Jean-François Allienne

Outbreak of urogenital schistosomiasis in Corsica (France): an epidemiological case study

BACKGROUND Schistosomiasis is a snail-borne parasitic disease endemic in several tropical and subtropical countries. However, in the summer of 2013, an unexpected outbreak of urogenital schistosomiasis occurred in Corsica, with more than 120 local people or tourists infected. We used a multidisciplinary approach to investigate the epidemiology of urogenital schistosomiasis in Corsica, aiming to elucidate the origin of the outbreak. METHODS We did parasitological and malacological surveys at nine potential sites of infection. With the snails found, we carried out snail-parasite compatibility experiments by exposing snails to schistosome larvae recovered from the urine of a locally infected Corsican patient. Genetic analysis of both mitochondrial (cox1) and nuclear (internal transcribed spacer) DNA data from the Schistosoma eggs or miracidia recovered from the infected patients was conducted to elucidate the epidemiology of this outbreak. FINDINGS We identified two main infection foci along the Cavu River, with many Bulinus truncatus snails found in both locations. Of the 3544 snails recovered across all sites, none were naturally infected, but laboratory-based experimental infections confirmed their compatibility with the schistosomes isolated from patients. Molecular characterisation of 73 eggs or miracidia isolated from 12 patients showed infection with Schistosoma haematobium, S haematobium-Schistosoma bovis hybrids, and S bovis. Further sequence data analysis also showed that the Corsican schistosomes were closely related to those from Senegal in west Africa. INTERPRETATION The freshwater swimming pools of the Cavu River harbour many B truncatus snails, which are capable of transmitting S haematobium-group schistosomes. Our molecular data suggest that the parasites were imported into Corsica by individuals infected in west Africa, specifically Senegal. Hybridisation between S haematobium and the cattle schistosome S bovis had a putative role in this outbreak, showing how easily and rapidly urogenital schistosomiasis can be introduced and spread into novel areas where Bulinus snails are endemic, and how hybridisation could increase the colonisation potential of schistosomes. Furthermore our results show the potential risk of schistosomiasis outbreaks in other European areas, warranting close monitoring and surveillance of all potential transmission foci. FUNDING WHO, ANSES, RICET, and the Ministry of Health and Consumption.

A family of variable immunoglobulin and lectin domain containing molecules in the snail Biomphalaria glabrata

Technical limitations have hindered comprehensive studies of highly variable immune response molecules that are thought to have evolved due to pathogen-mediated selection such as fibrinogen-related proteins (FREPs) from Biomphalaria glabrata. FREPs combine upstream immunoglobulin superfamily (IgSF) domains with a C-terminal fibrinogen-related domain (FreD) and participate in reactions against trematode parasites. From RNAseq data we assembled a de novo reference transcriptome of B. glabrata to investigate the diversity of FREP transcripts. This study increased over two fold the number of bonafide FREP subfamilies and revealed important sequence diversity within FREP12 subfamily. We also report the discovery of related molecules that feature one or two IgSF domains associated with different C-terminal lectin domains, named C-type lectin-related proteins (CREPs) and Galectin-related protein (GREP). Together, the highly similar FREPs, CREPs and GREP were designated VIgL (Variable Immunoglobulin and Lectin domain containing molecules).

Schistosoma mansoni and Echinostoma caproni excretory-secretory products differentially affect gene expression in Biomphalaria glabrata embryonic cells.

Biomphalaria glabrata embryonic (Bge) cells have been shown to be a valuable in vitro cellular model for the study of snail host-parasite interactions. They both promote the growth and differentiation of various trematode species including Schistosoma mansoni, and Echinostoma caproni and share some morphological and functional features with circulating haemocytes. As an approach to investigate snail genes potentially regulated following exposure to trematode excretory-secretory (ES) products, we compared gene expression profiles of Bge cells exposed to saline solution, or saline solution containing ES products from S. mansoni or E. caproni, two trematode species parasitizing B. glabrata. Following differential display RT-PCR analysis we characterized 23 differentially displayed cDNAs and we focussed on the 5 cDNAs showing sequence similarity to known genes for expression validation. Using RT-PCR, we confirmed that ES products from S. mansoni and E. caproni differentially affect the expression levels of 4 out of the 5 transcripts. These partial transcripts corresponded to novel B. glabrata sequences, and showed significant sequence similarity to genes coding for (i) cytochrome C, (ii) methyl-binding proteins, (iii) glutamine synthetases, and (iv) protease inhibitors from the Kunitz family. The possible significance of these gene expression changes in host-parasite molecular interactions is discussed.

Introgressive hybridizations of Schistosoma haematobium by Schistosoma bovis at the origin of the first case report of schistosomiasis in Corsica (France, Europe)

This study concerns the first urinary schistosomiasis case observed in Corsica (France, Europe) occurring in a 12-year-old German boy. The aim was to identify the relationship between this Schistosoma haematobium infection and other schistosomes of the Schistosoma group with terminal-spined ova. Morphological and molecular analyses were conducted on the ova. The results showed that the schistosome responsible for the emergence of schistosomiasis in Corsica was due to S. haematobium introgressed by genes from S. bovis.

Cheap, rapid and efficient DNA extraction method to perform multilocus microsatellite genotyping on all Schistosoma mansoni stages

Schistosomes are endoparasites causing a serious human disease called schistosomiasis. The quantification of parasite genetic diversity is an essential component to understand the schistosomiasis epidemiology and disease transmission patterns. In this paper, we propose a novel assay for a rapid, low costly and efficient DNA extraction method of egg, larval and adult stages of Schistosoma mansoni. One euro makes possible to perform 60,000 DNA extraction reactions at top speed (only 15 min of incubation and 5 handling steps).

Invasive species threat: parasite phylogenetics reveals patterns and processes of host-switching between non-native and native captive freshwater turtles.

One of the major threats to biodiversity involves biological invasions with direct consequences on the stability of ecosystems. In this context, the role of parasites is not negligible as it may enhance the success of invaders. The red-eared slider, Trachemys scripta elegans, has been globally considered among the worst invasive species. Since its introduction through the pet trade, T. s. elegans is now widespread and represents a threat for indigenous species. Because T. s. elegans coexists with Emys orbicularis and Mauremys leprosa in Europe, it has been suggested it may compete with the native turtle species and transmit pathogens. We examined parasite transfer from American captive to the two native species that co-exist in artificial pools of a Turtle Farm in France. As model parasite species we used platyhelminth worms of the family Polystomatidae (Monogenea) because polystomes have been described from American turtles in their native range. Phylogenetic relationships among polystomes parasitizing chelonian host species that are geographically widespread show patterns of diversification more complex than expected. Using DNA barcoding to identify species from adult and/or polystome eggs, several cases of host switching from exotic to indigenous individuals were illustrated, corroborating that parasite transmission is important when considering the pet trade and in reintroduction programmes to reinforce wild populations of indigenous species.

Correlating Early Evolution of Parasitic Platyhelminths to Gondwana Breakup

Investigating patterns and processes of parasite diversification over ancient geological periods should involve comparisons of host and parasite phylogenies in a biogeographic context. It has been shown previously that the geographical distribution of host-specific parasites of sarcopterygians was guided, from Palaeozoic to Cainozoic times, mostly by evolution and diversification of their freshwater hosts. Here, we propose phylogenies of neobatrachian frogs and their specific parasites (Platyhelminthes, Monogenea) to investigate coevolutionary processes and historical biogeography of polystomes and further discuss all the possible assumptions that may account for the early evolution of these parasites. Phylogenetic analyses of concatenated rRNA nuclear genes (18S and partial 28S) supplemented by cophylogenetic and biogeographic vicariance analyses reveal four main parasite lineages that can be ascribed to centers of diversity, namely Australia, India, Africa, and South America. In addition, the relationships among these biogeographical monophyletic groups, substantiated by molecular dating, reflect sequential origins during the breakup of Gondwana. The Australian polystome lineage may have been isolated during the first stages of the breakup, whereas the Indian lineage would have arisen after the complete separation of western and eastern Gondwanan components. Next, polystomes would have codiverged with hyloid sensu stricto and ranoid frog lineages before the completion of South American and African plate separation. Ultimately, they would have undergone an extensive diversification in South America when their ancestral host families diversified. Therefore, the presence of polystome parasites in specific anuran host clades and in discrete geographic areas reveals the importance of biogeographic vicariance in diversification processes and supports the occurrence and radiation of amphibians over ancient and recent geological periods.

Natural Interactions between S. haematobium and S. guineensis in the Republic of Benin

Schistosomiasis is a parasitic disease which affects millions of people around the world, particularly in Africa. In this continent, different species are able to interbreed, like Schistosoma haematobium and Schistosoma guineensis, two schistosome species infecting humans. The Republic of Benin is known to harbor S. haematobium, but its geographical situation in between Nigeria, Mali, and Burkina Faso, where S. guineensis was found, raises the question about the possible presence of S. haematobium/S. guineensis hybrids in this country. We conducted morphological analyses on schistosome eggs and molecular analyses on schistosome larvae (high resolution melting (HRM) analysis and gene sequencing) in order to detect any natural interaction between these two species of schistosomes. The morphological results showed the presence of three egg morphotypes (S. haematobium, S. guineensis, and intermediate). Three genotypes were detected by ITS2 rDNA HRM analysis: S. haematobium, S. guineensis, and hybrid, and their percentages confirmed the results of the morphological analysis. However, sequencing of the CO1 mtDNA gene showed that all the samples from Benin belonged to S. haematobium. Our results provide the first evidence of introgression of S. guineensis genes in S. haematobium in Benin.

Sex-Biased Transcriptome of Schistosoma mansoni: Host-Parasite Interaction, Genetic Determinants and Epigenetic Regulators Are Associated with Sexual Differentiation

Background Among more than 20,000 species of hermaphroditic trematodes, Schistosomatidae are unusual since they have evolved gonochorism. In schistosomes, sex is determined by a female heterogametic system, but phenotypic sexual dimorphism appears only after infection of the vertebrate definitive host. The completion of gonad maturation occurs even later, after pairing. To date, the molecular mechanisms that trigger the sexual differentiation in these species remain unknown, and in vivo studies on the developing schistosomulum stages are lacking. To study the molecular basis of sex determination and sexual differentiation in schistosomes, we investigated the whole transcriptome of the human parasite Schistosoma mansoni in a stage- and sex-comparative manner. Methodology/ Principal Findings We performed a RNA-seq on males and females for five developmental stages: cercariae larvae, three in vivo schistosomulum stages and adults. We detected 7,168 genes differentially expressed between sexes in at least one of the developmental stages, and 4,065 of them were functionally annotated. Transcriptome data were completed with H3K27me3 histone modification analysis using ChIP-Seq before (in cercariae) and after (in adults) the phenotypic sexual dimorphism appearance. In this paper we present (i) candidate determinants of the sexual differentiation, (ii) sex-biased players of the interaction with the vertebrate host, and (iii) different dynamic of the H3K27me3 histone mark between sexes as an illustration of sex-biased epigenetic landscapes. Conclusions/ Significance Our work presents evidence that sexual differentiation in S. mansoni is accompanied by distinct male and female transcriptional landscapes of known players of the host-parasite crosstalk, genetic determinants and epigenetic regulators. Our results suggest that such combination could lead to the optimized sexual dimorphism of this parasitic species. As S. mansoni is pathogenic for humans, this study represents a promising source of therapeutic targets, providing not only data on the parasite development in interaction with its vertebrate host, but also new insights on its reproductive function.

Follow-up of the genetic diversity and snail infectivity of a Schistosoma mansoni strain from field to laboratory

Schistosoma mansoni is an endoparasite causing a serious human disease called schistosomiasis. The quantification of parasite genetic diversity is an essential component to understand schistosomiasis epidemiology and disease transmission patterns but some studies on parasite genetic diversity are performed using parasite laboratory strains. However, a potential discrepancy in level of genetic variation between field populations and laboratory strains may have various implications in our deductions. In this study, 246 adult worms were analysed on 15 microsatellite markers to investigate variation of genetic diversity between a founder field isolate and the nine successive laboratory generations during three years of laboratory maintenance. In parallel, we measured a parasite life trait (snail infectivity) at each generation in order to test a potential link between inbreeding and snail infectivity. Our genetic analyses demonstrate a significant genetic differentiation between all parasite generations and a significant isolation by time associated with a decrease in neutral genetic diversity that is likely to be the result of successive bottleneck events. However, while snail infectivity decreases sharply between field isolate and the first laboratory generation, this parasite life trait does not evolve between other laboratory generations and appeared disconnected from this continuous neutral genetic diversity loss. We hypothesize that a sufficient level of compatibility polymorphism at a genomic level is maintained independently of an increase of inbreeding, ensuring the stability in the parasite life trait.