Morphological and molecular identification of Heterakis gallinarum in Gallus gallus and its phylogenetic analysis based on ITS1-5.8S-ITS2 rDNA fragment in Kashmir valley, India.

Abstract

Heterakis gallinarum is one of the most common round worms infecting wide range of terrestrial birds including domestic fowl. H. dispar and H. isolonche are the species of same genus which infect other hosts predominantly aquatic birds, but show morphological similarities mainly females of these species are difficult to identify. Here, we described the morphologically distinct features of H. gallinarum along with identification through molecular analysis using ITS1-5.8S-ITS2 ribosomal DNA. Four hundred gastrointestinal tracts of domestic fowl were analysed for a period of 2 years. Light microscopy and Scanning electron microscopy (SEM), was used for determining the morphometric measurements and characteristic morphological features of H. gallinarum. A PCR based DNA sequencing of ITS1-5.8S-ITS2 ribosomal DNA was used for molecular characterization and phylogenetic analysis was carried out through MEGA 11. Ninety gastrointestinal tracts were found to be infected with Heterakis gallinarum. The male worm displayed well developed lateral and caudal alae, caudal end had an anal sucker, 12 pairs of caudal papillae and cloaca from which 2 unequal spicules emerged. Female had visible vaginal bends, vulva present approximately in the middle of the body, and caudal end had a long pointed tail. Sequence analysis revealed that the PCR products of ITS1-5.8S-ITS2 rDNA were 600 bp in size and showed 96-98% similarity with the ITS1-5.8S-ITS2 sequences of H. gallinarum on BLAST analysis. Our ITS sequence of H. gallinarum showed a nucleotide divergence of 4.9% with H. gallinarum of Lucknow India. The phylogenetic analysis clearly differentiated H. gallinarum from the other representatives of the genus and revealed the relationship among different Heterakis species. The study revealed the infection status of Heterakis gallinarum prevailing among domestic fowl, and need of strategic control measures. The results of the study suggest that ITS rDNA would serve as a potential molecular marker in identification, diagnosis and systematics of Heterakis species.