In vivo and in vitro metacyclogenesis tests of two strains of Trypanosoma cruzi in the triatomine vectors Triatoma pseudomaculata and Rhodnius neglectus: short/long-term and comparative study

Metacyclogenesis of Trypanosoma cruzi of the Y and Berenice strains was studied in Triatoma pseudomaculata and Rhodnius neglectus. Results in vivo showed a higher production of metacyclic trypomastigotes in R. neglectus' digestive tube than in T. pseudomaculata. In vitro experiments were also carried out in order to compare the behavior of culture forms of T. cruzi incubated in extracts of different compartments (stomach, intestine, and rectum) of the digestive tract of both species of triatomines. A higher percentage of metacyclic trypomastigotes for both parasite strains, Y and Berenice, was detected in the rectum extract of R. neglectus in comparison to that from T. pseudomaculata. The same results were obtained with in vitro experiments, using parasites incubated in urine from each of those vectors. The adhesion of parasites to the incubated rectum epithelial cells was also compared. In incubations with the Y strain no significant differences were detected between the two triatomine species but, however, with the Berenice strain the mean percentage of cells with adhered parasites was higher in R. neglectus than in T. pseudomaculata.     

Comparative biological characterization of Berenice and Berenice-78 strains of Trypanosoma cruzi isolated from the same patient at different times

The Berenice-78 strain of T. cruzi is very different from the Berenice strain isolated 16 years earlier from the same patient. The authors verified its high infectivity and low virulence for C3H inbred mice that survived the acute phase of infection. In these animals, it was verified that the tropism of parasites was more accentuated for cardiac and skeletal musculature and the parasitaemic level progressively increased with successive blood passages with posterior stability. In relation to Berenice strain the same characteristics were observed as described by Brener, Chiari & Alvarenga (1974). The increase in its virulence for albino mice was again demonstrated. The authors discussed the possibility of reinfection of the patient called Berenice and the importance of knowledge about T. cruzi strains of low virulence for laboratory animals.     

The interaction of myotropic and macrophagotropic strains of Trypanosoma cruzi with myoblasts and fibers of skeletal muscle

The process of interaction of bloodstream trypomastigotes from the myotropic CL and Colombiana strains and the macrophagotropic Y strain of Trypanosoma cruzi with mouse myoblasts and myotubes was analysed. After 24 h of parasite-host cell interaction, parasites from the CL and Colombiana strains appeared to be more infective to myoblasts than those from the Y strain. Parasites from the Colombiana strain were more infective for myotubes than those from the Y strain, while those from the CL strain showed very a low ability to infect the cells. For all strains the infectivity was low for short periods of interaction, increasing with time. Myoblasts infected with parasites from the Y strain fused with other infected and uninfected cells to form myotubes. However, the process of fusion was blocked when the myoblasts were infected with parasites from the CL and Colombiana strains. These data indicate a different behavior of muscle cells when in contact with myotropic or non-myotropic strains of T. cruzi.     

Exacerbation of experimental Trypanosoma cruzi infection in mice by concomitant malaria.

The effect of malaria on the chronic phase of Chagas' disease was investigated in mice. The animals were given Plasmodium bergheri-infected red blood cells 2 to 12 months after their initial inoculation with trypomastigotes of 3 different strains of Trypanosoma cruzi (Y. CL and Gilmar). in all the experiments carried out with one of the strains (CL), a somewhat variable but always considerable percentage of mice (average 39%) relapsed in to the acute phase of Chagas' disease. This relapse was characterized by a significant increase in the number of circulating trypomastigotes. Recrudescence was observed also with a 2nd strain of T. cruzi (Gilmar), which is similar in many aspects to the CL strain, e.g. the morphology of blood stages, curved of parasitemia and susceptibility to antibodies in vitro. In mice whose chronic phase was induced by trypomastigotes of the Y strain, malaria infections did not induce a typical acute phas with high parasitemia by T. cruzi. Bloodstream forms of Y parasites differ from those of CL and Gilmar strains morphologically as well as immunologically, i.e. only the Y strain is easily agglutinated and partly inactivated by specific immune serum. In light of this and other known characteristics of the strains used in the present work, the author speculates on mechanisms which allow malaria infections selectively to suppress acquired host resistance to certain strains of T. cruzi.     

Lectin receptors distribution in the surface membrane of Trypanosoma cruzi blood forms collected from mice submitted to specific treatment.

The authors investigated the distribution of lectin receptors on Trypanosoma cruzi blood forms collected from mice inoculated with, respectively, the drug-resistant and drug-sensitive strains VL-10 and CL, and treated with the two standard active nitroheterocyclic compounds nifurtimox and benznidazole used for treatment of human Chagas' disease. Blood trypomastigotes purified in Fycoll-Hypaque were incubated with fluorescein-labelled lectins Con A, WGA, EE, WFA, TPA and PNA and then microscopically examined. Neither qualitative or quantitative differences in the fluorescence intensity could be detected between the parasites from VL-10 and CL strains submitted or not to treatment. The results suggest that both strains do not differ in their surface membrane carbohydrate moieties. Moreover, the rapid clearance of blood forms from the drug-sensitive strain in animals treated with single doses of both compounds is not likely to depend on membrane alterations expressed by changes in the carbohydrate components. Furthermore, resistance or sensitivity to drugs is not apparently related to carbohydrate distribution on T. cruzi blood forms.     

Exacerbation of Experimental Trypanosoma cruzi Infection in Mice by Concomitant Malaria*

SYNOPSIS. The effect of malaria on the chronic phase of Chagas’disease was investigated in mice. The animals were given Plasmodium berghei-infected red blood cells 2 to 12 months after their initial inoculation with trypomastigotes of 3 different strains of Trypanosoma cruzi (Y, CL and Gilmar). In all the experiments carried out with one of the strains (CL), a somewhat variable but always considerable percentage of mice (average 39%) relapsed in to the acute phase of Chagas’disease. This relapse was characterized by a significant increase in the number of circulating trypomastigotes. Recrudescence was observed also with a 2nd strain of T. cruzi (Gilmar), which is similar in many aspects to the CL strain, e.g. the morphology of blood stages, curve of parasitemia and susceptibility to antibodies in vitro. In mice whose chronic phase was induced by trypomastigotes of the Y strain, malaria infections did not induce a typical acute phase with high parasitemia by T. cruzi. Bloodstream forms of Y parasites differ from those of CL and Gilmar strains morphologically as well as immunologically, i.e. only the Y strain is easily agglutinated and partly inactivated by specific immune serum. In light of this and other known characteristics of the strains used in the present work, the author speculates on mechanisms which allow malaria infections selectively to suppress acquired host resistance to certain strains of T. cruzi.     

Trypanosoma cruzi: quantification in tissues of experimentally infected mice by limiting dilution analysis

A limiting dilution assay (LDA) was developed for the quantification of Trypanosoma cruzi in the heart and blood of infected mice. Three groups of swiss mice were injected ip with "CL", "Colombiana," and "Y" strains. At 1-day intervals after infection, blood and the heart were removed. Serial blood dilutions in LIT medium were performed and distributed in four groups of 24 microplate wells. The growth of parasite was visually checked in an inverted microscope. It was found that curves of parasitemia obtained by parasite counting in a hemocytometer or estimated by LDA were similar. A similar method was used to quantify parasites in the heart of mice. The heart was cut, washed, dried, and its weight was determined. The heart pieces were disrupted by passage through a mesh stainless-steel screen into LIT. Serial dilutions of the heart homogenate were made in LIT and added to at least 24 replicate microplate wells. Parasites were detectable earlier in the heart of mouse infected with Y strain when compared to CL and Colombiana strains. Parasites were detected in the heart of mice of all strains by 6 days after infection. This LDA for quantification of T. cruzi permits a more precise evaluation of the number of living parasites in infected tissues.     

Expression profile and subcellular localization of HslV, the proteasome related protease from Trypanosoma cruzi

Trypanosoma cruzi is a rare example of an eukaryote that has genes for two threonine proteases: HslVU complex and 20S proteasome. HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. In this study, we expressed and obtained specific antibodies to HslU and HslV recombinant proteins and demonstrated the interaction between HslU/HslV by coimmunoprecipitation. To evaluate the intracellular distribution of HslV in T. cruzi we used an immunofluorescence assay and ultrastructural localization by transmission electron microscopy. Both techniques demonstrated that HslV was localized in the kinetoplast of epimastigotes. We also analyzed the HslV/20S proteasome co-expression in Y, Berenice 62 (Be-62) and Berenice 78 (Be-78) T. cruzi strains. Our results showed that HslV and 20S proteasome are differently expressed in these strains. To investigate whether a proteasome inhibitor could modulate HslV and proteasome expressions, epimastigotes from T. cruzi were grown in the presence of PSI, a classical proteasome inhibitor. This result showed that while the level of expression of HslV/20S proteasome is not affected in Be-78 strain, in Y and Be-62 strains the presence of PSI induced a significantly increase in Hslv/20S proteasome expression. Together, these results suggest the coexistence of the protease HslVU and 20S proteasome in T. cruzi, reinforcing the hypothesis that non-lysosomal degradation pathways have an important role in T. cruzi biology.     

Active entry of bloodstream forms of Trypanosoma cruzi into macrophages.

The uptake of bloodstream forms of Trypanosoma cruzi, Y and CL stocks, by mouse peritoneal macrophages and their intracellular differentiation and multiplication has been compared in vitro. After 48 h the number of macrophages showing intracellular amastigote forms was higher when the Y stock was used. The number of parasitized cells increased with the time of contact between parasites and macrophages. Prior treatment of the parasites with anti-T. cruzi antibodies and/or complement increased the number of infected macrophages, but did not interfere with their subsequent differentiation within the macrophages. The number of parasitized cells was greater when macrophages were obtained from mice previously treated with lipopolysaccharide, peptone or thioglycollate. Uptake was not appreciably affected when macrophages were pre-treated with trypsin or anti-macrophage serum, or when the parasites and macrophages were incubated in the presence of cytochalasin B. In the same experimental conditions, epimastigotes of T. cruzi when not able to differentiate into amastigotes. Their uptake was potentiated by previous treatment with specific antibodies and/or complement and was blocked by cytochalasin B. These results confirm that epimastigotes derived from T. cruzi cultures are phagocytosed and suggest that bloodstream forms penetrate actively into macrophages.     

Kinetics of infection induced byYersinia

Yersinia enterocolitica of different serotypes andY. intermedia, Y. frederiksenii, andY. kristensenii, in a total of nine strains, were inoculated intragastrically and intravenously into Swiss mice. The animals were observed daily to check for clinical alterations. Groups of five were killed intermittently at 6-h, and 3-, 6-, 10-, 15-,and 21-day periods or more after the inoculation; possible macroscopic alterations of the organs and tissues were checked. Development of infection at these periods was followed by performing viable bacterial counts on homogenates of selected tissues and the kinetics of infection was established. Clinical and pathologic alterations occurred only in the animals inoculated with the human strains ofY. enterocolitica 0:3 and 0:8, independent of the route of infection. After intragastric inoculation, theY. enterocolitica strains considered to be adapted to man were isolated from all organs and tissues, with the exception of the blood, from which only serotype 0:8 was isolated; otherYersinia strains were found only in the cecal content. After intravenous challenge, all the strains infected the organs and tissues at different times and in varied intensity, with exception of Peyer's patches and mesenteric lymph nodes, which were not infected by all theYersinia strains.     

Leishmania major: histopathological responses before and after topical treatment in experimental animals

The cellular response in the cutaneous leishmaniasis lesion (CL), of BALB/c mice treated topically with an ointment composed of 15% paromomycin and 12% methylbenzethonium chloride (PR-ointment) was studied. In the infected, untreated control group, the lesion showed progressive necrosis with an increase in the number of parasites, macrophages, lymphocytes, and polymorphonuclear cells over a period of 18 weeks. In the PR ointment-treated group, complete healing of the lesion was observed 4 weeks after termination of treatment, but total elimination of the parasites from the lesion was observed only 2 weeks later. A marked reduction in the number of macrophages and polymorphonuclear cells was observed during the healing process. A similar phenomenon was observed with mice inoculated intraperitoneally with paromomycin alone, although total elimination of the parasites from the lesions of these mice was not demonstrated over a period of 18 weeks. Neither L3T4 helper T cells nor Ly2 cytotoxic suppressor T cells were detected in the CL lesion, either before or after treatment.     

Trypanosoma cruzi: infection patterns in intact and athymic mice of susceptible and resistant genotypes

Inbred strains of mice inoculated with the T cruzi Y strain behaved as susceptible (A/J, C3H/HeN), intermediate (BALB/c) or relatively resistant (C57BL/6) with respect to the magnitude of parasitaemia and mortality rate. C57BL/10 mice were susceptible in relation to parasitaemia but resistant when mortality was analyzed. Infection with T cruzi CL strain presented the same results, except for C57BL/6 which behaved as susceptible mice. Athymic mice of various backgrounds revealed no differences in susceptibility, presenting the same dramatic parasitaemia, tissue colonization pattern and no inflammatory reaction in any of the tissues studied. Infection of euthymic and athymic BALB/c mice elicited the production of parasite-specific antibodies, which reached similar levels on the first 9 days but differed after day 13. Serum transfer experiments in BALB/c mice did not show great differences in parasitaemia but altered T. cruzi polymorphism reducing the slender forms in athymic mice. Histopathology of athymic BALB/c mice showed the same tissue tropism when infected either with T cruzi Y or CL strain.     

Susceptibility of inbred mice to Leishmania tropica infection: correlation of susceptibility with in vitro defective macrophage microbicidal activities

Eleven mouse strains were inoculated in footpads with amastigotes of Leishmania tropica and observed for 12 weeks. Liver and spleen impression smears from infected mice were examined for the presence of intracellular parasites. Four strains (BALB/cJ, C57L/J, NZW/N, and P/J) failed to heal the subcutaneous lesion and showed evidence of systemic infection; the remaining seven strains (A/J, C3H/HeJ, C3H/HeN, C3HeB/FeJ, C57BL/6J, C57BL/10J, and C57BL/10ScN) were each resistant to infection and resolved their lesions by Week 10. Macrophages from the four susceptible strains could not be activated to kill L. tropica amastigotes by treatment with soluble lymphocyte products in vitro. In contrast, macrophages from all seven resistant strains responded to lymphokine treatment and eliminated 80-90% of intracellular parasites. These results suggest that in vitro macrophage microbicidal activities predict the course of systemic leishmanial disease.     

The effect of iron deficiency and iron overload on the evolution of Chagas disease produced by three strains of Trypanosoma cruzi in CFW mice.

1. CFW mice were fed either on control diet or on iron-deficient diet. 2. After 5 months the mice were infected with CL, Y or YuYu strain of Trypanosoma cruzi. 3. On the fifth day after the infection, the mice on control diet were divided in three groups: one group remained as controls, two groups were injected either with desferrioxamine or iron-dextran. 4. The severity of the disease was evaluated by parasitemia and mortality. 5. The experimental groups were compared with the infected group fed on the control diet. 6. In mice fed on the iron-deficient diet, the disease was more severe for CL strain and less severe for Y and YuYu strains. 7. Treatment with desferrioxamine produced a less severe disease with YuYu strain and no difference with the other strains. 8. On Treatment with iron-dextran, the disease became more severe with Y and CL strains; no effect was observed with YuYu strain. 9. These findings may be due to intraspecific differences among the strains.     

Improvements in obtaining New World Leishmania sp from mucosal lesions: Notes on isolating and stocking parasites

Tegumentary leishmaniasis is an endemic protozoan disease that, in Brazil, is caused by parasites from Viannia or Leishmania complex. The clinical forms of cutaneous disease comprise localized, disseminated, mucosal or mucocutaneous, and diffuse leishmaniasis. Viannia complex parasites are not easy to isolate from patient lesions, especially from mucosal lesions, and they are difficult to culture. The aim of the present study was to compare the efficiency of ex vivo (culture) and in vivo (IFNγ-deficient mice) parasite isolation methods to improve the isolation rate and storage of stocks of New World Leishmania sp that cause cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). Biopsy fragments from cutaneous or mucosal lesions were inoculated into culture medium or mouse footpads. We evaluated 114 samples (86 CL, 28 ML) using both methods independently. Samples from CL patients had a higher isolation rate in ex vivo cultures than in mice (34.1% vs. 18.7%, P<0.05). Nevertheless, almost twice the number of isolates from ML lesions was isolated using the mouse model compared to ex vivo cultures (mouse, 6/25; culture, 3/27). The overall rates of isolation were 40.2% for CL samples and 29.6% for ML samples. Of the 43 isolations, we successfully stocked 35 isolates (81.4%; 27 CL, 8 ML). Contaminations were more frequently detected in cultures of ML than CL lesions. For comparison, the use of both methods simultaneously was performed in 74 samples of CL and 25 samples of ML, and similar results were obtained. Of the eight ML isolates, five were isolated only in mice, indicating the advantage of using the in vivo method to obtain ML parasites. All parasites obtained from in vivo isolation were cryopreserved, whereas only 68% of ex vivo isolations from CL lesions were stocked. In conclusion, the use of genetically modified mice can improve the isolation of parasites from ML. Isolation and stocking of New World Leishmania parasites, especially those from ML that are almost absent in laboratory stocks, are critical for evaluating parasite genetic diversity as well as studying host-parasite interactions to identify biological markers of Leishmania. In this paper, we also discuss some of the difficulties associated with isolating and stocking parasites.     

Evaluation of the rabbit as a model for Chagas' disease. I. Parasitological studies

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, 72 animals have been inoculated by intraperitoneal and conjunctival route with bloodstream forms, vector-derived metacyclic trypomastigotes and tissue culture trypomastigotes of Trypanosoma cruzi strains Y, CL and Ernane. In 95.6% of the animals trypomastigotes had been detected at the early stages of infection by fresh blood examination. The course of parasitemia at the acute phase was strongly influenced by the parasite strain and route of inoculation. At the chronic phase parasites had been recovered by xenodiagnosis and/or hemoculture in 40% of the examined animals. The xenodiagnosis studies have shown selective interactions between the T. cruzi strains and the four species of vectors used, inducing significant variability in the results. The data herein present are consistent with the parasitological requirements established for a suitable model for chronic Chagas' disease.     

Trypanosoma cruzi surface molecule gp90 downregulates invasion of gastric mucosal epithelium in orally infected mice

Experiments were performed to elucidate why Trypanosoma cruzi isolates 573 and 587 differ widely in their efficiency to infect gastric mucosal epithelium when administered orally to mice. These isolates have the same surface profile and a similar capacity to enter host cells in vitro. Metacyclic forms of isolates 573 and 587 and the control CL isolate expressed similar levels of gp82, which is a cell invasion-promoting molecule. Expression of gp90, a molecule that downregulates cell invasion, was lower in the CL isolate. Consistent with this profile, approximately threefold fewer parasites of isolates 573 and 587 entered epithelial HeLa cells, as compared to the CL isolate. No difference in the rate of intracellular parasite replication was observed between isolates. When given orally to mice, metacyclic forms of isolate 573, like the CL isolate, produced high parasitemia (>10(6) parasites per ml at the peak), killing approximately 40% of animals, whereas infection with isolate 587 resulted in low parasitemia (<10(5) parasites per ml), with zero mortality. On the fourth day post-inoculation, tissue sections of the mouse stomach stained with hematoxylin and eosin showed a four to sixfold higher number of epithelial cells infected with isolate 573 or CL than with isolate 587. The rate of intracellular parasite development was similar in all isolates. Mimicking in vivo infection, parasites were treated with pepsin at acidic pH and then assayed for their ability to enter HeLa cells or explanted gastric epithelial cells. Pepsin extensively digested gp90 from isolate 573 and significantly increased invasion of both cells, but had minor effect on gp90 or infectivity of isolates 587 and CL. The profile of g82 digestion was similar in isolates 573 and 587, with partial degradation to a approximately 70 kDa fragment, which preserved the target cell binding domain as well as the region involved in gastric mucin adhesion. Gp82 from CL isolate was resistant to pepsin. Assays with parasites recovered from the mouse stomach 2 h after oral infection showed an extensive digestion of gp90 and increased infectivity of isolate 573, but not of isolate 587 or CL. Our data indicate that T. cruzi infection in vitro does not always correlate with in vivo infection because host factors may act on parasites, modulating their infectivity, as is the case of pepsin digestion of isolate 573 gp90.     

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.     

Disruption of the Toxoplasma gondii bradyzoite-specific gene BAG1 decreases in vivo cyst formation

The bradyzoite stage of the Apicomplexan protozoan parasite Toxoplasma gondii plays a critical role in maintenance of latent infection. We reported previously the cloning of a bradyzoite-specific gene BAG1/hsp30 (previously referred to as BAG5) encoding a cytoplasmic antigen related to small heat shock proteins. We have now disrupted BAG1 in the T. gondii PLK strain by homologous recombination. H7, a cloned null mutant, and Y8, a control positive for both cat and BAG1, were chosen for further characterization. Immunofluorescence and Western blot analysis of bradyzoites with BAG1 antisera demonstrated expression of BAG1 in the Y8 and the PLK strain but no expression in H7. All three strains expressed a 116 kDa bradyzoite cyst wall antigen, a 29 kDa matrix antigen and the 65 kDa matrix reactive antigen MAG1. Mice inoculated with H7 parasites formed significantly fewer cysts than those inoculated with the Y8 and the PLK strains. H7 parasites were complemented with BAG1 using phleomycin selection. Cyst formation in vivo for the BAG1-complemented H7 parasites was similar to wild-type parasites. We therefore conclude that BAG1 is not essential for cyst formation, but facilitates formation of cysts in vivo.     

The relationships of some viruses causing necrotic diseases of the potato

Potato virus B , and some other viruses with reactions in potato varieties different from any previously described, are strains of virus X . All produce intracellular inclusions which vary with different hosts and virus strains. Except with virus B, the inclusions are larger and more frequent in potato than in tobacco or tomato. All give systemic infection when inoculated to tobacco, tomato and potato varieties in which they are carried or cause mosaic symptoms; some give systemic infection when inoculated to varieties in which they cause top-necrosis, whereas others give only local lesions.
Potato virus C is a strain of virus Y: in tobacco and a few potato varieties both produce similar symptoms, but in those varieties in which Y causes leaf-drop streak, C causes top-necrosis. C causes systemic infection when inoculated to tobacco and to potato varieties in which it causes mosaic symptoms, but not when inoculated to potato varieties in which it causes top-necrosis. Virus C was not transmitted by M . persicae. Viruses C and Y produce a few small intracellular inclusions in potato and tobacco.
Virus A is not related to Y or X : no inclusions were found in plants infected with A alone.