A Paratuberculose,
uma doença causada pelo Mycobacterium avium subsp. paratuberculosis (Map), é caracterizada
por uma enterite crônica granulomatosa. Visamos registrar a
anatomopatologia de três casos de paratuberculose em bovinos leiteiros
autóctones oriundos de Resende-RJ. Esses animais apresentavam sinais
clínicos de paratuberculose e, após teste de ELISA positivo, foram
eutanasiados e necropsiados. Amostras de intestino, linfonodos
mesentéricos e válvula ileocecal foram colhidas, fixadas em formol
neutro tamponado a 10%, processadas
e coradas pelo HE (hematoxilina-eosina) e ZN (Ziehl-Neelsen).
Alterações macroscópicas, como espessamento da parede do intestino
delgado, mucosas congestas e corrugadas, foram observadas. A
linfadenomegalia e linfangectasia também foram evidentes, assim como a
congestão e edemaciação das válvulas ileocecais. Os principais achados
histopatológicos foram enterite, linfangite e linfadenite
granulomatosas. As lesões intestinais se resumiam à mucosa e submucosa,
principalmente do jejuno e do íleo e caracterizavam-se por infiltrado
inflamatório de linfócitos, eosinófilos e macrófagos epitelióides. A
coloração de ZN demonstrou bacilos álcool ácido resistentes (BAAR) nos
macrófagos, no ápice das vilosidades e lâmina própria e, ainda, no
parênquima dos linfonodos mesentéricos. A anatomopatologia foi
característica e considerada uma valiosa ferramenta no diagnóstico da
paratuberculose.
------------------------------
PALAVRAS-CHAVE: anatomopatológico; bovino;
Map; paratuberculose
INTRODUCTION
Paratuberculosis,
or Johne’s disease, is a chronic, infectious, granulomatous enteritis,
which affects domestic and wild ruminants (CHIODINI et al., 1984) and
whose etiologic agent is
Mycobacterium avium
subsp. paratuberculosis (
Map).
Paratuberculosis causes progressive emaciation, thickening
and corrugationof the small intestine’s wall,
particularly the last part of the small intestine, the ileum, besides
mesenteric
lymph node enlargement (RIEMANN et al., 1979; COLLINS,
1999). The histopathologic substrate of such inflammatory process is
constituted of an infiltrate of epithelioid cells and of scarce
Langhan’s giant cells present in the intestinal mucosa and submucosa,
which, however, do not reveal signs of necrosis or ulcerations (JONES
et al., 1996; CLARKE, 1997). Animals get infected with
Map
by contact with other infected animals’ manure, by
drinking milk or colostrum from an infected animal, or even from a
contaminated intrauterine environment (SWENEY, 1996).
Bovines
that develop this disease are, in general, those infected in their
first weeks of life. The disease manifests itself in animals older than
two years of age (SWENEY, 1996). Stressful situations as calf delivery
and nutritional deficiency may cause the appearance of the first
clinical signs of the disease (KREEGER, 1991). According to BLOOD et
al. (1989), the disease is rampant in confined animals, since the
environment is greatly contaminated with feces.
Paratuberculosis
is responsible for great economic losses in cattle breeding, due to
productivity reduction, greater susceptibility of the animals to other
diseases, rising of sanitary cost, and greater early discard rate. It
is considered nowadays one of the most serious and costly diseases to
affect dairy, beef, and, specially, pure animals (COLLINS, 1994;
HUTCHINSON, 1996).
The
most commonly used tests in the diagnosis of Paratuberculosis can be
divided in two main categories: methods to detect the causative
bacterium, such as bacterial culture, molecular methods with genetic
probes or PCR, and histological examination of target-tissues
confirming the presence of alcohol-acid resistant bacillus (BAAR); and
methods to detect antibodies in serum, being ELISA the most used one.
(BUERGELT & GINN, 2000). The isolation of the agent
from feces or tissues is the gold standard diagnostic method; however,
due to a lack of qualification of veterinary bacteriology laboratories
on mycobacteriology and to difficulties inherent to Map’s cultivation,
it is infrequently performed in Brazil (RISTOW
et al., 2007).
The
main macroscopic lesions appear in the small intestine characterized by
the thickening of the mucosa, which presents a typical corrugation of
mucosa, associated with mesenteric lymph node enlargement (GONZÁLEZ et
al., 2005). The most relevant histopathological
findings consist of granulomatous enteritis, lymphangitis and
lymphadenitis, associated with BAAR inside macrophages (CHIODINI et
al., 1984; KREEGER, 1991).
A
differential diagnosis of Paratuberculosis and other chronic processes
which lead to progressive weight loss (cachexia), such as malnutrition,
tuberculosis, iron and cobalt deficiency, molybdenum intoxication,
parasitosis, and chronic liver diseases is necessary (BLOOD et al.,
1989).
The
purpose of this work was to report the anatomopathology of three cases
of paratuberculosis in diagnosed bovines from Resende, Rio de Janeiro,
Brazil.
MATERIAL AND METHODS
Three
bovines from two dairy farms (A and B), located in Resende, Rio de
Janeiro, Brazil, with were euthanized and necropsied. Farm
(A), which produces type B milk through a semi-intensive system, had a
herd of approximately 340 half-breed Girolando cattle, where the
systems of stud farm and artificial insemination with semen imported
from Argentina and Uruguay are used. Farm (B) also produces milk, only
that with a semi-intensive system for the production of type C milk.
Its herd summed up approximately 150 half-breed Girolando cattle. A
significant number of cattle were bought from farm A.
The
animals showed clinical symptoms of the disease such as chronic
diarrhea and cachexia and were reactive to the
serum ELISA test. Serum was tested with a previously standardized
indirect ELISA (MARASSI et al., 2005). Briefly, the test used
paratuberculosis protoplasmatic antigen (PPA Allied Monitor), with
serum pre-absorbed with M. phlei, and an anti-bovine IgG conjugate
(Sigma–Aldrich Laboratories). Optical density (OD) was measured at a
wavelength of 405 nm, and a cut-off point of 0.350 was chosen as the
value giving the most accurate division of positive and negative sera.
The animals that presented characteristic clinical symptoms of
paratuberculosis and were seroreactive to ELISA were euthanized and
necropsied.
Samples
of small intestine (duodenum, jejunum, and ileum) and large intestine
(colon, cecum, and rectum), mesenteric lymph nodes, and ileocecal valve
were collected and fixed in 10% neutral buffered formalin, processed in
the automatic tissue processor Leica TP-1020 for the inclusion of
paraffin. Sections of 5 µm were cut and stained by the routine method,
haematoxilin-eosin (HE) and special, Ziehl-Neelsen (ZN).
RESULTS
Two
animals (animals 1 and 2) out of the three animals with suspicion of
paratuberculosis belonged to the same farm (farm A). According to the
farm owner, decreases in the production of milk along with chronic
diarrhea episodes in adult animals have been observed, at least over
the six years, followed by death due to diarrheic frame and progressive
weight loss.
Animals
1 and 2 were approximately five-and-six year old Girolando females,
which had been suffering from chronic diarrhea, progressive weight
loss, and emaciation for six months. Appetite and reproductive
functions were normal, and, despite vermifuged and treated with
antibiotics, the animals were cachectic, with bristled and dull fur,
and showed no clinical improvement.
Animal
3 belonged to farm B and was a two-year-old half-breed male. Despite
normal appetite, it had been giving away, for a month, evident signs of
cachexia, apathy, liquid, green and mucus diarrhea. The farm owner
stated the death of 12 animals with chronic diarrhea and cachexia from
2001 through 2002. The main clinical suspicion was of paratuberculosis.
The
three animals, in awful nutritional state, were necropsied. It was
possible to examine thickened intestine walls in all three of them,
especifically the small intestine. In animal 3, thickening occurred in
both small and large intestines. There were as well adherence between
intestinal loops, hyperemia, mucus and severe corrugation on mucosal
surface in various segments of the intestine (
Figure 1).
Lymphadenomegally and lymphangiectasia were evident in all three cases,
as well as congestion and emaciation in the ileocecal valves.
The main findings were observed in the
small intestine, in the mesenterics lymph vessels and lymph nodes,
representing granulomatous enteritis, lymphangitis and lymphadenitis.
Intestinal lesions were restricted to the mucosa and
submucosa, mainly in the jejunum and in the ileum and were
characterized by lymphocytes, eosinophils, and epithelioid macrophages
infiltrates (
Figura2). Granulomatous reaction
was evident mostly on the
top of villi and lamina propria (
Figura 3).
By special stain ZN, acid fast bacilli (AFB) were
observed in macrophages, most of the times in the form of cluster on
the intestinal mucosa, especially on the top of villi and lamina
propria (
Figure 4) and still on the
mesenteric lymph nodes parenchyma.
At microscopia of mesenteric lymph nodes revealed
lymphangitis with granulomatous inflammation on cortical and medullar
with cell infiltrate with numerous epithelioid macrophages (
Figura 5).
DISCUSSION
Clinical
suspicion of paratuberculosis in the three animals was confirmed based
on the clinical signs, positive ELISA tests, on necropsies and
histopathological findings and through gold diagnosis, that is
isolation of the etiologic agent, conventionally confirmed by
mycobactin J dependency tests. Clinical signs as well as macroscopic
and microscopic alterations in the three animals were similar to the
ones described in cases of bovine paratuberculosis (JONES
et al., 1996; CLARKE, 1997; COLLINS,1999). The chronic diarrhea
episodes, intermittent at times, were also evidenced in this study,
according to BLOOD et al. (1989), WHITLOCK and BUERGELT (1996).
In the described cases
of paratuberculosis, the presence of AFB in the macrophages and
epithelioid cells are common findings, whereas in these investigation
samples, a great quantity of AFB could be observed in the lesions of
the intestinal mucosas and submucosas, which clustered in bacilli,
similar to the ones observed by CARRIGAN and SEAMAN, 1990; CORPA et
al., 2000, RISTOW et al., 2008.
The paratuberculosis
characteristic clinical signs, based on macroscopic and microscopic
changes, which were present in the animals studied, were in accordance
with the ones that have already been described in other bovines
infected by
Mycobacterium avium subsp.
paratuberculosis
(CHIODINI et al., 1984; GONZÁLEZ et al., 2005). The paratuberculosis
case diagnosis in this study was based on epidemic data assessment of
the clinical signs, the necropsy and the microscopic findings. The
lesion localization and the granuloma constitution, associated with the
lesion type, were similar to the findings by RAMOS et al. (1994);
DREIMEIER et al. (1999); HUDA & JENSEN, (2003).
CONCLUSIONS
The thickening
of enteric wall, lymph nodes enlargement and the corrugated aspect of
the enteric mucosa were observed through necropsy. Histopathological
findings of the epithelioid granuloma with scarcely differentiating
Giant cells and presence of AFB have also been identified, mainly on
ileal mucosa and submucosa. The
clinical sign of chronic diarrhea is not specific
for paratuberculosis, and is observed in several other diseases.
Nevertheless, it is necessary to perform a differential diagnosis with
other chronic processes which lead to cachexia. The anatomopathology,
especially the ZN stain, revealed pathognomonic findings for
paratuberculosis.
Paratuberculosis
granuloma in bovine does not present caseification, mineralization and
it shows scarce gigantocitary answer, different from that of the
tuberculosis in this specie.
ACKNOWLEDGEMENTS
The authors
acknowledge the funding of the current study by the FAPERJ, Rio de Janeiro State Foundation for the
Endowment of Research
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Protocolado
em: 06 fev. 2009. Aceito em: 24 maio 2012.