Genetic Variation Of ITS RDNA Of Haemonchus Contortus Isolated From Goat And Establishment Of PCR Method For Detecting H.contortus Eggs

Haemonchu contortus,one of the important gastrointestinal nematodes,colonize the abdomen and small intestine of ruminants.Its host ranges extensively in ruminants such as sheep,goats,cattle,and camels.In some severely affected areas,the infection rate of H.contortus can be as high as 100%.It is a blood-feeding helminth which relies on blood capillary of ruminants’ abomasum,and once heavily infected the hosts may die of it.It is ubiquitous and prevalent in many regions in the world due to its high pathogenicity and strong fertility,which has has brought huge economic losses to the world’s sheep and goat industry.Accurate diagnosis of H.contortus in sheep,therefore,is of great significance to the effective prevention and control of the disease as well as the protection of healthy sheep breeding.In this study,to clearly understand the genetic variation of the ITS rDNA gene,PCR method was used to amplify the ITS gene sequence of H.contortus isolates from naturally infected goat or sheep in Shaanxi province.According to the characteristics of H.contortus ITS2 rDNA,a specific PCR method to differentiate and examine H.contortus was established.Meanwhile,the established PCR method was applied to clarify the infection rate of H.contortus in fecal samples from dairy goats clinically,while fecal microscopy was conducted and compared as well.The results are as follows:1.44 isolates of H.contortus from Shaanxi province were amplified and analyzed based on ITS rDNA in present study.The lengths of ITS1 rDNA of H.contortus was 400 or 404 bp,with the intra-specific genetic variations being 0.0-3.6%.Alignment of a total of 27 ITS1 genotypes with the reference sequence revealed 16 single nucleotide polymorphism and 4consecutive deletions in 270-273 sites as well.In comparsion,the lengths of ITS2 rDNA was231 bp,and the intra-specific genetic variations was 0.0-6.9%.15 single nucleotide polymorphism were observed by alignment of a total of 20 ITS2 genotypes with the reference sequence.The results showed that the intraspecific variation rate of the ITS rDNA sequence of H.contortus from 2015 to 2020 was relatively small in Shaanxi Province,and there was no population differentiation.The preliminary phylogeny analysis showed there was no significant correlation between variation of ITS rDNA of H.contortus and various geographical conditions,and there were high genetic similarities among the isolates from all over the world.2.The specific primers,named Hc.F and Hc.R,had good specificity and can distinguish H.contortus from other nematodes,including Nematodirus spp.,Haemonchus similis,Trichuris lani,Oxyuris spp.,Dictyocaulus spp.,Chabertia spp.,Trichostrongylus colubriformis,Oesophagostomum asperum,Trichuris spp.The optimal annealing temperature for PCR reaction was 53°C.H.contortus can still be detected with a low DNA template concentration of 0.298 pg,indicating its high sensitivity.A total of 23 H.contortus positive fecal samples(27.06%,23/85)were detected from 85 dairy goat fecal samples through fecal microscopy,while 38 H.contortus positive feces(44.71%,38/85)were detected by specific PCR microscopy.And the diagnosis results of microscopic examination between PCR method was generally consistent(Kappa=0.530).In summary,the genetic evolution degree of the ITS rDNA gene sequence of H.contortus in Xianyang Shaanxi is low.The established molecular biology method can accurately detect H.contortus eggs from fecal samples.This study provides a diagnostic method for clinical detection of H.contortus in sheep breeding.