Molecular detection and phylogenetic characterization of hybrid Fasciola in black Bengal goats from Bangladesh: β-tubulin isotype 3 polymorphisms and genetic diversity

Fasciolosis is a neglected tropical disease that poses a major threat to human and animal health, especially in low- and middle-income countries like Bangladesh, where climate change, widespread infection, emergence of hybrid Fasciola and rising benzimidazole (BZ) resistance have complicated disease control efforts. Therefore, this study aims to molecularly characterize and differentiate F. gigantica and hybrid Fasciola in black Bengal goats to better understand their genetic diversity and resistance patterns, ultimately supporting the development of more effective control strategies. A cross-sectional study was conducted from 2021-2022 across all eight divisions of Bangladesh, during which 3,134 liver flukes were collected from slaughterhouses. From these, 72 morphologically suspected hybrids were selected for molecular analysis. ITS2 sequencing and phylogeny confirmed all 72 as F. gigantica, revealing four haplotypes and low divergence from other Asian isolates. Hybrid status was confirmed in 76% of cases by pepck multiplex PCR and 39% by pold PCR-RFLP. Mitochondrial NAD1 analysis of 25 hybrids revealed four haplotypes, moderate genetic diversity, and phylogenetic clustering with hybrids from India, China, Vietnam, and Nepal. COX1 phylogeny supported these findings, placing Bangladeshi hybrids in a monophyletic F. gigantica clade, distinct from F. hepatica. Polymorphisms in the {beta}-tubulin isotype 3 gene were observed in both F. gigantica and hybrid forms, with phylogenetic clustering alongside BZ-resistant F. hepatica isolates from Europe and Australia, suggesting possible emerging resistance in Bangladesh. This is the first report of hybrid Fasciola and potential resistance-associated {beta}-tubulin variants in goats in Bangladesh. Whole genome sequencing and gene mapping related to pathogenicity and BZ resistance are imperative for future studies. Author summaryFasciolosis is a serious disease caused by parasitic worms that affects the health and productivity of both animals and people, particularly in poorer countries like Bangladesh. In our study, we focused on these parasites in native Black Bengal goats, which are an important source of income and nutrition for rural communities. We traveled across all eight divisions of Bangladesh and collected over 3,000 liver flukes from infected goats. We then used genetic tools to identify whether the parasites were a known species or a mix (hybrid) of two different types. We found that many of the parasites were hybrids, which are harder to detect and may resist common treatments. We also discovered genetic signs that these worms might be developing resistance to drugs commonly used to treat them. This is the first-time hybrid liver flukes have been reported in goats in Bangladesh. Our findings raise concern that current treatments may become less effective over time. We believe this work highlights the urgent need for better disease monitoring, improved treatment strategies, and further research using whole genome sequencing to understand how these parasites spread and survive.