Laboratory Diagnosis of Johne’s Disease

By Sree Rajeev, DVM, PhD, DACVM

Johne’s disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is an economically important disease of ruminants. The disease is characterized by chronic, progressive granulomatous enteritis and affects a wide variety of hosts including cattle, sheep, goats, llamas, alpacas, bison and deer. The U.S. dairy cattle industry estimates an annual economic loss of $220 million due to JD. The presence of MAP in milk, its resistance to pasteurization temperatures, and its possible association with Crohn’s disease in humans suggest a significant public health risk.

Rapid and accurate diagnosis of MAP infection is critical for JD control programs. Fecal culture has merit over serological methods because of its higher sensitivity and specificity, and it is the method of choice for the detection and removal of individual infected animals from a herd. The Tifton Veterinary Diagnostic and Investigational Laboratory (TVDIL) uses an automated liquid culture system (ESP® para-JEM System, TREK Diagnostic Systems Inc., Cleveland, OH) for culture of MAP. The cost of testing is $20 per sample. Our experience indicates that the sensitivity of this method is much higher than the previous culture method using solid media. The detection rate of this technique is 40–50% more in some heavily infected herds principally because of the higher chance of detecting low shedding animals.

In the MAP diagnostic procedure, fecal samples and tissues go through an extensive decontamination process for 48 hours, and then the samples are incubated in the media in the ESP system. As we obtain positive signal from the system, the samples are taken out and confirmed by acid-fast staining and PCR using a MAP-specific genomic target. Results from heavy shedding animals are obtained before the 25th day of incubation, and final results on all animals can be obtained by about 8 weeks after sample submission as opposed to 16 weeks previously required for incubation on solid media. Direct detection of MAP in fecal samples by PCR is available on request. However, direct detection by PCR is not done routinely due to low sensitivity in detecting low shedding animals. In order to assure our clients with quality services, TVDIL participates in annual proficiency testing for MAP culture offered by the National Veterinary Services Laboratory.

Tips for sample collection and shipping

Collect fresh fecal samples in a screw-capped plastic container and ship them overnight with ice packs. Freezing samples in a household refrigerator is NOT recommended since this may reduce the viability of the organism. If storage is required, it is advisable to store samples in a refrigerator. Herd-level detection of disease can be done either by culturing pooled fecal or environmental samples and by ELISA on serum samples. Pools of 5 fecal samples are recommended. Collection of environmental samples should be performed from high manure concentration areas. However, for the detection and removal of animals, individual fecal cultures must be done. It is important to note that detection and removal of the infected animals and management of the herd to prevent new infections are the only way to control this disease since no effective treatment or vaccines are available.

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