Multi-marker analysis of <i>Fasciola gigantica</i> from cattle and buffalo across Pakistan reveals high levels of genetic diversity and novel haplotypes

Molecular analyses of geographically dispersed <i>Fasciola</i> spp. isolates based on ribosomal, mitochondrial and nuclear molecular markers have revealed high levels of genetic diversity within liver fluke populations. To investigate the <i>Fasciola</i> population substructure across Pakistan 4 molecular markers were compared (fatty acid binding protein, <i>fabp;</i> phosphoenolpyruvate carboxykinase, <i>pepck</i>; random amplified polymorphic DNA, RAPD; mitochondrial NADH dehydrogenase, <i>mt-nd1</i>). Adult parasites (<i>n</i> = 595) were collected from buffalo and cattle across 4 provinces in Pakistan (Baluchistan, Gilgit Baltistan, Khyber Pakhtunkhwa, Punjab). Species classification of all 595 parasites was confirmed by the 3 gel-based markers (<i>pepck, fabp</i> and RAPD) as <i>F. gigantica</i>, except for the <i>fabp</i> marker which unexpectedly could not be amplified in 274 parasites (46%). Analysis of a subset of samples indicates the potential for mis-priming due to multiple genomic loci that match the <i>fabp</i> primer sequences resulting in negative PCR products in some cases. Sequence analysis of the <i>mt-nd1</i> PCR products identified 29 haplotypes within the samples from Pakistan, the majority of which are unique to this study. None of the 29 haplotype sequences were identified in samples from Africa, highlighting the genetic diversity between geographically disparate liver fluke populations. Inconsistencies between <i>Fasciola</i> spp. molecular markers in this study highlights the need for multiple markers, validated on large numbers of geographically disparate parasites, to generate robust analyses of liver fluke genetic diversity. This study echoes other <i>Fasciola</i> spp. population studies and highlights the genetic diversity of <i>F. gigantica</i> populations in Pakistan that is comparable to observations of diversity throughout Asia.