Proceedings of the 10th International Colloquium on Paratuberculosis

More documents

Recommendations

Info

(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
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 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 86 and 87: samples from the s
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 <
Page 136 and 137: #157 Study of <str
Page 138 and 139:
#163 Immunohistochemical expression
Page 140 and 141:
#193 Development of</strong
Page 142 and 143:
#209 Murine macrophages carrying an
Page 144 and 145:
#224 Application of</strong
Page 146 and 147:
Table 1 UK and Cyprus MAP survey Re
Page 148 and 149:
#228 Mycobacterium avium ssp. parat
Page 150 and 151:
#145 MERKAL AWARD LECTURE Multinomi
Page 152 and 153:
#50 Assessment of
Page 154 and 155:
Table 1. Posterior median (CrIs) <s
Page 156 and 157:
#55 Birth clusters of</stro
Page 158 and 159:
from their dam, se
Page 160 and 161:
#88 Influence of b
Page 162 and 163:
#104 Relation between faecal shedde
Page 164 and 165:
Epidemiology Poster Abstracts 109 1
Page 166 and 167:
#22 The Prevalence of</stro
Page 168 and 169:
CONCLUSION IS1311-based nested Ma/M
Page 170 and 171:
#42 Seroprevalence survey o
Page 172 and 173:
#77 Characterisation of</st
Page 174 and 175:
Environmental samples were collecte
Page 176 and 177:
REFERENCES 1) Chiodini RJ, Van Krui
Page 178 and 179:
#113 Effect of soi
Page 180 and 181:
#136 Error-adjusted, variance parti
Page 182 and 183:
Latium Region Viterbo districts RES
Page 184 and 185:
#144 The suitability of</st
Page 186 and 187:
#178 Age structure of</stro
Page 188 and 189:
A statistically significant lower f
Page 190 and 191:
#183 Sero-prevalence of</st
Page 192 and 193:
#218 Johne’s disease in t
Page 194 and 195:
#244 A survey to estimate t
Page 196 and 197:
Control Programs Keynote Lecture Re
Page 198 and 199:
#202 Milk quality assurance for par
Page 200 and 201:
#141 Control of pa
Page 202 and 203:
#198 Use of small
Page 204 and 205:
Control Programs Poster Abstracts 1
Page 206 and 207:
RESULTS Loss of al
Page 208 and 209:
#40 Managing Bovine Johnes Disease
Page 210 and 211:
#74 Economic analysis of</s
Page 212 and 213:
#87 Evaluation of
Page 214 and 215:
separately for each existing herd <
Page 216 and 217:
#98 An inter-laboratory ring-trial
Page 218 and 219:
#120 Paratuberculosis vaccine inter
Page 220 and 221:
The statement provides: • A stand
Page 222 and 223:
COMPLEMENTARY ASPECTS On-Farm Disea
Page 224 and 225:
#168 Paratuberculosis in Iran: past
Page 226 and 227:
#180 Behavioural change in owners <
Page 228 and 229:
the fact that <str
Page 230 and 231:
#182 An integrated risk based appro
Page 232 and 233:
etween 0 to 10, depending on <stron
Page 234 and 235:
#203 Milk quality assurance for par
Page 236 and 237:
#234 Johne’s disease vaccine: a c
Page 238 and 239:
Pathogenomics and MAP Biology Conve
Page 240 and 241:
#45 Identification of</stro
Page 242 and 243:
#117 Adsorption of
Page 244 and 245:
#222 Atypical structural features <
Page 246 and 247:
Pathogenomics and MAP Biology Persp
Page 248 and 249:
247
Page 250 and 251:
#52 Is ‘Indian Bison Type’ Myco
Page 252 and 253:
#69 In vitro susceptibility <strong
Page 254 and 255:
#118 Cloning, expression and purifi
Page 256 and 257:
#176 A reference proteomic pr<stron
Page 258 and 259:
#213 Construction the</stro
Page 260 and 261:
#230 Iron concentration dependent s
Page 262 and 263:
261
Page 264 and 265:
263
Page 266 and 267:
Abstract not available at press tim
Page 268 and 269:
#174 Sharing of
Page 270 and 271:
#177 Associations between CARD15 po
Page 272 and 273:
Perspectives Talk: Public Health My
Page 274 and 275:
273
Page 276 and 277:
A total of 206 qua
Page 278 and 279:
Feller, M., K. Huwiler, R. Stephan,
Page 280 and 281:
In 2008, the Ameri
Page 282 and 283:
#115 Crohn’s disease and ruminant
Page 284 and 285:
International John
Page 286 and 287:
#76 Towards improved reporting stan
Page 288 and 289:
#97 The EU ParaTBTools Project - Th
Page 290 and 291:
#241 US Vaccine Initiative through
Page 292 and 293:
Paratuberculosis ELISA Reliable, si
Page 294 and 295:
PRIONICS AG WAGISTRASSE 27A PHONE +
show all

Proceedings of the 10th International Colloquium on Paratuberculosis

Create successful ePaper yourself

Delete template?

Save as template?