Proceedings of the 10th International Colloquium on Paratuberculosis

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(MAA) (MAP87 and MAP3776) and 1 from an immunological hot spot region (MAP3783). Following overnight culture, <strong>the</strong> culture plates were centrifuged and <strong>the</strong> supernatants collected and stored at -20ºC until fur<strong>the</strong>r analysis. The antigen specific IFN-� production in supernatants were determined by an in-house ELISA as described elsewhere (Mikkelsen et al., 2009). The level <strong>of</strong> IFN-� (pg/ml) was calculated using linear regression on log-log transformed readings from <strong>the</strong> two-fold dilution series <strong>of</strong> a reference standard with known IFN-� concentration. Samples were excluded if <strong>the</strong> IFN-� value in <strong>the</strong> SEB-stimulated sample was below 1500 pg/ml or <strong>the</strong> PBS-stimulated sample was higher than 250 pg/ml. Based on <strong>the</strong>se exclusion criteria, one heifer was excluded at first and second sampling and two heifers were excluded at <strong>the</strong> third sampling. To determine <strong>the</strong> cut-<strong>of</strong>f for each <strong>of</strong> <strong>the</strong> 14 novel antigens and PPDj, blood samples were collected from 60 heifers 15-24 months <strong>of</strong> age from a non-infected herd. Samples from <strong>the</strong> non-infected herd heifer were excluded, if <strong>the</strong> IFN-� value in SEB-stimulated sample was below 1500 pg/ml or <strong>the</strong> PBS-stimulated sample was higher than 75 pg/ml. The remaining samples were used to calculate <strong>the</strong> cut-<strong>of</strong>f for each antigen. The cut-<strong>of</strong>f for each antigen was calculated as: mean IFN-� response + (1.96 � standard deviation <strong>of</strong> IFN-� response). RESULTS Figure 1 shows <strong>the</strong> percentage <strong>of</strong> positive calves detected by each antigen at <strong>the</strong> three sampling dates. e v i t i s o p t n e c r e P 90 80 70 60 50 40 30 20 10 0 90 80 70 60 50 40 30 0 ESAT-6 family members Sampling 1 Sampling 2 Sampling 3 20 Percent 10 positive calves Secreted proteins Sampling 1 Sampling 2 Sampling 3 MAP160 esxH esxK esxU MAP217 MAP1662 MAP2888 90 80 70 60 50 40 30 20 Percent 10 positive calves 0 90 80 70 60 50 40 30 20 0 Latency proteins Sampling 1 Sampling 2 Sampling 3 Percent 10 positive calves O<strong>the</strong>r antigens Sampling 1 Sampling 2 Sampling 3 MAP2487c MAP2768c 6 MAP3273c 7 MAP3701c 8 7 P 3 A P M A M , A , A MA M t nt en se e s re pr p t oImm hot spot, MAP3783 t No N Ag85b, MAP1609 PPDj Figure 1. Percent positive calves detected by each antigen by <strong>the</strong> IFN-� test. The novel antigens tested were divided into four groups: ESAT-6 family members, latency proteins, secreted proteins and o<strong>the</strong>r antigens. Cut<strong>of</strong>f for each antigen, to distinguish between test-positives and test-negatives, was calculated based on results from a negative herd. The same 30 heifers were tested at <strong>the</strong> three samplings. Data presented here are from 29 heifers at sampling 1 and 2, and 28 heifers at sampling 3, after exclusion <strong>of</strong> invalid samples. 87 2
PPDj detected 50% <strong>of</strong> <strong>the</strong> heifers as MAP positives at <strong>the</strong> first sampling, 60% at <strong>the</strong> second sampling and less than 20% on <strong>the</strong> last sampling date. In comparison <strong>the</strong> antigen 85B (Ag85B), which share high homology with o<strong>the</strong>r mycobacteria such as MAA, detected 50% as positive on <strong>the</strong> first sampling and 60% as positive on <strong>the</strong> second and third sampling. In general, <strong>the</strong> antigens detected <strong>the</strong> highest percentage <strong>of</strong> heifers as positives on <strong>the</strong> second and third sampling with exception <strong>of</strong> PPDj. The groups <strong>of</strong> latency proteins and secreted proteins detected 50% to 60% as positive on <strong>the</strong> second and third sampling. Similarly, <strong>the</strong> three ESAT-6 family peptides (MAP160, esxH, esxU) detected 50% to 60% as positive on <strong>the</strong> second and third sampling. The two protein antigens selected as not present in MAA, detected 30% to 50% <strong>of</strong> <strong>the</strong> heifers as positives, whereas <strong>the</strong> protein antigen selected from an immunological hot spot region detected 30% to 40% <strong>of</strong> <strong>the</strong> heifers as positive. DISCUSSION AND CONCLUSION The IFN-� responses fluctuated between <strong>the</strong> three sampling dates, which indicate <strong>the</strong> importance <strong>of</strong> repeated test for evaluation <strong>of</strong> novel antigen performance in <strong>the</strong> IFN-� test. Surprisingly, PPDj detected less than 20% as positive on <strong>the</strong> third sampling, but 50% to 60% at <strong>the</strong> first two sampling dates. For <strong>the</strong> majority <strong>of</strong> <strong>the</strong> antigens, <strong>the</strong> highest percentage <strong>of</strong> positive animals was detected at <strong>the</strong> second and third sampling dates. There is no evident explanation for <strong>the</strong> low percentage <strong>of</strong> animals detected by PPDj on <strong>the</strong> third sampling date and this result do not agree with <strong>the</strong> result for <strong>the</strong> majority <strong>of</strong> <strong>the</strong> antigens. On <strong>the</strong> o<strong>the</strong>r hand, <strong>the</strong> observed fluctuations may emphasise <strong>the</strong> need for an alternative to PPDj in <strong>the</strong> IFN-� test. For optimization <strong>of</strong> <strong>the</strong> IFN-� test well-characterised antigens should be included to induce specificity to MAP and reduce cross-reactions to environmental mycobacteria. To obtain high specificity <strong>of</strong> <strong>the</strong> IFN-� test a combination <strong>of</strong> perhaps three novel antigens should be included. The optimal combination <strong>of</strong> novel antigens to be included in a MAP specific IFN-� test remains to be selected. ACKNOWLEDEMENTS Technicians Abdellatif El Ghazi and Sardar Ahmad have done most <strong>of</strong> <strong>the</strong> ELISAs. This study was co-funded by <strong>the</strong> European Commission within <strong>the</strong> Sitxh Framework Programme, as part <strong>of</strong> <strong>the</strong> project ParaTBTools (contract no. 023106 (FOOD)). REFERENCES Cho, D., Collins, M.T., 2006. Comparison <strong>of</strong> <strong>the</strong> proteosomes and antigenicities <strong>of</strong> secreted and cellular proteins produced by Mycobacterium paratuberculosis. Clin. Vaccine Immunol. 13, 1155-1161. Leyten, E.M.S., Lin, M.Y., Franken, K.L.M.C., Friggen, A.H., Prins, C., van Meijgaarden, K.E., Voskuil, M.I., Weldingh, K., Andersen, P., Schoolnik, G.K., Arend, S.M., Ottenh<strong>of</strong>f, T.H.M., Klein, M.R., 2006. Human T-cell responses to 25 novel antigens encoded by genes <strong>of</strong> <strong>the</strong> dormancy regulon <strong>of</strong> Mycobacterium tuberculosis. Microb. Infet. 8, 2052-2060. Mikkelsen, H., Jungersen, G., Nielsen, S.S., 2009. Association between milk antibody and interferon-gamma responses in cattle from Mycobacterium avium subsp. paratuberculosis infected herds. Vet. Immunol. Immunopathol. 127, 235-241. van Pinxteren, L.A.H., Ravn, P., Agger, E.M., Pollock, J., Andersen, P., 2000. Diagnosis <strong>of</strong> tuberculosis based on <strong>the</strong> two specific antigens ESAT-6 and CFP10. Clin. Diagn. Lab. Immunol. 7, 155-160. Wood P.R., Kopsidas K., Milner A.R., Hill J., Gill I., Webb R. et al., 1989. The development <strong>of</strong> an in vitro cellular assay for Johne´s disease in cattle. In :Milner A.R., Wood P.R. (editors), Johne´s Disease: current trends in research, diagnosis and management, 164-167. 88 3
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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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Page 78 and 79: #146 Analysis of v
Page 84 and 85: #191 Potentiating day-old blood sam
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Page 100 and 101: Table 2. Paratuberculosis PCR resul
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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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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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Abstract not available at press tim
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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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