Special culture methods and new blood tests are used to diagnose Johne's disease in sheep.

Sheep are one of the more difficult species in which to diagnose this infection. Clinically, the only sign of infection may be weight loss which is frequently masked by the fleece. Even if noted, the weight loss is often attributed to other causes (enteroparasites, teeth problems, etc). Diarrhea is not a frequent sign of Johne's disease in sheep although it can occur in some cases.

The biology of this disease makes diagnosis challenging: the animal may not appear ill nor produce consistent, specific and long-lasting immunologic signs of the infection until months after the infection occurs. This means that test results may be negative although the animal is truly infected. For instance, a "false negative" fecal culture test result may occur since the organism is shed only intermittently. This means the particular manure sample collected may not contain M. paratuberculosis although the animal is truly infected. Another example is a negative blood test result for an animal with Johne's disease. This "false negative" result usually occurs because the element the test is looking for (antibody) is not produced by the animal until late in the disease: since the infected animal may be at an earlier phase of the infection and has not made any antibody yet, the test result is negative.

There are however a number of effective tests for Johne's disease diagnosis that have helped animal managers detect and control the infection, especially on a flock basis and for clinically affected animals.

While it may appear taxing to investigate all deaths on site, if there is any possibility of M. paratuberculosis infection in the herd it is worth the "up-front" effort to find all cases and block further transmission rather than suffer the much more costly process of controlling the infection once it has spread.

Blood tests:

The production of antibody is called humoral immunity. In Johne's disease, this type of immunity neither clears the infection nor slows its progress. Production of antibody is thought to be a late stage event in the course of the infection. When it is detected, it is probable that the sheep is or soon will show signs of disease and likely is shedding M. paratuberculosis in its manure and perhaps its milk/colostrum.

There are three blood tests available. They differ somewhat in their mechanics but each is designed to accomplish the same thing: detect antibody in serum produced by the animal in response to M. paratuberculosis infection. The tests are:

The most commonly used blood tests in sheep are the ELISA and the AGID. It is thought that AGID sensitivity is comparable to the ELISA for clinically affected sheep. Specificity may be higher for the AGID than the ELISA (false-positive results may occur more often with the ELISA due to cross-reacting antibodies caused by infection by other organisms such as Corynebacterium pseudotuberculosis, the cause of caseous lymphadenitis - "CLA", or other mycobacteria). The CF test remains the M. paratuberculosis infection screening test usually requested by the importing country for animals being shipped internationally.

Sample collection:
Blood should be collected in a serum separator or "red top" tube, centrifuged promptly and the serum submitted to the laboratory.

Isolation of the organism:

The organism causing Johne's disease, M. paratuberculosis, can be isolated ("cultured") from manure or tissues sampled from sheep. Because M. paratuberculosis is one of the slowest growing bacteria, it can take weeks to months for the organism to be cultured from the samples. This is particularly true for some of the strains infecting sheep - they can be even slower to grow to a detectable level in the culture than M. paratuberculosis strains affecting other species.

This assay provides the most direct "proof" of the infection because the actual organism causing the disease is found. The identity of the organism grown from the samples is confirmed with a genetic probe for an insertion element believed to be unique to M. paratuberculosis (IS900 - see below) or through mycobactin dependency testing. (Since the organism doesn"t grow unless provided a substance called mycobactin, labs can identify the bacteria using 2 types of growth media. If the bacteria grows only in media with mycobactin, it is M. paratuberculosis link photo. If it grows in media with mycobactin and media without it, the bacteria is not M. paratuberculosis)

This culture test is used in individual animals to confirm a Johne's disease diagnosis and is also used in groups of animals to assess the infection status of the flock. In Australia, pooling manure samples from large flocks has been shown to reduce the overall cost of testing while still providing managers with a good sense of the prevalence of the infection in the flock.

Sample collection:
Fresh pellets should be collected directly from the rectum or from the ground soon after defecation. They should be placed in a clean plastic bag that can be sealed, labeled, kept cool and shipped overnight to the diagnostic lab.

Identification of M. paratuberculosis DNA:

A component of genetic material, i.e. an insertion element called IS900, is believed to occur in M. paratuberculosis DNA only. This insertion element is therefore used as a marker for infection with this mycobacterial species. PCR amplification and molecular probes for IS900 can be applied directly to samples thought to contain the organism (manure, both formalin fixed and fresh tissue, water, milk, etc. ). As there may be PCR inhibitors in biological samples, this method is usually reserved for identifying mycobacteria obtained through culturing the samples first. Another approach is to use an immunomagnetic separation technique to concentrate the organisms prior to applying the probe.