02.02.2013 Views

Proceedings of the 10th International Colloquium on Paratuberculosis

Proceedings of the 10th International Colloquium on Paratuberculosis

Proceedings of the 10th International Colloquium on Paratuberculosis

SHOW MORE
SHOW LESS

Create successful ePaper yourself

Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.

#22<br />

The Prevalence <str<strong>on</strong>g>of</str<strong>on</strong>g> Possible Mycobacterium Avium Subspecies Avium<br />

in Fecal Sample from Dairy Cows<br />

M<strong>on</strong>if GRG 1 , Lin TL 2 , Williams JE 1 , Wu CC 2<br />

University <str<strong>on</strong>g>of</str<strong>on</strong>g> Florida College <str<strong>on</strong>g>of</str<strong>on</strong>g> Veterinary Medicine,<br />

Department <str<strong>on</strong>g>of</str<strong>on</strong>g> Infectious Diseases and Pathobiology<br />

Purdue University School <str<strong>on</strong>g>of</str<strong>on</strong>g> Veterinary Medicine, Department <str<strong>on</strong>g>of</str<strong>on</strong>g> Pathobiology<br />

ABSTRACT<br />

A comparative study <str<strong>on</strong>g>of</str<strong>on</strong>g> fecal culture, hspX Map real-time PCR and nested 1311-based PCR<br />

tests was undertaken to determine <str<strong>on</strong>g>the</str<strong>on</strong>g> incidence <str<strong>on</strong>g>of</str<strong>on</strong>g> positive fecal test results using IS1311base<br />

nest PCR primers relative to fecal culture and real-time PCR using hspX.<br />

Three hundred sixty-eight fecal samples from <str<strong>on</strong>g>the</str<strong>on</strong>g> Florida Johne’s Disease Dairy Herd<br />

Dem<strong>on</strong>strati<strong>on</strong> Project had been analyzed using fecal culture, real-time PCR and nested<br />

PCR for <str<strong>on</strong>g>the</str<strong>on</strong>g> detecti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> Mycobacterium avium subsp. paratuberculosis {Map}. Forty-<strong>on</strong>e<br />

fecal specimens tested positive by <str<strong>on</strong>g>the</str<strong>on</strong>g> direct fecal nested Map PCR test (FecaMap®}. In 34<br />

<str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> cases, <str<strong>on</strong>g>the</str<strong>on</strong>g> corresp<strong>on</strong>ding real time PCR test for Map was also positive. Mycobacterium<br />

isolates were achieved by fecal culture in 21 <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> 41 cases. In 20 <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> 21 cases <str<strong>on</strong>g>of</str<strong>on</strong>g> culture<br />

recovery <str<strong>on</strong>g>of</str<strong>on</strong>g> a mycobacterium, IS900-based primers c<strong>on</strong>firmed Map. In <str<strong>on</strong>g>the</str<strong>on</strong>g> remaining case,<br />

fecal culture dem<strong>on</strong>strated case heavy growth and <str<strong>on</strong>g>the</str<strong>on</strong>g> corresp<strong>on</strong>ding hspX real time PCR<br />

were both positive. In <str<strong>on</strong>g>the</str<strong>on</strong>g> remaining 6 direct nested PCR tests, no evidence <str<strong>on</strong>g>of</str<strong>on</strong>g><br />

mycobacterium growth was present. Assuming fecal culture to be 100% sensitive, <str<strong>on</strong>g>the</str<strong>on</strong>g> project<br />

herd false-positive incidence using IS1311 based nested primers would be approximately<br />

1.1%<br />

INTRODUCTION<br />

The Linda strain <str<strong>on</strong>g>of</str<strong>on</strong>g> Mycobacterium avium subspecies paratuberculosis (Map) that<br />

established <str<strong>on</strong>g>the</str<strong>on</strong>g> IS900 inserti<strong>on</strong> sequences as <str<strong>on</strong>g>the</str<strong>on</strong>g> definitive marker <str<strong>on</strong>g>of</str<strong>on</strong>g> mycobacterium that<br />

cause Johne’s diseases was deemed a centralist stain representative <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> group (Harris<br />

and Barletta, 2001). It has been subsequently argued that <str<strong>on</strong>g>the</str<strong>on</strong>g> IS900 inserti<strong>on</strong> sequence is a<br />

vertical cut through a horiz<strong>on</strong>tal evoluti<strong>on</strong>ary process emanating from Mycobacterium avium<br />

subspecies avium in which exist polymorphic variants <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>se two species that can cause<br />

Johne’s disease in herbivores and omnivores (Frothingham, 1999; Turenne et al., 2007). In<br />

horses, pigs, and dogs, Ma and Mycobacterium avium complex (Mac) are <str<strong>on</strong>g>the</str<strong>on</strong>g> causative<br />

agents <str<strong>on</strong>g>of</str<strong>on</strong>g> Johne’s disease (Turenne et al., 2007). The FecaMap® and LactoMap (Infectious<br />

Diseases Incorporated, Bellevue, NE) direct and nest PCR test primers were developed<br />

using <str<strong>on</strong>g>the</str<strong>on</strong>g> IS1311 inserti<strong>on</strong> sequence in order to effectively span <str<strong>on</strong>g>the</str<strong>on</strong>g> potential spectrum <str<strong>on</strong>g>of</str<strong>on</strong>g><br />

pathogenic mycobacterium as well as to provide tests applicable to avian species and zoo<br />

animals not identifiable using commercial Map ELISA tests.<br />

Despite dem<strong>on</strong>strated pathogenicity in horses, pigs, dogs, and selected zoo animals,<br />

<str<strong>on</strong>g>the</str<strong>on</strong>g>re is a tendency to c<strong>on</strong>sider Ma as an envir<strong>on</strong>mental mycobacterium ra<str<strong>on</strong>g>the</str<strong>on</strong>g>r than a<br />

pathogenic mycobacterium. The argument can be advanced that use <str<strong>on</strong>g>of</str<strong>on</strong>g> PCR tests based<br />

up<strong>on</strong> <str<strong>on</strong>g>the</str<strong>on</strong>g> IS1311 inserti<strong>on</strong> sequence would <strong>on</strong>ly result in a significant number <str<strong>on</strong>g>of</str<strong>on</strong>g> falsepositive<br />

results.<br />

The purpose <str<strong>on</strong>g>of</str<strong>on</strong>g> this study was to analyze to what degree using a IS1311-based PCR<br />

test would positive results be identified that were not substantiated by fecal culture or realtime<br />

PCR using hspX.<br />

MATERIALS AND METHODS<br />

Study populati<strong>on</strong>: Three hundred sixty-eight dairy cows within <str<strong>on</strong>g>the</str<strong>on</strong>g> Florida Johne’s Disease<br />

Dairy Herd Dem<strong>on</strong>strati<strong>on</strong> Project c<strong>on</strong>stituted <str<strong>on</strong>g>the</str<strong>on</strong>g> study populati<strong>on</strong>. Selecti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> a cow was<br />

predicated up<strong>on</strong> prior independent analysis <str<strong>on</strong>g>of</str<strong>on</strong>g> its feces using <str<strong>on</strong>g>the</str<strong>on</strong>g> FecaMap® nested Map<br />

PCR test.<br />

165

Hooray! Your file is uploaded and ready to be published.

Saved successfully!

Ooh no, something went wrong!