Assessment of genetic markers for multilocus sequence typing (MLST) of <i>Fasciola</i> isolates from Iran

<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Several markers have been described to characterise the population structure and genetic diversity of <jats:italic>Fasciola</jats:italic> species (<jats:italic>Fasciola hepatica</jats:italic> <jats:italic>(F. hepatica)</jats:italic> and <jats:italic>Fasciola gigantica</jats:italic> (<jats:italic>F. gigantica</jats:italic>). However, sequence analysis of a single genomic locus cannot provide sufficient resolution for the genetic diversity of the <jats:italic>Fasciola</jats:italic> parasite whose genomes are ∼1.3 GB in size.</jats:p></jats:sec><jats:sec><jats:title>Objectives</jats:title><jats:p>To gain a better understanding of the gene diversity of <jats:italic>Fasciola</jats:italic> isolates from western Iran and to identify the most informative markers as candidates for epidemiological studies, five housekeeping genes were evaluated using a multilocus sequence typing (MLST) approach.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>MLST analysis was developed based on five genes (<jats:italic>ND1</jats:italic>, <jats:italic>Pepck</jats:italic>, <jats:italic>Pold</jats:italic>, <jats:italic>Cyt b</jats:italic> and <jats:italic>HSP70</jats:italic>) after genomic DNA extraction, amplification and sequencing. Nucleotide diversity and phylogeny analysis were conducted on both concatenated MLST loci and each individual locus. A median joining haplotype network was created to examine the haplotypes relationship among <jats:italic>Fasciola</jats:italic> isolates.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Thirty‐three <jats:italic>Fasciola</jats:italic> isolates (19 <jats:italic>F. hepatica</jats:italic> and 14 <jats:italic>F. gigantica</jats:italic>) were included in the study. A total of 2971 bp was analysed for each isolate and 31 sequence types (STs) were identified among the 33 isolates (19 for <jats:italic>F. hepatica</jats:italic> and 14 for <jats:italic>F. gigantica</jats:italic> isolates). The STs produced 44 and 42 polymorphic sites and 17 and 14 haplotypes for <jats:italic>F. hepatica</jats:italic> and <jats:italic>F. gigantica</jats:italic>, respectively. Haplotype diversity was 0.982 ± 0.026 and 1.000 ± 0.027 and nucleotide diversity was 0.00200 and 0.00353 ± 0.00088 for <jats:italic>F. hepatica</jats:italic> and <jats:italic>F. gigantica</jats:italic>, respectively. There was a high degree of genetic diversity with a Simpson's index of diversity of 0.98 and 1 for <jats:italic>F. hepatica</jats:italic> and <jats:italic>F. gigantica</jats:italic>, respectively. While <jats:italic>HSP70</jats:italic> and <jats:italic>Pold</jats:italic> haplotypes from <jats:italic>Fasciola</jats:italic> species were separated by one to three mutational steps, the haplotype networks of <jats:italic>ND1</jats:italic> and <jats:italic>Cyt b</jats:italic> were more complex and numerous mutational steps were found, likely due to recombination.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Although <jats:italic>HSP70</jats:italic> and <jats:italic>Pold</jats:italic> genes from <jats:italic>F. gigantica</jats:italic> were invariant over the entire region of sequence coverage, MLST was useful for investigating the phylogenetic relationship of <jats:italic>Fasciola</jats:italic> species. The present study also provided insight into markers more suitable for phylogenetic studies and the genetic structure of <jats:italic>Fasciola</jats:italic> parasites.</jats:p></jats:sec>