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Abstract The three most important genera of snails for the transmission of schistosomes are Bulinus, Biomphalaria and Oncomelania. Each of these genera, found in two distantly related families, includes species that act as the intermediate host for one of the three most widespread schistosome species infecting humans, Schistosoma haematobium, S. mansoni and S. japonicum, respectively. An important step in the fight against schistosomiasis in Asia has been taken with the publication of the article “Chromosome-level genome assembly of Oncomelania hupensis: the intermediate snail host of Schistosoma japonicum”, which means that genomes for all three major genera, including species across three continents, are now available in the public domain. This includes the first genomes of African snail vectors, namely Biomphalaria sudanica, Bi. pfeifferi and Bulinus truncatus, as well as high-quality chromosome level assemblies for South American Bi. glabrata. Most importantly, the wealth of new genomic and transcriptomic data is helping to establish the specific molecular mechanisms that underly compatibility between snails and their schistosomes, which although diverse and complex, may help to identify potential targets dictating host parasite interactions that can be utilised in future transmission control strategies. This new work on Oncomelania hupensis and indeed studies on other snail vectors, which provide deep insights into the genome, will stimulate research that may well lead to new and much needed control interventions. Graphical Abstract

Abstract Background Control and elimination of schistosomiasis is an arduous task, with current strategies proving inadequate to break transmission. Exploration of genetic approaches to interrupt Schistosoma mansoni transmission, the causative agent for human intestinal schistosomiasis in sub-Saharan Africa and South America, has led to genomic research of the snail vector hosts of the genus Biomphalaria. Few complete genomic resources exist, with African Biomphalaria species being particularly underrepresented despite this being where the majority of S. mansoni infections occur. Here we generate and annotate the first genome assembly of Biomphalaria sudanica sensu lato, a species responsible for S. mansoni transmission in lake and marsh habitats of the African Rift Valley. Supported by whole-genome diversity data among five inbred lines, we describe orthologs of immune-relevant gene regions in the South American vector B. glabrata and present a bioinformatic pipeline to identify candidate novel pathogen recognition receptors (PRRs). Results De novo genome and transcriptome assembly of inbred B. sudanica originating from the shoreline of Lake Victoria (Kisumu, Kenya) resulted in a haploid genome size of ~ 944.2 Mb (6,728 fragments, N50 = 1.067 Mb), comprising 23,598 genes (BUSCO = 93.6% complete). The B. sudanica genome contains orthologues to all described immune genes/regions tied to protection against S. mansoni in B. glabrata, including the polymorphic transmembrane clusters (PTC1 and PTC2), RADres, and other loci. The B. sudanica PTC2 candidate immune genomic region contained many PRR-like genes across a much wider genomic region than has been shown in B. glabrata, as well as a large inversion between species. High levels of intra-species nucleotide diversity were seen in PTC2, as well as in regions linked to PTC1 and RADres orthologues. Immune related and putative PRR gene families were significantly over-represented in the sub-set of B. sudanica genes determined as hyperdiverse, including high extracellular diversity in transmembrane genes, which could be under pathogen-mediated balancing selection. However, no overall expansion in immunity related genes was seen in African compared to South American lineages. Conclusions The B. sudanica genome and analyses presented here will facilitate future research in vector immune defense mechanisms against pathogens. This genomic/transcriptomic resource provides necessary data for the future development of molecular snail vector control/surveillance tools, facilitating schistosome transmission interruption mechanisms in Africa.

Schistosomiasis is a neglected tropical disease (NTD) caused by infection with parasitic trematodes of the genus Schistosoma that can lead to debilitating morbidity and mortality. The World Health Organization recommend molecular xenomonitoring of Biomphalaria spp. freshwater snail intermediate hosts of Schistosoma mansoni to identify highly focal intestinal schistosomiasis transmission sites and monitor disease transmission, particularly in low-endemicity areas. A standardised protocol to do this, however, is needed. Here, two previously published primer sets were selected to develop and validate a multiplex molecular xenomonitoring end-point PCR assay capable of detecting S. mansoni infections within individual Biomphalaria spp. missed by cercarial shedding. The assay proved highly sensitive and highly specific in detecting and amplifying S. mansoni DNA and also proved highly sensitive in detecting and amplifying non-S. mansoni trematode DNA. The optimised assay was then used to screen Biomphalaria spp. collected from a S. mansoni-endemic area for infection and successfully detected S. mansoni infections missed by cercarial shedding as well as infections with non-S. mansoni trematodes. The continued development and use of molecular xenomonitoring assays such as this will aid in improving disease control efforts, significantly reducing disease-related morbidities experienced by those in schistosomiasis-endemic areas.

Interactions between Schistosoma mansoni and its snail host are understood primarily through experimental work with one South American vector species, Biomphalaria glabrata. However, 90% of schistosomiasis transmission occurs in Africa, where a diversity of Biomphalaria species may serve as vectors. With the long-term goal of determining the genetic and ecological determinants of infection in African snail hosts, we developed genetic models of Biomphalaria sudanica, a principal vector in the African Great Lakes. We determined laboratory infection dynamics of two S. mansoni lines in four B. sudanica lines. We measured the effects of the following variables on infection success and the number of cercariae produced (infection intensity): (i) the combination of parasite and snail line; (ii) the dose of parasites; and (iii) the size of snail at time of exposure. We found one snail line to be almost completely incompatible with both parasite lines, while other snail lines showed a polymorphism in compatibility: compatible with one parasite line while incompatible with another. Interestingly, these patterns were opposite in some of the snail lines. The parasite-snail combination had no significant effect on the number of cercariae produced in a successful infection. Miracidia dose had a strong effect on infection status, in that higher doses led to a greater proportion of infected snails, but had no effect on infection intensity. In one of the snail-schistosome combinations, snail size at the time of exposure affected both infection status and cercarial production in that the smallest size class of snails (1.5–2.9 mm) had the highest infection rates, and produced the greatest number of cercariae, suggesting that immunity increases with age and development. The strongest predictor of the infection intensity was the size of snail at the time of shedding: 1 ​mm of snail growth equated to a 19% increase in cercarial production. These results strongly suggest that infection status is determined in part by the interaction between snail and schistosome genetic lines, consistent with a gene-for-gene or matching allele model. This foundational work provides rationale for determining the genetic interactions between African snails and schistosomes, which may be applied to control strategies.

Bulinus senegalensis and Bulinus umbilicatus, two sympatric freshwater snails found in temporal ponds in Senegal, were thought to be involved in the transmission of Schistosoma haematobium and/or Schistosoma curassoni. To better understand the role of these Bulinus species in the transmission of human and animal Schistosoma species, B. senegalensis and B. umbilicatus were collected in 2015, during a malacological survey, from a temporal pond in Niakhar, central Senegal. Snails were induced to shed cercariae on two consecutive days. Individual cercariae from each snail were collected and preserved for molecular identification. Infected snails were identified by analysis of a partial region of the cytochrome c oxidase subunit 1 (cox1) gene. Six individual cercariae shed from each infected snail were identified by analyses of the cox1, nuclear ITS and partial 18S rDNA regions. Of the 98 snails collected, one B. senegalensis had a mixed infection shedding S. haematobium, S. bovis and S. haematobium-S. bovis hybrid cercariae and one B. umbilicatus was found to be shedding only S. haematobium. These data provide molecular confirmation for B. senegalensis transmitting S. bovis and S. haematobium-S. bovis hybrids in Senegal. The multiple Bulinus species involved in the human urogenital schistosomiasis in Senegal provides a high force of transmission warranting detailed mapping, surveillance and regular treatment of at-risk populations.

The World Health Organization's revised NTD Roadmap and the newly launched Guidelines target elimination of schistosomiasis as a public health problem in all endemic areas by 2030. Key to meeting this goal is elucidating how selective pressures imposed by interventions shape parasite populations. Our aim was to identify any differential impact of a unique cluster-randomized tri-armed elimination intervention (biannual mass drug administration (MDA) applied alone or in association with either mollusciciding (snail control) or behavioural change interventions) across two Zanzibarian islands (Pemba and Unguja) on the population genetic composition of Schistosoma haematobium over space and time. Fifteen microsatellite loci were used to analyse individual miracidia collected from infected individuals across islands and intervention arms at the start (2012 baseline: 1,522 miracidia from 176 children; 303 from 43 adults; age-range 6-75, mean 12.7 years) and at year 5 (2016: 1,486 miracidia from 146 children; 214 from 25 adults; age-range 9-46, mean 12.4 years). Measures of genetic diversity included allelic richness (Ar), Expected (He) and Observed heterozygosity (Ho), inbreeding coefficient (FST), parentage analysis, estimated worm burden, worm fecundity, and genetic sub-structuring. There was little evidence of differential selective pressures on population genetic diversity, inbreeding or estimated worm burdens by treatment arm, with only the MDA+snail control arm within Unguja showing trends towards reduced diversity and altered inbreeding over time. The greatest differences overall, both in terms of parasite fecundity and genetic sub-structuring, were observed between the islands, consistent with Pemba's persistently higher mean infection intensities compared to neighbouring Unguja, and within islands in terms of infection hotspots (across three definitions). These findings highlight the important contribution of population genetic analyses to elucidate extensive genetic diversity and biological drivers, including potential gene-environmental factors, that may override short term selective pressures imposed by differential disease control strategies. Trial Registration: ClinicalTrials.gov ISRCTN48837681.

BackgroundThe Zanzibar Archipelago (Pemba and Unguja islands) is targeted for the elimination of human urogenital schistosomiasis caused by infection with Schistosoma haematobium where the intermediate snail host is Bulinus globosus. Following multiple studies, it has remained unclear if B. nasutus (a snail species that occupies geographically distinct regions on the Archipelago) is involved in S. haematobium transmission on Zanzibar. Additionally, S. haematobium was thought to be the only Schistosoma species present on the Zanzibar Archipelago until the sympatric transmission of S. bovis, a parasite of ruminants, was recently identified. Here we re-assess the epidemiology of schistosomiasis on Pemba and Unguja together with the role and genetic diversity of the Bulinus spp. involved in transmission.Methodology/principal findingsMalacological and parasitological surveys were conducted between 2016 and 2019. In total, 11,116 Bulinus spp. snails were collected from 65 of 112 freshwater bodies surveyed. Bulinus species identification were determined using mitochondrial cox1 sequences for a representative subset of collected Bulinus (n = 504) and together with archived museum specimens (n = 6), 433 B. globosus and 77 B. nasutus were identified. Phylogenetic analysis of cox1 haplotypes revealed three distinct populations of B. globosus, two with an overlapping distribution on Pemba and one on Unguja. For B. nasutus, only a single clade with matching haplotypes was observed across the islands and included reference sequences from Kenya. Schistosoma haematobium cercariae (n = 158) were identified from 12 infected B. globosus and one B. nasutus collected between 2016 and 2019 in Pemba, and cercariae originating from 69 Bulinus spp. archived in museum collections. Schistosoma bovis cercariae (n = 21) were identified from seven additional B. globosus collected between 2016 and 2019 in Pemba. By analysing a partial mitochondrial cox1 region and the nuclear ITS (1-5.8S-2) rDNA region of Schistosoma cercariae, we identified 18 S. haematobium and three S. bovis haplotypes representing populations associated with mainland Africa and the Indian Ocean Islands (Zanzibar, Madagascar, Mauritius and Mafia).Conclusions/significanceThe individual B. nasutus on Pemba infected with S. haematobium demonstrates that B. nasutus could also play a role in the local transmission of S. haematobium. We provide preliminary evidence that intraspecific variability of S. haematobium on Pemba may increase the transmission potential of S. haematobium locally due to the expanded intermediate host range, and that the presence of S. bovis complicates the environmental surveillance of schistosome infections.

Abstract Background Urogenital schistosomiasis, caused by infection with Schistosoma haematobium, is endemic in Niger but complicated by the presence of Schistosoma bovis, Schistosoma curassoni and S. haematobium group hybrids along with various Bulinus snail intermediate host species. Establishing the schistosomes and snails involved in transmission aids disease surveillance whilst providing insights into snail-schistosome interactions/compatibilities and biology. Methods Infected Bulinus spp. were collected from 16 villages north and south of the Niamey region, Niger, between 2011 and 2015. From each Bulinus spp., 20–52 cercariae shed were analysed using microsatellite markers and a subset identified using the mitochondrial (mt) cox1 and nuclear ITS1 + 2 and 18S DNA regions. Infected Bulinus spp. were identified using both morphological and molecular analysis (partial mt cox1 region). Results A total of 87 infected Bulinus from 24 sites were found, 29 were molecularly confirmed as B. truncatus, three as B. forskalii and four as B. globosus. The remaining samples were morphologically identified as B. truncatus (n = 49) and B. forskalii (n = 2). The microsatellite analysis of 1124 cercariae revealed 186 cercarial multilocus genotypes (MLGs). Identical cercarial genotypes were frequently (60%) identified from the same snail (clonal populations from a single miracidia); however, several (40%) of the snails had cercariae of different genotypes (2–10 MLG’s) indicating multiple miracidial infections. Fifty-seven of the B. truncatus and all of the B. forskalii and B. globosus were shedding the Bovid schistosome S. bovis. The other B. truncatus were shedding the human schistosomes, S. haematobium (n = 6) and the S. haematobium group hybrids (n = 13). Two B. truncatus had co-infections with S. haematobium and S. haematobium group hybrids whilst no co-infections with S. bovis were observed. Conclusions This study has advanced our understanding of human and bovid schistosomiasis transmission in the Niger River Valley region. Human Schistosoma species/forms (S. haematobium and S. haematobium hybrids) were found transmitted only in five villages whereas those causing veterinary schistosomiasis (S. bovis), were found in most villages. Bulinus truncatus was most abundant, transmitting all Schistosoma species, while the less abundant B. forskalii and B. globosus, only transmitted S. bovis. Our data suggest that species-specific biological traits may exist in relation to co-infections, snail-schistosome compatibility and intramolluscan schistosome development.

Abstract Background Accurate diagnosis of urogenital schistosomiasis is vital for surveillance and control programmes. While a number of diagnostic techniques are available there is a need for simple, rapid and highly sensitive point-of-need (PON) tests in areas where infection prevalence and intensity are low. Recombinase Polymerase Amplification (RPA) is a sensitive isothermal molecular diagnostic technology that is rapid, portable and has been used at the PON for several pathogens. Results A real time fluorescence RPA assay (RT-ShDra1-RPA) targeting the Schistosoma haematobium Dra1 genomic repeat region was developed and was able to detect 1 fg of S. haematobium gDNA. Results were obtained within 10 minutes using a small portable battery powered tube scanner device that incubated reactions at 40 °C, whilst detecting DNA amplification and fluorescence over time. The assay’s performance was evaluated using 20 urine samples, with varying S. haematobium egg counts, from school children from Pemba Island, Zanzibar Archipelago, Tanzania. Prior to RPA analysis, samples were prepared using a quick crude field DNA extraction method, the Speed Extract Kit (Qiagen, Manchester, UK). Positive assay results were obtained from urine samples with egg counts of 1–926 eggs/10 ml, except for two samples, which had inconclusive results. These two samples had egg counts of two and three eggs/10 ml of urine. Conclusions The RT-ShDra1-RPA assay proved robust for S. haematobium gDNA detection and was able to amplify and detect S. haematobium DNA in urine samples from infected patients. The assay’s speed and portability, together with the use of crude sample preparation methods, could advance the rapid molecular diagnosis of urogenital schistosomiasis at the PON within endemic countries.

Abstract Background Sound knowledge of the abundance and distribution of intermediate host snails is key to understanding schistosomiasis transmission and to inform effective interventions in endemic areas. Methods A longitudinal field survey of freshwater snails of biomedical importance was undertaken in the Niger River Valley (NRV) between July 2011 and January 2016, targeting Bulinus spp. and Biomphalaria pfeifferi (intermediate hosts of Schistosoma spp.), and Radix natalensis (intermediate host of Fasciola spp.). Monthly snail collections were carried out in 92 sites, near 20 localities endemic for S. haematobium. All bulinids and Bi. pfeifferi were inspected for infection with Schistosoma spp., and R. natalensis for infection with Fasciola spp. Results Bulinus truncatus was the most abundant species found, followed by Bulinus forskalii, R. natalensis and Bi. pfeifferi. High abundance was associated with irrigation canals for all species with highest numbers of Bulinus spp. and R. natalensis. Seasonality in abundance was statistically significant in all species, with greater numbers associated with dry season months in the first half of the year. Both B. truncatus and R. natalensis showed a negative association with some wet season months, particularly August. Prevalences of Schistosoma spp. within snails across the entire study were as follows: Bi. pfeifferi: 3.45% (79/2290); B. truncatus: 0.8% (342/42,500); and B. forskalii: 0.2% (24/11,989). No R. natalensis (n = 2530) were infected. Seasonality of infection was evident for B. truncatus, with highest proportions shedding in the middle of the dry season and lowest in the rainy season, and month being a significant predictor of infection. Bulinus spp. and Bi. pfeifferi showed a significant correlation of snail abundance with the number of snails shedding. In B. truncatus, both prevalence of Schistosoma spp. infection, and abundance of shedding snails were significantly higher in pond habitats than in irrigation canals. Conclusions Evidence of seasonality in both overall snail abundance and infection with Schistosoma spp. in B. truncatus, the main intermediate host in the region, has significant implications for monitoring and interrupting transmission of Schistosoma spp. in the NRV. Monthly longitudinal surveys, representing intensive sampling effort have provided the resolution needed to ascertain both temporal and spatial trends in this study. These data can inform planning of interventions and treatment within the region.

Schistosoma bovis is a parasitic trematode of ungulates transmitted by freshwater snails in Sub-Saharan Africa causing bovine intestinal schistosomiasis that leads to chronic morbidity and significant agricultural economic losses. The recently reported occurrence of Bulinus globosus infected with S. bovis for the first time on Pemba Island (Zanzibar, United Republic of Tanzania) is a cause of concern for livestock/wildlife health and complicates the surveillance of Schistosoma haematobium. To confirm that local cattle are infected with S. bovis, fresh faecal samples were collected from six adult cows surrounding two schistosomiasis transmission sites in Kinyasini, Pemba Island. Schistosome eggs were concentrated, egg hatching stimulated and miracidia were individually captured and identified by analysis of the partial mitochondrial cytochrome c oxidase subunit 1 (cox1) and the partial nuclear internal transcribed spacer region (ITS1+5.8S+ITS2). Two S. bovis miracidia were collected from one faecal sample with two cox1 haplotypes, one matching cox1 data obtained from S. bovis cercariae, collected previously at the same site in Pemba, the other matching S. bovis cox1 data originating from coastal Tanzania. The findings conclude that S. bovis transmission has been established on Pemba Island and is likely to have been imported through livestock trade with East Africa. Increasing the sensitivity of non-invasive diagnostics for bovine schistosomiasis, together with wider sampling, will enable a better assessment on the epidemiology of S. bovis on Pemba Island.

Abstract Background Schistosomiasis is hyper-endemic in the Lake Victoria basin; with intestinal schistosomiasis plaguing communities adjacent to the lake, where the intermediate host snails live. The two intermediate host species of Schistosoma mansoni in the Mwanza region are Biomphalaria sudanica, found on the banks of the lakes, and B. choanomphala, found in the lake itself. There are few longitudinal surveys documenting changing abundance and differential transmission patterns of these Biomphalaria snails across seasons and years. We undertook 15 field surveys at 26 sites over four years to determine the parameters that influence Biomphalaria abundance, presence of S. mansoni-shedding snails and impact of schistosomiasis treatment interventions on transmission potential in the Mwanza region. Results Statistical analysis revealed seasonal difference in the abundance of B. sudanica with the highest number of snails found in the dry season (Kruskal-Wallis χ 2 = 37.231, df = 3, P

Schistosomiasis, a neglected tropical disease of medical and veterinary importance, transmitted through specific freshwater snail intermediate hosts, is targeted for elimination in several endemic regions in sub-Saharan Africa. Multi-disciplinary methods are required for both human and environmental diagnostics to certify schistosomiasis elimination when eventually reached. Molecular xenomonitoring protocols, a DNA-based detection method for screening disease vectors, have been developed and trialed for parasites transmitted by hematophagous insects, such as filarial worms and trypanosomes, yet few have been extensively trialed or proven reliable for the intermediate host snails transmitting schistosomes. Here, previously published universal and Schistosoma-specific internal transcribed spacer (ITS) rDNA primers were adapted into a triplex PCR primer assay that allowed for simple, robust, and rapid detection of Schistosoma haematobium and Schistosoma bovis in Bulinus snails. We showed this two-step protocol could sensitively detect DNA of a single larval schistosome from experimentally infected snails and demonstrate its functionality for detecting S. haematobium infections in wild-caught snails from Zanzibar. Such surveillance tools are a necessity for succeeding in and certifying the 2030 control and elimination goals set by the World Health Organization.

Accurate diagnosis of urogenital schistosomiasis is crucial for disease surveillance and control. Routine diagnostic methods, however, lack sensitivity when assessing patients with low levels of infection still able to maintain pathogen transmission. Therefore, there is a need for highly sensitive diagnostic tools that can be used at the point-of-care in endemic areas. Recombinase polymerase amplification (RPA) is a rapid and sensitive diagnostic tool that has been used to diagnose several pathogens at the point-of-care. Here, the analytical performance of a previously developed RPA assay (RT-ShDra1-RPA) targeting the Schistosoma haematobium Dra1 genomic region was assessed using commercially synthesised S. haematobium Dra1 copies and laboratory-prepared samples spiked with S. haematobium eggs. Clinical performance was also assessed by comparing diagnostic outcomes with that of a reference diagnostic standard, urine-egg microscopy. The RT-ShDra1-RPA was able to detect 1 × 101 copies of commercially synthesised Dra1 DNA as well as one S. haematobium egg within laboratory-spiked ddH2O samples. When compared with urine-egg microscopy, the overall sensitivity and specificity of the RT-ShDra1-RPA assay was 93.7% (±88.7–96.9) and 100% (±69.1–100), respectively. Positive and negative predictive values were 100% (±97.5–100) and 50% (±27.2–72.8), respectively. The RT-ShDra1-RPA therefore shows promise as a rapid and highly sensitive diagnostic tool able to diagnose urogenital schistosomiasis at the point-of-care.