Molecular investigation of Neorickettsia risticii in trematodes and snails in a region with serological evidence of this agent in horses, state of Rio de Janeiro / Pesquisa molecular de Neorickettsia risticii em trematódeos e moluscos em região com evidência sorológica do agente em equinos, estado do Rio de Janeiro

In Brazil, some studies have indicated that Neorickettsia risticii circulates in horses, but it is unclear which are the possible intermediate vectors of this bacterium in the country. The aim of this study was to use molecular techniques in order to analyze the presence of N. risticii in snails and larval stages of trematodes in farms in a region with a history of seroreactive horses towards this bacterium, in Rio de Janeiro, Brazil. Convenience sampling was used in the studied region. The collected snails were exposed to incandescent light (60W) for 2-4 hours in order to investigate trematodes in larval forms. Deoxyribonucleic acid (DNA) was extracted from snail tissue and trematode. Real-time PCR (qPCR) technique was used to investigate the presence of a 16S rRNA gene fragment of N. risticii. Snail specimens (n=410) were collected from 11 horse-breeding farms, and the following species were identified: Melanoides tuberculata, Pomacea sp., Biomphalaria tenagophila, Physa acuta, Drepanotrema anatinum and Biomphalaria straminea. Only 3.17% (n=13/410) of the collected snails were infected by trematodes. The cercariae obtained from these snails were classified as Megalourous cercariae, Pleurolophocercus cercariae and Furcocercous cercariae. There was no amplification of the target DNA of N. risticii in the snail and trematode samples tested by qPCR. Based on these data, the transmission of N. risticii by trematodes using these snail species in this region does not appear to occur or occurs at very low rates. Thus, further studies are needed in order to clarify which species of invertebrate hosts are infected by this bacterium and potentially participate in the transmission chain of equine neorickettsiosis in the state of Rio de Janeiro, Brazil.(AU)

No Brasil, estudos apontam a circulação de Neorickettsia risticii em equinos, contudo não estão claros quais os possíveis vetores intermediários dessa bactéria no país. O objetivo do presente estudo foi analisar a presença de N. risticii, utilizando-se técnicas moleculares, em caramujos e estágios larvais de trematódeos em propriedades rurais de uma região com histórico de equinos sororreativos para essa bactéria, no Rio de Janeiro, Brasil. Uma amostragem por conveniência foi utilizada na região de estudo. Os caramujos coletados foram expostos à luz incandescente (60W) durante duas-quatro horas para a investigação de trematódeos nas formas larvais. A extração de ácido desoxirribonucleico (DNA) foi realizada em tecidos de caramujos e trematódeos. A técnica de PCR em tempo real (qPCR) foi utilizada para investigar a presença de um fragmento do gene 16S rRNA de N. risticii. Foram coletados 410 espécimes de caramujos de 11 propriedades com criações de equinos, sendo identificadas as seguintes espécies: Melanoides tuberculata, Pomacea sp., Biomphalaria tenagophila, Physa acuta, Drepanotrema anatinum e Biomphalaria straminea. Apenas 3,17% (n=13/410) dos caramujos identificados estavam infectados por trematódeos. As cercárias obtidas desses caramujos foram classificadas em Megalourous cercariae, Pleurolophocercus cercariae e Furcocercous cercariae. Não foi observada a amplificação do DNA-alvo de N. risticii, por meio da qPCR, em nenhuma das amostras de caramujos e trematódeos testadas. Com base nesses dados, a transmissão de N. risticii por trematódeos que utilizam as espécies de caramujos nessa região parece não ocorrer ou ocorre a taxas muito reduzidas. Portanto, novos estudos são necessários para elucidar quais espécies de hospedeiros invertebrados se infectam por essa bactéria e potencialmente participam da cadeia de transmissão da neorickettsiose equina no estado do Rio de Janeiro, Brasil.(AU)

Isolation and characterization of cultivable fermentative bacteria from the intestine of two edible snails, Helixpomatia and Cornu aspersum (Gastropoda: Pulmonata)

The intestinal microbiota of the edible snails Cornu aspersum fSyn: H. aspersa), and Helix pomatia were investigated by culture-based methods, 16S rRNA sequence analyses and phenotypic characterisations. The study was carried out on aestivating snails and two populations of H. pomatia were considered. The cultivable bacteria dominated in the distal part of the intestine, with up to 5.10(9) CFU g -1, but the Swedish H. pomatia appeared significantly less colonised, suggesting a higher sensitivity of its microbiota to climatic change. All the strains, but one, shared ¡Ý 97 percent sequence identity with reference strains. They were arranged into two taxa: the Gamma Proteobacteria with Buttiauxella, Citrobacter, Enterobacter, Kluyvera, Obesumbacterium, Raoultella and the Firmicutes with Enterococcus, Lactococcus, and Clostridium. According to the literature, these genera are mostly assigned to enteric environments or to phyllosphere, data in favour of culturing snails in contact with soil and plants. None of the strains were able to digest filter paper, Avicel cellulose or carboxymethyl cellulose (CMC). Acetogens and methanogenic archaea were not cultivated, so the fate of hydrogen remains questionable. This microbiota could play important roles in the digestive process (fermentation) and the energy supply of the snail (L-lactate, acetate). The choice of cereals and plants by snail farmers should take into account the fermentative abilities of the intestinal microbiota.

Molecular characterisation of intermediate snail hosts and the search for resistence genes

The relationship between schistosomes and their intermediate hosts is an extremely intricate one with strains and species of the parasite depending on particular species of snail, which in turn may vary in their susceptibility to the parasites. In order to gain a better understanding of the epidemiology of the disease we have been investigating the use of molecular markers for snail identification and for studying host-parasite relationships. In this paper we will draw on examples concerning schistosomiasis in West and East Africa to illustrate how a molecular analysis can be used as part of a total evidence approach to characterisation of Bulinus species and provide insights into parasite transmission. Particular emphasis is given to ribosomal RNA genes (rRNA), random amplified polymorphic DNA (RAPDs) and the mitochondrial gene cytochrome oxidase I (COI). Snails resistant to infection occur naturally and there is a genetic basis for this resistance. In Biomphalaria glabrata resistance to Schistosoma mansoni is known to be a polygenic trait and we have initiated a preliminary search for snail genomic regions linked to, or involved in, resistance by using a RAPD based approach in conjunction with progeny pooling methods. We are currently characterising a variety of STSs (sequence tagged sites) associated with resistance. These can be used for local linkage and interval mapping to define genomic regions associated with the resistance trait. The development of such markers into simple dot-blot or specif PCR-based assays may have a direct and practical application for the identification of resistant snails in natural populations.