Trypanosoma cruzi in a caviomorph rodent: parasitological and pathological features of the experimental infection of Trichomys apereoides (Rodentia, Echimyidae)
Experimental Parasitology 107 (2004) 78–88 www.elsevier.com/locate/yexpr Trypanosoma cruzi in a caviomorph rodent: parasitological and pathological features of the experimental infection of Trichomys apereoides (Rodentia, Echimyidae) Leidi Herrera,a,b Samanta das Chagas Xavier,a Claudia Viegas,a Clara Martinez,c Paulo Marcelo Cotias,d Hernan Carrasco,e Servio Urdaneta-Morales,b and Ana Maria Jansena,* a Laboratory of Tripanosomatid Biology, Department of Protozoology, Oswaldo Cruz Institute, FIOCRUZ, Av Brasil 4365, Manguinhos, RJ, Brazil b Institute of Tropical Zoology, Science Faculty, Central University of Venezuela, 47058, Los Chaguaramos 1041-A, Caracas, Venezuela c Nutrition School, Medicine Faculty, Central University of Venezuela, Caracas, Venezuela d Evandro Chagas Research Institute, FIOCRUZ, RJ, Brazil e Tropical Medicine Institute, Medicine Faculty, Central University of Venezuela, Caracas, Venezuela Received December 2003; received in revised form 30 March 2004; accepted 21 April 2004 Available online Abstract To understand the interaction of Trypanosoma cruzi with caviomorph rodents, which supposedly have an ancient co-evolutionary history with this parasite, experimental infection of laboratory reared Trichomys apereoides with several isolates of both genotypes of the parasite was studied Parasitemia, pattern of hematic cells, specific humoral immune response, histopathological features and parasite clearance were appraised T apereoides maintained stable infections independent of the T cruzi genotype as demonstrated by positive PCR results in analyses of several tissues after a months follow-up The acute phase was characterized by abundant and disseminated presence of amastigotes, vacuolization and/or myocytolysis Lymphocytosis was a common feature The chronic phase was characterized mainly by lymphomacroeosinophilic infiltrates independent of the inoculated T cruzi isolate T cruzi of different genotypes did not show any tissular preference in T apereoides Ó 2004 Elsevier Inc All rights reserved Index Descriptors and Abbreviations: Trypanosoma cruzi; Changes’ disease; Echimyidae; Trichomys apereoides; Histopathology; bp, base pairs; DIG-labeled DNA, DNA probes with digoxygenin-labeled deoxynucleotides; DNA, deoxyribonucleic acid; Ethylene diamine tetra acetic acid; FITC, fluorescein isothiocyanate; IFA, immunofluorescence assay; HE, hematoxylin–eosin; IgG, immunoglobulin G; kDNA, kinetoplast deoxyribonucleic acid; LCSSP-PCR, low-stringency single specific primer PCR; LIT, liver infusion tryptose medium; NNN, Novy–Mc Neal–Nicole medium; PCR, polymerase chain reaction; PI, post-infection; SSC, saline sodium citrate solution; TAE, trisma acetate buffer; Taq, thermostable DNA polymerase Introduction American Trypanosomiasis (Chagas, 1909) is a autochthonous and ancient protozoan infection of wild mammals in which the heteroxenic hemoflagellate Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae) is transmitted by wild vectors (Hemiptera, Reduviidae, * Corresponding author Fax: +55-21-280-1589 E-mail address: jansen@ioc.fiocruz.br (A.M Jansen) 0014-4894/$ - see front matter Ó 2004 Elsevier Inc All rights reserved doi:10.1016/j.exppara.2004.04.008 Triatominae) in a primary sylvatic cycle (Pinto Dias, 2000) Trypanosoma cruzi comprises complex multiclonal populations, differing in genetic and biological attributes and different epidemiological, pathological, and clinical manifestations of the human disease Two main T cruzi groups, T cruzi I and T cruzi II, were recognized in the taxon, associated, in Brazil, with wild and domestic transmission cycles, respectively T cruzi I and T cruzi II are described as correlated with phenotypic subpopulations defined by its isozymic patterns or zymodemes 79 L Herrera et al / Experimental Parasitology 107 (2004) 78–88 Zymodeme and are correlated with T cruzi I and II, respectively; zymodeme is still a matter of debate (Anon., 1999; Araujo et al., 2002; Fernandes et al., 1999; Miles et al., 1977; Santos et al., 2002) The origin of T cruzi I and T cruzi II, respectively, in Didelphis marsupialis and in primates and/or caviomorph rodents, was suggested by Briones et al (1999) and Buscaglia and Di Noia (2003) Indeed, caviomorph rodents and primates have an ancient evolutive history in South America, since they arrived at the Southern continent, coming probably from Africa, in the Oligocene (38 millions of years ago) (Flynn and Wyss, 1998; Gaunt and Miles, 2000) Trichomys apereoides (Rodentia, Echimyidae) is a caviomorph rodent and therefore probably an ancient host of T cruzi The taxon is associated to xeric and rocky environments with frequent incursions into human dwellings The genus Trichomys has an ample distribution in savannahs (‘‘cerrado’’), white scrub (‘‘caatinga’’), and marshland (‘‘pantanal’’) biomes, typical of the Northeast and Central Brazil, occupying a vast extension of the country (Bandouk and dos Reis, 1995; Neiva and Penna, 1916) In spite of the recent hypothesis concerning divergence of T cruzi I and T cruzi II and the importance of caviomorph rodents in the evolutionary history of T cruzi only scarce data on the interaction of this parasite with these hosts are available In addition, studies have shown that T apereoides may act as a good reservoir of T cruzi, as demonstrated (Jansen, A.M., non-published data), by positive hemocultures of 50% (21/44) of specimens collected inside Serra da Capivara National Park a biological reserve in semi-arid biome of ‘‘caatinga,’’ in Piauı State, Brazil and an endemic area for Chagas disease In this context, we considered it necessary to better understand the association of T cruzi with this caviomorph, a possible reservoir in endemic areas of Chagas disease In this study, the parasitological and histopathological patterns of experimental infection of T apereoides with T cruzi I and T cruzi II genotypes were studied Our purpose was also to supply data that may clarify the putative association of caviomorph rodents with the T cruzi II genotype Materials and methods 2.1 Parasites Experimental infections were performed with four recently obtained T cruzi isolates, previously typified by analysis of the non-transcribed spacer of the mini-exon gene, in agreement with Fernandes et al (1999) Two isolates considered as references for T cruzi I (XXXX/ BR/1971/F) and T cruzi II (MHOM/BR/1957/Y) were also used (Deane et al., 1984b) Characteristics of the inoculated isolates are listed in Table Inocula derived from spontaneous metacyclogenesis in NNN medium with a LIT overlay were used throughout 2.2 Inoculation schedule Trichomys apereoides (250 g mean weight) were distributed in six batches of six animals; (n ¼ 36) The animals, born at animal facilities and gently supplied by Dr P D’Andrea from the Tropical Medicine Department, Oswaldo Cruz Institute, RJ, Brazil, were inoculated subcutaneously with 200 metacyclics/g body weight, for each isolate A group of non-infected animals was included as control (n ¼ 6) 2.3 Parasitological, hematological, and serological follow-up Fresh blood samples from the tail vein and Giemsa stained thin smears of infected and control animals were microscopically examined at 400, thrice a week until negativation of parasitemia and the following parameters were determined: pre-patent period, parasitemia (by countings in a Neubauer hemocytometer) and pattern and percentage of hematic cells (by differential countings of 100 microscopic fields) Mortality was recorded daily Humoral immune response was evaluated weekly by IFA until 63 days PI and reevaluated at the end of the follow-up, when necropsy was performed (5 months PI) IFA was performed by an FITC anti-mouse IgG Sigma conjugate and the test was performed as described by Camargo (1966) Table Trypanosoma cruzi in Trichomys apereoides: isolates from different origins and molecular groups used in this study Codea Host Locality T cruzi group MTRI/BR/1999/R4 MDID/BR/1999/M1 TBRA/BR/1999/JCA3 MLEO/BR/2000/M593 MHOM/BR/1957/Y XXXX/BR/1971/F Trichomys apereoides Didelphis albiventris Triatoma brasiliensis Leontopithecus rosalia Human Unknown primary sourceb Piauı, Northeast Brazil Piauı, Northeast Brazil Piauı, Northeast Brazil Rio de Janeiro, Brazil Maintained in Brazil as reference isolate b Maintained in Brazil as reference isolateb II I II II II I a b Anon (1999) Deane et al (1984b) 80 L Herrera et al / Experimental Parasitology 107 (2004) 78–88 Hemocultures were performed in all animals at necropsy, which was accomplished months PI Hemocultures were maintained at 28 °C and examined fortnightly during months 2.4 Necropsy Two animals in the patent phase infected with MTRI/ BR/1999/R4 isolate and two animals/batch in the subpatent phase of infection (5 months PI) of all groups were sacrificed by anesthetic overdose of ketamine (Ketaset HCl 100 mg/ml) Tissue samples were fixed in Formalin-Millonig (Carson et al., 1973) and routinely processed for paraplast embedding and hematoxylin– eosin staining At least two lm thick sections, at intervals of 60 lm, from heart, skeletal muscle, skin, small and large intestines, liver, spleen, lungs, kidneys, pancreas, urinary bladder, and brain were microscopically examined (1000) in a double blind test The tissular parasitism and histopathologic features were determined and photographed with a Nikon Microflex HPX-35 camera on Agfa APX-100 films 2.5 Extraction of DNA from T apereoides tissue sections Sections (5–10 lm thick) from blocks of fixed embedded heart, smooth muscle from urinary bladder, skeletal muscle and pancreas of subpatent animals (5 months PI) were dispensed with sterile toothpicks in Eppendorf tubes Each section was treated twice with octane or mixed xylene to remove the paraplast, washed twice with 100% ethanol, and rinsed with 2–3 drops of acetone Tissue digestion was done with 100 ll of digestion buffer (50 mM Tris, pH 8.5, mM EDTA, and 0.5% Tween 20) and ll of Proteinase K (200 lg/ml) incubating at 37 °C (Wright and Manos, 1990) DNA of the digested tissue was extracted using the Wizard Genomic DNA Purification System (Promega, Maddison, WI) An initial denaturizing step at 94 °C, for min, was followed by 35 cycles of 94, 55, and 72 °C, for each, and an extension at 72 °C for 10 in a Programmable Thermal Controller (Lane et al., 1997) To ensure that the product of DNA tissue isolation was amenable to DNA amplification, the b-actin protein for mice was simultaneously amplified with the primers 50 -GCTGTGCTATGTTGCCCTAGAATTCGAGC-30 and 50 -CGTACTCCTGCTTGCTGATCCACATGTG C-30 (Herwaldt et al., 2000), determining the integrity of the constitutive DNA A positive control of lg of T cruzi DNA; DNA of non-infected animals and negative control in absence of DNA template were included with every PCR run The PCR products were analyzed by electrophoresis on a 2.5% ethidium bromide stained agarose gel and visualized under ultraviolet light 2.7 Southern blot, labeling, and hybridization of PCR products The Southern blot was carried according to Holtke (1995) In short: the PCR products of tissular kDNA amplification, electrophorized in 2.5% agarose gel, were submitted to alkali denaturizing step (0.5 N NaOH; 1.5 M NaCl) and, subsequently, transferred to nylon membranes (capillary transfer) The membrane was neutralized with 1.5 M NaCl and 0.5 M NaOH, washed with SSC 10, and the transferred products were fixed with 120,000 mJ of ultraviolet light, using an ultraviolet cross-linking apparatus DIG-labeled DNA probes were generated with DIG-High Prime, according to the random primed labeling technique (DIG High Prime: DNA labeling and detection Starter Kit II, Roche) and used for hybridization with membrane blotted nucleic acids, according to standard methods, with high stringency The hybridized probes were immunodetected with antidigoxygenin phosphatase alkaline conjugated Fab fragments and visualized with a chemiluminescent substrate and recorded on X-ray Films (10 exposure time) 2.6 Detection of T cruzi DNA by polymerase chain reaction amplification Results Specific polymerase chain reaction (PCR) amplification of a nucleotide sequence of the 330 bp corresponding to the four variable regions of T cruzi kDNA minicircle in the digested tissue of T apereoides was performed with primers 121—50 -AAATAATGTACGG GKGAGATGCATGA-30 and 122—50 -GGTTCGATT GGGTTGGTGTAATATA-30 (Britto et al., 1995) Briefly, ll of DNA template was added to 15 ll of PCR mixture to give a final concentration of 20 mM Tris– HCl, pH 8.8; 50 mM KCl; 1.5 mM MgCl2 ; 10 lM dNTPs, 10 pM of each primer, and U of Taq DNA Polymerase 3.1 Parasitological follow-up Data concerning parasitological follow-up are summarized in Table In short, T apereoides was able to efficiently control the number of circulating parasites and maintain the infection with both T cruzi I and T cruzi II subpopulations Mortality (50%) was observed only in the rodents inoculated with MTRI/BR/1999/R4 isolate Death of the animals infected with MTRI/BR/ 1999/R4 isolate occurred, respectively, after 22, 25, and 27 days (mean value 25 days) The animals inoculated 81 L Herrera et al / Experimental Parasitology 107 (2004) 78–88 Table Trypanosoma cruzi in Trichomys apereoides: parasitological follow-up of laboratory reared animals inoculated with isolates with different origin and genotypes Isolates codea MTRI/BR/1999/R4 (T cruzi II) MDID/BR/1999/M1(T cruzi I) MHOM/BR/1957/Y(T cruzi II) TBRA/BR/1999/JCA3 (T cruzi II) MLEO/BR/2000/M593 (T cruzi II) XXXX/BR/1971/F (T cruzi I) a b Pre-patent period (days) Patent period (days) X SD X 18 22 29 45 38 22 1.6 1.83 2.5 12.2 1.83 10 14 SD 7.4 3.2 b — b — b — 17 10.6 Peak of parasitemia (tripomastigotes/ml blood)/day Mortality (%) 2.9 106 /27 1.8 105 /22 0.2 105 /32 0.3 105 /46 0.6 105 /44 2.3 105 /26 50 0 0 Anon (1999) Intermittent patent parasitemia expressed by recurring parasitemia, Fig with MTRI/BR/1999/R4, XXXX/BR/1971/F, and MDID/BR/1999/M1 isolates and those that survived MTRI/BR/1999/R4 inoculation displayed higher parasitemias and longer patent periods When inoculated with MHOM/BR/1957/Y, TBRA/BR/1999/JCA3, and MLEO/BR/2000/M593 isolates, respectively, T apereoides displayed intermittent patent parasitemias expressed by recurring parasitemia waves that reached 105 parasites/ml (Table 2; Figs 1A–C) Positive hemocultures (performed at necropsy) were observed only in animals infected with MTRI/BR/1999/ R4 and MHOM/BR/1957/Y isolates Positive PCR of viscera was observed in all infected animals (5 months PI) The kinetics of parasitemia in the animals infected with MTRI/BR/1999/R4, MDID/BR/1999/M1, and MHOM/BR/1957/Y isolates are shown in Figs 1A–C, respectively 3.2 Hematological findings Leukocytosis with high numbers of lymphocytes and neutrophils was the main feature of all infected animals An increase of 4% in the size of myeloid cells with presence of basophile granules was observed Band cells displayed increased nuclei with an amoeboid appearance Number of lymphocytes in infected animals increased significantly in relation to controls (95% of confidence by Student’s t test, p < 0:05), regardless of parasitemia levels The kinetics of the lymphocyte populations in relation to parasitemia in the animals infected with MTRI/BR/1999/R4, MDID/BR/1999/M1, and MHOM/ BR/1957/Y isolates are shown in Figs 2A–C tectable from the fifth day PI onwards) was observed in the rodents infected with MDID/BR/1999/M1 isolate In contrast, animals infected with the MTRI/BR/1999/R4 strain displayed positive IFA tests, only from the 22th day of infection onwards (Figs 1A–C) High serological titers (1:80–1:320) were observed in all animals at necropsy, performed months PI 3.4 Histopathological findings Histological analyses of ‘‘in extremis’’ killed animals, during the acute infection phase with MTRI/BR/1999/ R4 isolate showed parasites in viscera, muscles, and glands (8/12 organs examined) with pseudocysts in heart, skeletal, and smooth muscle Invasion of pancreas acini, adipocytes, and macrophages of the connective tissue adjacent to skeletal muscle was found Liver and spleen were also parasitized Parasites surrounded by diffuse myocarditis and abundant inflammatory lymphoeosinophylic infiltrates with extensive damage in cardiac fibers and different grades of vacuolization and/or myocytolysis in muscular tissue were observed No correspondence was observed between the profile of parasitemia and parasite load in the subpatent phase as accessed by the examination of HE stained tissues Indeed, the necropsied animals displayed only low numbers of amastigote nests found only in duodenum and heart of rodents inoculated with the MTRI/BR/ 1999/R4 isolate that resulted in high parasitema and the MHOM/BR/1957/Y isolate that induced intermittent parasitemia Additional histopathological findings are summarized in Table and Fig 3.5 Stability of the infection 3.3 Humoral immune response Infected T apereoides responded with a strong humoral immune response The onset of the humoral immune response varied according to the inoculated isolate: a precocious humoral immune response (de- The stability of infections of T apereoides inoculated with the T cruzi isolates was evidenced by amplified T cruzi kDNA (330 bp band) in at least one of the following tissues: heart, skeletal muscle, and pancreas (Fig 4) 82 L Herrera et al / Experimental Parasitology 107 (2004) 78–88 Fig Trypanosoma cruzi in Trichomys apereoides Kinetics of parasitemia (–––) and humoral immune response (IFA) Each point represents the mean values of parasite numbers/ml peripheral blood or IgG values plotted as log2 of the dilution titers of six animals infected with isolates SD MTRI/BR/1999/R4, T cruzi II genotype (A); MDID/BR/1999/M1, T cruzi I genotype (B); MHOM/BR/1957/Y, T cruzi II genotype (C) , IgG titer, months PI (standard deviation); and The Southern blot analyses of amplified PCR products confirmed that specific T cruzi kDNA sequences were present in all tissue samples studied Consistently, the amplified kDNA also hybridized with the probe No hybridization was observed in DNA from non-infected animals and negative controls from the PCR (data not shown) That T apereoides may display stable infections by T cruzi was also demonstrated by a two-year followup of one exemplar experimentally infected with MDID/ BR/1999/M1 (T cruzi I) isolate that remained infected during two years PI, as observed by positive hemoculture performed at necropsy L Herrera et al / Experimental Parasitology 107 (2004) 78–88 83 Fig Trypanosoma cruzi in Trichomys apereoides Kinetics of parasitemia (–––) and mean values of peripheral blood lymphocytes of infected (gray columns) and control animals (intermittent line) Infection with isolates: MTRI/BR/1999/R4, T cruzi II genotype (A); MDID/BR/1999/M1, T cruzi I genotype (B); and MHOM/BR/1957/Y, T cruzi II genotype (C) Each point represents the mean value of parasite numbers/ml peripheral blood on peripheral blood lymphocytes of six animals infected with isolates SD (standard error) Discussion Probably due to the difficulties in breeding wild mammals in captivity, they are rarely used as model hosts for T cruzi infection studies Nevertheless, peculiarities of a given host–parasite interaction may be better clarified through studying the original host Herein we are evaluating the interaction of T cruzi with T apereoides, a caviomorph rodent, a group claimed to be ancient hosts of this parasite (Briones et al., 1999) When inoculated with either of the two T cruzi genotypes, T apereoides displayed stable infections with 84 L Herrera et al / Experimental Parasitology 107 (2004) 78–88 Table Trypanosoma cruzi in Trichomys apereoides: tissular parasitism and histopathology in microscopy of H/E viscera sections (5 lm thick, at intervals of 60 lm, 1000) Isolates/organ MTRI/BR/ 1999/R4 (patent phase) MTRI/BR/ 1999/R4 (sub-patent phase) TBRA/BR/ 1999/JCA3 (chronic phase) MDID/BR/ 1999/M (chronic phase) MLEO/BR/2000/ M593 (chronic phase) MHOM/BR/ 1950/Y (chronic phase) XXXX/BR/ 1971/F (chronic phase) Heart Skeletal muscle Urinary bladder Pancreas Duodenum Colon Liver Spleen a, e, f, g, i c, h, i b, g, j, m c, l c, g, h c, g, h c c, k, l d, m, n d, m d, m, n d c, l d d d d, l d d d d d d d d, m, n d d d d d d d d, m, n d, m, n d, l d d d d d c, l, n d d d d d d d d, m, n d d d d d, m d d a–c, Abundant, moderate or scarce pseudocysts with amastigotes and/or trypomastigotes d, No pseudocysts observed e, Diffuse myocarditis; extensive degeneration of myocardial fibers f, Lymphomacroeosinobasophilic inflammatory infiltration Sarcoplasmic substitution g, Vacuolization and/or focal or diffuse myocytolysis, adjacent to parasite nests h, Focal or interstitial lymphocytic myocytis i, Scarce smooth connective tissue j, Extensive peri-vascular smooth connective tissue k, Focal vacuolization in red pulp; apparent normal strome l, Scarce focal inflammatory infiltrates m, Abundant focal or disseminated infiltrates in myocytic cells or in interstitia n, Focal or diffuse myocytolysis and or vacuolization hardly any mortality and a significant humoral immune response A trend for an inverse association between the rise of humoral immune response and the fall of parasitemia could be observed This was an expected feature since correspondence between humoral immune response and decrease of parasitemia has already been described in other placental and marsupial mammals (dos Reis and Lopes, 2000; Jansen et al., 1991) It is worth mentioning that IFA was performed with an antimouse (Muridae) conjugate Higher serological titers would be probably observed if a specific Trichomys (Echymidae) conjugate had been used The majority of the rodents was able to efficiently control the parasite population as confirmed by the scarce parasite burden observed in HE stained tissues after necropsy However, infection was not eliminated, as demonstrated by the persistence of specific antibodies as well as positive PCR tests in all inoculated rodents It is worth mentioning that when infected with the MHOM/BR/1957/Y isolate, recognized as extremely virulent and pathogenic to Mus musculus, T apereoides displayed only intermittent parasitemia and no mortality The T apereoides muscle microhabitat was the most appropriate niche for all T cruzi isolates, as shown by the frequency of colonization and PCR confirmed parasite persistence T cruzi was found colonizing almost every tissue of this rodent including pancreatic cells and adipocytes This pan-infectivity of the parasite in T apereoides confirms the parasite eclecticism, regarding its wide range of mammalian reservoirs and parasitized tissues (Hoare, 1972; Lenzi et al., 1996) Leukocytosis has already been described in other experimentally infected mammal species Furthermore, the high number of polymorphonuclear cells is probably a consequence of the rupture of parasitized cells and the subsequent liberation of chemoattractants as similarly proposed for other mammals (Monte on et al., 1996) The high number of neutrophils recorded in the initial phase of T apereoides infection is most likely explained by intense parasite phagocytosis The histopathological framework observed in T apereoides was comparable to other experimental animal models, in which myocytosis prevailed and myocytolysis was observed The characteristic inflammatory infiltrates of the colonized tissues were also present in the chronic phase, when the number of amastigote nests was scarce or absent, suggesting that inflammation acts as a control mechanism of tissular parasitism also in this rodent species, as well as in other hosts (Molina and Kierszenbaum, 1988; Soares et al., 2001; Teixeira et al., 2002) Given that the described pathological features observed in T apereoides were also noticed in other mammalian hosts including opossums, considered to be the most ancient T cruzi reservoir host (Schofield, 2000), these characteristics are, probably, ancient traits of the T cruzi survival strategy Higher parasitemia could not be associated to the parasite genotype since positive hemocultures were observed only in the animals infected with HOM/BR/1957/ Y and MTRI/BR/1999/R4 isolates, both characterized as T cruzi II and not with MLEO/BR2000/M593 and TBRA/BR/1999/JCA3 isolates, also of this genotype L Herrera et al / Experimental Parasitology 107 (2004) 78–88 85 Fig Parasitological and histopathological features from Trichomys apereoides experimentally infected with different isolates and strains of Trypanosoma cruzi (1) Degeneration of myocardial fibers showing inflammatory infiltrate, vacuolization, and myocytolysis; pseudocysts with amastigotes (a) and trypomastigotes (t), in the sarcoplasm of fibers (MTRI/BR/1999/R4, T cruzi II isolate, acute phase); (2) nest with amastigotes in smooth muscle (sm) of urinary bladder near to the lumen (lu) Myocytolysis and lymphocytic diffuse infiltrates are observed (MTRI/BR/1999/R4 T cruzi II isolate; acute phase); (3) nest with amastigotes, lymphocytic infiltrate, and cellular lysis in pancreas (MTRI/BR/1999/R4, T cruzi II isolate; acute phase); (4) urinary bladder: intense lymphocytic infiltrate and lysis in myocytic cells and interstitium, without parasites (MTRI/BR/1999/R4 T cruzi II isolate; chronic phase); (5) amastigotes’ nest in heart, with myocytolysis and infiltrate (MHOM/BR/1950/Y, T cruzi II reference strain; chronic phase); and (6) interstitial inflammatory infiltrate in skeletal muscle; parasites absents (MLEO/BR/2000/M593 T cruzi II isolate; chronic phase) (HE; scale bar ¼ 15 lm) 86 L Herrera et al / Experimental Parasitology 107 (2004) 78–88 Fig PCR amplification of the 330 bp fragment (black long arrow) from the conserved regions of kDNA extracted from tissues of Trichomys apereoides experimentally infected (chronic phase, months PI) with Trypanosoma cruzi isolates and reference strains, in agarose gel 2.5% electrophoresis (ethidium bromide stain): (A) heart; (B) skeletal muscle; and (C) pancreas Lane 1, migration of the markers of kb ladder (Gibco-BRL Life Technologies, Gaithersburg, MD) Molecular size from the bottom up: 506, 1118–1115, and 1600–1300 bp (black short arrow) Lane 2, negative animal control; lane 3, nude T cruzi DNA; lane 4, MTRI/BR/1999/R4, T cruzi II isolate; lane 5, MHOM/BR/1950/Y, T cruzi II reference strain; lane 6, TBRA/BR/ 1999/JCA3, T cruzi II isolate; lane 7, MDID/BR/1999/M1, T cruzi I isolate; lane 8, XXXX/BR/1971/F, T cruzi I reference strain; lane 9, MLEO/BR/2000/M593, T cruzi II isolate; and lane 10, no DNA in the reaction mixture for PCR amplification Mortality and an overall severe scenario observed in T apereoides infected with MTRI/BR/1999/R4, an isolate derived from a naturally infected T apereoides, is probably the consequence of the complexity of T cruzi transmission cycles in nature, where the parasite randomly infects several mammalian species through different routes and distinct inocula sizes Indeed, as observed in experimentally infected opossums, the oral route resulted always in milder infections in comparison to subcutaneously infected opossum (Jansen et al., 1991) Recent studies in light of more sensitive methodologies (LCSSP-PCR, RAPD, mini-exon polymorphism) have emphasized the importance of the genomic variation of T cruzi in definition of human disease Also, a putative association of T cruzi strains with their hosts, including humans, has been claimed (Andrade et al., 2002; Vago et al., 1996, 2000; Zingales et al., 1999) Indeed, it has been proven that living systems, i.e., animals and culture media, frequently act as biological filters of T cruzi subpopulations, with a consequent parasite subpopulation selection (Deane et al., 1984a; Jansen et al., 1991; Zingales et al., 1999) Moreover, at least in Brazil, T cruzi II is described as associated to primates and to the domestic transmission cycle, while T cruzi I is associated to the sylvatic transmission cycle (Fernandes et al., 1998, 1999) Nevertheless, this does not seem to be a very strict association considering that infection by T cruzi II has already been described in Procyon lotor (Procyoniidae—Carnivora), T apereoides (Echimyidae—Rodentia) (data not shown), Philander frenata, and Didelphis albiventris (Didelphidae—Marsupialia) (Pietrzak and Pung, 1998; Pinho et al., 2000) One should rather take into account that each animal species may exert distinct and particular selective pressures on the several T cruzi subpopulations according to factors such as origin and size of inocula, health status of the host animal, co-infection with other parasites, and even other subpopulations of T cruzi Here, laboratory reared animals submitted to one single inoculum did not display T cruzi tissue dependent tropism, since no striking differences in the pathological picture in the chronic phase could be associated to parasite genotype or isolate Consequently, histotropism in T cruzi is probably a non-fixed peculiarity influenced by several macro- and microenvironmental parameters in addition to parasite and host genetic background The observed pathological picture of T cruzi infection in T apereoides indicates that extreme caution is necessary when forecasting the outcome of infection or attributing virulence, morbidity and mortality to a given geno- or phenotype of T cruzi The presented results demonstrate that T apereoides may act as an efficient natural reservoir, since it maintains long-lasting infections by different T cruzi subpopulations of both genotypes T cruzi I and T cruzi II In addition, this host, gradually becoming synanthropic, may represent an important parasite source for human infection L Herrera et al / Experimental Parasitology 107 (2004) 78–88 Acknowledgments The authors are thankful to Marlene Rodriguez, Estefanıa Flores, Carlos Arde, and Alcidineia Ivo for the technical support, to Dr Paulo Sergio D’Andrea for the supply and management of the experimental caviomorph rodents, and to Dr Vera Bongertz for many helpful comments on the English version of the manuscript Supported by: IRD/CNPq No 910157-00-6, PAPES No 01250250108, CAPES, FUNDMHAM, FIOCRUZ-Brazil, CONICIT No S198000388, FONACYT S1-98000388, C.D.C.H.-U.C.V No 03314729-2000, and No 0934-4097-2001 The present work has the endorsement of the Ethical Commission for Experimentation with Animal Models (CEUA) from Fundacß~ ao Oswaldo Cruz—FIOCRUZ, RJ, Brazil Registration No P0007 References Andrade, L.O., Machado, C.R.S., Chiari, E., Pena, S.D., Macedo, A.M., 2002 Trypanosoma cruzi: role of host genetic background in the differential tissue distribution of parasite clonal populations Experimental Parasitology 100, 269–275 Anon., 1999 Recommendations from a satellite meeting Mem orias Instituto Oswaldo Cruz 94, 429–432 Araujo, C., Mello, C.B., Jansen, A.M., 2002 Trypanosoma cruzi I and Trypanosoma cruzi II: recognition of sugar 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