Molecular characterization of Paramphistomum cervi in buffaloes

Background: Paramphistomiasis (Rumen fluke disease) in ruminants is a major health problem, characterized by coarse hair, weakness, loss of appetite, weight retardations, intestine ulcers, inter-mandibular inflammation, causing substantial economic losses, and high mortality. In tropical and subtropical regions, the disease was neglected but has recently emerged as an important cause of production losses. While documented reports on Paramphistomum cervi, Paramphistomum ichikawai and Paramphistomum are limited in Asian countries and paramphistomosis has been considered the major health and economic problem in several countries. The present study aimed to identify paramphistomoid flukes that infects buffaloes with the goal of characterization of prevalence in Pakistan and its comparison with neighbor countries. Materials, Methods & Results: In 2018, a total of 178 slaughtered buffaloes aged four to six years were examined. After an immediate postmortem examination of each buffalo, flukes were collected from their infected rumen and reticulum using sterilized forceps and placed in a saline solution. DNA was extracted from adult Paramphistome species using the standard phenol chloroform method and used for amplification of partial fragment of 18S rRNA sequences using specific pair of primer. After amplification and sequencing of 18S rRNA partial fragment, the generated sequences were assembled and trimmed to remove any primer contaminations. Twenty-three randomly selected and morphologically identified adult Paramphistomum were used in species-level identification using specific primers for partial fragment of 18S rRNA sequences. The cleaned sequences (810 bp) were used to identify similar sequences using BLAST on the NCBI website. The GenBank retrieved sequences and new Paramphistomum species isolated sequences were aligned using CLUSTAL in the BioEdit Sequence Alignment Editor. In addition, a phylogenetic tree was constructed using maximum likelihood method in MEGA X. The 18S rRNA sequence was found 100% similar with Paramphistomum cervi of China and 98% with Paramphistomum epiclitum and other Paramphistomum species of India. The parasitic Pharamphistomum species was identified molecularly as Paramphistomum cervi. Discussion: Molecular studies provide insight into the biology and phylogenetic relationship among various parasites. These studies are reliable in the genetic-based identification and description of several disease causing agents. The 18S rRNA sequence of Paramphistomum cervi generated in this study was found closely identical to the P. cervi of the neighbor countries (China and India) which may be due to the similar geographical, environmental conditions and transboundary movement of infected hosts. This is the first nature of study which provides the molecular-based evidence of P. cervi existence in Pakistan and revealed the 18S rRNA as novel molecular marker for the identification and further characterization of Paramphistomum species across Pakistan. The submitted sequence of this study will provide a baseline for further molecular characterization and to compare with other Paramphistoma species from different regions of Pakistan.