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
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
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
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
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
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