Research Article | 31 Oct 2025

Molecular differentiation of Opisthorchis felineus and Metorchis bilis in cyprinid fish from Northern Kazakhstan: Epidemiological insights and diagnostic advances

Aiganym Bekaidarovna Bekenova1 , Aleksey V. Katokhin2 , and Kundyz B. Muratbekova1 Show more
VETERINARY WORLD | pg no. 3197-3207 | Vol. 18, Issue 10 | DOI: 10.14202/vetworld.2025.3197-3207
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Abstract

          
Background and Aim: Opisthorchiasis and metorchiasis are significant zoonotic fish-borne trematodiases caused by Opisthorchis felineus and Metorchis bilis. These parasites exhibit overlapping geographic ranges and morphologically similar larval stages, complicating species-level identification. In Kazakhstan, where raw or undercooked freshwater fish is widely consumed, opisthorchiasis remains an endemic concern. This study aimed to investigate the prevalence of O. felineus and M. bilis in cyprinid fish from Akmola Region and to establish molecular tools for their differential diagnosis.
Materials and Methods: A total of 818 freshwater cyprinid fish were collected from Lakes Sholak, Esey, and Karazhar between 2021 and 2023. Muscle tissue was examined using the compression method for metacercariae detection. Morphological identification was complemented with a newly designed multiplex polymerase chain reaction (PCR) assay targeting the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene. Selected amplicons were sequenced and subjected to phylogenetic analysis. Prevalence and infection intensity were calculated, and statistical comparisons were made among fish species and lakes.
Results: Opisthorchiidae metacercariae were detected in ide, bream, and roach, with prevalence varying across lakes. Lake Sholak exhibited the highest infection rate (42.9%), with ide showing the greatest susceptibility (40.4%). No infections were detected in fish from Lake Karazhar. Morphological differentiation between O. felineus and M. bilis was inconclusive due to overlapping features. Multiplex PCR successfully distinguished O. felineus (307 bp) from M. bilis (252 bp), with >99% sequence identity to GenBank references. Two representative sequences (PQ669120 and PQ669125) were deposited in GenBank. Phylogenetic analysis confirmed distinct clustering of both species.
Conclusion: This study provides the first molecular confirmation of O. felineus and M. bilis in freshwater fish of the Akmola region, Kazakhstan. The developed multiplex PCR assay offers a sensitive and reliable diagnostic tool for species-level differentiation. These findings highlight moderate to high prevalence in local fish, underline the zoonotic risks associated with fish consumption, and emphasize the need for integrated One Health surveillance to inform control strategies and food safety policies.
          
Keywords: cytochrome c oxidase subunit 1, Kazakhstan, metorchiasis, Metorchis bilis, multiplex polymerase chain reaction, opisthorchiasis, Opisthorchis felineus, zoonotic trematodes.