 2012 Apr;50(4):1209-18. 

Epub 2012 Feb 8. 

3. OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 

2010 Chapter 2.4.7. Bovine tuberculosis (Version adopted in May 2009) 

4. Boadella M., Lyashchenko K., Greenwald R., Esfandiari J., Jaroso R., 

Carta T., Garrido J. M., Vicente J., de la Fuente J., Gortázar C. (2011) 

Serologic tests for detecting antibodies against Mycobacterium bovis and 

Mycobacterium avium subspecies paratuberculosis in Eurasian wild boar 

(Sus scrofa scrofa). J Vet Diagn Invest. 23 (1): 77-83

S3 - P - 21 

USE OF EXPERIMENTAL JOHNIN IN THE GAMMA-INTERFERON TEST 

IN CATTLE INFECTED BY Mycobacterium avium subsp. paratuberculosis: PRELIMINARY DATA 

Piera Mazzone 1 , Nicoletta Vitale 2 , Matteo Ricchi 3 , Sara Corneli 1 , Piermario Mangili 1 , Paola Papa 1 , Massimo 

Biagetti 1 , Carla Sebastiani 1 , Angela Caporali 1 , Alex Raber 4 , Giovanni Pezzotti 1 , Norma Arrigoni 3 , Monica Cagiola 1 

1 

Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Italy 

2 

Istituto Zooprofilattico Sperimentale Piemonte, Liguria e Valle d’Aosta,Italy 

3 

Centro di 

Referenza Nazionale per la Paratubercolosi - Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna,Italy 4 Prionics AG Switzerland 

Paratuberculosis, γ-IFN test, Johnin 

Introduction 

The diagnosis of Paratuberculosis (PTB), due to Mycobacterium 

avium subsp. paratuberculosis (MAP), is usually based on 

serology and fecal culture, detecting advanced stages of 

infection. The Gamma Interferon (γ-IFN) test, used for antemortem 

diagnosis of bovine Tuberculosis (bTB), due to 

Mycobacterium bovis (MB), together with Skin Test (IDT) (1), 

may be used as additional test also for PTB diagnosis. Moreover, 

the development of an immunological test that, at the same time, 

may distinguish between animals infected with MB or MAP and 

enables an early diagnosis of PTB, could support the control of 

both diseases. The γ-IFN test detects the amount of cytokine 

produced by T lymphocytes of infected animals, in response to a 

stimulation carried out with the traditional bovine and avian 

purified protein derivatives, respectively extracted by MB (PPDB) 

and by M. avium (PPDA). Similarly, the Johnin PPD (PPDJ) is 

obtained from a culture of MAP (2). In this preliminary study, in 

order to achieve an early diagnosis of PTB, 3 new PPDJs were 

produced, used in the γ-IFN test and compared to classic PPDs, 

avoiding false positive reactions for bTB. 


Johnin production: 20 Italian MAP strains, candidates to be used 

for production of PPDJs, were genotyped by amplification of Mini 

and Microsatellite loci (3). Two strains were selected: the one 

with the most and the one with the least frequent genetic profile 

observed in Italy, identified as Strain A and Strain B respectively. 

The strain ATCC 19698 (Strain C) was used for the third batch of 

Johnin. The three MAP strains were cultured for 4 months at 

37°C in Watson-Reid modified broth, and then the bottles were 

autoclaved at 100°C for 3 hours. Cells were removed and the 

proteins extracted by precipitation with trichloroacetic acid. The 

precipitate was then washed and dissolved in phosphate 

phenolate buffer and glycerine, with a final protein content of 1 

mg/ml, as required for PPDB by the Decree of the Italian Ministry 

of Health June, 26 th 1981 and Regulation (EC) N o . 1226/2002. 

Field trials: 24 IDT tuberculosis negative bovines from 3 bTB 

officially free (OF) dairy herds were selected. In these herds, 

clinical cases of PTB had been reported. The animals were 

subjected in parallel to ELISA test for PTB (IDVet), PCR (4) and 

culture (2) for MAP from faeces. Animals with positive results in 

at least one of the tests were considered positive for PTB. Of the 

24 animals, 16 were positive to at least one of the tests and 8 

were negative for all. 

In vitro stimulation and γ-IFN test: The heparinized blood 

samples of the 24 bovines were dispensed in aliquots of 1 ml and 

stimulated respectively with: "Phosphate Buffered Saline" (PBS 

0.01 M, pH 7.2), considered as blank; 30 μg of PPDB and 30 μg 

of PPDA (AgriQuality Australia Pty Ltd, Victoria, Australia); 10 μg 

of Italian PPDB and 10 μg of Italian PPDA; three experimental 

PPDJ (strains A, B, C) with three different dilutions (1:5, 1:10 and 

1:15). The evaluation of γ-IFN was performed with the Elisa kit 

BOVIGAM ® in the plasma collected after 24 hours of incubation. 

The samples have been considered positive when the Optical 

Density value (OD450 nm) was, at least, twice the OD of the 

blank. To evaluate the "dilution factor" of Johnin (1:5, 1:10; 1:15) 

and the "strain factor" (A, B, C), a 2-way analysis of variance 

using the procedure Proc GLM of SAS ® 

v9.2 software was 

conducted. The relative specificity of PPDB and PPDJ in the 

diagnosis of bTB (bTB OF farms) was calculated using the IDT 

as Gold Standard (GS). The relative sensitivity of PPDJ in the 

diagnosis of PTB was calculated using as GS positivity in ELISA 

and/or PCR and/or Bacteriological test. 

Results 

The results of the lymphocyte stimulation with the different PPDs 

are shown in Figure 1. The analysis of variance showed 

statistically significant differences between the mean OD 450 of 

PPDJ for both the "dilution factor" of Johnin (F test = 28.44, 

p

S3 – P - 22 

COMPARISON OF THE PERFORMANCE OF FIVE DIFFERENT IMMUNOASSAYS TO DETECT SPECIFIC 

ANTIBODIES AGAINST EMERGING ATYPICAL BOVINE PESTIVIRUS 

Magdalena Larska 1,2 , Lihong Liu 3 , Mirosław P. Polak 1 , Stefan Alenius 2 , Åse Uttenthal 5 

1 National Veterinary Research Institute (NVRI), Virology Department, Puławy, Poland, 2 Department of Clinical Sciences, Swedish University of 

Agricultural Sciences (SLU), Uppsala, Sweden; 3 Department of Virology, Immunology and Parasitology (VIP), National Veterinary Institute (SVA), 

Uppsala, Sweden; 5 National Veterinary Institute, Technical University of Denmark, Lindholm, Denmark 

Atypical bovine pestivirus, antibodies, ELISA, Microsphere-based immunoassay (MIA), diagnosis 

Introduction 

Atypical bovine pestiviruses are a group of viruses which together 

with two bovine viral diarrhoea virus species (BVDV-1 and 

BVDV-2), border disease virus (BDV) and classical swine fever 

virus (CSFV) comprise the Pestivirus genus of the Flaviviridae 

family. The group was previously referred to as Hobi-like 

pestiviruses from the precursor strain isolated from Brazilian 

foetal bovine serum (FBS) (2). Increasing evidence of 

intercontinental spread of the atypical pestivirus raised the 

discussion over the emergence of the new BVDV species and 

prompts the search for the tools supporting testing and control 

programs. Serological test has not been validated to detect 

specific antibodies against those pestiviruses yet. 

0.0001) with ρ values between 0.82 for b-ELISA-2 and 0.92 for i- 

ELISA-2. 


The study was conducted using 140 sera from calves 

experimentally inoculated with Asian atypical pestivirus 

Th/04_KhonKaen strain (Group A); BVDV-1a strain Horton 916 

(Group B), with a mixture of both viruses (Group C) and 

uninfected controls (1). Serum samples were collected from all 

animals on 0, 7, 14, 21, 28, 35, 42 days post inoculation (dpi). 

Five serological tests including two commercial indirect BVDV 

ELISAs (i-ELISA-1 and 2), two blocking BVDV ELISAs (b-ELISA- 

1 and 2) and newly developed microsphere immunoassay (MIA) 

(3) using recombinant E rns protein of Th/04_KhonKaen pestivirus 

were evaluated for their robustness, sensitivity, specificity and 

receiver operating characteristic (ROC) using virus neutralization 

test (VNT) as ’gold’ standard. 

Results 

According to VNT using homologous to inoculum viruses the 

animals from groups A and B started to seroconvert against 

homologous viruses at 14 dpi, while Group C seroconverted 

independently to both viruses before 14 dpi. The earliest 

detection of antibodies in the Group A was observed at 14 dpi in 

the i-ELISA-1, b-ELISA-2, MIA. Using i-ELISA-2 the antibodies 

were detected first in two animals from Group A at 21 dpi and in 

all animals from 28 dpi. Detection of specific antibodies in Group 

A was the most delayed to the end of experiment in b-ELISA-2. 

100% sensitivity in detecting Th/04_KhonKaen virus specific 

antibodies in Group A was obtained by b-ELISA-2 and MIA, while 

their specificities were 82% and 91%, respectively. Other assays 

were characterised by 100% specificity, however the sensitivity 

varied from only 25% for b-ELISA-2 and BVDV-1 VNT to 83% for 

i-ELISA-1 when testing calves from Group A. Most assays 

detected 100% of seropositive animals from Group B which were 

categorised as positive in BVDV-1 VNT, except for b-ELISA-1 

which had only 72% sensitivity. The specificity of the tests was 

lower in respect to VNT test, possibly due to the lower sensitivity 

of the latter. The AUC for the methods detecting atypical 

pestivirus antibodies were the highest reaching 0.93 for MIA and 

the lowest (0.62) for b-ELISA-1 and BVDV-1 VNT. Evaluation of 

positive-negative cut-off values by ROC analysis showed that 

optimizing the recommended by manufacturer or SOP values 

could increase senstitivities and specificities of some 

immunoassays. Two tests: MIA and b-ELISA-2 showed more 

flexible cut-offs which allowed the values to fluctuate within a 

wider range without influencing their performance. 

The values of all ELISA tests were significantly correlated with 

the neutralizing titres against atypical pestivirus and BVDV-1 (P < 

0.0001) when tested by the Spearman rank correlation 

coefficients (ρ). Anti-Th/04_KhonKaen antibody titres showed the 

comparable correlations with all ELISAs tested, with the highest 

correlation to b-ELISA-2 (ρ= 0.84). The lowest correlation was 

observed between Th/04_KhonKaen antibody titres and VNT 

using BVDV-1 strain (ρ= 0.65). The correlations between BVDV-1 

antibody titres and ELISA results were also significant (P < 

Figure 1: Comparison of the sensitivity (Se) and specificity (Sp) 

(A) and AUC (area under ROC curve) values (B) of all 

immunoassays. 


The results have shown that all the tested immunoassays were 

suitable to detect antibodies raised in calves inoculated with 

atypical pestivirus, however only some could be used in the early 

detection of the seroconversion. Two ELISAs (i-ELISA-1 and b- 

ELISA-2) and novel MIA were considered to be superior (even in 

respect to BVDV-1 VNT) for the routine detection of atypical 

bovine pestivirus antibodies in cattle. The analysis of optimal cutoff 

values suggested that cautious result interpretation using the 

recommended cut-off values diminish the possibility of obtaining 

false results. Here the focus is on early detection of antibodies, 

as the immune system will initially produce IgM antibodies there 

is a natural preference of assays that are independent of IgG. 

Therefore, despite the conservative nature of NS3 protein, 

blocking b-ELISA-1 performed with the lowest sensitivity and the 

seroconversion appeared at the latest. Development of MIA 

using microspheres conjugated with mAbs against atypical 

pestivirus E rns protein resulted in high sensitivity and specificity of 

detection of antibodies against homologous virus comparable to 

VNT, which however did not promise much possibility for the 

pestivirus specific discrimination as it detected BVDV-1 

antibodies equally efficiently. 

References 

1. Larska et al., 2012. Kinetics of single and dual infection of calves with an 

Asian atypical bovine pestivirus and a highly virulent strain of bovine viral 

diarrhoea virus 1.Comp Immunol Microbiol Infec Dis. {Epub ahead of print] 

2. Schirrmeier et al., 2004. Genetic and antigenic characterization of an 

atypical pestivirus isolate, a putative member of a novel pestivirus species. 

J Gen Virol. 85, 3647–3652 

3. Xia et al., 2010. A microsphere-based immunoassay for rapid and 

sensitive detection of bovine viral diarrhoea virus antibodies. J Virol 

Methods 168, 18-21.

S3 - P - 23 

SCHMALLENBERG VIRUS (SBV) IN POLAND – PRELIMINARY RESULTS 

Magdalena Larska, Mirosław P. Polak, Jan F. Żmudziński 

National Veterinary Research Institute (NVRI), Virology Department, Puławy, Poland 

Schmallenberg virus, testing, Culicoides 

Introduction 

SBV was first detected in Germany by metagenomics analysis of 

samples collected from sick cows from the city of Schmallenberg 

in the autumn 2011 (1). Until now over 4000 cases were 

described in Northern Europe. Because Poland is a neighbouring 

country to the area covered by SBV epidemic, the risk of further 

spread to our territory is possible. European SBV is a reassortant 

of Sathuperi and Shamonda viruses belonging to 

Orthobunyavirus of the family Bunyaviridae which emerged 

around a decade ago in Japan (2). The source of SBV infection in 

Europe is not know, however the virus is known to spread by 

Culicoides sp. biting midges, similarly to bluetongue virus (BTV), 

what may suggest climate change or involvement of redistribution 

of vectors as a possible cause. 


Tissue and blood samples from fifteen animals from two cattle 

and one goat herds from different parts of Poland were submitted 

to NVRI by district official veterinarians under the suspicion of 

Schmallenberg virus infection. The clinical signs included 

abortions (no malformation observed), fever, depression and in 

older cows respiratory disorders with sudden death. Mortality in 

older cows was later confirmed to occur due to antibiotic resistant 

Mannheimia haemolytica infection. Additionally, 36 pools of 

midges including C. obsoletus, C. punctatus, C. chiropterus and 

C. pulicaris from the border zone with Germany and from northeastern 

part of Poland collected in September/October 2011 

were tested. Midge pools were homogenised in Ribolyzer tubes 

(Hybaid, UK) containing 0.6g beads with TRI Reagent (Sigma 

Adrich) for 80 sec. at 5m/sec. speed. Viral RNA was extracted 

using TRI Reagent. Real time RT-PCR was performed according 

to Friedrich-Loeffler Institut (FLI) protocol using primers and 

probes specific to S and L segment of SBV genome with β-actin 

as internal control. AgPath-ID One-Step RT-PCR Reagents 

(Ambion-Applied Biosystems) were used and the reaction was 

performed in StepOnePlus machine (Applied Biosystems). SBV 

RNA standard used as positive control and to determine 

sensitivity/specificity was acquired from FLI. 

Figure 2: Standard curve (correlation of Ct value and log dilution 

of SBV RNA standard) 


More samples have to be tested to verify the possibility of SBV 

introduction into Polish herds. However, Polish population of 

sheep and goats is scarce (approx. 250 000 and 130 000, 

respectively), the disease may already be present in the country, 

but SBV is not detected because of its mostly asymptomatic 

course in cattle. More research including some seroprevalence 

studies is planned. 

References 

1. Hoffmann et al., 2012. Novel orthobunyavirus in Cattle, Europe, 2011. 

Emerg Infect Dis. 18, :469-7 

2. Yanase et al., 2012. Genetic reassortment between Sathuperi and 

Shamonda viruses of the genus Orthobunyavirus in nature: implications for 

their genetic relationship to Schmallenberg virus. Arch Virol. [Epub ahead 

of print] 

Results 

All samples were negative for the presence of SBV genetic 

material. In all animal and insect samples amplification of β-actin 

was positive with Ct values ranging from 25 to 34. Specificity of 

PCR products was confirmed by sequencing. The specificity of 

the assays was 100%, while sensitivity was comparable to FLI 

(detection limit at 10 -7 

SBV RNA). The efficiency determined 

using the standard RNA was approx. 98% with the mean slope of 

3.38. 

Figure 1: Amplification plots of SBV RNA standard with segment 

S specific primers and TaqMan probe.

S3 - P - 24 

DEVELOPMENT OF A VIRUS NEUTRALISATION TEST TO DETECT ANTIBODIES AGAINST 

SCHMALLENBERG VIRUS AND FIRST RESULTS IN SUSPECT AND INFECTED HERDS 

W.L.A. Loeffen 1 , S. Quak 1 , E. de Boer-Luijtze 1 , W.H.M. van der Poel 1 , R. Bouwstra 2 , R. Maas 1 

1 

Central Veterinary Institute, of Wageningen University and Research Centre (CVI-Lelystad), Department of Virology, Lelystad, The Netherland. 

2 

Central Veterinary Institute, of Wageningen University and Research Centre (CVI-Lelystad), Department of Epidemiology, Crisis Management and 

Diagnostics, Lelystad, The Netherland. 

Schmallenberg, VNT, sensitivity, specificity 

Introduction 

On the 18th of November 2011, a new Orthobunya virus was 

reported to be found in Germany (3). This virus was closely 

related to Shamonda-, Aino- and Akabane-virus, which all belong 

to the Simbu serogroup of the genus Orthobunyavirus, family 

Bunyaviridae (1). The virus, provisionally called Schmallenberg 

virus (SBV), has since been associated with clinical signs of 

decreased milk production, watery diarrhoea, fever and 

congenital malformations (3,4,5). 

To determine the prevalence of the disease and carry out 

epidemiological studies, there was a need for a reliable and 

robust serological assay. A virus neutralisation test (VNT) was 

developed, as this also allows for a (semi)quantitative detection 

of antibodies against the virus. An initial validation (specificity, 

sensitivity, repeatability, reproducibility, and robustness) was 

carried out and the test was used in suspect (congenital 

malformation, no positive RT-PCR) and infected herds (positive 

RT-PCR). 


The VNT was carried out in flat bottomed 96 well micro titre 

plates. Serum samples were inactivated for 30 min at 56°C and 

from a starting dilution of 1:4, two-fold dilutions were made. 

Subsequently 500 TCID 50 of virus was added to each well. Serum 

and virus were pre-incubated at 37°C for 1 to 2 hours to allow 

neutralisation of the virus. Thereafter, 20,000 cells (VERO cells) 

per well were added. Plates were then incubated for 5 days at 

37°C and under 5% CO2. 

After 5 days, the plates were stained with amido black and read 

macroscopically. Wells with 25-100% staining were considered to 

have no or limited CPE only. Wells with less than 25% staining 

were considered to have extensive or full CPE. In each dilution 

series the last well with no or limited CPE was identified and the 

sample was assigned a titre that was the reciprocal of the dilution 

in that well. Control samples were included and a back titration of 

the virus was carried out for each test run. 

Initially, 351 blood samples (160 sheep, 191 cattle) from noninfected 

animals were tested in the final version of the assay. 

These samples originated from the Netherlands, from the period 

of 2000-2010, before the introduction of the Schmallenberg virus. 

They were used to set the initial cut-off value in the assay. 

Subsequently, 366 samples from suspect herds (being herds in 

which congenital malformations were observed, with no 

confirmation in the RT-PCR), and infected herds (herds where 

the infection was confirmed by RT-PCR). 

Results 

In serum samples from non-infected sheep, a higher background 

was seen than in sera from non-infected cattle (figure 1). 

Figure 1: Distribution of titres in non-infected sheep (n=160) and 

cattle (n=191). 

Based on testing 160 sheep and 191 cows, the cut-off values 

were set at a titre of 16 for sheep and 8 for cattle. Given this cutoff 

value, a specificity of 99.4% in sheep and 99.5% in cattle was 

estimated. 

Ninety-two percent of the cattle and 94% of the sheep from 

suspect and infected herds scored positive in the VNT. In sheep 

no titres of 8 and in cattle no titres of 4 were observed, thus 

clearly separating the peak of the positive samples from the peak 

of the negative samples (figure 2). This again corresponds to a 

cut-off value of 16 and 8 respectively from sheep and cattle, as 

determined already by testing serum samples from non-infected 

animals. Sensitivity of the assay is therefore at least 92% and 

94% in cattle and sheep respectively, assuming the worst case 

scenario that all were seropositive in reality. However, the clear 

separation of the negative and positive peak suggests that the 

sensitivity for both species is close to 100%. 

Figure 2: Distribution of titres in goats, sheep and cattle from 

suspect (in utero malformations, RT-PCR negative), and infected 

herds (infection confirmed by RT-PCR). 


A very robust VNT was developed with a very high specificity and 

sensitivity, both close to 100%, in sheep and cattle. The assay 

was shown to be highly repeatable and reproducible (results not 

shown). The VNT is easy to perform and can be read 

macroscopically, as a result of the amido black staining. With the 

current, optimized protocol, this staining results most of the times 

in a very sharp transition between wells with and without CPE. 

This makes it easy and very fast to read the plates, with a high 

reliability and reproducibility. 

In suspect and infected herds more than 90% of the sheep and 

cattle tested seropositive, suggesting that within herds with 

congenital malformations, the infection is wide spread with a high 

within-herd seroprevalence. High seroprevalences, both on a 

regional level, but also within herds, with and without obvious 

clinical signs, are not uncommon for viruses related to SBV. A 

very high overall seroprevalence of 70% in cattle was recently 

also reported in the Netherlands, using this VNT (2). 

References 

1. Causey OR, Kemp GE, Causey CE, Lee VH. 1972. Ann Trop Med 

Parasitol 66: 357-62. 

2. Elbers ARW, Loeffen WLA, Quak S, et al. 2012. Emerg Infect Dis. 

3. Hoffmann B, Scheuch M, Höper D, et al. 2012. Emerg Infect Dis. 

4. Muskens J, Smolenaars AJG, Van der Poel WHM, et al. 2012 Tijdschr 

Diergeneeskd. 137: 112-5. 

5 .Van den Brom R, Luttikholt SJM, Lievaart-Peteron K,et al. 2012 

Tijdschr Diergeneeskd 137: 106-11.

S3 - P - 25 

VIRAL DIAGNOSIS USING TRANSMISSION ELECTRON MICROSCOPY SCHMALLENBERG VIRUS 

David J. Everest and W. A. Cooley 

1 

Central Animal Health and Veterinary Laboratories Agency-Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK 

Most viruses are very small and visualisation by Transmission 

Electron Microscopy (TEM) has provided a major contribution in 

the discovery and detection of viral infections. The AHVLA’s Bioimaging 

unit provides a rapid viral diagnostic service, analysing 

fresh unfixed scab, lesion, wart and faecal materials by TEM. 

Species range from domestic farm animals, to wildlife, marine 

mammals and Zoo animals, covering red squirrels to Elephants. 

Historically, this form of microscopy has been a key application in 

the diagnosis and surveillance of several wildlife viral infections. 

These have included Squirrel pox and adenovirus, both 

contributing to the decline of the UK Red Squirrel, calicivirus from 

Rabbits and Hares, the causative agent of rabbit haemorrhagic 

disease and pox viruses in various marine mammal species.

S3 - P - 26 

DETECTION OF EQUINE FOAMY VIRUS INFECTIONS IN HORSES 

Magdalena Materniak, Jacek Kuzmak 

National Veterinary Research Institute, Department of Biochemistry, Pulawy, Poland 

Equine foamy virus, diagnostics, PCR, ELISA 

Introduction 

Foamy viruses (FVs) are the least known complex retroviruses. 

FVs infections are highly prevalent among several animal species 

including non-human primates (chimpanzees, gorillas, etc.), cats 

and cattle. Another foamy virus was isolated from horses (Equine 

foamy virus - EFV) (1). Similarly to other FVs, EFV shows 

characteristic ultrastructure and susceptibility to infect large 

number of cell lines. Furthermore, its molecular analysis exhibits 

genome organization typical for other known foamy viruses, but 

its sequence shows the highest similarity to bovine foamy virus. 

Although the first isolation of EFV was reported over ten years 

ago, still nothing is known about the prevalence of EFV infections 

in horses. Therefore we aimed to develop diagnostic tools and 

apply them to investigate the EFV infection in horses from 

Poland. 

References 

1. Tobaly-Tapiero J., Bittoun P., Neves M., Guillemin M.-C., Lecellier Ch.- 

H., Puvion-Dutilleul F., Gicquel B., Zientara S., Giron M.-L., The H. de, 

Saϊb A. (2000). Isolation and characterization of an eqiune foamy virus. J. 

Virol., 74, 4064-4073. 

2. Romen F., Backes P., Materniak M., Sting R., Vahlenkamp T. W., Riebe 

R., Pawlita M., Kuzmak J., Löchelt M. (2007). Serological detection 

systems for identification of cows shedding bovine foamy virus via milk. 

Virology, 364, 123-131. 


Blood samples were collected from 175 horses (4 -18 years old), 

including common breeds, Hucul ponies and randomly selected 

samples from slaughtered horses. Semi-nested PCR was 

developed and applied to detect EFV DNA in DNA extracted from 

peripheral blood leukocytes (PBLs). Selected primers flanked 275 

bp fragment within pol gene, the most conservative EFV region. 

GST ELISA with BFV specific Gag-GST fusion protein (2) was 

used for detection of foamy virus specific antibodies in horse 

serum samples. To confirm ELISA results immunoblotting with 

antigen prepared from Cf2Th/BFV 100 cells was applied. 

Additionally, attempts were made to isolate EFV from peripheral 

blood leukocytes of PCR positive animals by co-cultivation with 

BHK-21 and RK-13 cells. 

Results 

12.5% of samples were positive in semi-nested PCR. Specificity 

of amplified fragments was confirmed by sequencing and parwise 

identity of particular sequences ranged from 95.3% to 98.3%, 

when compared to EFV reference sequence (GenBank Acc. No. 

NC_002201). In contrast, almost 19% of the samples showed 

seroreactivity to BFV Gag protein. Most of PCR positive samples 

were detected in Hucul ponies, while only several in samples 

randomly collected from slaughtered animals. All attempts to 

isolate EFV from PBLs of PCR positive horses failed. 

Table 1: Summary of results. 

Origin 

PCR (+) ELISA (+) 

samples samples 

Hucul ponies 15 14 0 

Saddle-horses 3 6 0 

Slaughtered 

horses 

TOTAL 

4 

22 

13 

33 

Co-cultivation (+) 

samples 


This study presents the first report on prevalence of EFV infection 

in horses. The discordance between PCR and serological results 

can be due to application of BFV specific tests instead of EFV 

specific serological assays. Therefore further studies are 

essential to develop EFV specific serological tests to confirm 

obtained results. Nevertheless, our study clearly shows that EFV 

infections are present among horses in Poland. 


The authors would like to thank Dr. Ali Saïb and Dr. Joelle 

Tobaly-Tapiero for providing EFV positive DNA and serum 

controls. 

0 

0


“New techniques in bacteriology, 

parasitology and pathology” 

(4 th session)

S4 - P - 01 

MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS 

STRAINS IN A NATIONAL PARATUBERCULOSIS CONTROL PROGRAM 

Michaela Altmann 1 , Eva Sodoma 1 , Petra Möbius 2 , Michael Dünser 1 

1 

Institute for Veterinary Disease Control, Austria Agency for Health and Food Safety, Linz, Austria 

2 

Institute for Molecular Pathogenesis, Friedrich Loeffler Institute, Jena, Germany 

Mycobacterium avium subsp. paratuberculosis, RFLP, MIRU, VNTR, paratuberculosis 

Introduction 

In 2006 Austria started a national paratuberculosis (PTB) control 

programme based on government regulation which affects cattle, 

sheep, goat and farmed deer. PTB was declared a notifiable 

disease, so animals showing clinical signs have to be tested for 

MAP at the national reference laboratory. MAP strains isolated 

from different herds between 2006 and 2011 and strains from red 

deer were selected for molecular characterization. 


165 MAP field strains from 164 clinically diseased ruminants 

were analysed by IS900 RFLP with two restriction enzymes 

(BstEII, PstI) and MIRU-VNTR at 8 genomic loci (MIRU292,X3; 

VNTR25,47,3,7,10,32) according to MÖBIUS et al. (2008) and 

THIBAULT et al. (2007). These field strains included 141 MAP 

cattle isolates from 73 farms and different breeds (Angus, 

Aquitaine Blonde, Austrian Brown Mountain, Fleckvieh 

(Simmental), Tyrolean Grey Mountain, Holstein Friesian, Jersey, 

Limousin, Murbodner, Piemontese, Red Friesian, Danish 

Melkrace), 19 isolates from goat and 4 isolates from red deer. 

Results 

The combination of RFLP and MIRU-VNTR analysis allowed a 

differentiation of 18 MAP strain types classified AT1 to AT18. 

Detailed results and frequency in animals and herds are shown in 

Table 1. The occurrence of MAP strain types in different cattle 

breeds, goats and red deer is listed in Table 2. 

Table 1: RFLP and MIRU-VNTR results of 165 MAP field strains 

(INMV: INRA NOUZILLY MIRU-VNTR) 

RFLP INMV 

MIRU VNTR 

Σ 

292,X3 25,47,3,7,10,32 animals 

AT1 C1P1 1 4,2 3,3,2,2,2,8 16 

AT2 C1P1 2 3,2 3,3,2,2,2,8 59 

AT3 C18 2 3,2 3,3,2,2,2,8 18 

PnewIII 

AT4 C1P1 17 3,1 3,3,2,2,2,8 8 

AT5 C18 2 4,2 3,3,2,2,2,8 4 

PnewII 

AT6 C1 2 4,2 3,3,2,2,2,8 9 

PnewV 

AT7 C1P7 1 4,2 3,3,2,2,2,8 1 

AT8 C1P1 5 4,2 3,3,2,2,1,8 2 

AT9 C18 1 4,2 3,3,2,2,2,8 15 

PnewIII 

AT10 C1P1 new 2,2 5,2,2,2,2,6 1 

AT11 CnewI 2 4,2 3,3,2,2,2,8 1 

PnewIII 

AT12 C18P1 17 3,1 3,3,2,2,2,8 1 

AT13 C28 1 4,2 3,3,2,2,2,8 1 

PnewI 

AT14 C18P1 2 4,2 3,3,2,2,2,8 5 

AT15 C1 2 4,2 3,3,2,2,2,8 1 

PnewIV 

AT16 C1P1 12 2,2 5,2,2,2,2,8 3 

AT17 CnewIII 2 4,2 3,3,2,2,2,8 19 

PnewIV 

AT18 C14 

PnewIII 

2 4,2 3,3,2,2,2,8 1 

Table 2: The occurrence of MAP strain types AT1-18 

Breed 

AT MAP strain type (number) 

Angus 2(3) 

Aquitaine Blonde 5(2),6(3) 

Austrian Braun Mountain 1(1),2(3),6(1) 

Fleckvieh 1(2),2(10),3(1),4(5),5(1),8(2) 

Austrian Grey Mountain 1(2) 

Holstein Friesian 1(1),2(9),5(1),6(3),16(3) 

Jersey 2(3),3(3),7(1),15(1) 

Limousin 1(5),2(25),3(12),4(3),9(15),11(1) 

13(1),14(5),18(1) 

Murbodner 3(1),12(1) 

Piemontese 1(4) 

Red Friesian 2(2),3(1),6(1), 

Danish Melkrace 2(1),6(1),10(1) 

Goat 

Red Deer 

18(19) 

1(1), 2(3) 


Despite the fact that 18 different MAP types were detected in a 

comparatively large and diverse sample selection, 42% of all 

isolates were classified as MAP type AT2 (RFLP C1-P1; INMV 

2). MAP type AT2 was found in 8 of 12 PTB affected cattle 

breeds and in 2 of 4 isolates from red deer and is therefore the 

predominating MAP type strain in Austria. 9 new RFLP patterns 

and 1 new INMV profile could be determined within the 165 

analysed field strains. Some unique occurring strains showed 

association with certain cattle breeds, goat and red deer (Table 

2). This is the first study carried out in Austria providing data 

about MAP strain types on a national scale. Additional 

information about routes of transmission between herds and the 

epidemiology of PTB in Austria could be achieved by using the 

national cattle data base and the results of MAP typing. 

References 

1. MÖBIUS, P., LUYVEN, G., HOTZEL, H., KÖHLER, H. (2008): High 

Genetic Diversity among Mycobacterium avium subsp. paratuberculosis 

Strains from German Cattle Herds Shown by Combination of IS900 

Restriction Fragment Length Polymorphism Analysis and Mycobacterial 

Interspersed Repetitive Unit Variable Number Tandem Repeat Typing. J. 

Clin. Microbiology, 46, 972-980 

2. THIBAULT, V., GRAYON, M., BOSCHIROLI, M. L., HUBBANS, C., 

OVERDUIN, P., STEVENSON, K., GUTIERREZ, M.C., SUPPLY, P., BIET, 

F. (2007): New Variable Number Tandem Repeat Markers for Typing 

Mycobacterium avium subsp. paratuberculosis and M. avium Strains: 

Comparison with IS900 and IS1245 Restriction Fragment Length 

Polymorphism Typing. J. Clin. Microbiology, 45, 2404-2410

S4 - P - 02 

DEVELOPMENT AND VALIDATION OF A REAL-TIME PCR ASSAY FOR THE DETECTION OF 

LEPTOSPIRA BORGPETERSENII SEROVAR HARDJO IN URINE AND KIDNEY 

Zbigniew Arent 1 , Caroline Frizzell 2 , Colm Gilmore 1 , William Ellis 1 

1 

OIE Leptospirosis Reference Laboratory, Veterinary Sciences Division, AFBI, Belfast, Northern Ireland 

2 

Department of Veterinary Science, School of Agriculture and Food Science, The Queen’s University of Belfast 

Leptospira, leptospirosis, real-time PCR 

Introduction 

Leptospira borgpetersenii serovar Hardjo is the most common 

cause of leptospirosis in cattle throughout the world. Infection of 

cattle with Hardjo can cause abortion, stillbirth, birth of weak 

calves and decreased milk production (1, 2). Infected animals 

can act as lifelong renal carriers without clinical signs of the 

disease (3) and shed live organisms over a long period of time 

(4). 

There is no satisfactory method available for the rapid 

identification of carrier animals. Culturing of leptospires is the 

most definitive test. However this is possible only on fresh 

samples where viable leptospires are present. . PCR assays 

have the potential to detect both viable and non-viable 

leptospires and in recent years, several real-time PCR assays 

have been described. Levett et al. (5) developed a real-time 

assay using SYBR green chemistry, in which the target was the 

gene for the outer-membrane protein LipL32. Expression of the 

LipL32 gene is a unique virulence factor for pathogenic 

leptospires. LipL32 is an outer membrane lipoprotein that is 

conserved genetically and immunologically in pathogenic 

leptospires. It is one of the most abundant proteins in Leptospira. 

In this study we developed and validated a TaqMan PCR using 

novel primers which target a 200 bp fragment of the LipL32 gene. 


Eight animals that had no evidence of exposure to serovar Hardjo 

were experimentally infected with L. borgpetersenii serovar 

Hardjo type Bovis via conjunctival instillation of 2.5 x 10 6 

organisms on 3 consecutive days. A total of 8 urine samples 

were collected from each animal prior to and at intervals up to 40 

days after infection. In addition , 24 culture negative urine 

samples obtained from herds with no evidence of leptospirosis 

and 72 kidney samples collected post mortem from animals with 

serological evidence of Leptospira serovar Hardjo exposure were 

used in the study. All samples were cultured using Leptospira 

semisolid Tween 80/40 medium and biphasic culture medium 

with different variants of inhibitory substance addition. 

DNA was extracted from bacterial cultures and kidneys using the 

QIAamp DNA Mini kits (Qiagen) and from urines using the 

QIAamp Viral RNA Mini Kit (Qiagen) according to the 

manufacturer’s recommendations. 

The TaqMan assay to amplify a 200 bp fragment of the LipL32 

gene was optimized (primer/probe concentration, annealing 

temperature and incubation time with the QuantiTec Probe PCR 

Kit (Qiagen) for urine samples and kidney according to the 

manufacturer’s instructions. As a control for PCR inhibitors, 

specific primers and probe were designed to a housekeeping 

gene, β-actin, in cattle for kidney samples but in case of urine 

samples TaqMan Exogenous Internal Positive Control reagent 

(Applied Biosystems) was added to the amplification reaction. 

The limit of detection was ten template copies. Thirty two urine 

samples were used to determined diagnostic specificity (DSp). All 

of them were culture negative. Also thirty two urine samples and 

20 kidney samples that contained serovar Hardjo, as determined 

by culture were used to determine diagnostic sensitivity (DSe). 

The table below gives the results. 

Table 1: Diagnostic sensitivity and specificity of the LipL32 realtime 

PCR. 

Sensitivity % Specificity % 

Urine 84.4 90.6 

Kidney 95.0 Not tested 


This study evaluated the real-time PCR assay targeting LipL32 

gene for detection of Leptospira spp in bovine kidney and urine. 

The results indicate that the sensitivity of the diagnostic assay 

varies for kidney and urines. Presumably, some false negative 

results were caused by substances in the urine that inhibited 

extraction of the DNA, as the exogenous internal positive control 

did not show inhibition of the amplification reaction. 

In view of the difficulties associated with isolation of leptospires 

and the fact that serology does not imitate the carrier or shedding 

status, the LipL32 real-time PCR assay may be used as method 

for the detection of pathogenic leptospires in bovine urine and 

kidney samples 

References 

1. Ellis, W.A., (1984) Bovine Leptospirosis in the tropics: Prevalence, 

Pathogenesis and Control. Pre Vet Med 2: 411-421. 

2. Ellis, W.A., O’Brien, J.J., Bryson, D.G., Mackie, D.P., (1985) Bovine 

leptospirosis: some clinical features of serovar Hardjo infection. Vet Rec 

117: 101-104. 

3. Ellis, W.A., Songer, J.G., Montgomery, J., Cassells, J.A., (1986) 

Prevalence of Leptospira interrogans serovar Hardjo in the genital and 

urinary tracts of non-pregnant cattle. Vet Rec 118: 11-13. 

4. Ellis, W.A., (1994) Leptospirosis as a cause of reproductive failure. Vet 

Clin North Am Food Anim Pract 10: 463-478. 

5. Levett, P.N., Morey R.E., Galloway R.L., Turner D.E., Steigerwalt A.G., 

Mayer L.W. (2005) Detection of pathogenic leptospires by real-time 

quantitative PCR. J Med Microbiol 54: 45-49. 

The real time PCR was tested against a panel of bacterial 

species known to be ruminant pathogens to determine the 

analytical specificity of the assay. The analytical sensitivity was 

calculated using a dilution series of target DNA. A genome size of 

3.9 Mb was used to determine the genomic equivalent (GE) per 

microlitre of the purified DNA. The diagnostic sensitivity and 

specificity of the assay was determined by testing samples with 

only known culture status. 

Results 

The LipL32 target was amplified from 35 strains tested which 

belonged to 6 pathogenic leptospiral species (L.interrogans, 

L.borgpetersenil, L.kirschneri, L. santarosai, L.noguchii, L. weilii). 

The non-pathogenic Leptospira biflexa, intermediate L. fainei and 

other 20 bovine pathogens included were not amplified.

S4 - P - 03 

USEFULNESS OF LIVE/DEAD BACLIGHT BACTERIAL VIABILITY KIT TYPE 7007 MOLECULAR 

PROBES FOR EVALUATION OF VIABILITY ASCARIS SP., TOXOCARA SP. AND TRICHURIS SP. 

EGGS ISOLATED FROM SEWAGE SLUDGE 

Joanna Dąbrowska, Maciej Kochanowski, Krzysztof Stojecki, Jolanta Zdybel, Tomasz Cencek 

National Veterinary Research Insitute, Departament of Parasitology and Invasive Disease, Pulawy, Poland 

Viability, sludge, Ascaris, staining, fluorescence 

Introduction 

Sewage sludge and treated sewage from wastewater treatment 

plant despite the use of new ways of decontamination may 

contain live parasite eggs, which after entering the watering 

place, drinking water or into the fields as fertilizer can be a source 

of infection of humans and animals. For this reason, there is duty 

to monitor the safety of these substances, including the obligation 

to parasitological research. According the current rules 

parasitological examination includes detection of the Ascaris 

suum, Trichuris sp., and Toxocara sp. eggs and assess their 

viability.The assessment of their viability based only on the 

incubation and observation of isolated egg is long, time- and 

labour-consiuming and imprecise. 

The aim of this study was to develop sensitive and less labourintensive 

methods for assessing viability of Ascaris suum, 

Toxocara sp. and Trichuris sp. eggs in sewage sludge and 

organic fertilizers. 


Dehydrated sludge obtained from municipal wastewater 

treatment plant. Eggs of parasites isolated from the female 

nematode Ascaris suum, Trichuris ovis and Toxocara canis were 

used in the investigation (parasites were obtained from the 

intestines of animals killed in a slaughterhouse or excreted with 

faeces). Some of the eggs were inactivated by incubation in a 

water bath in 60 0 C for 30min. Eggs viability was confirmed by 

incubation in a moist chamber at 27 0 C. For differential staining of 

live and dead eggs were used diagnostic kit Live/Dead Bacterial 

of Molecular Probes, type 7007. 

The study was conducted in two experiments. 

Experiment 1. Live and inactivated parasite eggs were 

suspended in distilled water in separate test tubes. Then the 

eggs were stained using a set of Live/Dead Bacterial Kit 7007 

using the ratio: 1 ml of eggs suspended in water and 6 ml dye 

mixture (3 ml dye SYTO 9 and 3 ml of dye propiodium iodide). 

After thorough mixing, the suspension was incubated for 15 min 

at room temperature in the dark. Then it filtered through a 

polycarbonate filter, and so prepared sample coated with 

glycerol. The sample was viewed under a fluorescence 

microscope with suitable filters for wavelength settings excitation 

470 nm, emission 490 at the area (magnifcation 400x). 

Experiment 2. Live and dead eggs were added to separate 

samples of dewatered sewage sludge. Then the eggs were 

isolated from samples using own methods which is the 

combination of flotation and sedimentation methods (3) which is 

the combination of flotation and sedimentation methods. Before 

filtering stage of the match after flotation and sedimentation of 

sludge contained in the eggs of parasites were stained with 

Live/Dead Bacterial, type 7007 Molecular Probes. In the 

experiment used the proportion: in 20 ml of sludge was added 8 

ml mixture of dyes (4 ml SYTO 9 and 4 ml of dye propidium 

iodide). Then the sample was proceeded as in experiment 1. 

Results 

Experiment 1. The significant differences in staining of live and 

dead eggs of all three species of parasites were obtained. Strong 

green color of the live eggs and red color of inactivated eggs 

were observed under the fluorescence microscope (Figure 1). 

Experiment 2. With regard to the presence of organic matter 

derived from sewage sludge generated staining of eggs was 

similar as in experiment 1 although the colours were less 

intensive. Moreover, live eggs of Toxocara sp. took a green or 

blue than in experiment 1. In addition, live eggs of Toxocara sp. 

took a green or blue colour. Exemplary stained live and dead 

eggs obtained from the sewage sludge observed in fluorescence 

compared with the same eggs in bright field are shown in Figure 

2. 

Fig. 1 Example of stained live and dead eggs of Trichuris sp. 

suspended in water observed in fluorescence microscope 

Live egg in water 

Dead egg in water 

Fig. 2 Stained live and dead eggs obtained from sewage sludge 

observed in fluorescence and bright field microscope 

Live eggs 

Dead eggs 

Method of observation 

Method of observation 

Bright field Fluorescence Bright field Fluorescence 

1 1 

2 2 

3 3 

As is apparent from the images the biggest difference in the 

staining of live and dead eggs were obtained for eggs A. suum 

(images 1) and Trichuris (images 3). Slightly weaker results were 

obtained when stained eggs of nematodes- Toxocara (some of 

the living eggs stained in blue, and dead eggs were pink-blue, 

images 3). 


In most methods used in the world for parasitological 

examinations of sewage sludge eggs viability assessment is 

made by direct evaluation of the morphology of the eggs or 

observation and assessment their development during 

incubation. However, these methods are very subjective and in 

case of incubation very time and labour-consuming. Moreover 

the addition of glycerol (required to observe the microscopic filter 

papers) strongly inhibits the development of eggs. Diagnostic Kit 

Live/Dead Bacterial, the type of company Molecular Probes 

7007, composed of two components: SYTO 9 giving a green 

fluorescence, and propidium iodide (PI) - red fluorescence make 

use of a different ability of the two dyes to penetrate membrane 

of bacteria (1). 

The study showed that both dyes behave similarly also in relation 

to the cells of worm eggs. It was found that this method after the 

introduction of minor modifications by the nature of the test 

samples can be successfully used to distinguish between 

live and dead eggs of intestinal parasites in sewage sludge. This 

method is less dependent from subjective assessment of the 

investigator and less time consuming than other methods used to 

assess the viability of eggs of parasitic nematodes. 

References 

1. Biggerstaff J.P., Le Puil M., Weidow B.L., Prater J., Glass K., 

Radosevich M., White D. New methodology for viability testing in 

environmental samples. Mol Cell Probes. 2006 Apr;20(2):141-6. 

Epub 2006 Feb 14. 

2. Zdybel J, Karamon J., Cencek T.. Występowanie jaj nicieni 

pasożytniczych z rodzajow Ascaris, Trichuris i Toxocara 

w nawozach organicznych i organiczno-mineralnych oraz 

osadach ściekowych. Życie Weterynaryjne, rocznik 84 2009, 

rocznik 84

S4 - P - 04 

AUTOMATION OF A CAPTURE PROBE MAGNETIC BEAD DNA EXTRACTION METHOD FOR 3 GRAM 

FAECAL SAMPLES FROM RED FOX INTENDED FOR PCR-DETECTION OF ECHINOCOCCUS 

MULTILOCULARIS 

Isaksson, M 1 , Hagström, Å 1 , Lukacs, M 2 , Holmberg, A 3 , Juremalm, M 1 

1 

National Veterinary Institute, Department of Virology, Immunobiology and Parasitology, Uppsala, Sweden 

2 

Nordiag ASA, Research and development, Oslo, Norway 

3 

Nordiag AB, Research and development, Stockholm, Sweden 

Automation, capture probe, DNA extraction, faecal samples, Echinococcus multilocularis 

Introduction 

As part of the Echinococcus multilocularis (EM) surveillance in 

Sweden, 4000 red fox faecal samples will be collected and 

analyzed by PCR. This necessitates an automated DNA 

extraction method. Due to the heterogeneous presence of EMeggs 

in faeces, 3 grams of sample material is needed for the 

DNA extraction of this analysis. Earlier findings show that a 

capture probe magnetic bead extraction method (1) increases 

sensitivity of the assay compared to the standard egg-flotation 

and DNA extraction previously used at the National Veterinary 

Institute in Sweden (not published). In this study, we show how 

automation of the manual capture probe magnetic bead DNA 

extraction method capable of handling 3 grams of faeces per 

sample can increase the sample throughput capacity compared 

to manual extraction. 


The robot used in this study is a Nordiag Bullet (Nordiag ASA). It 

is a flexible robot for microplate format and includes a gripper for 

plate transfers, a splittable 1ml eight channel pipette head, 

magnetic separation station, bar code capability and external 

waste. To be able to automate the large sample volume of the 

three grams of faeces in lysis buffer, 10ml 24-well deepwell 

plates were used together with two high power magnet stations. 

Results 

Essentially the automation consists of the five wash steps of the 

magnetic beads and the magnetic pelleting between washes, 

eliminating the need to manually pipette the wash steps of the 

samples. The robot can be loaded with up to 48 individual 

samples consisting of 3 grams of homogenized fox faeces in 

buffer with biotinylated hybridization probes hybridized to the 

target and attached to streptavidin coated magnetic beads. When 

the robot is finished, the target DNA is melted off the probes and 

the probe/magnetic bead pellet is discarded. The manual capture 

probe magnetic bead DNA extraction method allows a laboratory 

technician to finish 12-20 samples in one day. The automation 

allows the same technician to finish 48 samples in one day, with 

time to do other things while the robot is working. 96 samples in 

one long day are also possible, but in this case with more or less 

100% hands on time, preparing the next batch of samples while 

the robot is processing the first batch. 

Tab 1. Throughput of samples per week per technician, time for 

weighing and division of samples not included. 

Samples per 

week per 

technician 

Manual extraction 

Automated extraction 

100 336 

Fig 1. The Nordiag Bullet robot used in the study 


The preparation of faecal samples before extraction, manual or 

automated, is difficult to automate. The samples have to be 

divided and put in the correct format tube or well before the 

extraction method itself can be initiated. Automating the washing 

steps of this method means that the sample throughput is 

increased enough for the hybridization capture probe method to 

be implemented as a routine, large scale DNA extraction method 

for the PCR detection of EM eggs in red fox faecal samples. 

Current work is aimed at modifying the automated capture probe 

DNA extraction method to be usable also for RNA targets in a 

standardized format, mixing RNA and DNA targets in the same 

robot run, or even in the same sample well. This would make the 

automated capture probe extraction method even more 

interesting for a number of difficult sample materials such as 

organ material, soil, sewage water etc. 


This project is funded by the Swedish Civil Contingencies 

Agency. 

References 

1. Opsteegh, M, Langlelaar, M, Sprong, H, den Hartog, L, De Craeye, S, 

Bokken, G, Ajzenberg, D, Kijlstra, A, van der Giessen, J. 2010. Direct 

detection and genotyping of Toxoplasma gondii in meat samples using 

magnetic capture and PCR. International Journal of Food Microbiology, 

139(3), 193-201.

S4 - P - 05 

EVALUATION OF THREE ELISAs FOR DETECTING SERUM ANTIBODIES AGAINST MYCOPLASMA 

HYOPNEUMONIAE 

Martos-Raich, Alba, Porquet-Garanto, Lourdes, Rebordosa-Trigueros, Xavier 

HIPRA, 17170 Amer, Girona, Spain 

1 Keywords: Mycoplasma hyopneumoniae, ELISA, vaccinated/challenged animals 

Introduction 

Mycoplasma hyopneumoniae (Mhyo) is an important component 

of the so called Porcine Respiratory Disease Complex (PRDC). 

Mhyo infection in pigs is usually monitored through serology. In 

this study, we compared three ELISAs developed to detect 

antibodies to Mhyo in swine serum. The main objectives of this 

study were to differentiate serum antibody profiles of four groups 

of animals: non-challenged/vaccinated, challenged/vaccinated, 

challenged/non-vaccinated and non-challenged/non-vaccinated 

animals. 


Seventy young piglets (10-day-old) serologically negative to 

Mhyo were divided into 7 groups of 10 animals each. Groups 1-5 

were vaccinated with 5 different commercial vaccines against 

enzootic pneumonia on D0, and D21, and subsequently 

challenged on D70 with Mhyo strain 3371. Groups 6 and 7 were 

used as non-vaccinated/challenged and non-vaccinated/nonchallenged 

control groups, respectively. Blood samples were 

taken from all animals at D0, D21, D42, D70 and D98. All 

samples were tested using the CIVTEST SUIS MHYO ELISA kit, 

a commercial indirect ELISA (ELISA-I), and a commercial 

competition ELISA based on a monoclonal antibody against the 

p74 (ELISA-C). 

Results 

All the kits showed good performance in the analysis of specificity 

(100%) (2). In regard to sensitivity, it varies depending on the 

type of sample analyzed: ELISA-C detected more positive 

samples from vaccinated animals (72,2%), followed by CIVTEST 

(41,67%) and the ELISA-I (14,81%). No differences in sensitivity 

were observed between ELISA-C and the CIVTEST (87,5% in 

both cases) in the samples from challenged animals (vaccinated 

and non-vaccinated); ELISA-I was less sensitive (50%) (Figure 

1). All three kits showed some common performance 

characteristics: they displayed maximum sensitivity at D42 after 

two doses of vaccine and sensitivity diminishes at D70. Also, 

sensitivity was lower when dealing with only challenged samples 

(2); in this case, CIVTEST and ELISA-C displayed the same 

sensitivity (33.3%) and detected all samples from 

vaccinated/challenged animals (100%); ELISA-I was the less 

sensitive (0% and 61.54%, respectively). 

Table 1. Kappa values obtained in the different comparisons. 

Comparison All Vacc. Chall. 

CIVTEST vs ELISA-C 0.7 0.4 1.0 

ELISA-I vs ELISA-I 0.54 0.39 0.25 

ELISA-C vs ELISA-I 0.34 0.12 0.25 

Figure 1. Quantitative representation of the Mhyo ELISA 

serology using three commercial kits. The results are shown as a 

mean (central point) and standard desviation (vertical bars). 


The most similar kits were the CIVTEST and the ELISA-C 

because both of them have a high sensitivity (Table 1). CIVTEST 

has better performance differentiating quantitatively samples from 

vaccinated animals and samples from vaccinated/challenged 

animals. The high sensitivity of the ELISA-C when detecting 

vaccination seems to make this test have more problems when 

distinguishing between samples from vaccinated animals and 

samples from vaccinated/challenged animals (Figure 1). 

References 

1. Ameri-Mahabadi, Mehrdad et al: 2055, J Vet Diagn Invest 17:61-65. 

2. Earlandson, KR et al: 2005, Journal of Swine Health and Production 

198:203.

S4 - P - 06 

SEPRION-COATED MICROCANTILEVER SENSORS FOR PRP SC DETECTION 

Daniela Meloni¹, Danilo Pitardi¹, Maria Mazza¹, Riccardo Castagna 2 , Ivan Ferrante 2 , Carlo Ricciardi 2 , Elena 

Bozzetta¹. 

¹Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Istopatologia e Test Rapidi, Turin, Italy; 

2 

Politecnico di Torino, Scienza dei Materiali e Ingegneria Chimica, Turin, Italy. 

Cantilever, Seprion, TSE, PrP sc detection 

Introduction 

The current state of technology to detect animal TSEs relies upon 

the post-mortem detection of prions, considering the EU trend in 

the age limit for testing [1] and in order to improve surveillance 

and food safety, new technologies should be developed to allow 

the ante mortem tests for PrP sc proteins. The new tests should be 

body fluids-based and performed on presymptomatic animals, but 

to be effective they would need to detect small amounts of PrP sc 

and to differentiate PrP c from PrP sc . 

Mechanical biosensors appear to be one of the most promising 

techniques for molecular diagnostics [2]. Dynamic-mode 

mechanical biosensors are oscillating devices with a resonance 

frequency that changes when molecules land on the cantilever. 

Thanks to the monitoring of different resonance modes over 

arrays of several cantilever-based microbalances, the selective 

identification and quantification of adsorbed mass of the order of 

few picograms with very high precision was recently 

demonstrated [3]. 

Here we propose the development of a biosensing platform for 

PrP sc 

detection based on microcantilever arrays coated with a 

selective polymer (Seprion from IDEXX Lab. Inc.). 


Microcantilever arrays were fabricated in a clean room making 

use of standard microtechnologies such as KOH bulk etching, 

optical lithography and Reactive Ion Etching; they were dipped 

into coating solution for few minutes inside a laminar flow bench, 

washed with PBS and MilliQ water, and finally dried on a plate 

dryer. 

An optical lever setup is used to monitor cantilever resonance 

curve while vibrating in vacuum environment; a piezoactuator is 

employed for the excitation. We calculated the arithmetic mean 

and uncertainty of relative frequency deviation Δf/f over the 

modes for each cantilever, and then we used the “weighted 

average”method to have the best estimation of the true value Δf/f 

of the array [3]. 

PrP sc 

was extracted from scrapie positive samples, brainstems 

were homogenised in lysis buffer and centrifuged at 22.000 g for 

20 minutes, the supernatant was collected and incubated with 

proteinase K (20 μg/ml) for 1 hour at 37° C. After centrifugation at 

215.000 g for 1 hour, the pellet was dissolved in distilled water. 

The coated arrays were incubated for 2h in a water-based 

solution containing pathologic PrP sc or simply water as negative 

control experiment (“blank”). Samples were then rinsed in DI 

water and finally dried in a steam of nitrogen. 

Fig. 2. Micro-cantilever array 

Fig3. PrP sc capture 

Results 

Microcantilever arrays were successfully treated with both 0.1% 

and 0.2% coating solution: the calculated mass surface densities 

seemed to be rather similar over the arrays and dilution, showing 

a good homogeneity of the functionalization procedure. 

Preliminary data exhibited a relevant negative relative frequency 

deviation Δf/f up to nearly 10 times higher respect to blank 

signals, showing that the polymer remained active in immobilizing 

the target molecule and thus an increment of mass occurred on 

cantilever surfaces when exposed to PrP sc solutions. 


Cantilever-based microbalances were successfully coated with 

Seprion polymer to selectively immobilize pathogenic PrP sc . 

Preliminary data showed that the technique looks promising, but 

more focused experiments are needed to exploit its potentiality. 

References 

1. European Commission, Brussels, 16.7.2010, COM (2010) 384 final. 

Communication from the Commission to the European Parliament and the 

Council. The TSE Road map 2, a Strategy paper on Transmissible 

Spongiform Encephalopathies for 2010-2015. 

2. Arlett, J.L., Myers, E.B., Roukes, M.L. (2011). Comparative advantages 

of mechanical biosensors. Nat. Nanotechnol. 6, 4:203-15. 

3. Ricciardi, C., Fiorilli, S., Bianco, S., Canavese, G., Castagna, R., 

Ferrante, I., Digregorio, G., Marasso, S.L., Napione, L., Bussolino, F. 

(2010). Development of microcantilever based biosensor array to detect 

Angiopoietin-1, a marker of tumor angiogenesis. Biosens. Bioelect., 

25:1193-1198 

Figure 1: Flow chart explaining the new technique

S4 - P - 07 

EVALUATION OF DIFFERENT EXTRACTION METHODS FOR THE DETECTION OF MYCOBACTERIUM 

AVIUM SUBSP. PARATUBERCULOSIS IN BOVINE FAECES 

Jean-Louis Moyen 1 , Laure Brugère 1 , Julie Charrot 2 , Eric Sellal 2 , Line Kirsty 3 , Adrian McGoldrick 3 

1 

Laboratoire départemental d’Analyse et de Recherche de la Dordogne,24660 Coulounieix-Chamiers France 

2 

Laboratoire Service International, Lisieux, France 

3 Animal Health and Veterinary Laboratories Agency Starcross, Exeter, United Kingdom 

M; avium susp. paratuberculosis, MAP, 

Introduction 

Johne’s disease or bovine paratuberculosis occurs throughout 

the world. It is a chronic, untreatable, intestinal disease of 

ruminants caused by Mycobacterium avium subspecies 

paratuberculosis (MAP) 1 . The profile and interest in the disease 

has risen in recent years, and consequently, it will be beneficial to 

have accurate information about prevalence in order to formulate 

policy and appropriate control strategies. Central to gaining 

accurate prevalence data in any country, is the availability of 

good diagnostic methods. In order to control the disease, MAP 

has to be diagnosed ante-mortem by either detecting the 

causative agent itself or by the detection of specific antibodies. 

The need of a rapid, low cost, and sensitive diagnostic test would 

be beneficial in the detection and control of the organism. The 

methods currently available include solid and liquid culture, 

serological methodologies and more recently, polymerase chain 

reaction (PCR) based tests. 

Culture (liquid and solid) is sensitive but the time to detection 

ranges from 6 to 24 weeks. Serological methods tend to be 

used in screening programmes since they are quick and cheap, 

however the sensitivity is low. Available PCR methods allow fast 

results (2 days) but reported sensitivity also remains low. 

Sensitivity and the time taken to reach diagnosis are critical 

points in MAP management control programmes. Indeed, these 

disease control strategies can often fail because new clinical 

cases are found even after culling of positive diagnosed animals. 

In this study, with specific reference to PCR, we hypothesise that 

the optimization of sample preparation and nucleic acids 

extraction can improve the sensitivity making PCR a viable option 

in the control of the disease. 


Faecal material from cows belonging to certified MAP free herds 

were used as negative controls. Positive samples originated 

from naturally infected animals as well as from spiking negative 

faeces with MAP homogenates. 

Naturally infected samples, as well as those spiked, were used to 

prepare a dilution series of samples. Each set of samples were 

prepared in duplicate. All samples, both naturally infected and 

those spiked, were thoroughly mixed to ensure homogeneity (as 

much as practically possible). 

For a field trial (epidemiological study), sampling was made from 

undiluted samples. The heterogeneity between samples is 

increased but the aliquots are more representative using real 

faeces. 

Sample preparation conditions were comparable (size, dilution, 

shaking, bead beating, pK incubation and spinning) 

In this study we compared 3 PCR methods. The first was an 

IS900 real time PCR test from LSI (Taqman) 2,3 using a Qiagen 

spin column extraction method with 1g faeces and 4 ml H2O. 

The alternative methods were: 

An ambion magnetic beads method on Magmax KF96 

developped by AHVLA 4 and an LSI magnetic beads (MV384) 

method on Magmax KF96 optimized by the LDAR24. 

Results 

Spiked and diluted faeces: 

All negative samples were negative 

Detection of higher dilutions depends on the methods 

Tab.1. spiked samples – CT values for serial dilutions for each 

method 

10 -2 1/2 10 -2 1/4 10 -2 1/8 

10 -2 

1/16 

10 -2 

1/32 

10 -2 

1/64 

Qiagen 

Spin 

columns 

33.52 34.44 34.73 undet 37.81 38.75 

35.54 35.03 35.57 35.53 34.59 undet 

33.26 33.97 36.09 37.85 undet 37.39 

33.58 38.09 36.97 38.13 undet undet 

mean 33.98 35.38 35.84 37.17 36.20 38.07 

s 1.05 1.86 0.94 1.43 2.28 0.96 

Soil kit 

24.96 26.39 27.73 28.26 29.33 31.24 

25.36 26.14 27.2 28.01 29.77 31.06 

25.3 25.81 27.16 28.17 29.4 33.1 

26.02 26.49 27.31 28.09 29.64 34.42 

mean 25.41 26.21 27.35 28.13 29.54 32.46 

s 0.44 0.30 0.26 0.11 0.21 1.60 

Ambion 

AHVLA 

28.94 28.08 30.09 30.46 31.3 33 

27.21 27.11 31.17 31.89 31.54 32.96 

26.9 29.23 30.04 31.31 31.2 32.72 

26.96 27.63 31.05 33.06 33.44 33.08 

mean 27.50 28.01 30.59 31.68 31.87 32.94 

s 0.97 0.90 0.61 1.09 1.06 0.15 


The heterogeneity of samples for field trial is important. 

The best method sensitivity is obtained with AHVLA 

method. 

This method is requires more time and needs a larger 

centrifuge. 

The LSI magnetic beads method gives a very good 

sensitivity too. 

The classic spin column method gives the worst 

sensitivity 

The epidemiological sensitivity is much better with the 

optimized methods and allows better eradication 

program. 

The choice between these methods depends on the customer’s 

requirement. (cost versus sensitivity) 

References 

1. N. Beth Harris, R. G. Barletta Clin. Microbiol. Rev. 2001 14(3): 489-512 

Mycobacterium avium ssp paratuberculosis in veterinary medicine 

2. E.P. Green, M.L.V. Tizard, M.T. Moss, J. Thompson, D.J. Winterhouse, 

J.J. McFadden and J. Hermon-Taylor: 1989, Sequence and characteristics 

of IS900, an insertion element identiefied in a human Crohn’s disease 

isolate of Mycobacterium paratuberculosis. Nucleic acid research volume 

17 number 22 9063-9073 

3.. LSI Kit TaqVetM. paratuberculosis advanced V003 

4. A. McGoldrich, K. Line 2012 (personnal communication) 

 

 

Sample preparation (sample size, dilution, shaking, 

bead beating and spinning conditions) is very important 

Soil kit (Fast Dna MPBIO) gives the best sensitivity but 

is time consuming and is not usable for field diagnosis

S4 - P - 08 

REAL-TIME PCR FOR DETECTION OF CAMPYLOBACTER JEJUNI, COLI AND LARI IN FOODS: TEST 

VALIDATION ACCORDING TO ISO16140:2003 

Nogarol C. 1 , Vencia W. 1 , Bianchi DM. 1 , Gallina S. 1 , Adriano D. 1 , Zuccon F. 1 , Decastelli L. 1 

1 

Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, S.C. Controllo Alimenti e Igiene delle Produzioni, Turin, Italy 

Campylobacter, ISO 16140:2003, Real Time PCR 

Introduction 

Several countries with foodborne outbreaks (FBO) reporting 

systems documented significant increases over the past few 

decades in the incidence of diseases caused by microorganisms 

in food, including pathogens such as Salmonella, Campylobacter 

jejuni and enterohaemorrhagic Escherichia coli (3). 

Changes in food habits, such as a preference for fresh and 

minimally processed foods and the longer shelflife of products, 

contributed to the increased incidence of FBO ascribed to 

microbiological organisms. 

The recent EU-policy bases the surveillance of foodborne 

zoonoses on laboratory isolation of bacteria considered sources 

of outbreaks (3). 

Althought standardized by international bodies, cultural methods, 

due to execution time, not always allow the pathogen isolation 

and identification before outbreak conclusion, in particular when 

sources of FBO are represented by slow growing bacteria. On 

the contrary, the identification of bacteria responsible of FBO 

should be ended in shortly, in order to guarantee a specific 

therapy of ill patients and to correctly implement report database 

systems. 

For this reason, the use of alternative methods able to rapidly 

identify the source of FBO is recommended: European 

regulations accept the use of these rapid methods as long as 

they have been validated towards standardized reference method 

according to ISO 16140:2003 (1). 

Data here reported describe the results of validation of a 

qualitative Real-Time PCR commercial kit for the detection of 

Campylobacter jejuni, coli and lari (JCL) in the food category 

“fruit and vegetable based products”. 


From January to August 2011, the Laboratorio Controllo Alimenti 

e Igiene delle Produzioni, in Istituto Zooprofilattico Sperimentale 

of Piemonte, Liguria and Valle d’Aosta (North-Western Italy) led a 

study aimed to verify the performances and to validate a 

Campylobacter JCL Real-Time detection kit. 

The commercial kit consists in two steps occurring in a semiautomated 

closed system: extraction of bacterial DNA and 

detection of Campylobacter JCL target genes. Reagents 

necessary for these two steps are included in the kit. According 

to the ISO 16140:2003, the validation was performed against the 

reference method (ISO 10272:2006) (2) and the following 

parameters were evaluated: Limit of Detection (LOD); relative 

accuracy, relative specificity and sensitivity; relative detection 

level (RDL); inclusivity and exclusivity. 

To evaluate LOD, a spiking inoculum was prepared with a 

concentration of Campylobacter jejuni of 1,5*10 7 

CFU/ml: eight 

decimal dilutions were prepared and one millilitre of each dilution 

was used to spike samples; after incubation, pre-enriched 

samples were tested with commercial detection kit. 

In order to determine relative accuracy, relative specificity and 

sensitivity a total of number of 60 test portions (50% positive, with 

three contamination levels and 50% negative samples) were 

analyzed. 

The relative detection level (RDL) is the smallest number of 

culturable microorganism that can be detected in the sample in 

50% of occasions by the alternative and reference methods: it 

was evaluated throught the contamination of 72 food products 

with the three different target microorganisms (C.jejuni, coli and 

lari), at three concentration levels (24 negative control, 24 

samples spiked with theoretical detection level and 24 samples 

spiked with a bacteria concentration just above LOD level). Each 

combination (food product-level of contamination) was replicated 

six times by both the alternative and reference methods. 

In order to evaluate the inclusivity of the alternative method, 50 

pure culture of target microorganism were tested. The inoculum 

level used was 10 to 100 times greater than the minimum RDL of 

the alternative method. 

The exclusivity was verified analyzing 30 pure cultures of nontarget 

strains (50% of Gram positive and 50% of Gram negative) 

Finally, to perform a complete validation according to ISO 

16140:2003 an interlaboratory study was organized, in order to 

determine the variability of the results obtained in different 

laboratories using identical samples. 

A total of 10 collaborative laboratories were included in the study: 

each of them received 24 blind samples, spiked with three 

contaminations levels (8 negative controls, 8 slightly above the 

LOD level and 8 samples 10 times greater than LOD level). 

Contamination was performed with reference material (lenticules 

discs) from HPA containing lyophilized C. jejuni pure culture. 

Guidelines and requirements for the interlaboratory study are 

given in the Annex H of ISO 16140:2003. 

Results 

The results obtained in our laboratory defined the LOD of Real- 

Time PCR protocol at 4 CFU/ 25 g or ml of food sample; the 

Table 1 resumes the results about relative accuracy, relative 

specificity and sensitivity. 

Table 1: Criteria percentage obtained with paired tabulate; LCL: 

lower confidence limit at 95% 

PARAMETERS % LCL 95% 

Relative Accuracy 98.33 96 

Relative Specificity 100 > 98 

Relative Sensitivity 96,66 93 

Only one sample resulted positive with alternative method and 

negative with the reference one. 

Interpretation of results obtained for the determination of RDL 

were calculated for each level and for each food/strain 

combination, comparing both methods performing exact Fisher 

tests. Results gave a p value=1 for each contamination levels: 

the probability that methods are able to detect the inoculum 

levels is 100% in all tested samples. Results of inclusivity, 

exclusivity and interlaboratory study were satisfactory, confirming 

all expected determinations. 


The alternative Real-Time PCR method was successfully 

validated according to ISO 16140:2003. All the criteria and 

parameters tested gave excellent values and allowed to consider 

this commercial kit suitable for official control on fruit and 

vegetable food samples. Finally the study also confirmed that this 

commercial kit is easy to use and handle by laboratory 

personnel. 


This study was founded by the Italian Health Ministry: Ricerca Corrente 

Ministero della Salute, anno 2009, IZS PLV 12/09. 

References 

1. ISO 16140:2003, Microbiology of food and animal feeding stuffs – 

Protocol for the validation of alternative methods. 

2. ISO 10272:2006, Microbiology of food and animal feeding stuffs- 

Horizontal method for detection and enumeration of Campylobacter spp. 

3. EFSA Journal 2012;10(3):2597

S4 - P - 09 

VALIDATION OF THE ID SCREEN SALMONELLA DUBLIN COMPETITIVE ELISA 

Pourquier, P. 1 , Loic, C. 1 , Pozet, F. 2 , Buthod-Garçon, M.P. 2 

1 


2 

LVD39, Poligny, France, 

Salmonella Dublin, diagnostics, serology, ELISA 

Introduction 

The most common type of Salmonella affecting cattle in many 

countries is Salmonella enteritica, subspecies enteritica serotype 

Dublin (Salmonella Dublin). Salmonella Typhimurium and 

Salmonella Montevideo are also frequently isolated from cattle. 

While antigens O:1 and O:12 are common to serotypes Dublin 

and Typhimurium, antigen O:9 characterises infection by S. 

Dublin. 

Salmonella Dublin is a bacteria which is host-adapted to cattle 

and which causes both economical and welfare losses to the 

cattle industry. It can potentially spread to humans, particularly 

through the consumption of contaminated, unpasteurised milk 

and cheese. 

Unlike most other types of Salmonella bacteria, Salmonella 

Dublin has a tendency to reside in the herds for years or 

decades, mainly due to its ability to produce persistently infected 

carriers that shed bacteria to the environment either continuously 

or periodically. In order to control infection in such endemically 

infected herds, it is necessary to cull the carriers and prevent 

infection of new carriers. 

Serology is therefore an important tool in order to limit the spread 

of the disease by identifying disease carriers, and eliminate 

excretors before contamination of bulk milk. 

IDvet, in collaboration with the Jura Departmental Veterinary 

Laboratory (LVD 39), has developed two ELISAs for the detection 

of antibodies against Salmonella Dublin in bovine milk, serum 

and plasma: ID Screen ® Salmonella Dublin Competition, and ID 

Screen ® Salmonella Dublin Indirect. 


The specificity of the test ID Screen ® 

Salmonella Dublin 

Competition was found to be (100%, IC95% 99.5-99.99%). No 

false positive results due Salmonella Typhimurium were 

observed. 

The ID Screen ® competitive ELISA is a cost-effective Salmonella 

Dublin diagnostic method which facilitates identification of herds 

at risk and transitory excretors. 

It should be noted that the analytical sensitivity of competitive 

ELISA method on bulk milk is limited by degree of excretion of 

the animal, the size of the bulk milk, and the fact that milk 

contains less antibodies than sera. 

A suggested testing protocol would be to screen bulk milks with 

the ID Screen Indirect ELISA, and test any positive samples 

individually with the competitive ELISA in order to maximize 

analytical sensitivity. 

References 

1. Hoorfar J, Feld NC, Schirmer AL, Bitsch V, Lind P. Serodiagnosis of 

Salmonella dublin infection in Danish dairy herds using O-antigen based 

enzyme-linked immunosorbent assay. Can J Vet Res. 1994 Oct;58(4):268- 

74. 

2. Hoorfar J, Lind P, Bitsch V. Evaluation of an O antigen enzyme-linked 

immunosorbent assay for screening of milk samples for Salmonella dublin 

infection in dairy herds. Can J Vet Res. 1995 Apr;59(2):142-8. 

3. Nielsen LR, Ersbøll AK. Age-stratified validation of an indirect 

Salmonella Dublin serum enzyme-linked immunosorbent assay for 

individual diagnosis in cattle. J Vet Diagn Invest. 2004 May;16(3):212-8. 

This study presents the test validation data for the competitive 

ELISA. 


The ID Screen ® 

Salmonella Dublin Competition ELISA is a 

competitive ELISA based on microplates coated with S. Dublin 

LPS and an anti-O:9 monoclonal antibody conjugate. 


Salmonella Dublin Indirect ELISA is based on 

microplates coated with S. Dublin LPS and an anti-ruminant-HRP 

conjugate. 

Specificity: 

176 individual milks, 84 bulk milks, and 180 sera from a diseasefree 

region (Brittany, France) were tested with both kits. 

Exclusivity: 

111 individual milks from a herd infected with Salmonella 

Typhimurium (bacteria were isolated from several animals), were 

tested. 

Sensitivity: 

158 paired milk and sera samples from 20 herds with a history of 

S. Dublin infection were tested. A bacteriological study of 

seropositive animals allowed for the identification of excretors in 

Herd 2. 

Results 

Specificity and exclusivity studies: All animals were found 

negative with the ID Screen ® 

Salmonella Dublin competitive 

ELISA. 

Sensitivity study: 

- Correlation between milk and serum results was excellent 

(100%). 

- The average herd seroprevalence was found to be: 12.9 % 

(3,8%-30,2%). 

- 89% (16/18) of excretors were also seropositive.

S4 - P - 10 

ID SCREEN ® INTERFERON GAMMA ELISA : IMPROVEMENT OF ANALYTICAL SENSITIVITY AND 

INTRODUCTION OF A STANDARD REFERENCE CONTROL TO IMPROVE RESULT INTERPRETATION 

Pourquier, P. 1 , Loic, C. 1 , Olagnon, L. 1 , Marion, S. 1, 

1 


Interferon gamma, diagnostics, ELISA 

Introduction 

Detection of Interferon gamma (IFN-g) by the sandwich ELISA 

method is widely used to detect the cellular response to 

pathogens such as Mycobacterium avium subsp paratuberculosis 

(Map) or Mycobacterium bovis by measuring the difference 

between activated and non-activated whole blood or peripheral 

Blood Mononuclear Cells (PBMC) IFN-g signals. Results, 

expressed as the difference between raw optical densities, are 

not generally linked to a stable reference control. 


Ruminant IFN-g ELISA, contrary to other 

commercial ELISAs, expresses the level of IFN-g with respect 

to a standardized, freeze-dried positive reference control. 

This relative expression of the measured quantity of IFN-g 

guarantees the standardization of results between runs and kit 

batches. 

Results are expressed as sample / positive reference control 

(S/P) ratios. 


ID Screen ® Interferon gamma Capture ELISA: Wells are coated 

with an anti-IFN-γ monoclonal antibody (Mab). The conjugate is 

anti-IFN-γ–HRP conjugate monoclonal antibody.Creation of 

standard reference controls: A freeze-dried positive control 

containing native bovine IFN-g was created and introduced onto 

each plate. All results were calculated with respect to this 

standard. A standard positive plasma reference sample was also 

created. 

Results 

Test baseline: Bovine and ovine sera and PBS-activated bovine 

and caprine plasmas were tested and found to have a low 

baseline with few heterophilic reactions. 

Analytical sensitivity: 5 bovine, 1 caprine and 1 ovine blood 

sample were non-specifically activated with 5 µg/ml of lectin, 24h 

at 37°C. Plasma was collected and each sample was serially 

diluted in non-activated plasma. Samples were then tested in 

parallel with the ID Screen ® 

ELISA and another commerciallyavailable 

IFN-g sandwich ELISA (kit A). 

- The ID Screen ® ELISA and Test A showed similar analytical 

sensitivity for ovine IFN-g, while the ID Screen ELISA 

demonstrated superior analytical sensitivity for bovine and 

caprine IFN-g. In addition, the ID Screen ® ELISA showed clearer 

separation of positive and negative samples. 

Multi-site field validation: 3 French laboratories tested previouslyactivated 

samples (n=216) using the ID Screen ® 

ELISA and 

another commercial ELISA. 

- Excellent correlation (98.6%) was found between the two tests, 

with the ID Screen ® test giving higher OD values for the positive 

samples. 

Field data from a Map-infected goat herd: 180 caprine blood 

samples from herd with a history of clinical paratuberculosis were 

activated with 9 different antigens, including 3 different Map 

extracts from a field caprine isolate, as well as PPD avium. 

- One of the IDvet caprine Map antigen preparations gave a 

similar result profile to the PPD avium antigen tested. 


The new ID Screen® Ruminant IFN-g ELISA has improved 

upon current IFN-g ELISA technology by incorporating a standard 

positive reference control for standardized result calculation and 


The improved analytical sensitivity allows for the efficient 

detection of small quantities of native IFN-g (threshold dilution on 

other techniques), giving optical densities clearly distinct from the 

test baseline, thus avoiding false positive results. The very low 

level of heterophilic reactions and the low baseline guarantee that 

result interpretation is possible for all samples to be tested. 

In conclusion, the ID Screen ® Ruminant IFN-g ELISA is a reliable 

tool for the measurement of ruminant interferon gamma. 

References 

1. Buddle BM, et al. Advances in ante-mortem diagnosis of tuberculosis in 

cattle. New Zeland Veterinary Journal. 2009, 57(4):173-180. 

2. Denis M, Weldock DN, McCarthy AR, Parlane NA, Cockle PJ, 

Vordermeier HM, Hewinson RG, Buddles BM, 2007. Enhancement of the 

sensitivity of the whole-blood gamma interferon assay for diagnosis of 

Mycobacterium bovis infections in cattle. Clinical and vaccine immunology, 

14(11): 1483-1489. 

3. Gormley E, et al. The effect of the tuberculin test and the consequences 

of a delay in blood culture on the sensitivity of a gamma-interferon assay 

for the detection of Mycobacterium bovis infection in cattle. Vet Immunol 

Immunopathol. 2004, Dec 28;102(4):413-20 

4. Lardizabal AA, Reichman BL, 2006. Interferon-gamma release assay for 

detection of Tuberculosis Infection. US respiratory Disease, 59-61. 

5. Pai M, Riley LW, Colford Jr JM, 2004. Interferon-g assays in the 

immunodiagnosis of tuberculosis: a systematic review. Infectious diseases, 

4:761-776. 

6. Robbe-Austermann S, et al. Time delay, temperature effects and 

assessment of positive controls on whole blood for the gamma interferon 

ELISA to detect Paratuberculosis. J Vet Med B Infect Dis Vet Public 

Health. 2006, 53(5):213-7. 

7. Ryan JT, et al. An evaluation of the gamma interferon test for detecting 

bovine tuberculosis in cattle 8 to 28 days after tuberculin skin testing. 

Research in Veterinary Science. 2000, 69(1) :57-61.

S4 - P - 11 

EFFECTIVE TESTING STRATEGY WITH PRIMAGAM ® FOR TUBERCULOSIS IN HUMAN PRIMATES 

Björn Schröder 2 , Christian Wenker 1 , Stefan Hoby 1 , Bettina Bernhard 2 , Alex J. Raeber 2 

1 Zoo Basel, Switzerland and 2 Prionics AG, Schlieren, Switzerland 

Introduction 

Primates infected with tuberculosis (TB) represent a serious 

regulatory, ethical and health concern as TB control requires 

accurate diagnostic methods. However, the primary test, the 

tuberculin skin test has serious limitations. Both false negative 

and false positive reactions are common, resulting in a spread of 

infection and devastating TB outbreaks in colonies. As a gold 

standard for TB diagnostic is not available, a combination of test 

systems has been suggested to reliably diagnose the disease. In 

primates the skin test should be conducted as a primary test but 

should always be accompanied in the early and mid term phases 

of the disease by the IFN- release assay as the most 

appropriate test system. As late stage diagnostic tools, 

serological and direct tests may provide additional diagnostic 

benefits. 

In 2010, the Basel ZOO relocated all human primates to an 

external facility during the construction phase of a new primate 

house. The occasion was used to test all animals for TB before 

movement of the animals and following their resettlement in the 

new primate house. The Guideline for the prevention and control 

of tuberculosis in non-human primates 1 was used as the basis for 

the test schedule 


Lelystad tuberculin PPD antigens (Prionics, The Netherlands) 

were used for stimulation of whole blood cultures. The release of 

IFN- from PBMCs was measured with the PRIMAGAM ® 

(Prionics, Switzerland) sandwich enzyme immunoassay (EIA). 

Diagnostic sensitivity was assessed in primate colonies of 

unknown Tb status. 

Results 

Here we report the TB test results from three human primate 

colonies from the Basel ZOO. Seven Orangutans, six Gorillas 

and eight Chimpanzees have been initially tested with two 

different skin tests, two IFN- tests (one was PRIMAGAM ® ), two 

antibody tests and thoracic x-ray. Animals which were negative 

for all test results were considered as TB free. Animals that 

tested positive in at least one test system were required to 

undergo further monitoring. One Chimpanzee was tested positive 

in more than one test. After relocation all reactors were tested a 

second time, whereas all animals were negative tested with 

PRIMAGAM ® . 


We have compared the diagnostic performance of tuberculin 

PPD for the stimulation of blood cultures in PRIMAGAM ® IFN- 

assay. In the presentation we will highlight the differences of 

different test systems and point out how and when the different 

tests should be conducted and how PRIMAGAM ® 

can help to 

optimize the TB diagnostic in primates. 

References 

1. Guideline for the prevention and control of tuberculosis in non-human 

primates: recommendations of the European primate veterinary 

association working group on Tuberculosis, M. Bushmitz; A. Lecu; F. 

Verreck; E. Preussing; S. Rensing; K. Rensing; J. Med Primatol, 38, 59-69, 

2009) 

Tuberculosis, Primagam, tuberculin,

S4 - P - 12 

COMPARISON OF THE EFFICIENCY OF DIFFERENT PARASITOLOGICAL DIAGNOSTIC METHODS 

USED IN ANALYSIS OF DEHYDRATED SEWAGE SLUDGES 

Jolanta Zdybel, Tomasz Cencek, Jacek Karamon 

National Veterinary Research Institute, Departament of Parasitology and Invasive Disease, Pulawy, Poland 

Ascaris, Toxocara, Trichuris, eggs, sewage sludge, fertilizers 

Introduction 

The use of sewage sludge as fertilizer is connected with 

microbiological and parasitological hazards in terms of public 

health safety. Therefore, it is necessary to carry on proper 

parasitological investigations of this type of samples. Majority, of 

current parasitological diagnostic procedures using in sewage 

sludge surveys are derived from soil samples investigation. This 

kind of procedure are characterized by low efficiency, due to 

occurrence of polyelectrolytes in sewage sludge samples. 

Therefore, new method of sewage sludge parasitological 

diagnostics was developed. The methods is focused on detection 

of parasitic nematodes eggs, belonging to Ascaris, Trichuris and 

Toxocara genus. It is combination of floatation and sedimentation 

method, preceded by sample prolonged dispersion. The purpose 

of this study is to compare the efficiency of the method developed 

in Department of Parasitology and Invasive Diseases to other 

methods which are used in laboratories worldwide. 

References 

1. Zdybel J., Karamon J., Cencek C.: The occurrence of eggs of 

parasitic roundworms (genera: Ascaris, Trichuris, Toxocara) in 

organic and mineral-organic fertilizers and in sewage sludge. 

Zycie Wet.2009,84(12),992-996. 

2. Simonart T., Roussel S., Gireaudot-Liepman M.F.: desk study 

on European standard for the enumeration of diable helminth ova 

in sludge, soil and solid waste. HORIZONTAL-WP3-5, France 

2003. 


For this study we used randomly chosen 10 dehydrated sewage 

sludge samples derived from Polish waste water treatment 

plants. Each sample weighed 10 grams (dry weight) and was 

examined by our own method, PN-Z-19000-4 method, flotation 

method described by Quinn, method according to Environmental 

Protection Agency (EPA) and Triple flotation (TF) method 

according to ANFOR XP X33-017 . Microscopy (X20) was used 

to estimate the number of nematode eggs. 

Results 

Results concerning percentage of positive samples and numbers 

of detected parasite eggs obtained with the use of different 

methods were presented in Table 1. 

Table. 1. Mean number of parasite eggs and % of positive 

samples detected in sewage sludges samples with the use of 5 

different methods. 

Methods 

Mean number of detected eggs 

(% of positive samples) 

Ascaris Toxocara Trichuris 

Quinn 

2.2 

(70%) 

2.1 

(90%) 

0.3 

(30%) 

PN-Z-19000-4 

0.3 

(40%) 

0.15 

(30%) 

0 

Triple flotation 

0.6 

(10% 

3.3 

(40%) 

0 

EPA 

1.7 

(80%) 

1.3 

(100%) 

1.3 

(20%) 

Own method 

16.4 

(100%) 

11.1 

(100%) 

1.7 

(100%) 


The results of the present study demonstrated that method 

developed in Department of Parasitology and Invasive Diseases 

is six-fold more efficient than flotation method devised by Quinn, 

seven-fold more efficient than TF method and EPA method, 65- 

fold more efficient then PN-Z-19000-4 method. The adaptation of 

described method in other parasitological laboratories is highly 

recommended.

List of Abstracts (in order of number) 

S1-K-01 VAN DER POEL WIM H. M. 

S1-K-02 SCHWARZ STEFAN 

S1-O-01 BOSS CHRISTINA 

S1-O-02 

STRUTZBERG- 

MINDER 

KATRIN 

S1-O-03 BOUWSTRA RUTH 

S1-O-04 GÓRNA KAMILA 

S1-O-05 NARDINI ROBERTO 

S1-O-06 BALLAGI ANDREA 

S1-O-07 VAN MAANEN KEES 

S1-O-08 

REVILLA- 

FERNÁNDEZ 

SANDRA 

S1-O-09 COOLEY WILLIAM 

S1-O-10 LANGEVELD JAN 

S1-O-11 

REBORDOSA- 

TRIGUEROS 

XAVIER 

S1-O-12 O'NEILL RONAN 

S1-O-13 SCHIRRMEIER HORST 

S1-O-14 BOSS CHRISTINA 

S1-O-15 PORQUET-GARANTO LOURDES 

S1-O-15 VILLA ALEIDA 

S1-O-17 BALLAGI ANDREA 

S1-O-18 LOPEZ PABLO 

S2-K-01 SOLDAN ANDREW 

S2-O-01 NORTH SARAH 

THE EMERGENCE OF SCHMALLENBERG VIRUS – HOW TO 

RESPOND TO NEW EPIZOOTICS IN EUROPE 

ASSESSING THE ANTIMICROBIAL SUSCEPTIBILITY OF 

BACTERIA OBTAINED FROM ANIMALS 

ORAL FLUIDS – SAMPLE MATRIX FOR EFFECTIVE HERD 

HEALTH MONITORING 

DIAGNOSTIC PROPERTIES OF SEVERAL PRRS ANTIBODY 

ELISAS IN THE CONTEXT OF A LONGITUDINALSTUDY OF 

A FARM USING DIFFERENT VACCINATION STRATEGIES 

INTRODUCTION RATE OF A LOW PATHOGENIC AVIAN 

INFLUENZA VIRUS INFECTION IN DIFFERENT DUTCH 

POULTRY SECTORS 

A DUPLEX ONE-STEP REAL TIME RT-PCR FOR DIAGNOSIS 

OF FOOT AND MOUTH DISEASE 

VALIDATION OF A COMPETITIVE ELISA FOR THE 

DETECTION OF ANTIBODIES ANTI-P26 OF EQUINE 

INFECTIOUS ANEMIA VIRUS IN EQUINE SERA 

DIAGNOSTIC PERFORMANCE OF A COMMERCIAL PRRS 

SERUM ANTIBODY ELISA ADAPTED TO ORAL FLUID 

SPECIMENS: FIELD SAMPLES 

SUBTYPING OF SWINE INFLUENZA VIRUSES BY 

MULTIPLEX REAL-TIME PCR 

IMPORTANCE OF CONTINUOUS VALIDATION OF 

MOLECULAR METHODS FOR ROUTINE DIAGNOSIS OF 

PRRSV RNA IN CLINICAL SAMPLES 

VIRAL DIAGNOSIS USING TRANSMISSION ELECTRON 

MICROSCOPY 

A BEAD BASED MULTIPLEX IMMUNOFLUOROMETRIC 

ASSAY FOR SCREENING AND CONFIRMATION OF ALL 

MAJOR PRION TYPES IN SHEEP 

USING ORAL FLUID FOR THE SEROLOGICAL 

MONITORIZATION OF PRRSV CIRCULATION IN A GROUP 

OF INFECTED GILTS 

ENHANCED DETECTION OF BOVINE RESPIRATORY 

VIRUSES BY INCLUSION OF COHORT ANIMALS 

THE FIRST YEAR OF OBLIGATORY BVD CONTROL IN 

GERMANY – DIAGNOSTIC STRATEGIES, RESULTS AND 

EXPERIENCES 

EUROPEAN PRRSV – DIAGNOSTIC SOLUTIONS FOR A 

RAPIDLY MUTATING VIRUS 

SEROLOGICAL ANALYSIS AND MONITORING OF IBR IS IT 

POSSIBLE TO CONTROL IBRGE ANTIBODIES IN A BULK 

TANK MILK? 

DEVELOPMENT AND VALIDATION OF A REAL-TIME PCR 

ZEN GEL MIX FOR THE DIAGNOSIS AND QUANTIFICATION 

OF COXIELLA BURNETII 

RING TEST EVALUATION FOR THE DETECTION OF PRRSV 

ANTIBODIES IN ORAL FLUID SPECIMENS USING A 


REAL TIME PCR, MYCOPLASMA GALLISEPTICUM, 

MYCOPLASMA SYNOVIAE, MYCOPLASMA MELEAGRIDIS 

WHEN DO YOU WANT THE RESULT? HOW MUCH DO YOU 

WANT TO PAY! 

DEVELOPMENT OF A RAPID ISOTHERMAL ASSAY TO 

DETECT TAYLORELLA EQUIGENITALIS, THE CAUSATIVE 

AGENT OF CONTAGIOUS EQUINE METRITIS

S2-O-02 VRANCKEN ROBERT 

S2-O-03 SOCHA WOJCIECH 

S2-O-04 VALLS LAURA 

S3-K-01 GAVIER-WIDÉN DOLORES 

S3-O-01 BOUWSTRA RUTH 

S3-O-02 KOOI BART 

S3-O-03 BÖTTCHER JENS 

S3-O-04 CARTER CRAIG N. 

S3-O-05 EGLI C. 

LABORATORY VALIDATION OF AN 

IMMUNOCHROMATOGRAPHIC TEST FOR THE RAPID 

DETECTION OF KOI HERPESVIRUS (CYHV-3) IN GILL 

SWABS 

EVALUATION OF RAPID HRSV STRIP TESTS FOR 

DETECTION OF BOVINE RESPIRATORY SYNCYTIAL VIRUS 

EVALUATION OF A LATEX AGGLUTINATION TEST FOR THE 

IDENTIFICATION OF CLOSTRIDIUM DIFFICILE OF PORCINE 

ORIGIN 

EMERGING AND RE-EMERGING WILDLIFE DISEASES: 

PATHOLOGY AND RELATED TECHNIQUES 

SCHMALLENBERG VIRUS OUTBREAK IN THE 

NETHERLANDS: ROUTINE DIAGNOSTICS AND TEST 

RESULTS 

25 YEARS OF PASSIVE SURVEILLANCE OF BATS IN THE 

NETHERLANDS. MOLECULAR EPIDEMIOLOGY AND 

EVOLUTION OF EBLV-1 

DIAGNOSIS OF Q FEVER IN DAIRY CATTLE BY PHASE- 

SPECIFIC MILK-SEROLOGY 

EQUINE NOCARDIOFORM PLACENTITIS & ABORTION 

OUTBREAK AND FARM-BASED RISK FACTOR STUDY, 

2010-2011 

A NEW DIAGNOSTIC TOOL FOR BOVINE TUBERCULOSIS- 

IDEXX M.BOVIS ANTIBODY TEST KIT 

S3-O-06 LA ROCCA ANNA DETECTION OF SCHMALLENBERG VIRUS IN THE UK 

S3-O-07 CHAINTOUTIS SERAFEIM C. 

S3-O-08 SCHIRRMEIER HORST 

S3-O-09 FOERSTER CHRISTINE 

S3-O-10 STEINRIGL ADOLF 

S3-O-11 POURQUIER PHILIPPE 

DETECTION OF WNV ENZOOTIC CIRCULATION IN HORSES 

WITH NEUROLOGICAL SIGNS AND IN CAPTIVE SENTINEL 

CHICKENS IN THE PREFECTURE OF THESSALONIKI, 

GREECE 

SCHMALLENBERGVIRUS: SEROLOGICAL STUDIES IN 

GERMAN HOLDINGS 

DIFFERENT DIAGNOSTIC TOOLS FOR A BROAD RANGE 

OF MACAVIRUSES AND THEIR RESERVOIR AND 

SUSCEPTIBLE HOSTS 

DIAGNOSTIC ASPECTS OF SUID HERPESVIRUS 1 

INFECTION IN WILD BOAR 

PRELIMINARY VALIDATION OF THE ID SCREEN® 

SCHMALLENBERG VIRUS INDIRECT ELISA 

S4-K-01 KOSTRZEWA MARCUS MALDI-TOF AND OTHER NEW DIAGNOSTIC 

S4-O-01 RIPP ULRIKE 

S4-O-02 WRAGG PETER 

S4-O-03 RAEBER ALEX 

S4-O-04 HEUVELINK ANNET 

S4-O-05 OVERESCH GUDRUN 

S4-O-06 SAWYER JASON 

S1-P-01 AHOLA HEIKKI 

S1-P-02 BALLAGI ANDREA 

SUITABILITY OF RECOMBINANT PROTEINS FOR THE 

DIAGNOSIS OF LEPTOSPIROSIS IN PIGS 

BIOLOG GENERATION III, MATRIX ASSISTED LASER 

DESORPTION/IONISATION TIME-OF-FLIGHT (MALDI-TOF) 

MASS SPECTROMETRY AND 16S RRNA GENE 

SEQUENCING FOR THE IDENTIFICATION OF BACTERIA OF 

VETERINARY INTEREST 

EVALUATION OF THE DIAGNOSTIC PERFORMANCE OF 

PEPTIDE COCKTAILS IN THE INTERFERON GAMMA ASSAY 

FOR DIAGNOSIS OF TUBERCULOSIS IN CATTLE 

MATRIX-ASSISTED LASER DESORPTION IONIZATION-TIME 

OF FLIGHT MASS SPECTROMETRY (MALDI-TOF MS) IN A 

VETERINARY DIAGNOSTIC LABORATORY 

RAPID IDENTIFICATION OF BOVINE MASTITIS 

PATHOGENS USING MALDI TOF 

15 MINUTE ELISA USING A LOW COST COMMERCIAL 

BIOSENSOR 

LAWSONIA INTRACELLULARIS IN BLUE FOXES IN 

FINLAND. A CASE REPORT 

DIAGNOSTIC PERFORMANCE OF A COMMERCIAL PRRS 

SERUM ANTIBODY ELISA ADAPTED TO ORAL FLUID: 

LONGITUDINAL RESPONSE IN EXPERIMENTALLY- 

INOCULATED POPULATIONS

S1-P-03 BALLAGI ANDREA 

S1-P-04 BENITO ALFREDO 

S1-P-05 BLANCHARD BEATRICE 

S1-P-06 BLANCHARD BEATRICE 

S1-P-07 BOSS CHRISTINA 

S1-P-08 BÖTTCHER JENS 

S1-P-09 VAN MAANEN KEES 

S1-P-10 NARDINI R. 

S1-P-11 CHERNYSHOV ANATOLIY 

S1-P-12 CHERNYSHOVA ELENA 

S1-P-13 COCCHI MONIA 

S1-P-14 CORNAGLIA ESTELA 

S1-P-15 EIRAS CARMEN 


S1-P-17 METREVELI GIORGI 

S1-P-18 GEROLIMETTO ELISA 


S1-P-20 HIRVELÄ-KOSKI VARPU 

S1-P-21 KOCHANOWSKI MACIEJ 

S1-P-22 KOVAC GABRIEL 

S1-P-23 KÜHN TILMAN 

S1-P-24 MAGNEE DAMIEN 

DETECTION OF PRRSV ANTIBODY IN ORAL FLUID 

SPECIMENS FROM INDIVIDUAL BOARS USING A 


DIAGNOSIS OF MAIN HAEMOPARASITIC DISEASES OF 

CATTLE BY REAL-TIME PCR 

DEVELOPMENT AND EVALUATION OF A NEW AND 

ORIGINAL EXTRACTION PROTOCOL TO DETECT 

MYCOBACTERIUM AVIUM SUBSP PARATUBERCULOSIS IN 

BOVINE FECES BY REAL TIME 

VALIDATION OF REAL-TIME PCR TEST FOR THE 

DETECTION AND QUANTIFICATION OF COXIELLA 

BURNETII FOR ABORTION CONTROL PROGRAM 

ANALYTICAL EVALUATION OF A SWINE INFLUENZA VIRUS 

PCR ON AVIAN SAMPLES 

QUALITY CONTROL OF PRRSV VACCINATION BY 

PARAMETERS OF IMMUNITY 

PTS FOR BVDV ANTIGEN DETECTION BY GD - ANIMAL 

HEALTH SERVICE 

PRESENTATION OF THE RESULTS OF THE ANNUAL 

PROFICIENCY TEST FOR THE SEROLOGICAL DIAGNOSIS 

OF EQUINE INFECTIOUS ANEMIA CONDUCTED IN ITALY 

BETWEEN 2006-2010 

ANTIMICROBIAL SUSCEPTIBILITY OF AVIBACTERIUM 

(HAEMOPHILUS) PARAGALLINARUM ISOLATES 

RECOVERED IN THE RUSSIAN TERRITORY 

COMPARISON BETWEEN ANALYTICAL SENSITIVITY OF 

RABIES VIRUS ISOLATION IN NEUROBLASTOMA CELL 

CULTURE WITH ANALYTICAL SENSITIVITY OF MOUSE 

INOCULATION TEST 

IDENTIFICATION OF GLOBICATELLA SANGUINIS 

ISOLATED FROM PNEUMONIA IN A GOAT 

KAIZEN PROCESS APPLIED AT THE DIAGNOSTIC SERVICE 

OF FACULTÉ DE MÉDICINE VÉTÉRINAIRE, UNIVERSITÉ DE 

MONTREAL 

ACCURACY OF PARATUBERCULOSIS ANTIBODY ELISA AT 

PREDICTING FECAL SHEDDING OF MYCOBACTERIUM 

AVIUM SUBSP PARATUBERCULOSIS IN CATTLE 

EVALUATION OF AN ELISA ASSAY AS AN ANCILLARY 

METHOD IN A HERD WITH A NATURAL TUBERCULOSIS 

INFECTION 

VIROLOGICAL SURVEILLANCE AND CHARACTERIZATION 

OF AVIAN-LIKE REASSORTANT H1N2 SWINE INFLUENZA 

VIRUSES IN SWEDEN 

A TOOL TO ASSESS ANTIMICROBIAL RESISTANCE 

PATTERNS IN DAIRY FARMS 

CHARACTERIZATION OF EXTENDED-SPECTRUM ß- 

LACTAMASE (ESBL)-PRODUCING ESCHERICHIA COLI 

BOVINE ISOLATES IN NORTHWEST SPAIN 

OVINE ABORTION CAUSED BY YERSINIA 

PSEUDOTUBERCULOSIS – A CASE REPORT 

ANALYSIS OF THE EFFICIENCY OF THE MCMASTER 

METHOD IN RAYNAUD’S MODIFICATION IN DETECTION OF 

TOXOCARA SP. AND TRICHURIS SP. EGGS IN 

CARNIVORES FAECES 

MILK AMYLOID A AND ITS USEFULNESS IN THE 

LABORATORY DIAGNOSIS OF MASTITIS 

RELIABLE AND EARLY DETECTION OF SALMONELLA IN 

PIGS 

DETECTION OF BVD VIRUS ON PERSISTENTLY INFECTED 

CALVES BY REAL TIME REVERSE TRANSCRIPTION PCR 

ON EAR NOTCHES

S1-P-25 MARQUES SARA 

S1-P-26 MICHALSKI MIROSLAW 

S1-P-27 MICHALSKI MIROSLAW 

S1-P-28 MELONI D. 

S1-P-29 PRITZ-VERSCHUREN SYLVIA 

S1-P-30 PROHASKA SARAH 

S1-P-31 SATTLER TATJANA 

S1-P-32 SCHROEDER CARSTEN 

S1-P-33 

SCICLUNA 

MARIA 

TERESA 

S1-P-34 KOVAC GABRIEL 

S1-P-35 WIDÉN FREDERIK 

GENOTYPIC VARIABILITY DETERMINATION OF 

PATHOGENIC PROTOTHECA 

DETECTION OF SAXITOXINE (PSP MARINE BIOTOXIN) IN 

MOLLUSCS BY ELISA 

PROFICIENCY TESTING (PT) FOR CHEMICAL 

LABORATORIES PERFORMING ANALYSIS OF MEAT AND 

MEAT PRODUCTS ORGANISED BY NVRI IN PULAWY 

SPECIFIED RISK MATERIAL REMOVAL PRACTICES: CAN 

WE REDUCE THE BSE HAZARD TO HUMAN HEALTH 

LOW PATHOGENIC AVIAN INFLUENZA VIRUS INFECTIONS 

ON POULTRY FARMS IN THE NETHERLANDS 

DETECTION OF BRACHYSPIRA HYODYSENTERIAE AND 

BRACHYSPIRA PILOSICOLI IN SWISS PIGS USING A 

COMBINATION OF CULTURE AND PCR 

PRRSV DIAGNOSTICS BY RT-PCR AND DIFFERENT ELISA 

SYSTEMS IN SERUM AND ORAL FLUID OF PRRSV 

VACCINATED PIGS 

RELIABLE DETECTION AND TYPING OF PRRSV USING 

MULTIPLEX REAL-TIME RT-PCR 

APPLICATION OF A HERD PROGRAMME BASED ON 

CONTROL MEASURES AND LABORATORY MONITORING 

TO ERADICATE A LEPTOSPIRA OUTBREAK IN A LARGE 

DAIRY CATTLE HERD 

THE SERUM PROTEIN ELECTROPHORETIC PATTERN IN 

CALVES WITH CHRONIC RESPIRATORY DISEASES 

DEVELOPMENT OF A BROADLY DETECTING ASSAY FOR 

FLAVIVIRUS USING A NOVEL CONCEPT 

S1-P-36 O'NEILL RONAN EXTENDING THE SHELF LIFE OF COMMERCIAL ELISA KITS 

S1-P-37 POLAK MIROSŁAW P. 

S1-P-38 SZCZOTKA MARIA 

S1-P-39 SZCZOTKA MARIA 

S1-P-40 SZCZOTKA ANNA 

S2-P-01 OLOFSON ANN-SOPHIE 

S2-P-02 SANZ ANTONIO 

S2-P-03 LEBLANC NEIL 

S3-P-01 ACHTERBERG RENÉ 

S3-P-02 ARENT ZBIGNIEW 

S3-P-03 BLOME SANDRA 

S3-P-04 SCHELP CHRISTIAN 

S3-P-05 KARAMON JACEK 

MONITORING OF BOVINE VIRAL DIARRHEA VIRUS (BVDV) 

IN DAIRY CATTLE HERDS IN POLAND 

DETECTION OF BOVINE LEUKAEMIA VIRUS PROVIRAL 

DNA IN DENDRITIC CELLS BY IN SITU PCR 

BOVINE BLOOD DENDRITIC CELLS IN CATTLE INFECTED 

WITH BOVINE LEUKAEMIA VIRUS (BLV 

PORCINE CIRCOVIRUS TYPE 2 IN POSTWEANING PIGS 

WITH ANTIBIOTIC NON-RESPONSIVE DIARRHEA 

EVALUATION OF FIELD BASED METHODS FOR 

DETECTION OF CLASSICAL SWINE FEVER BY PCR 

NEW IMMUNOASSAYS FOR DIAGNOSIS OF ASFV BASED 

ON VP72: CAPTURE ELISA FOR DETECTION OF IGM 

SPECIFIC ANTIBODIES AND PEN-SIDE TEST FOR BLOOD 

SAMPLES 

PORTABLE PLATFORMS FOR THE DETECTION OF 

AFRICAN SWINE FEVER VIRUS TESTED IN FIELD 

CONDITIONS IN NORTHERN UGANDA 

BEAD-BASED SUSPENSION ARRAY FOR THE 

SIMULTANIOUS DETECTION OF ANTIBODIES AGAINS THE 

RIFT VALLEY FEVER VIRUS NUCLEOCAPSID AND GN 

GLYCOPROTEIN 

LIPOPOLYSACCHARIDE-CAPTURE ELISA FOR THE 

DETECTION OF LEPTOSPIRA BORGPETERSENII SEROVAR 

HARDJO IN SHEEP 

EVALUATION OF DIRECT BLOOD POLYMERASE CHAIN 

REACTION FOR RAPID DETECTION OF AFRICAN SWINE 

FEVER VIRUS 

DEVELOPMENT OF A SCHMALLENBERG VIRUS ANTIBODY 

ELISA 

DETECTION OF ECHINOCOCCUS MULTILOCULARIS IN 

FOXES’ FAECES BY PCR WITH THE USE OF DILUTED DNA 

SAMPLES – COMPARISON OF DIFFERENT METHODS OF 

DNA ISOLATION

S3-P-06 KELLER SELINA 

S3-P-07 KÖNIG MATTHIAS 

COMPARISON OF CULTURE AND PCR FOR DETECTION 

OF MYCOBACTERIUM AVIUM SSP. PARATUBERCULOSIS 

IN BOVINE FAECES 

HEPATITIS E VIRUS IN DOMESTIC SWINE AND WILD BOAR 

FROM GERMANY 

S3-P-08 MAGNEE DAMIEN EMERGENCE OF SCHMALLENBERG VIRUS: 

S3-P-09 NARDINI ROBERTO 

S3-P-10 ŚMIETANKA KRZYSZTOF 

S3-P-11 PELETTO SIMONE 

S3-P-12 POURQUIER PHILIPE 

S3-P-13 RABALSKI LUKASZ 

S3-P-14 RANZ ANA 

S3-P-15 REID SCOTT M. 

S3-P-16 REID SCOTT M. 

S3-P-17 KOLBASOV DENIS 

S3-P-18 SKRZYPCZAK TERESA 

S3-P-19 WELLENBERG GERARD 

S3-P-20 MAZZONE PIERA 

S3-P-21 MAZZONE PIERA 

S3-P-22 LARSKA MAGDALENA 

S3-P-23 LARSKA MAGDALENA 

S3-P-24 LOEFFEN W.L.A. 

S3-P-25 COOLEY W.A. 

S3-P-26 MATERNIAK MAGDALENA 

S4-P-01 ALTMANN MICHAELA 

S4-P-02 ARENT ZBIGNIEW 

PRELIMINARY VALIDATION OF A SOLID- PHASE 

COMPETITIVE ELISA FOR THE DETECTION OF 

ANTIBODIES AGAINST WEST NILE DISEASE VIRUS IN 

HORSE SERA 

SIMULTANEOUS DETECTION AND DIFFERENTIATION OF 

INFLUENZA A VIRUS AND NEWCASTLE DISEASE VIRUS BY 

ONE STEP RT-PCR 

DOLPHIN MORBILLIVIRUS INFECTION IN A CAPTIVE 

HARBOR SEAL (PHOCA VITULINA) 

PRELIMINARY VALIDATION OF THE ID SCREEN AFRICAN 

SWINE FEVER INDIRECT ELISA BASED ON THREE 

RECOMBINANT ASF PROTEINS 

ONE STEP RT-PCR FOLLOWED BY MSSCP AS A NOVEL 

METHOD FOR A FAST AND RELIABLE DETECTION AND 

DIFFERENTIATION OF MIXED INFECTION WITH 

NEWCASTLE DISEASE VIRUS OF DIFFERENT ORIGIN 

DEVELOPMENT OF MULTISPECIES SEROLOGICAL 

ASSAYS TO DETECT ANTIBODIES SPECIFIC FOR 

MYCOBACTERIUM BOVIS IN SERUM SAMPLES 

FIRST REPORTED LOW PATHOGENCITY AVIAN 

INFLEUNZA VIRUS SUBTYPE H9 NFECTION OF DOMESTIC 

FOWL IN ENGLAND 

DETECTION OF IBV QX IN COMMERCIAL POULTRY 

FLOCKS IN THE UNITED KINGDO 

APPLICATION OF REVERSE TRANSCRIPTION REAL-TIME 

PCR TO DETECT THE SCHMALLENBERG VIRUS GENOME 

PREVALENCE OF COXIELLA BURNETII ANTIBODIES IN 

SHEEP AND GOATS IN FINLAND 

DETECTION OF SCHMALLENBERG VIRUS IN BOVINE 

SEMEN BY ONE-STEP REAL-TIME RT-PCR 

INTERFERON-GAMMA ASSAY TO DIAGNOSE 

MYCOBACTERIUM BOVIS INFECTION IN PIGS 

USE OF EXPERIMENTAL JOHNIN IN THE GAMMA- 

INTERFERON TEST IN CATTLE INFECTED BY 

MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS: 

PRELIMINARY DATA 

COMPARISON OF THE PERFORMANCE OF FIVE 

DIFFERENT IMMUNOASSAYS TO DETECT SPECIFIC 

ANTIBODIES AGAINST EMERGING ATYPICAL BOVINE 

PESTIVIRUS 

SCHMALLENBERG VIRUS (SBV) IN POLAND – 

PRELIMINARY RESULTS 

DEVELOPMENT OF A VIRUS NEUTRALISATION TEST TO 

DETECT ANTIBODIES AGAINST 

VIRAL DIAGNOSIS USING TRANSMISSION ELECTRON 

MICROSCOPY SCHMALLENBERG VIRUS 

DETECTION OF EQUINE FOAMY VIRUS INFECTIONS IN 

HORSES 

MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM 

AVIUM SUBSP. PARATUBERCULOSIS STRAINS IN A 

NATIONAL PARATUBERCULOSIS CONTROL PROGRAM 

DEVELOPMENT AND VALIDATION OF A REAL-TIME PCR 

ASSAY FOR THE DETECTION OF LEPTOSPIRA 

BORGPETERSENII SEROVAR HARDJO IN URINE AND 

KIDNEY

S4-P-03 DĄBROWSKA JOANNA 

S4-P-04 ISAKSSON MATS 

S4-P-05 MARTOS RAICH ALBA 

S4-P-06 MELONI DANIELA 

S4-P-07 MOYEN JEAN-LOUIS 

S4-P-08 NOGAROL CHIARA 

S4-P-09 POURQUIER PHILIPPE 

S4-P-10 POURQUIER PHILIPPE 

S4-P-11 SCHROEDER BJOERN 

S4-P-12 ZDYBEL JOLANTA 

USEFULNESS OF LIVE/DEAD BACLIGHT BACTERIAL 

VIABILITY KIT TYPE 7007 MOLECULAR PROBES FOR 

EVALUATION OF VIABILITY ASCARIS SP., TOXOCARA SP. 

AND TRICHURIS SP. EGGS ISOLATED FROM SEWAGE 

SLUDGE 

AUTOMATION OF A CAPTURE PROBE MAGNETIC BEAD 

DNA EXTRACTION METHOD FOR 3 GRAM FAECAL 

SAMPLES FROM RED FOX INTENDED FOR PCR- 

DETECTION OF ECHINOCOCCUS MULTILOCULARIS 

EVALUATION OF THREE ELISAS FOR DETECTING SERUM 

ANTIBODIES AGAINST MYCOPLASMA HYOPNEUMONIAE. 

SEPRION-COATED MICROCANTILEVER SENSORS FOR 

PRPSC DETECTION 

EVALUATION OF DIFFERENT EXTRACTION METHODS 

FOR THE DETECTION OF MYCOBACTERIUM AVIUM 

SUBSP. PARATUBERCULOSIS IN BOVINE FAECES 

REAL-TIME PCR FOR DETECTION OF CAMPYLOBACTER 

JEJUNI, COLI AND LARI IN FOODS: TEST VALIDATION 

ACCORDING TO ISO16140:2003 

VALIDATION OF THE ID SCREEN SALMONELLA DUBLIN 

COMPETITIVE ELIS 

ID SCREEN® INTERFERON GAMMA ELISA : 

IMPROVEMENT OF ANALYTICAL SENSITIVITY AND 

INTRODUCTION OF A STANDARD REFERENCE CONTROL 

TO IMPROVE RESULT INTERPRETATION 

EFFECTIVE TESTING STRATEGY WITH PRIMAGAM® FOR 

TUBERCULOSIS IN HUMAN PRIMATES 

COMPARISON OF THE EFFICIENCY OF DIFFERENT 

PARASITOLOGICAL DIAGNOSTIC METHODS USED IN 

ANALYSIS OF DEHYDRATED SEWAGE SLUDGES

List of abstracts (alphabetical order of presenting authors) 

ACHTERBERG RENÉ S3-P-01 

AHOLA HEIKKI S1-P-01 

ALTMANN MICHAELA S4-P-01 

ARENT ZBIGNIEW S3-P-02 

ARENT ZBIGNIEW S4-P-02 

BALLAGI ANDREA S1-O-06 

BALLAGI ANDREA S1-O-17 

BALLAGI ANDREA S1-P-02 

BALLAGI ANDREA S1-P-03 

BENITO ALFREDO S1-P-04 

BLANCHARD BEATRICE S1-P-05 

BLANCHARD BEATRICE S1-P-06 

BLOME SANDRA S3-P-03 

BOSS CHRISTINA S1-O-01 

BOSS CHRISTINA S1-O-14 

BOSS CHRISTINA S1-P-07 

BÖTTCHER JENS S3-O-03 

BÖTTCHER JENS S1-P-08 

BOUWSTRA RUTH S1-O-03 

BOUWSTRA RUTH S3-O-01 

CARTER CRAIG N. S3-O-04 

CHAINTOUTIS SERAFEIM C. S3-O-07 

CHERNYSHOV ANATOLIY S1-P-11 

CHERNYSHOVA ELENA S1-P-12 

COCCHI MONIA S1-P-13 

COOLEY WILLIAM S1-O-09 

COOLEY W.A. S3-P-25 

CORNAGLIA ESTELA S1-P-14 

DĄBROWSKA JOANNA S4-P-03 

EGLI C. S3-O-05 

EIRAS CARMEN S1-P-15 



FOERSTER CHRISTINE S3-O-09 

GAVIER-WIDÉN DOLORES S3-K-01 

GEROLIMETTO ELISA S1-P-18 

GÓRNA KAMILA S1-O-04 

HEUVELINK ANNET S4-O-04 

HIRVELÄ-KOSKI VARPU S1-P-20 

ISAKSSON MATS S4-P-04 

KARAMON JACEK S3-P-05 

KELLER SELINA S3-P-06 

KOCHANOWSKI MACIEJ S1-P-21 

KOLBASOV DENIS S3-P-17 

KÖNIG MATTHIAS S3-P-07 

KOOI BART S3-O-02 

KOSTRZEWA MARCUS S4-K-01 

KOVAC GABRIEL S1-P-22 

KOVAC GABRIEL S1-P-34 

KÜHN TILMAN S1-P-23 

LA ROCCA ANNA S3-O-06 

LANGEVELD JAN S1-O-10 

LARSKA MAGDALENA S3-P-22 

LARSKA MAGDALENA S3-P-23 

LEBLANC NEIL S2-P-03 

LOEFFEN W.L.A. S3-P-24 

LOPEZ PABLO S1-O-18 

MAGNEE DAMIEN S1-P-24 

MAGNEE DAMIEN S3-P-08 

MARQUES SARA S1-P-25 

MARTOS RAICH ALBA S4-P-05 

MATERNIAK MAGDALENA S3-P-26 

MAZZONE PIERA S3-P-20 

MAZZONE PIERA S3-P-21 

MELONI D. S1-P-28 

MELONI DANIELA S4-P-06 

METREVELI GIORGI S1-P-17 

MICHALSKI MIROSLAW S1-P-26 

MICHALSKI MIROSLAW S1-P-27 

MOYEN JEAN-LOUIS S4-P-07 

NARDINI ROBERTO S1-O-05 

NARDINI R. S1-P-10 

NARDINI ROBERTO S3-P-09 

NOGAROL CHIARA S4-P-08 

NORTH SARAH S2-O-01 

OLOFSON ANN-SOPHIE S2-P-01 

O'NEILL RONAN S1-O-12 

O'NEILL RONAN S1-P-36 

OVERESCH GUDRUN S4-O-05 

PELETTO SIMONE S3-P-11 

POLAK MIROSŁAW P. S1-P-37 

PORQUET- 

GARANTO LOURDES S1-O-15 

POURQUIER PHILIPPE S3-O-11 

POURQUIER PHILIPE S3-P-12 

POURQUIER PHILIPPE S4-P-09 

POURQUIER PHILIPPE S4-P-10 

PRITZ- 

VERSCHUREN SYLVIA S1-P-29 

PROHASKA SARAH S1-P-30 

RABALSKI LUKASZ S3-P-13 

RAEBER ALEX S4-O-03

RANZ ANA S3-P-14 

REBORDOSA- 

TRIGUEROS XAVIER S1-O-11 

REID SCOTT M. S3-P-15 

REID SCOTT M. S3-P-16 

REVILLA- 

FERNÁNDEZ SANDRA S1-O-08 

RIPP ULRIKE S4-O-01 

SANZ ANTONIO S2-P-02 

SATTLER TATJANA S1-P-31 

SAWYER JASON S4-O-06 

SCHELP CHRISTIAN S3-P-04 

SCHIRRMEIER HORST S1-O-13 

SCHIRRMEIER HORST S3-O-08 

SCHROEDER CARSTEN S1-P-32 

SCHROEDER BJOERN S4-P-11 

SCHWARZ STEFAN S1-K-02 

SCICLUNA MARIA TERESA S1-P-33 

SKRZYPCZAK TERESA S3-P-18 

SOCHA WOJCIECH S2-O-03 

SOLDAN ANDREW S2-K-01 

STEINRIGL ADOLF S3-O-10 

STRUTZBERG- 

MINDER KATRIN S1-O-02 

SZCZOTKA MARIA S1-P-38 

SZCZOTKA MARIA S1-P-39 

SZCZOTKA ANNA S1-P-40 

ŚMIETANKA KRZYSZTOF S3-P-10 

VALLS LAURA S2-O-04 

VAN DER POEL WIM H. M. S1-K-01 

VAN MAANEN KEES S1-O-07 

VAN MAANEN KEES S1-P-09 

VILLA ALEIDA S1-O-15 

VRANCKEN ROBERT S2-O-02 

WELLENBERG GERARD S3-P-19 

WIDÉN FREDERIK S1-P-35 

WRAGG PETER S4-O-02 

ZDYBEL JOLANTA S4-P-12

Abstract Book of EAVLD2012 - eavld congress 2012

Create successful ePaper yourself

Delete template?

Save as template?