Development of environmental loop-mediated isothermal amplification (eLAMP) diagnostic tool for Bulinus truncatus field detection.

BACKGROUND: Global changes are reshaping the distribution of vector-borne diseases by spreading vectors to previously non-endemic areas. Since 2013, urogenital schistosomiasis has emerged in Corsica and threatens European countries. Gastropod vectors release schistosome larvae that can infect humans who come into contact with freshwater bodies. Monitoring schistosomiasis host vectors is a prerequisite to understand and subsequently to control this pathogen transmission. Because malacological surveys are time consuming and require special expertise, the use of a simple molecular method is desirable. METHODS: The aim of this study is to develop a ready-to-use protocol using the LAMP (loop-mediated isothermal amplification) method to detect environmental DNA of Bulinus truncatus, vector of Schistosoma haematobium. Interestingly, LAMP method possesses all the characteristics required for adaptability to field conditions particularly in low-income countries: speed, simplicity, lyophilized reagents, low cost and robustness against DNA amplification inhibitors. We have tested this new method on Corsican water samples previously analysed by qPCR and ddPCR. RESULTS: We demonstrate that our diagnostic tool B. truncatus eLAMP (Bt-eLAMP) can detect the eDNA of Bulinus truncatus as effectively as the two other methods. Bt-eLAMP can even detect 1/4 of positive samples not detectable by qPCR. Moreover, the complete Bt-eLAMP protocol (sampling, sample pre-process, amplification and revelation) does not require sophisticated equipment and can be done in 1 ½ h. CONCLUSIONS: LAMP detection of environmental DNA provides large-scale sensitive surveillance of urogenital schistosomiasis possible by identifying potentially threatened areas. More generally, eLAMP method has great potential in vector-borne diseases and ecology.

Simultaneous genotyping of snails and infecting trematode parasites using high-throughput amplicon sequencing.

Several methodological issues currently hamper the study of entire trematode communities within populations of their intermediate snail hosts. Here we develop a new workflow using high-throughput amplicon sequencing to simultaneously genotype snail hosts and their infecting trematode parasites. We designed primers to amplify four snail and five trematode markers in a single multiplex PCR. While also applicable to other genera, we focused on medically and economically important snail genera within the superorder Hygrophila and targeted a broad taxonomic range of parasites within the class Trematoda. We tested the workflow using 417 Biomphalaria glabrata specimens experimentally infected with Schistosoma rodhaini, two strains of Schistosoma mansoni and combinations thereof. We evaluated the reliability of infection diagnostics, the robustness of the workflow, its specificity related to host and parasite identification, and the sensitivity to detect co-infections, immature infections and changes of parasite biomass during the infection process. Finally, we investigated its applicability in wild-caught snails of other genera naturally infected with a diverse range of trematodes. After stringent quality control the workflow allows the identification of snails to species level, and of trematodes to taxonomic levels ranging from family to strain. It is sensitive to detect immature infections and changes in parasite biomass described in previous experimental studies. Co-infections were successfully identified, opening the possibility to examine parasite-parasite interactions such as interspecific competition. Together, these results demonstrate that our workflow provides a powerful tool to analyse the processes shaping trematode communities within natural snail populations.

No pre-zygotic isolation mechanisms between Schistosoma haematobium and Schistosoma bovis parasites: From mating interactions to differential gene expression.

Species usually develop reproductive isolation mechanisms allowing them to avoid interbreeding. These preventive barriers can act before reproduction, "pre-zygotic barriers", or after reproduction, "post-zygotic barriers". Pre-zygotic barriers prevent unfavourable mating, while post-zygotic barriers determine the viability and selective success of the hybrid offspring. Hybridization in parasites and the underlying reproductive isolation mechanisms maintaining their genetic integrity have been overlooked. Using an integrated approach this work aims to quantify the relative importance of pre-zygotic barriers in Schistosoma haematobium x S. bovis crosses. These two co-endemic species cause schistosomiasis, one of the major debilitating parasitic diseases worldwide, and can hybridize naturally. Using mate choice experiments we first tested if a specific mate recognition system exists between both species. Second, using RNA-sequencing we analysed differential gene expression between homo- and hetero-specific pairing in male and female adult parasites. We show that homo- and hetero-specific pairing occurs randomly between these two species, and few genes in both sexes are affected by hetero-specific pairing. This suggests that i) mate choice is not a reproductive isolating factor, and that ii) no pre-zygotic barrier except spatial isolation "by the final vertebrate host" seems to limit interbreeding between these two species. Interestingly, among the few genes affected by the pairing status of the worms, some can be related to pathways affected during male and female interactions and may also present interesting candidates for species isolation mechanisms and hybridization in schistosome parasites.

Genetic diversity and relationships of the liver fluke Fasciola hepatica (Trematoda) with native and introduced definitive and intermediate hosts.

Fasciolosis is a worldwide spread parasitosis mainly caused by the trematode Fasciola hepatica. This disease is particularly important for public health in tropical regions, but it can also affect the economies of many developed countries due to large infections in domestic animals. Although several studies have tried to understand the transmission by studying the prevalence of different host species, only a few have used population genetic approaches to understand the links between domestic and wildlife infections. Here, we present the results of such genetic approach combined with classical parasitological data (prevalence and intensity) by studying domestic and wild definitive hosts from Camargue (southern France) where fasciolosis is considered as a problem. We found 60% of domestic hosts (cattle) infected with F. hepatica but lower values in wild hosts (nutria, 19%; wild boars, 4.5%). We explored nine variable microsatellite loci for 1,148 adult flukes recovered from four different populations (non-treated cattle, treated cattle, nutria and wild boars). Populations from the four groups differed, though we found a number of migrants particularly non-treated cattle and nutria. Overall, we detected 729 different multilocus genotypes (from 783 completely genotyped individuals) and only 46 genotypes repeated across samples. Finally, we experimentally infected native and introduced intermediate snail hosts to explore their compatibility with F. hepatica and assess the risks of fasciolosis expansion in the region. The introduced species Galba truncatula and Pseudosuccinea columella attained the higher values of overall compatibility in relation to the European species. However, concerning the origin, sympatric combinations of G. truncatula were more compatible (higher prevalence, intensity and survival) than the allopatric tested. According to our results, we should note that the assessment of epidemiological risks cannot be limited to a single host-parasite system, but should focus on understanding the diversity of hosts in the heterogeneous environment through space and time.

A rapid diagnostic multiplex PCR approach for xenomonitoring of human and animal schistosomiasis in a 'One Health' context.

Studying the epidemiology of schistosomiasis-the most prevalent gastropod-borne human disease and an economic burden for the livestock industry-relies on adequate monitoring tools. Here we describe a molecular assay for detecting human and animal African schistosome species in their planorbid gastropod host (xenomonitoring) using a two-step approach. First, schistosome infections are detected and discriminated from other trematode infections using a multiplex polymerase chain reaction (PCR) that includes a trematode-specific marker (in 18S rDNA), a Schistosoma genus-specific marker (in internal transcribed spacer 2 [ITS2]) and a general gastropod marker (in 18S rDNA) as an internal control. Upon Schistosoma sp. detection, a second multiplex PCR is performed to discriminate among Schistosoma haematobium, Schistosoma mansoni, Schistosoma mattheei and Schistosoma bovis/Schistosoma curassoni/Schistosoma guineensis using markers of differential lengths in the cytochrome c oxidase subunit 1 (COX1) gene. The specificity of these assays was validated with adult worms, naturally infected gastropods and human urine and stool samples. Sensitivity was tested on experimentally infected snail specimens that were sacrificed 10 and 40 days post-infection in order to mimic a natural prepatent and mature infection, respectively. The assay provides a diagnostic tool to support the xenomonitoring of planorbid gastropods for trematode infections in a One Health context, with a focus on the transmission monitoring of schistosomiasis.

Persistent establishment of a tropical disease in Europe: the preadaptation of schistosomes to overwinter.

BACKGROUND: Global changes promote the spread of infectious diseases worldwide. In this context, tropical urogenital schistosomiasis is now permanently established in Corsica since its first emergence in 2013. The local persistence of the tropical pathogens (schistosomes) responsible for urogenital schistosomiasis at such latitudes might be explained by (i) the presence of its intermediate host, the snail Bulinus truncatus, (ii) the recurrent local reseeding of schistosomes by their vertebrate hosts (either human or animal) every summer, and/or (iii) the maintenance and survival of schistosomes within their snail hosts over winter. METHODS: In this study we conducted an ecological experiment to assess the ability of temperate and tropical schistosome strains to survive in classical winter temperatures in Corsican rivers when infecting temperate (local) snail strains. We also quantified the ability of the schistosomes to complete their life-cycle post-overwintering when returned to classical summer water temperatures. RESULTS: Our results show that Mediterranean molluscs are locally adapted to winter conditions compared to tropical molluscs. Moreover, temperate and tropical schistosome strains equally survived the cold and produced viable offspring when returned to optimal temperatures. These results indicate that schistosomes can overwinter under temperate climates when infecting locally adapted snails and might partly explain the establishment and maintenance of schistosomes in Corsica from year to year. CONCLUSIONS: The observed broader thermal range of schistosomes compared to that of their snail hosts was unexpected and clearly indicates that the spread and establishment of schistosomiasis in temperate countries relies primarily on the presence of the locally adapted snail host lineages, currently known to be present in France, Italy, Portugal, Spain and Greece.

Epidemiological surveillance of schistosomiasis outbreak in Corsica (France): Are animal reservoir hosts implicated in local transmission?

Environmental and anthropogenic changes are expected to promote emergence and spread of pathogens worldwide. Since 2013, human urogenital schistosomiasis is established in Corsica island (France). Schistosomiasis is a parasitic disease affecting both humans and animals. The parasite involved in the Corsican outbreak is a hybrid form between Schistosoma haematobium, a human parasite, and Schistosoma bovis, a livestock parasite. S. bovis has been detected in Corsican livestock few decades ago raising the questions whether hybridization occurred in Corsica and if animals could behave as a reservoir for the recently established parasite lineage. The latter hypothesis has huge epidemiological outcomes since the emergence of a zoonotic lineage of schistosomes would be considerably harder to control and eradicate the disease locally and definitively needs to be verified. In this study we combined a sero-epidemiological survey on ruminants and a rodent trapping campaign to check whether schistosomes could shift on vertebrate hosts other than humans. A total of 3,519 domesticated animals (1,147 cattle; 671 goats and 1,701 sheep) from 160 farms established in 14 municipalities were sampled. From these 3,519 screened animals, 17 were found to be serologically positive but were ultimately considered as false positive after complementary analyses. Additionally, our 7-day extensive rodent trapping (i.e. 1,949 traps placed) resulted in the capture of a total of 34 rats (Rattus rattus) and 4 mice (Mus musculus). Despite the low number of rodents captured, molecular diagnostic tests showed that two of them have been found to be infected by schistosomes. Given the low abundance of rodents and the low parasitic prevalence and intensity among rodents, it is unlikely that neither rats nor ruminants play a significant role in the maintenance of schistosomiasis outbreak in Corsica. Finally, the most likely hypothesis is that local people initially infected in 2013 re-contaminated the river during subsequent summers, however we cannot definitively rule out the possibility of an animal species acting as reservoir host.

Persistence of schistosomal transmission linked to the Cavu river in southern Corsica since 2013.

Seven cases of urogenital schistosomiasis occurred in Corsica in 2015 and 2016. The episodes were related to exposure to the same river and involved the same parasite strain as an outbreak with 106 cases in summer 2013. The connection calls for further investigations on the presence of an animal reservoir and the survival of infested snails during winter. However, recontamination of the river from previously infected bathers remains the most likely hypothesis.