Molecular Identification And Genetic Variation Study Of Nematodirus Spp. In Sheep And Goats

Nematodirus spp. parasitize in small intestines of ruminants, with species variety andwide distribution. N. oiratianus, N. spathiger and N. helvetianus are considered as thepredominant species in sheep and goats in northern China. Animals infected withNematodirus spp. could cause digestive disorder, gastrointestinal inflammation, diarrhea,inappetence, anemia, and even death with serious infections, which could result in greateconomic losses to the livestock industry. Therefore, the accurately identification ofNematodirus spp., as well as studies on the genetic variation and population structure, have animportant significance for controlling and vaccine development of Nematodirosis. In thispaper, the following four aspects were studied for molecular identification and geneticvariation of Nematodirus spp..1. ITS rDNA fragments of three Nematodirus species, including N. oiratianus, N.spathiger, and N. helvetianus, from naturally infected goats or sheep from four regions ofInner Mongolia Autonomous Region and Shaanxi province in China were amplified andanalyzed in the present study. The lengths of ITS rDNA of N. oiratianus, N. spathiger and N.helvetianus were772bp,766bp, and764bp-766bp, respectively. The respectiveintra-specific genetic variations in ITS1and ITS2rDNA regions were0-4.4%and0-6.1%inN. oiratianus,0.3%-1.8%and0-0.4%in N. spathiger, and0-6.5%and0-5.4%in N.helvetianus from China. The inter-specific genetic variations in ITS1and ITS2rDNA of threespecies were5.2%-9.7%and5.2%-12.8%. Alignments of ITS rDNA fragments showed98mutation sites among different species, consisting of50transitions,32transversions,10multiple substitutions, and furthermore a gap of6bases were observed only in the ITS rDNAsequences of N. oiratianus, which would be effective markers to differentiate the threeNematodirus species.2. According to the mutation sites of three Nematodirus species, two effective molecularidentification approaches to differentiate Nematodirus spp. were established. The resultsshowed that PCR-RFLP could distinguished the three Nematodirus species, with allamplicons of N. spathiger digested into two fragments by Apal I and Bstp I, and amplicons ofN. helvetianus only digested into two fragments (approximately600bp and300bp) by Apal I,but no obvious change for amplicons of N. oiratianus. Using the specific mutation sites of N. oiratianus, the specific-PCR assay allowed the high sensitive and specific identification of N.oiratianus with a low template concentration of0.69pg. Those results provided a newapproach for the identification of Nematodirus spp..3. In this study, the partial mitochondrial cytochrome c oxidase subunit1genes (pcox1)of N. spathiger and N. oiratianus from four regions in two provinces in China were amplifiedby PCR, and then sequenced and analyzed. The results showed that the lengths for pcox1were400bp in both of two species. The intra-specific sequence variations among N.oiratianus and N. spathiger were0.3%-4.0%and0-1.5%, respectively, while the inter-specificsequence differences among Nematodirus species was9.0%-12.8%. A phylogenetic analysisbased on mtDNA pcox1fragments indicated that N. oiratianus and N. spathiger isolates weregrouped in two solid clades, and could effectively distinguish the two close-related species ofNematodirus spp.. These results showed that the pcox1could provide an ideal molecularmarker for studying genetic variation and population evolution of Nematodirus spp..4. In the present study, the complete mitochondrial (mt) genomes of N. oiratianus and N.spathiger were obtained using Long-range PCR and sequencing. The complete mt genomes ofN. oiratianus and N. spathiger were13765bp and13519bp in length, respectively. Both mtgenomes were circular and consisted of36genes, including12genes encoding proteins(cox1-3, nad1-6, nad4L, atp6and cytb),22genes encoding tRNA, and2genes encodingrRNA, but without the gene of atp8, and the gene orders of both two Nematodirus specieswere consistent. The mt DNA sequences of two Nematodirus spp. showed that the sequencedifference between N. oiratianus and N. spathiger was16.3%. Phylogenetic analyses basedon the concatenated amino acid sequence data of all12protein-coding genes indicated thatthe two Nematodirus species were grouped in one solid clade, and closely related toDictyocaulidae.In conclusion, ITS rDNA, pcox1and mt DNA of the dominant Nematodirus species innorthern China were analyzed in this study, and then two molecular biological methods fordifferentiating Nematodirus spp. were established based on the data differences. These resultsare detailed analysis of the genetic variation and population structure of Nematodirus spp.,which not only enrich the study of parasitology of sheep and goats, but also provide atheoretical basis for controlling of disease and vaccine research.