Molecular approaches to differentiate three species of Nematodirus in sheep and goats from China based on internal transcribed spacer rDNA sequences

Abstract

Internal transcribed spacer (ITS) rDNA sequences of three Nematodirus species from naturally infected goats or sheep in two endemic provinces of China were analysed to establish an effective molecular approach to differentiate Nematodirus species in small ruminants. The respective intra-specific genetic variations in ITS1 and ITS2 rDNA regions were 0.3-1.8% and 0-0.4% in N. spathiger, 0-6.5% and 0-5.4% in N. helvetianus, and 0-4.4% and 0-6.1% in N. oiratianus from China. The respective intra-specific variations of ITS1 and ITS2 were 1.8-4.4% and 1.6-6.1% between N. oiratianus isolates from China and Iran, 5.7-7.1% and 6.3-8.3% between N. helvetianus samples from China and America. For N. spathiger, compared with samples from China, sequence differences in ITS1 rDNA were 0.3-2.4% in isolates from America, 0.3-2.9% in New Zealand and 2.1-2.4% in Australia. Genetic variations in ITS2 rDNA of N. spathiger were 0-0.4% between samples from China and America, and 0-0.8% between samples from China and New Zealand. Using mutation sites, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and specific PCR techniques were developed to differentiate these three Nematodirus species. The specific PCR assay allowed the accurate identification of N. oiratianus from other common nematodes with a sensitivity of 0.69 pg and further examination of Nematodirus samples demonstrated the reliability of these two molecular methods.

Characterization of the complete mitochondrial genomes of Nematodirus oiratianus and Nematodirus spathiger of small ruminants

“…N. oiratianus and N. spathiger samples in the present study clustered together and were highly closely related to the family Dictyocaulidae with high clade support (Bpp = 1.00; Bf = 100), confirming the results of previous studies using the morphological features and ITS rDNA as the genetic marker [ 2 , 9 , 42 ]. However, amino acid sequence-based inferences of the relationship between the two Nematodirus species gave slightly different results to those using ITS rDNA data in which they clustered in sister clades with high support, possibly due to the different taxa inclusions and types of analysis performed [ 2 , 11 - 13 , 46 ].…”