Molecular identification of Kalicephalus isolated from snakes in Hunan province, subtropical China

Parasite classification and identification are central to controlling parasitosis. Traditional methods for identifying parasite species are based on morphological features, but these are time-consuming and inaccurate, especially for cryptic species. The purpose of the present study was to select molecular markers to promote the development of molecular systematic for parasites. The internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA) falls in between 18S, 5.8S, and 28S rDNA sequences, including ITS-1 and ITS-2 sequences. Previous studies have demonstrated that rDNA ITS sequences provide useful genetic markers for identifying parasitic nematodes. With the ultimate goal of controlling parasite transmission, we identified Kalicephalus belonging to three species using ITS rDNA genes. The ITS genes (750-797 bp) of 21 Kalicephalus belonging to 3 species were cloned and sequenced. Intra- and interspecific identities were 98.4% and 80%-89%, respectively. The phylogenetic tree reconstructed with the neighbour-joining (NJ) method revealed that congener Kalicephalus form the same branch, which is far apart from other branches of other nematodes. This is consistent with morphological classifications, demonstrating the accuracy of our molecular method. This is the first report stating that ITS genes can be used to classify Kalicephalus, and it lays the foundation for identification, molecular epidemiology, and phylogenetics of Kalicephalus and related parasitic nematodes.