Trypanosoma cruzi in the opossum Didelphis marsupialis: a study of the correlations and kinetics of the systemic and scent gland infections in naturally and experimentally infected animals.

The genus Didelphis (Marsupialia, Didelphidae) has the unique capacity of supporting both multiplication cycles of Trypanosoma cruzi simultaneously; besides the intracellular forms, the epimastigotes can be found multiplying and differentiating abundantly in the lumen of the scent glands. The biological significance of the life cycle of T. cruzi within the scent glands of Didelphis marsupialis, as well as its contribution to the epidemiology of the disease, is presently unclear. In order to clarify the mechanisms involved in the colonization of this singular habitat by T. cruzi, as well as to understand its biological role, we have carried out a serological and parasitological follow-up of both natural and experimental infections of young and adult opossums. Although all natural infections were stable and long lasting, no infected scent glands were found, indicating that the stability of the systemic infections does not depend on the presence of flagellates in the scent gland. In 84% of the experimentally infected animals the colonization of the scent glands was preceded by a period of patent parasitemia. Parasitism of the scent glands was essentially permanent and bilateral, and its maintenance was independent of circulating parasites. Moreover, the course of the infection differed depending on the source (scent glands versus axenic culture-derived) of the metacyclic forms. Our results suggest that parasitism of the SG of D. marsupialis is most likely a secondary acquisition, a step toward independence from the insect vector, similarly to what is accepted for Trypanosoma equiperdum.

Development of Endotrypanum (Kinetoplastida:Trypanosomatidae) in experimentally infected phlebotomine sand flies (Diptera:Psychodidae).

The developmental biology (parasite establishment, migration, and differentiation) of Brazilian strains of Endotrypanum are reported for 3 sand fly species: Lutzomyia longipalpis Lutz & Neiva, L. shannoni Dyar, and Phlebotomus papatasi Scopoli. Laboratory-reared sand flies were infected by feeding on a promastigote suspension through a chick-skin membrane. Infections within the insect gut were examined at various times after feeding by staining fresh and fixed specimens. Development of Endotrypanum varied for each parasite-host species association. After feeding on culture forms of E. schaudinni Mesnil & Brimont (strain ISHA/BR/80/IM1111), significantly more L. shannoni (100%, 9/9) became infected than did L. longipalpis (62.3%, 33/53) or P. papatasi (27.3%, 15/55). The greatest number of infections were in the midgut and hindgut from 6 to 16 d after feeding, but flagellates also were present in the Malpighian tubules. Moreover, distinct development patterns in the sand fly gut were obtained when the Callejon L. longipalpis colony was fed on cultures of other Endotrypanum strains. Significantly fewer sand flies became infected with strain MCHO/BR/85/IM2259 (18.2%, 4/22) than with strain ISHA/BR/80/IM1111 (55.6%, 20/36). There were also individual variation in the distribution and survival of parasites within the guts of flies in each group. These data indicate that there is variation in the susceptibility to infection with Endotrypanum among and within sand fly species.

Histopathological study of experimental and natural infections by Trypanosoma cruzi in Didelphis marsupialis.

Didelphis marsupialis, the most important sylvatic reservoir of Trypanosoma cruzi, can also maintain in their anal scent glands the multiplicative forms only described in the intestinal tract of triatomine bugs. A study of 21 experimentally and 10 naturally infected opossums with T. cruzi was undertaken in order to establish the histopathological pattern under different conditions. Our results showed that the inflammation was predominantly lymphomacrophagic and more severe in the naturally infected animals but never as intense as those described in Chagas' disease or in other animal models. The parasitism in both groups was always mild with very scarce amastigote nests in the tissues. In the experimentally infected animals, the inflammation was directly related to the presence of amastigotes nests. Four 24 days-old animals, still in embryonic stage, showed multiple amastigotes nests and moderate inflammatory reactions, but even so they survived longer and presented less severe lesions than experimentally infected adult mice. Parasites were found in smooth, cardiac and/or predominantly striated muscles, as well as in nerve cells. Differing from the experimentally infected opossums parasitism in the naturally infected animals predominated in the heart, esophagus and stomach. Parasitism of the scent glands did not affect the histopathological pattern observed in extraglandular tissues.

Enzyme polymorphism in Endotrypanum and numerical analysis of isoenzyme data.

In this study, we have analysed enzyme polymorphism among a group of protozoan parasites of the genus Endotrypanum (Kinetoplastida: Trypanosomatidae). Seventeen stocks of Endotrypanum spp. isolated from sloths (Choloepus didactylus and C. juruanus) in the Amazon Region of Brazil were analysed by enzyme electrophoresis, and their electromorphic profiles were compared with reference strains reported previously. The 16 enzymic loci were analysed, and the strains were classified into zymodemes, each representing parasites with unique enzyme profiles. Each zymodeme was considered as an elementary taxon, and using numerical analyses (cladistic, agglomerative hierarchical and ordination techniques) the genus was shown to be monophyletic and the 12 zymodemes characterized could be divided into 3 groups (A, B, C). The heterogeneous population (which may represent a complex of parasite species or strains variants) showed, however, no correlation with the origin (i.e. host species involved or geographic area of isolation) of Endotrypanum stocks. Eight isolates of Endotrypanum sp. from Rondônia State (Brazil) and a parasite strain from Panama were clustered together into a zymodeme, which was phenetically closely related to the E. monterogeii from Costa Rica. The data indicate that E. schaudinni is a species complex.

The influence of sugars and amino acids on the blood-feeding behaviour, oviposition and longevity of laboratory colony of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae, Phlebotominae).

Schneider's Drosophila medium, a complex amino acid rich medium was tested alone and with seven different sugars for some aspects of the biology of Lutzomyia longipalpis. Statistically significant results were obtained when sucrose was used alone, indicating that among the sugars tested, this is still the most suitable and practical one for the maintenance of L. longipalpis colonies. However, the addition of Schneider's medium to a pool of different sugars, was suggested to be related with the acceptance of the first and second blood meals and to longevity, these being, obviously, quite relevant aspects when transmission experiments are contemplated.

Trypanosoma cruzi infection in the opossum Didelphis marsupialis: absence of neonatal transmission and protection by maternal antibodies in experimental infections.

The high rate of natural Trypanosoma cruzi infection found in opossums does not always correlate with appreciable densities of local triatomid populations. One alternative method which might bypass the invertebrate vector is direct transmission from mother to offspring. This possibility was investigated in five T. cruzi infected females and their litters (24 young). The influence of maternal antibodies transferred via lactation, on the course of experimental infection, was also examined. Our results show that neonatal transmission is probably not responsible for the high rate of natural T. cruzi infection among opossums. In addition antibodies of maternal origin confer a partial protection to the young. This was demonstrated by the finding of a double prepatency period and 4, 5 fold lower levels of circulating parasites, in experimentally infected pouch young from infected as compared to control uninfected mothers. On the other hand, the duration of patent parasitemia was twice as long as that observed in the control group.

Ultrastructural aspects of Trypanosoma (Megatrypanum) freitasi in co-cultivation with mammalian cells.

Cultures of Trypanosoma (Megatrypanum) freitasi with L929 mouse fibroblasts at 27.5 degrees C were examined by scanning and transmission electron microscopy in an attempt to clarify the processes of colony formation by the epimastigotes and of their attachment to substrata. It was seen that the flagellates occupy intercellular spaces and do not associate with intact fibroblasts. As the trypanosome population increases, ever larger portions of the substratum are cleared of fibroblasts and occupied by conglomerates of epimastigote colonies consisting of about a dozen organisms that attach to the substratum by their anterior extremities and form pyramidal clusters. Attachment of the epimastigotes involves the flagellar membrane, which becomes extraordinarily enlarged and assumes various aspects of broad sheets, filaments and loops over the substratum or along the flagellum, which exhibits a shortened axoneme. Desmosome or hemidesmosome plaques are present when the attachment takes place between membranes or between the membrane and the substratum.

Trypanosoma cruzi: course of infection in platelets-depleted mice.

The effect of platelet depletion on the course of Trypanosoma cruzi infection in BALB/c mice was investigated. Thrombocytopenia was achieved by inoculation of rabbit anti-platelet IgG during the parasitemic phase of the infection. The number of parasites in the blood of anti-platelet IgG treated was significantly higher than that of non-treated control mice, during the phase of high parasitemia. Cumulative mortality of platelet-depleted mice was consistently but not significantly higher than that of control mice up to the 32nd day of infection; from the 33rd day on they were equivalent, no mortalities occurring from then on, until observations were discontinued on the 60th day. These results suggest that platelets participate of the mechanisms of parasites removal from the bloodstream, but do not have an effective role in the mechanisms of defence against T. cruzi, during the acute phase of infection.

Trypanosoma cruzi in the opossum Didelphis marsupialis: parasitological and serological follow-up of the acute infection.

The opossum Didelphis marsupialis is known to be among the most important wild reservoirs of Trypanosoma cruzi and one in which the trypanosome may go through both the usual vertebrate intracellular cycle in its tissues and an extracellular cycle in the lumen of its scent glands. The species is highly resistant to heavy inocula and, depending on the parasite strain, experimental infections may be permanent or self limited. Aiming to understand the mechanisms involved in this parasite-host interaction we made a study of the acute phase of infection with different T. cruzi strains. Strains F, G-49 and G-327 produced durable infections with relatively high parasitemia and invasion of the scent glands, while equivalent inocula of the Y strain resulted in scanty parasitemia of short duration, no invasion of the SG, and no evidence of persistent parasitism. A smaller inoculum of G-49 produced only subpatent though persistent parasitemia and no invasion of the scent glands. The humoral immune response was less marked in the Y group; among the other groups IgM and IgG antibodies increased to high levels, higher in the G-49 group. The increase in IgG coincided with a drop of parasitemia to subpatent levels. Two opossums inoculated directly in the scent glands with culture forms of the Y strain had a short-lived subpatent parasitemia, but the parasites remained in the glands and serum Ig antibodies reached high levels. Immunoblot analysis showed that the sera of the inoculated opossums recognized few T. cruzi antigens (more in the F strain) in comparison with those of mice. However, with the only exception of those subcutaneously inoculated with the Y strain and including two naturally infected specimens, all the opossum's sera recognized a 90-kDa peptide in all T. cruzi strains. Our results confirm that opossums are able to selectively eliminate some strains of T. cruzi and indicate that the mechanism involved in this selection is probably not related to the humoral immune response. In infections by strains that are able to establish a permanent foothold in opossum tissues, there are indications that IgG antibodies participate in the control of the parasite population of the acute phase but are unable to prevent the chronic phase. It was once more demonstrated that the opossum infected scent glands function as diffusion chambers for parasite antigens but that, on the other hand, the parasites are here protected against the mechanisms developed by the host to control their population.

Developmental stages of Trypanosoma (Megatrypanum) freitasi Rego, Magalhães & Siqueira, 1957 in the opossum Didelphis marsupialis (Marsupialia, Didelphidae).

Trypanosoma (Megatrypanum) freitasi, a parasite of marsupials of the genus Didelphis, has been found to undergo in the lumen of the scent (anal) glands of its vertebrate host, a cycle such as usually occurs in the intestinal tract of the insect vectors of trypanosomatids and similar to what has been reported for Trypanosoma (Schizotrypanum) cruzi. The invertebrate host of Trypanosoma freitasi is still unknown. Developmental stages of the trypanosome in its mammalian host, especially the dividing epimastigotes, multinucleate plasmodial forms and rosettes found in the lumen of the scent glands of a naturally infected Didelphis marsupialis are described and illustrated.

Continuous growth and differentiation of Trypanosoma (Megatrypanum) freitasi Rego, Magalhães & Siqueira, 1957, in vitro.

Trypanosoma (Megatrypanum) freitasi, a parasite of didelphid opossum, was known to be very difficult to cultivate in conventional media. Co-cultivation with L929 cell line in Baltz's medium at 27.5 degrees C resulted in luxuriant growth of the trypanosome with the production of epimastigote colonies that adhered to the surface of culture flasks or tubes, and transformation into metacyclics. Further transformation was stimulated by raising the incubation temperature. At 37 degrees C the population was of the bloodstream type and resistant to lysis by complement.

Infection of a mammal by monogenetic insect trypanosomatids (Kinetoplastida, Trypanosomatidae).

Monogenetic insect trypanosomatids of the genera Crithidia, Leptomonas and Herpetomonas, multiplied as in axenic cultures, for many months, in the lumen of the scent glands of the opossum Didelphis marsupialis. Specific antibodies were detected in the serum of the animals but there was no evidence of invasion of their tissues by the parasites.

From a mono to a digenetic life-cycle: how was the jump for flagellates of the family Trypanosomatidae?

It has been found that species of the digenetic genus Trypanosoma as well as species of monogenetic trypanosomatids of insects can grow in the lumen of the scent glands of opossums reproducing the cycle they perform in the intestinal tract of their insect hosts. Based on these findings, speculations are made on the incompletely known cycles of many mammalian trypanosomes and on the evolution of the family Trypanosomatidae.

Are Asian monkeys the original mammalian hosts of Trypanosoma conorhini?

It is hypothesized that Asian monkeys were the original hosts of Trypanosoma conorhini because they have been found naturally infected, the vector among rats is a tropicopolitan triatomine bug that belongs to a complex of Asian species, and primates were shown to be more susceptible than rats.

Another Trypanosoma, distinct from T. cruzi, multiplies in the lumen of the anal glands of the opossum Didelphis marsupialis.

Epimastigotes were found multiplying in the anal glands and in hemocultures of an opossum; rare metacyclics were seen in the cultures. The flagellate is possibly T. (Megatrypanum) freitasi Rego, Magalhães & Siqueira, 1957, but its final identification is still pending.

Trypanosoma cruzi in the opossum Didelphis marsupialis: an indirect fluorescent antibody test for the diagnosis and follow-up of natural and experimental infections.

The use of an indirect fluorescent antibody test (IFAT) performed in a "sandwich" technique has demonstrated: (i) the usefulness of the test for the diagnosis of Trypanosoma cruzi infection in the opossum Didelphis marsupialis; (ii) the existence of differences in the serological response of the opossum, that were related to the parasite strain and were clearly evident during the follow-up of experimental infections in laboratory born specimens; (iii) that, despite a good correlation between serological and parasitological examinations, IFAT was the most sensitive diagnostic test used, followed by xenodiagnosis; and, (iv) that in general, the opossum D. marsupialis seems to be a good responder to T. cruzi antigens.