Development of sporogonic cycle of Plasmodium vivax in experimentally infected Anopheles albimanus mosquitoes

The sporogonic cycle of Plasmodium vivax was established and maintained under laboratory conditions in two different strains of Anopheles albimanus mosquitoes using as a parasite source blood from human patients or from Aotus monkeys infected with the VCC-2 P.vivax colombian isolate. Both the Tecojate strain isolate from Guatemala and the Cartagena strain from the colombian Pacific coast were susceptible to infections with P.vivax. A higher percentage of Cartagena mosquitoes was infected per trial, however the Tecojate strain developed higher sporozoite loads. Intravenous inoculation of Aotus monkeys with sporozoites obtained from both anopheline strains resulted in successful blood infections. Animals infected with sporozoites from the Tecojate strain presented a patent period of 21-32 days whereas parasitemia appeared between days 19-53 in monkeys infected with sporozites from Cartagena strain.

Optimization of a mouse immunization protocol with Paracoccidioides brasiliensis antigens

The objectives of the present study were to optimize the protocol of mouse immunization with Paracoccidioides brasiliensis antigens (Rifkind's protocol) and to test the modulation effect of cyclophosphamide (Cy) on the delayed hypersensitivity response (DHR) of immunized animals. Experiments were carried out using one to four immunizing doses of either crude particulate P. brasiliensis antigen or yeast-cell antigen, followed by DHR test four or seven days after the last immunizing dose. The data demonstrated that an immunizing dose already elicited response; higher DHR indices were obtained with two or three immunizing doses; there were no differences between DHR indices of animals challenged four or seven days after the last dose. Overall the inoculation of two or three doses of the yeast-cell antigen, which is easier to prepare, and DHR test at day 4 simplify the original Rifkind's immunization protocol and shorten the duration of the experiments. The modulation effect of Cy on DHR was assayed with administration of 2.5, 20 and 100 mg/kg weight at seven day intervals starting from day 4 prior to the first immunizing dose. Only the treatment with 2.5 mg Cy increased the DHR indices. Treatment with 100 mg Cy inhibited the DHR, whereas 20 mg Cy did not affect the DHR indices. Results suggest an immunostimulating effect of low dose of Cy on the DHR of mice immunized with P. brasiliensis antigens.

Trypanosoma cruzi in the anal glands of urban opossums: I- isolation and experimental infections

Opossums (Didelphis marsupialis) captured in intensely urbanized areas of the city of Caracas, Venezuela, were found infected with Trypanosoma cruzi. The developmental cycle of trypomastigote-epimastigote-metacyclic infective trypomastigote, usually occurring in the intestine of the triatomine vector, was taking place in the anal odoriferous glands of the opossums. Material from the glands, inoculated in young, healthy opossums and white mice by different routes, subcutaneously, intraperitoneally, orally, and into the eye, induced T. cruzi infections in all animals. Parasitemia, invasion of cardiac and skeletal muscle, and intracellular multiplication of amastigotes were observed. Inoculation of metacyclics from anal glands, cultured in LIT medium, gave equivalent results. All opossums survived; all mice died. Excreta of opossums may thus transmit Chagas' disease by contamination, even in urban areas where insect vectors are not present.

Chromoblastomycosis murine model and in vitro test to evaluate the sensitivity of Fonsecaea pedrosoi to ketoconazole, itraconazole and saperconazole

An experimental model of murine chromoblastomycosis and in vitro tests with Fonsecaea pedrosoi were used to test the sensitivity of this fungus to three different antimycotics. The experimental model was standardized in BALB/c mice inoculated intraperitoneally with a 10(6) CFU/ml suspension of a F. pedrosoi isolate. Clinical infection was evident after 5 days of inoculation. Three groups of 27 mice each were used in the experiment. One group was treated with ketoconazole (KTZ), another with itraconazole (ITZ) and the other with saperconazole (SPZ). Antimycotic therapy was continued for 21 days. The control group consisted of 40 mice which were inoculated, but not treated. Infection was documented by macroscopic and microscopic examination of affected tissue in addition to culture of tissue macerates. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) for the F. pedrosoi strain used were done. The in vitro results showed that SPZ was the most active with MIC 0.01 mg/ml and MFC 0.1 mg/ml, followed by ITZ. SPZ was also the most effective in vivo since 63% of the treated animals (p=0.01) showed a curative effect after the observation period. We concluded that SPZ had the best in vitro and in vivo activity against F. pedrosoi.

Does the eosinophil have a protective role in amebiasis?

While normal human eosinophils are destroyed in vitro by virulent Entamoeba histolytica, notwhistanding the presence of antibodies and complement, activated eosinophils promptly destroy the parasite although dying also at the end of the process. To study the possible in vivo participation of eosinophils in invasive amebiasis, we compared the induction of experimental amebic abscess of the liver (AAL) in gerbils (Meriones unguiculatus) previously made eosinophilic through Toxocara canis antigen injection and in normal control gerbils. After intraportal inoculation of 10(5) ameba trophozoites (6 and 24 hr), the ratio of gerbils with AAL, as well as the number and size of the microabscesses was comparable in eosinophilic and control gerbils. However, at 96 hr the number and size of the microabscesses were significanly smaller (p<0.05) in eosinophilic gerbils. On the other hand the actuarial AAL survival curve up to 45 days post-amebic inoculation was signficantly (p<0.05) shifted to the right in controls. These results suggest that antigen-induced eosinophilia may exert a protective effect against AAL in gerbils.

Experimental Infection of Calomys callosus (Rodentia, Cricetidae) by Toxoplasma gondii

Calomys callosus, Rengger 1830 (Rodentia, Cricetidae), a wild rodent found in Central Brazil, was studied to investigate its susceptibility to Toxoplasma gondii experimental infection and its humoral immune response against this protozoa. The electrophoretic profile of the serum proteins of C. callosus showed that IgG, which shows no affinity to Protein A, has higher cross reactivity with rat IgG than with IgG from other rodents. The susceptibility assay was performed by inoculation groups of animals with various suspensions of T. gondii tachyzoites from 102 to 106 parasites. All animals died between 3 and 9 days after infection and the kinetics of antibody synthesis was determined. Basically, they recognized predominantly the immunodominant antigen SAG-1 (P30). The immunohistochemistry assays revealed that the liver was the most heavily infected organ, followed by the spleen, lungs, intestine, brain and kidneys. It can be concluded that C. callosus is an excellent experimental model for acute phase of Toxoplasma infection

Seroprevalence of Trypanosoma cruzi Infection in Students at the Seven-Fourteen Age Range, Londrina, PR, Brazil, in 1995

Seropositivity for Chagas disease was evaluated in 834 children aged between 7 and 14 from the Municipal Teaching System in the district of Londrina, State of Paraná. A seroprevalence rate of 0.1% was found through the use of an indirect immunofluorescent test and an enzyme-linked immunosorbent assay. This low rate of seroprevalence provides evidence that the vectorial transmission of Chagas disease has been eliminated in Londrina. The main reason for the elimination of vectorial transmission of Trypanosoma cruzi infection, as evaluated by serological tests, may be a remarkable change in the economic structure of the northern region of Paraná in the 1960's. At that time coffee production was almost completely replaced by soy beans, wheat and grazing in the rural areas. This change deeply affected the rural ecology and caused an exodus of the population from rural to urban areas as well as a decrease in the total number of the population of that region. The measures introduced for controlling the disease through the Program of Chagas Disease Control established by the Fundação Nacional de Saúde of the Brazilian Ministry of Health, certainly, had a positive impact on the reduction of American trypanosomiasis prevalence in the area under study. However, it does not seem that this was the most relevant factor responsible for the elimination of vectorial transmission of Chagas disease in Londrina.

A dhfr-ts- Leishmania major Knockout Mutant Cross-protects against Leishmania amazonensis

E10-5A3 is a dhfr-ts- Leishmania major double knockout auxotrophic shown previously to induce substantial protection against virulent L. major infection in both genetically susceptible and resistant mice. We investigated the capacity of dhfr-ts- to protect against heterologous infection by L. amazonensis. The degree of protection was evaluated by immunization of BALB/c or C57BL/6 mice with E10-5A3, followed by L. amazonensis challenge. Whether immunized by subcutaneous (SC) or intravenous (IV) inoculation, susceptible and resistant mice displayed a partial degree of protection against challenge with virulent L. amazonensis. SC-immunized BALB/c mice developed lesions 40 to 65% smaller than non immunized mice, while IV immunization led to protection ranging from 40 to 75% in four out of six experiments compared to non immunized animals. The resistant C57BL/6 mice displayed comparable degrees of protection, 57% by SC and 49% by IV immunization. Results are encouraging as it has been previously difficult to obtain protection by SC vaccination against Leishmania, the preferred route for human immunization.

Polymorphism in Trypomastigotes of Trypanosoma (Megatrypanum) minasense in the blood of experimentally infected squirrel monkey and marmosets

Experimental infections by Trypanosoma (Megatrypanum) minasense were performed in primates - Saimiri sciureus and Callithrix penicillata - with the objective of searching for morphological variations of the blood trypomastigotes with respect to hosts and time of infection. We carried out morphological and morphometric analysis of blood trypomastigotes. Illustrations are given. Both the squirrel monkey and marmoset became infected after the injection of blood trypomastigotes of T. minasense , although the parasitaemia were briefer in the squirrel monkey. The parasites detected in the later host were narrower and shorter than those found in the inoculated marmoset. In the marmoset, the blood stream parasites derived from culture metacyclic trypomastigotes were considerably smaller than those derived from the inoculation of infected blood. Stronger evidence of polymorphism was found when, at the same time of infection, the blood trypomastigotes found in squirrel monkey had smaller length, body width and the distance from posterior end of the body to the kinetoplast almost four times smaller than the parasite found in the marmoset. Therefore, conflicting results on morphology and morphometry of T. minasense obtained by previous investigators could be due to polymorphism.

Experimental toxoplasmosis in Balb/c mice. Prevention of vertical disease transmission by treatment and reproductive failure in chronic infection

In a study of congenital transmission during acute infection of Toxoplasma gondii, 23 pregnant Balb/c mice were inoculated orally with two cysts each of the P strain. Eight mice were inoculated 6-11 days after becoming pregnant (Group 1). Eight mice inoculated on the 10th-15th day of pregnancy (Group 2) were treated with 100 mg/kg/day of minocycline 48 h after inoculation. Seven mice inoculated on the 10th-15th day of pregnancy were not treated and served as a control (Group 3). Congenital transmission was evaluated through direct examination of the brains of the pups or by bioassay and serologic tests. Congenital transmission was observed in 20 (60.6%) of the 33 pups of Group 1, in one (3.6%) of the 28 pups of Group 2, and in 13 (54.2%) of the 24 pups of Group 3. Forty-nine Balb/c mice were examined in the study of congenital transmission of T. gondii during chronic infection. The females showed reproductive problems during this phase of infection. It was observed accentuated hypertrophy of the endometrium and myometrium. Only two of the females gave birth. Our results demonstrate that Balb/c mice with acute toxoplasmosis can be used as a model for studies of congenital T. gondii infection. Our observations indicate the potential of this model for testing new chemotherapeutic agents against congenital toxoplasmosis.

Ultrastructural study of the TG180 murine sarcoma cell invasion by Toxoplasma gondii: comparison between in vivo and in vitro cell cultures

Infection of non-adherent TG180 murine sarcoma cells with Toxoplasma gondii was compared, at the ultrastructural level, in both in vivo and in vitro conditions. Suspensions of 3.0 x 10(6) TG180 cells infected in vitro with 1.0 x 10(6) parasites of the RH strain were harvested between the first and 6th day post-infection and processed for transmission electron microscopy. In vivo infection was made by intraperitoneal inoculation in mice of 1.0 x 10(6) TG180 cells, that were co-inoculated with a parasite suspension at the same cell concentration. Cells were harvested 10, 20, 30 min and 24, 48 h post-inoculation and processed for transmission electron microscopy at the same conditions of the in vitro culture. It was observed TG180 murine sarcoma cells with intense and equivalent intracellular parasitism in both conditions. Host cells with parasitophorous vacuoles containing up to 16 parasites, as well as parasites undergoing mitoses or presenting a bradyzoite-like morphology, were frequently seen in both culture methods.

Development of an enzyme-linked immunosorbent assay for detection of IgM antibodies to Babesia bigemina in cattle

A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina), or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days). The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.

Effect of interferon-alpha on experimental septal fibrosis of the liver - study with a new model

Interferon-alpha is used in antiviral therapy in humans, mainly for viral hepatitis B and C. An anti-fibrotic effect of interferon has been postulated even in the absence of anti-viral response, which suggests that interferon directly inhibits fibrogenesis. Rats infected with the helminth Capillaria hepatica regularly develop diffuse septal fibrosis of the liver, which terminates in cirrhosis 40 days after inoculation. The aim of this study was to test the anti-fibrotic effect of interferon in this experimental model. Evaluation of fibrosis was made by three separate methods: semi-quantitative histology, computerized morphometry and hydroxyproline measurements. Treatment with interferon-alpha proved to inhibit the development of fibrosis in this model, especially when doses of 500,000 and 800,000 IU were used for 60 days. Besides confirming the anti-fibrotic potential of interferon-alpha on a non-viral new experimental model of hepatic fibrosis, a clear-cut dose-dependent effect was observed.

Anesthesia of Biomphalaria spp. (Mollusca, Gastropoda): sodium pentobarbital is the drug of choice

The anesthetic effect of some water-soluble anesthesic or narcotic drugs currently used in mice was tested in molluscs of the Biomphalaria genus. Sodium thiopental was very toxic to the snails resulting in high rates of mortality in all the treatment schedules tested. Cetamine base, at concentration of 0.25 mg/ml of water, resulted in partial snail anesthesia (40% of snails were anesthetized) only after 20 h of exposition. The association of Cetamine base with Tiazine chloridrate did not improve the anesthesic effect, and higher concentrations of these drugs were toxic to the snails. Sodium pentobarbital at 0.4 mg/ml in water for 8 h was the best treatment schedule to anesthetize Biomphalaria snails. In this schedule, the snails were anesthetized without any toxic effect. The procedure provides a powerful tool for in vivo studies that demande a complete state of snail anesthesia.

Experimental transmission of Trypanosoma cruzi through the genitalia of albino mice

Trypanosoma cruzi is usually transmitted by contact with the excreta of infected Triatominae; among non-vectorial infections, direct transmission through coitus has been proposed. We investigated this possibility by instilling, through the external meatus of the vagina and the penis of previously anesthetized NMRI albino mice, blood of mice infected with strains isolated from Didelphis marsupialis (opossum, strain CO57), Rattus rattus (rat, strain CO22) and human (strain EP). Some animals were allowed to copulate the same day of the instillation. In other experiments, the strains were inoculated in the scrotum. To determine the effect of immunosuppression, some mice were treated with cyclophosphamide 30 days post-instillation. Controls were instilled orally and ocularly. Vaginal instillation with strain CO22 produced systemic infection with tropism to the heart, skeletal muscle, skin, duodenum, pancreas, ovary and sternum. Scrotal inoculation with strain EP likewise invaded liver, spleen, lung, lymph nodes and urogenital organs; while strain CO57 invaded skeletal and cardiac muscle, pancreas, testis, and vas deferens. Penile infection with strain CO22 was detected by xenodiagnosis. Immunosuppression did not increase parasitemia of vaginally infected mice or controls. Mating did not produce infection. Our results show that contact of blood trypomastigotes of T. cruzi with genital mucosa can produce blood and tissue infections. These results are discussed in relation to reports of frequent experimental tropism of T. cruzi toward urogenital organs.