Proceedings of the 10th International Colloquium on Paratuberculosis

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RESULTS Of <strong>the</strong> 327 fecal specimens analyzed by all three techniques, 22 animals were identified as heavy shedders based upon <strong>the</strong>ir fecal culture results. Of <strong>the</strong> 22 heavy fecal Map shedders identified by fecal culture results alone, 7 fecal specimens had corresponding confirmative real-time and nested PCR tests, 5 specimens had ei<strong>the</strong>r a positive real-time or nested PCR test. Ten fecal specimens (45%) failed to be confirmed by ei<strong>the</strong>r <strong>the</strong> corresponding real-time or nested PCR tests (Table 1). Of <strong>the</strong> 6 cows with positive correspondence between fecal culture, real-time PCR, and nested PCR, 4 were culled. Two had diagnostic ELISA titer at <strong>the</strong> time <strong>of</strong> <strong>the</strong>ir removal from <strong>the</strong> herd. Of <strong>the</strong> remaining two cows, one cow had an initial high suspicious titer as determined by ParaChek® Map ELISA testing that subsequently corrected. The o<strong>the</strong>r cow had no evidence <strong>of</strong> having had a serological response. DISCUSSION The three methods used to identify Map required separate test samples being taken from a given fecal specimen. The concurrence <strong>of</strong> all three tests decreases <strong>the</strong> probability <strong>of</strong> sample error due to clumping; whereas non-concurrence between <strong>the</strong> three tests argues for nonuniform distribution <strong>of</strong> organisms. The importance <strong>of</strong> correctly identifying heavy fecal shedding <strong>of</strong> Map is two-fold: 1) animals identified as heavy shedders are considered to be primary disseminators <strong>of</strong> pathogenic mycobacterium into <strong>the</strong> environment; and 2) heavy Map replication in feces documents advanced mucosal disease and increased probability <strong>of</strong> systemic progression. The lack <strong>of</strong> concurrence between a positive fecal culture and two sensitive PCR tests puts into question <strong>the</strong> designation <strong>of</strong> “heavy or moderate shedding” as identified by <strong>the</strong> culture. It argued that fecal culture results can be significantly colored by sampling error. CONCLUSION Forty-five percent <strong>of</strong> <strong>the</strong> cows identified in this study as heavy shedders by Trek® Diagnostic System were documented by real-time and nested PCR to be light fecal shedders. Decision makers may be well advised to seek additional confirmation <strong>of</strong> fecal heavy shedding status before culling an animal from <strong>the</strong> herd based upon this criterion alone. REFERENCES Collins M.T., Gardner I. A., Garry F. B., Roussel A.J., Wells S.J, 2006. Consensus recommendations on diagnostic testing for <strong>the</strong> detection <strong>of</strong> paratuberculosis in cattle in <strong>the</strong> United States. JAVMA, 229, 912-919. Harris N. B., Barletta R. G.: Mycobacterium avium subsp. paratuberculosis in veterinary medicine. Clin. Microbiol. Reviews 2001; 14:489-512. ACKNOWLEDGEMENT The authors acknowledge <strong>the</strong> gracious collaboration and support received from <strong>the</strong> Florida Department <strong>of</strong> Agriculture and Consumer Services and <strong>the</strong> United States Department <strong>of</strong> Agriculture. 43
#9 Comparative IS900 and IS1311 Direct Fecal Mycobacterium Avium Subspecies Paratuberculosis Nested PCR Tests: Significance <strong>of</strong> Disparities Williams JE, Pinedo PJ, Monif GRG University <strong>of</strong> Florida, College <strong>of</strong> Veterinary Medicine Department <strong>of</strong> Infectious Diseases and Pathobiology, Gainesville, FL ABSTRACT To challenge <strong>the</strong> hypo<strong>the</strong>sis <strong>of</strong> genomic polymorphism, two direct fecal nested polymerase chain reaction (PCR) tests based upon <strong>the</strong> IS900 and <strong>the</strong> IS1311 insertion sequences were constructed and tested in parallel in three United States Department <strong>of</strong> Agriculture (USDA) Laboratory Certification tests. The sensitivities for P90-P91 and J1-J2 IS900 direct and nested primers were 21.7% and 76.7% whereas those for <strong>the</strong> IS1-IS2 and IS3-IS4 primers were 38.3% and 86.7%. The ability <strong>of</strong> 1311 base insertion sequence primers to better identify Map in <strong>the</strong> USDA laboratory certification tests over IS900 base insertion sequence primers argues for <strong>the</strong> existence <strong>of</strong> a degree <strong>of</strong> genetic polymorphism among culture-positive isolates <strong>of</strong> Map used in USDA’s laboratory certification testing. INTRODUCTION Mycobacterium avium subspecies paratuberculosis is <strong>the</strong>orized to have evolved from Mycobacterium avium subsp. avium (Ma) (Frothingham, 1999; Turenne et al., 2007). Map and Ma, by genetic criteria, are classified as subsets <strong>of</strong> <strong>the</strong> same species (Harris and Barletta, 2001; Thorel et al., 1990). Research on diagnosis and epidemiological findings relative to Map has <strong>of</strong>ten denied <strong>the</strong> existence <strong>of</strong> closely related Map phenotypic variants more closely related to MA (Cousins et al., 1999). Some mycobacteria, more Ma-like than Map-like, contain <strong>the</strong> IS900 insertion sequence (Bolske and Johansson, 2002; Cousins et al., 1999; England et al., 2002). IS1311 is present in <strong>the</strong> vast majority <strong>of</strong> pathogenic mycobacterium. A long evolutionary time span is suggested by <strong>the</strong> presence <strong>of</strong> mutations in some <strong>of</strong> <strong>the</strong> IS1311 elements (Whittington et al., 1998). Genomic polymorphism is to be anticipated within species evolution. IS1311 is present in Ma and Map (9). Primers based upon <strong>the</strong> IS1311 insertion sequence that identify Ma variants and Map are encompassed in <strong>the</strong> direct and nested fecal FecaMap® patented primers. The purpose <strong>of</strong> this study was to compare <strong>the</strong> respective abilities <strong>of</strong> IS1311- and IS900-based PCR primers for identifying Map isolates within three USDA Laboratory Certification Tests. MATERIALS AND METHODS Population studied: The fecal samples were obtained from two dairy herds that participated in <strong>the</strong> Florida Johne’s Disease Dairy Herd Prevention Program. The number <strong>of</strong> fecal samples analyzed was determined by <strong>the</strong> number in which nested PCR data was available from <strong>the</strong> Diagnostic Laboratory <strong>of</strong> <strong>the</strong> Department <strong>of</strong> Infectious Diseases, University <strong>of</strong> Florida College <strong>of</strong> Veterinary Medicine. Fecal culturing: The fecal culture testing using <strong>the</strong> Trek® Diagnostic System was done at Purdue University School <strong>of</strong> Veterinary Medicine in accordance with <strong>the</strong> manufacturer’s instructions. Real-time Map PCR testing: The direct fecal PCR testing using <strong>the</strong> Tetracore® Map Diagnostic System were done at Purdue University School <strong>of</strong> Veterinary Medicine in accordance with <strong>the</strong> manufacturer’s instructions. 44
Page 1 and 2: Book of Abstracts
Page 4 and 5: Proceedings <stron
Page 6 and 7: Grant Support and Sponsorship <stro
Page 8 and 9: Scott Wells - Planning Committee Ch
Page 10 and 11: Monday, August 10, 2009 - All sessi
Page 12 and 13: Kari Røste Lybeck, Anne Kristine S
Page 14 and 15: 1120-1140 #88: Influence of
Page 16 and 17: Concurrent Session B - Rm 175 Wille
Page 18 and 19: Thursday, August 13 Thursday, Augus
Page 20: Diagnostics and Genotyping Convenor
Page 23 and 24: #229 Detecting Johne’s Disease he
Page 25 and 26: Kalis CHJ, Hesselink JW, Barkema HW
Page 27 and 28: MATERIALS AND METHODS Sampling For
Page 29 and 30: To better determine the</st
Page 31 and 32: #107 Multilocus Short Sequence Repe
Page 33 and 34: Fig. 1. Dendogram (showing genetic
Page 35 and 36: #105 MIRU-VNTR genotyping o
Page 37: Diagnostics and Genotyping Poster A
Page 40 and 41: Missouri). Serum from a cow with do
Page 42 and 43: #7 Significance of
Page 46 and 47: Direct fecal nested Map PCR testing
Page 48 and 49: #16 Histopathological and immunohis
Page 50 and 51: #19 Development of
Page 52 and 53: #36 Genetic diversity of</s
Page 54 and 55: #60 Evaluation of
Page 56 and 57: #66 Comparison of
Page 58 and 59: (b) IS900 Nested PCR, using p90-p91
Page 60 and 61: (d) f57 Real Time PCR. The figure o
Page 62 and 63: • MIRU (3 loci); • VNTR-MIRU (7
Page 64 and 65: indexes, compared to each single an
Page 66 and 67: #108 Evaluation of
Page 68 and 69: #110 Detection and molecular confir
Page 70 and 71: endoscopic criteria and histopathol
Page 72 and 73: Bull TJ, McMinn EJ, Sidi-Boumedine
Page 74 and 75: #131 Seroprevalence of</str
Page 76 and 77: could be partly attributed to a sma
Page 78 and 79: #146 Analysis of v
Page 84 and 85: #191 Potentiating day-old blood sam
Page 86 and 87: samples from the s
Page 88 and 89: (MAA) (MAP87 and MAP3776) and 1 fro
Page 90 and 91: #196 Inter- and intra-subtype genot
Page 92 and 93: RESULTS In 601 (29%) of</st
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#211 Culture of my
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#242 Application and field validati
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#251 Diagnosis of
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Table 2. Paratuberculosis PCR resul
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#254 Programmes on Paratuberculosis
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Fig. 1. European countries specifyi
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A bulk milk quality assurance progr
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Republic of Lithua
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#276 Elimination of</strong
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other projects and
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Woodbine KA, Schukken YH, Green LE,
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Host Response and Immunology Keynot
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#33 MERKAL AWARD LECTURE No interfe
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#75 Interleukin 17 and interleukin
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#173 Local and systemic roles for b
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#23 Role of Mycoba
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Host Response and Immunology Poster
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#28 Intestinal MAP Infection via Pe
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#73 Age susceptibility of</
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#95 Role of nitric
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#134 Analysis of <
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#157 Study of <str
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#163 Immunohistochemical expression
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#193 Development of</strong
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#209 Murine macrophages carrying an
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#224 Application of</strong
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Table 1 UK and Cyprus MAP survey Re
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#228 Mycobacterium avium ssp. parat
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#145 MERKAL AWARD LECTURE Multinomi
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#50 Assessment of
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Table 1. Posterior median (CrIs) <s
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#55 Birth clusters of</stro
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from their dam, se
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#88 Influence of b
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#104 Relation between faecal shedde
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Epidemiology Poster Abstracts 109 1
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#22 The Prevalence of</stro
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CONCLUSION IS1311-based nested Ma/M
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#42 Seroprevalence survey o
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#77 Characterisation of</st
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Environmental samples were collecte
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REFERENCES 1) Chiodini RJ, Van Krui
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#113 Effect of soi
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#136 Error-adjusted, variance parti
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Latium Region Viterbo districts RES
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#144 The suitability of</st
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#178 Age structure of</stro
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A statistically significant lower f
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#183 Sero-prevalence of</st
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#218 Johne’s disease in t
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#244 A survey to estimate t
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Control Programs Keynote Lecture Re
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#202 Milk quality assurance for par
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#141 Control of pa
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#198 Use of small
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Control Programs Poster Abstracts 1
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RESULTS Loss of al
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#40 Managing Bovine Johnes Disease
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#74 Economic analysis of</s
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#87 Evaluation of
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separately for each existing herd <
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#98 An inter-laboratory ring-trial
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#120 Paratuberculosis vaccine inter
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The statement provides: • A stand
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COMPLEMENTARY ASPECTS On-Farm Disea
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#168 Paratuberculosis in Iran: past
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#180 Behavioural change in owners <
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the fact that <str
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#182 An integrated risk based appro
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etween 0 to 10, depending on <stron
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#203 Milk quality assurance for par
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#234 Johne’s disease vaccine: a c
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Pathogenomics and MAP Biology Conve
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#45 Identification of</stro
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#117 Adsorption of
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#222 Atypical structural features <
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Pathogenomics and MAP Biology Persp
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247
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#52 Is ‘Indian Bison Type’ Myco
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#69 In vitro susceptibility <strong
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#118 Cloning, expression and purifi
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#176 A reference proteomic pr<stron
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#213 Construction the</stro
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#230 Iron concentration dependent s
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Abstract not available at press tim
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#174 Sharing of
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#177 Associations between CARD15 po
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Perspectives Talk: Public Health My
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273
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A total of 206 qua
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Feller, M., K. Huwiler, R. Stephan,
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In 2008, the Ameri
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#115 Crohn’s disease and ruminant
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International John
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#76 Towards improved reporting stan
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#97 The EU ParaTBTools Project - Th
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#241 US Vaccine Initiative through
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Paratuberculosis ELISA Reliable, si
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PRIONICS AG WAGISTRASSE 27A PHONE +
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