Molecular characterisation of hybrid Fasciola from central Vietnam

Abstract

Central Vietnam is a “hotspot” for fasciolosis, a re-emerging hyperendemic trematodiasis of both livestock and humans. Despite this, the main causative agent, i.e. Fasciola hepatica, F. gigantica or hybrid forms, remains unknown. Here, a multi-faceted approach was used to characterise approximately 555 flukes from the central Phu Yen and Binh Dinh provinces. Molecular screening revealed that parthenogenetic hybrids (~87%) were the dominant form followed by F. gigantica (~13%). Multiplex-PCR and PCR-RFLP analysis of the nuclear markers Pepck and Pold, respectively, displayed 100% correct identification of 504 co-analysed flukes. In contrast, whilst ITS-1 and -2 PCR-RFLP methods, commonly used for Fasciola species identification in Vietnam, accurately identified 100% of parental Fasciola spp., ITS-1 and -2 PCR-RFLP only correctly identified ~25% and ~83% of hybrid fluke samples, respectively. Sequencing revealed recombination within hybrid ITS regions; reducing the frequency of heterozygous bases and underlining their limited value for identification purposes. Phylogenetic analysis of mitochondrial NAD1 and COX1 markers revealed high diversity and geneflow of F. gigantica within Vietnam and its surrounding nations; having nine and four haplotypes, respectively. Meanwhile, hybrids showed uniformity with all mtDNA sequences grouping within the parthenogenetic hybrid ‘Fg-C2’ haplogroup that dominates Southeast Asia, indicating they were likely imported into Vietnam via infected livestock. Finally, the use of MALDI-ToF mass spectrometry as a high-throughput molecular identification tool for Fasciola spp. was explored. Here, representative spectral profiles of flukes from six different Fasciola spp. populations were obtained that could distinguish between them. Whilst reproducibility and statistical analyses revealed their suitability for automated detection, overlaps in PCA/PLS-DA, limited resolution for infra-populations and lack of reference databases limit its immediate implementation for Fasciola spp. identification.

Thesis is embargoed until 31 July 2028.

Date of Award	Jul 2025
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy
Supervisor	Mark Robinson (Supervisor) & Eric Morgan (Supervisor)