Genetic analysis confirms the presence of Dicrocoelium dendriticum in the Himalaya ranges of Pakistan.

Lancet liver flukes of the genus Dicrocoelium (Trematoda: Digenea) are recognised parasites of domestic and wild herbivores. The aim of the present study was to address a lack of knowledge of lancet flukes in the Himalaya ranges of Pakistan by characterising Dicrocoelium species collected from the Chitral valley. The morphology of 48 flukes belonging to eight host populations was examined in detail and according to published keys, they were identified as either D. dendriticum or Dicrocoelium chinensis. PCR and sequencing of fragments of ribosomal cistron DNA, and cytochrome oxidase-1 (COX-1) and NADH dehydrogenase-1 (ND-1) mitochondrial DNA from 34, 14 and 3 flukes revealed 10, 4 and 1 unique haplotypes, respectively. Single nucleotide polymorphisms in these haplotypes were used to differentiate between D. chinensis and D. dendriticum, and confirm the molecular species identity of each of the lancet flukes as D. dendriticum. Phylogenetic comparison of the D. dendriticum rDNA, COX-1 and ND-1 sequences with those from D. chinensis, Fasciola hepatica and Fasciola gigantica species was performed to assess within and between species variation and validate the use of species-specific markers for D. dendriticum. Genetic variations between D. dendriticum populations derived from different locations in the Himalaya ranges of Pakistan illustrate the potential impact of animal movements on gene flow. This work provides a proof of concept for the validation of species-specific D. dendriticum markers and is the first molecular confirmation of this parasite species from the Himalaya ranges of Pakistan. The characterisation of this parasite will allow research questions to be addressed on its ecology, biological diversity, and epidemiology.