Spatial analysis of areas at risk for schistosomiasis in the Alto Tietê Basin, São Paulo, Brazil.

The present study identified locations with the presence of Biomphalaria and the areas at risk for Schistosoma mansoni, the etiological agent of schistosomiasis, in the Metropolitan Region of São Paulo (MRSP), located in the Alto Tietê Basin (ATB), São Paulo, Brazil. The study area comprises floodplains of the Tietê River from its source, in Salesópolis, to Rasgão Dam, in Pirapora do Bom Jesus, covering 39 municipalities. Data from the planorbidic chart of the malacology laboratory of the Endemic Control Superintendence for 2008-2016 were used, as well as data from the notification forms of cases of human schistosomiasis in 2006-2015 made available by the Epidemiological Surveillance Groups of Mogi das Cruzes and Osasco, and by the Health and Surveillance Coordination of São Paulo. These data were used to generate maps of the distribution of Biomphalaria, of the scanning analysis, of the Gi statistics of the autochthonous and imported schistosomiasis cases, and of the flow of cases imported from other Brazilian states and municipalities in the state of São Paulo to the study area. A total of 21,618 specimens of the genus Biomphalaria were captured and six species were identified. Biomphalaria tenagophila was the predominant species in the region and of greatest epidemiological importance as it is the main responsible for the transmission of the disease in the state of São Paulo. A total of 3,623 cases of schistosomiasis were reported in the study area, and São Paulo and Guarulhos were the municipalities with the highest number of autochthonous and imported cases during the study period. The imported cases originated from 21 Brazilian states plus the Federal District and from 51 municipalities in São Paulo state located outside the study area. Autochthonous clusters with high risk for the occurrence of schistosomiasis were identified in the municipalities of São Paulo, Guarulhos, Santa Isabel/Mogi das Cruzes/Arujá, and Cotia/Itapevi. Clusters of imported cases were also identified in Itaquaquecetuba, at the border of Cotia/Itapevi and Suzano/São Paulo/Mogi das Cruzes. The presence of B. tenagophila in the ATB and in the clusters of autochthonous cases herein identified, although not carrying schistosome larvae, represents a risk for the transmission of infectious agents. The human population in the MRSP mainly occupies the lowlands of the Tietê River, which are locations with poor coverage of basic sanitation. Therefore, measures for the control and surveillance of schistosomiasis such as educational actions, improvements in basic sanitation, and the malacological and epidemiological monitoring of these areas are extremely important.

Molecular and morphological identification of Biomphalaria species from the state of São Paulo, Brazil.

DNA barcoding and morphological characters were used to identify adult snails belonging to the genus Biomphalaria from 17 municipalities in the state of São Paulo, Brazil. The DNA barcode analysis also included twenty-nine sequences retrieved from GenBank. The final data set of 104 sequences of the mitochondrial cytochrome oxidase I (COI) gene was analyzed for K2P intraspecific and interspecific divergences, through tree-reconstruction methods (Neighbor-Joining, Maximum Likelihood and Bayesian inference), and by applying different models (ABGD, bPTP, GMYC) to partition the sequences according to the pattern of genetic variation. Twenty-seven morphological parameters of internal organs were used to identify specimens. The molecular taxonomy of Biomphalaria agreed with the morphological identification of specimens from the same collection locality. DNA barcoding may therefore be a useful supporting tool for identifying Biomphalaria snails in areas at risk for schistosomiasis.

Biodiversidade das Biomphalaria (Mollusca, Planorbidae) na região metropolitana de São Paulo como complemento à carta planorbídica do estado de São Paulo / Biodiversity of Biomphalaria (Mollusca, Planorbidae) in Metropolitam Region of São Paulo as a complement to the planorbid chart of the state of São Paulo

A fauna planorbídica do gênero Biomphalaria foi inventariada em 39 municípios da Região Metropolitana de São Paulo (RMSP), contribuindo para a complementação da carta planorbídica do estado. As coletas foram realizadas no período de agosto de 2008 a março de 2016. Os moluscos foram identificados e submetidos a exame parasitológico. Foram encontradas seis espécies; Biomphalaria occidentalis, Biomphalaria oligoza, Biomphalaria peregrina, Biomphalaria schrammi, Biomphalaria straminea e Biomphalaria tenagophila, as quais tiveram sua distribuição geográfica atualizada na área de estudo. B. tenagophila demonstrou estar bem adaptada nos ambientes pesquisados. É a espécie com maior representatividade e predomínio em grande parte dos municípios. A presença de bionfalárias portando larvas de trematódeos, que não esquistossomo, constituem risco de transmissão de parasitas de interesse veterinário. A presença de B. tenagophila na RMSP faz desta uma área de importância para a transmissão de esquistossomose, sendo necessário, como forma de controle e prevenção, o monitoramento da área, ações educativas e melhorias no saneamento básico.

Developmental toxicity, acute toxicity and mutagenicity testing in freshwater snails Biomphalaria glabrata (Mollusca: Gastropoda) exposed to chromium and water samples.

A protocol combining acute toxicity, developmental toxicity and mutagenicity analysis in freshwater snail Biomphalaria glabrata for application in ecotoxicological studies is described. For acute toxicity testing, LC50 and EC50 values were determined; dominant lethal mutations induction was the endpoint for mutagenicity analysis. Reference toxicant potassium dichromate (K2Cr2O7) was used to characterize B. glabrata sensitivity for toxicity and cyclophosphamide to mutagenicity testing purposes. Compared to other relevant freshwater species, B. glabrata showed high sensitivity: the lowest EC50 value was obtained with embryos at veliger stage (5.76mg/L). To assess the model applicability for environmental studies, influent and effluent water samples from a wastewater treatment plant were evaluated. Gastropod sensitivity was assessed in comparison to the standardized bioassay with Daphnia similis exposed to the same water samples. Sampling sites identified as toxic to daphnids were also detected by snails, showing a qualitatively similar sensitivity suggesting that B. glabrata is a suitable test species for freshwater monitoring. Holding procedures and protocols implemented for toxicity and developmental bioassays showed to be in compliance with international standards for intra-laboratory precision. Thereby, we are proposing this system for application in ecotoxicological studies.

Schistosomiasis control using piplartine against Biomphalaria glabrata at different developmental stages.

BACKGROUND: Schistosomiasis is one of the most significant diseases in tropical countries and affects almost 200 million people worldwide. The application of molluscicides to eliminate the parasite's intermediate host, Biomphalaria glabrata, from infected water supplies is one strategy currently being used to control the disease. Previous studies have shown a potent molluscicidal activity of crude extracts from Piper species, with extracts from Piper tuberculatum being among the most active. METHODS AND FINDINGS: The molluscicidal activity of P. tuberculatum was monitored on methanolic extracts from different organs (roots, leaves, fruit and stems). The compounds responsible for the molluscicidal activity were identified using (1)H NMR and ESIMS data and multivariate analyses, including principal component analysis and partial least squares. These results indicated that the high molluscicidal activity displayed by root extracts (LC50 20.28 µg/ml) was due to the presence of piplartine, a well-known biologically-active amide. Piplartine was isolated from P. tuberculatum root extracts, and the molluscicidal activity of this compound on adults and embryos of B. glabrata was determined. The compound displayed potent activity against all developmental stages of B. glabrata. Next, the environmental toxicity of piplartine was evaluated using the microcrustacean Daphnia similis (LC50 7.32 µg/ml) and the fish Danio rerio (1.69 µg/ml). The toxicity to these organisms was less compared with the toxicity of niclosamide, a commercial molluscicide. CONCLUSIONS: The development of a new, natural molluscicide is highly desirable, particularly because the commercially available molluscicide niclosamide is highly toxic to some organisms in the environment (LC50 0.25 µg/ml to D. similis and 0.12 µg/ml to D. rerio). Thus, piplartine is a potential candidate for a natural molluscicide that has been extracted from a tropical plant species and showed less toxic to environment.

Current distribution of Achatina fulica, in the State of São Paulo including records of Aelurostrongylus abstrusus (Nematoda) larvae infestation / Distribuição atual de Achatina fulica Bowdich, 1822 no Estado de São Paulo com registro de infestação por larvas de Aelurostrongylus abstrusus (Nematoda)

The currently known distribution range of Achatina fulica Bowdich, 1822, in the state of São Paulo, Brazil, is presented. The record of A. fulica naturally infested with Aelurostrongylus abstrusus larvae (Railliet, 1898) (Nematoda: Metastrongylidae) can be found in the city of Guaratinguetá. It was found A. fulica with Metastrongylidae larvae without known medical and veterinary importance in the cities of Carapicuíba, Embu-Guaçu, Itapevi, São Caetano do Sul, São Paulo and Taboão da Serra.

É apresentada a distribuição de Achatina fulica Bowdich, 1822 no Estado de São Paulo, Brasil. É fornecido o registro de A. fulica naturalmente infestada por larvas de Aelurostrongylus abstrusus (Railliet, 1898) (Nematoda: Metastrongylidae) no município de Guaratinguetá. Foi encontrada A. fulica portando larvas de Metastrongylidae sem importância médica e veterinária conhecida nos municípios de Carapicuíba, Embu Guaçu, Itapevi, São Caetano do Sul, São Paulo e Taboão da Serra.

The effects of the experimental infection from a focus of transmission of Schistosoma mansoni in a population of Biomphalaria tenagophila (D'Orbigny, 1835) in the region of "Vale do Ribeira de Iguape", Brazil.

To observe the effects of the parasitic infection on the biology of B. tenagophila, field and laboratory populations of this mollusk from Itariri, in Vale do Ribeira, Brazil, were experimentally infected. Each mollusk received 10 miracidia of Schistosoma mansoni (SJ lineage) and was observed throughout the parasite's development. The biological variables were compared according to the criteria "group" and "infectious phase". The main damage caused by the parasitic infection manifested itself in reproduction, longevity and lesions on the shell of the mollusks in the patent phase. An infection rate of 58.8% was observed. Microanatomical study of the mollusk's digestive gland and ovotestis revealed the presence of evolving larval forms and cercariae. It was concluded that the effects of the parasitic infection on both populations were moderate, despite the low survival rate of the infected mollusks, the damage did not prevent either reproduction or the elimination of cercariae, which continued for a long time.

The effects of the experimental infection from a focus of transmission of Schistosoma mansoni in a population of Biomphalaria tenagophila (D'Orbigny, 1835) in the region of "Vale do Ribeira de Iguape", Brazil / Efeitos da infecção experimental de um foco de transmissão de Schistosoma mansoni em população de Biomphalaria tenagophila (D'Orbigny, 1835) na região do Vale do Ribeira de Iguape, Brasil

To observe the effects of the parasitic infection on the biology of B. tenagophila, field and laboratory populations of this mollusk from Itariri, in Vale do Ribeira, Brazil, were experimentally infected. Each mollusk received 10 miracidia of Schistosoma mansoni (SJ lineage) and was observed throughout the parasite's development. The biological variables were compared according to the criteria "group" and "infectious phase". The main damage caused by the parasitic infection manifested itself in reproduction, longevity and lesions on the shell of the mollusks in the patent phase. An infection rate of 58.8 percent was observed. Microanatomical study of the mollusk's digestive gland and ovotestis revealed the presence of evolving larval forms and cercariae. It was concluded that the effects of the parasitic infection on both populations were moderate, despite the low survival rate of the infected mollusks, the damage did not prevent either reproduction or the elimination of cercariae, which continued for a long time.

Com objetivo de observar os efeitos da infecção parasitária na biologia de B. tenagophila, foram realizadas infecções experimentais em populações de campo e laboratório, ambas procedentes de Itariri, Vale do Ribeira, Brasil. Cada molusco recebeu 10 miracídios de Schistosoma mansoni (linhagem SJ), sendo observado durante o desenvolvimento dos parasitos. As variáveis biológicas foram comparadas segundo os critérios "grupo" e "fase de infecção". Os principais danos decorrentes do parasitismo se manifestaram na reprodução, na longevidade e em lesões na concha dos moluscos na fase patente. Foi encontrada uma taxa de infecção de 58,8 por cento. O estudo microanatômico da glândula digestiva e do ovoteste do molusco revelou a presença de formas larvárias em evolução e cercárias. Concluiu-se que os efeitos da infecção parasitária, sobre as duas populações, foram moderados, uma vez que os danos não impediram a reprodução e a eliminação de cercárias que se manteve por um longo período, apesar da baixa sobrevivência dos moluscos parasitados.

Current distribution of Achatina fulica, in the state of São Paulo including records of Aelurostrongylus abstrusus (Nematoda) larvae infestation.

The currently known distribution range of Achatina fulica Bowdich, 1822, in the state of São Paulo, Brazil, is presented. The record of A. fulica naturally infested with Aelurostrongylus abstrusus larvae (Railliet, 1898) (Nematoda: Metastrongylidae) can be found in the city of Guaratinguetá. It was found A. fulica with Metastrongylidae larvae without known medical and veterinary importance in the cities of Carapicuíba, Embu-Guaçu, Itapevi, São Caetano do Sul, São Paulo and Taboão da Serra.

Schistosoma mansoni and other larval trematodes in Biomphalaria tenagophila (Planorbidae) from Guarulhos, São Paulo State, Brazil.

A total of 909 Biomphalaria tenagophila were collected from two areas in Guarulhos (Metropolitan area of São Paulo, São Paulo State, Brazil) to assess larval trematode infections. In all collection sites, only this species was found and 183 (20.13%) harbored trematode infections. In these collections, four morphologically distinguishable types of cercariae were identified by confocal microscopy. Xiphidiocercaria (Cercaria lutzi) was the most common type of cercaria recovered, contributing 76.5% of all infections. Schistosoma mansoni cercariae were recovered and comprised the total of 13.11%. Strigea cercaria (Cercaria caratinguensis) and Brevifurcate pharyngeate Clinostomatoide cercaria (Cercaria ocellifera) contributed 8.33% and 2.22% of all infections, respectively. Double infections (S. mansoni and C. lutzi) were found in twelve snails, contributing 6.55% of all infections. In all sites studied, small vertebrates were found in snail habitats and it was observed human contact with the water. The presence of trematode infected snails in large cities has public health implications. It further provides a starting point for some comprehensive studies on snail-related aspects of transmission and biology of trematode of medical and veterinary importance.

Schistosoma mansoni and other larval trematodes in Biomphalaria tenagophila (Planorbidae) from Guarulhos, São Paulo State, Brazil / Infecção por Schistosoma mansoni e outros trematódeos em Biomphalaria tenagophila (Planorbidae) de Guarulhos, São Paulo, Brasil

A total of 909 Biomphalaria tenagophila were collected from two areas in Guarulhos (Metropolitan area of São Paulo, São Paulo State, Brazil) to assess larval trematode infections. In all collection sites, only this species was found and 183 (20.13%) harbored trematode infections. In these collections, four morphologically distinguishable types of cercariae were identified by confocal microscopy. Xiphidiocercaria (Cercaria lutzi) was the most common type of cercaria recovered, contributing 76.5% of all infections. Schistosoma mansoni cercariae were recovered and comprised the total of 13.11%. Strigea cercaria (Cercaria caratinguensis) and Brevifurcate pharyngeate Clinostomatoide cercaria (Cercaria ocellifera) contributed 8.33% and 2.22% of all infections, respectively. Double infections (S. mansoni and C. lutzi) were found in twelve snails, contributing 6.55% of all infections. In all sites studied, small vertebrates were found in snail habitats and it was observed human contact with the water. The presence of trematode infected snails in large cities has public health implications. It further provides a starting point for some comprehensive studies on snail-related aspects of transmission and biology of trematode of medical and veterinary importance.

Um total de 909 exemplares de Biomphalaria tenagophila foi coletado de duas regiões em Guarulhos (área Metropolitana de São Paulo, Estado de São Paulo, Brasil) a fim de que fosse verificada infecção por larvas de trematódeos. Em todos os locais de coleta, somente essa espécie foi encontrada e 183 (20,13%) caramujos estavam infectados. Nestes locais, quatro tipos de cercárias foram identificadas com microscópio confocal. Xiphidiocercaria (Cercaria luzti) foi o tipo de cercária mais comum, contribuindo com 76,5% de toda infecção. Cercárias de Schistosoma mansoni foram encontradas, obtendo um total de 13,11%. Strigea cercaria (Cercaria caratinguensis) e Brevifurcate pharyngeate Clinostomatoide cercaria (Cercaria ocellifera) contribuíram com 8,33% e 2,22% de toda infecção, respectivamente. Dupla infecção foi encontrada em doze caramujos, contribuindo com 6,55% de toda infecção. Em todos os locais, pequenos vertebrados foram encontrados e foi observado contato humano com a água. A presença de caramujos infectados por trematódeos que infectam o homem em grandes cidades tem implicações na saúde pública. Deve-se salientar a importância de mais estudos epidemiológicos e biológicos destes parasitas de importância médica e veterinária.

Establishment of the dominant lethal test in the freshwater mollusk Biomphalaria glabrata (Say, 1818).

Mutagens in the environment may represent a long-term risk for ecosystems. The reproductive potential of populations can be affected by alterations in the fecundity and offspring viability caused by germ cell mutations. Despite the ecological relevance of these effects, there are few studies on germ cell mutagenicity in natural populations. Biomphalaria glabrata was chosen for this study because of the scarcity of data on freshwater invertebrates and the ecological importance of this group. The aim of this study was to establish a germ cell mutagenicity test in B. glabrata by using a similar approach to that used in the dominant lethal test in rodents. Mitomycin C was used as a direct mutagen and cyclophosphamide as a mutagen that requires metabolic activation. Wild-type snails were exposed for 10 days to three concentrations of each agent and crossed with non-exposed albino snails at the end of the treatment. The total frequencies of malformations were analyzed in the offspring of wild-type snails; among the offspring of albino snails, only the heterozygous wild-type embryos were analyzed for malformations. Both agents induced germ cell mutations. The analysis of the offspring of the wild-type snails showed an effect of the exposure up to approximately 5 days after the end of the treatment with cyclophosphamide; the effect of mitomycin C was observed until 45 days after the end of the exposure. There was an increase in the frequencies of malformations in the wild-type offspring of the non-exposed albino snails crossed with the wild-type snails exposed to both agents. The dominant lethal test in B. glabrata proposed in this work is easy to perform, efficient, specific and sensitive in the evaluation of germ cell mutations induced by reference mutagens. The possibility of expanding its use to environmental biomonitoring studies seems very promising and worth trying.

Biomphalaria tenagophila (Orbigny, 1835) (Mollusca): adaptation to desiccation and susceptibility to infection with Schistosoma mansoni Sambon, 1907.

Experiments were carried out to test the susceptibility of Biomphalaria tenagophila to the infection with strain SJ of Schistosoma mansoni in the F1, F2 and non-selected parental generation. The potential adaptation of B. tenagophila to desiccation, in healthy mollusks and those exposed to the larvae of S. mansoni of the F1, F2 and non-selected parental generations was also studied. The presence of mucus and soil, at the shell opening, protected the snails against desiccation, favoring survival. The healthy mollusks performed more attempts against desiccation than those exposed to the larvae of the parasite. The mortality rate, during desiccation, was higher among mollusks that remained buried and with the shell opening unobstructed. During the desiccation period the stage of development of the parasite was influenced by the weight loss and the survival of the snails. The longer the period of desiccation, the greater was the weight loss observed, abbreviating survival. The non-selected parental generation was more sensitive to desiccation than the F1 and F2 generations, both in healthy mollusks and in those exposed to S. mansoni larvae. Healthy mollusks were more resistant to desiccation than those exposed to the larvae of the S. mansoni. Desiccation did not interrupt the development of S. mansoni larvae in mollusks, causing a delay in the cercariae elimination. The susceptibility of B. tenagophila to the SJ strain of S. mansoni, in mollusks maintained in water during the larvae incubation period, was similar in all three generations.

Biomphalaria tenagophila (Orbigny, 1835) (Mollusca): adaptation to desiccation and susceptibility to infection with Schistosoma mansoni Sambon, 1907

Experiments were carried out to test the susceptibility of Biomphalaria tenagophila to the infection with strain SJ of Schistosoma mansoni in the F1, F2 and non-selected parental generation. The potential adaptation of B. tenagophila to desiccation, in healthy mollusks and those exposed to the larvae of S. mansoni of the F1, F2 and non-selected parental generations was also studied. The presence of mucus and soil, at the shell opening, protected the snails against desiccation, favoring survival. The healthy mollusks performed more attempts against desiccation than those exposed to the larvae of the parasite. The mortality rate, during desiccation, was higher among mollusks that remained buried and with the shell opening unobstructed. During the desiccation period the stage of development of the parasite was influenced by the weight loss and the survival of the snails. The longer the period of desiccation, the greater was the weight loss observed, abbreviating survival. The non-selected parental generation was more sensitive to desiccation than the F1 and F2 generations, both in healthy mollusks and in those exposed to S. mansoni larvae. Healthy mollusks were more resistant to desiccation than those exposed to the larvae of the S. mansoni. Desiccation did not interrupt the development of S. mansoni larvae in mollusks, causing a delay in the cercariae elimination. The susceptibility of B. tenagophila to the SJ strain of S. mansoni, in mollusks maintained in water during the larvae incubation period, was similar in all three generations