Germfree (GF) and conventional (CV) CFW (LOB) mice and Wistar and Sprague-Dawley rats were infected with Trypanosoma cruzi. The disease was more severe in the GF than in the CV animals as revealed by: (1) an earlier and more intense parasitemia; (2) a more precocious mortality; (3) a twice enlarged spleen: (4) a more intense cell and tissue parasitism; (5) visceral signs of cardiac failure.Camundongos CFW (LOB) e ratos Wistar e Sprague-Dawley isentos de germes (GF) e convencionais (CV) foram infectados com Trypanosoma cruzi. A doença foi mais grave nos animais GF do que nos CV, o que foi demonstrado por: (1) uma parasitemia mais precoce e mais intensa: (2) uma mortalidade mais precoce: (3) baço duas vezes maior; (4) um parasitismo celular e tissular mais intenso; (5) sinais viscerais de insuficiência cardíaca

Bambirra, Eduardo A.

Evangelista, Elizio A.

Nicoli, Jacques R.

Silva, Marcelo E.

Vieira, Enio C.

English

Camundongos CFW (LOB) e ratos Wistar e Sprague-Dawley isentos de germes (GF) e convencionais (CV) foram infectados com Trypanosoma cruzi. A doença foi mais grave nos animais GF do que nos CV, o que foi demonstrado por: (1) uma parasitemia mais precoce e mais intensa: (2) uma mortalidade mais precoce: (3) baço duas vezes maior; (4) um parasitismo celular e tissular mais intenso; (5) sinais viscerais de insuficiência cardíaca.Germfree (GF) and conventional (CV) CFW (LOB) mice and Wistar and Sprague-Dawley rats were infected with Trypanosoma cruzi. The disease was more severe in the GF than in the CV animals as revealed by: (1) an earlier and more intense parasitemia; (2) a more precocious mortality; (3) a twice enlarged spleen: (4) a more intense cell and tissue parasitism; (5) visceral signs of cardiac failure

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ReD. Inst. Med. trop. Stjo pau¿o29(51: 284.288, s etembr o-oÌLtubro, 1987l:.' :,:,.'ttt'i:, :,AMERICAN TRYPANOSOMIASIS (CHAGAS' DISEASE} IN CONVENTIONAL AND GERMFREERATS 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 SprâgueDawley rats were infected with Trypanosoma cruzi. The disease was more severein the cF than in the CV animals as revealed by: (1) an ea¡lier ând mo¡e intenseparasitemiâ; (2) a more precocious mortality; (3) â twice enlarged spleen: (4) a moreintense 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) andconventional (CV) animals of the same speciesis not restricted to the absence of the normalflora i¡ the former. To this châracteristics, thelack ofreaction of the host to germs and a seriesofdirect effects ofthese germs on the host shouldbe added (cOR,DON & pESTr, 19?1).GF animals, v/hen infected with pathogensmay exhibit higher or loq¡er resistance to theinfecting organism (GORDON & PESTI, 19?1;PLEASANTS, 1974). cF mice are five tÍmes mo_re resistant and show more intense mitogenieând immunogenlc rqsponses to bâcterial endoto-xins than their CV counterpa¡ts (KlyONO etal., 1980). FINERTY et al. (19?2)infected cF andCV mice with Plasmodium berghei; the rise inparasitemia was more Þrecocious in CV animalseven âlthough antibodies could be detected eârSUMMARYINTRODUCTIONUer in cF mice which also had higher plasma immu,noglobulin titers. On infection with Eperythro-zoon coccoides, CV reacted more efficien y thanGF mice(HYDE et al., 19?2). VIEIRA et aI., (1985)presented preliminary evidences thât schistoso-miasis mansoni was less severe in G!' than inCV mice.In the present work, the infection with Try.panosoma cruzi s/as studied in GF and CV miceand rats.MATERIAL AND METHODS{1) This wolk was Ðrtlacted Îloln the M. S. thesis ofMES.12) Plesent adalress: DeÞârtâmento de Nutrição, Escolâ cle Fârmácia, unive¡sidacte Fealerât de ouro preto, ouro preto, Mc.,Brâsil-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 weremaintained in CV mice and in irradiated CV rats.To obtain the inocula, the animâls were killedunder ether anesthesia. Blood was collectedftom t,I:e axillary f,lexus of mice or directly ftoln theSILVA. 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). Theadjustment of the number of parasites to thedesired inocula was done by dilution with bloodobtâined from uninfected animals. The manipulâtions were performed in a laminar flow hood.The blood v¡as transferred to sterile amfroules.A sâmple was seeded in thioglycollate mediumand brain-heart broth for control of asepsis. Theampoules 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 cFWistar râts were supplied by Dr. Morris pollard,University of Notre Dâme, Notre Dame, Indiana,USA. A breeding nucleus of GF Sprague-Dawleyrats 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 GFcolony. Rats andmice support very well the con-ventionalization.malswere killed under ether anesthesia. The âni-mals were opened ventrally from the neck to theend of the abdomen and immersed in 4q. fc.rrnal-dehyde solution. Fragments meâsuring 2-4mmwere removed from the thymus, liver, spleen,lymphonodes, brain, and skeletal, cardiac, andsmooth muscles, The fragments were fixed in4% formaldehyde solution and processed for parâffin embedding. The sect.ions were stainedwith hematoxylin-eosin. The slides v¡ere exami-ned by only one person who did not have accessto 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 according to WAGNER (1959).RESULTSMiceGF rats and mice were maintained in flexi-ble f¡lâstic isolators (TREXLER, 1959) an¿t manipulated according to established procedures(PLEASANTS, 1974). Unless otherwise stated,tåe CV counterpârts were also maintained ih isolâtors and handled accordingly.GF ând CV 21 and 56 days otd Wistâr ratsand 14 months old Sprague-Dâv¡ley rats we¡einoculated inhaperitoneâlly (i. p.)with t0s trypo:mâstigotqs per g¡âm of body weight of Colom-biân strain ofT. cruzi. GF ând CV three monthsold CFür (LOB) mice were inoculated i. p. withI x 104 trypomastigotes of Y strâin of T. cruzi.GF and CV 21 days old CFW (LOB) mice wereinoculated similarly with 1.0-1.3 x 10a trypomâstigotes of Y strain of T. cruzi.Blood trypomastigotes were counted daily,as described by BRENER (1962).The expetiment was run until the death ofthe animâls. In experiments with rats, some ani-Figure 1 shows the evolutior of the pa¡asitêmiâand the cumulative mortality ofsix GF and fourCV three months old male C¡'W mice infectedwith I x 104 blood forms of trypomastigotes ofT. cruzi, Y strain. An earlier ând more intense58 r50aÈ50Fig. I - Pârâsitemia and cumulâtive morialiùy in groups ofeelmfree {GF)' and conventionâl (CV)¡ th¡ee months oldCFw mice inoculated with 8xl0r blood fo¡lDs ofTrypa¡osomacruri, Y strai¡.285SILVA 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. Themortality also was more precocious in ¿he cFthan in CV group, even though two cF ânimalssurvived up to the 15ù day âftet infection. Atthe 12t¡ day, aU CV and 66Ea of tine cF ânimalswere dead.The experimentwas repeated with fourmaleand ñve fema-le GF and six male and tqrelve fe_male CV, 21 dâys old mice infected with 1.0-1.3x 10" tryÞomastigotes of y strain of T. cruzi-Again, both pa.rasitemia and mortality were moreÞrecociousin the GF group, aìthough thepeâkof parasitemia was highet in CV mice. In thisexperiment, the CV animâls were kept in theCV animal room. After the beginningofthe expe_riment, therewas â drop in the temperature thataffeCted mostly the CV mice.RatsA preliminary experiment with one femaleând one male GF a¡d two female CV 14 monthsold Sprague-Dâv¡ley rats was caFied out. A hi_gher pârasitemia and a more precocious deathì¡/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 davsold rats v,¡eighing approximately 60g and inocu_lâted with 6 x 106 trypomâstigotes of Colombianshain of T. c¡.uzi. The patasitemia was muchhigher in the cF group reaching 150.000 trypo_mastigotes/mm3 of blood. Themortâlity was alsomore precocious in the GF eroup. At the 21srday, all cF had died whereas one CV lived ufrto the 47h dây, when it was sacrifrced.The histopâthological findings were similarin rats and mice from the above mentionedgroups. Macroscopicâlty: (1) the spleens from eFanimals were twice the size of CV animals: (2)pronounced signs of congestive cardiac insuffi,ciency (such âs, ascites ând generalized visceralcongestion) i¡ GF animals. Microscopically: G)more intense cell ând tissue pa¡asitism in GFthan in CV animals; (2) the parasitism was muchmore intense in,the organs rich in celts of themononuclea¡ phagocytic system ând in the pa_renchyma of liver, âdrenâls and muscles (car_diâc, skeletal ând smooth) of both eF and CVânimâls. In the central nervous system. therewere sparse and discrete inflammatory lesionsa¡d ceuular parasitism.Figute 3 shov/s â sample of the histopatho_logic firdings in hearts of GF' and CV mice. Themore Ìntense agtessiveness of the disease in GFânimals is evident.The experimentrÃ¡as repeated with four maleand four female GF and three male and threefemale CV, 56 days old Wistal rats weighing ap_proximately 1109, The animals were Ínoculatedwith 12 x 106 trypomastigotes of Colombianshain ofT. ctuzi, The f,arasitemia was nore pre-cocious and higher in the cF groups. Atl animalssurvived to the ?1"t day, when they were killed.On histopathologicâl examination, no diffe_rence between the GF and the CV animals couldbe detected. A discrcte focal parasitism in themusculâr and macrophagic phagocytic systemcould be visuâlized.gI3soEñEl H t¡t¡eqnnsfl l.{, er}lF¡€!Fis. 2 Parasitemia and cumulâtive mortãliùy of semfteesand conventionalt days old WistâÌ male mts weighing 60g,inoculated with 6x10' blood forms ofU strâin of T¡ypåDosoEåcruri.2863I'Mice a-re the ânimals most widely used ashosts in experimental Chagas' disease. They de-velop an acute and a chronic phase followi¡ginfection with T. cruzi. Figure 1 shows that GFDISCUSSIONSILVA. M E.: EVANGELISTA. E. A.: NICOLL J. R.: BAMBIRRA. E. A. & VIEIRA. E. C. - AùrericÂn t¡\'påììosonì ia sis , Châgâsdisease) ¡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 thanthet CV counterparts These ¡esults may be explained by the fact that GF mice have a ìessdeveloped lymphoid system when comparedwith 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 beexplained by the smaller number of cells involved in immunological defense such as:'tymphocytes (OLSON& WOSTMAN, 1966a ) and neutrophils, monocytes. and eosinophils (OLSON &wosTMANN. 1966b).Moreover peritoneal macrophâges from GFmice a¡e possibìy less activâted th,an those ob,tained from CV animals. Macrophages ftom GFanimals 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 bepossibly related to the lack of stimuli from intes-tinål flora {WOODWARD, 19?8).The highe¡ parasitemia found in CV mice inthe second experiment might be explained bythe lower tempera¿ure of the ¡oom where theanimals were housed. lt is well known that thereis a raise in parasitemiâ at lower temperaturesrKOLODNY, 1940). Nevertheless. the death ¡âteand the histopathological findings confirmedthe ¡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 fourout five CV râts died. The high mortality ofyoung Iats infected- with T. cruzi conhÌms theresults ofKOLODNY (1940) and CULBERTSON& KESSLER (1942), who showed that the disease 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¡ eithergroup of animals. The pârasitemia, again. wasmore precocious and higher in the GF than int}le CV rats. The histopathological findings weresimilar for both groups.The ¡esults ¡eported herein show that Chagas'disease is more severe in GF than in CVrats and mice. Further q,ork will be ca¡ried outto elucidâte the reason fo¡ the observed diffe-rences.241globulin synthesis is 50 times higher in CV tha[i¡ GF mice (SELL & FAHEY, 1964). These dataale also suggestive of a slower humoral immuneresponse in GF than in CV mice. The histopatho-logicâl dâtâ conh¡m that the disease is muchmore seve¡e in the GF g¡oup (Fig. 3). The higheriì.1i'.,:1SILVA, 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€rmesCamundongos CFIJÍ¡ (LOB) e ratos Wistar eSprague Dawley isentos de germes (GF) e con-vencionais (CV) foram infectados com TrypÀno.soma cruzi. A doençâ foi mais grave nos animaiscF do que nos Cv, o que foi demonstrado por:(1) uma parasitemia mais precoce e mais intensa;(2) uma mortalidade mâis precoce; (3) baço duasvezes maior; (4) um parasitismo celular e tissularmais intenso: (5) si¡âis viscerais de insuficiênciacardÍaca.ACKNOWLEDGEMENTSRESUMOThis work vt'as supported by Financiadorade Estudos e Projetos (FINEP) and by conselhoNacionâl de Desenvolvimento Cientlflco e Tec-nológico (CNPq). The technical help of RonildaM- 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 âgâinsttlypanosome infecbion. L Theresistance of¡ats ofdifferentages 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) ofrâts. Am€¡. J. Hys.,32: 2l-23, 1940-OLSON, G. B. & WOSTMANN, B. S. - Lymphocytopuiesis, 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 ofcureonmiceexpe 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.-Aniibodyand 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. CellsrespondÍng riitogenicålly to 2 mercâptoetl¡ânol are typical 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_REFERENCBSPLEASANTS, J. R. - Onotobiotlcs. ID: MELBY JR., E.C. & ALTMAN, N. H. ed. - Hândbook oflaboratory åDi.hat scie¡ce. Clevelând, CRC Press, 1974. p. rrg 1?4.POLLARD, M. vlIâl status of germi:ee mice. Natl. cân-c€r Inst. MoDogl¡Þh.,20: 16?-1?2, 1965.ROGERS, T. J. & BALISI¡, E. Efrect oI the systemlccandidiasis on blastosenesis of lympbocytes tom gelm'Aee and conventional rabs. Infect. Immu¡.. 20: 142-150.19,r8.SELL, S- & F'AHEY, J. L. * Relâtionship- between " globulin merâbohsm ând low serum ? slobulir in gerrnfreemice. J.ImD¡¡¡oI.. 93: 8l-8?. 1964-SZER,I, I.; ÀNDERLi{, P-: BANOS, Z. & R,ADNAI, B,- Decæâsed cellulâl lespohse lÌ1 gelm-fiee ânimâls. ActâMicrobiol. Acâal. Sci. h\ns.,Zst 23r 234, 1978.TRE)iLER, P. C. - The use of plastics in the desigz¡ ofùolator systems. Ann. N. Y, Acad. Sci., ?8:29-36, 1959.VIEIE¡., L- Q.; MORAES E SAN"OS, T. & VIEIRA, E.C. - AlteIabions in cell numbe¡, size and couâgercontentiÌ livers ofconventionâl ând germfree mice beùlng Schistosoma nansoni.In: WOSTMANN. B. S.. ed. - C€¡mfr€ères€årcb: Microflora. Cobtrol ând lts âppllcatron to thebiomedlcal sclences, New York, Aìan R. Liss, 1985. p.235 238.14.I]YDE, C- L.i FINERTY, J. F. & EVANS, C. B. -å.ntibodyand immunogtobulin synthesis in germtee and conventional mice i¡fected with EDerythroroor coccoides. Âmer.J. troD. Med. HyA.,2l: 506'511, I9?2.I<IYONO, H.: McGHEE, J. R. & MICHALEK, s. M. -LPs Ëeulâtion of the immune response: compâ¡iron ofrcs-1.2881'1.WAGNER, M. - Determi4âtion of germftee status. AnD,N. Y. Acad. Sci., ?E:89'101, 1950.wooDwARD, B. -A st€rdological ultrast¡ucturâI Âtudyoipelitonea] macrophages Aom gelmtree and conventionally'reaÌed mice. CeU Tiis. Rês.,192: l5?'166. r978.Redebldo pùa publicaçáo em 16/1/8?.

Tripanosomíase americana (doença de Chagas) em ratos e camundongos convencionais e isentos de germes

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Tripanosomíase americana (doença de Chagas) em ratos e camundongos convencionais e isentos de germes

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