Clinically, the only sign of M. paratuberculosis infection may be weight loss despite a good appetite and adequate rations. The weight loss is often attributed to other causes (enteroparasites, teeth problems, etc), some of which may exist concurrently. Diarrhea may not be observed, although it does occur in some cases. These clinical signs usually appear months to years after the animal is infected. This clinical phase may last for some weeks to months, although the progression of the disease to death appears to faster in younger animals.		Johne's disease, or infection by M. paratuberculosis, can be diagnosed in elk by isolating the organism from manure or tissue collected at necropsy and by blood tests in some cases. Your veterinarian can assist you in choosing the best diagnostic test(s) for your situation.

Diagnostic tests are valuable tools used to control the spread of Johne's disease. It is true however that the biology of this disease makes diagnosis challenging: again, the animal may not appear ill nor produce consistent, specific and long-lasting 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 in the manure only intermittently. This means the particular manure sample collected may not contain M. paratuberculosis. An animal with this infection may also produce a negative blood test result. 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 deer/elk may be at an earlier phase of the infection and has yet to make any antibody, of course none is found and the test result is negative.

False-positive results can also occur since other infections may elicit the same response in the animal as M. paratuberculosis. In deer/elk, this may occur if the animal is infected with a close relative of M. paratuberculosis such as M. avium. There are however a number of effective Johne's disease tests, especially when used on a herd basis and for clinically affected animals, that have helped animal managers detect and control this infection.

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 deer/elk is showing 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 on the market, but they were developed to be used in cattle. 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. None of these blood tests have been validated for cervid species. The tests are:

The current lack of assay validation means that the ability of the tests to distinguish accurately between infected and uninfected deer/elk has not been statistically determined, a process that requires testing hundreds of known-infected and known-uninfected animals. (Research is underway to accomplish this for some cervid species. It is not known if an assay that works well for one cervid species will work equally well for another.)

Despite the lack of assay validation to date, the ELISA and AGID have been used to provide additional information about a clinically affected animal. A positive test result on one or both of these assays raises the index of suspicion, especially if the animal has clinical signs consistent with Johne's disease and comes from a herd with confirmed cases of M. paratuberculosis infection. Interpreters of these tests in elk should always consider the possibility of cross-reacting antibodies caused by an infection with a different organism. For this reason, blood test positive deer/elk should be tested by fecal culture to confirm the diagnosis by isolation the organism.

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 deer/elk. Because M. paratuberculosis is one of the slowest growing bacteria, it can take weeks for the organism to be obtained from the samples.

This assay provides the most direct "proof" of the infection because the living organism causing the disease is obtained. The identity of the organism obtained from the samples is confirmed with a PCR probe for a genetic 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 bacterium using 2 types of growth media. If the bacteria grows only in media with mycobactin, it is M. paratuberculosis. If it grows in media with mycobactin and media without it, the bacteria are not M. paratuberculosis.) This culture test is used in individual animals to confirm a Johne's disease diagnosis (based on clinical signs or a blood test) and is also used in groups of animals to assess the infection status of the herd. Pooling of samples from 4-5 animals reduces the cost of herd testing, although pooling may also reduce the sensitivity of the test so that fewer infected animals may be detected.

Sample Collection:
Approximately one tablespoon of fresh manure should be collected directly from the rectum or from the ground soon after defecation. It should be placed in a clean plastic bag that can be sealed, labeled with the animal's ID, 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. ). The accuracy of this assay depends on the lab performing it, the type of sample for which the probe was developed and the number of mycobacterial organisms in the sample.

Effective surveillance for Johne's disease includes collection of tissue samples from at least the ileum and mesenteric lymph node(s) (for both microscopic examination and for culture) from animals dying on-site. Protocols have been developed to use the PCR IS900 probe with histopathology slides in some cases. These tissues should be collected even if no lesions are visible at gross necropsy, especially if there have been any prior confirmed or suspected cases of Johne's disease in the herd.

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.