Fasciolosis, a foodborne zoonotic trematode infection in cattle in Bangladesh: multifaceted validation of parthenogenecity and anthelmintic efficacy

Parthenogenic Fasciola (Trematoda: Fasciolidae) flukes have been developed by the hybridization of Fasciola hepatica and Fasciola gigantica. They are aspermic (asF) but capable of clonal expansion through parthenogenesis and are spreading rapidly throughout the globe. Here, we unambiguously prove the occurrence of parthenogenic Fasciola in cattle in Bangladesh, along with their ex vivo culture protocol and anthelmintic efficacy. By employing multiple conventional and molecular tools, we confirmed the presence of both the spermic F. gigantica (sFg) (31.7%; 814/2575) and asF (68.3%; 1761/2575) in Bangladesh. Both the adult sFg and asF survived well in DMEM supplemented with 20% bovine serum and 20% bovine bile. Using a DMEM-based ex vivo culture protocol, we found that nitroxynil (NTX) and oxyclozanide (OCZ) efficiently killed both sFg and asFg in a concentration and time-dependent manner. Surprisingly, triclabendazole (TCBZ) and clorsulon (CRL) killed neither sFg nor asF. Also, praziquantel, albendazole, and levamisole did not affect the viability of the flukes. We found that all TCBZ survivors had more than one mutation, both in nucleotides (G440A, G643A, and G788A) and amino acids (R147K, E215K, and R263K) of the binding pocket of carboxylesterase B (CestB), providing molecular evidence of TCBZ resistance in Fasciola. Taken together, asF constitutes more than two-thirds of the Fasciola population in Bangladesh. This study unambiguously proved the ineffectiveness of TCBZ against both asF and sFg circulating in Bangladesh. Therefore, only OCZ and NTX remain effective against fasciolosis, which thus poses ongoing public health risks of infection in humans with TCBZ-tolerant strains of fasciolosis.