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  Not all tests are validated for all animal species. Check the web pages for specific animal species for their testing information. Types | Accuracy | Standard culture | BACTEC culture | DNA probe | CF test | AGID | ELISA | Skin test | Interferon | Which to use | When to test |
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How many and what types of tests are there?
It is a common
misconception that there are no accurate diagnostic tests for Johne's disease.
There are, in fact, 8 available tests: 3 methods for detecting the bacterium that
causes Johne's disease (Mycobacterium paratuberculosis), 4 blood tests
for detecting serum antibody, and 2 assays for cellular immunity. Five of these
tests are commercially available from multiple companies for use by veterinary
diagnostic laboratories worldwide. A few private laboratories have tests they
develop themselves. Some tests are simple enough to be able to be done in a veterinary
clinic. However, most require sophisticated laboratory equipment and skilled laboratory
technicians to be performed. In addition, factors such as the amplitude of a test
result (numerical value) and herd infection rate (prevalence) can influence interpretation
of Johne's disease test results. Consequently, it is recommended test interpretation
be made by veterinarians who are experienced in laboratory diagnostics. Most research
has been completed and hence most information is available about Johne's disease
in cattle so the diagnosis topic is most extensively discussed under the "Dairy"
and "Beef"
areas of interest. Few blood tests have been validated for animals other than
cattle and fecal culture (standard or BACTEC methods) remains the most well-accepted
diagnostic test for most non-bovine species.
In general, tests for Johne's disease have a high specificity : low rate of false-positive results. To avoid making management decisions based on false-positive blood tests, fecal culture can be completed as a "confirmatory" test for blood test positive animals . This, however, has some draw backs that are discussed on the Dairy-Diagnosis pages. Test sensitivity (percentage of infected animals that test positive) varies among tests, but due to the biology of this slowly progressing chronic disease is often less than 50%. In the very early stages of M. paratuberculosis infection, before animals start shedding the bacterium in feces or begin an immune response to the infection, all animals will be test-negative. As the infection progresses, most tests will eventually become positive as the animal begins to produce the signals of infection detected by the tests (e.g. antibody in the blood, the organism in manure). Tests have maximum sensitivity when used for animals with clinical signs of the infection: diarrhea and/or weight loss. Unfortunately, exceptions to these generalizations are common since individual animals don't "read the book" and follow the disease pattern exactly. 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 for maximum infection detection sensitivity.
Tests for the causative bacterium, Mycobacterium 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 manure of uninfected animals, if detected, it is assumed to be causing an infection and pathology in the animal. There are three ways of detecting M. paratuberculosis: two for isolating the entire organism with culture (standard or BACTEC [radiometric]) and one for detecting the genetic material (DNA probe, sometimes called PCR).
Standard bacterial
culture has been used for almost 100 years. Many laboratories around the world
can perform this test although methods used in The Netherlands and Denmark are
different 
from
those used in most other countries. 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 $20-$30/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 and perhaps other non-bovine
species may 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 (another mycobacterial disease) in humans. The culture medium is commercially available but requires supplementation with additional nutrients to support M. paratuberculosis growth. The main advantage of this method is that it can detect low numbers of M. paratuberculosis, can detect the bacterium faster than standard culture methods (8 weeks compared to 16 weeks). In addition, it 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
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 earlier versions of this test have not been 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-$35 per test (unsubsidized).
Tests for antibodies to M. paratuberculosis in serum (blood tests)
Detection of serum antibody to M. paratuberculosis is good 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) , and 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 among 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. These three assays have primarily been assessed for cattle, plus sheep and goats to a lesser extent. Overall conclusions about the relative accuracy of each method are incorporated into recommendations made on this website under the Diagnosis topic for each animal species of interest.
This test is one of the oldest tests used for blood testing cattle for Johne's disease. It is falling out of favor and being replaced by newer methods that are more accurate and more easily standardized. Some countries, however, still require this test before allowing importation of cattle to their country.
Agar-gel
immunodiffusion (AGID)
The AGID was developed at the University of Minnesota. It is sold as a commercial kit (Rapid Johne's Test®, ImmuCell) and has also been adapted as a "home-made" assay in several laboratories. It is a simple test to run and requires no special equipment. The AGID also can be used on a wider variety of animal species although it has been more thoroughly evaluated on cattle. It rarely makes a mistake when it is positive (high specificity). However, it has a lower sensitivity in cattle and its use is primarily restricted to confirmation of a clinical diagnosis: i.e. to verify a diagnosis on animals with clinical signs of disease that look like Johne's disease (diarrhea and weight loss).
Enzyme-linked immunosorbent assay (ELISA)
ELISA is a technology for detection of antibodies. It is more sensitive than either the CF or AGID in cattle. It has a high specificity and is almost as sensitive as fecal culture at detecting infected animals. It is designed for testing large numbers of samples quickly and this makes it a low-cost test. Another advantage is that the test produces numerical results that are a measure of the quantity of antibodies to M. paratuberculosis in the serum sample tested. Research has shown that the level of antibody is useful in predicting the stage of infection (proven so far only in cattle using a single commercial ELISA kit). A disadvantage of the ELISA is that it requires skilled laboratory technicians and specialized laboratory equipment. Therefore it is only done at diagnostic laboratories vs. veterinary clinics. The ELISA has only been validated for cattle but work is underway to adapt this test to other animal species.
Tests for antibodies to M. paratuberculosis in milk (milk ELISA)
Revised 20-April-2006
No ELISA kit for paratuberculosis has been approved by the USDA for use with milk samples. One kit made in France by Institute Pourquier, http://www.institut-pourquier.fr/, claims to work effectively on both serum and milk samples. A U.S. company called AntelBio® http://www.antelbio.com/ offers ELISA testing on milk samples as a service. They do not, however, sell a diagnostic kit nor have they provided any data about the performance characteristics of the assay. One peer-reviewed scientific study evaluated the accuracy of the milk ELISA for paratuberculosis offered by AntelBio. The assay was tested along side four serum ELISA kits with 364 cows with culture-confirmed cases of M. paratuberculosis infection and 352 cows in herds proven free of paratuberculosis. The study concluded that the accuracy of the milk ELISA for individual milk samples as performed by AntelBio was equal to or better than that of serum ELISA kits for the same cattle. (The results were reported June 2005 in the Journal Clinical and Diagnostic Laboratory Immunology. Click here to print a copy of that publication - link to pdf).
Important notes: Because AntelBio does not use a diagnostic kit that is USDA-licensed, there is no way to know if changes are made to the assay that affect its performance. It is also uncertain if other laboratories will be able to reproduce their assay and achieve the same level of accuracy. When testing milk samples from high-producing dairy cattle, it is best if the samples are collected at the beginning or end of lactation to avoid the peak milk production phase during which antibodies can be diluted leading to false-negative ELISA results.
Tests for a 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 at the injection site 2-3 days later. Skin testing has been valuable for diagnosing tuberculosis, another mycobacterial disease, in humans and animals. Early studies indicated skin testing did not work well for diagnosis of Johne's disease however. Investigators who have re-evaluated this question suggest that skin testing may indeed be valuable. More studies are needed to substantiate this finding.
A newer, 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 a chemical called gamma interferon released from white blood cells. The assay is now available as a diagnostic kit for cattle called Bovigam®. Few studies have been published yet evaluating its diagnostic accuracy. However, reports from scientific meetings indicate that the gamma interferon test will be a useful addition to the laboratory tools for diagnosis of M. paratuberculosis infections in a variety of species.
When choosing among available tests there are many considerations. Some are listed here and some of the more subtle ones are described on other pages under specific animal species. Still more considerations are understood by your veterinarian - use these animal health professionals to help you tackle a Johne's disease problem. Before testing, think about...
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1. | the animal species being tested |
| 2. | the age of the animal(s) being tested | |
| 3. | the clinical history of the herd (have any animals shown signs of Johne's disease) | |
| 4. | the goals of testing (control? eradication? diagnosis of an individual animal?) and the economics of the animal industry involved | |
| 5. | the accuracy of each test when used on individual animals or herds/flocks | |
| 6. | which would be more costly to you: false-positive or false-negative test results ? | |
| 7. | the epidemiology of Johne's disease. |
This website briefly explains the types of tests available for Johne's disease. How each test is performed is discussed briefly so that readers can better understand how to interpret the results. No specific commercial product endorsement is intended. For information about some of our sponsor's products please visit the sponsor's page, diagnostic products section.
When to use a laboratory test for Johne's disease:
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1. | Confirmation of a clinical diagnosis of Johne's disease |
| 2. | Confirmation of a positive result on a different laboratory test | |
| 3. | Estimation of the infection rate in a herd or flock | |
| 4. | Operation of a Johne's disease control program | |
| 5. | Herd certification | |
| 6. | Screening young animals (herd replacements) raised on the owner's farm | |
| 7. | Pre-purchase testing | |
| 8. | Export testing |
Diagnosis of Johne's disease can be challenging. Research and commercial interests are leading to rapid progress in this field. Watch for new and improved tests to become available in the near future.
Now, if you still want more information, read about Johne's disease diagnosis in dairy cattle or other species of interest.
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