A duplex tetra-primer ARMS-PCR assay to discriminate three species of the Schistosoma haematobium group: Schistosoma curassoni, S. bovis, S. haematobium and their hybrids.

BACKGROUND: The use of applications involving single nucleotide polymorphisms (SNPs) has greatly increased since the beginning of the 2000s, with the number of associated techniques expanding rapidly in the field of molecular research. Tetra-primer amplification refractory mutation system-PCR (T-ARMS-PCR) is one such technique involving SNP genotyping. It has the advantage of amplifying multiple alleles in a single reaction with the inclusion of an internal molecular control. We report here the development of a rapid, reliable and cost-effective duplex T-ARMS-PCR assay to distinguish between three Schistosoma species, namely Schistosoma haematobium (human parasite), Schistosoma bovis and Schistosoma curassoni (animal parasites), and their hybrids. This technique will facilitate studies of population genetics and the evolution of introgression events. METHODS: During the development of the technique we focused on one of the five inter-species internal transcribed spacer (ITS) SNPs and one of the inter-species 18S SNPs which, when combined, discriminate between all three Schistosoma species and their hybrid forms. We designed T-ARMS-PCR primers to amplify amplicons of specific lengths for each species, which in turn can then be visualized on an electrophoresis gel. This was further tested using laboratory and field-collected adult worms and field-collected larval stages (miracidia) from Spain, Egypt, Mali, Senegal and Ivory Coast. The combined duplex T-ARMS-PCR and ITS + 18S primer set was then used to differentiate the three species in a single reaction. RESULTS: The T-ARMS-PCR assay was able to detect DNA from both species being analysed at the maximum and minimum levels in the DNA ratios (95/5) tested. The duplex T-ARMS-PCR assay was also able to detect all hybrids tested and was validated by sequencing the ITS and the 18S amplicons of 148 of the field samples included in the study. CONCLUSIONS: The duplex tetra-primer ARMS-PCR assay described here can be applied to differentiate between Schistosoma species and their hybrid forms that infect humans and animals, thereby providing a method to investigate the epidemiology of these species in endemic areas. The addition of several markers in a single reaction saves considerable time and is of long-standing interest for investigating genetic populations.

Genetic profiles of Schistosoma haematobium parasites from Malian transmission hotspot areas.

BACKGROUND: Although schistosomiasis is a public health issue in Mali, little is known about the parasite genetic profile. The purpose of this study was to analyze the genetic profile of the schistosomes of Schistosoma haematobium group in school-aged children in various sites in Mali. METHODS: Urine samples were collected from 7 to 21 November 2021 and subjected to a filtration method for the presence S. haematobium eggs. The study took place in two schistosomiasis endemic villages (Fangouné Bamanan and Diakalèl), qualified as hotspots according to the World Health Organization (WHO) definition. Molecular genotyping on both Cox1 and ITS2/18S was used for eggs' taxonomic assignation. RESULTS: A total of 970 miracidia were individually collected from 63 school-aged children and stored on Whatman FTA cards for molecular analysis. After genotyping 42.0% (353/840) and 58.0% (487/840) of miracidia revealed Schistosoma bovis and S. haematobium Cox1 profiles, respectively; 95.7 (885/925) and 4.3% (40/925) revealed S. haematobium and S. haematobium/S. curassoni profiles for ITS/18S genes, respectively. There was a significant difference in the Cox1 and ITS2/18S profile distribution according to the village (P < 0.0001). Overall, 45.6% (360/789) were hybrids, of which 72.0% (322/447) were from Diakalèl. Three hybrids' profiles (Sb/Sc_ShxSc with 2.3%; Sb/Sc_ShxSh with 40.5%; Sh_ShxSc with 2.8%) and one pure profile (Sh_ShxSh with 54.4%) were identified. CONCLUSION: Our findings show, for the first time to our knowledge, high prevalence of hybrid schistosomes in Mali. More studies are needed on population genetics of schistosomes at the human and animal interface to evaluate the parasite's gene flow and its consequences on epidemiology of the disease as well as the transmission to humans.

Mating Interactions between Schistosoma bovis and S. mansoni and Compatibility of Their F1 Progeny with Biomphalaria glabrata and Bulinus truncatus.

Contrary to the majority of other Trematoda, Schistosoma species are gonochoric. Consequently, in endemic areas where several schistosome species overlap and can co-infect the same definitive host, there may be frequent opportunities for interspecific pairing. Our experimental study provides novel insight on the pairing behavior between Schistosoma bovis and S. mansoni in mixed infections in mice. We used six mate choice experiments to assess mating interactions between the two schistosome species. We show that mating between the two Schistosoma species is not random and that S. mansoni exhibits greater mate recognition compared to S. bovis. We also performed reciprocal crosses (male S. mansoni × female S. bovis) and (female S. mansoni × male S. bovis) that produce active swimming miracidia. These miracidia were genotyped by ITS2 sequencing and proposed for mollusc infection. Molecular analyses show that all the miracidia are parthenogenetically produced (i.e., their harbor the mother ITS2 genotype) and as a consequence can only infect the mollusc of the maternal species. Offspring produced by male S. mansoni × female S. bovis pairing can only infect Bulinus truncatus whereas offspring produced by female S. mansoni × male S. bovis can only infect Biomphalaria glabrata snails. Evolutionary and epidemiological consequences are discussed.