Laboratory Diagnosis of Johne's Disease

Important general information on tests their accuracy

It is a common misconception that there are no accurate diagnostic tests for Johne's disease. There are, in fact, 8 tests in common use today: 3 methods for detection of the bacterium that causes Johne's disease (M. paratuberculosis), 4 techniques for serum antibody detection, and 1 assay for gamma interferon (cell-mediated immunity). Five of these tests are commercially available for use by veterinary diagnostic laboratories. Only one is simple enough to be able to be done in a veterinary clinic. Most tests for Johne's disease require sophisticated laboratory equipment and skilled laboratory technicians to be performed. In addition, factors such as the strength of a test result (numerical value) and herd infection rate can influence interpretation of Johne's disease test results. Consequently, assistance with interpretation of tests by persons experienced in laboratory diagnostics is recommended.

In general, tests for Johne's disease have a high specificity: most have a false-positive rate M. paratuberculosis.

Test sensitivity (percentage of infected animals that test positive) varies due to the biology of Johne's disease. In the very early stages of M. paratuberculosis infection, before animals start shedding the bacterium in feces or begin an immune response, all animals will be negative, even if infected. As the infection progresses, most tests will become positive. Tests have maximum sensitivity when used on animals with clinical signs of the infection, diarrhea and/or weight loss. Unfortunately, exceptions to these generalizations are common. For this reason, when confidence in the absence of M. paratuberculosis infection is desired, use of two or more different types of tests at the same time is recommended.

No single diagnostic test is sufficient for all purposes, but accurate and cost-effective tests are available for the many different Johne's disease diagnosis and control needs. When choosing among available tests, it is important to have a clear understanding of:

This web page will briefly explain the available tests for Johne's disease, then make recommendations as to which tests to use in the various situations. How each test is performed will be discussed briefly so that readers can better understand the interpretation of the results.

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Tests for the bacterium that causes Johne's disease; M. paratuberculosis.

An excellent way of diagnosing Johne's disease is to detect the bacterium that causes the infection. Since this bacterium is not normally found in the feces, if detected, it is assumed to be causing an infection and pathology in the animal. There are three ways of detecting M. paratuberculosis:
  • Standard bacterial culture
  • BACTEC (radiometric) culture
  • DNA probe

Standard bacterial culture has been used for almost 100 years. Many laboratories around the world can perform this test. The standard culture method for M. paratuberculosis requires little equipment and is technically simple to perform. It does, however, require experienced technologists who are able to identify the growth (bacterial colonies) seen on the culture medium. This test is moderately expensive, costing roughly $12.00/test, and takes at least 16 weeks to complete because of the extremely slow growth rate of M. paratuberculosis. A problem with this test is that strains of M. paratuberculosis from sheep frequently fail to grow on standard culture media.
BACTEC (radiometric) culture is a radioisotope-based detection method adapted from the one used to isolate the cause of tuberculosis in humans. The culture medium is commercially available but requires supplementation with additional nutrients to make it able to grow M. paratuberculosis. The main advantage of this method is that it can detect low numbers of M. paratuberculosis and can detect the bacterium faster than standard culture methods (7 weeks compared to 16 weeks). Another advantage is that the BACTEC method can grow M. paratuberculosis from a wide variety of animal species, including sheep. Disadvantages are that the BACTEC method is more expensive, requires an instrument to read the culture vials, and involves handling of radioisotopes. (Bactec culturing is offered by the Johne's Testing Center.)
DNA probes allow detection of M. paratuberculosis without having to grow the bacterium. Therefore, they are much faster. A DNA probe test can be done in less than 3 days. The first version of this test is not as sensitive as culture methods for detection of M. paratuberculosis in fecal samples (even though it uses a technique known as PCR amplification). Improvements in the technology, however, can be expected in the near future. The main disadvantage of the DNA probe is cost. Most laboratories estimate the break-even charge for a DNA probe for M. paratuberculosis to be roughly $25.00 per test.
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Tests for antibodies to M. paratuberculosis in serum (blood tests)

Detection of antibodies to M. paratuberculosis in serum is strong evidence the animal is infected. There are three techniques for detection of serum antibodies in common use today:
  • Complement fixation (CF)
  • Agar-gel immunodiffusion (AGID)
  • Enzyme-linked immunosorbent assay (ELISA)
All three tests are fundamentally measuring the same thing, antibodies to M. paratuberculosis, but use different types of technology and reagents. Differences between these three serological methods for diagnosis of Johne's disease are too complex for accurate discussion in this web page. There is a large body of scientific literature, evaluating each of these tests and comparing their accuracy. References are provided at the end of this page for the interested reader. Overall conclusions about the relative accuracy of each method are incorporated into the section on recommendations. (The ELISA is offered by the Johne's Testing Center.)
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Tests for antibodies to M. paratuberculosis in milk (milk ELISA)

It would be convenient to use milk instead of blood samples to test for Johne's disease. To date, however, no published data suggest that tests on milk for diagnosis of M. paratuberculosis infections are sufficiently accurate to warrant their use.
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Tests for a gamma interferon (cell-mediated immune) response to M. paratuberculosis

Cell-mediated immune responses are considered to be the first and most important response of animals to infection with mycobacteria. A long standing way of measuring cell-mediated immune responses is skin testing: injection of tiny amounts of mycobacterial extracts under the skin and observation for swelling and redness at the injection site 2-3 days later. Skin testing works well for diagnosis of tuberculosis in humans and animals. However, it does not work well for diagnosis of Johne's disease.

A new, more sophisticated, laboratory test for cellular immunity to mycobacteria was developed by Paul Wood at CSIRO in Australia. This test is done on blood samples and measures release of a chemical from white blood cells called gamma interferon. The assay has been available as a diagnostic kit licensed by the US Department of Agriculture for several years. Few studies have been published yet evaluating its diagnostic accuracy, however. Early reports from scientific meetings indicate hope that the gamma interferon test will be a useful addition to the laboratory tools for diagnosis of M. paratuberculosis infections in cattle, sheep and goats. (Gamma interferon testing is offered by the Johne's Testing Center.)
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Testing recommendations - Choosing the right test for the right purpose:

  1. Confirmation of a clinical diagnosis of Johne's disease
    The most rapid, accurate and least costly test to confirm a diagnosis of Johne's disease in cattle is the ELISA. Over 85% of M. paratuberculosis-infected cattle with diarrhea and weight loss will test ELISA-positive, and false-positive results are rare. Given that about 15% of infected animals may not be antibody positive, even in late-stage infections, it is prudent to submit a fecal sample for culture on the ELISA-negative but clinically affected animals. Also, if Johne's disease has not been confirmed by culture in the herd previously, a fecal culture should be performed.

    The AGID test is available as a USDA-licensed kit for confirmation of Johne's disease in cattle with compatible clinical signs. It is 100% specific (a positive test is always right) but less sensitive than the ELISA (is positive on roughly half the Johne's cases that the ELISA finds as positive).

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  2. Confirmation of a positive laboratory test
    If a positive Johne's test is not consistent with clinical assessment of an animal or herd by a veterinarian, regardless of whether the test is fecal culture, DNA probe, serum antibody ELISA or gamma interferon, the best course of action is to confirm the diagnosis. Greater confidence can be placed in all diagnostic tests for Johne's disease once a herd has had a culture-confirmed case and/or when animals in the herd have clinical signs compatible with Johne's disease. It is advisable to use more than one type of test, for example fecal culture and ELISA, when attempting to confirm a diagnosis. Once a herd is confirmed to be infected, routine confirmation of ELISA tests by fecal culture is, in the opinion of this author, not necessary.

    In surveillance programs for paratuberculosis, animals may test positive. In this situation, if test-positive animals are particularly valuable, as in the case of bulls of proven genetic merit, or if the test result has serious implications for paratuberculosis-free status of the herd, confirmation of the diagnosis should be attempted by histopathology and culture of tissues collected surgically. Care must be taken to get an adequate sample size if this procedure is to be meaningful. A full thickness biopsy (0.5 cm x 2 cm) of the terminal ileum, as close to the ileocecal junction as possible, and one or more local lymph nodes are the tissues of choice. At least 1.0 gram of each tissue should be homogenized and cultured and the remainder fixed, sectioned and stained with an acid-fast stain. Multiple tissue sections should be examined by a veterinary pathologist. If the infection is not confirmed by biopsy, the ELISA test result should be classified as a false-positive.

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  3. Estimation of the infection rate in a herd or flock
    The fastest and easiest way to measure the M. paratuberculosis infection rate (prevalence) in a herd is by doing an ELISA on all animals two years and older in a herd. Results are most reliable for herds already confirmed to be infected by isolation of M. paratuberculosis from at least one animal. The percentage of a herd that is ELISA-positive (apparent prevalence) should be doubled to get a rough estimate of the real prevalence of paratuberculosis (given a test sensitivity of roughly 50%, only half of infected animals will test positive at a single point in time).

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  4. Johne's disease control program
    Culling test-positive animals should be part of any Johne's control program. The frequency of testing and number of different types of tests used will be governed by several factors such as:
    • the type of business in which the animals are used, the estimated herd prevalence of Johne's disease,
    • the owner's perception of the importance of Johne's disease to herd productivity,
    • the capacity of the owner to pay for diagnostic tests,
    • the speed with which the owner wants to achieve control of Johne's disease, and
    • whether the goal is control or eradication of the disease.
    For most commercial dairy herds, a whole-herd ELISA is the first step in a paratuberculosis control program. Once the ELISA-positive animals are culled from a herd, but within 12 months of the first test, a second whole-herd test should be done. For this second test the ELISA could be used again or a fecal culture could be done instead. The ELISA is less expensive, but the fecal culture may be able to detect animals missed by the ELISA. There is no proven "best" approach. Economics favor continuing to use the ELISA in a control program, but studies on Johne's disease indicate that every second or third test should be a fecal culture to maximize the likelihood of detecting infected animals.

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  5. Herd certification
    A model herd certification program for paratuberculosis was developed by a task force of the Johne's Disease Committee of the U.S. Animal Health Association in 1993. The goal of the task force was to devise a system that was scientifically sound and one that would permit greater uniformity of herd certification standards among states. Specific details of the model certification program are in the Proceedings of the 1993 Annual Meeting of the USAHA. In general terms, the model herd certification program is based on using a serum antibody test, such as the ELISA, and an organism detection test, such as fecal culture, on an alternating basis annually on all cattle more than 20 months old in a herd. Herds are given levels of certification reflecting the number of whole-herd negative tests up to level 5. After attaining a level 5 paratuberculosis-free certification, herds can maintain certification by retesting annually with either a serum antibody or M. paratuberculosis detection-based test. Veterinarians and herd owners should consult their government regulatory veterinarians in their state or country.

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  6. Screening young animals (herd replacements) raised on the owner's farm
    Testing herd replacements raised on the farm to avoid use of animals infected with M. paratuberculosis in the adult herd is very cost-effective means of controlling the disease. Recognizing this, herd owners frequently request tests on their replacement heifers. Unfortunately, because of the slow rate of progression of M. paratuberculosis infections, at present no test for paratuberculosis has demonstrated sufficient sensitivity in young cattle to warrant recommendation. The test with the greatest potential is the gamma interferon assay, however, more studies are needed to determine if the test is sufficiently sensitive and specific to warrant using it.

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  7. Pre-purchase testing
    Herds become infected with M. paratuberculosis by introduction of infected, although clinically normal (asymptomatic) animals. Biosecurity is of paramount importance in the prevention of Johne's disease. While the best way to stay free of Johne's is to maintain a closed herd, most animal owners will occasionally buy or lease animals. With each animal added to a herd, the owner runs the risk of introducing M. paratuberculosis. An estimate of that risk for dairy cattle bought from a sale barn or dealer in the USA is 10% per animal. The risk can be reduced to zero by only using Johne's-free herds as sources of replacement animals.

    In the absence of available certified paratuberculosis-free herds, owners wanting to add animals to their herds should seek out reputable sellers of cattle with herds having no clinical evidence of Johne's disease. Then, they should select a pre-purchase testing plan based on the degree of cooperation of the seller, the cost of testing, and the level of risk of M. paratuberculosis infection they are willing to tolerate. When sellers are unwilling to have pre-purchase testing as condition of the sale, the owner may elect to purchase the animal and quarantine it on the farm until testing is completed.

    Purchase of animals from herds 100% negative by a whole-herd ELISA or fecal culture, with testing done in the previous 12 months, gives a high level of confidence any individual animal raised in the herd is free of M. paratuberculosis infection. Whole herd test results, using reliable tests done by qualified laboratories, gives far more confidence about the true infection status of animals than do tests run on individual animals. Given how common the M. paratuberculosis infection is in dairy cattle world-wide, it is probably less risky to buy test-negative cattle from herds with a low percentage of test-positive animals than it is to buy untested animals.

    Pre-purchase testing of individual animals for paratuberculosis is the absolute minimum that should be done to prevent introduction of M. paratuberculosis into a herd. The cost of testing is so small in comparison with the cost incurred once the infection is established in the herd, that it is illogical not to test.

    Today, the fastest, most accurate, and least costly pre-purchase test is the ELISA. Minimally, the animal(s) purchased should be tested. Preferably the entire herd from which the animal(s) originate should be tested. Even greater confidence in the Johne's-free status of purchased animals could be had by doing fecal cultures on the purchased animals in addition to the ELISA, even though results would not normally be available until after the animal(s) were already on the buyers farm.

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  8. Export testing
    Testing requirements for export of animals are set by the receiving country. While the CF test is the one most commonly required, the ELISA likely will soon be accepted as an alternative. Requirements to use the intradermal or intravenous Johnin test should be resisted because of the high rate of false-negative and false-positive results.

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Print references

  1. Bech-Nielsen, S., J. B. Jorgensen, P. Ahrens, and N. C. Feld. 1992. Diagnostic accuracy of a Mycobacterium phlei-absorbed serum enzyme-linked immunosorbent assay for diagnosis of bovine paratuberculosis in dairy cows. J.Clin.Microbiol. 30:613-618.

  2. Benedictus, G. and J. Haagsma. 1986. The efficacy of mesenteric lymph node biopsy in the eradication of paratuberculosis from an infected farm. Veterinary Quarterly 8:5-11.

  3. Burnside, D.M. and B. O. Rowley. 1994. Evaluation of an enzyme-linked immunosorbent assay for diagnosis of paratuberculosis in goats. Am.J.Vet.Res. 55:465-466.

  4. Collins, D.M., D. M. Stephens, and G. W. de Lisle. 1993. Comparison of polymerase chain reaction tests and faecal culture for detecting Mycobacterium paratuberculosis in bovine feces. Vet.Microbiol. 36:289-299.

  5. Collins, M.T. 1995. Diagnosis of paratuberculosis. In Paratuberculosis. RW Sweeney (ed.) Vet.Clin.North Am.Food Anim.Pract. 12:357-371.

  6. Collins, M.T., K. B. Kenefick, D. C. Sockett, R. S. Lambrecht, J. McDonald, and J. B. Jorgensen. 1990. Enhanced radiometric detection of Mycobacterium paratuberculosis using filter concentrated fecal specimens. J.Clin.Microbiol. 28:2514-2519.

  7. Collins, M.T., D. C. Sockett, S. Ridge, and J. C. Cox. 1991. Evaluation of a commercial enzyme-linked immunosorbent assay for Johne's disease. J.Clin.Microbiol. 29:272-276.

  8. Cox, J.C., D. P. Drane, S. L. Jones, S. Ridge, and A. R. Milner. 1991. Development and evaluation of a rapid absorbed enzyme immunoassay test for the diagnosis of Johne's disease in cattle. Austr.Vet.J. 68:157-160.

  9. de Lisle, G.W., B. S. Samagh, and J. R. Duncan. 1980. Bovine paratuberculosis II. A comparison of fecal culture and the antibody response. Can.J.Comp.Med. 44:183-191.

  10. de Lisle, G.W., P. Seguin, B. S. Samagh, A. H. Corner, and J. R. Duncan. 1980. Bovine paratuberculosis I. A herd study using complement fixation and intradermal tests. Can.J.Comp.Med. 44:177-182.

  11. Dubash, K., W. P. Shulaw, S. Bech-Nielsen, H. F. Stills, and R. D. Selmons. 1996. Evaluation of an agar gel immunodiffusion test kit for detection of antibodies to Mycobacterium paratuberculosis in sheep. J.Am.Vet.Med.Assoc. 208:401-403.

  12. Fawcett, A.R., P. J. Goddard, W. A. C. McKelvey, D. Buxton, H. W. Reid, A. Greig, and A. J. Macdonald. 1995. Johne's disease in a herd of farmed red deer. Vet.Rec. 136:165-169.

  13. Hietala, S.K. 1992. The options in diagnosing ruminant paratuberculosis. Vet.Med. 87:1122-1131.

  14. Milner, A.R., W. N. Mack, and K. J. Coates. 1989. A modified ELISA for detection of goats infected with Mycobacterium paratuberculosis. Austr.Vet.J. 66:305-306.

  15. National Veterinary Services Laboratory Staff, 1986. Microtitration complement fixation techniques, p.1-18. National Veterinary Services Laboratory, Ames, Iowa.

  16. Sherman, D.M. 1985. Current concepts in Johne's disease. Vet.Med. 80:77-84.

  17. Sherman, D.M., J. M. Gay, D. S. Bouley, and G. H. Nelson. 1990. Comparison of the complement fixation and agar gel immunodiffusion tests for the diagnosis of subclinical bovine paratuberculosis. Am.J.Vet.Res. 51:461-465.

  18. Sockett, D.C., D. J. Carr, and M. T. Collins. 1992. Evaluation of conventional and radiometric fecal culture and a commercial DNA probe for diagnosis of Mycobacterium paratuberculosis infections in cattle. Can.J.Vet.Res. 56:148-153.

  19. Sockett, D.C., T. A. Conrad, C. B. Thomas, and M. T. Collins. 1992. Evaluation of four serological tests for bovine paratuberculosis. J.Clin.Microbiol. 30:1134-1139.

  20. Sweeney, R.W., R. H. Whitlock, C. L. Buckley, P. Spencer, A. E. Roseberger, and L. J. Hutchinson. 1994. Diagnosis of paratuberculosis in dairy cattle, using enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in milk. Am.J.Vet.Res. 55:905-909.

  21. Sweeney, R.W., R. H. Whitlock, C. L. Buckley, and P. A. Spencer. 1995. Evaluation of a commercial enzyme-linked immunosorbent assay for the diagnosis of paratuberculosis in dairy cattle. J.Vet.Diagn.Invest. 7:488-493.

  22. Vary, P.H., P. R. Andersen, E. Green, J. Hermon-Taylor, and J. J. McFadden. 1990. Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's disease. J.Clin.Microbiol. 28:933-937.

  23. Whipple, D.L., D. R. Callihan, and J. L. Jarnagin. 1991. Cultivation of Mycobacterium paratuberculosis from bovine fecal specimens and a suggested standardized procedure. J.Vet.Diagn.Invest. 3:368-373.

  24. Yokomizo, Y. 1986. Evaluation on an enzyme-linked immunosorbent assay (ELISA) using Mycobacterium phlei-absorbed serum for the diagnosis of bovine paratuberculosis in a field study. Jpn.Ag.Res.Quarterly 20:60-67.
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Last revised April 9, 1999.
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