VAAM-Jahrestagung 2012 18.–21. März in Tübingen

154OTP074Comparison of Faecal Culture and Real-Time QuantitativePCR Methods for Detection of Mycobacterium avium subsp.paratuberculosis in Bovine Faecal SamplesA.A. Hassan* 1 , H. van Weering 1 , A. Heuvelink 1 , M. Zschöck 2 , î Akineden 31 GD-Animal Health Service, Bacteriology, Deventer, Netherlands2 Landesbetrieb Hessisches Landeslabor, Bacteriology, Giessen, Germany3 Oemer.Akineden@vetmed.uni-giessen.de, Professur fürMilchwissenschaften, Institut für Tierärztliche Nahrungsmittelkunde,Giessen, GermanyMycobacterium avium subsp. paratuberculosis (MAP) is a robustmicroorganism, which causes incurable chronic enteritis in cattle. Thepresent study compared the efficacy of two different faecal cultureprocedures and Taq-Man PCR assay (Applied Biosystem) for detection ofMAP in faecal samples. Sixty one faecal samples were collected from twoDutch cattle herds (n=40, and n=21, respectively) which are known to beMAP positive. For culturing, all individual samples were decontaminatedusing 0.75% HPC and cultured on HEYM agar (Harold’s Egg YolkMedium containing Mycobactin J and AVN, Becton Dickinson). Thesecond cultural method in sequentially two decontamination steps used 4%NaOH and malachite green-oxalic acid cultured on HEYM agar and on LJagar (modified Löwenstein-Jensen media contain Mycobactin J). For theTaq-Man real-time PCR method, all faecal samples were tested in twodifferent laboratories using the same PCR kit. The sensitivity of the twocultural methods were 1.6% (n=1/61), 4.9% (n=3/61) and 8.2% (5/61) ofHEYM/ 0.75% HPC; HEYM/ 4% NaOH/malachite green-oxalic acid andLJ/ 4% NaOH/malachite green-oxalic acid, respectively. The sensitivity ofthe Taq-Man real-time PCR in two different laboratories were 13.1%(n=8/61) and 16.4% (n=10/61). The results revealed that cultural methodusing LJ/ 4% NaOH/malachite green-oxalic acid is more sensitive thanothers and the Taq Man PCR assay had higher specificity than the culturalmethods. The results showed a significant deference between Taq-Manreal-time PCR assay and two cultural methods. In conclusion, Taq-Manreal-time PCR on bovine faecal samples is a fast reliable method and couldbe applied in routine screening of MAP, leading to the improvement of theefficiency of MAP control strategies.OTP075Multilocus Sequence Typing (MLST) for the infra-generictaxonomic classification of entomopathogenic RickettsiellabacteriaA. Leclerque* 1 , K. Hartelt 2 , C. Schuster 1 , K. Jung 1 , R.G. Kleepies 11 Julius Kühn-Institut (JKI), Institut für Biologischen Pflanzenschutz,Darmstadt, Germany2 Landesgesundheitsamt Baden-Württemberg, Ref. 93, MRE-Netzwerk ,Suttgart, GermanyThe taxonomic genus Rickettsiella comprises intracellular bacteriaassociated with a wide range of arthropods that are currently classified infour recognized species - namely the nomenclatural type species,Rickettsiella popilliae (Dutky & Gooden), as well as Rickettsiella grylli(Vago & Martoja), Rickettsiella chironomi (Weiser), and Rickettsiellastethorae (Hall & Badgley) - and numerous further pathotypes. Both thedelineation of species and the synonymization of pathotypes with speciesare highly problematic.In the sequel of a previous phylogenomic study at the supra-generic level,nine selected genes - the 16S and 23S rRNA genes and the proteinencodinggenes dnaG, ftsY, gidA, ksgA, rpoB, rpsA, and sucB - wereevaluated for their potential as markers for the generic and infra-generictaxonomic classification of Rickettsiella-like bacteria. A methodologicalapproach combining phylogenetic reconstruction with likelihood-basedsignificance testing was employed on the basis of sequence data from theRickettsiella popilliae - synonymized pathotypes `Rickettsiellamelolonthae’ and `Rickettsiella tipulae´ as well as the species R. grylli.The study identified two genetic markers, gidA and sucB, for MLSTanalysis within the bacterial genus Rickettsiella. In contrast, rpsA and ftsYgene sequences were found to be sufficiently phylogeny-informative toproduce a significant genus-level classification of Rickettsiella-likebacteria. Both the gidA and sucB genes were shown to be highlyphylogeny informative at the infra-generic taxonomic level and have beensubject to functional selection as concluded from their non-synonymous :synonymous site substitution frequencies (d N/d S) of 0.21 and 0.31,respectively. Moreover, being located at a distance of 570 kbp from eachother in the R. grylli genome (app. 1.5 Mbp), the simultaneous use of bothmarkers will make it likely that possible LGT events will not have affectedboth genes at a time. In particular, on the basis of the above analysis andwithin the range of infra-generic diversity covered by the present study,these markers’ reliability and resolution potential for taxonomic studieswithin the genus Rickettsiella appear higher than those of thecorresponding 16S rRNA-encoding sequences.Reference: Leclerque A, Hartelt K, Schuster C, Jung K, Kleespies RG(2011) Multilocus sequence typing (MLST) for the infra-generictaxonomic classification of entomopathogenic Rickettsiella bacteria.FEMS Microbiology Letters 324:125-134.OTP076Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) forSpecies Identification of Bacteria of Genera Arcanobacteriumand TrueperellaM. Hijazin 1 , J. Alber 1 , C. Lämmler 1 , A.A. Hassan 2 , M. Timke 3 ,M. Kostrzewa* 3 , E. Prenger-Berninghoff 4 , M. Zschöck 51 Justus-Liebig-Universität Gießen, Institut für Pharmakologie und Toxikologie,Gießen, Germany2 De Gezondheidsdienst voor Dieren (Animal Health Service), Deventer, TheNetherlands, Netherlands3 Bruker Daltonik GmbH, Entwicklung Bioanalyse, Bremen, Germany4 Justus-Liebig-Universität Gießen, Institut für Hygiene undInfektionskrankheiten der Tiere, Gießen, Germany5 Landesbetrieb Hessisches Landeslabor, Gießen, GermanyGenus Arcanobacterium (A.) consisted of nine species, it was split in twodistinct phylogenetic lineages Arcanobacterium and Trueperella (T.) in2011. Species A. phocae, A. pluranimalium, A. hippocoleae, T. pyogenes,T. bonasi, T. bialowiezensis and T. abortisuis were mainly recovered frominfections of various animals and A. haemolyticum and T. bernardiaegenerally cause diseases in humans. In the present study Matrix-AssistedLaser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) was evaluated to identify 121 isolates and 11 reference strains ofgenus Arcanobacterium and genus Trueperella. All 121 isolates wererecovered from different animals and previously classified phenotypicallyand genotypically to six species of both genera. Species identification byMALDI-TOF MS was carried out by comparing the main spectrum of eachisolate with the main spectra of 11 Arcanobacterium or Trueperellareference strains obtained in the present study and 3740 database entriesincluded in the MALDI Biotyper 2.0 software package (version 3.1.1.0)(Bruker Daltonik GmbH, Bremen, Germany). MALDI-TOF MS correctlyidentified (log (score) values 2.0) 22 of 23 T. abortisuis isolates and allinvestigated isolates of the species A. haemolyticum (n= 10), A.pluranimalium (n = 1), T. bialowiezensis (n = 3), T. bonasi (n = 7), and T.pyogenes (n = 77). According to the present results MALDI-TOF MS is apromising tool for fast and reliable identification of species ofArcanobacterium and Trueperella. Further studies with additional isolates,also including Arcanobacterium and Trueperella species commonlyrelated with human infections, would underline the robustness of MALDI-TOF MS for identification of bacteria of both genera.OTP077Will not be presented!OTP078Identification of Campylobacter Species from Zoo Animals byMatrix-Assisted Laser Desorption Ionization-Time of FlightMass SpectrometryA.A. Hassan 1 , A. Heuvelink 1 , E. van Engelen 1 , M. Hijazin 2 , C. Lämmler 2 ,M. Zschöck 3 , M. Kostrzewa* 4 , M. Timke 41 De Gezondheidsdienst voor Dieren (Animal Health Service), Deventer,The Netherlands, Netherlands2 Justus-Liebig-Universität Gießen, Institut für Pharmakologie undToxikologie, Gießen, Germany3 Landesbetrieb Hessisches Landeslabor, Gießen, Germany4 Bruker Daltonik GmbH, Entwicklung Bioanalyse, Bremen, GermanyThe identification of genus Campylobacter at species level in routinediagnostic laboratories using conventional methods is still problematic dueto their poor biochemical activity. In this study, a total of 32 faecalsamples from 32 wild animals were examined during routinemicrobiological diagnosis. Five isolates were suspected Campylobacterstrains isolated from five animals (monkey, trumpeter swan, leopard andtwo meerkats). For species identification, Matrix-Assisted LaserDesorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOFMS) and DNA sequencing techniques were used. Two Campylobacterupsaliensis affiliated and two Campylobacter jejuni strains were identified.MALDI Biotyper software resulted in no reliable identification for isolateOV50-1. Indeed, this isolate may represent a new species of the genusCampylobacter. Partial 16S rRNA gene sequence similarity was only97.7% to C. upsaliensis, the best match of GenBank database comparison.This underlines that there are no false-positive identification results byMALDI Biotyper software. According to the present results MALDI-TOFMS is a fast and reliable method for identification of bacteria of genusCampylobacter at the species level in routine diagnostic laboratories andmight help to elucidate the role of Campylobacter in infections even ofexotic species.BIOspektrum | Tagungsband 2012