Morphological and genomic characterisation of the Schistosoma hybrid infecting humans in Europe reveals admixture between Schistosoma haematobium and Schistosoma bovis

Schistosomes cause schistosomiasis, the world’s second most important parasitic disease after malaria in terms of public health and social-economic impacts. A peculiar feature of these dioecious parasites is their ability to produce viable and fertile hybrid offspring. Originally only present in the tropics, schistosomiasis is now also endemic in southern Europe. Based on the analysis of two genetic markers the European schistosomes had previously been identified as hybrids between the livestock- and the human-infective species Schistosoma bovis and Schistosoma haematobium, respectively. Here, using PacBio long-read sequencing technology we performed genome assembly improvement and annotation of S. bovis, one of the parental species for which no satisfactory genome assembly was available. We then describe the whole genome introgression levels of the hybrid schistosomes, their morphometric parameters (eggs and adult worms) and their compatibility with two European snail strains used as vectors (Bulinus truncatus and Planorbarius metidjensis). Schistosome-snail compatibility is a key parameter for the parasites life cycle progression, and thus the capability of the parasite to establish in a given area. Our results show that this Schistosoma hybrid is strongly introgressed genetically, composed of 77% S. haematobium and 23% S. bovis origin. This genomic admixture suggests an ancient hybridization event and subsequent backcrosses with the human-specific species, S. haematobium, before its introduction in Corsica. We also show that egg morphology (commonly used as a species diagnostic) does not allow for accurate hybrid identification while genetic tests do.

Spillover, hybridization, and persistence in schistosome transmission dynamics at the human–animal interface

Zoonotic spillover and hybridization of parasites are major emerging public and veterinary health concerns at the interface of infectious disease biology, evolution, and control. Schistosomiasis is a neglected tropical disease of global importance caused by parasites of the Schistosoma genus, and the Schistosoma spp. system within Africa represents a key example of a system where spillover of animal parasites into human populations has enabled formation of hybrids. Combining model-based approaches and analyses of parasitological, molecular, and epidemiological data from northern Senegal, a region with a high prevalence of schistosome hybrids, we aimed to unravel the transmission dynamics of this complex multihost, multiparasite system. Using Bayesian methods and by estimating the basic reproduction number (R0), we evaluate the frequency of zoonotic spillover of Schistosoma bovis from livestock and the potential for onward transmission of hybrid S. bovis × S. haematobium offspring within human populations. We estimate R0 of hybrid schistosomes to be greater than the critical threshold of one (1.76; 95% CI 1.59 to 1.99), demonstrating the potential for hybridization to facilitate spread and establishment of schistosomiasis beyond its original geographical boundaries. We estimate R0 for S. bovis to be greater than one in cattle (1.43; 95% CI 1.24 to 1.85) but not in other ruminants, confirming cattle as the primary zoonotic reservoir. Through longitudinal simulations, we also show that where S. bovis and S. haematobium are coendemic (in livestock and humans respectively), the relative importance of zoonotic transmission is predicted to increase as the disease in humans nears elimination.

Hybridized Zoonotic Schistosoma Infections Result in Hybridized Morbidity Profiles: A Clinical Morbidity Study amongst Co-Infected Human Populations of Senegal

Hybridization of infectious agents is a major emerging public and veterinary health concern at the interface of evolution, epidemiology, and control. Whilst evidence of the extent of hybridization amongst parasites is increasing, their impact on morbidity remains largely unknown. This may be predicted to be particularly pertinent where parasites of animals with contrasting pathogenicity viably hybridize with human parasites. Recent research has revealed that viable zoonotic hybrids between human urogenital Schistosoma haematobium with intestinal Schistosoma species of livestock, notably Schistosoma bovis, can be highly prevalent across Africa and beyond. Examining human populations in Senegal, we found increased hepatic but decreased urogenital morbidity, and reduced improvement following treatment with praziquantel, in those infected with zoonotic hybrids compared to non-hybrids. Our results have implications for effective monitoring and evaluation of control programmes, and demonstrate for the first time the potential impact of parasite hybridizations on host morbidity.

Application of a Genus-Specific LAMP Assay for Schistosome Species to Detect Schistosoma haematobium x Schistosoma bovis Hybrids

Schistosomiasis is a disease of great medical and veterinary importance in tropical and subtropical regions caused by different species of parasitic flatworms of the genus Schistosoma. The emergence of natural hybrids of schistosomes indicate the risk of possible infection to humans and their zoonotic potential, specifically for Schistosoma haematobium and S. bovis. Hybrid schistosomes have the potential to replace existing species, generate new resistances, pathologies and extending host ranges. Hybrids may also confuse the serological, molecular and parasitological diagnosis. Currently, LAMP technology based on detection of nucleic acids is used for detection of many agents, including schistosomes. Here, we evaluate our previously developed species-specific LAMP assays for S. haematobium, S. mansoni, S. bovis and also the genus-specific LAMP for the simultaneous detection of several Schistosoma species against both DNA from pure and, for the first time, S. haematobium x S. bovis hybrids. Proper operation was evaluated with DNA from hybrid schistosomes and with human urine samples artificially contaminated with parasites’ DNA. LAMP was performed with and without prior DNA extraction. The genus-specific LAMP properly amplified pure Schistosoma species and different S. haematobium-S. bovis hybrids with different sensitivity. The Schistosoma spp.-LAMP method is potentially adaptable for field diagnosis and disease surveillance in schistosomiasis endemic areas where human infections by schistosome hybrids are increasingly common.

Mastomys natalensis (Smith, 1834) as a natural host for Schistosoma haematobium (Bilharz, 1852) Weinland, 1858 x Schistosoma bovis Sonsino, 1876 introgressive hybrids

Cercarial emission of schistosomes is a determinant in the transmission to the definitive host and constitutes a good marker to identify which definitive host is responsible for transmission, mainly in introgressive hybridization situations. Our goal was to test the hypothesis that micro-mammals play a role in Schistosoma haematobium, S. bovis, and/or S. haematobium x S. bovis transmission. Small mammal sampling was conducted in seven semi-lacustrine villages of southern Benin. Among the 62 animals trapped, 50 individuals were investigated for Schistosoma adults and eggs: 37 Rattus rattus, 3 Rattus norvegicus, 9 Mastomys natalensis, and 1 Crocidura olivieri. Schistosoma adults were found in four R. rattus and two M. natalensis, with a local prevalence reaching 80% and 50%, respectively. Two cercarial chronotypes were found from Bulinus globosus experimentally infected with miracidia extracted from naturally infected M. natalensis: a late diurnal and nocturnal chronotype, and an early diurnal, late diurnal, and nocturnal chronotype. The cytochrome C oxidase subunit I mtDNA gene of the collected schistosomes (adults, miracidia, and cercariae) belonged to the S. bovis clade. Eleven internal transcribed spacer rDNA profiles were found; four belonged to S. bovis and seven to S. haematobium x S. bovis. These molecular results together with the observed multi-peak chronotypes add M. natalensis as a new host implicated in S. haematobium x S. bovis transmission. We discuss the origin of the new chronotypes which have become more complex with the appearance of several peaks in a 24-h day. We also discuss how the new populations of offspring may optimize intra-host ecological niche, host spectrum, and transmission time period.

Caractérisation biologique de Schistosoma haematobium, S. bovis et leurs hybrides chez l’homme et chez les mollusques Bulinus truncatus naturellement infestés, au Centre et Nord de la Côte d’Ivoire.

Objectif : Identifier les espèces Schistosoma haematobium, S. bovis et leurs hybrides et, évaluer la compatibilité des schistosomes avec les mollusques hôtes intermédiaires et la souris blanche (Mus musculus albinos), hôte définitif, en infestation expérimentale. Méthodologie et résultats : Des schistosomes ont été obtenus à partir de bulins infestés naturellement ou expérimentalement avec des miracidiums provenant des urines de l’homme. Ils ont permis d’étudier la compatibilité de quatre populations de Bulinus truncatus avec deux souches du groupe S. haematobium. La chronobiologie cercarienne a été étudiée à partir de quatre tranches horaires : 6-10h, 10-12h, 12-15h et 15- 18h. Des souris blanches infestées expérimentalement, ont été perfusées pour dénombrer les vers adultes. Une meilleure compatibilité schistosome-mollusque a été observée au niveau des infestations sympatriques. Le pic d’émergence cercarienne pour les mollusques naturellement infestés a été observé entre 6-10h, 10h15h et à 15-18h, tandis que celui des bulins infestés expérimentalement a été majoritairement observé entre 10-15h. En termes de nombre de vers collectés, les souris blanches ont été plus permissives aux schistosomes provenant des mollusques naturellement infestés. Conclusion et application des résultats : Les pics d’émergence cercarienne précoce (6-10h) et tardif (15-18h) pourraient être liés à S. bovis, tandis que celui de 10-15h correspondrait à S. haematobium. De même, en raison de la très faible compatibilité connue entre Mus musculus albinos et S. haematobium, les schistosomes auxquels les souris blanches ont été plus permissives sont fort probablement des S. bovis et/ou hybrides S. bovis x S. haematobium. Ainsi, les espèces anthropophiles et celles zoophiles du groupe Schistosoma haematobium peuvent être distinguées de manière routinière par deux approches. La première, en disséquant des souris de laboratoire (Mus musculus albinos) 4 mois après leur mise au contact de l’eau contenant des cercaires obtenues après exposition à la lumière de mollusques infestés prélevés sur le terrain ; la seconde en comparant les profils obtenus après dénombrement des cercaires émises au cours de quatre tranches horaires bien choisies. Mots clés : Bulinus truncatus, Schistosoma haematobium, Schistosoma bovis, Caractérisation, Chronobiologie. Glitho et al., J. Appl. Biosci. 2021 Caractérisation biologique de Schistosoma haematobium, S. bovis et leurs hybrides chez l’homme et chez les mollusques Bulinus truncatus naturellement infestés, au Centre et Nord de la Côte d’Ivoire. 16341 ABSTRACT Objective: Identify the species Schistosoma haematobium, S. bovis and their hybrids; to evaluate the compatibility of schistosomes with intermediate host snails and the white mouse (Mus musculus albinos), the definitive host for experimental infestation. Methodology and results: Schistosomes have been obtained from naturally or experimentally infested snails with miracidiums from human urine. The compatibility of four populations of Bulinus truncatus with two strains of the S. haematobium group was studied. The chronobiology of cercariae was studied in four time slots: 6- 10h, 10-12h, 12-15h and 15-18h. Experimentally infested white mice were perfused and adult worms were collected. A better schistosome-snail compatibility was observed in sympatric infestations. The peak of cercarial emergence for naturally infested snails was at 6-10h, 10h-15h and 15-18h, while that of the snails experimentally infested was mostly observed at 10-15h. In terms of number of worms collected, white mice were more permissive to schistosomes from naturally infested snails. Conclusion and application of results: Early (6-10h) and late (15-18h) cercarial emergence peaks can be related to S. bovis, while the 10-15h peak correspond to S. haematobium. Due to the reported low compatibility between the white mice Mus musculus albinos and S. haematobium, the observed schistosomes permissive to the white mice are most likely S. bovis and/or hybrids S. bovis x S. haematobium. Therefore, anthropophilic and zoophilic species of the Schistosoma haematobium group can be routinely distinguished by two approaches. The first, dissecting laboratory mice (Mus musculus albinos) 4 months after their contact with water containing cercariae obtained after exposure to light from infested snails collected in the field; the second by comparing the profiles obtained after enumeration of cercariae emitted during four well-chosen time slots. Keywords: Bulinus truncatus, Schistosoma haematobium, Schistosoma bovis, hybrid, Characterization, Chronobiology.

Diverging patterns of introgression from Schistosoma bovis across S. haematobium African lineages

Hybridization is a fascinating evolutionary phenomenon that raises the question of how species maintain their integrity. Inter-species hybridization occurs between certain Schistosoma species that can cause important public health and veterinary issues. In particular hybrids between Schistosoma haematobium and S. bovis associated with humans and animals respectively are frequently identified in Africa. Recent genomic evidence indicates that some S. haematobium populations show signatures of genomic introgression from S. bovis. Here, we conducted a genomic comparative study and investigated the genomic relationships between S. haematobium, S. bovis and their hybrids using 19 isolates originating from a wide geographical range over Africa, including samples initially classified as S. haematobium (n = 11), S. bovis (n = 6) and S. haematobium x S. bovis hybrids (n = 2). Based on a whole genomic sequencing approach, we developed 56,181 SNPs that allowed a clear differentiation of S. bovis isolates from a genomic cluster including all S. haematobium isolates and a natural S. haematobium-bovis hybrid. All the isolates from the S. haematobium cluster except the isolate from Madagascar harbored signatures of genomic introgression from S. bovis. Isolates from Corsica, Mali and Egypt harbored the S. bovis-like Invadolysin gene, an introgressed tract that has been previously detected in some introgressed S. haematobium populations from Niger. Together our results highlight the fact that introgression from S. bovis is widespread across S. haematobium and that the observed introgression is unidirectional.

Development of a Molecular Snail Xenomonitoring Assay to Detect Schistosoma haematobium and Schistosoma bovis Infections in their Bulinus Snail Hosts

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.

Molecular Markers for Detecting Schistosoma Species by Loop-Mediated Isothermal Amplification

Schistosomiasis is considered a neglected parasitic disease. Around 280,000 people die from it annually, and more than 779 million people are at risk of getting infected. The schistosome species which infect human beings are Schistosoma mansoni, Schistosoma haematobium, Schistosoma intercalatum, Schistosoma japonicum, Schistosoma guineensis, and Schistosoma mekongi. This disease is also of veterinary significance; the most important species being Schistosoma bovis since it causes the disease in around 160 million livestock in Africa and Asia. This work was aimed at designing and developing a genus-specific loop-mediated isothermal amplification (LAMP) method for detecting the most important schistosome species affecting humans and for the species-specific detection of S. bovis. Bioinformatics tools were used for primer design, and the LAMP method was standardised for detecting the ITS-1 region from S. intercalatum, S. haematobium, S. mansoni, S. japonicum, and S. bovis DNA (generic test) and the NADH 1 gene for specifically detecting S. bovis (at different DNA concentrations). Detection limits achieved were 1 pg DNA for S. mansoni, 0.1 pg for S. haematobium, 1 pg for S. intercalatum, and 10 pg for S. bovis. No amplification for S. japonicum DNA was obtained. The LAMP designed for the amplification of S. bovis NADH-1 worked specifically for this species, and no other DNA from other schistosome species included in the study was amplified. Two highly sensitive LAMP methods for detecting different Schistosoma species important for human and veterinary health were standardised. These methods could be very useful for the diagnosis and surveillance of schistosome infections.