A potential vector of Schistosoma mansoni in Uruguay

Susceptibily experiments were carried out with a Biomphalaria straminea-like planorbid snail (Biomphalaria aff. straminea, species inquirenda) from Espinillar, near Salto (Uruguay), in the area of the Salto Grande reservoir, exposed individually to 5 miracidia of Schistosoma mansoni (SJ2 and BH2 strains). Of 130 snails exposed to the SJ2 strain, originally infective to Biomphalaria tenagophila, 30 became infected (23%). The prepatent (precercaria) period ranged from 35 to 65 days. The cercarial output was irregular, following no definite pattern, varying from 138 to 76,075 per snail (daily average 4.3 to 447.5 and ending up with death. Three specimens that died, without having shed cercarie, on days 69 (2) and 80 after exposure to miracidia, had developing secondary sporocysts in their tissues, justifying the prospect of a longer precercarial period in these cases. In a control group of 120 B. teangophila, exposed to the SJ2 strain, 40 became infected, showing an infection rate (33.3%) not significantly different from that of the Espinillar snail (X [raised to the power of] 2 = 3.26). No cercarie were produced by any of the Espinilar snails exposed to miracidia of the BH2 strain, originally infective to Biomphalaria glabrata. Four specimens showed each a primary sporocyst in one tentacle, which disappeared between 15 and 25 days post-exposure, and two others died with immature, very slender sporocysts in their tissues on days 36 and 54. In a control group of 100 B. glabrata exposed to BH2 miracidia, 94 shed cercariae (94%) and 6 remained negative. Calculation of Frandsen's (1979a, b) TCP/100 index shows that "Espinillar Biomphalaria-SJ2 S. mansoni" is a vector-parasite "compatible" combination. Seeing that tenagophila-borne schistosomiasis is prevalent in Rio de Janeiro and São Paulo states and has recently spread sothwards to Santa Catarina state, and the range of B. tenagophila overlaps taht of the Espinillar Biomphalaria, the possibility of schistosomiais establishing itself in Uruguay, although not imminent, is not to be disregarded.

Resistance of schistosomes to hycanthone and oxamniquine

Genetic crosses between phenotypically resistant and sensitive schistosomes demonstrated that resistance to hycanthone and oxamniquine behaves like a recessive trait, thus suggesting that resistance is due to the lack of some factor. We hypothesized that, in order to kill schistosomes, hycanthone and oxamniquine need to be converted into an active metabolite by some parasite enzyme wich, if inactive, results in drug resistance. Esterification of the drugs seemed to be the most likely event as it would lead to the production of an alkylating agent upon dissociation of the ester. An artificial ester of hycanthone was indeed active even in resistant worms, thus indirectly supporting our hypothesis. In addition, several lines of evidence demonstrated that exposure to hycanthone and oxamniquine results in alkylation of worm macromolecules. Thus, radioactive drugs formed covalent bonds with the DNA of sensitive (but not of resistant) schistosomes; an antiserum raised against hycanthone detected the presence of the drug in the purified DNA fraction of sensitive (but not of resistant) schistosomes; a drug-DNA adduct was isolated from hycanthone-treated worms and fully characterized as hycanthone-deoxyguanosine.

Effects of irradiation and tunicamycin on the surface glycoproteins Schistosoma mansoni

The cercarial glycocalyx and schistosomulum surface contains a number of glycoproteins which are expressed in very variable amounts within a parasite population. Tunicamycin inhibits glycoprotein synthesis of schistosomula if the parasites are incubated for 24hr with the drug (10µg ml[raised to the power of -1]). An unexpected increase in lectin binding to the parasite surface was observed but no other changes were detected. Schistosomula treated in this way did not develop in the host past the lung stage. Ultraviolet irradiation (400µW min cm[raised to the power of-2]) also inhibited glycoprotein synthesis. Synthesis of other proteins, and in particular heat shock proteins, were also inhibited. Sera from mice (NIH strain) infected with irradiated cercariae contained antibodies which bound to normal schistosomula with lower affinity than to irradiated parasites. This is evidence that irradiation modifies the surface and secreted glycoproteins of schistosomula, so they are processed in a different way to normal glycoproteins by the host's immune system. The effects of irradiation on heat shock protein synthesis may allow the parasite to release a variety of proteins and glycoproteins in abnormal conformations. This may explain the enhanced immunogenicity of irradiated cercariae.

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-346) 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 (2x 10 [raised to the power of] 4 and 5x 10 [raised to the power of] 4 blood forms respectively). The rate of morality 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 evaluated in the present study. Chemotherapy was performed with Benznidazole (N-benzil-2-nitro-1-imidazolacetamide) in the dose of 100mg/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, subinovulation 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 mitritial components and collagen fibers, macrophages and fibroblasts appeared at the ultra structural examination. Deposits of fibronectin, laminin, pro-III and IV collagens were seen, most intense in those infected with the Colombian strain. Treated nice, 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 nice infected with the 21 SF with Benznidazole determined parasitological cure of all animals, that showed mild inflammation and fibrosis of the myocardium. The cured mice of all groups and treated but uncured showed collagen degradation at electronmicroscopy and decrease of immunofluorescence pattern of the matrix.

Inhibition of gastric secretion by a water extract from Baccharis triptera, Mart

Baccharus triptera Mart, is a widespread Compositae used in Brazilian folk medicine to treat gastrointestinal disturbances, rheumatic disease, mild fever, diabetes and as an anti-helminthic. Water extract of small branches of the plant (WE) administered to mice and rats (0.1 to 2 g/Kg, p.o) did not alter spontaneous motor activity, sleeping time induced by barbiturates or the tailflick response in mice. The extract decreased by 40 por cento the number of writhings induced by 0.8 por cento scetic acid, i.p., but did not influence paw edema induced by carrageenan or dextran in rats WE (2g/Kg, p.o.) decreased the intestinal transit of charcoal in mice by 20//. Gastric secretion in pylorus ligated rats was reduced after treatment with WE (1 and 2 g/Kg. i.p. or intraduodenal and the gastric pH was raised. The extract (1 g/Kg, p.o.) prevented gastric ulcers induced in rats by immobilization at 4ºC, but not those induced by indomethacin (10 mg/Kg, s.c.). The results indicate that WE may relieve gastrointestinal disorders by reducing acid secretion and gastrointestinal hiperactivity. Neither analgesic nor anti-inflammatory activities were detectable.

Proteinase inhibitors in Brazilian leguminosae

Serine proteinase inhitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil), were studied using bovine trypsin, a digestive enzyme, Factor XIIa and human plasma Kallikrein, two blood clotting factors. The inhibitors were purified from Enterolobium contortisiliquum (Mr=23,000), Torresea cearensis (Mr = 13,000), Bauhinia pentandra (Mr = 20,000) and Bauhinia bauhinioides (Mr = 20,000). E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XSSa, but does nor affect plasma kallikrein; both Bauhinia inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the Bpentandra inhibitor affects Factor XIIa. Ki values were calculated between 10 [raised to the power of] -7 and 10 [raised to the power of] -8 M.

The Pf332 gene codes for a megadalton protein of Plasmodium falciparum asexual blood stages

We characterized the Plasmodium falciparum antigen 332 (Ag332) which is specifically expressed during the asexual intraerythrocytic cycle of the parasite. The corresponding Pf332 gene has been located in the subtelomeric region of chromosome 11. Furthermore, it is present in all strais so far analyzed and shows marked restriction length fragment polymorphism. Partial sequence and restriction endonuclease digestion of cloned fragments revealed that the Pf332 gene is composed of highly degenerated repeats rich is glutamic acid. Mung been nuclease digestion and Northern blot analysis suggested that Pf332 gene codes for a protein of about 700 kDa. These data were further confirmed by Western blot and immunoprecipitation of parasites extracts with an antiserum raised against a recombinant clone expressing part of the Ag332. Confocal immunofluorescence showed that Ag332 is translocated from the parasite to the surface of infected red blood cells within vesicle-like structures. In addition, Ag332 was detected on the surface of monkey erythrocytes infected with Plasmodium falciparum.

Protection of aouts monkeys after immunization with recombinant antigens of Plasmodium falciparum

The genus Aotus spp. (owl monkey) is one of the WHO recommended experimental models for Plasmodium falciparum blood stage infection, especially relevant for vaccination studies with asexual blood stage antigens of this parasite. For several immunization trials with purified recombinant merozoite/schizont antigens, the susceptible Aouts kenotypes II, III, IV and VI were immunized with Escherichia coli derived fusion proteins containg partial sequences of the proteins MSAI (merozoite surface antigen I), SERP (serine-strech protein) and HRPII (histidine alanine rich protein II) as well as with a group of recombinant antigens obtained by an antiserum raised against a protective 41 kD protein band. The subcutaneous application (3x) of the antigen preparations was carried out in intact animals followed by splenectomy prior to challange, in order to increase the susceptibility of the experimental hosts to the parasite. A partial sequence of HRPII, the combination of three different fusion proteins of the 41 kD group and mixture of two sequences of SERP in the presence of the modified Al(OH)3 adjuvant conferred significant protection against a challange infection with P. falciparum blood stages (2-5 x 10 (elevado a sexta potência) i. RBC). Monkey immunized with the MS2-fusion protein carrying the N-terminal part of the 195 kD precursor of the major merozoite surface antigens induced only marginal protection showing some correlation between antibody titer and degree of parasitaemia. Based on the protective capacity of these recombinant antigens we have expressed two hybrid proteins (MS2/SERP/HRPII and SERP/MSAI/HRPII) in E. coli containing selected partial sequences of SERP, HRPII and MSAI. Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding schizont polypeptides. In two independent immunization trials using 13 animals (age 7 months to 3 years) we could show that immunization of Aotus monkeys with either of the two hybrid proteins administered in an oil-based well tolerated formulation protected the animals frm a severe experimental P. falciparum (strain Palo Alto) infection.

Prevalence of human papillomavirus DNA in female cervical lesions from Rio de Janeiro, Brazil

A hundred-sixty paraffin-embedded specimens from female cervical lesions were examined for human papillomavirus (HPV) types 6, 11, 16 and 18 infections by non-isotopic in situ hybridization. The data were compared with histologic diagnosis. Eighty-eight (55) biopsies contained HPV DNA sequences. In low grade cervical intraepithelial neoplasias (CIN I), HPV infection was detected in 78.7 of the cases, the benign HPV 6 was the most prevalent type. HPV DNA was detected in 58 of CIN II and CIN III cases and in 41.8 of squamous cell carcinomas (SCC). Histologically normal women presented 20 of HPV infection. Oncogenic HPV was found in 10 of these cases, what may indicate a higher risk of developing CINs and cancer. Twenty-five percent of the infected tissues contained mixed infections. HPV 16 was the most common type infecting the cervix and its prevalence raised significantly with the severity of the lesions, pointing its role in cancer pathogenesis. White women presented twice the cervical lesions of mulatto and African origin women, although HPV infection rates were nearly the same for the three groups (approximately 50). Our results showed that HPV typing by in situ hybridization is a useful tool for distinguishing between low and high risk cervical lesions. Further studies are required to elucidate risk factors associated with HPV infection and progression to malignancy in Brazilian population.

A novel merozoite surface antigen of Plasmodium falciparum (MSP-3) identified by cellular-antibody cooperative mechanism antigenicity and biological activity of antibodies

We report the identification of a 48kDa antigen targeted by antibodies which inhibit Plasmodium falciparum in vitro growth by cooperation with blood monocytes in an ADCI assay correlated to the naturally acquired protection. This protein is located on the surface of the merozoite stage of P. falciparum, and is detectable in all isolates tested. Epidemiological studies demonstrated that peptides derived from the amino acid sequence of MSP-3 contain potent B and T-cell epitopes recognized by a majority of individuals living in endemic areas. Moreover human antibodies either purified on the recombinant protein, or on the synthetic peptide MSP-3b, as well as antibodies raised in mice, were all found to promote parasite killing mediated by monocytes.

Biochemical, immunological and toxicological characteristics of the crystal proteins of Bacillus thuringiensis subsp. medellin

Characterization of the insecticidal and hemolytic activity of solubilized crystal proteins of Bacillus thuringiensis (Bt) subsp. medellin (Btmed) was performed and compared to solubilized crystal proteins of isolates 1884 of B. thuringiensis subsp. israelensis (Bti) and isolate PG-14 of B. thuringiensis subsp. morrisoni (Btm). In general, at acid pH values solubilization of the Bt crystalline parasporal inclusions (CPI) was lower than at alkaline pH. The larvicidal activity demonstrated by the CPI of Btmed indicated that optimal solubilization of CPI takes place at a pH value of 11.3, in Bti at pH values from 5.03 to 11.3 and in Btm at pH values from 9.05 to 11.3. Hemolytic activity against sheep red blood cells was mainly found following extraction at pH 11.3 in all Bt strains tested. Polyacrylamide gel electrophoresis under denaturing conditions revealed that optimal solubilization of the CPI in all Bt strains takes place at the alkaline pH values from 9.05 to 11.3. An enriched preparation of Btmed crystals was obtained, solubilized and crystal proteins were separated on a size exclusion column (Sephacryl S-200). Three main protein peaks were observed on the chromatogram. The first peak had two main proteins that migrate between 90 to 100 kDa. These proteins are apparently not common to other Bt strains isolated to date. The second and third peaks obtained from the size exclusion column yielded polypeptides of 68 and 28-30 kDa, respectively. Each peak independently, showed toxicity against 1st instar Culex quinquefasciatus larvae. Interestingly, combinations of the fractions corresponding to the 68 and 30 kDa protein showed an increased toxicity. These results suggest that the 94 kDa protein is an important component of the Btmed toxins with the highest potency to kill mosquito larvae. When crystal proteins of Bti were probed with antisera raised independently against the three main protein fractions of Btmed, the only crystal protein that showed cross reaction was the 28 kDa protein. These data suggest that Btmed could be an alternative bacterium for mosquito control programs in case mosquito larval resistance emerges to Bti toxic proteins.

Cloning, Expression and Toxicity of a Mosquitocidal Toxin Gene of Bacillus thuringiensis subsp. medellin

Bacillus thuringiensis (Bt) subsp. medellin (Btmed) produces parasporal crystalline inclusions which are toxic to mosquito larvae. It has been shown that the inclusions of this bacterium contain mainly proteins of 94, 68 and 28-30 kDa. EcoRI partially digested total DNA of Btmed was cloned by using the Lambda Zap II cloning kit. Recombinant plaques were screened with a mouse policlonal antibody raised against the 94 kDa crystal protein of Btmed. One of the positive plaques was selected, and by in vivo excision, a recombinant pBluescript SK(-) was obtained. The gene encoding the 94 kDa toxin of Btmed DNA was cloned in a 4.4 kb DNA fragment. Btmed DNA was then subcloned as a EcoRI/EcoRI fragment into the shuttle vector pBU4 producing the recombinant plasmid pBTM3 and used to transform by electroporation Bt subsp. israelensis (Bti) crystal negative strain 4Q2-81. Toxicity to mosquito larvae was estimated by using first instar laboratory reared Aedes aegypti, and Culex quinquefasciatus larvae challenged with whole crystals. Toxicity results indicate that the purified inclusions from the recombinant Bti strain were toxic to all mosquito species tested, although the toxicity was not as high as the one produced by the crystal of the Btmed wild type strain. Poliacrylamide gel electrophoresis indicate that the inclusions produced by the recombinant strain Bti (pBTM3) were mainly composed of the 94 kDa protein of Btmed, as it was determined by Western blot

Expression of Recombinant Antigens in Escherichia coli: Application on Immunochemical Studies of Schistosoma mansoni Tegumental Antigens

Sm15 and Sm13 are recognized by antibodies from mice protectively vaccinated with tegumental membranes, suggesting a potential role in protective immunity. In order to raise antibodies for immunochemical investigations, the genes for these antigens were expressed in pGEX and pMal vectors so that comparisons could be made among different expression systems and different genes. The fusion proteins corresponding to several parts of the gene for the precursor of Sm15 failed in producing antibodies recognizing the parasite counterpart. On the other hand, antibodies raised against Sm13 MBP-fusion proteins recognized the 13 kDa tegumental protein. Thus the peculiarities of the gene of interest are important and the choice of the expression system must sometimes be decided on an empirical basis

Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase

The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins

Dynamics of Haematobia irritans irritans (Diptera: Muscidae) infestation on Nelore cattle in the Pantanal, Brazil

From June 1993 to May 1995, horn fly counts were conducted twice a month on untreated Nelore cattle raised extensively in the Pantanal. Horn fly population showed a bimodal fluctuation and peaks were observed every year after the beginning (November/December) and at the end (May/June) of the rainy season, which coincided with mid-late spring and mid-late fall, respectively. Horn flies were present on cattle throughout the year in at least 64% of the animals. Mean horn fly numbers on animals did not exceed 85 flies/cow during peaks and were under 35 flies/cow in most of the remaining periods. The highest infestations (population peaks) were short and dropped suddenly within two weeks. Less than 15% of the animals in both herds could be considered as "fly-susceptible" - showing consistently higher infestations, or "fly-resistant" - showing consistently lower infestations.