Colombian and Y st¡ains of T. cluzi were

ReD. Inst. Med. trop. Stjo pau¿o 29(51: 284.288, s etembr o-oÌLtubro, 1987

l:.' :

,:,. 'ttt'

i:, :,

AMERICAN TRYPANOSOMIASIS (CHAGAS' _{DISEASE} IN CONVENTIONAL AND GERMFREE}

RATS AND MICE (1}

''''

Marcelo E. SILVA (2). _{Elizio A. EVANGELTSTA (3)1acgì¡êslR. NICOLT (3), Eduardo A. BAMBTRRA (4) & Dnio}

C.

Germf.ree (cF) and conventional (CV) CFW (LOB) _{mice and Wistar and Sprâgue} Dawley rats were infected _{with Trypanosoma cruzi. The disease was more severe}

in

the

cF

than

in

the CV animals as revealed by: (1) _{an ea¡lier ând mo¡e intense} parasitemiâ; (2) a more precocious mortality; (3) _{â twice enlarged spleen: (4) a more} intense cell and tissue pa¡asitism; (5) _{visceral signs of cardiac failure.}

KEY WORDS: Chagas'disease; Trypanosomiasis; Germfree rats; Germfi:ee mice.

The difference between germfree (GF) and

conventional (CV) animals of the same species

is not restricted

to

the absence of the normal flora

i¡

the former. To this châracteristics, the lack ofreaction of the host to germs and a series

ofdirect effects ofthese germs on the host should

be added (cOR,DON & pESTr, 19?1).

GF animals, v/hen infected with pathogens

may exhibit higher or loq¡er resistance

to

the

infecting organism (GORDON

&

PESTI, 19?1; PLEASANTS, 1974).

cF

mice are five tÍmes mo_ re resistant and show more intense mitogenie ând immunogenlc rqsponses to bâcterial

endoto-xins than their CV counterpa¡ts

(KlyONO

_et

al., 1980). FINERTY _{et al.} (19?2)infected

_cF

_and CV mice with Plasmodium berghei; the rise in

parasitemia was more Þrecocious in CV animals even âlthough antibodies could be detected eâr

SUMMARY

INTRODUCTION

Uer in cF mice which also had higher plasma immu, noglobulin titers. On infection with

Eperythro-zoon coccoides, CV reacted more efficien y than

GF mice(HYDE et al., 19?2). VIEIRA et aI., (1985) presented preliminary evidences thât

schistoso-miasis mansoni was less severe

in G!'

than in

CV mice.

In the present work, the infection with Try.

panosoma cruzi s/as studied in GF and CV mice

and rats.

MATERIAL AND METHODS

{1) This wolk was Ðrtlacted Îloln the M. S. thesis ofMES.

12) _Brâsil-Plesent adalress: DeÞârtâmento de Nutrição, Escolâ cle Fârmácia, unive¡sidacte Fealerât de ouro preto, ouro preto, Mc.,

13) Depârtamento de Bioqu¡mica lmunologia, Instituto de ciêncras Biolóeicâs, Unjversictâ.te F.ederât de Minâs ceraÍs (UI¡MG),

Belo Honzonte, MA., B¡asit.

(4) Departâmenio de Anatomia pâtotógica, Faculdade de Mealicina, UFMG, Bero Ho¡izonre. Mo.. B¡âß

Colombian and Y st¡ains of T.

cluzi

were

maintained in CV mice and in irradiated CV rats.

To obtain the inocula, the animâls were killed under ether anesthesia. Blood was collected

SILVA. M. E : EVANGELISTA, E. A.: NICOLI, J. R.i BAMBIR,R,A. E- A, & VIEIR,A, E. C _ ArnE¡ICAN TIYPANOSOMiASß fChâSAS, diseâse) in conventionâl and germftee rats ând mice. Rev. I¡sú. Med. t¡op. Sáo paulo, 29:284 2BB. r98?.

heaú of rats

with

syringes containing hepârin. The evâluation of the number of

trypomastigo-tes'was done according to BRENER (1962). _The

adjustment of the number of parasites

to

the

desired inocula was done by dilution with blood obtâined from uninfected animals. The manipu lâtions were performed

in

a laminar flow hood.

The blood v¡as transferred to sterile amfroules.

A sâmple was seeded in thioglycollate medium

and brain-heart broth for control of asepsis. The

ampoules were then seâled in a flâme and

intro-duced into the isolâtor. Contamination wâs ne-ver detected in the control samples after

incuba-tion at 28.C and 3?"C.

Breeding nuclei of

cF

(LOB) mice ând

cF

Wistar râts were supplied by Dr. Morris pollard,

University of Notre Dâme, Notre Dame, Indiana,

USA. A breeding nucleus of GF Sprague-Dawley rats was obtained from Dr. Edward Balish,

Uni-versity of lryisconsin, Mâdison, Wisco¡si¡, USA.

Those ânimals are free of áll demonstrable

mi-crobes. Mice, however, bear a leukemogenic

vi-rus acquired prenâtally which remains latent un-less activated by radiâtion (POLLARD, 1965).

The CV animâls were derived from the GF

colony. Rats andmice support very well the

con-ventionalization.

malswere killed under ether anesthesia. The âni-mals were opened ventrally from the neck to the

end of the abdomen and immersed in 4q.

fc.rrnal-dehyde solution. Fragments meâsuring 2-4mm

were removed from the thymus, liver, spleen,

lymphonodes, brain, and skeletal, cardiac, and

smooth muscles, The fragments were fixed in

4% formaldehyde solution and processed for pa

râffin

embedding. The sect.ions were stained

with hematoxylin-eosin. The slides v¡ere

exami-ned by only one person _{who did} not have access

to the codification of the slides, whose

identifi-cation was done only after each reporthâd alrea-dy been written.

The control of isolator asepsis was done ac cording to WAGNER (1959).

RESULTS Mice

GF rats and mice were maintained in

flexi-ble _{f¡lâstic isolators} (TREXLER, _{1959) an¿t mani}

pulated according

to

established procedures

(PLEASANTS, 1974). Unless otherwise stated,

tåe CV counterpârts were also maintained ih iso

lâtors and handled accordingly.

GF ând CV 21 and 56 days otd Wistâr rats and 14 _{months old Sprague-Dâv¡ley rats we¡e} inoculated inhaperitoneâlly (i. p.)with _{t0s trypo:} mâstigotqs per g¡âm of body weight of

Colom-biân strain ofT. cruzi. GF ând CV three months

old CFür (LOB) mice were inoculated i. p. with

I

x

104 trypomastigotes of Y strâin of T. cruzi.

GF and CV 21 days old CFW (LOB) mice were

inoculated similarly with 1.0-1.3 x 10a trypomâs tigotes of Y strain of T. cruzi.

Blood trypomastigotes were counted daily,

as described by BRENER (1962).

The expetiment was run

until

the death of

the animâls. In experiments with rats, some

ani-Figure 1 shows the evolutior _{of the} pa¡asitêmiâ and the cumulative mortality ofsix GF and four

CV three months old male C¡'W mice infected

with

I

x

104 blood forms of trypomastigotes _of

T. cruzi, Y strain. An earlier ând more intense

5

8

r50

a

È50

SILVA M E ; EvANGELrsrA. E. A.. Nrcolr, J. R.;BAMBTRRA, E. A. & vIEr'.A. E. o. _{Ame¡ican ,.ypanosomiåsis {châgas,}

dFease) in conventionâì alrd gelmftee râts and mice Rev. Ins¿. Me.t. t¡op. Sáo pãulo, z9:2g4 288. l98z. parasitemia q'âs _{observed in the GF group. The}

mortality _{also was more precocious}

_in

_¿he

_cF

than in CV group, even though two

cF

ânimals

survived up

to

the 15ù day âftet infection.

_At

the 12t¡ day, aU CV and 66Ea of tine

cF

ânimals

were dead.

The experimentwas repeated with fourmale

and ñve fema-le GF and six male and tqrelve fe_ male CV, 21 dâys old mice infected

with

1.0-1.3

x

10" tryÞomastigotes of

y

strain of T.

cruzi-Again, both pa.rasitemia and mortality were mo reÞrecociousin the GF group, aìthough thepeâk

of parasitemia _{was highet}

_in

_{CV mice.}

_In

_this experiment, the CV animâls were kept

in

the CV animal room. After the beginningofthe expe_ riment, therewas â drop in the temperature

that

affeCted mostly the CV mice.

Rats

A preliminary experiment

with

one female ând one male GF a¡d two female CV 14 months

old Sprague-Dâv¡ley rats was caFied out. A hi_ gher pârasitemia _{and a more precocious death}

ì¡/ere observed in GF group.

Figure 2 sho\Ã's the pÐasitemia and the

morta-ìity

of five GF and six CV Wistar mâle

2l

davs

old rats v,¡eighing approximately 60g and inocu_

lâted with 6 x 106 trypomâstigotes _{of Colombian}

shain

of T. c¡.uzi. The patasitemia was much

higher in the

cF

group reaching 150.000 trypo_

mastigotes/mm3 of blood. Themortâlity was also

more precocious

in

_{the GF eroup.}

_At

the 21sr day, all

cF

had died whereas one CV lived ufr

to the 47h dây, when it was sacrifrced.

The histopâthological _{findings were similar}

in

rats

and mice from

the

above mentioned

groups. Macroscopicâlty: (1) _{the spleens from}

eF

animals were twice the size of CV animals: (2) pronounced signs of congestive cardiac insuffi,

ciency (such âs, ascites ând generalized visceral congestion)

i¡

GF animals. Microscopically: _G)

more intense cell ând tissue pa¡asitism

_in

_GF

than in CV animals; (2) _the parasitism was much

more intense in,the organs rich

in

celts of the

mononuclea¡ phagocytic system ând

in

the pa_

renchyma of liver, âdrenâls and muscles (car_ diâc, skeletal _{ând smooth) of both}

_eF

_{and CV} ânimâls.

In

the central nervous system. there

were sparse and discrete inflammatory lesions

a¡d ceuular parasitism.

Figute 3 shov/s â sample of the histopatho_ logic firdings in hearts of GF' and CV mice. The

more Ìntense agtessiveness of the disease in GF

ânimals is evident.

The experimentráas repeated with four male and four female GF and three male and three

female CV, 56 days old Wistal rats weighing ap_

proximately 1109, The animals were Ínoculated

with

12

x

106 trypomastigotes of Colombian

shain ofT. ctuzi, The _f,arasitemia was nore pre-cocious and higher in the

cF

groups. Atl animals

survived to the ?1"t day, when they were killed.

On histopathologicâl _{examination, no diffe_}

rence between the GF and the CV animals could

be detected.

A

discrcte focal parasitism

_in

the musculâr and macrophagic phagocytic _system

could be visuâlized. g

I 3so

E

ñ

El H t¡t¡eqnns fl l.{, er}lF¡€!

Fis. 2 _{Parasitemia and cumulâtive mortãliùy of semftees} and conventionalt days old WistâÌ male mts weighing 60g, inoculated with 6x10' blood forms ofU strâin of T¡ypåDosoEå cruri.

3

I'

Mice a-re the ânimals most widely used as hosts in experimental Chagas' disease. They

de-velop an acute and a chronic phase

followi¡g

infection with T. cruzi. Figure 1 shows

that

GF

SILVA. M E.: EVANGELISTA. E. A.: NICOLL J. R.: BAMBIRRA. E. A. & VIEIRA. E. C. _- AùrericÂn t¡\'påììosonì ia sis , Châgâs disease) ¡n conventjonal and gemìfree rats and mjce Rer. Inst. ìled. t¡op. Siào P¡ulÒ. 29:281.288. t98?

mice had an ea¡lie¡ and higher parasitemia than

thet

CV counterparts These ¡esults may be ex

plained by the fact

that

GF mice have a ìess

developed lymphoid system when compared

with CV mice, impâiring the cell mediated im-mune lesponse (SZERI et â1., 19?6). ROGERS

&

BALISH (1978) _ând GOODMAN et al. (19?8)

reported evidences fo¡ the "immunological

im-maturity'of

GF animals. The rate of

gamma-cell and tissue parasitism

in

GF mice may be explained by the smaller number of cells invol ved in immunological defense such as:'tympho

cytes (OLSON& WOSTMAN, 1966a ) and neutro

phils, monocytes. and eosinophils (OLSON & wosTMANN. 1966b).

Moreover peritoneal macrophâges from GF

mice a¡e possibìy less activâted th,an those ob,

tained from CV animals. Macrophages ftom GF

animals have smaller and more spherical nuclei, fewer mitochondria and a higher number

oflyso-some-like granules per unit voìume ofcytoplasm

(WOODWARD, 19?8). These differences may be possibly related to the lack of stimuli from

intes-tinål flora {WOODWARD, 19?8).

The highe¡ parasitemia found in CV mice in

the second experiment might be explained by

the lower tempera¿ure of the ¡oom where the

animals were housed.

lt

is well known that there

is a raise in parasitemiâ at lower temperatures rKOLODNY, 1940). Nevertheless. the death ¡âte

and the histopathological findings confirmed

the ¡esults of the hrst experimenl.

Figue 2 shows tÌìat, in rats, the leveìs

ofparasi-temia were higher in the GF than in CV group.

ln 21 days oìd animals, the mo¡tality was earlie¡ and totâl in the GF group.

All

six GF and four

out

five CV

râts

died. The high

mortality

of

young Iats infected-

with

T. cruzi conhÌms the

results ofKOLODNY (1940) and CULBERTSON & KESSLER (1942), _{who showed} that the disea se is more seve¡e in yolrnger animals. The mor-phological findings a¡e comÞâtible with the

pa-rasitological data, i. e., the disease was more se-vere in GF than in CV rats.

In older rats there was no mortality

i¡

either group of animals. The pârasitemia, again. was

more precocious and higher

in

the GF than in

t}le CV rats. The histopathological findings were

similar for both groups.

The ¡esults ¡eported herein show that Cha

gas'disease is more severe

in

GF than

in

CV

rats and mice. Further q,ork

_will

be ca¡ried out

to

elucidâte the reason fo¡ the observed diffe-rences.

globulin synthesis is 50 times higher in CV

tha[

i¡

GF mice (SELL & FAHEY, 1964). These data

ale also suggestive of a slower humoral immune response in GF than in CV mice. The

histopatho-logicâl dâtâ conh¡m

that

the disease is much

iì.1

i'.,:

1

SILVA, M. E.; EVANGELISTA, E. A.: NICOLI, J- R.: BAMBIRR'A, E. A. & VIEIRA. E. C- Amencan hypanosomiâsis (Chagâs'

clisease) ìn conventionâl and germftee rats ând mice. Rev. Inst. Med. ¿¡op. Siio Paulo, 29:284 288.

198?-Tdpanosom¡ase americana (doença de Chagas) em rÀtos e camundongos convencionais e

isen-tos de g€rmes

Camundongos CFIJÍ¡ (LOB) e ratos Wistar e Sprague Dawley isentos de germes (GF) e

con-vencionais (CV) foram infectados com TrypÀno.

soma cruzi. A doençâ foi mais grave nos animais

cF

do que nos Cv, o que foi demonstrado por: (1) _uma parasitemia _{mais precoce} e mais intensa; (2) uma mortalidade mâis precoce; (3) _{baço duas} vezes maior; (4) _um parasitismo celular e tissular

mais intenso: (5) si¡âis viscerais de insuficiência

cardÍaca.

ACKNOWLEDGEMENTS RESUMO

This work vt'as supported by Financiadora

de Estudos e Projetos (FINEP) and by conselho Nacionâl de Desenvolvimento Cientlflco e

Tec-nológico (CNPq). The technical help of Ronilda

M- de Paulâ and Carlos Gomes Silva is

acknow-ledged.

ponse to LPS in germfree, Eschêrichiâ coli

monoâssôc¡â-ted ând conventionâl mice. J. Immunol.,124:36 41, 1980. KOLODNY, M. H. _- Studies on age resistance âgâinst tlypanosome infecbion. L Theresistance of¡ats ofdifferent ages to infection with T. cruzi. Am€¡. J. Hyci,,29: 13-24, 1939.

KOIODNY, M. H. _- The efrect ofenvironmental üempe

!âtule upon expedrnentâl llypânosomiasis (T. r¡uzi) of

râts. Am€¡. J. Hys.,32: 2l-23,

1940-OLSON, G. B. & WOSTMANN, B. S. _- Lymphocytopuie

sis, plâsmocytopoiesis âîd ceuul prclife¡ation in non-antÍgenical¡y stimulåted germfree mice. J. Immunol., 9?: 287 274, lgß8à.

ol-soN, G. B. & wOSTMANN, B. s. _- cellulù andhumo-ral immune response of germftee mice stimulated 1l'lth

?s IIGGor Salmoneua ¿ypbi¡r¡rrium. J. Immunol., 97: 2?5-286, 1966b_

BRENER, Z.-Thempeutic activity ând cliteûon ofcure onmiceexpe meDtauyinfectedwithTrypânosor¡acruzi. ney.Ihst. Meal. trop. S. Påulo,4:389 398,1962.

CULBEF,TSON, J. t. & KESSLER, W. R. _- Age re8is-tance of mice to TrypanosoDå cruri. J, Paråsi¿., 28: 155,158, 1942.

FINERTY, J. F.; TOBIE,J. E. d¿ EVANS, C. B.-Aniibody

and lÌÌlmunoglobuliì synthesis 1¡ gelmflee ând conven-tional mice infected with Plasmo¿lium bc¡sh€i- Âne¡. J. trop. Meit. Hyg., Z1:499 505, 19?2.

GOODMAN, M- G.i FIDLER, J. M. & {¡EIGLE, W. O.

-Nonspeciñc aclivation of EuÌinelys¡Þhocytes. ûr. Cells respondÍng riitogenicålly to 2 mercâptoetl¡ânol are typi cal unstimulated Iymphocytes. J. Immunol., t2l:

1899-1904,1978:

GORDON, H. A. & PESTI, L. _- the gnotobiotic anÍma¡

as a toolin the study ofhosi miclobial relâtionship. Bâct. Rev..35:390 429. 19?1.

10_

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