Morphological and Molecular Charactrization of Gastrointestinal Parasites of wild water Buffalo of Chitwan National Park

Abstract

The microscopy-based identification of gastrointestinal parasites can be further strengthened at species-level, which shows the importance of molecular based identification.The main aim of this study is the molecular characterization of the gastrointestinal parasites in Wild Water buffalo of Chitwan National Park. A wide range of helminth and protozoan species can infect or colonize the gastrointestinal tract of humans and animals. Intestinal parasitic infections caused mainly by protozoa and helminths are most prevalent in Wild Water buffalo, which are of relevance to critically endangered species. Twenty-five fecal samples were collected from groups of Wild Water buffalo. The fecal samples were examined microscopically by direct wet mount method, sedimentation method, floatation method, and molecularly by PCR, sequencing and, analysis. An integrative taxonomic approach is followed, where conspicuous morphological traits and phylogenetic trees based on DNA sequences are used to test the validity of the species. Nine intestinal parasites were detected microscopically, where the infection rate was 96%, of which 88% and 84% were due to protozoa and helminths respectively. The identified protozoan parasites were Entamoeba (80%), Eimeria (60%), Balantidium (8%), and Cryptosporidium (4%) and helminths were Oxyruid (64%) Strongyloides (28%), Strongyle (36%), Eurytrema (8%), and Paramphistomum (4%). The PCR assays were conducted using Entamoeba spp. 18S specific primers. Ribosomal genome sequence (550 bp) was obtained and DNA sequence analysis enabled more precise identification of E. bovis. Entamoeba was successfully sequenced and it was separated from those of other Entamoeba species/genotypes in phylogenetic analysis by the Neighbor Joining method. Also the amplification of Eimeria was done by using genus-specific primers targeting 18S ribosomal region. This investigation gives benchmark data of GI parasites and their conveyance in wild buffalo. This is the primary report for the molecular discovery of E. bovis in translocated wild buffalo in Nepal. Keywords: Bubalus arnee, Chitwan National Park, Gastrointestinal Parasite, Protozoa, Helminthes, 18S rDNA