Phylogenetic analysis of Biomphalaria tenagophila (Orbigny, 1835) (Mollusca: Gastropoda).

Mitochondrial DNA of Biomphalaria tenagophila, a mollusc intermediate host of Schistosoma mansoni in Brazil, was sequenced and characterised. The genome size found for B. tenagophila was 13,722 bp and contained 13 messenger RNAs, 22 transfer RNAs (tRNA) and two ribosomal RNAs (rRNA). In addition to sequencing, the mitochondrial DNA (mtDNA) genome organization of B. tenagophila was analysed based on its content and localization of both coding and non-coding regions, regions of gene overlap and tRNA nucleotide sequences. Sequences of protein, rRNA 12S and rRNA 16S nucleotides as well as gene organization were compared between B. tenagophila and Biomphalaria glabrata, as the latter is the most important S. mansoni intermediate host in Brazil. Differences between such species were observed regarding rRNA composition. The complete sequence of the B. tenagophila mitochondrial genome was deposited in GenBank (accession EF433576). Furthermore, phylogenetic relationships were estimated among 28 mollusc species, which had their complete mitochondrial genome deposited in GenBank, using the neighbour-joining method, maximum parsimony and maximum likelihood bootstrap. B. tenagophila was positioned at a branch close to B. glabrata and Pulmonata molluscs, collectively comprising a paraphyletic group, contrary to Opistobranchia, which was positioned at a single branch and constituted a monophyletic group.

Evaluation of a linear spectral mixture model and vegetation indices (NDVI and EVI) in a study of schistosomiasis mansoni and Biomphalaria glabrata distribution in the state of Minas Gerais, Brazil.

This paper analyses the associations between Normalized Difference Vegetation Index (NDVI) and Enhanced Vegetation Index (EVI) on the prevalence of schistosomiasis and the presence of Biomphalaria glabrata in the state of Minas Gerais (MG), Brazil. Additionally, vegetation, soil and shade fraction images were created using a Linear Spectral Mixture Model (LSMM) from the blue, red and infrared channels of the Moderate Resolution Imaging Spectroradiometer spaceborne sensor and the relationship between these images and the prevalence of schistosomiasis and the presence of B. glabrata was analysed. First, we found a high correlation between the vegetation fraction image and EVI and second, a high correlation between soil fraction image and NDVI. The results also indicate that there was a positive correlation between prevalence and the vegetation fraction image (July 2002), a negative correlation between prevalence and the soil fraction image (July 2002) and a positive correlation between B. glabrata and the shade fraction image (July 2002). This paper demonstrates that the LSMM variables can be used as a substitute for the standard vegetation indices (EVI and NDVI) to determine and delimit risk areas for B. glabrata and schistosomiasis in MG, which can be used to improve the allocation of resources for disease control.

The Estrada Real project and endemic diseases: the case of schistosomiasis, geoprocessing and tourism.

Geographical Information System (GIS) is a tool that has recently been applied to better understand spatial disease distributions. Using meteorological, social, sanitation, mollusc distribution data and remote sensing variables, this study aimed to further develop the GIS technology by creating a model for the spatial distribution of schistosomiasis and to apply this model to an area with rural tourism in the Brazilian state of Minas Gerais (MG). The Estrada Real, covering about 1,400 km, is the largest and most important Brazilian tourism project, involving 163 cities in MG with different schistosomiasis prevalence rates. The model with three variables showed a R(2) = 0.34, with a standard deviation of risk estimated adequate for public health needs. The main variables selected for modelling were summer vegetation, summer minimal temperature and winter minimal temperature. The results confirmed the importance of Remote Sensing data and the valuable contribution of GIS in identifying priority areas for intervention in tourism regions which are endemic to schistosomiasis.

Schistosomiasis risk mapping in the state of Minas Gerais, Brazil, using a decision tree approach, remote sensing data and sociological indicators.

Schistosomiasis mansoni is not just a physical disease, but is related to social and behavioural factors as well. Snails of the Biomphalaria genus are an intermediate host for Schistosoma mansoni and infect humans through water. The objective of this study is to classify the risk of schistosomiasis in the state of Minas Gerais (MG). We focus on socioeconomic and demographic features, basic sanitation features, the presence of accumulated water bodies, dense vegetation in the summer and winter seasons and related terrain characteristics. We draw on the decision tree approach to infection risk modelling and mapping. The model robustness was properly verified. The main variables that were selected by the procedure included the terrain's water accumulation capacity, temperature extremes and the Human Development Index. In addition, the model was used to generate two maps, one that included risk classification for the entire of MG and another that included classification errors. The resulting map was 62.9% accurate.

Spatial distribution of Schistosoma mansoni infection before and after chemotherapy with two praziquantel doses in a community of Pernambuco, Brazil.

Praziquantel chemotherapy has been the focus of the Schistosomiasis Control Program in Brazil for the past two decades. Nevertheless, information on the impact of selective chemotherapy against Schistosoma mansoni infection under the conditions confronted by the health teams in endemic municipalities remains scarce. This paper compares the spatial pattern of infection before and after treatment with either a 40 mg/kg or 60 mg/kg dose of praziquantel by determining the intensity of spatial cluster among patients at 180 and 360 days after treatment. The spatial-temporal distribution of egg-positive patients was analysed in a Geographic Information System using the kernel smoothing technique. While all patients became egg-negative after 21 days, 17.9% and 30.9% reverted to an egg-positive condition after 180 and 360 days, respectively. Both the prevalence and intensity of infection after treatment were significantly lower in the 60 mg/kg than in the 40 mg/kg treatment group. The higher intensity of the kernel in the 40 mg/kg group compared to the 60 mg/kg group, at both 180 and 360 days, reflects the higher number of reverted cases in the lower dose group. Auxiliary, preventive measures to control transmission should be integrated with chemotherapy to achieve a more enduring impact.

Proteome analysis of the cardiac and pericardial tissue of Biomphalaria tenagophila populations susceptible and resistant to Schistosoma mansoni infection.

For a better comprehension of the parasite-host interaction, proteins expressed by the cardiac and pericardial tissues were compared between susceptible (Cabo Frio) and resistant (Taim) Biomphalaria tenagophila populations, challenged (c) and non-challenged (nc) with Schistosoma mansoni. Proteins were separated by two-dimensional gel electrophoresis (2DE) and stained with Coomassie blue. A total of 146 and 135 spots were observed in Cabo Frio (CFnc) and in Taim (Tnc) non-challenged populations, respectively, whereas 153 spots were detected in both Cabo Frio (CFc) and Taim (Tc) challenged populations. Regarding comparisons between CFnc and CFc, the numbers of exclusive spots obtained were one and nine, respectively, whereas Tnc yielded 17 and Tc eight exclusive spots. By comparing the total of spots in CF (nc+c) with T (nc+c) populations, we obtained: four exclusive spots for CFc; zero for CFnc; four for Tc and; one for Tnc. A quantitative comparison (reason>2.5) of the total spots of CF (nc+c) with T (nc+c) populations allowed us to distinguish five more intense spots for Tc, 14 for Tnc, 15 for CFnc and 11 for CFc. In the CFnc population, two proteins were identified: actin and ATP synthase alpha chain; in the CFc population, four proteins: actin, calmodulin, HSP70, and dehydrogenase; in the Tnc population, five proteins: matrilin, HSP70, actin, ATP synthase alpha chain and intermediate filament of the protein; and in the Tc population, three proteins: actin, alpha-S1 casein and ATP synthase alpha chain. Out of a total of 79 spots, only nine proteins were identified due to the low number of available nucleotide sequences in the GenBank. Nevertheless, knowing proteins regarded as differentially expressed is indispensable for hitherto unidentified genes implicated in B. tenagophila resistance and or susceptibility to S. mansoni infection.

Isolation and detection of Fasciola hepatica DNA in Lymnaea viatrix from formalin-fixed and paraffin-embedded tissues through multiplex-PCR.

Detection of Fasciola hepatica infection in Lymnaea viatrix through analysis of histological cuts is based upon morphological characters of the parasite during the intra-mollusk phase of parasitism. At this stage, trematode forms are very similar and, thus, very difficult to differentiate. Specific detection may also be impaired by the presence of other helminthes in the mollusk. Histological samples are usually fixed in formalin, embedded in paraffin, sectioned and HE stained. In the current study, a method for the extraction of DNA from formalin-fixed, paraffin-embedded tissues was standardized by means of deparaffinizing with xylol and digesting with proteinase K. Extracted DNA was amplified in a multiplex-PCR, by using simultaneous primers in a single reaction under high stringency conditions. Results showed specific amplification of DNA from the trematode and the snails. The technique was sensitive enough to detect F. hepatica infections in L. viatrix, in histological sections in which the presence of larval stages could not be observed through brightfield microscopy. The profiles generated were: stair bands referring to F. hepatica DNAmt amplification; a band of 1200 bp referring to L. viatrix ITS and another of 1300 bp referring to F. hepatica ITS and other trematodes. Multiplex-PCR has shown to be a fast, safe, highly sensitive and specific method, which is able to amplify DNA from fixed tissues, despite a low DNA quantity and its degradation caused by fixation processes. Such methodology may be useful in studies on fascioliasis epidemiology, enabling the use of material from histological collections.

Microarray analysis of gene expression induced by sexual contact in Schistosoma mansoni.

BACKGROUND: The parasitic trematode Schistosoma mansoni is one of the major causative agents of Schistosomiasis, a disease that affects approximately 200 million people, mostly in developing countries. Since much of the pathology is associated with eggs laid by the female worm, understanding the mechanisms involved in oogenesis and sexual maturation is an important step towards the discovery of new targets for effective drug therapy. It is known that the adult female worm only develops fully in the presence of a male worm and that the rates of oviposition and maturation of eggs are significantly increased by mating. In order to study gene transcripts associated with sexual maturation and oviposition, we compared the gene expression profiles of sexually mature and immature parasites using DNA microarrays. RESULTS: For each experiment, three amplified RNA microarray hybridizations and their dye swaps were analyzed. Our results show that 265 transcripts are differentially expressed in adult females and 53 in adult males when mature and immature worms are compared. Of the genes differentially expressed, 55% are expressed at higher levels in paired females while the remaining 45% are more expressed in unpaired ones and 56.6% are expressed at higher levels in paired male worms while the remaining 43.4% are more expressed in immature parasites. Real-time RT-PCR analysis validated the microarray results. Several new maturation associated transcripts were identified. Genes that were up-regulated in single-sex females were mostly related to energy generation (i.e. carbohydrate and protein metabolism, generation of precursor metabolites and energy, cellular catabolism, and organelle organization and biogenesis) while genes that were down-regulated related to RNA metabolism, reactive oxygen species metabolism, electron transport, organelle organization and biogenesis and protein biosynthesis. CONCLUSION: Our results confirm previous observations related to gene expression induced by sexual maturation in female schistosome worms. They also increase the list of S. mansoni maturation associated transcripts considerably, therefore opening new and exciting avenues for the study of the conjugal biology and development of new drugs against schistosomes.

Corrigendum: Whole genome analysis of a schistosomiasis-transmitting freshwater snail.

This corrects the article DOI: 10.1038/ncomms15451.

Whole genome analysis of a schistosomiasis-transmitting freshwater snail.

Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis.

Multiplex PCR for both identification of Brazilian Biomphalaria species (Gastropoda: Planorbidae) and diagnosis of infection by Schistosoma mansoni (Trematoda: Schistosomatidae).

A simple and single-step technique based on multiplex PCR (multiplex polymerase chain reaction) has been developed for simultaneous identification of Brazilian Biomphalaria species, the intermediate hosts of Schistosoma mansoni, and their diagnosis of infection by the trematode. We used species-specific primers directed both to the internal transcribed spacer 2 (ITS2) of ribosomal DNA from 3 of the S. mansoni host species and to the mitochondrial DNA (mtDNA) from the trematode. Those primers were used simultaneously in a single multiplex-PCR reaction, and template DNA was obtained from S. mansoni-infected and noninfected snails. The results were visualized in silver stained polyacrylamide gels, revealing the presence of specific bands. The methodology has shown to be efficient, fast, and reproducible for Biomphalaria species identification and diagnosis of snails infected by S. mansoni during prepatent periods.

Infection with Schistosoma mansoni correlates with altered immune responses to Ascaris lumbricoides and hookworm.

Studies were performed on humoral and cellular immune responses of patients from areas in Brazil endemic for hookworm and Ascaris lumbricoides, and either endemic or non-endemic for Schistosoma mansoni. Humoral and cellular responses were evaluated by enzyme-linked immunosorbant assay (ELISA) and peripheral blood mononuclear cell (PBMC) proliferation assays against larval hookworm antigens, A. lumbricoides egg antigens, and soluble egg antigens (SEA) or soluble whole adult antigenic preparation (SWAP) from S. mansoni. Patients from S. mansoni-endemic areas, who currently had only hookworm or Ascaris infections, expressed lower humoral and cellular responses to hookworm or Ascaris antigens, respectively, than did their counterparts from areas not endemic for S. mansoni. Individuals from S. mansoni endemic area, although without detectable S. mansoni infection, do mount humoral and cellular responses to SEA and SWAP. This group of individuals has been probably in contact with S. mansoni antigens, since the groups harboring A. lumbricoides or hookworm infections from non-S. mansoni endemic areas do not have detectable anti-S. mansoni responses. PBMC proliferative responses discriminated well between patients with active hookworm infections versus ascariasis, if they were from areas not endemic for S. mansoni.

Spatial distribution of Biomphalaria spp., the intermediate host snails of Schistosoma mansoni, in Brazil.

Schistosomiasis mansoni remains an important parasitic disease of man, endemic in large parts of sub-Saharan Africa, the Middle East, South America and the Caribbean. The aetiological agent is the trematode Schistosoma mansoni, whereas aquatic snails of the genus Biomphalaria act as intermediate hosts in the parasite life cycle. In Brazil, the distribution of Biomphalaria spp. is closely associated with the occurrence of schistosomiasis. The purpose of this study was to map and predict the spatial distribution of the intermediate host snails of S. mansoni across Brazil. We assembled snail "presenceonly" data and used a maximum entropy approach, along with climatic and environmental variables to produce predictive risk maps. We identified a series of risk factors that govern the distribution of Biomphalaria snails. We find that high-risk areas for B. glabrata are concentrated in the regions of Northeast and Southeast and the northern part of the South region. B. straminea are found in the Northeast and Southeast regions, and B. tenagophila are concentrated in the Southeast and South regions. Our findings confirm that the presence of the intermediate host snails is correlated with the occurrence of schistosomiasis mansoni. The generated risk maps of intermediate host snails might assist the national control programme for spatial targeting of control interventions and to ultimately move towards schistosomiasis elimination in Brazil.

Use of indicator kriging to investigate schistosomiasis in minas gerais state, Brazil.

Geographic Information Systems (GISs) are composed of useful tools to map and to model the spatial distribution of events that have geographic importance as schistosomiasis. This paper is a review of the use the indicator kriging, implemented on the Georeferenced Information Processing System (SPRING) to make inferences about the prevalence of schistosomiasis and the presence of the species of Biomphalaria, intermediate hosts of Schistosoma mansoni, in areas without this information, in the Minas Gerais State, Brazil. The results were two maps. The first one was a map of Biomphalaria species, and the second was a new map of estimated prevalence of schistosomiasis. The obtained results showed that the indicator kriging can be used to better allocate resources for study and control of schistosomiasis in areas with transmission or the possibility of disease transmission.

Utilizing environmental, socioeconomic data and GIS techniques to estimate the risk for ascariasis and trichuriasis in Minas Gerais, Brazil.

The impact of intestinal helminths on human health is well known among the population and health authorities because of their wide geographic distribution and the serious problems they cause. Geohelminths are highly prevalent and have a big impact on public health, mainly in underdeveloped and developing countries. Geohelminths are responsible for the high levels of debility found in the younger population and are often related to cases of chronic diarrhea and malnutrition, which put the physical and intellectual development of children at risk. These geohelminths have not been sufficiently studied. One obstacle in implementing a control program is the lack of knowledge of the prevalence and geographical distribution. Geographical information systems (GIS) and remote sensing (RS) have been utilized to improve understanding of infectious disease distribution and climatic patterns. In this study, GIS and RS technologies, as well as meteorological, social, and environmental variables were utilized for the modeling and prediction of ascariasis and trichuriasis. The GIS and RS technologies specifically used were those produced by orbital sensing including the Moderate Resolution Imaging Spectroradiometer (MODIS) and the Shuttle Radar Topography Mission (SRTM). The results of this study demonstrated important factors related to the transmission of ascariasis and trichuriasis and confirmed the key association between environmental variables and the poverty index, which enabled us to identify priority areas for intervention planning in the state of Minas Gerais in Brazil.

Molecular epidemiology of Brazilian Biomphalaria: a review of the identification of species and the detection of infected snails.

The three vector species of Schistosoma mansoni in Brazil, Biomphalaria glabrata, B. tenagophila and B. straminea show different susceptibility levels to the trematode besides a wide geographical distribution. The identification of such molluscs is important to further understand the disease epidemiology. Considering the fact that morphological identification may become difficult or even impossible under particular circumstances, the use of molecular-based methods have permitted the generation of more consistent information concerning the population structure of Biomphalaria furthering knowledge on taxonomy and diagnosis of infection. We have developed methodologies in order to provide simultaneous species identification of the intermediate host and diagnosis of infection with S. mansoni.

Spatial distribution of Biomphalaria mollusks at São Francisco River Basin, Minas Gerais, Brazil, using geostatistical procedures.

Geostatistics is used in this work to make inferences about the presence of the species of Biomphalaria (B. glabrata, B. tenagophila and/or B. straminea), intermediate hosts of Schistosoma mansoni, at the São Francisco River Basin, in Minas Gerais, Brazil. One of these geostatistical procedures, known as indicator kriging, allows the classification of categorical data, in areas where the data are not available, using a punctual sample set. The result is a map of species and risk area definition. More than a single map of the categorical attribute, the procedure also permits the association of uncertainties of the stochastic model, which can be used to qualify the inferences. In order to validate the estimated data of the risk map, a fieldwork in five municipalities was carried out. The obtained results showed that indicator kriging is a rather robust tool since it presented a very good agreement with the field findings. The obtained risk map can be thought as an auxiliary tool to formulate proper public health strategies, and to guide other fieldwork, considering the places with higher occurrence probability of the most important snail species. Also, the risk map will enable better resource distribution and adequate policies for the mollusk control. This methodology will be applied to other river basins to generate a predictive map for Biomphalaria species distribution for the entire state of Minas Gerais.

Development of Angiostrongylus costaricensis Morera and Céspedes 1971 (Nematoda: Angiostrongylidae) larvae in the intermediate host Sarasinula marginata (Semper 1885) (Mollusca: Soleolifera).

In life cycle of Angiostrongylus costaricensis, veronicellidae mollusks participate as the invertebrate host while rodents as the main vertebrate host. The current work shows a sequential larval development of A. costaricensis in Sarasinula marginata, individually killed and digested from day 1 to 43, post infection. Some larvae, recovered from sedimentation, were submitted to selective staining after paraffin embedded or inclusion in JB-4 to study inner structures. As control, four slugs were used, two killed at the beginning of infection and the others at the end of the experiment. At day 2 post infection, larvae were motionless and thick, presenting initial retention of granules. At day 4, L2 were detected, persisting until 43 days post infection. Larvae L2 displayed a large amount of granules rich in lipids and carbohydrates through its overall body, with more accumulation at the medial third corresponding to the esophagus-intestine transition site. Lipid granules, the main energetic source, were located at the basal and apical regions of intestinal cells. Both L1 and L3 presented bilateral alae, which is also common in other nematodes. Transition forms between L2 to L3 molts were also observed.

Schistosomiasis risk estimation in Minas Gerais State, Brazil, using environmental data and GIS techniques.

The influence of climate and environmental variables to the distribution of schistosomiasis has been assessed in several previous studies. Also Geographical Information System (GIS), is a tool that has been recently tested for better understanding the spatial disease distribution. The objective of this paper is to further develop the GIS technology for modeling and control of schistosomiasis using meteorological and social variables and introducing new potential environmental-related variables, particularly those produced by recently launched orbital sensors like the Moderate Resolution Imaging Spectroradiometer (MODIS) and the Shuttle Radar Topography Mission (SRTM). Three different scenarios have been analyzed, and despite of not quite large determination factor, the standard deviation of risk estimates was considered adequate for public health needs. The main variables selected as important for modeling purposes was topographic elevation, summer minimum temperature, the NDVI vegetation index, and the social index HDI91.