Proceedings of the 10th International Colloquium on Paratuberculosis

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Table 2. Paratuberculosis PCR results <strong>of</strong> herds from a dairy region in Colombia Nested PCR a Real-Time PCR b Herd District Elisa A- positive positive negative positive negative 1 A 3 1 2 1 2 2 B 0 nd nd nd nd 3 C 3 2 1 0 3 4 1 0 1 0 1 5 6 D 0 3 nd 0 nd 3 nd 0 nd 3 7 1 0 1 0 1 8 E 4 1 3 0 4 9 6 1 5 0 6 10 F 5 0 5 1 4 11 G 1 0 1 0 1 12 0 nd nd nd nd 13 H 4 0 4 0 4 14 I 0 nd nd nd nd 31 5 26 2 29 a Performed only to positive samples with Elisa A b Performed only to positive samples with Elisa A c Not determined DISCUSSION Our preliminary hypo<strong>the</strong>sis was <strong>the</strong> diagnosis <strong>of</strong> paratuberculosis in a higher proportion <strong>of</strong> animals by using <strong>of</strong> indirect and direct methods. In <strong>the</strong> same way it was expected a higher concordance between <strong>the</strong> different tests used. However confirmation <strong>of</strong> all ELISA Aseropositive animals with ELISA B was not achieved. Differences in antigens LAM in ELISA A and protoplasmic antigen in ELISA B is a possible explanation <strong>of</strong> <strong>the</strong> poor concordance between both tests. In addition <strong>the</strong> use <strong>of</strong> <strong>the</strong> pre-absorption phase in <strong>the</strong> ELISA B could reduce <strong>the</strong> number <strong>of</strong> false positives, but also <strong>the</strong> detection <strong>of</strong> true positives (McKenna et al., 2005). On <strong>the</strong> o<strong>the</strong>r hand, a cross-reaction with o<strong>the</strong>r mycobacteria could have played an important role in <strong>the</strong> positive results obtained with <strong>the</strong> ELISA A. The capacity <strong>of</strong> o<strong>the</strong>r mycobacteria to interfere with pre-absorbed ELISA serological test in cattle has been demonstrated (Muskens et al., 2007). The poor agreement between PCR and serology contradicted <strong>the</strong> proposed idea that <strong>the</strong> combined use <strong>of</strong> ELISA and fecal PCR has <strong>the</strong> potential to increase <strong>the</strong> overall sensitivity for <strong>the</strong> diagnosis <strong>of</strong> paratuberculosis infection in dairy cows. The low shedding rate <strong>of</strong> MAP organisms in subclinical animals can make real time-PCR equal or less sensitive than ELISA (Wells et al., 2006). On <strong>the</strong> o<strong>the</strong>r hand, although some primers use in <strong>the</strong> PCR systems have shown adequate results (e.g. F57), o<strong>the</strong>rs have shown limitations (e.g. ISMav2). In addition, single-round PCR has been evaluated as very reliable, but <strong>the</strong> used <strong>of</strong> nested PCR assays could lead to high contaminations risks (Mobius et al., 2008). Regarding <strong>the</strong> cultural results, negative samples from ELISA-positive herds could be explained by <strong>the</strong> low or still absent MAP shedding rate in seropositive animals, by <strong>the</strong> low sensitivity <strong>of</strong> culture or by <strong>the</strong> culture <strong>of</strong> samples from false positive animals in ELISA A. In fact, in herds without previous diagnosis or clinical cases <strong>of</strong> paratuberculosis, <strong>the</strong> positive predictive value <strong>of</strong> an ELISA test is expected to be low (Muskens et al., 2003). ELISA can predict better <strong>the</strong> presence <strong>of</strong> culture positive animals when <strong>the</strong>se are heavy shedders. The sensitivity <strong>of</strong> <strong>the</strong> ELISA is higher when a heavy instead a low shedder is tested. Negative results or false positive results in a cross-sectional study have a low probability <strong>of</strong> deliver a positive culture, if just a single sampled is planned (Sweeney et al., 2006). Finally <strong>the</strong> conservation <strong>of</strong> serum and fecal samples at -20°C for several weeks could have affected ELISA and culture results. Serum-ELISA results can change from positive to negative (Alinovi et al., 2009) and fecal samples can undergo a significantly reduction <strong>of</strong> 99
viable microorganisms, changing <strong>the</strong> status from positive to negative, in animals with a extremely low number <strong>of</strong> detectable MAP organisms (Khare et al., 2008). CONCLUSION Results confirm <strong>the</strong> presence <strong>of</strong> paratuberculosis in dairy herds in Colombia and <strong>the</strong> limitations <strong>of</strong> <strong>the</strong> currently available diagnostic tests for <strong>the</strong> detection <strong>of</strong> subclinical paratuberculosis infections in dairy cattle. They demonstrate <strong>the</strong> determinant influence <strong>of</strong> <strong>the</strong> ELISA type used, <strong>the</strong> low bacterial shedding on <strong>the</strong> feces <strong>of</strong> <strong>the</strong> examined cattle, <strong>the</strong> roll <strong>of</strong> o<strong>the</strong>r mycobacteria and <strong>the</strong> possible effect <strong>of</strong> conservation on <strong>the</strong> diagnosis <strong>of</strong> paratuberculosis. Fur<strong>the</strong>r paratuberculosis research in Colombia is needed. ACKNOWLEDGMENTS This study was partially financed by <strong>the</strong> German Academic Exchange Service (DAAD), Colciencias and Ministerio de Educación Nacional de Colombia. Authors would like to thank Michael Palacio, Ferney Agudelo and Julian Reyes for <strong>the</strong> technical support in Colombia. REFERENCES Alinovi CA, Ward MP, Lin TL, Wu CC, 2009. Sample handling substantially affects Johne's ELISA. Prev Vet Med, 90, 278-283. Bull TJ, McMinn EJ, Sidi-Boumedine K, Skull A, Durkin D, Neild P, Rhodes G, Pickup R, Hermon-Taylor J, 2003. Detection and verification <strong>of</strong> Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease. J Clin Microbiol, 41, 2915-2923. Khare S, Adams LG, Osterstock J, Roussel A, David L, 2008. Effects <strong>of</strong> shipping and storage conditions <strong>of</strong> fecal samples on viability <strong>of</strong> Mycobacterium paratuberculosis. J Clin Microbiol, 46, 1561-1562. McKenna S L, Keefe GP, Barkema HW, Sockett DC, 2005. Evaluation <strong>of</strong> three ELISAs for Mycobacterium avium subsp. paratuberculosis using tissue and fecal culture as comparison standards. Vet Microbiol, 110, 105-111. Mobius P, Hotzel H, Rassbach A, Kohler H, 2008. Comparison <strong>of</strong> 13 single-round and nested PCR assays targeting IS900, ISMav2, f57 and locus 255 for detection <strong>of</strong> Mycobacterium avium subsp. paratuberculosis. Vet Microbiol, 126, 324-333. Muskens J, Mars MH, Elbers AR, van Maanen K, Bakker D, 2003. The results <strong>of</strong> using faecal culture as confirmation test <strong>of</strong> paratuberculosis-seropositive dairy cattle. J Vet Med B Infect Dis Vet Public Health, 50, 231-234. Osterstock JB, Fosgate GT, Norby B, Manning EJ, Collins MT, Roussel AJ, 2007. Contribution <strong>of</strong> environmental mycobacteria to false-positive serum ELISA results for paratuberculosis. J Am Vet Med Assoc, 230, 896-901. Schonenbrucher H, Abdulmawjood A, Failing K, Bulte M, 2008. New triplex real-time PCR assay for detection <strong>of</strong> Mycobacterium avium subsp. paratuberculosis in bovine feces. Appl Environ Microbiol, 74, 2751-2758. Sweeney R W, Whitlock RH, McAdams S, Fyock T, 2006. Longitudinal study <strong>of</strong> ELISA seroreactivity to Mycobacterium avium subsp. paratuberculosis in infected cattle and culture-negative herd mates. J Vet Diagn Invest, 18, 2-6. Wells SJ, Collins MT, Faaberg KS, Wees C, Tavornpanich S, Petrini KR, Collins JE, Cernicchiaro N, Whitlock RH, 2006. Evaluation <strong>of</strong> a rapid fecal PCR test for detection <strong>of</strong> Mycobacterium avium subsp. paratuberculosis in dairy cattle. Clin Vaccine Immunol, 13, 1125-1130. 100
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Book of Abstracts
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Proceedings <stron
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Grant Support and Sponsorship <stro
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Scott Wells - Planning Committee Ch
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Monday, August 10, 2009 - All sessi
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Kari Røste Lybeck, Anne Kristine S
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1120-1140 #88: Influence of
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Concurrent Session B - Rm 175 Wille
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Thursday, August 13 Thursday, Augus
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Diagnostics and Genotyping Convenor
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#229 Detecting Johne’s Disease he
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Kalis CHJ, Hesselink JW, Barkema HW
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MATERIALS AND METHODS Sampling For
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To better determine the</st
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#107 Multilocus Short Sequence Repe
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Fig. 1. Dendogram (showing genetic
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#105 MIRU-VNTR genotyping o
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Diagnostics and Genotyping Poster A
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Missouri). Serum from a cow with do
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#7 Significance of
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RESULTS Of the 327
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Direct fecal nested Map PCR testing
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#16 Histopathological and immunohis
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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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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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the fact that <str
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etween 0 to 10, depending on <stron
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#234 Johne’s disease vaccine: a c
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Pathogenomics and MAP Biology Conve
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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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#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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#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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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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