Proceedings of the 10th International Colloquium on Paratuberculosis

More documents

Recommendations

Info

(d) f57 Real Time PCR. The figure on <strong>the</strong> left shows <strong>the</strong> amplifications <strong>of</strong> <strong>the</strong> positive sample and <strong>the</strong> positive control. The figure on <strong>the</strong> right shows <strong>the</strong> melting analysis results <strong>of</strong> amplicons. Note <strong>the</strong> single peak at 84.6 °C corresponding to <strong>the</strong> single specific amplification product. DNA from MAP ATCC 19698 was used as positive control. Data were in duplicate. Positive control Positive sample CONCLUSIONS In order to compare <strong>the</strong> sensitivity <strong>of</strong> four PCR methods for <strong>the</strong> detection <strong>of</strong> MAP, we tested bulk milk samples derived for presumably infected herds (ELISA positive). As expected <strong>the</strong> most sensitive method was Nested PCR, while conventional PCR and Real Time PCR showed lower sensitivity. Although more sensitive, Nested PCR method remains difficult to apply because is time consuming and <strong>the</strong>re are high risks <strong>of</strong> cross-contamination. REFERENCES 1. Greenstein RJ. 2003. Is Crohn's disease caused by a mycobacterium? Comparisons with leprosy, tuberculosis, and Johne's disease. Lancet Infect Dis.; 3:507-14. 2. Dow TC. 2006. Paratuberculosis and Type I diabetes: is this <strong>the</strong> trigger? Med Hypo<strong>the</strong>ses. 67:782-5. 3. Donaghy JA, Rowe MT, Rademaker JL, Hammer P, Herman L, De Jonghe V, Blanchard B, Duhem K, Vindel E. 2008. An inter-laboratory ring trial for <strong>the</strong> detection and isolation <strong>of</strong> Mycobacterium avium subsp. paratuberculosis from raw milk artificially contaminated with naturally infected faeces. Food Microbiol.;25:128-35. 4. Slana I, Kralik P, Kralova A, Pavlik I. 2008. On-farm spread <strong>of</strong> Mycobacterium avium subsp. paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. Int J Food Microbiol. 10;128:250-7. 5. Cousins, D. V., Whittington R., Marsh I., Masters A., Evans R. J., Kluver P. 1999. Mycobacteria distinct from Mycobacterium avium subsp. paratuberculosis isolated from <strong>the</strong> faeces <strong>of</strong> ruminants possess IS900-like sequences detectable by IS900 polymerase chain reaction: implications for diagnosis. Mol. 9 Cell. Probes 13: 431-442. 6. Taddei R, Barbieri I, Pacciarini ML, Fallacara F, Belletti GL, Arrigoni N. 2008. Mycobacterium porcinum strains isolated from bovine bulk milk: implications for Mycobacterium avium subsp. paratuberculosis detection by PCR and culture. Vet Microbiol. 2008;130:338-47. 59
#86 Typing <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (MAP) strains isolated from different Italian regions by four Variable-Number Tandem Repeat (VNTR) methods alone or in association Ricchi M, Taddei R, Barbieri I, Belletti GL, Pacciarini ML and Arrigoni N Istituto Zoopr<strong>of</strong>ilattico Sperimentale della Lombardia e dell’Emilia Romagna (IZSLER), Italy ABSTRACT The control <strong>of</strong> Paratuberculosis requires <strong>the</strong> knowledge <strong>of</strong> <strong>the</strong> causative agent, Mycobacterium avium subsp. paratuberculosis (MAP), both in terms <strong>of</strong> epidemiology and biodiversity within different strains. One <strong>of</strong> <strong>the</strong> most widely used method for MAP typing is IS900 sequence restriction fragment length polymorphism. However it is applicable only on cultivable strains, is technically demanding and has limited discriminatory power. More recently, tandem-repeat PCR based methods overcame <strong>the</strong>se problems and, at present, are considered <strong>the</strong> emerging techniques for MAP typing. The aim <strong>of</strong> this study was to evaluate <strong>the</strong> discriminatory power <strong>of</strong> four PCR typing methods. We selected ten different strains from various Italian regions. We used MIRU (3 loci), VNTR-MIRU (7 loci), MLSSR (11 loci) and MLVA (5 loci) analysis to differentiate <strong>the</strong> bacterial DNA. Both MIRU and VNTR-MIRU gave 2 clusters (DI 0.556), while MLVA and MLSSR gave respectively 5 (DI 0.667) and 9 (DI 0.978) clusters. The combinations gave different results: MIRU+MLVA and VNTR-MIRU+MLVA gave 5 clusters (DI 0.806), MLSSR+MIRU or MLSSR+VNTR-MIRU did not increase <strong>the</strong> discriminatory results <strong>of</strong> MLSSR alone (9 clusters); MLSSR+MLVA gave 10 clusters (maximum <strong>the</strong>oretic DI value i.e. 1.00). Although a limited number <strong>of</strong> strains was used in this study, our data suggest that applying a single analysis, MLSSR provides <strong>the</strong> highest DI. Moreover, MLSSR coupled with MLVA showed <strong>the</strong> best discriminatory power. Finally, <strong>the</strong> combination between MIRU or VNTR-MIRU and MLVA enhanced <strong>the</strong> indexes as compared to single analysis. INTRODUCTION The control <strong>of</strong> Paratuberculosis requires <strong>the</strong> knowledge <strong>of</strong> <strong>the</strong> causative agent, Mycobacterium avium subsp. paratuberculosis (MAP), both in terms <strong>of</strong> epidemiology and biodiversity within different strains. Many methods have been proposed to type MAP: multiplex PCR for IS900 integration loci (MPIL) 1 , IS900 sequence restriction fragment length polymorphism (RFLP) 2 , amplified fragment length polymorphism (AFLP) 3 and Pulsed Field Gel Electrophoresis (PFGE) 4 . The most popular is IS900 RFLP, however it is applicable only on cultivable strains, is technically demanding and has limited discriminatory power. More recently, tandem-repeat PCR based methods (TR-PCR) overcame many <strong>of</strong> <strong>the</strong>se problems and, at present, are considered <strong>the</strong> emerging techniques for MAP typing. The aim <strong>of</strong> this study was to evaluate <strong>the</strong> discriminatory power <strong>of</strong> four PCR typing methods alone or in association. We evaluated: mycobacterial interspersed repetitive units (MIRU) 5 , variable number tandem repeat - mycobacterial interspersed repetitive units (VNTR-MIRU) 6 , multilocus variable-number tandem-repeat (MLVA) 7 and multilocus short sequence repeat (MLSSR) 8 techniques. METHODS We selected ten different strains from various Italian regions. Seven samples derived from faeces, two from milk and one from ileum. Eight strains derived from cattle and two from goats. When <strong>the</strong> strain grown in culture was available, <strong>the</strong> DNA was extracted suspending one colony in 100 μl <strong>of</strong> distilled sterile water and boiling for 20 min. For <strong>the</strong> strains 9 and 10, since <strong>the</strong> culture was not possible, we extracted MAP DNA respectively from faeces and ileum tissue with DNA QIAmp Tissue Kit (Qiagen, Milan, Italy). In order to differentiate <strong>the</strong> bacterial DNA, we used <strong>the</strong> following Tandem Repeat-PCR methods: 60
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 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 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 <
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?