Proceedings of the 10th International Colloquium on Paratuberculosis

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#22 The Prevalence <strong>of</strong> Possible Mycobacterium Avium Subspecies Avium in Fecal Sample from Dairy Cows Monif GRG 1 , Lin TL 2 , Williams JE 1 , Wu CC 2 University <strong>of</strong> Florida College <strong>of</strong> Veterinary Medicine, Department <strong>of</strong> Infectious Diseases and Pathobiology Purdue University School <strong>of</strong> Veterinary Medicine, Department <strong>of</strong> Pathobiology ABSTRACT A comparative study <strong>of</strong> fecal culture, hspX Map real-time PCR and nested 1311-based PCR tests was undertaken to determine <strong>the</strong> incidence <strong>of</strong> positive fecal test results using IS1311base nest PCR primers relative to fecal culture and real-time PCR using hspX. Three hundred sixty-eight fecal samples from <strong>the</strong> Florida Johne’s Disease Dairy Herd Demonstration Project had been analyzed using fecal culture, real-time PCR and nested PCR for <strong>the</strong> detection <strong>of</strong> Mycobacterium avium subsp. paratuberculosis {Map}. Forty-one fecal specimens tested positive by <strong>the</strong> direct fecal nested Map PCR test (FecaMap®}. In 34 <strong>of</strong> <strong>the</strong> cases, <strong>the</strong> corresponding real time PCR test for Map was also positive. Mycobacterium isolates were achieved by fecal culture in 21 <strong>of</strong> <strong>the</strong> 41 cases. In 20 <strong>of</strong> <strong>the</strong> 21 cases <strong>of</strong> culture recovery <strong>of</strong> a mycobacterium, IS900-based primers confirmed Map. In <strong>the</strong> remaining case, fecal culture demonstrated case heavy growth and <strong>the</strong> corresponding hspX real time PCR were both positive. In <strong>the</strong> remaining 6 direct nested PCR tests, no evidence <strong>of</strong> mycobacterium growth was present. Assuming fecal culture to be 100% sensitive, <strong>the</strong> project herd false-positive incidence using IS1311 based nested primers would be approximately 1.1% INTRODUCTION The Linda strain <strong>of</strong> Mycobacterium avium subspecies paratuberculosis (Map) that established <strong>the</strong> IS900 insertion sequences as <strong>the</strong> definitive marker <strong>of</strong> mycobacterium that cause Johne’s diseases was deemed a centralist stain representative <strong>of</strong> <strong>the</strong> group (Harris and Barletta, 2001). It has been subsequently argued that <strong>the</strong> IS900 insertion sequence is a vertical cut through a horizontal evolutionary process emanating from Mycobacterium avium subspecies avium in which exist polymorphic variants <strong>of</strong> <strong>the</strong>se two species that can cause Johne’s disease in herbivores and omnivores (Frothingham, 1999; Turenne et al., 2007). In horses, pigs, and dogs, Ma and Mycobacterium avium complex (Mac) are <strong>the</strong> causative agents <strong>of</strong> Johne’s disease (Turenne et al., 2007). The FecaMap® and LactoMap (Infectious Diseases Incorporated, Bellevue, NE) direct and nest PCR test primers were developed using <strong>the</strong> IS1311 insertion sequence in order to effectively span <strong>the</strong> potential spectrum <strong>of</strong> pathogenic mycobacterium as well as to provide tests applicable to avian species and zoo animals not identifiable using commercial Map ELISA tests. Despite demonstrated pathogenicity in horses, pigs, dogs, and selected zoo animals, <strong>the</strong>re is a tendency to consider Ma as an environmental mycobacterium ra<strong>the</strong>r than a pathogenic mycobacterium. The argument can be advanced that use <strong>of</strong> PCR tests based upon <strong>the</strong> IS1311 insertion sequence would only result in a significant number <strong>of</strong> falsepositive results. The purpose <strong>of</strong> this study was to analyze to what degree using a IS1311-based PCR test would positive results be identified that were not substantiated by fecal culture or realtime PCR using hspX. MATERIALS AND METHODS Study population: Three hundred sixty-eight dairy cows within <strong>the</strong> Florida Johne’s Disease Dairy Herd Demonstration Project constituted <strong>the</strong> study population. Selection <strong>of</strong> a cow was predicated upon prior independent analysis <strong>of</strong> its feces using <strong>the</strong> FecaMap® nested Map PCR test. 165
Fecal cultures: Fecal cultures were done at Animal Disease Diagnostic Laboratory, School <strong>of</strong> Veterinary Medicine, Purdue University using <strong>the</strong> Trek® Map Culture System in accordance with <strong>the</strong> manufacturer’s instructions. Real-time Map PCR tests: Real-time Map PCR tests were done at Animal Disease Diagnostic Laboratory, School <strong>of</strong> Veterinary Medicine, Purdue University using Tetracore® Map Extraction and DNA test kit in accordance with <strong>the</strong> manufacturer’s instructions. Direct fecal nested Map PCR tests: Direct fecal nested Map PCR tests were done at University <strong>of</strong> Florida College <strong>of</strong> Veterinary Medicine using <strong>the</strong> FecaMap® system in accordance with <strong>the</strong> manufacturer’s instructions. The FecaMap® direct primers recognize a 242 base pair sequence <strong>of</strong> Map IS1311 and its nested primers overlap and span a 104 base pair region within <strong>the</strong> insertion sequence. RESULTS Three hundred sixty-eight fecal samples from <strong>the</strong> Florida Johne’s Disease Dairy Herd Demonstration Project had been analyzed. Out <strong>of</strong> 368 fecal samples, Ma/Map was identified by <strong>the</strong> IS1311 primer <strong>of</strong> <strong>the</strong> FecaMap® test (1.1%). Forty-one fecal specimens tested positive by <strong>the</strong> nested Map PCR test. In 34 <strong>of</strong> <strong>the</strong> cases, <strong>the</strong> corresponding real time PCR test for Map was also positive (Table 1). Mycobacterium isolates were achieved by fecal culture in 21 <strong>of</strong> <strong>the</strong> 41 cases. In 20 <strong>of</strong> <strong>the</strong> 21 cases <strong>of</strong> culture recovery <strong>of</strong> a mycobacterium, IS900-based primers confirmed Map. In <strong>the</strong> remaining case, fecal culture demonstrated case heavy growth and <strong>the</strong> corresponding hspX real time PCR was positive. The animal was culled before <strong>the</strong> need to retest was identified. Six fecal samples identified by <strong>the</strong> IS1311 nested PCR were not substantiated by ei<strong>the</strong>r fecal culture or real-time PCR using hspX. Table 1. Analysis <strong>of</strong> dairy cows in <strong>the</strong> Florida Johne’s Disease Prevention Dairy Herd Demonstration Project for prevalence <strong>of</strong> Map/Ma DNA in fecal samples as determined by <strong>the</strong> FecaMap® direct nest fecal Map PCR test. # <strong>of</strong> fecal # culture +/ # RT PCR +/ # non-Map + cultures/ specimens # nested - nested + RT & nested + 368 20/41 34/41 1/1 DISCUSSION Despite covering only 6-8 copies, <strong>the</strong> direct IS1311 direct and nested primers were demonstrated to be more sensitive in identifying Map contained within USDA’s Laboratory Certification Tests. The overall incidence <strong>of</strong> a positive IS1311-based nested Map PCR test was 1.1%. A positive nested reaction had an 85% probability <strong>of</strong> identifying Map; however <strong>the</strong> remaining 14% <strong>of</strong> positive nested PCR tests were not confirmed by <strong>the</strong>ir corresponding fecal real-time PCR and culture tests. More importantly, <strong>the</strong> nest Map PCR identified a non-IS900 mycobacterium whose test pr<strong>of</strong>ile was that <strong>of</strong> being a heavy shedder in <strong>the</strong> Trek® culture system and <strong>of</strong> testing positive in <strong>the</strong> Tetracore® PCR system. These observations coupled with early culling makes it, more likely than not that this animal had a significant mycobacterium infection. Culture data demonstrating heavy shedding would have been disregarded had diagnostic confirmation been done using IS900 primers. To what degree o<strong>the</strong>r non-IS900 potentially pathogenic mycobacteria have been dismissed as being environmental contaminants is unknown. In this small series, <strong>the</strong> presence <strong>of</strong> a non-IS900 presumably pathogenic mycobacterium was calculated to be 2.4%. 166
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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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#19 Development of
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#36 Genetic diversity of</s
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#60 Evaluation of
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#66 Comparison of
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(b) IS900 Nested PCR, using p90-p91
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(d) f57 Real Time PCR. The figure o
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• MIRU (3 loci); • VNTR-MIRU (7
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indexes, compared to each single an
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#108 Evaluation of
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#110 Detection and molecular confir
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endoscopic criteria and histopathol
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Bull TJ, McMinn EJ, Sidi-Boumedine
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#131 Seroprevalence of</str
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could be partly attributed to a sma
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#146 Analysis of v
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#162 Comparison of
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#187 Comparison of
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#191 Potentiating day-old blood sam
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samples from the s
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(MAA) (MAP87 and MAP3776) and 1 fro
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#196 Inter- and intra-subtype genot
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RESULTS In 601 (29%) of</st
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#211 Culture of my
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#242 Application and field validati
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#251 Diagnosis of
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Table 2. Paratuberculosis PCR resul
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#254 Programmes on Paratuberculosis
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Fig. 1. European countries specifyi
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A bulk milk quality assurance progr
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Republic of Lithua
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#276 Elimination of</strong
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other projects and
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Woodbine KA, Schukken YH, Green LE,
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Page 118 and 119: #33 MERKAL AWARD LECTURE No interfe
Page 120 and 121: #75 Interleukin 17 and interleukin
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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
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Page 148 and 149: #228 Mycobacterium avium ssp. parat
Page 150 and 151: #145 MERKAL AWARD LECTURE Multinomi
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Page 154 and 155: Table 1. Posterior median (CrIs) <s
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Page 158 and 159: from their dam, se
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Page 162 and 163: #104 Relation between faecal shedde
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Page 168 and 169: CONCLUSION IS1311-based nested Ma/M
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Page 176 and 177: REFERENCES 1) Chiodini RJ, Van Krui
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Page 188 and 189: A statistically significant lower f
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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
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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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#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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