Enabuele, E. E.; Platt, R. N.; Adeyemi, E. E.; Aisien, M. S. O.; Ajakaye, O. G.; Ali, M. U.; Amaechi, E. C.; Atalabi, T. E.; Auta, T.; Awosolu, O. B.; Dagona, A. G.; Edo-Taiwo, O.; Ejikeugwu, C. P.; Igbeneghu, C.; Njom, V. S.; Onwude-Agbugui, M.; Orji, M.-K. N.; Oyinloye, F. O.; Oyemade, E.; Ozemoka, H. J.; Pam, C. R.; Ugah, U. I.; Hulke, J. M.; Arya, G. A.; Anderson, T. J.

The nuclear, internal transcribed spacer (ITS) and mitochondrial cox1 markers are widely used to differentiate Schistosoma haematobium from its livestock counterparts, S. bovis and S. curassoni. Schistosoma isolated from humans that have ITS and cox1 markers from livestock schistosomes are typically inferred as zoonotic infections, those with mixed species, heterozygous ITS are classified as F1s or recent hybrids, while those with discordant ITS and cox1 markers are considered to reflect older hybridization events. We evaluated the reliability of this classification scheme by genotyping ITS and cox1 from 132 parasites isolated from human urine, and from 37 adult schistosomes collected from cattle at 14 Nigerian locations. We also genome sequenced each sample to empirically determine livestock schistosome ancestry. ITS/cox1 genotyping suggested extensive recent hybridization and zoonotic infection. Among parasites from humans, 10.1% carried both S. curassoni and S. haematobium ITS, consistent with F1 or early generation hybrids, 21% had livestock schistosome markers at both cox1 and ITS suggesting zoonotic infection, while 13.7% carried S. bovis cox1 alongside mixed S. curassoni and S. haematobium ITS, suggesting more complex ancestry. Genome sequencing revealed a very different picture. All parasites from humans formed a tight cluster regardless of ITS or cox1 genotype, while all worms from cattle were well differentiated. We found no schistosomes containing 50% livestock parasite ancestry consistent with F1s. Instead, we observed regionally varying levels of S. bovis introgression, with modest levels in southern Nigeria (mean = 4.9%) and low levels in northern Nigeria (mean = 0.06%). These results demonstrate that: (i) two-locus genotyping is uninformative for detecting zoonotic infection or recent hybridization between S. haematobium and livestock schistosomes and (ii) previous data generated using this approach requires reinterpretation. These findings reveal the limitations of widely-used approaches for documenting zoonotic infection and hybridization between S. haematobium and livestock schistosome species.