First report on Molecular Characterization of Oesturs ovis in sheep from India

Abstract

Purpose Upon post mortem examination of sheep (from SRRC, Mannavanur, South India) having the history of natural death, the nasal sinuses of the animals contained dipteran larvae. The objective of the present study is to characterize the dipteran larvae species infesting the sheep being maintained at SRRC, Mannavanur, by means of COI gene based PCR. Methods During the last week of May 2021, an Avikalin male sheep (20 months old) died suddenly. Upon Post mortem examination, the skull of the dead sheep was having of larvae in the nasal sinuses. The larvae were washed in PBS (pH 7.2) and preserved in 70 % alcohol. The larvae taken out of 70 % alcohol were thoroughly ground to make a fine powder using liquid Nitrogen in a sterile mortar and pestle. The maggot in powder form was then used for the isolation of total genomic DNA isolation. Using the isolated genomic DNA from the larvae as a template, Cytochrome c oxidase subunit I (COI) gene based PCR was employed using the primers designed based on the COI gene of reference isolate of Oestrus ovis available in the GenBank. Full length COI gene (1534 bp) gene of Oestrus ovis in sheep from South India was targeted in the PCR experiment. The pTZ57R/T vector was used for the cloning of the PCR amplified fragment and the confirmed recombinant plasmid was subjected to sequencing experiments. The resultant nucleotide sequences of COI gene of the O.ovis in sheep from South India was analysed with that of 29 dipteran species from different different geographical areas of the world (available in GenBank) by using standard bioinformatics tools. Results In addition to morphological examination, based on COI gene based PCR, eventual sequencing experiments and BLAST analysis, it was confirmed that the larvae in the nasal sinuses of sheep from South India were Oestrus ovis. The South Indian isolate of Oestrus ovis is sharing 100% sequence identity both at nucleotide and amino acid levels with that of O.ovis from Spain. The North Indian isolate of O.ovis (from Jammu) exhibited 92 & and 99 % identity at respective nucleotide and amino acid levels with South Indian isolate. With other members of the subfamily Oestrinae, the share of per cent nucleotide and amino acid identities of South Indian O.ovis ranged from 85-86 % and 95-96 %, respectively. O.ovis from South India was grouped with the other members of Oestrinae from different geographical areas of the globe in the analysis of phylogenetic tree based on COI amino acid sequences. ConclusionBased on the research findings, it is concluded that Oestrus ovis is the dipteran species infesting the sheep at Mannavanur, Tamil Nadu, India. To our knowledge, this is the first report on full length nucleotide sequences of COI gene of O.ovis in sheep from Indian subcontinent. An investigation on the report of human ocular infections and the accidental hosts for O ovis in the area of the present study has to be explored.