Ana Maria Jansen

Trypanosoma cruzi: vertebrate and invertebrate cycles in the same mammal host, the opossum Didelphis marsupialis

Epimastigotes multiplying extracellularly and metacyclic trypomastigotes, stages that correspond to the cycle of Trypanosoma cruzi in the intestinal lumen of its insect vector, were consistently found in the lumen of the anal glands of opossums Didelphis marsupialis inoculated subcutaneously with infective feces of triatomid bugs.

Domestic and Wild Mammalian Reservoirs

The perception of the attributes of a reservoir changed significantly during the last decades and, consequently, the necessity of studying zoonosis under a large perspective emerged. This is particularly the case of trypanosomiasis due to Trypanosoma cruzi since this taxon is characterized by its extreme adaptability to almost all tissues in hundreds of mammal host species. In fact, the transmission cycle of T. cruzi among wild and domestic mammal hosts still represents an epidemiological “puzzle,” as several links may or may not be connected and are far from being completely understood. T. cruzi is highly enzootic, which is probably due to its extreme heterogeneity. Several transmission strategies besides vectorial transmission, predation of infected bugs or small mammals, fights, and direct transmission by anal glands of Didelphis sp. should also be considered. This chapter presents some of the mammalian species that are able to maintain the parasite in nature (and, therefore, serve as potential reservoirs), and some of the most relevant studies in each one of the eight orders of mammals—Marsupialia, Cingulata, Pilosa, Rodentia, Primata, Carnivora, Chiroptera, and Artiodactyla—responsible for maintaining T. cruzi in nature.

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 opossums 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.

Trypanosoma cruzi infection in Leontopithecus rosalia at the Reserva Biológica de Poço das Antas, Rio de Janeiro, Brazil

Wild golden lion tamarins (Leontopithecus rosalia) - endangered primates that are native to the Brazilian Atlantic coastal forest - were surveyed for the presence of Trypanosoma cruzi with the use of Giemsa-stained blood smears, hemocultures and an indirect immunofluorescence assay (IFAT). Positive IFAT with titers ranging from 1:20 to 1:1280 were observed in 52% of the 118 wild tamarins examined and the parasite was isolated from 38 tamarins. No patent parasitemia was observed among the tamarins from which T. cruzi was isolated. Serum conversion and positive hemoculture was observed for three animals that had yielded negative results some months earlier, which indicates that T. cruzi is actively transmitted among tamarins. In contrast to observations with other sylvatic isolates, those from the tamarins were significantly more virulent and most of them produced mortality in experimentally infected Swiss mice. Some variation in the kDNA restriction profiles among the isolates was observed. Electrophoresis with GPI, G6PDH, IDH, MDH and ME enzymes showed a Z2 profile.

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.

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.

The use of immunoblot analysis in the diagnosis of canine visceral leishmaniasis in an endemic area of Rio de Janeiro.

In this article, we describe the findings obtained using immunoblot analysis in the diagnosis of canine visceral leishmaniasis (VL) and its correlation with serological titer and clinical status. We found that all animals bearing amastigote forms recognized antigens with 29 and 32 kDa and that this pattern can be exploited for diagnostic and epidemiological purposes. The recognition of the 29- and 32-kDa antigens was verified even in seronegative dogs and preceded seroconversion in periods ranging from several months to 2 yr. We found a correlation between serological titer and parasite burden. Although no correlation between antigenic recognition pattern and clinical status was observed, immunoblot analysis proved to be a reliable test to detect antibodies against Leishmania sp. antigens in dogs from areas with endemic VL.

Modulating variables of Trypanosoma cruzi and Trypanosoma evansi transmission in free-ranging Coati (Nasua nasua) from the Brazilian Pantanal region.

This is a long-term follow-up of infection by Trypanosoma cruzi (TC) and Trypanosoma evansi (TE) in the free-ranging coatis (Procyonidae: Nasua nasua) from Pantanal region (Mato Grosso do Sul, Brazil). We evaluated TC and TE infection by immunofluorescence assay, hemoculture (HC), and microhematocrit centrifuge techniques (MHCT). We also examined coatis health by quantifying hematological parameters including packed cell volume (PCV), white blood cell (WBC) count, and differential leukocyte count. TC isolates thought HC were typed by miniexon gene. Mixed infections by both parasites and the two main lineages of TC (76% TCI, 3% TCII, and 14% TCI/TCII) were observed. Trypanosoma rangeli was also isolated (7%). Overall, seroprevalence of TC and TE infection were 53.5% and 42.0%, respectively. Positive HC (indicating high TC parasitemia) occurred in 34% of seropositive coatis for TC, and positive MHCT (high TE parasitemia) were observed in 36.4% of seropositive coatis for TE. We detected higher prevalence of positive HC in females (72%) than males (43%), and also during the dry season, indicating a seasonal potential of this host species on TC transmission. These features did not occur for TE infection. However, prevalence of TE based on serology and MHCT was higher among adults than subadults. Coatis with positive HC or MHCT displayed a slight decrease in their WBC. In contrast to the animals with positive HC, coatis with positive MHCT displayed a decrease on their PCV. Moreover, concurrent high TC and TE parasitemia caused a larger decrease of PCV values. This study corroborates the importance of coatis in the maintenance of TC and TE transmission cycles in the southern Pantanal and shows a seasonal character of TC transmissibility to its vector by the coati population from the study area.

Trypanosoma cruzi Infection Modulates the Expression of Triatoma brasiliensis def1 in the Midgut

Antimicrobial peptides are an essential component of the insect immune system. One of the most ubiquitous is defensin, which has been identified in all examined insect orders. Triatoma brasiliensis (Heteroptera, Triatominae), the main Trypanosoma cruzi vector in semi-arid regions of north-eastern Brazil, expresses def1, a defensin encoding gene, predominantly in the anterior region (cardia and stomach) of the midgut. In the present study, we compared the transcript abundance of T. brasiliensis def1 in the anterior (stomach) and posterior midgut (small intestine) regions of naïve bugs with those infected with a familiar T. cruzi isolate. In the stomach, only slight differences between the two insect groups were visible, whereas in the small intestine wide differences (up to 9.6-fold) between infected and noninfected bugs become apparent. The highly increased def1 transcript abundance at 20 days after the infective blood meal might be a response to the T. cruzi infection, suggesting a potential function of intestinal defensin in the parasite population control.

Distinct Leishmania species infecting wild caviomorph rodents (Rodentia: Hystricognathi) from Brazil.

Background Caviomorph rodents, some of the oldest Leishmania spp. hosts, are widely dispersed in Brazil. Despite both experimental and field studies having suggested that these rodents are potential reservoirs of Leishmania parasites, not more than 88 specimens were analyzed in the few studies of natural infection. Our hypothesis was that caviomorph rodents are inserted in the transmission cycles of Leishmania in different regions, more so than is currently recognized. Methodology We investigated the Leishmania infection in spleen fragments of 373 caviomorph rodents from 20 different species collected in five Brazilian biomes in a period of 13 years. PCR reactions targeting kDNA of Leishmania sp. were used to diagnose infection, while Leishmania species identification was performed by DNA sequencing of the amplified products obtained in the HSP70 (234) targeting. Serology by IFAT was performed on the available serum of these rodents. Principal findings In 13 caviomorph rodents, DNA sequencing analyses allowed the identification of 4 species of the subgenus L. (Viannia): L. shawi, L. guyanensis, L. naiffi, and L. braziliensis; and 1 species of the subgenus L. (Leishmania): L. infantum. These include the description of parasite species in areas not previously included in their known distribution: L. shawi in Thrichomys inermis from Northeastern Brazil and L. naiffi in T. fosteri from Western Brazil. From the four other positive rodents, two were positive for HSP70 (234) targeting but did not generate sequences that enabled the species identification, and another two were positive only in kDNA targeting. Conclusions/Significance The infection rate demonstrated by the serology (51.3%) points out that the natural Leishmania infection in caviomorph rodents is much higher than that observed in the molecular diagnosis (4.6%), highlighting that, in terms of the host species responsible for maintaining Leishmania species in the wild, our current knowledge represents only the “tip of the iceberg.”