The Animal Health Diagnostic Center (AHDC) offers a Johne’s Direct Fecal PCR test. This test is available to veterinarians and is intended to replace individual cow or goat cultures for most herds controlling Johne’s disease through management interventions and testing.

What sample is needed?

At least 15 grams (approx. 3 teaspoons) of bovine or caprine feces from an individual animal. Environmental manure samples, milk samples, or composite samples of multiple cows are not acceptable. Fecal cups sold by our diagnostic supply center, filled half-full, are ideal for handling and shipping. Containers must be leak-proof. Keep refrigerated until shipped overnight on ice. Ship these samples as UN3373 Biological Substance Category B.

What is the cost of this test?

The cost to the veterinarian for samples submitted directly to the AHDC will depend upon if the herd being tested is a NYSCHAP herd, an in-state herd or an out of state herd. Please consult AHDC Test and Submissions for current pricing. Samples must be submitted through a licensed veterinarian.

What is the lag time?

Results will normally be available 7-10 business days after the AHDC receives the sample.

Is advanced scheduling required for this test?

Yes. The lab should be called (Johne’s Lab, Monica Carey, 607-253-4473) in advance of collecting samples for this test, to schedule herd testing. Single clinical suspect samples do not require advanced scheduling. The AHDC anticipates being able to meet testing needs for this new assay, but large numbers of incoming samples will require scheduling to minimize delays for all of our clients.

What are the details of the test?

The Johne’s Direct Fecal PCR test is a real-time polymerase chain reaction assay with an internal amplification standard control for every sample. It is validated for high throughput in a 96-well plate format. It detects amplified DNA of Mycobacterium avium subsp. paratuberculosis extracted from fecal samples of shedding cows. PCR assays are run in thermal cyclers, which produce results as the numbers of cycles to positive (Ct) versus not detected. Validation of this assay included a quantitative evaluation of the relationship between cycles to positive and the number of colony counts (CFU) on solid HEY agar culture, which has been used as the gold standard for MAP detection. Since a statistically significant relationship exists between Ct and CFU, a quantitative result will also be reported, suggesting shedding at the “Light”, “Moderate”, or “Heavy” level.

In what situations might this test be used?

This test may be used in a herd control program, and it may also be used as a primary diagnostic test in individual cows or goats with clinical signs compatible with Johne’s disease. As has always been the case, Johne’s disease needs to be approached by first addressing the areas of management that allow susceptible animals to be exposed to an infective dose of MAP. An overall Johne’s disease control strategy and herd health plan should be in place for a given herd. Diagnostic testing is only one piece of the control strategy aimed at identifying animals that are shedding or have the potential to shed MAP at a time when they would expose susceptible animals such as at parturition.

High sensitivity, rapid turnaround and reasonable fees, even for non-NY cattle, will make this test the most appropriate test for clinical suspects. Clinical Johne’s disease, which occurs in advanced MAP- infected cattle and goats, is accompanied by heavy shedding. This PCR assay has detected 100% of all heavy shedders in two different validation sample sets of 601 and 804 cows from high prevalence Map-infected herds, with 17 and 58 heavy shedders, respectively.

For dairy herds in which young stock are separated from adults at birth, removal of heavy and moderate shedders has been identified as important in helping to reduce transmission of MAP within herds. The current recommendation of the AHDC and NYSDAM is to consider using this test as an individual cow level test to detect heavy and moderate shedders. Internal validation with 804 samples collected in high prevalence populations estimates the relative sensitivity of this direct fecal PCR assay for detection of heavy and moderate shedders, as compared to solid HEY agar culture, at 95.45%. For light (few) shedders, it is 74.43%*. Therefore, herds managing light shedders by immediate culling may want to continue to use culture methods. Specificity of the assay components have been determined to be 100% on an NVSL proficiency test with known negative samples.

For beef cattle and meat goat herds and other livestock herds in which young stock remain suckling on their dams until weaning or remain with the adult herd even later, allowing light shedders to remain in the population is not a feasible control strategy. A control strategy should be devised to maximize detection at or prior to shedding, which often entails using both fecal detection tests such as a fecal PCR or culture, and a serum antibody detection test. The goal is to remove all current shedder or animals that are could become shedders prior to the onset of calving/kidding/lambing.

Validation work has not been completed for pooled fecal PCR detection, or for PCR detection in environmental samples, non-fecal specimens, or non-bovine/non-caprine species. Some regulatory/export agencies may not allow substitution of the fecal PCR for accepted culture procedures. If PCR testing is desired for samples from non-bovine/non-caprine species, we strongly recommend the use of simultaneous culture of the samples. We will perform the PCR test on samples from non-bovine/ non-caprine animals and will report the results with a disclaimer that the assay has not been validated for these species.

This test will be the default test for individual sample testing of bovine samples comprising positive pools as detected by culture. This will improve both the cost and turn-around time for pooled testing when positive pools are detected.

Can this test be used in the National Voluntary Johne’s Test Negative Status Program (TNSP) for cattle herds?

Yes. The AHDC has passed the USDA Johne’s PCR check-test. Direct fecal PCR testing is one of the approved methods for testing in TNSP herds. In the USDA program standards, this test is referred to as a "DNA probe."

Can the values for the direct fecal PCR be compared to the liquid culture?

The AHDC conducted a study comparing direct fecal PCR, liquid culture, and solid HEY agar culture on 804 samples in 2009. A statistical correlation exists between PCR and solid HEY culture. Previously, the AHDC has internally validated liquid culture in comparison to solid HEY culture, demonstrating a similar statistical correlation. It appears that the PCR assay will perform similarly to culture in detecting heavy and moderate shedders.

Since culture also has limitations in sensitivity, relative to detecting all shedding or infected cows, it is difficult to determine which testing platform has the best sensitivity in detecting all shedding cows*.

How do I interpret the test results on an individual cow?

A “not detected” result does not necessarily indicate that the cow is not infected or not shedding as evidenced by the sensitivity of 74.43%* on a population of cows positive on fecal culture. False or unconfirmed positive results are also possible when single samples are tested by different assays, as there may be within-sample variation in the distribution of the MAP organisms. The primer set selected by the reagent manufacturer is expected to be specific to MAP, and has undergone testing to assure non-detected results when tested against known negative samples provided in the NVSL proficiency test. The AHDC has not evaluated the performance of the assay in advanced test-negative status herds.

For more information on Johne’s disease diagnostic testing strategies please talk with your NYSDAM field veterinarian or contact the AHDC and speak with one of our VSS Diagnostic Intelligence Officers (607-253-3900).

*It is possible that this PCR assay may actually have superior sensitivity in detecting shedding, as compared to current culture techniques. In our work, there were also samples detected by PCR that were not detected by culture. Study design did not allow for Bayesian statistical evaluation. Long-term study of infected animals, or other techniques, such as evaluation compared to detection in post-mortem tissue via multiple techniques, would be needed to define sensitivity greater than that detected by culture. Since adoption of this assay at the AHDC, it appears that most herds on a long term testing program experienced a slight increase (~1-2%) in positive results when PCR testing was adopted.

Selected references:

1. Scott HM, Fosgate GT, Libal MC, Sneed LW, Erol E, Angulo AB, Jordan ER. Field testing of an enhanced direct-fecal polymerase chain reaction procedure, bacterial culture of feces, and a serum enzyme- linked immunosorbent assay for detecting Mycobacterium avium subsp paratuberculosis infection in adult dairy cattle. Am J Vet Res. 2007 Mar;68(3):236-45
2. Alinovi CA, Ward MP, Lin TL, Moore GE, Wu CC. Real-time PCR, compared to liquid and solid culture media and ELISA, for the detection of Mycobacterium avium ssp. paratuberculosis. Vet Microbiol. 2009 Apr 14;136(1-2):177-9.
3. Clark DL Jr, Koziczkowski JJ, Radcliff RP, Carlson RA, Ellingson JL. Detection of Mycobacterium avium subspecies paratuberculosis: comparing fecal culture versus serum enzyme-linked immunosorbent assay and direct fecal polymerase chain reaction. J Dairy Sci. 2008 Jul;91(7):2620-7.