Immunocytochemical study of tissue parasitism of dog adrenal

Immunocytochemical study of tissue parasitism of dog adrenal

Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/1994014397
IMMUNOCYTOCHEMICAL STUDY OF TISSUE PARASITISM OF DOG ADRENAL GLANDS
IN EXPERIMENTAL CHAGAS' DISEASE
CALIARI M.V.*, LANA M.**, OLIVEIRA E.R.***, BARBOSA A.J.A.**** and TAFURI W.L.***
Summary :
Résumé
In human beings, the central vein of the adrenal glands has been
LA GLANDE ADRÉNALE DU CHIEN DURANT LA MALADIE DE CHAGAS EXPÉRI-
considered by some authors to be one of the preferential sites of
MENTALE.
Trypanosoma cruzi settling in the chronic form of Chagas' disease.
Chez l'humain, la veine centrale de la glande adrénale est considé-
: ÉTUDE IMMUNOCYTOCHIMIQUE DU PARASITISME TISSULAIRE DE
This appears to be due to the local corticoid-rich environment that
rée par certains auteurs comme un des sites préférentiels de l'établis-
may protect the parasite against the immunological defences of
sement de Trypanosoma cruzi durant la phase chronique de la
the host. An immunocytochemical study of the adrenal glands of
maladie de Chagas. Cela semble dû à l'environnement local riche
15 dogs was carried out for the detection of tissue parasitism in
en corticoides qui peut protéger le parasite contre les défenses
the acute and chronic phases of experimental T. cruzi infection.
immunologiques de l'hôte. Une étude immunocytochimique des
No amastigote nests or isolated amastigotes were detected in the
glandes adrénales de 15 chiens a été conduite pour détecter le
adrenal parenchyma or adrenal central vein of the animals stu-
parasitisme tissulaire des phases aiguë et chronique de l'infection
died. Minimal nonspecific inflammatory changes were found in
expérimentale par Trypanosoma cruzi. Aucun nid d'amastigote ou
some glands of both infected and noninfected animals. The pre-
amastigote isolé n'a pu être détecté dans le parenchyme adrénal ou
sent results show that the adrenal glands of the dog are not injured
la veine adrénale centrale des animaux étudiés. Des changements
and do not constitute a site of escape or reservoir of parasites in
inflammatoires non spécifiques minimaux ont été trouvés dans
experimental Chagas' disease.
quelques glandes d'animaux infectés ou non. les résultats obtenus
ici montrent que les glandes adrénales du chien ne sont pas lésées
KEYWORDS : Chagas' disease. Trypanosoma cruzi. adrenal glands, dog. et ne constituent pas
MOTS CLES : maladie de Chagas. Trypanosoma cruzi. glandes surrénales,
chien.
INTRODUCTION
I
n most patients in the chronic phase o f Chagas'
disease (American trypanosomosis) the number o f
T. cruzi in tissue is very scarce and inconsistent
with the intensity o f the inflammatory response that
o c c u r s , e s p e c i a l l y in the heart. T h e s e o b s e r v a t i o n s
have led many authors to propose that the progression o f the inflammatory p h e n o m e n o n in the chronic
phase o f Chagas' disease, mainly the myocarditis, may
occur regardless of the p r e s e n c e of T. cruzi. However,
o t h e r w o r k e r s have recently indicated that in s o m e
cases of Chagas' disease, the inflammatory lesions of
chronic chagasic cardiopathy are associated with the
presence o f parasites (Almeida et al., 1981 ; Barbosa et
* Departamento de Patologia Geral, Instituto) de Ciências
Biológicas, Universidade Federal de Minas Gerais.
** Departamento de Análises Clínicas, Escola de Farmácia,
Universidade Federal de Ouro Preto.
*** Departamento de Ciências Biológicas, Instituto) de Ciências
Biológicas. Universidade Federal de Ouro Preto.
**** Departamento de Anatomia Patológica e Medicina Legal,
Faculdade de Medicina, Universidade Federal de Minas Gerais.
Address for correspondence : Prof. Marcelo Vidigal Caliari, Depto.
de Patologia Geral, Institute de Ciências Biológicas, Universidade
Federal de Minas Gerais. Av. Antônio Carlos 6627, Pampulha, Belo
Horizonte-MG-Brasil. 31.270.010; Fax (031) 441-1412.
Supported by CNPq, FAPEMIG and FINEP.
Parasite, 1994, 1, 397-400
un site d'échappement ou un réservoir des
parasites lors de la maladie de Chagas expérimentale.
al., 1 9 8 6 ; Higuchi et al., 1 9 9 3 ) . Therefore, the possibility o f the existence of a reservoir o f T. cruzi in host
tissues that may act as a source of periodic discharge
parasite, would b e very important for the study of the
pathogenesis of Chagas' disease. Examining this possibility in human chronic Chagas' disease, Teixeira et al.
( 1 9 8 6 ) o b s e r v e d intense parasitism o n the adrenal
gland central vein wall, which differed from the myoc a r d i u m a n d o t h e r t i s s u e s e x a m i n e d . It w a s then
hypothesized that the glucocorticoid-rich environment
o f the adrenal glands may explain the preferential tropism of T. cruzi for this site, allowing its perpetuation
in host tissues and then representing a factor respons i b l e for t h e p r o g r e s s i o n o f c h r o n i c m y o c a r d i t i s .
H o w e v e r , in a study o f adrenal glands o f rats with
experimental trypanosomiasis, Machado et al. ( 1 9 9 0 )
obtained discordant results.
T h e objective of the present investigation was to study
the parasitism of the central vein and parenchyma of
adrenal glands in dogs inoculated with the Be-62 and
B e - 7 8 strains of T. cruzi and killed during the acute
and chronic phases of the disease. T h e dog, in contrast
to the rat, can develop a chronic cardiomyopathy with
an anatomoclinical pattern similar to that observed in
human beings (Lana et al., 1 9 9 2 ; Lana et al., 1 9 8 8 ;
Tafuri et al., 1988) and thus it may be considered a
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397
CALIARI M.V., LANAM.,OLIVEIRA E.R., BARBOSA A.J.A. and TAFURI W.L.
Fig. 1. - Dog inoculated with the Be-78 strain of
T. cruzi and killed during the acute phase of
the infection, (a) Acute, diffuse myocarditis
with intense exsudation of mononuclear cells
and amastigote nests (arrows), (b) Presence of
strongly stained clusters of amastigotes (large
arrows) and numerous isolated form of amastigotes (small arrows) scattered through out the
section of cardiac fibers, a. HE staining b. PAP
staining.
Fig. 1. - Dog inoculated with the Be-78 strain of
T. cruzi and killed during the acute phase of the
infection. Medullar and cortical regions of adrenal gland showing the central vein (arrowheads)
without parasites or inflammation. HF staining.
398
Note de recherche
Parasite, 1994, 1, 397-400
TISSUE PARASITISM IN EXPERM
I ENTAL CHAGAS' DISEASE
more suitable animal model for the study of adrenal
gland parasitism and its possible role as a T. cruzi tissue reservoir in the c h r o n i c phase o f Chagas' disease
as it is s u p p o s e d to occur in human disease.
MATERIALS A N D M E T H O D S
N
ineteen young mongrel dogs (65-80 days old)
born and kept under experimental conditions
were used. Fifteen dogs were inoculated with
2 , 0 0 0 m e t a c y c l i c trypomastigotes/kg weight by the
conjunctival route, six with the Be-62 strain and nine
with the B e - 7 8 strain. T h e s e two different strains of T.
cruzi were isolated from patient Berenice in 1962 and
1978, respectively (Salgado et al., 1 9 6 2 ; Lana & Chiari,
1986). Berenice is considered to b e the first reported
human case of Chagas' disease (Chagas, 1909). Four
dogs served as control. T h e animals were kept on an
ad libitum
diet a n d u n d e r daily o b s e r v a t i o n in an
appropriate kennel. Parasitemia was evaluated daily
during the acute p h a s e by parasite c o u n t s in fresh
b l o o d ( B r e n e r , 1962). Eleven dogs, four inoculated
with the Be-62 strain and seven with the B e - 7 8 strain,
w e r e killed 37 to 4 4 days after i n o c u l a t i o n ( a c u t e
p h a s e ) and submitted to n e c r o p s y . T h e o t h e r four
dogs, two inoculated with Be-62 and two with B e - 7 8 ,
were sacrificed 3 to 6 6 months after infection.
T h e adrenal glands w e r e c o l l e c t e d in toto, fixed in
b u f f e r e d neutral f o r m a l d e h y d e , a n d p r o c e s s e d for
paraffin e m b e d d i n g . A total o f 120 4 µm thick s e c tions w e r e obtained from each pair o f g l a n d s ; 60 o f
them w e r e stained with hematoxylin and eosin (HE)
for histological analysis and the other 60 w e r e stained
by the p e r o x i d a s e - a n t i p e r o x i d a s e ( P A P ) m e t h o d
( B a r b o s a , 1 9 8 5 ) f o r s p e c i f i c s t a i n i n g o f T.
cruzi.
Antiserum p r o d u c e d in rabbits against the CL strain o f
T. cruzi was used at 1/1,000 dilution. HE-stained s e c tions w e r e e x a m i n e d for both inflammatory reaction
and amastigote nests, while the PAP-stained sections
w e r e used to determine and evaluate the p r e s e n c e of
amastigotes in tissue. Previous w o r k had s h o w n that
the PAP m e t h o d is m o r e sensitive than HE for amastigote quantification in tissue sections ( B a r b o s a . 1986).
Several paraffin sections of the heart atrial wall w e r e
used for histopathological analysis o f the myocardium
(HE staining) and for evaluation o f tissue parasitism
(PAP staining).
RESULTS
A
cute myocarditis characterized by diffuse or
multiple foci of m o n o n u c l e a r cells was
o b s e r v e d in all d o g s sacrified d u r i n g t h e
Parasite, 1994,1, 397-400
acute phase o f the disease. In these animals the card i a c fibers p r e s e n t e d a b u n d a n t T. cruzi
parasitism
that w a s well defined by both staining procedures,
HE and PAP (fig. 1 A and B ) . T h e two dogs inoculated with the B e - 7 8 strain and killed during the chronic p h a s e o f the d i s e a s e presented c h r o n i c fibrous
myocarditis with sparse tissue parasitism only visualized in PAP-stained sections. In the animals inoculated
with the B e - 6 2 strain and killed during the chronic
p h a s e , n e i t h e r c a r d i a c fibrosis n o r parasitism w a s
observed although small foci o f myocarditis could b e
detected. T. cruzi parasitism was not detected in the
adrenal glands o f any animals killed during the acute
or c h r o n i c p h a s e o f C h a g a s ' d i s e a s e (fig. 2 ) . Small
foci o f m o n o n u c l e a r inflammatory cells were observed in parenchyma o f the glands o f a few animals of
both the chagasic and non-chagasic groups.
DISCUSSION
h e h u m a n adrenal glands p r e s e n t a central
vein with a well d e v e l o p e d s m o o t h m u s c l e
layer that a p p e a r s to b e the most intensely
parasitized tissue in human chronic Chagas' disease
( A l m e i d a et al., 1 9 8 1 ; T e i x e i r a et al., 1 9 8 6 ) . In
contrast, the central vein o f the rat adrenal gland is
very delicate, without a distinguishable muscle coat
and this may explain the negative results obtained in
studies of adrenal parasitism during the chronic phase
o f e x p e r i m e n t a l t r y p a n o s o m o s i s c r u z i in r a t s
( M a c h a d o et al., 1990). A very delicate central adrenal
vein containing a hardly d e v e l o p e d s m o o t h muscle
layer w a s also o b s e r v e d in the d o g s studied h e r e .
However, while Machado et al. ( 1 9 9 0 ) found intense
p a r a s i t i s m d u r i n g t h e a c u t e p h a s e in rat a d r e n a l
glands, we could not detect
the p r e s e n c e of
Trypanosoma
cruzi in the adrenal tissues o f clogs
either in the acute or the chronic phase. Machado et
al. ( 1 9 9 0 ) concluded that their results did not support
the h y p o t h e s i s that the c o r t i c o i d - r i c h e n v i r o n m e n t
would favour local T. cruzi colonization. T h e present
r e s u l t s l e d us t o r e a c h t h e s a m e c o n c l u s i o n .
N e v e r t h e l e s s , w e d o not k n o w if o u r results w e r e
influenced b y the scarceness of smooth muscle fibers
in the adrenal central vein. T h e r e f o r e , e v e n though
w e can not e x c l u d e c o m p l e t e l y the h y p o t h e s i s o f
A l m e i d a et al. ( 1 9 8 1 ) a n d T e i x e i r a et al. ( 1 9 8 6 )
c o n c e r n i n g the adrenal parasitism in human Chagas'
disease, other organs from different animals should
b e studied to c o n c l u d e for eventual sites o f T. cruzi
predilection. In addition, the e c o l o g i c a l interaction
b e t w e e n different strains o f T. cruzi with different
a n i m a l s p e c i e s s h o u l d b e b e t t e r s t u d i e d . In t h e
Brazilian o p o s s u m Didelphis
albiventris
inoculated
Note de recherche
399
CALIARI M. V., LANA M., OLIVEIRA E.R.. BARBOSA A.JA. and TAFURI W.L.
with T. cruzi,
w e h a v e o b s e r v e d tissue parasitism
de A., R i o s LEITF V . H . , BARBOSA A.J.A., TOLEDO M . J . O . and
without a n y e v i d e n c e o f an i n f l a m m a t o r y r e a c t i o n
CHIARI E . Ease crônica cardïaca fibrosante da tripanosomiase cruzi experimental no cào. Revista da Sociedade
Brasileira de Medicina Tropical, 1988, 21, 113-121.
(unpublished data).
T h e present results indicate that in c a n i n e e x p e r i m e n tal trypanosomiasis cruzi t h e adrenals glands d o not
MACHADO L . R . , MACHADO C . R . S . , CARDOSO J . E . and BARBOSA
may c o n c l u d e that these glands are not a preferential
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site for parasite survival a n d e s c a p e from t h e host
SALGADO J.A., GARCEZ P.N., OLIVEIRA C . A . and GALIZZI J .
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resulting from the p r e s e n c e o f T. cruzi.
Therefore, w e
d e f e n c e s . This negative finding is m o r e significant if
w e consider that t w o dogs in the chronic p h a s e devel o p e d c a r d i a c l e s i o n s similar t o t h o s e o b s e r v e d in
h u m a n c h a g a s i c c a r d i o p a t h y . T h u s , t h e local corticoid-rich
environment m a y not b e sufficient to
explain t h e frequent parasitism observed in the adrenal glands o f s o m e patients with
chronic chagasic
cardiopathy.
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