Proceedings of the 10th International Colloquium on Paratuberculosis

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samples from <strong>the</strong> same animals on different sample days; hence repeated testing is required for validation <strong>of</strong> an optimized IFN-� protocol. The fluctuations may be caused by variation in a number <strong>of</strong> factors, such as stress response <strong>of</strong> animals during sample collection, number <strong>of</strong> leukocytes in <strong>the</strong> blood samples or perhaps <strong>the</strong> fluctuations are higher when using a complex antigen mixture such as PPDj compared to well-defined antigens. The variation in IFN-� responses to <strong>the</strong>se novel antigens remains to be fur<strong>the</strong>r investigated and <strong>the</strong> application <strong>of</strong> a dayold protocol using o<strong>the</strong>r antigens needs to be fur<strong>the</strong>r explored Acknowledgements 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 Buza, J.J., Hikono, H., Mori, Y., Nagata, R., Hirayama, S., Bari, A.M., Aodon-geril, Shu, Y.J., Tsuji, N.M., Momotani, E., 2004. Neutralization <strong>of</strong> interleukin-10 significantly enhances gamma interferon expression in peripheral blood by stimulation with Johnin purified protein derivative and by infection with Mycobacterium avium subsp. paratuberculosis in experimentally infected cattle with paratuberculosis. Infect. Immun. 72, 2425-2428. Jungersen G., Grell S.N., Clemensen A., Roust T., Huda A., Howard C.J., 2005. Interleukin- 12 potentiation <strong>of</strong> <strong>the</strong> interferon-gamma test rescues day-old blood samples for diagnosis <strong>of</strong> paratuberculosis PPD specific cellular mediated immune response. In :Manning E.J.B., Nielsen S.S. (editors), <strong>Proceedings</strong> <strong>of</strong> <strong>the</strong> 8th <strong>International</strong> <strong>Colloquium</strong> on Paratuberculosis, Copenhagen, Denmark, August 14 2005-August 17 2005, 501-506. 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. 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. 85 3
#192 Novel antigens used to detect cell-mediated immune responses over time in Mycobacterium avium subsp. paratuberculosis infected cattle Heidi Mikkelsen 1,2 , Claus Aagaard 3 , Søren Saxmose Nielsen 2 , Gregers Jungersen 1 1 National Veterinary Institute, Technical University <strong>of</strong> Denmark, Copenhagen, Denmark; 2 Faculty <strong>of</strong> Life Sciences, University <strong>of</strong> Copenhagen, Frederiksberg, Denmark, 3 State Serum Institute, Copenhagen, Denmark. ABSTRACT Early stage Mycobacterium avium subsp. paratuberculosis (MAP) infection <strong>of</strong> cattle can be detected by measuring specific cell mediated immune responses, using <strong>the</strong> interferon gamma (IFN-�) test. Available IFN-� tests are using purified protein derivatives <strong>of</strong> MAP (PPDj) which are crude products consisting <strong>of</strong> undefined antigens with possible cross reactions toward o<strong>the</strong>r environmental bacteria. The objective <strong>of</strong> <strong>the</strong> study was to optimize <strong>the</strong> IFN-� test using different types <strong>of</strong> novel antigens for stimulation. Fourteen novel antigen candidates were selected for testing, including 4 peptides <strong>of</strong> <strong>the</strong> ESAT-6 family and 10 hypo<strong>the</strong>tical proteins: 4 latency proteins, 3 secreted proteins, 2 proteins not present in Mycobacterium avium subsp. avium (MAA) and 1 from an immunological hot spot region. To determine variation <strong>of</strong> IFN-� responses, three repeated tests was done with 4 and 5 week intervals on <strong>the</strong> same 30 heifers from a known MAP infected herd. Determination <strong>of</strong> cut-<strong>of</strong>f for each antigen was based on samples from a non-infected herd, including 60 heifers. Based on PPDj stimulations, more than 50% <strong>of</strong> <strong>the</strong> heifers tested MAP positive at <strong>the</strong> first two samplings, whereas only 20% tested positive at third sampling. The resulted showed that PPDj detect a high percentage as MAP positive animals, as this crude antigen mixture is expected to induce non-specific IFN-� production. However, <strong>the</strong> tested latency antigens, some secreted proteins and some peptides <strong>of</strong> <strong>the</strong> ESAT-6 family detected a comparable high percentage <strong>of</strong> animals as MAP positives. By combining novel antigens higher specificity might be obtained. INTRODUCTION Early MAP specific cell-mediated immune responses can be measured using <strong>the</strong> IFN-� test (Wood et al., 1989). The IFN-� test is a whole-blood proliferation assay, in which blood is cultured overnight with MAP antigens followed by collection <strong>of</strong> supernatant. The IFN-� level in supernatants is <strong>the</strong>n detected by an IFN-� specific enzyme linked immunosorbent assay (ELISA). Available whole IFN-� tests for MAP diagnosis are using PPDj, which is a crude undefined extract <strong>of</strong> MAP antigens. Lack <strong>of</strong> standardized PPDj is a major concern, as <strong>the</strong> preparation <strong>of</strong> PPDj and <strong>the</strong>refore <strong>the</strong> antigen composition varies between laboratories. In addition, PPDj are known to cross-react with environmental mycobacteria such as MAA leading to low specificity <strong>of</strong> <strong>the</strong> IFN-� test. To induce <strong>the</strong> specificity <strong>of</strong> <strong>the</strong> IFN-� test, well-defined and MAP specific antigens are needed. In this study 14 novel antigen candidates were selected for testing with <strong>the</strong> aim to increase specificity <strong>of</strong> <strong>the</strong> IFN-� test. The novel antigen candidates included peptides <strong>of</strong> <strong>the</strong> ESAT-6 family (van Pinxteren et al., 2000), latency proteins (Leyten et al., 2006), secreted proteins (Cho and Collins, 2006), proteins not present in MAA and a protein from an immunological hot spot region. MATERIALS AND METHODS Blood samples were collected 3 times with 4 and 5 week intervals from <strong>the</strong> same 30 heifers 15-24 months <strong>of</strong> age in a Danish dairy herd known to be infected with MAP. On each sample date <strong>the</strong> whole blood samples incubated for 20-22 hours at 37ºC in 5% CO2 with <strong>the</strong> 14 novel antigens, PPDj, a negative (PBS) and a positive control (Staphylococcal enterotoxin B; SEB) in parallel cultures. The 14 novel antigens candidates included 4 peptides <strong>of</strong> <strong>the</strong> ESAT-6 family (MAP160, esxH, esxK and esxU) and 10 hypo<strong>the</strong>tical proteins: 4 latency proteins (MAP2487c, MAP2768c, MAP3273c, and MAP3701c), 3 secreted proteins (MAP217, MAP1662 and MAP2888) 2 proteins not present in Mycobacterium avium subsp. avium 86 1
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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
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 44 and 45: RESULTS Of the 327
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 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
Page 94 and 95: #211 Culture of my
Page 96 and 97: #242 Application and field validati
Page 98 and 99: #251 Diagnosis of
Page 100 and 101: Table 2. Paratuberculosis PCR resul
Page 102 and 103: #254 Programmes on Paratuberculosis
Page 104 and 105: Fig. 1. European countries specifyi
Page 106 and 107: A bulk milk quality assurance progr
Page 108 and 109: Republic of Lithua
Page 110 and 111: #276 Elimination of</strong
Page 112 and 113: other projects and
Page 114 and 115: Woodbine KA, Schukken YH, Green LE,
Page 116 and 117: Host Response and Immunology Keynot
Page 118 and 119: #33 MERKAL AWARD LECTURE No interfe
Page 120 and 121: #75 Interleukin 17 and interleukin
Page 122 and 123: #173 Local and systemic roles for b
Page 124 and 125: #23 Role of Mycoba
Page 126 and 127: Host Response and Immunology Poster
Page 128 and 129: #28 Intestinal MAP Infection via Pe
Page 130 and 131: #73 Age susceptibility of</
Page 132 and 133: #95 Role of nitric
Page 134 and 135: #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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#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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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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