Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania) amazonensis

In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania) amazonensis (LLa) with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guérin). The antigenicity of these vaccines was measured by their ability to induce the production of IFN-g by lymphocyte from subjects vaccinated with Leishvacinâ . The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 mg of each protein at 15 days interval. One hundred mg of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 105 infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86% of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57.14% of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43% protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7.60) and 10.77UI/ml of IFN-g production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14% of protection was found after the first challenge and 28.57% after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that would protect mice against cutaneous leishmaniasis.

Analysis of proteins from membrane and soluble fractions of Giardia duodenalis trophozoites of two Brazilian axenic strains

In the present study, we have analyzed by sodium docecyl sulphate - polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting and Concanavalin A blotting (Con A blotting) proteins of membrane fractions and soluble fractions obtained from Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic (BTU-11) and an asymptomatic patient (BTU-10), as compared to the reference strain Portland 1. Both Brazilian strains showed a complex and homogeneous electrophoretic pattern of proteins, but some differences could be observed. Several glycoproteins were detected, particularly the proteins of 81, 72, 59 kDa and the protein of 62 kDa in the membrane proteins and cytosol, respectively. Many antigenic components were revealed by anti-Giardia rabbit IgG antibodies in the immunoblotting analysis. Among these components, the membrane protein of 32 kDa and the cytosol protein of 30 kDa could be related to giardin, as previously demonstrated.

Melanoides tuberculatus (Gastropoda: Thiaridae) as intermediate host of Heterophyidae (Trematoda: Digenea) in Rio de Janeiro metropolitan area, Brazil

In the late 1960s, Melanoides tuberculatus snails were introduced in Brazil from North/East Africa and Southeast Asia. The first records of specimens infected with cercariae were registered in Rio de Janeiro State in 2001. The present study reports the occurrence of M. tuberculatus infected with larval trematodes in Rio de Janeiro City. Bottom sediment was collected with dip nets and sieved through 0.25 inch-mesh screening. Snails were transported to the laboratory in vials with stream water, then measured and individually isolated in glass vials with distilled water. They were exposed to artificial light and temperature to induce cercarial emergence. The most actively emerging cercariae were processed by differential staining and silver nitrate impregnation methods. Negative snails were subsequently dissected. Approximately 700 snails were collected. Snail total lengths ranged from 1.2 to 3.3 cm. The prevalence rate was 15.76% although 53.76% of the snails were found infected in one of the sites. Infected snails were infected with rediae and pleurolophocercous cercariae. Cercarial morphology and chaetotaxy were consistent with those of the family Heterophyidae mostly due to the presence of median dorsal and ventral fins on the tail and the absence of CI dorsal sensory receptors.

Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3 - 10 days was approximately 1.08 ± 0.04 µg/female and 0.1 ± 0.05 µg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

Indications of carbapenem resistance evolution through heteroresistance as an intermediate stage in Acinetobacter baumannii after carbapenem administration

We describe an in vivo evolution of an antimicrobial profile from susceptibility to full-resistance to carbapenems, with heteroresistance as an intermediate stage, in an Acinetobacter baumannii strain. Heteroresistance was characterized by the growth of sub-populations within the susceptibility halo in both disk-diffusion and Etest. PCRs for the main A. baumannii carbapenemases were negative. The exact resistance mechanism, diagnostic methods and clinical relevance of heteroresistance in A. baumannii warrant further investigations. This is the first description of such phenomenon in vivo and the second report of heteroresistance to carbapenems in A. baumannii.

Melanoides tuberculata (Mollusca: Thiaridae) as an intermediate host of Centrocestus formosanus (Trematoda: Heterophyidae) in Brazil

Pleurolophocercous cercariae emerged from naturally infected Melanoides tuberculata from Minas Gerais State, Brazil, were used to perform experimental infection of laboratory-reared Poecilia reticulata. Mature metacercariae were obtained from the gills of fishes and force-fed to Mus musculus. The adult parasites which recovered from small intestines of mice were identified as Centrocestus formosanus. This is the first report of M. tuberculata as intermediate host of this heterophyid in Brazil.

Melanoides tuberculata as intermediate host of Philophthalmus gralli in Brazil

Melanoides tuberculata that naturally harbored trematode larvae were collected at the Pampulha dam, Belo Horizonte (Minas Gerais, Brazil), during malacological surveys conducted from 2006 to 2010. From 7,164 specimens of M. tuberculata collected, 25 (0.35%) were infected by cercariae, which have been morphologically characterized as belonging to the Megalurous group, genus Philophthalmus. Excysted metacercariae were used for successful experimental infection of Gallus gallus domesticus, and adult parasites recovered from the nictitating membranes of chickens were identified as Philophthalmus gralli. This is the first report of P. gralli in M. tuberculata in Brazil.

SPATIAL DISTRIBUTION OF LYMNAEIDAE (MOLLUSCA, BASOMMATOPHORA), INTERMEDIATE HOST OF Fasciola hepatica LINNAEUS, 1758 (TREMATODA, DIGENEA) IN BRAZIL

Snails of the family Lymnaeidae act as intermediate hosts in the biological cycle of Fasciola hepatica, which is a biological agent of fasciolosis, a parasitic disease of medical importance for humans and animals. The present work aimed to update and map the spatial distribution of the intermediate host snails of F. hepatica in Brazil. Data on the distribution of lymnaeids species were compiled from the Collection of Medical Malacology (Fiocruz-CMM, CPqRR), Collection of Malacology (MZUSP), “SpeciesLink” (CRIA) network and through systematic surveys in the literature. Our maps of the distribution of lymnaeids show that Pseudosuccinea columella is the most common species and it is widespread in the South and Southeast with few records in the Midwest, North and Northeast regions. The distribution of the Galba viatrix, G. cubensis and G. truncatula showed a few records in the South and Southeast regions, they were not reported for the Midwest, North and Northeast. In addition, in the South region there are a few records for G. viatrix and one occurrence of Lymnaea rupestris. Our findings resulted in the first map of the spatial distribution of Lymnaeidae species in Brazil which might be useful to better understand the fasciolosis distribution and delineate priority areas for control interventions.

Anopheles (Kerteszia) cruzii (DIPTERA: CULICIDAE) IN PERIDOMICILIARY AREA DURING ASYMPTOMATIC MALARIA TRANSMISSION IN THE ATLANTIC FOREST: MOLECULAR IDENTIFICATION OF BLOOD-MEAL SOURCES INDICATES HUMANS AS PRIMARY INTERMEDIATE HOSTS

Anopheles (Kerteszia) cruzii has been implicated as the primary vector of human and simian malarias out of the Brazilian Amazon and specifically in the Atlantic Forest regions. The presence of asymptomatic human cases, parasite-positive wild monkeys and the similarity between the parasites infecting them support the discussion whether these infections can be considered as a zoonosis. Although many aspects of the biology of An. cruzii have already been addressed, studies conducted during outbreaks of malaria transmission, aiming at the analysis of blood feeding and infectivity, are missing in the Atlantic Forest. This study was conducted in the location of Palestina, Juquitiba, where annually the majority of autochthonous human cases are notified in the Atlantic Forest of the state of São Paulo. Peridomiciliary sites were selected for collection of mosquitoes in a perimeter of up to 100 m around the residences of human malaria cases. The mosquitoes were analyzed with the purpose of molecular identification of blood-meal sources and to examine the prevalence of Plasmodium. A total of 13,441 females of An. (Ker.) cruzii were collected. The minimum infection rate was calculated at 0.03% and 0.01%, respectively, for P. vivax and P. malariae and only human blood was detected in the blood-fed mosquitoes analyzed. This data reinforce the hypothesis that asymptomatic human carriers are the main source of anopheline infection in the peridomiciliary area, making the probability of zoonotic transmission less likely to happen.

MOLLUSCICIDAL AND LARVICIDAL ACTIVITIES OF Atriplex inflata AERIAL PARTS AGAINST THE MOLLUSK Galba truncatula, INTERMEDIATE HOST OF Fasciola hepatica

Fasciolosis is a widespread parasitosis of farm live-stock in many developing countries. For this reason, it is necessary to search for new substances against parasitic diseases caused by flukes. Indeed, a wide variety of terrestrial plants have been subjected to chemical and pharmacological screening in order to discover their potential for human medicinal use. The molluscicidal and larvicidal activities of Atriplex inflata were tested on Galba truncatula and Fasciola hepatica larval stages infecting this snail in Tunisia. Phytochemical tests were conducted on extracts in order to establish a meaningful relationship with molluscicidal and larvicidal activities. The molluscicidal activity was evaluated by subjecting snails to sample aqueous solutions. Accordingly, hexane, ethyl acetate, methanol and methanol-water (8:2, v-v) were used as extraction solvents. As a result, hexane and ethyl acetate extracts showed potent activity, according to the World Health Organization, giving LC50 = 7.59 mg/L and 6.69 mg/L for hexane extracts of leaves and fruits, respectively. Ethyl acetate extracts gave LC50 = 5.90 mg/L and 7.32 mg/L for leaves and fruits, successively. Molluscicidal activities of powders were less potent on snails, but active according to the World Health Organization. Hexane and ethyl acetate extracts from leaves and fruits gave potent larvicidal activities with a delay rate exceeding 45.50% (45.50- 98.92%). Phytochemical tests showed that these activities may be attributed to the presence of triterpenoids and/or sterols.

Angiostrongylus costaricensis and the intermediate hosts: observations on elimination of L3 in the mucus and inoculation of L1 through the tegument of mollucs

Human accidental infection with Angiostrongylus costaricensis may result in abdominal disease of varied severity. Slugs from the Veronicellidae family are the main intermediate hosts for this parasitic nematode of rodents. Phyllocaulis variegatus, Phyllocaulis soleiformis and Phyllocaulis boraceiensis were experimentally infected to describe the kinetics of L3 elimination in the mucus secretions of those veronicelid species. A maximum of 2 L3/g/day was found in the mucus, while the number of L3 isolated from the fibromuscular tissues varied from 14 to 448. Productive infection was established by inoculations in the hyponotum or in the body cavity, through the tegument. Intra-cavity injection is a less complex procedure and permits a better control of inocula. A preliminary trial to titrate the infective dosis for P. variegatus indicated that inocula should range between 1000 and 5000 L1. The data also confirmed the importance of P. variegatus as an intermediate host of A. costaricensis.

Report on the ocurrence of Angiostrongylus costaricensis in southern Brazil, in a new intermediate host from the genus Sarasinula (Veronicellidae, Gastropoda)

Veronicellid slugs are the main intermediate hosts for Angiostrongylus costaricencis. In a rural locality in Nova Itaberaba (SC, southern Brazil) Sarasinula linguaeformis was identified as a crop pest. The parasitological examination revealed A. costaricencis infection in 43 out ot 50 slugs. The prevalence of 86% and the individual parasitic burdens are the highest sofar reported in Brazil and S. linguaeformis is the first species from the genus Sarasinula to be identified as intermediate host for A. costaricencis in southern Brazil.

Genetic diversity and differentiation in natural Plasmodium falciparum populations inferred by molecular typing of the merozoite surface proteins 1 and 2

Genetic diversity and differentiation, inferred by typing the polymorphic genes coding for the merozoite surface proteins 1 (Msp-1) and 2 (Msp-2), were compared for 345 isolates belonging to seven Plasmodium falciparum populations from three continents. Both loci yielded similar estimates of genetic diversity for each population, but rather different patterns of between-population differentiation, suggesting that natural selection on these loci, rather than the transmission dynamics of P. falciparum, determines the variation in allele frequencies among populations.