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JD testing and diagnosis

Animal Health Australia (AHA) coordinates the National Johne’s Disease Project for Australia’s livestock industries.

How to diagnose Johne’s disease (JD)

The diagnostic challenge of JD is detecting animals that are subclinically infected (asymptomatic carriers of the disease-causing bacteria) before they start to become infectious. This is one of the reasons that the Australian program focuses more on herd risk and herd-level assurance.

Tests for JD indicate the presence of the actual bacteria or the animal’s response to bacteria in their system.

Animals are tested in two stages:

  1. screening test of a large sample of mature animals from the herd
  2. test of animals that showed a reaction to the bacteria.

Tests are available for JD in cattle, including tests on blood, faeces and tissues. But most tests have very poor sensitivity (ability to correctly identify infected animals) early in the onset of the disease or in young animals.

False positive results can occur in the blood test, more commonly in northern Australia due to cross-reactions with other bacteria that cattle have picked up from their environment.

To improve the accuracy of diagnosis, tests are often used in combination. For example, initial screening by blood test, followed by faecal culture, PCR (polymerase chain reaction) or a post-mortem tissue test on reactor animals.

The sensitivity of these tests increases as the disease progresses, even before signs of disease are noticeable.

Types of screening tests for JD in cattle

Screening tests to test for JD in live animals.

Antibody tests (serological or blood tests)

The main antibody-screening test used for JD is the absorbed ELISA (enzyme-linked immunosorbent assay) test in cattle. There is also a gel or AGID (agar gel immunodiffusion) test for goats. The blood test detects antibodies to the Johne’s disease bacteria, and results are reported relatively quickly (1–2 weeks).

Individual faecal culture test

Dung or faecal samples collected from individual animals are cultured for the bacteria. This is more expensive and slower than antibody tests (2–5 months), depending on the culture method used.

Faecal culture is used as a definitive test to confirm infection in cattle that react to the ELISA, and it can be used with antibody tests in cattle herds to increase the chance of detecting infected animals. Faecal culture is used for alpaca and deer, which have no other approved antibody tests.

Pooled faecal culture (PFC) test

Faecal samples from up to five cattle may be cultured in one pool as a herd-screening test. While the PFC test still takes 2–5 months, it is a cheaper herd test option than individual faecal cultures. If a positive pool is identified, then the individual animals need to be cultured to identify the source of infection.

Herd environmental culture (HEC) test

HEC is another type of faecal test that has been approved by Animal Health Committee’s former Sub-Committee on Animal Health Laboratory Standards (SCAHLS) for use in dairy herds as a:

High Throughput-Johne’s (HT-J) PCR test

Faecal samples from up to five animals may be tested in one pool as a herd-screening test. A much quicker test than the PFC with similar accuracy.

Follow-up tests

Any animals or groups of animals that test positive to an antibody test are tested again with a definitive test to confirm the result. For instance, ELISA reactors are tested by individual faecal culture or by a post-mortem examination, microscopic examination of the gut tissue and possibly culture of gut tissue. These tests detect the actual bacteria or the characteristic microscopic lesions that they cause.

Testing of herds

Check Test

A Check Test is either:

• 50 adult animals in the herd (or all eligible animals in a herd of less than 50 adult animals) biased to increase the probability of detecting infection, tested by pooled faecal culture, pooled faecal HT-J PCR or ELISA [not for WA entry]; or

• a HEC test in dairy herds that comply with the requirements above.

A Check Test is positive only if infection is confirmed in the herd.

Sample Test

Screening of the adult herd or a large representative sample of the adult herd by an approved test (pooled faecal culture or pooled faecal HT-J PCR, or ELISA [not for WA entry]), with follow-up faecal culture or tissue culture and histopathological investigation of any reactors (if appropriate). The cattle to be tested are selected from the herd in accordance with Appendix 2 of the JD in cattle Definitions and Guidelines document. Where a Sample Test comprises a screening test, the test is not complete until any reactors have been further investigated using a definitive test to establish the infection status of the herd (i.e. once infection is confirmed in one animal on one definitive test, it is not necessary to continue testing all reactors with definitive tests).

A Sample Test is positive only if infection is confirmed in the herd.

Maintenance Test (only for Western Australia)

Maintenance testing requires random sample selection of 50 adult cattle that are 4 years of age and older. Maintenance testing is done every 34-38 months after the previous negative Sample Test or Maintenance Test.

All testing by (pooled) faecal culture or (pooled) HT-J faecal PCR.

 

Table 1: Advantages and disadvantages of approved tests to diagnose JD in cattle

Test Use Advantages Disadvantages
Blood test (ELISA)—tests for disease antibodies Screening animals older than 2 years for export or clinical cases Rapid result

Relatively cheap

Useful herd test (but not reliable as an individual animal test)

Low sensitivity1 for individual animal tests; can fail to detect infection when it is present

Moderate sensitivity for herd tests

Imperfect specificity2 results in false positive reactors, requiring further investigation (especially in northern Australia)

Faecal culture—tests for bacteria in faeces Confirming infection in suspect3 animals Highly specific

Better sensitivity than blood test

Use as pooled test to reduce cost and increase throughput

Takes 2–5 months to get a result

Relatively expensive compared to ELISA

HT-J PCR— tests for DNA of bacteria in faeces Confirming infection in suspect3 animals Highly specific

Better sensitivity than blood test

Rapid result (one to two weeks)

Use as pooled test to reduce cost and increase throughput

Expensive compared to ELISA (similar to faecal culture)

Requires follow-up tests to confirm infection in herds not previously confirmed or suspect

Histology—microscopic examination of tissue samples from gut Confirming infection in suspect animals High specificity

High sensitivity

Requires animal slaughter and immediate collection of samples at abattoir or farm
Tissue culture—culturing tissue samples from gut Confirming infection in suspect animals High specificity

High sensitivity

Requires animal slaughter and immediate collection of samples at abattoir or farm

1 Sensitivity is the ability to give a positive result in an infected animal.

2 Specificity is the ability to give a negative result in an animal that is not infected.

3 Suspect animals are those with clinical signs of JD or who have returned a positive blood test (ELISA).

FAQs about Johne’s disease testing and diagnosis

How accurate are current Johne’s disease tests?

To be accurate, a test must not only be good at detecting infected animals (sensitivity) but also at correctly giving non-infected animals a negative result (specificity).

The sensitivity of Johne’s diease (JD) tests overall is low, (i.e. the rate of false negatives is high), especially in young or recently infected animals. Sensitivity increases as the infection progresses in the animal, so the probability of missing the infection (false negative results) decreases in older animals or in animals in the advanced stage of the disease.

Testing groups of adult animals increases the probability of detecting the infection in the herd or flock to an acceptable level.

Current JD tests have adequate sensitivity when used as a herd or flock test, but a negative test on an individual animal gives very little assurance that the animal is not infected.

The specificity of JD tests is very good, so false positive results are very rare. On average in Australia:

  • AGID test (used mainly in sheep) produces about 1 false positive in 1000 tests
  • ELISA (used mainly in cattle) produces about 2 false positives in 1000 tests.

One ELISA test (Parachek®) is approved for use in sheep and goats, but has a higher false positive rate (about 1 false positive per 100 tests).

There is a very small chance of a result being false positive; so all reactors to screening tests are followed-up.

The faecal tests have better sensitivity than the blood tests (ability to find diseased animals) and a very low chance of a false positive result.

Why aren’t animals tested when they are young?

Even though most animals become infected when they are young, the Johne’s bacterium, Mycobacterium paratuberculosis, grows very slowly.

In the early stages of the disease, the immune system is not stimulated strongly enough for it to produce detectable antibodies, so blood tests are usually negative.

Similarly, little or no shedding of bacteria in the faeces occurs early in infection, so faecal culture tests will also be negative.

So, although most animals get infected when they are young, it’s not useful to test an animal until it’s at least 2 years old when the current tests are more likely to detect the disease. (Research is being undertaken to see if the minimum age for testing can be lowered.)

The exceptions to this are deer and alpaca, where disease has progressed more quickly in cases in Australia.

Goats are tested for the GoatMAP at a minimum age of 12 months, but the check test applies to goats 2-years-old and over.

Why are groups of animals tested, rather than individual animals?

Sampling rates for the MAPs and for surveillance programs are designed to give a high probability of detecting infection, even if a small proportion of the tested animals are infected. Finding one infected animal indicates that there is JD infection in the herd or flock, as there will almost certainly be other infected animals in the early stages of the disease that have not reacted to the test, or were not tested.

Related links

 

Page reviewed: May 19, 2017