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Johne's Disease

Johne's Disease is a disease of cattle resulting from a prolonged course of infection caused by a bacterium recently reclassified as Mycobacterium paratuberculosis sbsp. avium. It is an acid-fast organism that can also affect sheep, goats, other ruminants, swine and horses. However, lesions in swine and horses are minimal to none at all. In cattle, the disease has a long progression with no clinical signs for months to years after initial infection. It can eventually progress to a chronic wasting form with diarrhea, emaciation, and eventual death.

The infection is usually acquired in young animals by fecal-oral contamination. Some cattle can be infected in utero if born from infected cows and contaminated mother's milk can be a source of infection. A larger initial dose causes a shorter incubation period. Once infected, the organism replicates slowly in the intestinal mucosa. Over time, as macrophages are recruited to the area and the organisms continue to slowly multiply, the intestinal mucosa and submucosa become thickened and less able to absorb nutrients. The thickened wall eventually begins to leak proteins resulting in a hypoproteinemia. Weight loss and diarrhea result from the malabsorption and the albumin leakage.

In the first stage of infection, the adults that were affected as young still show no clinical signs, but antibodies may be detectable. Immunosuppression may also occur predisposing the animal to other diseases. Though the fecal culture is usually negative in this stage, shedding to the environment is possible. These animals pose an infection risk for other animals.

In the third stage of infection, clinical signs begin to appear. Gradual weight loss and diarrhea ensue. Emaciation can develop gradually and milk production decreases. Organism numbers are higher and more likely to be found in the feces. Antibody levels are also higher making them more readily detectable.

In the last stage of Johne's disease, clinical signs are more evident and the animal's condition becomes more severe. Weakness, emaciation and "pipestream" diarrhea are usually present.

Diagnosing Mycobacterium paratubercu-losis infection can be very difficult depending on the stage of infection. Obviously, since earlier (non-clinical) stages have lower organism counts, fewer organisms are present to be shed in feces or to trigger higher antibody production. This translates to decreased detection in the early stages. By the time the numbers are high enough for detection in the later stages, the likelihood of organism spread to the environment and to other animals is already high. For every cow that shows clinical signs, there are potentially several others that are infected due to environmental contamination. Moreover, since treatment is not efficient, infected animals are culled causing further economic losses.

Despite the problems associated with disease staging, there are several tests available to diagnose Johne's Disease in cattle. Fecal culture is one of these tests. It is considered the "gold standard" for diagnosis. The high specificity of the culture makes it a popular test. The culture test has been shown to detect organisms 1-4 years prior to the development of clinical signs, giving it a relatively high sensitivity. Yet, if an infected animal is not shedding at the time of sampling or is shedding low numbers, the culture may miss the true status of the animal. Another major disadvantage of the test is that completion takes from 12-16 weeks.

Other methods used for diagnosis involve serological testing (ELISA, AGID and complement fixation). These tests take a relatively short time to produce results and several samples can be processed each day. But the earlier the stage of infection, the lower the sensitivity will be. It is not until later stages that serum changes become adequate enough for these tests to gain respectable sensitivities.

Enzyme-linked immunosorbent assay (ELISA) measures serum antibodies directed against Mycobacterium paratuberculosis. Because cattle are exposed to mycobacterial antigens from several different mycobacterial species, the specificity of this test is relatively low. Sensitivity, however, is considered relatively higher (50%) with ELISA than with the other serum tests and, if used with other diagnostic tests (e.g. fecal culture, DNA probe), is considered a good tool for herd screening.

Agar gel immunodiffusion (AGID) is considered highly specific (100%). Positive test results correlate well with clinical signs. But the sensitivity is low for this test. If the animal is not showing clinical signs, the likelihood of a positive result is low (less than 50%). When comparing AGID with ELISA, AGID is better for diagnosing infection in animals that are already showing clinical signs. On the other hand, ELISA is better for detecting infection in animals without clinical signs, with localized infections and low-level mycobacterial burden.

Complement fixation also has low sensitivity in lightly infected animals; however, positive results correlate well with active shedding of organisms. It must be noted that false positives have been experienced when using this test.

Cell-Mediated Immunity (CMI) is the first and strongest response to mycobacterial infections. Tests to detect CMI, which is mediated by T-lymphocytes, have low specificity but high sensitivity. Skin testing often misidentifies M. tuberculosis infections since the organism shares several antigens with other environmental mycobacterial species and the potential for cross-reactions with these is great. Tests that measure paratuberculosis specific gamma interferon, a sensitized T-lymphocyte mediator, have been shown to have higher sensitivities than other serological tests, but specificity is still low.

PCR testing represents a newer form of diagnosis and is used to detect Mycobacterium parabuterculosis in the feces of infected animals. At the Purdue ADDL, we have combined PCR with culturing to give better accuracy and shorter time for results. It is also highly specific (nearly 100%). However, it is dependent upon active shedding in the feces and subclinical infections may be missed.

In more than half of affected cattle, gastrointestinal lesions have been shown to extend to the rectal mucosa and colon. This occurrence offers the opportunity for diagnosis via rectal scrapings. Gently scraped mucosa is applied to and air dried on glass slides, then stained for acid-fast organisms (e.g. Ziehl-Neelsen stain). Microscopic exam should be able to detect the presence of clumps of acid-fast organisms still inside macrophages. Histopathology of the ileocolic lymph node and terminal ileum should also show the same acid-fast clumps inside the macrophages of an infected animal. These microscopic signs are enough to declare a positive diagnosis. These microscopic diagnostic methods are useful when testing an individual animal suspected of having Johne's disease. Also, herd culling can influence test performance. For example, if all of the positive and/or clinical animals have been removed from the herd, the remaining population will be all non-clinical and more difficult to diagnose. These and other factors (such as testing expense) must be considered when deciding on diagnostic testing to identify mycobacterial infection in a herd.

-by Lee Winnette, Class of 2002
-edited by Marlon Rebelatto, ADDL Graduate
Student

References
Collins MT, 1996: Diagnosis of Paratuber-culosis. Vet Clin North America: Food Animal Practice. 12: 357-369.

Hope AF, Kluver PF, Jones SL, Condron RJ, 2000: Sensitivity and Specificity of Two Serological Tests for the Detection of Ovine Paratuberculosis. Australian Vet J. 78: 850-855.

Jones T, Hunt R, King N, 1997: Veterinary Pathology, 6th ed. 498-500

Smith BP, 1996: Large Animal Internal Medicine 2nd ed. 899-903.

Step DL, Streeter RN, Kirkpatrick JG, 2000: Johne's Disease Update. Bovine Practitioner. 34: 6-11.

 

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West Lafayette, IN 47907
Phone: 765-494-7440
Fax: 765-494-9181

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Dubois, IN 47527
Phone: (812) 678-3401
Fax: (812) 678-3412

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