Authors

Discussion

Schistosomiasis is a parasitic disease caused by blood flukes in the genus Schistosoma that infect human and animal hosts. Reports of ongoing hybridization between human and animal species of schistosomes in many parts of Africa suggest the existence of a species complex within the Schistosoma haematobium group, comprised of S. haematobium, S. bovis, S. currasoni, S. intercalatum, S. guinnesis, and S. mattheei. 

As proof-of-concept, we developed a chromosome-wide Nanopore Multiplex Amplicon Sequencing (NMA-Seq) platform using 13 genetic markers of varying phylogenetic strength to generate high-resolution data for studying schistosome genetic diversity. The NMA-Seq multiplexing assay combines dual barcoding and sample pooling of PCR amplicons to generate high depth of coverage per dual barcode using MinION sequencing. Pooled MinION sequences were deconvoluted by aligning to a custom database of all sequence types across the species complex. This methodology was used to screen for schistosome hybrids among 95 parasite isolates obtained through urine filtration or from hatched miracidia collected from two pastoral and two non-pastoral communities in Nigeria. 58 of these isolates were also Sanger sequenced to assess accuracy of the NMA-Seq results. All 95 schistosome isolates resolved as hybrids. Isolates that had been characterized as S. haematobium based solely on mtCOX1 and rITS all possessed S. bovis alleles at other markers establishing that current markers are insufficiently resolved to infer the population genetic structure within this species complex. All isolates from one non-pastoral site possessed only S. bovis mitochondria.  Two of these isolates possessed homozygous S. bovis alleles at several nuclear markers supporting a model of ongoing hybridization between S. bovis and S. hematobium in Nigeria. Furthermore, we recorded 99.9% agreement between our Sanger and NMA-Seq data, with a 70% cost reduction for NMA-Seq. Our approach demonstrates the utility and cost-effectiveness of this field-deployable, NMA-Seq strategy to distinguish between S. haematobium, S. bovis, and hybrids from both species. 

Keywords: NMA-Seq, Schistosomiasis, Hybridization, Species, Diversity, Nigeria