First genotyping of Trypanosoma cruzi from naturally infected Triatoma juazeirensis,Triatoma melanica and Triatoma sherlocki from Bahia State,Brazil

Many previous studies have shown a great phylogenetic and biological variability of Trypanosoma cruzi using different molecular and biochemical methods. Populations of T. cruzi were initially clustered into two main lineages called TcI and TcII by the size of the mini‐exon PCR product. In the present study, 33 isolates derived from three triatomine taxa, which belong to the Triatoma brasiliensis species complex (Triatoma juazeirensis, Triatoma melanica and Triatoma sherlocki); collected in three distinct areas of Bahia state were characterized by PCR. The isolates were identified by the size of the mini‐exon gene, 18S rRNA and 24Sα rRNA amplicons. T. cruzi isolates obtained in sylvatic and intradomiciliar ecotopes, derived from T. juazeirensis and T. melanica, were identified as TcI while the parasites originated from T. sherlocki were characterized as TcI and TcII genotypes, respectively. Those species are present in sylvatic ecotopes but are able to infest intradomiciliar areas. Therefore, it would be important to maintain studies in those localities of Bahia and further investigate the possibilities of Chagas disease transmission. Human disease may occur by any T. cruzi genotype and not only by TcII as it is the case in Amazonia.     

Defecation index and reproductive success of Triatoma maculata (Hemiptera: Reduviidae) under laboratory conditions

The reproductive and defecating behavior of Triatoma maculata (Erichson 1848) was studied on animals from an university culture in Venezuela. This species does not reach the importance of Rhodnius prolixus Stal 1859 as Chagas disease vector in Venezuela. This study addressed the role of defecating frequency, an index of how dangerous the animals are for the human population, and its relationship with why T. maculata is a less important vector than R. prolixus. Human blood was fed to the insects through an artificial feeding device. The 2nd and 3rd instar nymphs defecated more frequently (Id= 0.6, n=40), and our Vth instar nymphs did not complete sexual differentiation. Fertility was 55% (n=865) and fecundity 8 eggs/female/week (n=26). Egg incubation lasted 22 days (n=477). Female longevity was 51 days (n=26). Intermould time grew progressively from 35 days for 1st to 40 days for 4th instar nymphs (n=40). Mould percentage varied from 0% for Vth to 63% for 3rd instar nymphs. Mortality varied from 8% for 3rd to 100% for Vth instar nymphs. These results support evidences explaining the lesser vectorial capacity and low density of T. maculata in human domiciles, including reduced reproduction and defecation when the animal feeds on human blood.     

Genitourinary changes in hamsters infected and reinfected with Trypanosoma cruzi

Authors describe genitourinary changes in male hamsters infected and reinfected with Trypanosoma cruzi. Changes in genital organs have been described in human and in experimental chagasic infection. Genital dysfunctions in chronic chagasic patients affect ejaculation, libido and sexual potency, and testis biopsies may show arrested maturation of germ cells, oligozoospermia and azoospermia. Sixty-five male hamsters were inoculated and reinoculated with 2x10 trypomastigotes of T. cruzi VIC strain, and 22 non-infected animals constituted the control group. Animals were necropsied and fragments from testis, epididymis, seminal vesicle and bladder were collected and stained with hematoxylin-eosin. Peroxidase anti-peroxidase procedure was utilized to detect tissue parasitism. T. cruzi nests were found in testis, epididymis and seminal vesicle of these hamsters. Such parasitism plays a role in the origin of genital lesions observed in humans and laboratory animals during chronic chagasic infection.     

Breeding of wild-caught rodent cricetidae Holochilus brasiliensis under laboratory conditions

The breeding of wild-caught rodent Holochilus brasiliensis (Desmarest, 1819) was studied under laboratory conditions. The mean gestation length was 28.4 days. Litter size ranged from one to five. Males matured at 2-3 months and females at 2-4.5 months of age. The oestrous cycle lasted 6-8 days. Eleven pairs observed over 6-13 months increased to a population of 276 individuals (153 males and 123 females).     

Ecology of Triatoma brasiliensis in northeastern Brazil: seasonal distribution,feeding resources,and Trypanosoma cruzi infection in a sylvatic population

We assessed some ecological parameters of Triatoma brasiliensis in rock piles in the state of Ceará during the rainy and dry seasons. The greatest density was in April (median = 12.5 triatomines/site). The greatest abundance was in December, when the insects were more dispersed and the density per site was lower (6 triatomines/site). The nutritional status of females and 5^th instar nymphs was increased in July. The rate of T. cruzi infection reached its highest peak in July (10.9%). ELISA revealed that the principal food sources were birds (33.1%), followed by armadillos (18.8%). Food sources were more frequently identified during the rainy season. T. brasiliensis specimens collected in the drought tended to: i) present lower rates of T. cruzi infection and gut content reactivity to tested antisera, ii) have a poorer nutritional status, iii) exhibit lower fecundity, iv) be more dispersed among the studied collection sites, and v) be more abundant and easily collected in the surface of the rocks, possibly reflecting an increased searching for blood meals. Such findings underscore epidemiological concerns and allow inferences about the season when triatomines can more frequently invade the peridomestic environment in search of food and recolonize artificial structures.     

Growth and differentiation of Trypanosoma cruzi cultivated with a Triatoma infestans embryo cell line.

Trypanosoma cruzi strain Peru was cultivated in the presence of an established cell line of Triatoma infestans embryo cells (TI- 32). Bloodstream trypomastigotes differentiated into amastigote-like cells (first differentiation phase) which multiplied to form large clusters of cells. Because of their clustering nature, a new term, "staphylomastigotes," has been proposed for this stage. After 10 days of cultivation, 90% of the staphylomastigotes underwent differentiation (2nd differentiation phase) to trypomastigotes (approximately 98%) or epimastigotes (approximately 2%). Bloodstream trypomastigotes cultivated without TI-32 cells underwent the first, but not the 2nd differentiation phase, although occasional epimastogotes were seen (less than 1%). The evidence presented suggests that TI-32 cells produce a labile factor(s) important not only for initiation of the 2nd differentiation phase but also for maintaining the parasites in the trypomastigote stage. The pH of the culture medium was not the initiating factor for the 2nd differentiation phase. Infectivity studies indicated that staphylomastigotes were as infective as blood stream trypomastigotes, but that metacyclic trypomastigotes isolated from culture after the 2nd differentiation phase were slightly more infective than bloodstream forms. Electromicrographs of styphylomastigotes do not provide any evidence of exchange of genetic material between cells.     

Growth and Differentiation of Trypanosoma cruzi Cultivated with a Triatoma infestans Embryo Cell Line*

SYNOPSIS. Trypanosoma cruzi strain Peru was cultivated in the presence of an established cell line of Triatoma infestans embryo cells (TI-32). Bloodstream trypomastigotes differentiated into amastigote-like cells (first differentiation phase) which multiplied to form large clusters of cells. Because of their clustering nature, a new term, “staphylomastigotes,” has been proposed for this stage. After 10 days of cultivation, 90% of the staphylomastigotes underwent differentiation (2nd differentiation phase) to trypomastigotes (?98%) or epimastigotes (?2%). Bloodstream trypomastigotes cultivated without TI-32 cells underwent the first, but not the 2nd differentiation phase, although occasional epimastigotes were seen (< 1%). The evidence presented suggests that TI-32 cells produce a labile factor(s) important not only for initiation of the 2nd differentiation phase but also for maintaining the parasites in the trypomastigote stage. The pH of the culture medium was not the initiating factor for the 2nd differentiation phase. Infectivity studies indicated that staphylomastigotes were as infective as bloodstream trypomastigotes, but that metacyclic trypomastigotes isolated from culture after the 2nd differentiation phase were slightly more infective than bloodstream forms. Electromicrographs of styphylomastigotes do not provide any evidence of exchange of genetic material between cells.     

Trypanosoma cruzi: differentiation after interaction of epimastigotes and Triatoma infestans intestinal homogenate

Incubation of Trypanosoma cruzi epimastigotes with Triatoma infestans intestinal homogenate leads to differentiation to the metacyclic trypomastigote. Features of this interaction are presented. The morphogenetic mechanism was triggered almost at once; for the minimum interaction period assayed (15 min), the degree of differentiation achieved in Grace medium by Day 6 was 70.0 +/- 9.0%. Longer interaction periods failed to improve differentiation. The morphogenesis became irreversible at 4 hr after interaction. Epimastigotes incubated for 4 hr with T. infestans intestinal homogenate and then washed reached significant differentiation, while those washed before this time failed to do so. Treatment of epimastigotes with albumin improved the experimental conditions thereby hastening morphogenesis, the same percentage of metacyclics occurring in only 4 days. The factors capable of triggering differentiation were adsorbed by T. cruzi epimastigotes, as expected, but also by Leishmania mexicana and, to a lesser degree, by sheep red blood cells. Once the morphogenetic mechanism had been triggered following interaction of epimastigotes with intestinal homogenate for 15 min, metacyclic forms developed when parasites were transferred to Grace but not to other media. Treatment of epimastigotes with trypsin abolished their capacity to differentiate, which was completely reversed following a 5 hr incubation in LIT medium.     

Heterogeneous infectiousness in guinea pigs experimentally infected with Trypanosoma cruzi

Guinea pigs are important reservoirs of Trypanosoma cruzi, the causative parasite of Chagas disease, and in the Southern Cone of South America, transmission is mediated mainly by the vector Triatoma infestans. Interestingly, colonies of Triatoma infestans captured from guinea pig corrals sporadically have infection prevalence rates above 80%. Such high values are not consistent with the relatively short 7–8 week parasitemic period that has been reported for guinea pigs in the literature. We experimentally measured the infectious periods of a group of T. cruzi-infected guinea pigs by performing xenodiagnosis and direct microscopy each week for one year. Another group of infected guinea pigs received only direct microscopy to control for the effect that inoculation by triatomine saliva may have on parasitemia in the host. We observed infectious periods longer than those previously reported in a number of guinea pigs from both the xenodiagnosis and control groups. While some guinea pigs were infectious for a short time, other “super-shedders” were parasitemic up to 22 weeks after infection, and/or positive by xenodiagnosis for a year after infection. This heterogeneity in infectiousness has strong implications for T. cruzi transmission dynamics and control, as super-shedder guinea pigs may play a disproportionate role in pathogen spread.     

Molecular Individual-Based Approach on Triatoma brasiliensis: Inferences on Triatomine Foci,Trypanosoma cruzi Natural Infection Prevalence,Parasite Diversity and Feeding Sources

We used an individual-based molecular multisource approach to assess the epidemiological importance of Triatoma brasiliensis collected in distinct sites and ecotopes in Rio Grande do Norte State, Brazil. In the semi-arid zones of Brazil, this blood sucking bug is the most important vector of Trypanosoma cruzi—the parasite that causes Chagas disease. First, cytochrome b (cytb) and microsatellite markers were used for inferences on the genetic structure of five populations (108 bugs). Second, we determined the natural T. cruzi infection prevalence and parasite diversity in 126 bugs by amplifying a mini-exon gene from triatomine gut contents. Third, we identified the natural feeding sources of 60 T. brasiliensis by using the blood meal content via vertebrate cytb analysis. Demographic inferences based on cytb variation indicated expansion events in some sylvatic and domiciliary populations. Microsatellite results indicated gene flow between sylvatic and anthropic (domiciliary and peridomiciliary) populations, which threatens vector control efforts because sylvatic population are uncontrollable. A high natural T. cruzi infection prevalence (52–71%) and two parasite lineages were found for the sylvatic foci, in which 68% of bugs had fed on Kerodon rupestris (Rodentia: Caviidae), highlighting it as a potential reservoir. For peridomiciliary bugs, Galea spixii (Rodentia: Caviidae) was the main mammal feeding source, which may reinforce previous concerns about the potential of this animal to link the sylvatic and domiciliary T. cruzi cycles.     

Agglutination of Trypanosoma cruzi in infected cells treated with serum from chronically infected mice

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. The chronic stage of infection is characterized by a production of neutralizing antibodies in the vertebrate host. A polyclonal antibody, anti-egressin, has been found to inhibit egress of parasites from the host cell late in the intracellular cycle, after the parasites have transformed from the replicative amastigote into the trypomastigote. It has also been found that BALB/c mouse fibroblasts in the late stages of parasite infection become permeable to molecules as large as antibodies, leading to the possibility that anti-egressin affects the intracellular parasites. This project addresses the fate of the intracellular trypomastigotes that have been inhibited from egressing the host cell. Extended cultures of infected fibroblasts treated with chronic mouse serum reduced parasite egress at all time points measured. Parasites released from infected fibroblasts treated with chronic serum had a reduced ability to infect fibroblasts in culture, yet did not lose infectivity entirely. Absorption of chronic serum with living trypomastigotes removed the anti-egressin effect. The possibility that the target of anti-egressin is a parasite surface component is further indicated by the agglutination of extracellular trypomastigotes by chronic serum. The possibility that cross-linking by antibody occurs intracellularly, thus inhibiting egress, was reinforced by cleaving purified IgG into Fab fragments, which did not inhibit egress when added to infected cultures. From this work, it is proposed that the current, best explanation of the mechanism of egress inhibition by anti-egressin is intracellular agglutination, preventing normal parasite-driven egress.     

Reversibility of cardiac fibrosis in mice chronically infected with Trypanosoma cruzi, under specific chemotherapy.

This investigation was performed to verify the effect of specific chemotherapy (Benznidazole or MK-436) on the inflammatory and fibrotic cardiac alterations in mice chronically infected with the strains 21 SF (Type II) and Colombian (Type III) of Trypanosoma cruzi. To obtain chronically infected mice, two groups of 100 Swiss mice each, were infected with either the 21 SF or the Colombian strain (2 x 10(4) and 5 x 10(4) blood forms respectively). The rate of mortality in the acute phase was of 80% for both groups. Twenty surviving mice chronically infected with the 21 SF strain and 20 with the Colombian strain were then divided in treated and untreated groups. Excluding those that died during the course of treatment, 14 mice chronically infected with the 21 SF strain and 15 with the Colombian strain were finally evaluated in the present study. Chemotherapy was performed with Benznidazole (N-benzil-2-nitro-1-imidazolacetamide) in the dose of 100 mg/k.b.w/day, for 60 days, or with the MK-436 (3(1-methyl-5 nitroimidazol-2-yl) in two daily doses of 250 mg/k.b.w, for 20 days. Parasitological cure tests were performed (xenodiagnosis, haemoculture, subinoculation of the blood into newborn mice), and serological indirect immunofluorescence test. The treated and untreated mice as well as intact controls were killed at different periods after treatment and the heart were submitted to histopathological study with hematoxilineosin and picrosirius staining; ultrastructural study; collagen immunotyping, fibronectin and laminin identification by immunofluorescence tests. Results: the untreated controls either infected with 21 SF or Colombian strain, showed inflammatory and fibrotic alterations that were mild to moderate with the 21 SF strain and intense with the Colombian strain. Redpicrosirius staining showed bundles of collagen in the interstitial space and around cardiac fibers. Increased deposits of matritial components and collagen fibers, macrophages and fibroblasts appeared at the ultrastructural examination. Deposits of fibronectin, laminin, pro-III and IV collagens were seen, most intense in those infected with the Colombian strain. Treated mice, parasitologically cured, presented clear-cut regression of the inflammatory lesions and of the interstitial matrix thickening. Mice infected with the Colombian strain and treated with MK-436, was parasitologically cured in 5/6 cases and showed mild inflammatory infiltration and fibrosis. The mice treated with Benznidazole (Colombian strain) did not cure and showed moderate fibrosis and inflammation. Treatment of the mice infected with the 21 SF with Benznidazole determined parasitological cure of all animals, that showed mild inflammation and fibrosis of the myocardium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution and pathogenicity of Trypanosoma cruzi isolated from peridomestic populations of Triatoma infestans and Triatoma guasayana from rural Western Argentina

We assessed the distribution of Trypanosoma cruzi infection in peridomestic triatomines collected manually at a district-wide scale in rural villages around Olta, Western Argentina, and typed the isolated strains according to their pathogenicity to laboratory mice. Of 1623 triatomines examined, only 14 (0.9%) were infected with T. cruzi based on microscopical examination of feces. The prevalence of T. cruzi infection was 0.8% in Triatoma infestans, 2.3% in T. guasayana, and nil in T. garciabesi, T. platensis, and T. eratyrusiformis. Local transmission occurred in kitchens, store-rooms and goat corrals or nearby, though at very low levels. T. cruzi was detected by at least one parasitological method in 11 (79%) of 14 microscope-positive bugs. Hemoculture was the most sensitive method (67%) followed by culture of organ homogenates, histopathology or xenodiagnosis of inoculated suckling mice (55-58%), and culture of microscope-positive bug feces (46%). The evidence suggests that most of the isolated T. cruzi strains would be myotropic type III. Our study establishes for the first time that peridomestic, microscope-positive T. guasayana nymphs were actually infected with T. cruzi, and may be implicated as a putative secondary vector of T. cruzi in domestic or peridomestic sites.