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Proceedings of the 10th International Colloquium on Paratuberculosis

Proceedings of the 10th International Colloquium on Paratuberculosis

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Direct fecal nested Map PCR testing: Direct fecal nested Map PCR tests were d<strong>on</strong>e at<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 using <str<strong>on</strong>g>the</str<strong>on</strong>g> FecaMap® system in<br />

accordance with <str<strong>on</strong>g>the</str<strong>on</strong>g> manufacturer’s instructi<strong>on</strong>s.<br />

Statistical analysis: The Fisher's Exact Test was used to test whe<str<strong>on</strong>g>the</str<strong>on</strong>g>r <str<strong>on</strong>g>the</str<strong>on</strong>g>re was any n<strong>on</strong>random<br />

associati<strong>on</strong> between variables <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> two direct fecal, nested Map PCR test results<br />

and provided culture results. Kappa coefficient, sensitivity was estimated using Win Episcope<br />

2.0 s<str<strong>on</strong>g>of</str<strong>on</strong>g>tware (Win Episcope 2.0). Ninety-five percent c<strong>on</strong>fidence intervals (CI) were<br />

c<strong>on</strong>structed for all estimates.<br />

RESULTS<br />

The direct IS900 and IS1311 PCR primers had a sensitivity <str<strong>on</strong>g>of</str<strong>on</strong>g> 21.7 and 38.3% respectively;<br />

whereas <str<strong>on</strong>g>the</str<strong>on</strong>g> corresp<strong>on</strong>ding nested PCR primers had sensitivities <str<strong>on</strong>g>of</str<strong>on</strong>g> 76.6% and 86.7% (Table<br />

1). The P90-P91 primers did not identify <str<strong>on</strong>g>the</str<strong>on</strong>g> Ma -spiked culture as being positive whereas<br />

<str<strong>on</strong>g>the</str<strong>on</strong>g> direct IS1311, nested IS900 and IS1311 primers correctly identified 2 <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> 3 M. avium<br />

spiked cultures as being positive respectively.<br />

The dem<strong>on</strong>strati<strong>on</strong> that <str<strong>on</strong>g>the</str<strong>on</strong>g> IS900 nested primers identified <str<strong>on</strong>g>the</str<strong>on</strong>g> M. avium-spiked fecal<br />

specimens in a manner comparable to <str<strong>on</strong>g>the</str<strong>on</strong>g> IS1311 nested primers necessitated excluding<br />

<str<strong>on</strong>g>the</str<strong>on</strong>g>se samples in <str<strong>on</strong>g>the</str<strong>on</strong>g> overall calculati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> comparative sensitivity. The spiked fecal samples<br />

were deleted in <str<strong>on</strong>g>the</str<strong>on</strong>g> statistical comparis<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> direct IS900 and IS1311 primers. The ability <str<strong>on</strong>g>of</str<strong>on</strong>g><br />

<str<strong>on</strong>g>the</str<strong>on</strong>g> nested IS900 PCR primers to extend <str<strong>on</strong>g>the</str<strong>on</strong>g> test sensitivity to M. avium was also a c<strong>on</strong>found<br />

variable in <str<strong>on</strong>g>the</str<strong>on</strong>g> comparis<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> nested IS900 vs. IS1311 nested primers (Table 1).<br />

Table 1. Statistical comparis<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> IS900 versus IS1311 direct and nested primers <strong>on</strong> fecal<br />

specimens with three USDA Laboratory Certificati<strong>on</strong> Tests<br />

P90-P91 IS1-IS2<br />

Sensitivity 21.7% (13+/46-) 38.3%(23+/37-)<br />

Specificity 100% (19/19) 94.7% (18/19)<br />

Kappa Coefficient 0.11 0.2<br />

P90-P91/J1-J2 IS1-IS2/IS3-IS4<br />

Sensitivity 76.7% (46+/18-) 86.7% (52+/9-)<br />

Specificity 94.7% (18/19) 100% (19/19)<br />

Kappa Coefficient 0.2 0.76<br />

Interpretati<strong>on</strong> good agreement very good agreement<br />

DISCUSSION<br />

Given that <str<strong>on</strong>g>the</str<strong>on</strong>g> IS1311 based primers, IS1/IS2, identify <strong>on</strong>ly 6-8 copies where as <str<strong>on</strong>g>the</str<strong>on</strong>g> P90/P91<br />

primers based up<strong>on</strong> <str<strong>on</strong>g>the</str<strong>on</strong>g> IS900 sequence identify 14-18 copies, <str<strong>on</strong>g>the</str<strong>on</strong>g>re should have been no<br />

reas<strong>on</strong> to anticipate that <str<strong>on</strong>g>the</str<strong>on</strong>g> IS1311 base primers would exhibit superior sensitivity unless<br />

<str<strong>on</strong>g>the</str<strong>on</strong>g> sequences covered had greater antigenic representati<strong>on</strong>.<br />

As expected, fecal specimens spiked with M. avium were not detected by P90-P91<br />

inserti<strong>on</strong> sequence, but were detected by <str<strong>on</strong>g>the</str<strong>on</strong>g> IS1311 direct primers. What was not<br />

anticipated was that <str<strong>on</strong>g>the</str<strong>on</strong>g> nested IS900-based J1/J2 in two <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> three Ma spiked samples as<br />

well as <str<strong>on</strong>g>the</str<strong>on</strong>g> IS1311 nested IS3/IS4 primers identified M. avium.<br />

Clinical relevance is inferred by a herd study <str<strong>on</strong>g>of</str<strong>on</strong>g> 341 dairy cows in which IS1311basedFecaMap®<br />

identified a n<strong>on</strong>-IS900 mycobacterium from a cow whose feces<br />

dem<strong>on</strong>strated heavy shedding and tested positive with <str<strong>on</strong>g>the</str<strong>on</strong>g> Tetracore® hspX PCR test and<br />

who was an early cull (Williams et al., 2009).<br />

45

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