Deep sequencing of small RNAs reveals the repertoire of miRNAs and piRNAs in Biomphalaria glabrata

BACKGROUND Biomphalaria glabrata snails are widely distributed in schistosomiasis endemic areas like America and Caribe, displaying high susceptibility to infection by Schistosoma mansoni. After the availability of B. glabrata genome and transcriptome data, studies focusing on genetic markers and small non-coding RNAs have become more relevant. The small RNAs have been considered important through their ability to finely regulate the gene expression in several organisms, thus controlling the functions like cell growth, metabolism, and susceptibility/resistance to infection. OBJECTIVE The present study aims on identification and characterisation of the repertoire of small non-coding RNAs in B. glabrata (Bgl-small RNAs). METHODS By using small RNA sequencing, bioinformatics tools and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we identified, characterised, and validated the presence of small RNAs in B. glabrata. FINDINGS 89 mature miRNAs were identified and five of them were classified as Mollusk-specific. When compared to model organisms, sequences of B. glabrata miRNAs showed a high degree of conservation. In addition, several target genes were predicted for all the mature miRNAs identified. Furthermore, piRNAs were identified in the genome of B. glabrata for the first time. The B. glabrata piRNAs showed strong conservation of uridine as first nucleotide at 5' end, besides adenine at 10th position. Our results showed that B. glabrata has diverse repertoire of circulating ncRNAs, several which might be involved in mollusk susceptibility to infection, due to their potential roles in the regulation of S. mansoni development. MAIN CONCLUSIONS Further studies are necessary in order to confirm the role of the Bgl-small RNAs in the parasite/host relationship thus opening new perspectives on interference of small RNAs in the organism development and susceptibility to infection.

Prevalence of infection of biomphalaria glabrata by schistosoma mansoni and the risk of urban schistosomiasis mansoni in Salvador, Bahia, Brazil

Abstract INTRODUCTION: Biomphalaria glabrata is considered to be responsible for the incidence of schistosomiasis in Brazil. Therefore, surveillance of areas where schistosomiasis is prevalent is fundamental for public health planning. This study was aimed to evaluate B. glabrata populations in water bodies of the city of Salvador, determine their distribution, estimate the prevalence of Schistosoma mansoni infections, characterize shed cercariae, and identify transmission foci. METHODS: Malacological surveys were carried out in 17 water collections from Salvador. Snail species were identified based on shell and mantle characteristics. Snails were evaluated for S. mansoni infection by exposure to light and via real time polymerase chain reaction (qPCR) using S. mansoni-18S rRNA subunit specific primers. RESULTS: 1,403 B. glabrata were collected. Classical cercarial shedding indicated that 5 snails (0.4%) were positive for S. mansoni. A higher prevalence of infections was found in Horta de Saramandaia (5.5%) and Lagoa do IAT (1.9%). Non-Schistosoma larvae, such as Xiphidiocercaria, Strigeidae, Spirorchiidae and Clinostomidae, were observed in 3.2% of the snails. S. mansoni DNA was detected in 6.2% snails via qPCR. CONCLUSIONS: B. glabrata is widely distributed in Salvador, as indicated by 7 water collections associated with a risk of schistosomiasis transmission. To our knowledge, this is the first study to identify B. glabrata eliminating cercariae of Clinostomidae, Strigeidae, and Spirorchiidae in Salvador. We propose that qPCR may be employed in combination with classical cercarial shedding. Estimating S. mansoni prevalence in snails by only considering the results of light exposure method classical into account may underestimate the problem.

Genome-wide identification, characterisation and expression profiling of the ubiquitin-proteasome genes in Biomphalaria glabrata

BACKGROUND Biomphalaria glabrata is the major species used for the study of schistosomiasis-related parasite-host relationships, and understanding its gene regulation may aid in this endeavor. The ubiquitin-proteasome system (UPS) performs post-translational regulation in order to maintain cellular protein homeostasis and is related to several mechanisms, including immune responses. OBJECTIVE The aims of this work were to identify and characterise the putative genes and proteins involved in UPS using bioinformatic tools and also their expression on different tissues of B. glabrata. METHODS The putative genes and proteins of UPS in B. glabrata were predicted using BLASTp and as queries reference proteins from model organism. We characterised these putative proteins using PFAM and CDD software describing the conserved domains and active sites. The phylogenetic analysis was performed using ClustalX2 and MEGA5.2. Expression evaluation was performed from 12 snail tissues using RPKM. FINDINGS 119 sequences involved in the UPS in B. glabrata were identified, which 86 have been related to the ubiquitination pathway and 33 to proteasome. In addition, the conserved domains found were associated with the ubiquitin family, UQ_con, HECT, U-box and proteasome. The main active sites were lysine and cysteine residues. Lysines are responsible and the starting point for the formation of polyubiquitin chains, while the cysteine residues of the enzymes are responsible for binding to ubiquitin. The phylogenetic analysis showed an organised distribution between the organisms and the clades of the sequences, corresponding to the tree of life of the animals, for all groups of sequences analysed. The ubiquitin sequence was the only one with a high expression profile found in all libraries, inferring its wide range of performance. MAIN CONCLUSIONS Our results show the presence, conservation and expression profile of the UPS in this mollusk, providing a basis and new knowledge for other studies involving this system. Due to the importance of the UPS and B. glabrata, this work may influence the search for new methodologies for the control of schistosomiasis.

Development of a natural molluscicide prototype kit (MoluSchall) for the control of schistosomiasis mansoni transmission

Abstract INTRODUCTION In Brazil, Biomphalaria glabrata, B. tenagophila, and B. straminea are intermediate hosts of Schistosoma mansoni, the etiological agent of schistosomiasis mansoni. Molluscicide use is recommended by the WHO for controlling the transmission of this parasite. Euphorbia milii latex has shown promising results as an alternative molluscicide. Thus, a natural molluscicide prototype kit based on freeze-dried E. milii latex was developed and evaluated against Biomphalaria spp. METHODS E. milii latex was collected, processed, and lyophilized. Two diluents were defined for freeze-dried latex rehydration, and a prototype kit, called MoluSchall, was produced. A stability test was conducted using prototype kits stored at different temperatures, and a toxicity assay was performed using Danio rerio. Additionally, MoluSchall was tested against B. glabrata under semi-natural conditions according to defined conditions in the laboratory. RESULTS MoluSchall was lethal to three Brazilian snail species while exhibiting low toxicity to D. rerio. Regardless of storage temperature, MoluSchall was stable for 24 months and was effective against B. glabrata under semi-natural conditions, with the same LD100 as observed under laboratory conditions. CONCLUSIONS MoluSchall is a natural, effective, and inexpensive molluscicide with lower environmental toxicity than existing molluscicides. Its production offers a possible alternative strategy for controlling S. mansoni transmission.

Accumulation of oocysts of Cryptosporidium parvum in Biomphalaria glabrata (Pulmonata: Planorbidae) under experimental conditions

Abstract INTRODUCTION: Cryptosporidium oocysts are easily transported to various aquatic environments. The objective of this study was to evaluate B. glabrata mollusks exposed to food containing C. parvum oocysts. METHODS: Six experimental groups were used with B. glabrata either exposed or not to C. parvum oocysts. Microscopic and molecular diagnostics were conducted in water samples and tissues of B. glabrata. RESULTS: By light microscopy, C. parvum oocysts were identified in the water of the exposed groups. C. parvum DNA was not detected in water but was detected in tissue samples. CONCLUSIONS: Further studies should be conducted under natural conditions.

Histopathological and ultrastructural studies on biomphalaria alexandrina snails infected with schistosoma mansoni miracidia and treated with plant extracts

Background: Biomphalaria alexandrina snails are the intermediate host of Schistosoma mansoni in Egypt

Aim of the work: this study aimed to evaluate the molluscicidal activity of the methanol extract of the plants Anagallis arvensis and Viburnum tinus against B. alexandrina [Normal and S. mansoni infected]

Results: the present results proved high activity for both plant extracts [LC[50] and LC[90] which reached 45and 60 ppm and 38 and 59 ppm for A. arvensis and V. tinus, respectively]. The effect of sub-lethal concentration, 1/2 LC[5], of the two plant extracts [26 and 11 ppm, respectively] affected B. alexandrina survival rate to be in the following order, control > V. tinus treated > A. arvensis treated > infected > infected-A. arvensis treated > infected- V. tinus treated. On the other hand, exposure to those sub-lethal doses caused considerable reduction in the infection percentages. In addition, the histopathological effects of the examined sub-lethal concentrations on hepatopancreatic tubules of the treated snails showed cells vacuolation, presence of hyaline substances filled the lumens of the tubules and necrotic focal areas in case of A. arvnsis and vacuolar degeneration with the necrotic changes in case of V. tinus. While, alterations in the hermaphrodite glands of the treated snails included: degeneration and necrotic changes in the acini. The severity of lesions was progressed with infection as a result of invading of snail tissue by developmental stages of the S. mansoni cercariae. The ultrastructural micrographs were used to explain and confirm the recorded histopathological alterations in the hermaphrodite glands of the infected-treated snails. In comparison with the control and infected snail groups, infected-treated snails showed degeneration with severe deformation and destruction in their reproductive units, degeneration in developmental stages tissues of S. mansoni cercariae and accumulation of the toxic agents

Conclusion: the two examined plants, A. arvensis and V. tinus plant extracts showed high activity against B. alexandrina and provide a considerable scope in exploiting local indigenous resources for snail's molluscicidal agents. The sub-letal concentrations, 1/2 LC[5], of the two plant extracts caused a considerable reduction in survival rate and infection rate among S. mansoni infected snails. Histopathological changes in the digestive glands showed cells vacuolation, hyaline substance filled lumens of the tubules and necrotic focal areas in the digestive glands. Histopathological effects explained and confirmed by TEM images showed degeneration with severe deformation and destruction in the reproductive units

Assessment of the impact of changes in temperature in Biomphalaria glabrata (Say, 1818) melanic and albino variants infected with Schistosoma mansoni (Sambon, 1907) / Avaliação de impacto de alterações na temperatura em Biomphalaria glabrata variantes melânica e albina infectadas com Schistosoma mansoni

Abstract Fluctuations in population density of planorbid hosts of S. mansoni are influenced by climatic factors. The knowledge about interference from changes in water temperature in these populations is an important aspect of the epidemiology of schistosomiasis. In this experiment, it is explored the influence of different temperatures on the development of Schistosoma mansoni in Biomphalaria glabrata melanic and albino variants. The results indicated an intrinsic relationship between temperature and development of the parasite in the intramollusc phase, independent of the pigmentation of the mantle of the molluscs. The higher the temperature, the shorter the period necessary for the development of the parasite was while the higher the mortality of infected mollusks. It is concluded that, in the presence of climate change, the increasement of temperature in cold and flooded regions may encourage the establishment of new foci of transmission of schistosomiasis by changing the geographic extent and extending the epidemiological transmission potential. In warm climates, higher temperatures, however, could compromise the transmission of the disease because of biological stress suffered by parasite and host. Under these conditions, it can result in the death of the parasite or a change in their ability to infect new host species of molluscs in new areas. Mantle pigmentation patterns in molluscs have not shown significant interference in the development of the parasite.

Resumo Flutuações na densidade populacional de planorbídeos hospedeiros do S. mansoni são influenciadas por fatores climáticos. O conhecimento sobre a interferência de alterações na temperatura da água nestas populações é um aspecto importante da epidemiologia da esquistossomose. Neste experimento avaliou-se a influência de diferentes temperaturas no desenvolvimento de Schistosoma mansoni em Biomphalaria glabrata variantes melânica e albina. Os resultados indicaram uma relação intrínseca entre temperatura e desenvolvimento do parasito na fase intramolusco, independente da pigmentação do manto dos moluscos. Quanto mais elevada a temperatura, menor o período necessário para desenvolvimento do parasito e maior mortalidade dos moluscos infectados. Conclui-se que, na presença de alterações climáticas, o aumento da temperatura em regiões frias e alagadas poderá favorecer o estabelecimento de novos focos de transmissão da esquistossomose alterando a extensão geográfica e ampliando o potencial epidemiológico da transmissão. Em regiões de clima quente, o aumento da temperatura, por sua vez, poderá comprometer a transmissão da doença em virtude do estresse biológico sofrido por parasito e hospedeiro. Nestas condições, poderá ocorrer a morte do parasito ou uma alteração na sua habilidade de infectar novas espécies de moluscos hospedeiros em novas áreas. Padrões de pigmentação do manto nos moluscos não demonstraram interferência significativa no desenvolvimento do parasita.

Host-parasite relationship between Biomphalaria amazonica (Paraense, 1966) and Schistosoma mansoni (Sambon, 1907) / Relação parasito-hospedeiro entre Biomphalaria amazonica e Schistosoma mansoni

Abstract Biomphalaria amazonica is a planorbid species considered a potential host of Schistosoma mansoni. It is widely distributed in the Neotropical zone, particularly in the North and Centre-West of Brazil and in the North of Bolivia. The aim of the present study was to determine the host-parasite relationship between B. amazonica and S. mansoni (BH and SJ strains). Specimens of B. amazonica and their snail-conditioned water were examined in terms of their ability to attract miracidia. The infectivity of the mollusks was determined by exposing them to 20 miracidia of both strains. Sporocyst development and amebocyte reactions were studied after each mollusk specimen was exposed to 100 miracidia. Although no cercariae were eliminated, specimens of B. amazonica proved capable of attracting 77% of the miracidia they were exposed to. Viable sporocysts with no amebocyte reaction were found 96 hours after the exposure to miracidia. These results indicate the susceptibility of B. amazonica to the BH and SJ strains of S. mansoni, and therefore demonstrate the importance of this planorbid species as a potential vector of the trematode in the areas where it occurs.

Resumo Biomphalaria amazonica é uma espécie de planorbídeo considerada vetora potencial do Schistosoma mansoni. É amplamente distribuída na zona neotropical, especialmente no Norte e Centro-Oeste do Brasil e Norte da Bolívia. O presente trabalho teve por objetivo estudar a relação parasito-hospedeiro entre B. amazonica e S. mansoni (linhagens BH e SJ). Espécimes de B. amazonica e sua água de condicionamento foram examinados em relação à sua capacidade de atração miraxonal. A infectividade dos moluscos foi testada expondo-os a 20 miracídios de ambas as linhagens. A viabilidade dos esporocistos e o desenvolvimento de reações amebocitárias foram estudados após cada molusco ser exposto a 100 miracídios. Apesar de não eliminarem cercárias, B. amazonica provou ser capaz de atrair 77% dos miracídios a que foram expostos. Esporocistos viáveis sem reação amebócitaria foram encontrados 96 horas após a exposição aos miracídios. Esses resultados indicam a suscetibilidade de B. amazonica às linhagens BH e SJ de S. mansoni e, portanto, demonstram a importância desta espécie de planorbídeo como um vetor potencial do trematodeo na área onde ele ocorre.

The Geographic Information System applied to study schistosomiasis in Pernambuco / Os Sistemas de Informação Geográfica em estudo sobre a esquistossomose em Pernambuco

ABSTRACT OBJECTIVE Diagnose risk environments for schistosomiasis in coastal localities of Pernambuco using geoprocessing techniques. METHODS A coproscopic and malacological survey were carried out in the Forte Orange and Serrambi areas. Environmental variables (temperature, salinity, pH, total dissolved solids and water fecal coliform dosage) were collected from Biomphalaria breeding sites or foci. The spatial analysis was performed using ArcGis 10.1 software, applying the kernel estimator, elevation map, and distance map. RESULTS In Forte Orange, 4.3% of the population had S. mansoni and were found two B. glabrata and 26 B. straminea breeding sites. The breeding sites had temperatures of 25ºC to 41ºC, pH of 6.9 to 11.1, total dissolved solids between 148 and 661, and salinity of 1,000 d. In Serrambi, 4.4% of the population had S. mansoni and were found seven B. straminea and seven B. glabrata breeding sites. Breeding sites had temperatures of 24ºC to 36ºC, pH of 7.1 to 9.8, total dissolved solids between 116 and 855, and salinity of 1,000 d. The kernel estimator shows the clusters of positive patients and foci of Biomphalaria, and the digital elevation map indicates areas of rainwater concentration. The distance map shows the proximity of the snail foci with schools and health facilities. CONCLUSIONS Geoprocessing techniques prove to be a competent tool for locating and scaling the risk areas for schistosomiasis, and can subsidize the health services control actions.

RESUMO OBJETIVO Diagnosticar ambientes de risco para esquistossomose em localidades litorâneas de Pernambuco utilizando técnicas de geoprocessamento. MÉTODOS Foi realizado inquérito coproscópico e malacológico nas localidades Forte Orange e Serrambi. Foram coletadas variáveis ambientais (temperatura, salinidade, pH, sólidos totais dissolvidos e dosagem de coliformes fecais da água) relacionadas aos criadouros ou focos de Biomphalaria. A análise espacial foi realizada no software ArcGis 10.1, aplicando-se o estimador kernel, mapa de elevação e mapa de distância. RESULTADOS No Forte Orange, 4,3% da população estava com S. mansoni e existem dois criadouros de B. glabrata e 26 de B. straminea. Os criadouros apresentaram temperaturas de 25ºC a 41ºC, pH de 6,9 a 11,1, sólidos totais dissolvidos de 148 a 661 e salinidade de 1.000 d. Em Serrambi, 4,4% da população estava com S. mansoni e há sete criadouros de B. straminea e sete de B. glabrata. Os criadouros apresentaram temperaturas de 24ºC a 36ºC, pH de 7,1 a 9,8, sólidos totais dissolvidos de 116 a 855 e salinidade de 1.000 d. O estimador de kernel mostra os aglomerados de pacientes positivos e de focos de Biomphalaria, e o mapa de elevação digital indica áreas de concentração de águas pluviais. O mapa de distância mostra a proximidade dos focos dos caramujos em relação às escolas e unidades de saúde. CONCLUSÕES As técnicas de geoprocessamento se mostraram como importantes ferramentas para a localização e dimensionamento das áreas de risco para esquistossomose, podendo subsidiar as ações de controle por parte dos serviços de saúde.

Transmissão urbana da esquistossomose: novo cenário epidemiológico na Zona da Mata de Pernambuco / Urban transmission of schistosomiasis: new epidemiological situation in the forest area of Pernambuco

RESUMO: Introdução: A esquistossomose é considerada uma endemia em Vitória de Santo Antão, Pernambuco, o qual apresenta há décadas altas incidências e prevalências para essa doença. Nesse município ocorre a transmissão clássica da doença por meio do contato da população de áreas rurais com águas contaminadas durante o desenvolvimento de suas atividades de vida diárias. Recentemente surgiram indícios da presença do caramujo vetor na área urbana da cidade, o que pode configurar um novo modelo de transmissão para esquistossomose nesse município. Objetivo: Identificar novo cenário epidemiológico para ocorrência da transmissão urbana da esquistossomose em Vitória de Santo Antão, Pernambuco. Métodos: Foi conduzido um inquérito malacológico, investigando-se todas as coleções hídricas do perímetro urbano quanto à presença do caramujo vetor da esquistossomose (Biomphalaria spp.). Os caramujos coletados foram examinados para identificação taxonômica e de infecção pelo Schistosoma mansoni. Todos os criadouros (CRs) foram georreferenciados para construção de mapas de risco por meio dos software TrackMaker-Pro e ArcGIS. Resultados: Foram identificados 22 CRs da espécie Biomphalaria straminea, nos quais foram coletados 1.704 caramujos. Desses CRs, um foi identificado como foco de transmissão da doença e sete como focos potenciais para transmissão. Os mapas construídos identificaram duas áreas de risco para transmissão urbana da esquistossomose, bem como áreas de expansão dos CRs, configurando um aumento no risco de transmissão para a população. Conclusão: Os resultados constatam a existência de um novo cenário epidemiológico, no qual a possibilidade de transmissão urbana dessa doença foi confirmada.

ABSTRACT: Introduction: Schistosomiasis is considered an endemic disease in Vitória de Santo Antão, Pernambuco, a district which has presented both high incidence and prevalence of it for decades. Poor environmental conditions lead to contamination of water sources in rural areas, which are used by the population during daily activities, resulting in typical transmission. Recently, there has been evidence of vector snails in urban areas, which could set a new model for schistosomiasis transmission in this district. Objective: To identify the new epidemiological situation for the urban transmission of schistosomiasis in Vitória de Santo Antão, Pernambuco. Methods: A malacological survey was conducted in all water sources in the city limits to investigate schistosomiasis vector snails (Biomphalaria spp.). The collected snails were examined for taxonomic identification and Schistosoma mansoni infection. All breeding sites were georeferenced to build risk maps through the TrackMaker PRO program and ArcGIS software. Results: We identified 22 Biomphalaria straminea breeding sites and collected 1,704 snails. One of these breeding sites was identified as a source of transmission and seven as potential sources of transmission. The designed maps identified two risk areas of urban transmission of schistosomiasis and expansion areas for breeding sites, establishing an increased risk of transmission to the population. Conclusion: This study verified the existence of a new epidemiological situation in which the possibility of the urban transmission of the disease was confirmed.

Total protein composition of young and adult Biomphalaria alexandrina snails with different compatibilities to Schistosoma mansoni infection / Composición total de proteína en caracoles Biomphalaria alexandrina juveniles y adultos con distintas compatibilidades a la infección por Schistosoma mansoni

Abstract:Schistosomiasis remains a disease of major global public health concern since it is a chronic and debilitating illness. The widely distributed Schistosoma mansoni that causes intestinal schistosomiasis represents a great threat. Its world-wide distribution is permitted by the broad geographic range of the susceptible species of its intermediate host, Biomphalaria, which serves as an obligatory host for the larval stage, at which humans get infected. The objectives were to identify the proteins responsible for the snails' compatibility outcome through differentiation between the total proteins among Biomphalaria alexandrina snails at different ages. The work was conducted on snails that differ in age and genetic backgrounds. Four subgroups (F1) from the progeny of self-reproduced susceptible and resistant snails (F0) were studied. Infection rates of these subgroups (young susceptible, adult susceptible, young resistant and adult resistant) were 90 %, 75 %, 40 % and 0 %, respectively. Using Sodium Dodecyl Sulphate Polyacrylamide Gel electrophoresis (SDS-PAGE), differences in protein expression were evaluated between adult and young snails of different subgroups. Dice similarity coefficient was calculated to determine the percentage of band sharing among the experimental subgroups. The results showed that the combination of similarities between age and compatibility status of the snails, lead to the highest similarity coefficient, followed by the combination of similarities of both genetic origin and age, even though they differ in the compatibility status. On the other hand, the differences in the genetic background, age and compatibility status, lead to the least similarity index. It was also found that the genetic background in young snails plays a major role in the determination of their compatibility, while the internal defense system has the upper hand in determining the level of adult compatibility. In conclusion, the findings of the present work highlight the great impact of the snail age in concomitance with the genetics and the internal defense in the determination of B. alexandrina/S.mansoni compatibility. Future works are recommended, as further characterization of the shared protein bands among the studied subgroups is needed to clarify their role in host-parasite relationship. Rev. Biol. Trop. 64 (4): 1747-1757. Epub 2016 December 01.

Resumen:La esquistosomiasis es una enfermedad crónica y debilitante que constituye una problemática de salud pública a nivel mundial. Debido a que Schistosoma mansoni está ampliamente distribuida y a que es el causante de la esquistosomiasis intestinal representa una gran amenaza. Biomphalaria es el hospedero intermedio y obligatorio para el estado larval, presenta una amplia distribución geográfica e infecta al ser humano. El objetivo fue identificar las proteínas responsables del efecto de compatibilidad en caracoles Biomphalaria alexandrina de distintos estadios a través de la diferenciación del total de proteínas en ellos. La investigación se llevó a cabo con caracoles de diferentes edades y antecedentes genéticos. Se estudiaron cuatro subgrupos (F1) de la progenie de caracoles susceptibles y resistentes reproducidos asexualmente (F0). Las tasas de infección de estos subgrupos (juvenil susceptible, adulto susceptible, juvenil resistente, adulto resistente) fueron 90 %, 75 %, 40 % y 0 %, respectivamente. Con la electroforesis en gel de poliacrilamida en presencia de dodecilsulfato sódico (SDS-PAGE) se evaluaron las diferencias en la expresión proteica entre los caracoles juveniles y adultos de los distintos subgrupos. Se calculó el coeficiente de similitud de Dice para determinar el porcentaje de bandas compartidas entre los subgrupos experimentales. Los resultados mostraron que la combinación de similitudes entre la edad y el estado de compatibilidad de los caracoles genera el mayor coeficiente de similitud seguido por el de la combinación de similitudes tanto de la edad como del origen genético aunque varían en el estado de compatibilidad. Por otra parte, las diferencias en los antecedentes genéticos, la edad y el estado de compatibilidad generan el índice de similitud más bajo. También se encontró que el antecedente genético en caracoles juveniles es importante en la determinación de su compatibilidad, mientras que el sistema de defensa interno es el que determina el nivel de compatibilidad en adultos. En conclusión, los resultados de este trabajo resaltan la importancia de la edad del caracol en conjunto con la genética y la defensa interna para determinar la compatibilidad de B. alexandrina/S.mansoni. Se recomienda realizar futuros trabajos así como una mayor caracterización de las bandas proteicas compartidas entre los subgrupos estudiados para esclarecer su papel en la relación hospedero-parásito.

Irrigation canals in Melo creek basin (Rio Espera and Capela Nova municipalities, Minas Gerais, Brazil): habitats to Biomphalaria (Gastropoda: Planorbidae) and potential spread of schistosomiasis / Canais de irrigação na bacia do ribeirão do Melo (Municípios de Rio Espera e Capela Nova, Minas Gerais, Brasil): habitats para a proliferação de Biomphalaria (Gastropoda: Planorbidae) e potencial disseminação da esquistossomose

Abstract This study analyzed the presence of Biomphalaria in Melo creek basin, Minas Gerais state, and its relationship to irrigation canals. Seventeen of these canals were used to determine a limnological, morphological and hydrological characterization during an annual seasonal cycle. Biomphalaria samples were sent to René Rachou Research Center/FIOCRUZ for identification and parasitological examination. Six canals were identified as breeding areas for mollusks and in one of them it was registered the coexistence of B. tenagophila (first report to this basin) and B. glabrata species. Results indicated that the low flow rate and speed of water flow were the main characteristics that contributed to this specific growth of the mollusks in the area. These hydraulic characteristics were created due to anthropogenic action through the canalization of lotic areas in Melo creek, which allowed ideal ecological conditions to Biomphalaria outbreak. The results emphasize the need of adequate handling and constant monitoring of the hydrographic basin, subject to inadequate phytosanitary conditions, aiming to prevent the occurrence and propagation of schistosomiasis.

Resumo Neste estudo avaliou-se a presença de espécies de Biomphalaria na bacia do Ribeirão do Melo, municípios de Rio Espera e Capela Nova, sudeste do estado de Minas Gerais, e sua relação com os canais de irrigação presentes na região. Em 17 desses canais foi realizada uma caracterização limnológica, morfológica e hidrológica durante um ciclo sazonal anual. Espécimes de Biomphalaria foram coletados e encaminhados ao Centro de Pesquisas René Rachou/FIOCRUZ (Belo Horizonte, MG) para identificação e exame parasitológico. Dos 17 canais estudados, foram identificados seis como criadouros do caramujo, sendo que em um dos canais coexistiam as espécies B. tenagophila (primeiro registro para a bacia) e B. glabrata. Os resultados indicaram que a baixa vazão e a velocidade do fluxo foram os fatores que contribuíram para a ocorrência pontual dos caramujos na bacia. Estas características hidrológicas foram modificadas por ação antropogênica, através da canalização de trechos lóticos do ribeirão do Melo. Os resultados destacam a necessidade do manejo adequado e monitoramento constante da bacia hidrográfica, sujeita a condições sanitárias inadequadas, como forma de prevenção da ocorrência e propagação da esquistossomose.

Caracterização hemocitária de uma linhagem resistente de Biomphalaria straminea (Dunker, 1848) exposta a schistosoma mansoni sambon, 1907 / Haemocyte characterization of a resistant lineage of biomphalaria straminea (Dunker, 1848) exposed to schistosoma mansoni sambon, 1907

A esquistossomose mansônica ainda constitui um grave problema de saúde pública no Brasil e, portanto, o conhecimento dos diversos aspectos da interação Schistosoma mansoni-Biomphalaria, são pertinentes e relevantes como subsídios à medidas de controle e prevenção desta parasitose. Estudos in vitro têm mostrado que a suscetibilidade dos hospedeiros intermediários de S. mansoni, Biomphalaria straminea e Biomphalaria glabrata está relacionado à presença de lectinas. Importante transmissor da Esquistossomose no Brasil, B. straminea apresenta baixos índices de infecção em condições experimentais. Apesar disto, existem poucos estudos a respeito das características morfológicas e do comportamento in vitro dos hemócitos deste molusco frente à infecção por S. mansoni. Com o objetivo de caracterizar a resposta hemocitária de B. straminea (cepa Souza-PB) expostas a miracídios de S. mansoni, os moluscos foram expostos em massa e expostos individualmente a cinco miracídios. Exemplares de B. glabrata foram utilizadas como controle positivo. A hemolinfa foi coletada, corada em Azul de Tripan e os hemócitos foram contados em câmara de Neubauer. Alguns hemócitos foram colocados em placa de cultura para observação da interação das células com os parasitos e outros foram submetidos à marcação por lectinas fluoresceinadas de Griffonia simplicifolia e Lens culinaris conjugadas a FITC. As imagens foram obtidas e gravadas através de microscópio AxioObserver e câmera McR5 Zeiss. Os moluscos expostos em massa foram sacrificados logo após a penetração inicial do parasito (0 minuto), 24, 48, 72 horas e 30 dias após exposição (dpe)...

Schistosomiasis still remain as an important parasitic disease under the public health point of view.Thus, studies that involve the interaction between Schistosoma mansoni-Biomphalaria are pertinentand useful to the disease prevention and control. In vitro studies have shown that the susceptibility ofBiomphalaria straminea and Biomphalaria glabrata, intermediate hosts of Schistosoma mansoni, isrelated to the presence of lectins. As an important carrier of schistosomiasis in Brazil, B. stramineapresents low infection rates under laboratory conditions. Despite of that, there are few studiesregarding the morphological features and in vitro behavior of this mollusc's haemocytes challenged byS. mansoni miracidia. For this purpose, we analyzed the haemocyte response of B. straminea (Sousa– PB strain) challenged by the parasite, so the molluscs were put under mass exposure and individualexposure to five miracidia. B. glabrata molluscs were used as positive controls. The molluscs had theirhaemolymph drawn, the haemocytes were stained in Trypan Blue and counted in Neubauer chamber.Some haemocytes were put into cell culture plates in order to observe the interaction between thecells and the parasites and other cells were stained by FITC conjugated lectins from Griffoniasimplicifolia and Lens culinaris. The images and films were made at the microscope AxioObserver withMcR5 camera from Zeiss. The mass exposed molluscs were killed at 0 minutes (first penetration of themiracidia), 24, 48, 72 hours and 30 days after exposure (dae)...

Perfil proteico da hemolinfa de Biomphalaria glabrata e linhagens de B. straminea, frente à exposição ao Schistosoma mansoni / Protein profile of hemolymph of Biomphalaria glabrata and strains of B. straminea, against the exposure to the Schistosoma mansoni

O gênero Biomphalaria possui espécies de grande relevância médica uma vez que atuam como hospedeiros intermediários naturais do parasita Schistosoma mansoni, causador da esquistossomose. Dentro desse gênero de moluscos, três espécies são tidas como hospedeiros naturais do parasita, Biomphalaria glabrata, B. straminea e B. tenagophila. O perfil de suscetibilidade à infecção por S. mansoni dentro do gênero é muito variado e muitas pesquisas buscam elucidar a dinâmica da relação parasita-hospedeiro intermediário na finalidade de criar novas medidas de controle da doença. Por isso, esse estudo tem como objetivo determinar o perfil bidimensional de proteínas que podem estar envolvidas na resposta imune contra o S. mansoni comparando duas espécies com diferentes perfis de susceptibilidade B. glabrata, B. straminea além de uma refratária ao S. mansoni, a B. straminea R3. Para isso, os caramujos de cada espécie foram divididos em dois grupos: Infectado, expostos aos miracídios do S. mansoni; e Controle, submetidos ao estresse do processo de infecção livre de miracídios. A hemolinfa foi retirada 24 horas após a exposição. Foi feito o extrato proteico total e determinada a concentração das proteínas totais para cada grupo investigado. As proteínas foram separadas por eletroforese bidimensional onde foi obtido o ponto isoelétrico e peso molecular de todos os spots nos géis...

The Biomphalaria has species of great medical relevance since that act as natural intermediate hosts of the parasite Schistosoma mansoni, which causes schistosomiasis. Within this kind of mollusks, three species are considered natural hosts of the parasite, Biomphalaria glabrata, B. stramineaand B. tenagophila. The profile of usceptibility to S. mansoni infection within the genre is very varied and many studies seek to elucidate the dynamics of host-parasite relationship intermediary in order to create new disease control easures. Therefore, this study aims to determine the two-dimensional profile of proteins that may be involved in the immune response against S. mansonicomparing two species with different susceptibility profiles B. glabrata, B. straminea and a refractory to S. mansoni, B. straminea R3. For that, the snails of each species were divided into two groups: Infected exposed to iracidia of S. mansoni; and control, subjected to stress the miracidia free infection process. The hemolymph was removed 24 hours after exposure. It was made the total protein extract and determined the concentration of total protein for each group investigated. Proteins were separated by two-dimensional electrophoresis was obtained where the isoelectric point and molecular weight of all the spots in the gels...

Impact of the age of Biomphalaria alexandrina snails on Schistosoma mansoni transmission: modulation of the genetic outcome and the internal defence system of the snail

Of the approximately 34 identified Biomphalariaspecies,Biomphalaria alexandrinarepresents the intermediate host of Schistosoma mansoniin Egypt. Using parasitological and SOD1 enzyme assay, this study aimed to elucidate the impact of the age of B. alexandrinasnails on their genetic variability and internal defence against S. mansoniinfection. Susceptible and resistant snails were reared individually for self-reproduction; four subgroups of their progeny were used in experiment. The young susceptible subgroup showed the highest infection rate, the shortest pre-patent period, the highest total cercarial production, the highest mortality rate and the lowest SOD1 activity. Among the young and adult susceptible subgroups, 8% and 26% were found to be resistant, indicating the inheritance of resistance alleles from parents. The adult resistant subgroup, however, contained only resistant snails and showed the highest enzyme activity. The complex interaction between snail age, genetic background and internal defence resulted in great variability in compatibility patterns, with the highest significant difference between young susceptible and adult resistant snails. The results demonstrate that resistance alleles function to a greater degree in adults, with higher SOD1 activity and provide potential implications for Biomphalariacontrol. The identification of the most susceptible snail age enables determination of the best timing for applying molluscicides. Moreover, adult resistant snails could be beneficial in biological snail control.

A comparative cross-sectional study on the prevalence and morbidity of schistosomiasis in a community in northeastern Brazil (1979-2010)

A cross-sectional study on the prevalence and morbidity of schistosomiasis was conducted in the main settlement of the municipality of Alhandra, in the southern coastal region of the state of Paraíba, in 2010. The results of this study were compared with the results of a previous study conducted in the same area in 1979. The systematic sampling per family conglomerate included approximately 10% of the resident population in the urban area of Alhandra. Faecal examinations were performed using the Kato-Katz method. The clinical forms of the disease were classified in accordance with FS Barbosa as Type I - intestinal form, Type II - hepatointestinal form and Type III - hepatosplenic form. The prevalence of the infection in 2010 was 10.05%, whereas in 1979 it was 46.6% among untreated patients. The percentages of the three clinical forms in 2010 were as follows: 95.3% Type I, 4.6% Type II and 0% Type III; in 1979, the percentages were 94.4%, 3% and 2.6% for Types I, II and III, respectively. In 1979, 6.07% of the Biomphalaria glabrata specimens (the intermediate host in this area) excreted cercariae, where in 2010 only 1.27% of the specimens caught excreted the parasite.

Avaliação bioquímica, hematológica e histopatológica da infecção experimental por Angiostrongylus cantonensis no hospedeiro definitivo Rattus norvegicus (Wistar) e estudo da interação Angiostrongylus cantonensis / Echinostoma paraensei no hospedeiro intermediário Biomphalaria glabrata / Biochemical, hematological and histopathological evaluation of experimental infection with A. cantonensis in Rattus norvegicus definitive host (Wistar) and study the interaction A. cantonensis / Echinostoma paraensei the intermediate host Biomphalari

A meningite eosinofílica é uma doença caracterizada pelo aumento de eosinófilos no líquidocefalorraquidiano (LCR), cuja causa mais comum é a invasão do sistema nervoso central porhelmintos. Entre os helmintos, destaca-se o Angiostrongylus cantonensis. O roedor Rattusnorvegicus é o hospedeiro natural definitivo e o homem atua como hospedeiro acidental e que podese infectar através da ingestão de moluscos crus ou mal cozidos ou alimentos contaminados com alarva L3. Recentemente, no Brasil houve quatro casos de meningite eosinofílica, devido à ingestãoou manipulação de Achatina fulica infectada. Foi realizado uma avaliação das alteraçõesmetabólicas, fisiológicas e histopatológicas em R. norvegicus experimentalmente infectados com100 larvas L3 de A. cantonensis e o estudo do efeito da infecção concomitante por Echinostomaparaensei e A. cantonensis sobre a atividade das aminotransferases ALT e AST, na concentração deproteínas totais, ácido úrico, ureia e análise histopatológica para compreender o efeito da infecçãoconcomitante em Biomphalaria glabrata. Os resultados obtidos demonstraram que a infecçãocausada por A. cantonensis em R. norvegicus promoveu alterações metabólicas hepáticas ehistopatológicas, caracterizadas pelo aumento da atividade das enzimas ALT, AST, GGT e ALKP,alteração no metabolismo glicídico, presença de infiltrado inflamatório, alterações hematológicassignificativas, além disso promoveu alterações histopatológicas no parênquima pulmonardemonstraram nos roedores a ocorrência de áreas de necrose e extensa fibrose, podendo estardiretamente relacionada com o desenvolvimento de hipóxia celular...

Eosinophilic meningitis is a disease characterized by increased eosinophils in the cerebrospinalfluid (CSF), which is the most commonly caused by invasion of the central nervous system byhelminths, as occurs in Angiostrongylus cantonensis infections. The rodent Rattus norvegicus is thedefinitive natural host and humans act as accidental hosts and can become infected by eating raw orundercooked snails or food contaminated with infective L3 larvae. Recently in Brazil there havebeen four cases of eosinophilic meningitis due to ingestion of infected Achatina fulica. Anevaluation of metabolic, physiological and pathological changes in R. norvegicus experimentallyinfected with 100 L3 larvae of A. cantonensis and study the effect of co-infection with Echinostomaparaensei and A. cantonensis on the activity of aminotransferases ALT and AST was performed inthe concentration total protein, uric acid, urea and histopathologic analysis to understand the effectof co-infection in Biomphalaria glabrata. The results showed that the infection caused by A.cantonensis in R. norvegicus promoted histopathological changes in liver metabolism ischaracterized by increased activity of ALT, AST, GGT and ALKP enzymes in glucose metabolism,inflammatory infiltrate, hematological changes significant in addition in addition histopathologicalalterations in lung parenchyma in rodents have shown the occurrence of extensive areas of necrosisand fibrosis, which can be directly related to the development of cellular hypoxia. The infectedsnails showed altered levels of aminotransferases and increased excretion of nitrogen products,histopathological findings showed a change in the distribution of A. cantonensis in the presence ofE. paraensei, indicating that the presence of this trematode may interfere with the dynamics of A.cantonensis...

Biology of Kalipharynx sp. (Trematoda: Digenea) metacercariae in Biomphalaria (Gasteropoda: Planorbidae) from Northeastern Argentina / Biología de metacercarias Kalipharynx sp. (Trematoda: Digenea) en Biomphalaria (Gasteropoda: Planorbidae) del noreste argentino

In Argentina, no ecological studies have been reported on the infection parameters of Kalipharynx sp. metacercariae in planorbid snails. To this end, the aims of this study were: (i) to provide information on the population biology of Kalipharynx sp. metacercariae in the planorbid snails Biomphalaria tenagophila and B. occidentalis through the study of prevalence and intensity of larval infection during a seasonal cycle; (ii) to evaluate the effects of host shell size on prevalence and infection intensity, (iii) to evaluate the effect of infection intensity on cyst size. Samples were taken between June 2010 and April 2011 (encompassing all seasons) from a subtropical permanent pond in Corrientes City, Corrientes, Argentina. A total of 362 metacercariae (n=262 and n=100; from B. tenagophila and B. occidentalis respectively) were collected from 616 snails (n=466 and n=150 from B. tenagophila and B. occidentalis respectively). The metacercarial cysts were found in the digestive gland, mantle cavity, intestine and ovotestis. B. tenagophila showed a range of infection from 1 to 60 cysts per snail (mean=4.5±SD=9.9), and cyst diameter ranging between 255 and 705μm (466.4±119); while, B. occidentalis showed a range of infection from 1 to 23 (5.5±5.6), and cyst diameter ranging between 310 to 900μm (554.5±150). Results obtained indicated that, although absent in autumn, metacercariae of Kalipharynx sp. were present most of the year in both species of Biomphalaria, showing high values in both warm-season. Furthermore, both the infection intensity and host shell size varied significantly between seasons, although no seasonal variation was observed with respect to metacercarial cyst size, suggesting the possibility of more than one peak of cercariae emergence during the year. The prevalence of infection was significantly and positively correlated with snail size in both host species (p<0.05). The smallest host size class harbouring a metacercarial infection was 7.1-8.0mm and 11.1-12.0mm in B. tenagophila and B. occidentalis, respectively. The mean intensity of infection was positively correlated with snail size, but this relation was significant only in B. tenagophila (p<0.05). However, non-significant negative correlations were observed for intensity of infection vs cyst size in both host species (p>0.05). The results of this study show a significant influence of host size on prevalence and infection intensity, and a tendency towards density-dependent reductions in the growth of cysts. This is the first study in Argentina analyzing the population biology of Kalipharynx sp. metacercariae.Rev. Biol. Trop. 61 (4): 1647-1656. Epub 2013 December 01.

En Argentina, no hay estudios ecológicos que hayan reportado los parámetros de infección de metacercarias Kalipharynx sp. en caracoles planorbídeos. Por lo que los objetivos de este estudio fueron: (i) proporcionar información sobre la biología de la población de metacercarias Kalipharynx sp. en los planorbídeos Biomphalaria tenagophila y B. occidentalis, a través del estudio de la prevalencia e intensidad de la infección por larvas durante un ciclo estacional, (ii) evaluar los efectos del tamaño del hospedador sobre la prevalencia y la intensidad de la infección (iii) evaluar el efecto de la intensidad de infección sobre el tamaño del quiste. Entre junio 2010 y abril 2011 se recolectaron en una laguna semipermanente de Corrientes, Argentina, un total de 616 caracoles (n=466 y n=150 de B. tenagophila y B. occidentalis, respectivamente) de los cuales se extrajeron 362 metacercarias (n=262 y n=100; de B. tenagophila y B. occidentalis, respectivamente). Se encontraron quistes en glándula digestiva, cavidad del manto, intestino y ovotestis. B. tenagophila y B. occidentalis estuvieron infectados con metacercarias la mayor parte del año, excepto en otoño, con valores altos en las estaciones cálidas (primavera-verano). La intensidad de infección y el tamaño del hospedador mostraron diferencias significativas entre las tres estaciones analizadas (invierno-verano), aunque no se observaron variaciones estacionales con respecto al tamaño de los quistes, sugiriendo la posibilidad de más de un pico de emergencia de las cercarias durante el año. La prevalencia se correlacionó positiva y significativamente con el tamaño del hospedador en ambas especies (p<0.05). La intensidad media de infección se correlacionó positivamente con el tamaño del hospedador, pero esta relación fue significativa sólo en B. tenagophila (p<0.05). Sin embargo, se observaron correlaciones negativas no significativas entre la intensidad de infección y el tamaño de los quistes en ambas especies (p>0.05). Los resultados obtenidos muestran una influencia significativa del tamaño del hospedador sobre la prevalencia y la intensidad de infección y una tendencia hacia reducciones denso-dependientes en el crecimiento de los quistes. Es el primer estudio que analiza la biología poblacional de metacercarias Kalipharynx sp. en caracoles planorbídeos de Argentina.

OVICIDAL EFFECT OF PIPERACEAE SPECIES ON Biomphalaria glabrata, Schistosoma mansoni HOST / Efeito ovicida de espécies de Piperaceae em Biomphalaria glabrata, hospedeiro do Schistosoma mansoni

SUMMARY Schistosomiasis is a neglected disease with public health importance in tropical and subtropical regions. An alternative to the disease control is the use of molluscicides to eliminate or reduce the intermediate host snail population causing a reduction of transmission in endemic regions. In this study nine extracts from eight Piperaceae species were evaluated against Biomphalaria glabrata embryos at blastula stage. The extracts were evaluated in concentrations ranging from 100 to 10 mg/L. Piper crassinervium and Piper tuberculatum extracts were the most active (100% of mortality at 20 mg/L and 30 mg/L respectively). .

RESUMO A esquistossomose é uma doença negligenciada de importância para a saúde pública em regiões tropicais e subtropicais. Uma alternativa para o controle da doença é o uso de moluscicidas para eliminar ou reduzir a população de caramujo hospedeiro, acarretando uma redução da transmissão da doença nas regiões endemicas. Neste estudo, nove extratos vegetais provenientes de oito espécies de Piperaceae foram expostos a embriões de Biomphalaria glabrata no estágio de blástula. Os extratos foram avaliados em concentrações que variaram entre 100 e 10 mg/L, sendo Piper crassinervium e Piper tuberculatum os extratos mais ativos (100% de mortalidade a 20 mg/L e 30 mg/L respectivamente). .

Susceptibility of Argentinean Biomphalaria tenagophila and Biomphalaria straminea to infection by Schistosoma mansoni and the possibility of geographic expansion of mansoni schistosomiasis

Introduction Human migration and the presence of natural vectors (mollusks) of Schistosoma mansoni are the primary causes of the expansion of mansoni schistosomiasis into southern areas of South America. Water conditions are favorable for the expansion of this disease because of the extensive hydrographic network, which includes the basins of the Paraná and Uruguay rivers and favors mollusk reproduction. These rivers also aid agriculture and tourism in the area. Despite these favorable conditions, natural infection by S. mansoni has not yet been reported in Argentina, Uruguay, or Paraguay. Methods Two species of planorbid from Argentina, Biomphalaria straminea and B. tenagophila, were exposed to the miracidia of five Brazilian strains of S. mansoni. Results Biomphalaria tenagophila (Atalaya, Buenos Aires province) was infected with the SJS strain (infection rate 3.3%), confirming the experimental susceptibility of this Argentinian species. Biomphalaria straminea (Rio Santa Lucía, Corrientes province) was susceptible to two Brazilian strains: SJS (infection rate 6.7%) and Sergipe (infection rate 6.7%). Conclusions These results demonstrate that species from Argentina have the potential to be natural hosts of S. mansoni and that the appearance of foci of mansoni schistosomiasis in Argentina is possible. .