Characterization of plasma menbrane polypeptides of trypanosoma from bats

Cell surface proteins of Trypanosoma dionisii, Trypanosoma vespertilionis and Trypanosoma sp. (M238) were radiodinated and their distribution both in the detergent-poor (DPP) and dertergent-enriched phase (DRP) was studied using a phase separation technique in Triton X-114 as well as polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE). Significant differences were observed in the proteins present in the DRP when the three species of trypanosoma were compared. Two major bands with 88 and 70 KDa were observed in T. sp. (M238) but were not detectable in T. dionisii and T. vespertilionis. Three polypeptides whith 96, 77 and 60 KDa were identified in the DRP of T. vespertilionis. Three major bands with 84, 72 and 60 KDa were observed in the DRP of T. dionisii. Two polypeptides with 34-36 KDa present in the DPP, were observed in the three Trypanosome species analyzed. Our observations show that T. sp. (M238) has characteristic surface polypeptides not found in T. vespertilionis.

Evaluation of antibody responses in american visceral leishmaniasis by ELISA and immunoblot

American visceral leishmaniasis (AVL) is an important disease among children of northeast Brazil. In order to characterize antibody responses during AVL, sera of hospitalized patients were analyzed by ELISA and Western blot using a Leishmania chagasi antigen preparation. The ELISA was positive (asorbance [greater than or equals to] 0.196) at a serum dilution of 1:1024 in all patients at presentation, and fell to ward control levels over the following year. Only one of 72 control subjects tested positive, and that donor had a sibling with AVL. Immunoblots of the patients' sera recognized multiple bands, the most frequent of which were at approximately 116 kDa, 70 kDa, and 26 kDa. Less frenquently observed were bands at approximately 93 kDa, 74 kDa, 62 kDa, 46 kDa and 32 kDa. The ELISA responses and patterns of banding were distinctive for AVL, and could be used to differentiate patients with AVL from those with Chagas' disease of cutaneous leishmaniasis. Sera from six AVL patients followed for up to six weeks after treatment identified no new bands. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of surface iodinated parasite proteins showed one major band and four minor bands, whereas SDS-PAGE of biotinylated prarasite proteins revealed a banding pattern similar to those of patient sera. AVL appears to produce characteristic immunoblot patterns which can be used along with a sensitive screening ELISA to diagnose AVL.

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28§C and 37§C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-Page). Several proteins with molecular weights ranging from 34 kDa to 7 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could aldso be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discuted.

Use of an indirect haemagglutination test, for the detection of Clostridium perfringens type A enterotoxin

An indirect haemagglutination (IH) test is described for the detection of Clostridium perfringens type A enterotoxin, produced by strains isolated from human cases of food poisoning and from contaminated food. Though no strict relationship could be observed between titers in the IH test and the time it took mice to die from the intravenous inoculation of mice (IIM), results of the supernatants examined by both methods demonstrated that the IH test was more sensitive than the ILM one. No unspecific reaction was obtanined int he IH wirh a negative control and the inhibitions of the IH and IIM tests by specific antiserum against C. perfringens enterotoxin showed that the IH test is very spcific. The IH assay is recommended for its sensitivity and easy performance by less-equipped laboratories, by these and other data.

Pharmacological screening of Ageratum conyzoides L. (mentrasto)

The pharmacological activities of a water extract (WE) of Ageratum conyzoides L, a plant populary known for its analgesic and anti-inflamatory properties, were studied in vivo and in vitro preparations. Oral administration (p.o.) of the water extract (WE, 0.1 to 5 g/Kg) to rats and mice induced quietness and reduced the spontaneous motility. the sleeping time induced by sodium pentobarbital (50 mg/Kg, i.p.) in mice was not altered by previous treatment with We (2 g/Kg, p.o.). The same treatment did not influence the paw edema induced by carrageenan or dextran, nor did it reduce the chronic paw edema induced by complete Freund's adjuvant or formaldehyde in rats. The tail flick response in immersion test and writhings induced by 0.8%acetic acid in mice were not altered by WE either. In isolated guinea-pig ilea WE (0.4 to 4 mg/ml) did not alter the EC50 values of histamine or acetylcholine, but reduced the maximal response to the agonists by 20 to 50%. We (0.01 to 10 mg/ml) produced tonic contractions of the ileal smooth muscle proportional to the doses, reaching a maximum of 75% relatively to the maximum obtained with histamine. Those contractions were blocked by diphenhydramine (10 nM) and reduced by 32% in presence of atropine (10 nM). The results indicated that oral treatment of rodents with A. conyzoides L neither reduced the inflammatory edema nor did it decrease the reaction to pain stimuli. In vitro the extract presented an unexpected histamine-like activity characteristic of a partial agonist. The results did not confirm the popular medicinal indications of the plant.

Analgesic activity of a triterpene isolated from Scoparia dulcis L. (vassourinha)

Analgesic and anti-inflammatory activities of water (WE) and ethanolic (EE) extracts of Scoparia dulcis L. were investigated in rats and mice, and compared to the effects induced by Glutinol, a triterpene isolated by purification of EE. Oral adminsitration (p.o.) of either WE or EE (up to 2 g/Kg) did not alter the normal spontaneous activity of mice and rats. The sleeping time induced by sodium pentobarbital (50 mg/Kg, i.p.) was prolonged by 2 fold in mice pretreated with 0.5 g/Kg EE, p.o. Neither extract altered the tail flick response of mice in immersion test, but previous administration of EE (0.5 g/Kg, p.o.) reduced writhings induced by 0.8% acetic acid (0.1 ml/10 g, i.p.) in mice by 47% EE (0.5 and 1 g/Kg, p.o.) inhibited the paw edema induced by carrageenan in rats by respectively 46% and 58% after 2 h, being ineffective on the paw edema induced by dextran. No significant analgesic or anti-edema effects were detected in animals pretreated with WE (1 g/Kg, p.o.). Administration of Glutinol (30 mg/Kg, p.o.) reduced writhing induced by acetic acid in mice by 40% and the carrageenan induced paw edema in rats by 73%. The results indicate that the analgesic activity of S dulcis L. may be explained by explained by an anti-inflammatory activity probably related to the triterpene Glutinol.

Evaluation of the molluscicidal properties of Euphorbia splendens var. hislopii (n. e. b.) (Euphorbiaceae) - 1: experimental test in a lentic habitat

The latex of Euphorbia splendens var. hislopii, at concentrations between 5 to 12 mg/l, kills 100% of the population of Biomphalaria glabrata in a lentic habitat, after 24 h. The lyophilized latex, stocked for 18 months, killed only 34.2% of the snails, at the concentration of 5 mg/l, and 96.0% at 12 mg/l. No lethal effect was observed among Pomacea haustrum exposed to the same concentrations of the molluscicide.

Malaria seroepidemiology: comparison between indirect fluorescent antibody test and enzyme immunoassay using bloodspot eluates

Blood sampling on filter paper is a current practice seroepidemiological studies by indirect fluorescent antibody test (IFAT). There is, however, scant comparative information about the use of bloodspot eluates for detection of malarial IgG antibodies simultaneously by IFAT and enzyme immunoassay (ELISA). Here we report data obtained by both serological methods done on 219 bloodspot eluate samples collected in a rural community in Brazilian Amazon Basin (Alto Paraíso, Ariquemes municipality) where malaria is endemic. Plasmodium falciparum and P. vivax thick smear antigens were used in the IFAT; a detergent-soluble P. falciparum antigen was prepared for ELISA. Substantial agreement of results (Kappa coefficient k = 0.686) was observed when P. falciparum antigen was used in both tests, and IFAT titers were found to be strongly correlated ELISA antibody units (Spearman correlation coeficient rs = 0.818, p < 0.0001). Only moderate agreement (k = 0.467) between IFAT with P. vivax antigen and ELISA with P. falciparum antigen was observed. Spearman correlation coefficient value between quantitative results (IFAT titers and ELISA antibody units) in this case was numerically lowe (rs = 0.540, p < 0.0001). Our results suggest that, with P. falciparum antigen, both IFAT and ELISA performed on bloodspot eluates are equivalent for seropidemiological purposes.

A simple and economic slide micro-immunoenzymatic (Micro-SIA) test for epidemiological studies of toxoplasmosis

A slide micro-immunoenzymatic assay (micro-SIA) to detectantibodies to non-particulate Toxoplasma gondii antigens is described. This assay allows the diagnosis of toxoplasmosis infection in about 1 hr. Twenty-four determinations can be performed per slide. Five hundred ng of antigen and 5 or 10 µl drop of each reactive are necessary per well. The clear contrast of colours obtained for negative and positive sera after the test is finished, allows direct discrimination of the results. However, it is possible to quantify the results of the reaction using a minireader. Sera dilution cutoff value, determined as themost frequent titre for the general population, is 1:100. The toxoplasma micro-SIA correlates well with indirect immunofluorescence (IIF), its sensitivity is atleast three times as much as IIF. The test has an intra and inter assay variation coefficient of 5.46 per cent and of 6.24 per cent respectively. Sera obtained at random from argentinian people were analyzed and a 56 per cent of infection was found. The main features of the Toxoplasma micro-SIA are its simplicity, sensitivity, reproducibility, and the virtual absence of background making it very suitable for screening tests.

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.

Attempts to Improve Xenodiagnosis: Comparative Test of Sensibility Using Rhodnius neglectus, Panstrongylus megistus, Triatoma vitticeps and Triatoma infestans in Endemic Areas of Brazil

From June 1984 to July 1992, 392 xenodiagnostic tests were applied on 264 patients with chronic Chagas disease from Brazilian endemic areas of Virgem da Lapa and Coronel Murta, situated in the Jequitinhonha Valley, in the State of Minas Gerais. The susceptibilities of Rhodnius neglectus, Panstrongylus megistus, Triatoma vitticeps and Triatoma infestans were compared. Most of the time 20 nymphs (fourth instar) of each species were applied to 161 women and 103 men aged between 5 and 83 years of age. The tests were prepared to compare the susceptibilities of two species at a time, using the same patients for each test. Results showed a xenopositiveness of 26.28% (103 tests) being 27.98% in women (68 positive in 243 applied tests) and 23.49% in men (35 positive in 149 applied tests). The relative frequency of xenopositiveness displayed a great superiority of P. megistus and T. vitticeps. In tests from type I, for example, P. megistus was the unique responsible for 10.73% of positive xenodiagnosis vs. only 0.98% in T. infestans. Other parameters analized in this work confirm this superiority, and corroborate that T. infestans can be replaced by P. megistus and /or T. vitticeps in order to upgrade the efficacy of xenodiagnosis

Tuberculin Skin Test and CD4+/CD8+ T Cell Counts in Adults Infected with the Human Immunodeficiency Virus in Medellín, Colombia

To evaluate the effect of BCG vaccination and T lymphocyte subpopulations on the reactivity to the tuberculin skin test, 113 asymtomatic HIV+ individuals were tuberculin tested by intradermal injection of 5TU of purified protein derivative and the levels of circulating lymphocyte (CD3, CD4 and CD8) subpopulations determined by indirect immunofluorescence. Ninety-two percent of the subjects included in the study were males. The mean age of the group was 32.1±7.4 years. Sixty-two percent presented a BCG scar. However, only 22% exhibited positive tuberculin reactions (³5mm) irrespective of the presence of the BCG scar. Tuberculin positive individuals exhibited higher CD4+ cell counts (p=0.004) and CD4+/CD8+ ratios (p=0.006) than tuberculin negative (<5mm) HIV+ individuals. The number of individuals with positive tuberculin reactions was significantly higher in subjects with more than 500 CD4+ lymphocytes/ml (p=0.02) or CD4+/CD8+ ratios ³1.12 (p=0.002). These results suggest that a prior BCG vaccination does not influence the reactivity to the tuberculin skin test in HIV+ asymptomatic individuals and that the number of CD4+ lymphocytes and the CD4+/CD8+ ratio positively correlate with the tuberculin reactivity