Establishment and evaluation of a LAMP-CRISPR-based nucleic acid detection method for Schistosoma japonicum / 中国热带医学

@#Abstract: Objective To establish a sensitive and specific nucleic acid detection method for Schistosoma japonicum based on loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) technology. Methods The LAMP primers, gRNA and ssDNA probe that target Schistosoma japonicum SjR2 genes were designed according to the principles of LAMP and CRISPR. The LAMP-CRISPR reaction system was established and optimized. The sensitivity and specificity of the method were evaluated against the ten-fold serial dilutions of plasmid containing SjR2 target sequences, as well as genomic DNA at different stages of Schistosoma japonicum and other parasites, including Fasciola hepatica, Schistosoma mansoni, Taenia saginata, Clonorchis sinensis, Ascaris lumbricoides, Necator americanus, Paragonimus westermani, and Echinococcus granulosus. Additionally, 15 schistosome-infected snail and 30 uninfected samples were tested by LAMP-CRISPR and LAMP methods, respectively, to evaluate the potential of this method for screening for infected snails. Results　The developed LAMP-CRISPR method was able to specifically amplify and detect the SjR2 gene of S. japonicum. The optimal reaction temperature was 37 ℃, and the optimal reaction concentrations were both 40 nmol/L for gRNA and Cas12a protein. No cross-reaction was observed with genomic DNA from other parasites such as F. hepatica. The detection limit of the method was 10 copies/μL when testing 10-fold dilutions of recombinant plasmids as a template. Furthermore, the LAMP-CRISPR method was able to accurately detect genomic DNA from S. japonicum at various stages of development, including eggs, cercariae, schistosomula, juvenile worms, and adult worms. The results of testing 45 snail samples showed no significant difference between the LAMP-CRISPR and LAMP methods for detecting infected snails (χ2=0.05, P>0.05). The sensitivity and specificity of the LAMP-CRISPR method were 100.00% (15/15) and 96.67% (29/30), respectively, compared to the gold standard, while the sensitivity and specificity of the LAMP method were 100.00% (15/15) and 93.33% (28/30), respectively. Conclusions　This established LAMP-CRISPR detection method presented good sensitivity, specificity and reliability, making it a promising tool for rapid detection and risk monitoring of S. japonicum.

The risk surveillance of schistosomiasis in Yunnan, 2021 / 中国热带医学

@#Abstract: Objective　To evaluate the potential transmission risk of schistosomiasis in Yunnan Province, and to provide strategic basis for the prevention and control. Methods　Based on the prevalence of schistosomiasis, the social and environmental factors that may lead to the epidemic, 1-3 villages from 3 provincial-level and 15 county-level counties (cities and districts) were selected as the evaluated villages in 2021. The risk of schistosomiasis spread was analyzed comprehensively by consulting, reviewing and collecting routine surveillance data of schistosomiasis in the villages, combined with snail and wild feces survey. The risk level was evaluated for the positive snails, positive wild feces, resident infection, average density of live snails and snail frame occurrence rate. Results　Totally 7 snail counties schistosomiasis transmission was blocked of 18 epidemic counties and the rest were eliminated counties. A total of 152 447 snail frames were investigated and 3 043 frames with snails, 15 895 snails were captured and included 15 727 live snails in the 32 evaluated villages. The total area of snail was 58.87 hm2 and the area of reoccurrence was 34.19 hm2 with snail frame occurrence rate of 2.00% and average density of live snails 0.103 2/0.11 m2, and no positive snails were found by loop-mediated isothermal amplification (LAMP) assay. A total of 1 374 wild feces were collected in 27 evaluated villages of 14 epidemic counties, mainly from cattle, dogs, sheep, equine animals, pigs and so on, all of which were negative. According to the risk assessment of epidemic spread, Yongle Village and Yongsheng Village in Eryuan County, Zhiming Village in Chuxiong City were Ⅱ risk, and the rest were Ⅲ risk. Conclusions　Although the risk of transmission is low in Yunnan Province, the risk of transmission and spread still exists. It is necessary to strengthen the risk monitoring, control of snail and effective management of livestock to prevent the rebound of the epidemic.

Effects of urban construction on Oncomelania snails control in Wuhan City / 中国血吸虫病防治杂志

Objective To evaluate the effectiveness of urban construction projects on Oncomelania snails control in Wuhan City, so as to provide insights into the development of an integrated control strategy for urban schistosomiasis. Methods The data pertaining to the endemic situation of schistosomiasis, schistosomiasis control data, the construction of the Yangtze River Bridge, marshland management and park constructions were collected from Wuhan City from 1990 to 2020. The changes of areas of snail habitats and high-risk settings were compared before and after implementation of these urban construction projects to evaluate the effectiveness of urban construction projects on Oncomelania snails control. Results The number of schistosomiasis cases decreased by 97.35%, and the number of cattle with schistosomiasis decreased by 100% in Wuhan City from 1990 to 2020, with a 41.99% reduction in areas of snail habitats, including 94.97% and 34.40% reductions in snail habitats inside and outside the embankment decreased. During the period from 1990 through 2020, a total of 10 Yangtze River bridges were built in Wuhan City, and areas of snail habitats around the bridges reduced from 11 699.05 hm2 before the bridge building to 8 726.14 hm2 after the building (a 25.41% reduction), while the areas of high-risk settings reduced from 411.69 hm2 before the bridge building to 276.78 hm2 after the building (a 32.77% reduction). Following treatment of three marshlands in Hankou, Hanyang and Wuchang, the areas of snail habitats reduced from 225.80 hm2 before the management to 199.47 hm2 after the management (a 11.66% reduction), and the areas of high-risk settings reduced from 23.14 hm2 before the treatment to 17.73 hm2 after the building (a 23.38% reduction). Following the building of 5 parks in snail-infested settings, the areas of snail habitats reduced from 319.61 hm2 before the construction to 280.80 hm2 after the construction (a 12.14% reduction), and the areas of high-risk settings reduced from 35.00 hm2 before the construction to 27.73 hm2 after the construction (a 20.77% reduction). Conclusion Simultaneous implementation of urban construction projects and schistosomiasis control measures is effective to shrink snail-infested setting and high-risk settings and control the endemic situation of schistosomiasis in endemic foci in Wuhan City.

Efficiency of Three Extracts of Carica papaya as Molluscicidal and Anti-schistosomal Agents against Biomphalaria alexandrina and Schistosoma mansoni by Flow Cytometry

Schistosomiasis a prevalent parasitic disease in tropical and sub-tropical areas, which comes in the second place in terms of socioeconomic and public health burden. Around 600 million people in 74 countries are infected yearly, predominantly in the developing world. The aim of this work was to assess the efficiency of three extracts from Carica papaya (methanol, ethanol and butanol extracts) for their molluscicidal and anti-schistosomal activities. The LC50of methanol, ethanol and butanol extracts of Carica papaya against B. alexandrinea were 180, 499.3 and 509.1 mg/L while the respective LC90values were 220.3, 700.6, 769.6 mg/L respectively. The effect of these extracts on Biomphalaria alexandrina snails and larval stages of Schistosma mansoni, for miracidia the LC50of methanol, ethanol and butanol extracts ofCaricapapaya against miracidia were 3.4, 15.4and 8.1 mg/L, respectively, while the respective LC90values were 8.4, 38.2, 11.2 mg/L, and for cercariae the LC50of methanol, ethanol and butanol extracts ofCaricapapaya were 2, 20and 4mg/L, while the respective LC90values were 13.5, 80.5, 18.5mg/L respectively was evaluated, in addition to flowcytometric analysis of CD4, CD25, FOXP3 and TGF-βlevels during S. mansoniinfection in mice. The in-vivo results showed that the three extracts have variable potential against snails and miracidia and cercariae ofS. mansoni. The mortality rate in B. alexandrinasnails for methanol, ethanol and butanol extracts ofCarcia papaywere 86%, 45% and 64%, respectively. While it was 83%, 35% and 66%, respectively in miracidia and 92%, 40% and 70%, respectively in cercariae. The results indicated that methanol extract from Carica papaya recorded higher activity against snails, miracidia and cercariae. The levels of CD4, CD25, FOXP3 regulatory T (Treg) cells were decreased significantly (p<0.001) in infected mice compared to healthy controls. However, there was a significant (p<0.001) increase in levels of TGF-β. A significant increase in the levels of CD4, CD25, and FOXP3 Treg in Carica papaya treated group compared to infected control group, with a significant (p<0.001) decrease in TGF-βlevel than infected group. In conclusion, methanol extract was more effective at concentration of LC50 180 and LC90 220.3than ethanol and butanol extracts ofCaricapapaya therefore controlling B. alexandrinasnails by methanol extract is a promising way as it is an eco-friendly strategy in rural areas of developing countries, where schistosomiasis is endemic. Moreover, the increased immune defense mechanism in treated group with the same extracts is a promising target for new immune modulatory strategies against schistosomiasis

Identificación molecular y relaciones evolutivas de Pomacea nobilis, base para la autenticación específica del churo negro de la Amazonia peruana / Molecular identification and evolutionary relationships of Pomacea nobilis, basis for the specific authentication of the black apple snail from Peruvian Amazonia

Resumen En la Amazonia Peruana los caracoles dulceacuícolas de la familia Ampullariidae son conocidos como churos y originalmente han sido descritas para Perú alrededor de 20 especies. Aunque son muy usadas para alimentación, medicina tradicional y objeto de muchos estudios para su cultivo e industrialización, solamente es mencionada en la literatura la especie Pomacea maculata. Se llevó a cabo la identificación molecular sobre la base del marcador mitocondrial COI, de individuos de churos negros (Pomacea) comercializados en los mercados de Iquitos, así como los usados en platos a la carta en la ciudad de Lima, contrastados con otros individuos de procedencia de su hábitat natural. Se encontró que estos especímenes expendidos corresponden a la especie Pomacea nobilis (Reeve, 1856). El análisis filogenético molecular mostró que P. nobilis es especie hermana de P. guyanensis, en el grupo de P. glauca, distantemente relacionada de P. maculata. Las distancias no corregidas encontradas entre ellas, para el marcador mitocondrial COI, fueron de 11.33% a 13.17%, mientras que con P. maculata fueron de 13.67% a 15.33%. Estos resultados demostraron la eficacia del código de barras de ADN para la identificación y autenticación de la especie, lo que le da un valor agregado para su eventual comercio de exportación.

Abstract In the Peruvian Amazon, freshwater snails of the Ampullariidae family are known as churos, and around 20 species have originally been described for Peru. Although they are widely used for food, traditional medicine and the object of many studies for their cultivation and industrialization, only the species Pomacea maculata is mentioned in the literature. Molecular identification was carried out based on the mitochondrial marker COI of individuals of "churo negro" apple snails (Pomacea) commercialized in the markets of Iquitos, as well as those used in restaurant dishes in the city of Lima, and contrasted with specimens from their natural habitat. It was found that these specimens, correspond to the species Pomacea nobilis (Reeve, 1856). The molecular phylogenetic analysis showed P. nobilis as the sister species of P. guyanensis, in the P. glauca group, distantly related to P. maculata. The uncorrected distances found between them, for the mitochondrial marker COI, were from 11.33% to 13.17%, while with P. maculate were from 13.67% to 15.33%. These results demonstrated the effectiveness of the DNA barcode for the identification and authentication of the species, which gives it added value for its eventual export trade.

Reporte de Pseudosuccinea columella infectados con Fasciola hepática en Sierra de los Cuchumatanes, Guatemala / Report ofPseudosuccinea columella,infected withFasciola hepaticaat Sierra de los Cuchumatanes, Guatemala

RESUMEN Objetivo. Identificar la infección natural de caracoles de agua dulce con Fasciola hepatica en una localidad del altiplano de Guatemala. Materiales y métodos. Se colectaron caracoles de agua dulce en una aldea endémica de fascioliasis, en la localidad de Paquix, en el departamento de Huehuetenango, ubicado a ~3.300 msnm. La identificación de la especie de caracol se basó en las características morfológicas observadas por estereoscopio usando una clave de identificación. La búsqueda de fases del trematodo se realizó mediante la inspección visual externa con fuente de luz y la disección e inspección visual dentro del molusco bajo estereoscopio y microscopio. Resultados. 260 caracoles fueron encontrados e identificados como Pseudosuccinea columella. Se examinaron los especímenes colectados y se encontraron dos caracoles infectados de forma natural con fases larvarias de F. hepatica. Conclusiones . Este es el primer reporte de este hospedero intermediario portando formas de F. hepatica en Guatemala. Es necesario aumentar los reportes de este parásito y de sus interacciones ecológicas en otras áreas no estudiadas de Guatemala y Centro América, debido al riesgo latente de infección para poblaciones de animales herbívoros y humanos.

ABSTRACT Objective. Identify the natural infection of freshwater snails as an intermediate host of Fasciola hepatica in Guatemala. Materials and methods. Freshwater snails were collected in a high-altitude village (3.000 mamsl) from Huehuetenango department, where fasciolasis is endemic. The identification of the snail species was based on the morphological characteristics observed under the stereoscope using an identification key. The trematode phases were searched externally by visual inspection using a light source, and internally by dissection and visual inspection of the tissues under a stereoscope and microscope. Results. 260 snails were found and identified as Pseudosuccinea columella. Two were found naturally infected with larval phases of F. hepatica. Conclusions. This is the first report of P. columella carrying phases of F. hepatica in the highlands of Guatemala. It is necessary to increase the research of the ecology of this parasite in other understudied areas in Guatemala and Central America, due to the latent risk of infection for populations of herbivorous animals and humans.

Microhabitat selection, and seasonal and daily activity of the snail Tikoconus costarricanus (Stylommatophora: Euconulidae) in tropical montane wet forest understory / Selección de microhábitat, y actividad diaria y estacional del caracol Tikoconus costarricanus (Stylommatophora: Euconulidae) en el sotobosque de bosques tropicales húmedos de montaña

Introduction: The integument of terrestrial mollusks is highly susceptible to dehydration; therefore, microhabitat selection, seasonality and behavior around the day are crucial to their survival. However, they are still poorly understood, especially for tropical montane wet forest species. Objetive: To analyze activity patterns and microhabitat selection on shrubs of the land snail Tikoconus costarricanus, according to season, daytime and weather. Methods: I conducted the study near "El llano" water dam in a tropical montane wet forest in Costa Rica. I observed daily activity of T. costarricanus, during the rainy and the dry season on a 2 km long trail. I observed 167 specimens and made 781 observations in total. I took note of: season, time, activity, weather, temperature and relative humidity inside the forest, and part of the leaf and height where snails were. Active snails had optical tentacles extended. I analysed daily rainfall data from a nearby meteorological station. Results: This species can be found mainly on understory leaves between 1.1 and 2 m above the floor. They are active the day around during the whole year. Only 24 % of the snails were on the upper side of leaves, but 92 % of them were active. The following behaviors were season dependent: activity peaks, leaf side selection according to daytime and weather, quantity of snails aestivating and vertical distribution. During the dry season, I found more active snails in rainy and cloudy days. During the rainy season I found more active snails in sunny days. During rainy season light hours, active snail percentage on the upper side of leaves decreased with rainfall increase, while in the dry season decreased with temperature increase and relative humidity decrease. During night hours of both seasons, the number of snails on the upper side of leaves decreased considerably in relation to active snails, especially in the rainy season. This snail aestivated on the underside of leaves during periods shorter than 21 h, mainly around noon and afternoons during the dry season. High humidity in this forest allowed snail activity around the day at any season. However, this species aestivated when moisture reached the lowest values, mainly, around noon and in the afternoons in the dry season. The rainfall pattern during the rainy season light hours explains the specimen decrease on the upper side of leaves, but in the dry season it is better explained by the temperature increase and the decrease in relative humidity. During dark hours, the almost complete snail absence on the upper side of leaves during the rainy season is explained if the effect of canopy drip produced by rain and dew condensed on leaves is added to the rain pattern. The situation is slightly different in dark hours of the dry season, in this case, snail decrease on the upper side of leaves is explained by canopy drip from dew and fog precipitation. Seasonal difference in vertical distribution may be a way to avoid rain and rain splash out on the soil. Conclusions: Moisture, temperature, rain, and canopy drip from dew, fog and rain, affect behavior and substrate selection of small terrestrial mollusks that inhabit shrubs in wet tropical rainforests.

Introducción: El integumento de los moluscos terrestres es susceptible a desecación, por eso la selección de microhábitats, la estacionalidad y el comportamiento a lo largo del día son cruciales para su sobrevivencia. No obstante, es poco lo que se ha investigado al respecto, en especial para moluscos tropicales de arbustos del sotobosque en bosques húmedos de montaña. Objetivo: Analizar los patrones de actividad y de la selección de microhábitats del caracol terrestre Tikoconus costarricanus según la estación del año, la hora del día y el clima. Métodos: El trabajo lo realicé en un bosque tropical húmedo de montaña cerca de la represa hidrológica "El Llano", Costa Rica. Analicé la actividad diaria de especímenes de T.costarricanus en un trayecto de 2 km: observé 167 especímenes e hice 781 observaciones. Tomé nota de: estación del año, hora, actividad, parte de la hoja donde estaba el caracol, altura en la planta, clima y humedad relativa y temperatura dentro del bosque. Consideré que un espécimen estaba activo cuando los tentáculos ópticos estaban extendidos. Analicé la precipitación pluvial por horas con datos de una estación meteorológica cercana. Resultados: Encontré caracoles principalmente en las hojas de arbustos entre 1.1 y 2 m sobre el suelo; encontré individuos activos a todas horas y en cualquier época del año. Solo el 24 % de los individuos estaban en el haz, de esos el 92 % estaban activos. Encontré variación estacional en el patrón de actividad, la selección del lado de la hoja de acuerdo a la hora del día y el clima, la cantidad de especímenes estivando, y la distribución vertical. En la época seca encontré más caracoles activos con clima húmedo, por el contrario, en la estación lluviosa encontré más caracoles activos con clima seco. Durante las horas de luz de la estación lluviosa, la cantidad de caracoles activos sobre el haz de las hojas disminuyó con el aumento de precipitación pluvial, en tanto que en la estación seca disminuyó con el aumento de la temperatura y la disminución de la humedad relativa. Durante las horas nocturnas de ambas estaciones, la cantidad de caracoles sobre el haz de las hojas disminuyó considerablemente en relación a los caracoles activos, especialmente en la época lluviosa. Este caracol estivó principalmente a mediodía y en las tardes de la época seca en el envés de las hojas por periodos inferiores a 21 h seguidas. La elevada humedad del bosque tropical húmedo de montaña permite que estos caracoles estén activos en cualquier momento. Sin embargo, la estivación les permite protegerse de la desecación durante los periodos más secos. El patrón de lluvias durante las horas de luz de la época lluviosa explica la disminución de especímenes presentes en el haz de las hojas, pero en la época seca lo explica mejor el aumento de la temperatura y la disminución de la humedad relativa. Durante las horas oscuras, la casi ausencia de caracoles en el haz de las hojas durante la estación lluviosa se explica si al patrón de lluvia se le agrega el efecto del goteo foliar producido por la lluvia y el rocío que se condensa sobre las hojas. La situación es un poco diferente en las horas oscuras de la estación seca, en este caso, la disminución de caracoles en el haz, se explica por el goteo foliar derivado del rocío y de la precipitación de neblina. Es posible que la diferencia en la preferencia vertical se deba a que evitan la lluvia y la salpicadura de la lluvia al caer en el suelo. Conclusiones: La humedad, la temperatura, la lluvia y el goteo foliar derivado de rocío, neblina y lluvia, afectan el comportamiento y la selección de sustrato de moluscos terrestres pequeños que habitan en arbustos de bosques tropicales húmedos de montaña.

Occurrence of BIOMPHALARIA GLABRATA and PHYSA ACUTA (gastropoda: mollusca) snails in Quinta da Boa Vista municipal park, Rio de Janeiro, RJ, Brazil

The human species has an emotional connection with nature called "biophilia" (or, love for life). This connection goes from generation to generation, being one of the reasons why green spaces are present in large cities. Quinta da Boa Vista is a municipal park in Rio de Janeiro located in the São Cristóvão neighborhood in the northern part of the city. Its main attraction is the artificial lake system, a leisure attraction for the local community. This article reports the presence of Biomphalaria glabrata and Physa acuta snails around the main lake in the Quinta da Boa Vista Municipal Park in Rio de Janeiro, RJ. Seventy-one B. glabrata and twenty-seven P. acuta snails were collected. The snails were measured and examined for the presence of trematode larvae according to standardized procedures. The prevalence rate of B. glabrata was estimated at 15.5%. Rediae and cercariae resembling those of the species Typhlocoelum cucumerinum (Typhlocoelidae, Digenea) which parasitizes aquatic birds of the order Anseriformes were found. This study reinforces the importance of monitoring park lakes and ponds to better understand the risk of helminth transmission by molluscan vectors.

Effects of the environmental temperature and humidity on the survival and reproduction of Oncomelania snails in field experimental base / 公共卫生与预防医学

Objective To study the effects of environmental temperature and humidity on the survival and reproduction of Oncomelania snails, and to provide a basis for the development of scientific strategies and measures for snails control. Methods Based on the field experimental base of Oncomelania snails in Hubei Province, through long-term and systematic ecological observation of snails, the living conditions of snails at different temperatures were recorded, and the average densities of live snails and young snails as well as the amount of spawning were calculated. Results In a certain temperature range (15°C-30°C), the average densities of live snails and young snails were positively correlated with the changes of temperature and humidity, and the effect of temperature on the spawning of snails was also significant. The optimal breeding temperature of Oncomelania snails was 25°C. Conclusion In a certain temperature range (15°C-30°C), the environmental temperature and humidity have a significant impact on the survival and reproduction of snails in the field.

Molecular Method vs. Traditional Methods for Estimating the Prevalence of Larval Trematode Infections in Some Red Sea Snails

Peat reserves of the Russian Federation are estimated at more than 170.0 billion tons, it makes more than one-third of theworld resources. The use of peat as a component for the preparation of composts is an important factor in increasing theproductivity of agricultural ecosystems. During two cycles of four-course crop rotation ‘potatoes – barley – annual grasses– winter wheat’, we studied the influence of lowland peat, composted peat-poultry manure, cattle manure, their combinedapplication with mineral fertilizers on soil physicochemical properties, nitrifying and cellulosolytic activity of soil andproductivity of crop rotation. Application of cattle manure and composted peat-poultry manure contributes to the increase inthe content of humus in soil by 0.07-0.16%, when combined with mineral fertilizers – by 0.14-0.22%, respectively. Onaverage, the highest yield was noted in the variants with cattle manure and composted peat-poultry manure – 10.75-12.13 tha -1 of grain units (g.u.), while in the check (without fertilizers) it was 8.03 g.u. (t ha -1). That is why peat is more profitableto use in the form of composts, especially as a component of composted peat-poultry manure

Diversity of Biomphalaria spp. freshwater snails and associated mollusks in areas with schistosomiasis risk, using molecular and spatial analysis tools / Diversidade de caramujos de água doce Biomphalaria spp. e outros moluscos associados, em áreas de risco da esquistossomose, empregando ferramentas moleculares e espaciais

Abstract: The Middle Paranapanema River region of São Paulo, Brazil is home to significant diversity of Biomphalaria species and is very vulnerable to health and environmental impacts such as schistosomiasis. This study updates freshwater malacological surveys for ecosystems in one portion of the Middle Paranapanema River Basin, with emphasis on the genus Biomphalaria. Snails were collected from 114 distinct bodies of water between 2015 and 2018. Biomphalaria specimens were identified according to morphological and molecular characteristics, while animals in other genera (Drepanotrema, Lymnaea, Melanoides, Physa and Pomacea) were identified solely according to shell characteristics. A geographic information system was used to update intermediate host colonization sites and consequently assist in identifying probable hotspots for intermediate hosts of schistosomiasis. The sequences of the COI gene relating to the DNA barcode stretch were tested for similarity against sequences found in GenBank, for monophyly through Maximum Likelihood phylogenetic inference, and analyzed in ABDG, bPTP and GMYC for the delimitation of putative species. Of the 10,722 snails collected, 86.7% were in the Planorbidae family (75.5% Biomphalaria and 11.2% Drepanotrema) and 13.3% were other non-Planorbidae species (Lymnaea, Melanoides, Physa and Pomacea). The taxonomic COI reference sequences in the NCBI nucleotide database used for DNA sequence comparison, and phylogenetic analysis used to test the monophyly of the groups, resulted in more reliable taxonomic units than delimitation of the COI sequences in MOTUs using statistical taxonomic models. Analysis of the species distribution shows that B. glabrata and B. tenagophila are heterogeneously distributed in the study area. B. glabrata colonizes only five water bodies, in the study area, most of them in Ourinhos, while B. tenagophila predominates in water bodies in Ipaussu. Contrasting with this, B. straminea, B. occidentalis and B. peregrina are evenly distributed throughout the study area.

Resumo: A região do Médio Rio Paranapanema, em São Paulo, Brasil abriga uma diversidade significativa das espécies de Biomphalaria. É também uma região vulnerável a impactos ambientais e de saúde, como a esquistossomose. Este estudo atualiza dados sobre a distribuição de caramujos de água doce em ecossistemas de uma porção da Bacia do Médio Rio Paranapanema, com ênfase no gênero Biomphalaria. Os caramujos foram coletados de 114 corpos distintos de água doce, entre 2015 e 2018. Exemplares pertencentes ao gênero Biomphalaria foram identificados de acordo com características morfológicas e moleculares, enquanto animais de outros gêneros (Drepanotrema, Lymnaea, Melanoides, Physa e Pomacea) foram identificados somente de acordo com características da concha. Ferramentas de análise geoespaciais foram utilizadas para atualizar os sítios de colonização dos caramujos e, consequentemente, auxiliar na identificação de possíveis pontos críticos para hospedeiros intermediários da esquistossomose. As sequências do gene COI relacionadas ao DNA barcode foram testadas quanto à similaridade com sequências encontradas no GenBank, por análise filogenética sob maxima verossimilhança, e analisadas em ABDG, bPTP e GMYC para a delimitação de espécies putativas. Dos 10.722 moluscos coletados, 86,7% pertenciam a família Planorbidae (75,5% Biomphalaria e 11,2% Drepanotrema) e 13,3% a Lymnaea spp., Melanoides spp., Physa spp. e Pomacea spp. A comparação das sequências taxonômicas de COI com o banco de dados de nucleotídeos do NCBI, e a análise filogenética usada para testar a monofilia dos grupos, resultaram em delimitações taxonômicas comparáveis à delimitação morfológica. As espécies B. glabrata e B. tenagophila estão heterogeneamente distribuídas ao longo da área de estudo. B. glabrata foi identificada em apenas cinco coleções de água doce, quatro delas em Ourinhos, enquanto B. tenagophila predominou em Ipaussu. Por outro lado, B. straminea, B. occidentalis e B. peregrina estão distribuídas uniformemente na área de estudo.

Echinostoma macrorchis Metacercariae in Cipangopaludina chinensis malleata from Xiengkhuang Province, Lao PDR and Morphologies of Adults from Experimental Animals

We identified the echinostome metacercariae in Chinese mystery snails, Cipangopaludina chinensis malleata, from Xiengkhuang Province, Lao PDR with morphologies of adult worms recovered. Total 20 snails were examined with artificial digestion method and then the collected metacercariae were orally infected to a mouse and a rat. Adult worms recovered from experimental animals were observed with a light microscope and a SEM. The metacercariae were round, 125×123 μm in average size, with a moderately thick cyst wall, collar spines distributed in the head collar and excretory granules in 2 canals of excretory tube. Adult flukes (3-week-old in a rat) were elongated, ventrally curved and 5.310×1.023 mm in average size. Head collar distinct, bearing 43 collar spines with 5 end group ones on each side. Oral sucker subterminal, prepharynx very short, pharynx well developed, and esophagus relatively short. Cirrus sac well developed, with a saccular seminal vesicle, and ventral sucker very large. Ovary round and on the median line of the body. Testes tandom and elongated. Eggs operculated, elliptical and 90×57 μm in average size. In the SEM observation, the head crown prominent, with 43 collar spines resembled with horns of younger stag. Scale-like tegumental spines were densely distributed on the surface between the head collar and ventral sucker, and their densities were decreased posteriorly. Conclusively, the metacercariae detected in C. chinensis malleata from Lao PDR were identified as those of Echinostoma macrorchis based on the morphological characteristics of adult worms.

Subnicho etario y biología reproductiva de Parachondria neglectus (Littorinimorpha: Annulariidae) en Manzanillo, Cuba / Age-related subniche and reproductive biology of Parachondria neglectus (Littorinimorpha: Annulariidae) in Manzanillo, Cuba

Resumen Parachondria neglectus es un molusco terrestre de la familia Annulariidae, endémico de Cuba y microlocalizado en la provincia de Granma. En la presente investigación se caracterizaron la dinámica temporal del subnicho etario y algunos aspectos de la biología reproductiva de una población de P. neglectus localizada en Manzanillo, GranmaCuba. Se realizaron 30 expediciones entre diciembre 2013 y febrero 2015. Las comparaciones del largo (t = 8.05, P = 0.007) y diámetro mayor (t = 6.24, P = 0.0001) de las conchas de los juveniles truncados y los juveniles completos mostraron diferencias estadísticas significativas, indicando que ambos grupos representan dos clases de edades diferentes. La comparación del diámetro mayor de las conchas de los adultos truncados y los adultos completos (t = 1.50, P = 0.16) demostró que conforman un misma clase de edad en la población. La existencia de diferencias estadísticas significativas entre el largo (t = 19.45, P = 0.0003) y el diámetro mayor (t = 13.19, P = 0.006) de las conchas de las hembras y los machos reafirmaron el dimorfismo sexual en la especie, resultando las hembras de mayores dimensiones respecto a los machos. La mancha circular blancuzca en las conchas es una característica morfológica externa válida para diferenciar a las hembras adultas y ya apareadas en ocasiones anteriores, del resto de los miembros de la población. El período reproductivo comprendió de julio a diciembre 2014. Las cópulas se observaron de julio a octubre 2014 y estuvieron asociadas a condiciones de precipitaciones abundantes y temperaturas altas del verano. El período de reclutamiento ocurrió de septiembre a diciembre 2014.(AU)

Abstract Parachondria neglectus is a land snail of the Annulariidae family, endemic to Cuba and microlocalized in Granma province. In the present research, the temporal dynamics of age-related subniche and some aspects of the reproductive biology of a P. neglectus population, located in ManzanilloGranma-Cuba, were characterized. Thirty expeditions were performed from December 2013 to February 2015. The comparisons between the length (t = 8.05, P = 0.007) and biggest diameter (t = 6.24, P = 0.0001) of the shells of truncated juveniles and the shells of full juveniles showed significant statistical differences, indicating that both groups represent two kinds of different ages. The comparison between the biggest diameter of the shells of truncated adults and the shells of full adults (t = 1.50, P = 0.16) showed that both groups represent the same kind of age in the population. The existence of significant statistical differences between the length (t = 19.45, P = 0.0003) and the biggest diameter (t = 13.19, P = 0.006) of the females' shells and the males' shells verify the sexual dimorphism in the species, proving to be the females of bigger dimensions in relation to the males. The whitish circular spot in the shells is a valid external morphological characteristic to differentiate the adult females already mated in previous occasions from the rest of the members of the population. The reproductive period was from July to December 2014. Copulations were observed from July to October 2014 and they were associated to conditions of abundant precipitations and high temperatures of summer. The recruitment period took place from September to December 2014.(AU)

Infección con Fasciola hepatica en el ganado y hospederos intermediarios en 10 vaquerías de un municipio occidental de Cuba / Natural Infection with Fasciola hepatica in Host-Snails and Cattle in Ten Dairy Farms from a Western Municipality in Cuba / Infecção com Fascíola hepática no gado e hospedeiros intermediários em 10 leiterias de um município ocidental de Cuba

Resumen La fasciolosis es una trematodosis que afecta fuertemente al ganado de Cuba. Se estudiaron 10 vaquerías del municipio más occidental de Cuba para determinar la prevalencia de esta parasitosis en el ganado y en los hospederos intermediarios. Se recolectaron y estudiaron muestras fecales de 455 vacas lecheras seleccionadas al azar. Las muestras fueron conservadas a -20 oC hasta su estudio. La detección de la infección por Fasciola hepatica fue realizada a partir de un método de Elisa denominado FasciDIG®. El análisis e interpretación de los resultados demostró que 146 muestras fueran positivas a F. hepatica (32,09 %). Todas las vaquerías presentaron animales infectados con rangos de prevalencia entre las vaquerías que oscilaron de 9,5 % al 84 %. También fue posible encontrar las dos especies de hospederos intermediarios de este parásito en Cuba, Galba cubensis y Pseudosuccinea columella. Debe prestarse atención a la prevalencia en bovinos encontrada en este estudio para prevenir la aparición de brotes humanos de esta enfermedad.

Abstract Fasciolosis is a foodborne trematodosis affecting many cattle farms in Cuba. Ten dairy farms in western Cuba were studied to assess the prevalence in both intermediate and definitive hosts. A single stool sample from 455 dairy cows was taken and studied. The animals were randomly selected and the samples were kept at -20oC until infection was determined. Detection of the Fasciola hepatica infection was carried out using a non-commercial sandwich-ELISA called FasciDIG®. Results showed that 146 samples were positive for F. hepatica (32.09%) with every farm testing positive for F. hepatica infection, while prevalence ranged from 9.5% to 84% among farms. It was also possible to detect the presence of the two species of intermediate hosts of this trematode in Cuba, Galba cubensis and Pseudosuccinea columella. Main attention must be given to bovine prevalence found in this study in order to prevent eventual human outbreaks of this disease.

Resumo A fasciolose é uma trematoda que afeta fortemente ao gado de Cuba. Foram estudadas 10 leiterias do município mais ocidental de Cuba para determinar a prevalência desta parasitose no gado e nos hospedeiros intermediários. Se coletaram e estudiaram amostras fecais de 455 vacas leiteiras selecionadas aleatoriamente. As amostras foram conservadas a −20 oC até seu estudo. A detecção da infecção por Fascíola hepática foi realizada a partir de um método de Elisa denominado FasciDIG®. A análise e interpretação dos resultados demonstrou que 146 amostras foram positivas à F. hepática (32,09 %). Todas as leiterias apresentaram animais infectados com rangos de prevalência entre as leiterias que oscilaram de 9,5 % a 84 %. Também foi possível encontrar a duas espécies de hospedeiros intermediários deste parasita em Cuba, Galba cubensis e Pseudosuccinea columella. Deve prestar-se atenção à prevalência em bovinos encontrada neste estudo para prevenir a aparição de surtos humanos desta enfermidade.

Cultivo in vitro de larvas L3 de nematodos obtenidas del caracol gigante africano Lissachatina fulica (Mollusca: Gastropoda) en Santa Fe de Antioquia / In vitro culture of L3 larvae of nematodes obtained from the African giant snail Lissachatina fulica (Mollusca: Gastropoda) in Santa Fe de Antioquia

Introducción. Más de 170 municipios colombianos están invadidos por Lissachatina fulica, caracol africano que puede portar larvas de nematodos de interés en salud humana y veterinaria. Los parásitos entran al caracol huésped intermediario en el estadio de larva L1, y allí cambian a L2 y L3, formas estas capaces de infectar a vertebrados. Objetivo. Estandarizar el cultivo in vitro de las L3 portadas por especímenes de L. fulica recolectados en Santa Fe de Antioquia. Materiales y métodos. Entre julio y noviembre de 2014 se recolectaron 10 caracoles, se sacrificaron y se digirieron con ácido clorhídrico al 0,7 %. Las larvas se recuperaron mediante la técnica de Baermann; se cultivaron 36 días en los medios Schneider, mínimo esencial de Eagle modificado por Dulbecco (Dulbecco's Modified Eagles Minimal Essential Medium, DMEM), y Roswell Park Memorial Institute (RPMI), con suero fetal bovino (SFB) al 20 % y sin este, y agua destilada con SFB al 20 %. Los medios de cultivo se cambiaron cada 36 horas. Las larvas se midieron con el microscopio utilizando reglilla ocular, y se evaluaron la supervivencia, la longitud y el ancho. Se calcularon datos estadísticos de resumen y se hicieron gráficos de cajas y bigotes, así como la prueba t de Student. El nivel de significación (p) se estableció como menor de 0,05. Resultados. El 50 % de las larvas sobrevivió, 85 % en DMEM con SFB al 20 %, el 70 % con RPMI más SFB al 20 %, el 60 % en RPMI, el 50 % en Schneider más SFB al 20 %, el 45 % en Schneider y el 40 % en DMEM. El control sobrevivió diez días. Hubo diferencias significativas entre la longitud inicial promedio de las larvas y la longitud final promedio en los medios con suplementos: inicial, 645,83 µm; final en DMEM más SFB al 20 %, 732,65 µm (p<0,001); en RPMI más SFB al 20 %, 718,79 µm (p<0,001), y en Schneider más SFB al 20 %, 696,12 µm (p<0,01). No hubo diferencias significativas entre la anchura inicial promedio, de 24,99 µm, y la final. Conclusiones. El mejor medio para cultivar las L3 de L. fulica fue el DMEM más SFB al 20 %. En la evaluación del crecimiento larval, la longitud fue más informativa que la anchura. Las larvas estudiadas no correspondieron a Angiostrongylus cantonensis, A. costaricensis ni Aelurostrongylus abstrusus.

Introduction: Over 170 municipalities in Colombia have been invaded by Lissachatina fulica, an African snail that can carry larvae of nematodes of interest in human and veterinary health. Nematodes enter the host snail as larvae L1 and then change to L2 and L3, the infectious form for vertebrates. Objective: To standardize culture in vitro of L3 carried by L. fulica from Santa Fe de Antioquia. Materials and methods: Between July and November, 2014, 10 snails were collected, killed, and conserved with HCl 0.7%. Larvae were recovered using the Baermann technique and cultured for 36 days in Schneider, DMEM and RPMI media, with and without SFB 20% and distilled water with SFB 20%. Replacements were made every 36 hours; larvae were measured with an ocular micrometer on a microscope. Summary statistics were estimated; box and whisker plots were made; the t Student test was performed in SPSS 18™. A p-value below 0.05 was assumed as significant. Results: Fifty per cent of the larvae survived. The highest survival and growth was 85% in supplemented DMEM. The final average length of larvae in supplemented media exceeded the initial one. There were significant differences between the average length of larvae cultured in supplemented media and the initial length. The initial width of larvae did not change. Conclusions: The best medium for the culture of L3 larvae was supplemented DMEM. The length provided more information than the width for the larval growth evaluation. The larvae studied did not correspond to Angiostrongylus cantonensis, A. costaricensis or Aelurostrongylus abstrusus.

Cercariae (Digenea: Strigeidae, Diplostomidae) in Biomphalaria straminea (Planorbidae) from a rice field in Northeastern Argentina / Cercarias (Orden: Strigeidae, Diplostomidae) en Biomphalaria straminea (Planorbidae) de un campo de arroz en el Nordeste Argentino

AbstractThe rice fields can provide habitats for many species of aquatic invertebrates, as insects, molluscs, crustaceans; and vertebrates, as fish, amphibians, reptiles and birds, which may act as hosts in the life cycles of digenean parasites. In this context, the goal of the present study was to describe the cercariae found in Biomphalaria straminea from a rice field in Northeastern Argentina. This freshwater snail inhabits a wide variety of environments, favoring shallow, temporary and standing or slow-flowing freshwater bodies. For this study, snails were collected from the cultivated parcels and irrigated channels during the flooding periods (from the time of sowing to soon after harvesting of the rice) between December 2010 and May 2011 and December 2011 and April 2012, in a rice field from Corrientes province, one of the main rice producers of Argentina. A total of 5 510 snails were examined of which 26 were infected with three different larval trematodes belonging to Strigeidae, Furcocercaria V (0.40 %) and Furcocercaria XX (0.04 %), and Diplostomidae, Furcocercaria XIX (0.04 %). Furcocercaria XX and Furcocercaria XIX were present in one rice cultivation cycle, while Furcocercaria V was present in both rice cultivation cycles. The prevalence of the different furcocercariae was somewhat higher in the second rice cultivation cycle. The cercariae described are new records for Argentina that added to 53 cercariae previously reported for Biomphalaria spp. in different aquatic environments of country. The study of the digenean larval in rice fields is important because contribute to the knowledge of the biodiversity of these environments.

ResumenLos campos de arroz pueden proporcionar hábitats para muchas especies de invertebrados acuáticos, como insectos, moluscos, crustáceos; y vertebrados, como peces, anfibios, reptiles y aves, los cuales pueden actuar como hospedadores en los ciclos de vida de los parásitos digeneos. En este contexto, el objetivo del presente estudio fue describir las cercarias encontradas en Biomphalaria straminea de un campo de arroz en el Nordeste Argentino. Este caracol de agua dulce habita una amplia variedad de ambientes, principalmente cuerpos de agua superficiales, estancados o de flujo lento. Para este estudio, los caracoles fueron recolectados de las parcelas cultivadas y los canales de riego durante los periodos de inundación (desde la siembra hasta poco después de la cosecha del arroz) entre diciembre 2010 y Mayo 2011 y Diciembre 2011 y Abril 2012, en un campo de arroz de la provincia de Corrientes, una de las principales productoras de arroz de Argentina. Un total de 5 510 caracoles fueron examinados de los cuales 26 estuvieron infectados con tres trematodes larvales pertenecientes a Strigeidae, Furcocercaria V (0.40 %) y Furcocercaria XX (0.04 %), y Diplostomidae, Furcocercaria XIX (0.04 %). Furcocercaria XX y Furcocercaria XIX estuvieron presentes en un ciclo de cultivo de arroz, mientras que Furcocercaria V estuvo presente en ambos ciclos de cultivo. La prevalencia de las diferentes furcocercarias fue algo mayor en el segundo ciclo de cultivo de arroz. Las cercarias descritas son nuevos registros para Argentina que se adicionan a las 53 cercarias previamente registradas para Biomphalaria spp. en diferentes ambientes acuáticos del país. El estudio de digeneos larvales en los campos de arroz es importante porque contribuye al conocimiento de la biodiversidad en estos ambientes.

Centrocestus formosanus (Trematoda: Heterophyidae) in Melanoides tuberculata (Gastropoda: Thiaridae) from Vila do Abraão, Ilha Grande, Rio de Janeiro, Brazil / Centrocestus formosanus (Trematoda: Heterophyidae) em Melanoides tuberculata (Gastropoda, Thiaridae) da Vila do Abraão, Ilha Grande, Rio de Janeiro, Brasil

Abstract Pleurolophocercous cercariae found in the invasive gastropod Melanoides tuberculata (Müller, 1774) collected in a stream of the Vila do Abraão, Ilha Grande, Angra dos Reis, Rio de Janeiro, Brazil were used for experimental infection that enabled the identification of the heterophyid trematode Centrocestus formosanus (Nishigori, 1924). The parasite has been found in the locality since 2007, after two years of the introduction of M. tuberculata. Recently, from a sample of 483 specimens collected in June 2013, 101 (21%) were found infected with parasite. The potential environmental impacts caused by the parasite occurrence could be underestimated in the country, and actions to monitor and control both the parasite and the mollusk are necessary.

Resumo Cercárias do tipo pleurolofocerca encontradas no gastrópode invasor Melanoides tuberculata (Müller, 1774) coletados em um riacho da Vila do Abraão em Ilha Grande, Angra dos Reis, Rio de Janeiro, Brasil foram utilizadas em estudos de infecção experimental que possibilitaram a identificação do trematódeo heterofiídeo Centrocestus formosanus (Nishigori, 1924). O parasito vem sendo encontrado na localidade desde 2007, dois anos após a introdução do molusco. Recentemente, de uma amostra de 483 espécimes coletados em junho de 2013, 101 (21%) apresentavam-se infectados pelo parasito. Os potenciais impactos ambientais ocasionados pela ocorrência deste parasito podem estar sendo subestimados no país, sendo necessárias ações visando o monitoramento e controle tanto do parasito quanto do molusco.

Changes in energetic metabolism of Biomphalaria glabrata (Mollusca, Planorbidae) in response to exogenous calcium / Alterações no metabolismo energético de Biomphalaria glabrata em resposta ao cálcio exógeno

Abstract Calcium is considered an essential element for the metabolism of aquatic snail Biomphalaria glabrata (Say, 1818), intermediate host of Schistosoma mansoni Sambon, 1907 in Brazil, and represents a limiting factor to its distribution and adaptation to the environment. This study investigated the effect of different concentrations of exogenous CaCO3 on the energetic metabolism of B. glabrata for better understanding the physiological interference of chemical elements dissolved in the environment with the physiology of this species. Sixty-day-old snails were distributed into six groups, five exposed to different concentrations of CaCO3 (20, 40, 60, 80 and 100 mg/L) and a control group. The exposure to CaCO3 was assessed over time, with analysis of 15 snails of each group in the following intervals: 1, 14, 21 or 30 days for hemolymph extraction. Concentrations of calcium and glucose in the hemolymph were determined by commercial kits, and organic acids were extracted using an ion exchange column and analyzed by high-performance liquid chromatography. Concentration of calcium in the hemolymph showed no significant difference (p>0.05) from the control group and between the concentrations tested. Concentration of glucose decreased (p<0.05) in the treatments of exposure to 20 and 40 mg/L and increased when exposed to 80 and 100 mg/L CaCO3 compared to control and to other concentrations tested over 30 days. The organic acids pyruvate, oxaloacetate, citrate, succinate, fumarate, beta-hydroxybutyrate and lactate presented increased concentrations, while propionate and acetoacetate, decreased concentrations, when exposed to CaCO3 compared to control. Considering the influence of different periods of exposure to CaCO3, on the 14th day, there were stronger alterations in the metabolism of B. glabrata. In conclusion, exposure to CaCO3 reduced the concentration of glucose, which is metabolized into pyruvate, the final product of glycolysis, and also influenced the energetic metabolism pathways, indicating an aerobic or partially anaerobic functioning.

Resumo O cálcio é considerado um elemento essencial no metabolismo do molusco aquático Biomphalaria glabrata (Say, 1818), principal hospedeiro intermediário de Schistosoma mansoni Sambon, 1907 no Brasil e, tem sido descrito como um fator limitante na distribuição e adaptação desse molusco no ambiente. O presente trabalho avaliou o efeito de diferentes concentrações de carbonato de cálcio (CaCO3) exógeno ao metabolismo energético de B. glabrata, a fim de subsidiar uma melhor compreensão da interferência de elementos químicos dissolvidos no meio aquático na fisiologia destes moluscos. Foram utilizados moluscos com sessenta dias de vida, distribuídos em seis grupos, cinco expostos a diferentes concentrações de CaCO3 (20, 40, 60, 80 e 100mg/L) e um controle. A exposição ao CaCO3 foi avaliada em função do tempo, sendo retirados 15 moluscos de cada grupo nos seguintes intervalos: 1, 14, 21 ou 30 dias para extração da hemolinfa. As concentrações de cálcio e glicose na hemolinfa foram determinadas usando-se kits comercial e os ácidos orgânicos foram extraídos por meio da coluna de troca iônica e analisados através cromatografia líquida de alta eficiência. Os resultados demonstraram que a concentração de cálcio na hemolinfa não apresentou diferença significativa (p>0,05) em relação ao controle e nas concentrações testadas. A concentração de glicose diminuiu (p<0,05) nas exposições a 20 mg e 40 mg/L e aumentou nas exposições a 80 mg e 100 mg/L de CaCO3 em relação ao controle e demais concentrações testadas ao longo de 30 dias. Os ácidos orgânicos piruvato, oxaloaceato, citrato, succinato, fumarato, β-hidroxibutirato e lactato tiveram suas concentrações aumentadas, enqunato, propionato e acetoacetato tiveram suas concentrações diminuídas na exposição ao CaCO3 comparada ao controle. Quanto a influência dos diferentes períodos de exposição ao CaCO3, aos 14 dias, as alterações no metabolismo de B. glabrata foram mais expressivas. Conclui-se que as exposições ao CaCO3 influenciaram na redução de glicose, sendo esta metabolizada a piruvato, produto final da glicólise e alteraram as vias de metabolismo energético, indicando um funcionamento aeróbio ou parcialmente anaeróbio.

Evaluation of techniques for recovery of Angiostrongylus vasorum from Achatina fulica, a potential intermediate host

Mollusks are intermediate hosts of Angiostrongylus vasorum, which is a parasite with zoonotic potential for which canids are the definitive host. In this study, 180 specimens of Achatina fulica that had been experimentally infected with 1,000 L1 of A. vasorum were finely chopped to evaluate four tissue digestion techniques: 1% potassium hydroxide (group I); 3% hydrochloric acid (group II); 1% hydrochloric acid and pepsin (group III) with procedures at 37 ºC in a double boiler; and Baermann at 42 °C (group IV). On the first, eighth and thirtieth days after infection, L1, L2 and L3 were obtained, and 240 L3 recovered from group III were offered to a dog. The results were the following: group I: 59% L1, 7% L2, 13% L3; group II: 31% L1, 13% L2, 27% L3; group III: 23% L1, 22% L2, 30% L3; and group IV: 0.3% L1, 0.02% L2, 5% L3. Live larvae wereobserved in group III: 15.5% L1, 8.22% L3; group II: 9.8% L3; and group IV: 100% L1, L2 and L3. Larvae were detected 56 days after the dog became infected. All larvae stages of A. vasorum were recovered and identified through the techniques evaluated, thus confirming the potential of A. fulica as an intermediate host of the nematode

Epidemiological situation and molecular identification of cercarial stage in freshwater snails in Chao-Phraya Basin, Central Thailand

Objective: To investigate the prevalence of cercarial trematode infection in snails and to examine the reconstruction of the phylogenetic relationship to explain the molecular system of cercarial stage trematodes to estimate the infection rate of in the definite host from the Chao-Phraya Basin. Methods: The snails were collected from 10 provinces of the Chao-Phraya Basin, Thailand by stratified sampling method. The snails were examined for cercarial infection by the crushing method. All DNA specimens were amplified with internal transcribed spacer 3 (ITS3) and ITS4 primer based on PCR technique. The sequence data were aligned and used to reconstruct the phylogenetic tree by unweighted pair-group method with arithmetic means with 10 000 bootstraps. Results: The overall rate of cercarial infection was found to be 5.90%(122/2 067). Snails in the family Thiaridae were found to be in the highest prevalence followed by Lym-naeidae, Bithyniidae, Planorbidae, Viviparidae, and Ampullariidae, respectively, while the Buccinidae family (Clea helena) did not reveal any infections. The frequently found species of cercariae were parapleurolophocercous cercariae, cercariae and megarulous cercariae. The monophyletic tree separated the snails into five groups comprised of Heterophyidae, Strigeidae, Lecithodendriidae, Philophthalmidae and Echinostomatidae using the sequence of Angiostrongylus cantonensis as an out-group. Conclusions: This study was the first to report on cercarial infection in the Chao-Phraya Basin, Thailand. This revealed that a high variety of freshwater snails were infected by cercariae stage trematodes with a high prevalence. The sequence data of ITS2 can be used to investigate the phylogenetic relationships of trematodes at the family level and in each clade of different families separated by the definitive hosts.