Proceedings of the 10th International Colloquium on Paratuberculosis

#7
Significance <strong>of</strong> Heavy Fecal Shedding <strong>of</strong> Mycobacterium Avium Subspecies
Paratuberculosis (Map): Comparison <strong>of</strong> Fecal Culture, Real-Time and Nested
PCR Testing
Monif GRG 1 , Tsang Long Lin 2 , Williams JE 1 , Wu CC 2
University <strong>of</strong> Florida College <strong>of</strong> Veterinary Medicine, Gainesville, FL, USA 1 ; Purdue
University School <strong>of</strong> Veterinary Medicine, West Lafayette, IN 2 , USA
ABSTRACT
The potential that clumping by Mycobacterium avium subspecies paratuberculosis can
influence <strong>the</strong> quantity <strong>of</strong> organisms identified by fecal culturing was analyzed in a
prospective, blinded study using comparative fecal culture, hspX real-time PCR and direct
and nested IS1311-based PCR testing. Of <strong>the</strong> 22 fecal samples identified as coming from
“heavy shedders” by fecal culture, only 7 fecal cultures had positive correspondence with
real-time and nested PCR. Clumping by Map within fecal samples can cause quantitative
misrepresentation <strong>of</strong> <strong>the</strong> degree <strong>of</strong> fecal shedding within a given fecal specimen.
INTRODUCTION
A major herd management tool in controlling Johne’s disease has been <strong>the</strong> ability to quantify
<strong>the</strong> amount <strong>of</strong> Map present in a given fecal specimen. As a general rule, animals identified as
having heavy Map fecal shedding are considered to represent a significant threat to overall
herd health and are frequently culled (Collins et al., 2006).
Map differs from o<strong>the</strong>r pathogenic mycobacterium, such as Mycobacterium bovis and
Mycobacterium avium subspecies avium, in that organism replication results in <strong>the</strong> tight
clumping <strong>of</strong> individual mycobacterium (Harris and Barletta, 2001). Depending upon <strong>the</strong>
portion selected for testing within a given fecal specimen, Map clumping <strong>the</strong>oretically
introduces sampling error. No studies have been done to analyze whe<strong>the</strong>r sample site bias
occurs.
The purpose <strong>of</strong> this paper is to present corresponding fecal culture, real-time PCR and
nested PCR Map fecal test results as <strong>the</strong>y relate to validating or challenging <strong>the</strong> diagnostic
category <strong>of</strong> heavy shedding as defined by fecal culture.
MATERIALS AND METHODS
Study population: The fecal samples were obtained from two dairy herds that participated in
<strong>the</strong> Florida Johne’s Disease Dairy Herd Prevention Program. The fecal samples were sent
via Federal Express next day shipment in coolers with ice packs. The number in which
nested PCR data was available from <strong>the</strong> Diagnostic Laboratory <strong>of</strong> <strong>the</strong> Department <strong>of</strong>
Infectious Diseases, University <strong>of</strong> Florida College <strong>of</strong> Veterinary Medicine, determined <strong>the</strong>
number <strong>of</strong> fecal samples analyzed in <strong>the</strong> study.
Fecal culture tests: The fecal culture testing using <strong>the</strong> Trek® Diagnostic System was done at
<strong>the</strong> Animal Disease Diagnostic Laboratory, School <strong>of</strong> Veterinary Medicine, Purdue University.
Fecal PCR tests: The direct fecal polymerase chain reaction (PCR) testing using <strong>the</strong>
Tetracore® Map Diagnostic System was done at <strong>the</strong> Animal Disease Diagnostic Laboratory,
School <strong>of</strong> Veterinary Medicine, Purdue University. The direct and nested fecal PCR testing
using <strong>the</strong> FecaMap® Map Diagnostic System was done at <strong>the</strong> Veterinary Diagnostic
Laboratory <strong>of</strong> <strong>the</strong> Department <strong>of</strong> Infectious Diseases, College <strong>of</strong> Veterinary Medicine,
University <strong>of</strong> Florida.
All tests were done in accordance to <strong>the</strong> manufacturer’s instructions.
The Trek®, Tetracore ® Diagnostic Systems, and FecaMap Test sampled 2, 2, and
0.25 grams <strong>of</strong> fecal material respectively.
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