Revista da Sociedade Brasileira de Medicina Tropical 24(1): 5-11, jan-mar, 1991
A R T I G O S
IM M U NO CYT O CH EM ICA L ID E N T IF IC A T IO N O F
L E I S H M A N I AA N D
T R Y P A N O S O M A C R U Z I
A M A ST IG O T E S
I N S I T UW ITH H O M O LO G O US A N D
H ET ER O L O G O US PO LY CLO NA L A N T IB O D IE S
A .J .A . B a r b o s a , C .A . d a C o s ta , M .S .M . M ic h a lic k , W . M a y r in k , R .T . G a z z in e lli a n d W .L . T a fu r i.
T h e u n la be lle d a n tib o d y p ero xida se an tipe rox id a se m e th o d w as u se d to s tu d y th e im m u -n o c y to c h e m ic a lp r o p e r tie s o/Leishmania a n d Trypanosoma cruzi a m a stig o te s in situ a fte r tissu es h a d bee n s u b m itte d to d iffe r e n t f ix a tio n p ro ce du re s. A n tis e r a were o b ta in e d fr o m ra bb its c h r o n ic a lly in fe c te d w ith d iffe re n t str a in s o f T. cruzi o r im m u n iz e d w ith L. mexicana amazonensis a n d L. braziliensis guyanensis, a n d were a p p lie d on 5 /.im th ic k sections. T. cruzi a n tig e n s were well s ta in e d b y th e thre e a n ti-T . cruzi se ra a n d th e tw o a nti-he is.h m a m a .sera a t o p tim u m d ilu tio n between 1 :1 ,0 0 0 a n d 1:2,000, re ga rdless th e p a r a s ite strain. D iffe re n tly , th e le is h m a n ia l a n tig e n s were re v ea le d b y Leishmania se ra o n ly a t low d ilu tio n s (b etw e e n 1:6 0 - 1 : 1 6 0 ) , w he rea s th e a n ti-T . cruzi sera, a t th e se low d ilu tio n s, g a v e ra th e r w e a k sta in ing s. A lth o u g h there is no c le a r e x p la n a tio n f o r th is im m u n o c y to c h e m ic a l “re v e rse -m o n o d ire c tio n a l” c ross-re a c tiv ity between Leishmania a n d T. cruzi, th e p r e s e n t re su lts sh o w th a t p o ly c lo n a l a n tib o d ie s a g a in s Leishmania species, w he n u se d f o r im m u n o c y to c h e m ic a l d ete c tio n o f th e se p a r a s ite s in situ, re a ct m o re stro n g ly w ith T. cruzi a m a s tig o te s th a n w ith th e h o m o lo g o u s am a stig ote s.
Key-words: Leishmania. Trypanosoma cruzi. A n tig e n ic ity o f a m a stigo tes. P e ro x id a se -a n tip e ro x id -a se m ethod .
Polyclonal antibodies have been used in immu nocytochemical techniques for identification of both,
L e i s h m a n i a and T r y p a n o s o m a c r u z i amastigotes in sections of tissue specimens. Two types of staining methods have been reported: (a) the peroxidase- labelled antibody for staining leishmanial antigens1*’ 24 and (b) the unlabelled antibody peroxidase-antipero xidase (PAP) method for T. c r u z iamastigotes1 2. The titres of the w i a .- L e i s h m a n i a sera used in the former method ranged from 10 to 160 whereas the anti-T! c r u z i
sera had titres above 1,000 using the PAP method. The higher sensitivity of the unlabeled antibody PAP method in comparison to the labeled antibody immu- noperoxidase technique is well known^ 18 21 and could be an explanation for the differences of the antibody titres used. Concerning this, Croker & Kuhn6 found that an avidin-biotine-peroxidase complex technique was five to ten-fold more sensitive (best titres between 640 and 1,280) than peroxidase-labelled antibody method
Trabalho realizado nos Departamentos de Anatomia Pato lógica e Medicina Legal e de Parasitologia da Universidade Federal de Minas Gerais e no Centro de Pesquisas René Rachou, Belo Horizonte, MG, Brasil.
Auxílio financeiro da FINEP, CNPq e FAPEMIG. A d d re s s f o r correspondence: Dr. Alfredo J. A. Barbosa,
Facuidade de Medicina da UFMG, Av. Alfredo Balena 190, 30130, Belo Horizonte, MG, Brasil.
Recebido para publicação em 28/08/90.
(best titres between 80 and 160) for revealing T. c r u z i
amastigotes in tissue sections. The different titres of the primary antibody could also be due to different affini ties of the first antibody to the L e i s h m a n i a and T. c r u z i
antigens. In addition, the tissue fixation could interfere in both parasite epitopes, changing differently their immunoreactivity.
A nother question to be considered is how the antigenic cross-reactivity between T. c r u z i and
L e i s h m a n i a , observed when serological methods are
em ployed for diagnostic purpose4 5 23 25 js expressed in the tissue sections. Com parative studies on the immunoperoxidase staining of these two microorga nism s are not available in literature so far. The present work was carried out to study, by the P A P method, the T . c r u z i s a d L e i s h m a n i a immunoreactive properties in tissue sections using species-specific and heterologous antisera and different tissue fixation procedures.
M ATERIAL AND M ETHODS
A ntisera against L e i s h m a n i a
Antisera against promastigotes ofL . b r a z i l ie n s is
g u y a n e n s i s (code: M H O M /B R /70/M 1176) were
B arbosa A J A , Costa CA, M ic h a lic k M S M , M a y r in k W, G azzin elli R T , T afuri W L. Im m un oc y to c he m ic al identification o f
Leishmania a n d Trypanosoma Cruzi a m astigotes in situ with hom ologous a n d heterologous p oly c lo n a l antibodies. R e vista da Socied ade B rasileira de M edicina Tropical 24: 5-11, jan-m ar, 1991.
animals were bled 7 days afterwards. Antisera against
L . m e x ic a n a a m a z o n e n s is amastigotes (code: IFL A / BR/67/PH8) were obtained by immunization of rabbits with an amastigote suspension isolated from hamsters, purified as described by Shaw & Laison25 and dis rupted by ultrason. One ml of this suspension, con taining 2 mg of protein, emulsified with an equal volume of Freund’s complete adjuvant was used for the first inoculation of the animals and boosted as descri bed above. The indirect immunofluorescence test (using promastigotes as antigen) was used to detect the presence of anti- L e is h m a n ia antibodies in both sera.
A n tis e r a a g a in s t T. cruzi
Antisera against T. c r u z i were obtained from rabbits chronically infected with CL, Emani and Y strains. The test of indirect immunofluorescence was used for monitoring the titres of circulating specific antibodies.
C o lle c tin g a n d h a n d lin g o f tis s u e s p e c im e n s
L e i s h m a n i a infected tissues were obtained from nasal ulcers of hamsters infected several weeks before with promastigotes of the following species: L . m e x i c a n a m e x ic a n a , code M HOM /BR/60/BH6; L . m e x i c a n a a m a z o n e n s is , code IFLA /B R /67/PH 8 a n d i.
b ra z ilie n sis g u y a n e n s is , code MHOM /BR/70/M 1176. Different samples from each animal were fixed in neutral, buffered 4% formaldehyde and in Bouin’s fluid for 18-24 hours and then processed routinely for paraffin inclusion. Other samples from animals in fected with L . b r a z ilie n s is g u y a n e n s is and L . m e x i c a n a a m a z o n e n s is , were snap frozen in melting Freon immediately after the biopsy procedure; freeze-dried (Freeze Dryer “ Edwards do Brasil”) for 6 - 8 hours at -40°C and then divided into three sets: one set was embedded directly in paraffin wax under vacuum, at 60°C for 40 minutes (unfixed tissue); the others were left at 60°C under vapour of either benzoquinone crystals or paraformaldehyde, for 3 hours. After that, they were embedded in paraffin wax under vacuum, at 60°C for 40 minutes.
Tissues infected with T. c r u z i were obtained as follows: (a) heart fragments from two chagasic patients who died with congestive heart failure. Both presented chronic myocarditis with numerous amastigote nests (relapsing myocarditis)); (b) fragments of placenta, from a case of spontaneous abortion from a mother with chronic Chagas’ disease. On routine histology the placental fragments presented chorioamnionites and frequent nests of amastigotes; ( c) myocardial and liver tissues from mice inoculated with the Y strain of T. c r u z i and from dogs infected with B e r e n ic e and
C o lo m b ia n a strains. Tissue specimens infected with
T. c r u z i were fixed in either, 4% formaldehyde or Bouin’s fluid, then embedded in paraffin wax.
I m m u n o c y t o c h e m i c a l s ta in in g m e th o d
The PA P m eth o d ^ was used for identification
o i L e i s h m a n i a and T. c ru zi. Phosphate buffered saline (PBS) 0.01M, pH 7.2, was used throughout as the washing buffer after each step and as a diluent The sections were treated withO.3% hydrogenperoxyde to block endogenous peroxidase activity, then incubated with 1:30 diluted normal swine serum to reduce non specific binding of the link antibody. The sections were incubated with the primary antibody (sera anti-
L e i s h m a n i a or anti-7! c r u z i) at 4°C overnight after wards. A range of dilutions from 20 to 4,000 times was used. Swine anti-rabbit immunoglobulins (Dako Laboratories, Copenhagen) at 1:80 dilution was used as the second (link) antibody layer and rabbit pero- xidase-antiperoxidase complex (Miles-Yeda Ltd, Israel), at 1:200 dilution as the third layer. Visualization of theperoxidase-antiperoxidase complex was achieved by the diaminobenzidine method. Finally, the sections were counterstained with hematoxylin and mounted for examination.
For negative control of the T. c r u z i staining, the anti-T. c r u z i and anti- L e i s h m a n i a sera were adsorbed in trypomastigote-sepharose affinity column as pre viously reported^. The a n ti- L e is h m a n ia sera were adsorbed by incubation overnight with promastigotes of L . b r a z ilie n s is g u y a n e n s is .
Aliquotes of a n M -L e is h m a n ia and anti-T. c r u z i
sera were also pre-adsorbed with dried red blood cells of cattle, goat and rabbit and then used as primary antibodies to stain tissue samples known to be positive for T. c r u z i in order to verify the possibility of a false- positive reaction due to attachment of heterophyle antibodies to the surface membrane of T. c r u z i 15. Sera obtained from rabbits after immunization against
L e i s h m a n i a / T . cruzz'-unrelated antigens, using the same immunization schedule as described above for
L e i s h m a n i a antibodies production, were used for control of cross-reactivity between T. c r u z i and cons tituents of the Freund’s adjuvant
RESULTS
Leishmania s ta in in g w ith a n ti-Leishmania se ra.
B a r b o s a A J A , C osta CA, M ic h a l ic k M S M , M a y r i n k W , G a z z in e lli R T , T a fu r i W L . lm m u n o c y to c h e m ic a l id e n tific a tio n o f Leishmania a n d Trypanosoma Cruzi a m a stig o te s in situ w ith h o m o lo g o u s a n d hete ro log o us p o ly c lo n a l a n tib od ie s. R e v is ta d a S o c ie d a d e B ra sile ir a d e M e d ic in a T ro p ic a l 24: 5-11, ja n - m a r , 1991
Leishmania s t a i n i n g w i th a n t i - T . cruzi s e r a
N o p o s i t i v e s p e c i f i c s t a i n i n g o f L e i s h m a n i a amastigotes could be seen when serum anti-Y strain of T . c r u z i was used. The sera anti-CL and a n t i - E r n a n e strains gave a weak positive staining ofL. b r a z i l i e n s i s g u y a n e n s i s and L . m e x i c a n a a m a z o n e n s i s attitres up to 80 (Table 1). Nevertheless, these reactions were frequently dubious, due to nonspecific background staining observed mainly in freeze-dried tissues.
T. cruzi s t a i n i n g w i t h a n t i - T . cruzi s e r a
T . c r u z i amastigotes in tissue specimens of dogs and mice with experimental trypanosomiasis, and man naturally infected, were specifically stained with the three different anti-T! c r u z i sera. The best stainings were obtained with titres between 1,000 and 2,000 (Table 1). The tissue sections displayed amastigotes as strong, dark-brown bodies within the cells (Figure 2A). The nest of parasites could be seen easily at xlOO magnification of the light microscope. The nonspecific background staining was minimal or absent with titres above 1,000.
T. cruzi s t a i n i n g w i t h a n t i -Leishmania s e r a
T. c r u z i amastigotes in all tissue specimens were positively stained with the two anti- L e i s h m a n i a sera. The best titres were 1,000 (anti-L. m e x i c a n a a m a z o n e n s i s ) and 2,000 (anti-X. b r a z i l i e n s i s g u y a n e n s i s ) (Table 1). The amastigotes were stained strongly as conspicuous, dark-brown bodies, against the hema toxylin contrasted host cells (Figure 2B, C, D). On examination, these tissue sections were not distin guishable from those stained with the anti-T . c r u z i sera. The amastigotes were immunoreactive to antisera
T a b l e 1 - T i t r e s o f t h e anf/-L eishm ania a n d a n t i - T . cruzi s e r a f o r d e m o n s t r a t i n g Leishm ania a n d T. cruzi a n t i g e n s in f o r m a l d h e y d e a n d B o u i n ’s f l u i d f i x e d t i s s u e s e c t i o n s . P A P m e t h o d .
A ntisera to
L e i s h m a n i a antigens* T . c r u z i antigens**
80 160 320 1000 2000 4000
T . c r u z i
C L + / - — — + + /
-E m an i + / - — —
+
+
•Y — - + + + /
-L e i s h m a n ia
Lbg + + / - + + + /
-Lma + + / - + + / - —
* L . b r a z i l i e n s i s g u y a n e n s i s (Lbg); L . m e x i c a n a a m a z o n e n s i s (Lma) and L . m e x i c a n a m e x i c a n a . ** Y , C o l o m b i a n a a n d B e r e n i c e strains (anim al infections) and human tissue (natural infection).
+ = positive staining; + / — = weak staining; — = negative/very wèak staining.
7 in freeze-dried tissues, the parasites were identified
B a rb osa A J A , Costa CA, M ic h a lic k M S M , M a y rin k W, G a zz in e lliR T , T afuri W L. Im m un oc y to c he m ic al identification o f
Leishmania a n d Trypanosoma Cruzi am astigotes in situ with hom ologous a n d heterologous p oly c lon a l antibodies. R ev ista da Sociedade B rasileira de M e d ic in a Tropical 24: 5-11, ja n -m ar, 1991
with titres up to 2,000 ( anti-L. m e x ic a n a a m a z o n e n s is) and 4,000 (anti-L. b r a z ilie n s is g u y a n e n s is ) .
C o n tr o ls
The pre-adsorptions of heterophyle antibodies resulted in a slightly weaker staining of T. c r u z i
amastigotes in most sections, however it did not change sigmficatively the titres. The pre-adsorption of both anti-T. c r u z i and a n ti- L e is h m a n ia sera in
try-pomastigote-sepharose column and the a n ti-L e is h m a n ia sera with L . b r a z ilie n s is g u y a n e n s is promas- tigotes, abolished almost completely the stainings of T. c r u z i amastigotes and of T. c r u z i and L e i s h m a n i a
amastigotes, respectively. Antisera from rabbits that were immunized against L e i s h m a n i a / T . c r u z i u r a e
-lated antigens, using Freund’s adjuvant, were negative for staining of T. c r u z i amastigotes.
F ig u re 2 - H isto lo g ic a l se c tio n s s ta in e d b y th e P A P m e th o d f o r d e m o n stra tin g T. cruzi a m a s tig o te s in c a r d ia c m u sc le f i b e r f r o m a m a n with c h ro n ic C h a g a s ’ d ise a se ( D ) a n d in liv e r a n d m y o c a rd iu m tis su e s f r o m m ic e in th e a c u te p h a s e o f e x p e r im e n ta l try p a n o so m ia sis (A , B, C ) u sin g sp e cie sspe c ific a n d h e te ro lo g o u s a n tib od ie s. T h e p a r a s ite s a p p e a r a s d a rk
B a rb osa A J A , Costa CA, M ic h a lic k M S M , M a y rin k W, G a zzin e lli R T , T qfuri W L. Im m un o cy toc h em ica l identification o f
Leishmania a n d Trypanosoma C ruzi am astigotes in situ with h om ologous a n d he te rologouspolyc lonal antibodies. R e vista d a Sociedade B rasile ira de M e d ic in a Tropical 24: 5-11, ja n -m a r, 1991
D IS C U S S IO N
The specific staining o f amastigotes by the imm unoperoxidase techniques has considerable po tential for research and routine diagnostic in trypa nosomiasis. The high degree o f contrast between the am astigotes and the host tissue allows a quick asses- m ent o f the infection. However, in the case o f leish m aniasis the low ratio of the specific staining signal to background interferes with the quality o f the prepa rations. O ur present results show th a t T . c r u z i am as tigotes are detected by homologous (anti-T. c r u z i ) or heterologous (anti- L e i s h m a n i a ) sera at high dilutions (up to 4,000). However, detection o f L e i s h m a n i a species in s i t u was only possible at titres excessively low for application in imm unoperoxidase methods.
Two anti- l e i s h m a n i a sera were used, (anti-L. m e x i c a n a a m a z o n e n s i s and anti-L. b r a z i l i e n s i s
g u y a n e n s i s ) , both presenting antibodies which bound
amastigotes o f different kinetoplastid species. Indeed, m any antigens are shared by different species and evolutionary stages o f L e i s h m a n i a and T . c r u z i, consequently cross-immunity reactions between these parasites are expected^ 8 9 10 13 14 16 17 19 20 22 24
The polyclonal antibodies that we used in P A P technique to identify L e i s h m a n i a and T . c r u z i am as tigotes in s i t u dem onstrate by exam ination o f all sections stained th at antibodies against L e i s h m a n i a react more strongly with T. c r u z i amastigotes than with the homologous amastigotes. O n the other hand, antibodies against T . c r u z i did not react efficiently with leishmanial antigens. Also, the anti- L e i s h m a n i a serum react with L e i s h m a n i a a t low titres reducing its application in imm unoperoxidase methods because the weak staining signal yelded and the high degree of nonspecific background. Thus, the low titres o f the
m i i - L e i s h m a n i a sera in detecting homologous am as
tigotes, previously reported in literature16 24 was not a result of a lower sensitivity o f the labelled-antibody peroxidase technique in com parison to the P A P method. In addition, deleterious effect o f fixatives on leishm anial antigens, as observed in relation to other type o f cells27, was rulled out because different fixatives in liquid and vapour phases as well as freeze- dried unfixed tissues were used. O n the other hand, the presence o f heterophyle antibodies in the rabbit anti- L e i s h m a n i a sera, which could bind to T. c r u z i am as tig o te s ^ , was discarded by pre-adsorptions with eri- trocyte o f various origens.
The m t i - L e i s h m a n i a sera stained well the T.
c r u z i am astigotes in tissue sections with a stronger
affinity for T. c r u z i than for L e i s h m a n i a antigens. T here is n o clear explanation for this immunocyto-
c h e m i c a l “ reverse-m onodirectional” cross-reactivity
phenom enon. T his could be due to intrinsic properties
o f the leishm anial surface antigens. T. c r u z i amasti- gote epitopes recognized by a n t i - L e i s h m a n i a antibo dies m ay have different positions and a lower density in
L e i s h m a n i a surface. Also, specific host-parasite
chem ical interactions could m ask the leishmanial epitopes presentation. In favour of these hypothesis, is the recent report th a t antisera from mice infected and/or immunized with L e i s h m a n i a did not stained leishm anial am astigotes in tissue sections using the imm unoperoxidase technique. High titres o f antibo dies in these sera were, however, detected by E L IS A against various species and sub-species ofL e i s h m a n i a and T . c r u z i ^ .
Immunologic similarities between L e i s h m a n i a
and T . c r u z i are im portant in regions of the world
where both trypanosom iasis and leishmaniasis are endemic. T he immunologic diagnosis could give a false positive result o f Chagas disease in patients with leishmaniasis. T he possibility th at immunization against leishmaniasis would be effective against C hagas’ disease should be investigated based on our dem onstration of cross-reactive intra-specific antigens. In the case o f T. c r u z i, protective antibodies from experimental and from hum an Chagas’ disease were shown to destroy trypom astigotes in v itr o and in
v i v o 11 12. O n the other hand, immunity to leishma
niasis is dom inated by cell-mediated reactions although antibodies are fo rm e d ^ . These antibodies are not thought to be o f decisive im portance in protection against leishmaniasis. In favour of a possible increased resistance against T. c r u z i by anti- L e i s h m a n i a antibodies is the high degree o f cross protection against fatal murine C hagas’ disease in animals previously infected with L e i s h m a n i a b r a z i l i e n s i s p a n a m e n s i s ^ . W hether this protection results or not from cross-reactive antibodies is yet to be investigated.
R E S U M O
O m é to d o d a p e r o x id a s e -a n tip e r o x id a s e fo i u tiliza d o p a r a e s tu d a r a s p ro p rie d a d e s im u n o c ito q u im ic a s d e Leish- manias e d e a m a stig otas d o Trypanosoma cruzi, in situ, ap ós o s te cidos te rem sid o s u b m e tid o s a d ife re n te s tip o s de fix a ç ã o . A n ti- so ro s f o r a m o b tid o s de c oe lho s c ron ic a m e n te in fe c ta d o s com trê s c e p a s d e T. cruzi o u im u n iz a d o s c o m L. mexicana ámazonensis e L. braziliensis guyanensis e a p li c a d o s n o s co rtes h isto ló g ic o s d e 5 /xm d e espessura. O s a n tíg e n o s d e T. cruzi f o r a m c o ra do s m u ito b e m p e lo s três so ro s a h ti-T . cruzi e p e lo s d o is so ros anfí-Leishmania com d ilu iç õ e s e n tre 1 :1 .0 0 0 e 1:2.000. D ife ren te m e nte , o s a n tí g e n o s d ç Leishmania fo r a m re ve lad os p e lo s so ros a n
i-B arbo sa A J A , Costa CA, M ic h a lic k M S M , M a y r in k W, G azzin e lli R T , T afuri W L. Im m un oc y to c he m ica l identification o f
Leishmania a n d Trypanosoma Cruzi am astigotes in situ with hom ologous a n d heterologous p o ly clo na l antibodies. R ev ista da S ociedade B rasileira de M e d ic in a Tropical 24: 5-11, ja n-m ar, 1991
m ic a c ru za d a “re v e rsa -m o n o d ir e c io n a l” entre Leishmania e a m a stig o ta s de T. cruzi o s re su lta d o s do p re se n te tra ba lh o m o stra m q u e a n tic o rp o s p o lic lo n a is c o ntra d ife re ntes espé cies d e Leishmania, q u a n d o u sa d o s p a r a detec ção im u n o -c ito q u ím i-c a d e Leishmania e T. cruzi in situ, re agem m a is fo rte m e n te c o m a m a stig o ta s d e T. cruzi d o qu e c om espécies
hom óloga s.
Palavras-chaves: Leishmania. Trypanosoma cruzi. A n tig e n ic id a d e d e a m astig ota s. M é to d o p e r o x id a se -a n ti-p e ro xid ase .
ACK N O W LED G M EN TS
The authors are indebted to Z. Brener and L. E. Ramirez Giraldo for providing the anti-T. c r u z i sera, to A. de Oliveira Lima for helpful advice and to A. U. Krettli andF. E. L. Pereira for the critical review of the manuscript.
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