Establishment And Application Of PCR Method For Detection Of Three Gastrointestinal Nematodes Of Sheep And Goats Based On ITS Gene

Gastrointestinal nematodiasis,a chronic wasting disease,is a common parasitic disease of sheep and goats.The common gastrointestinal nematode species of sheep and goats are Oesophagostomum spp.,Bunostomum spp.,Trichuris spp.,Haemonchus spp.,Ostertagia spp.,Nematodirus spp.,etc.Sheep and goats infected with gastrointestinal nematodes generally manifest clinical symptoms such as loss of appetite,weak spirits,anemia,emaciation,diarrhea,and even death,which seriously threaten the healthy development of the sheep and goat industry.The increasing drug resistance of nematodes,plus geographical conditions,natural environment,social and cultural factors,have all,to some extent,contributed to the evolutionary differences among conspecific nematodes from different hosts and regions.Therefore,the study on the phylogenetic development of nematodes is of great significance for the analysis of population genetic structure,the law of evolution and the selection of related antiparasitic drugs.ITS gene as a reliable genetic marker has been widely used in many aspects such as phylogenetic study,species identification,and epidemic monitoring of nematodes.The purpose of this study was to conduct an amplification and sequence analysis on the ITS1 and ITS2 genes of O.asperum and B.trigonocephalum collected from Xianyang Shaanxi Province,and to find out the genetic evolution relationship and other related molecular characteristics of the two kinds of nematodes.In addition,in order to accurately identify the species of nematode eggs in goats’fecal samples,three common genera of gastro-intestinal parasitic nematodes,namely,Oesophagostomum,Bunostomum and Trichuris were selected as the research objects,and their respective genus-specific single PCR detection methods were established based on the ITS gene sequence.On this basis,the duplex PCR method was established for the simultaneous detection of Oesophagostomum and Trichuris,Bunostomum and Trichuris,Oesophagostomum and Bunostomum.The above PCR methods were applied to the detection of nematode eggs in clinical fresh feces of dairy goats.Results are as follows:1.The ITS sequence analysis of O.asperum and B.trigonocephalum showed that the ITS sequence size of 40 O.asperum isolates was about 786 bp.The ITS1 sequence consisted of 9 genotypes,and the intra-specific variation rate was 0-1.7%.The ITS2 sequence had 7genotypes,and the intra-specific variation rate was 0-1.9%.Among the sequences,the ITS1fragment was about 367 bp in length,and it contained 5 base mutation sites,including 4conversion sites and 1 translocation site.The ITS2 fragment was about 268 bp in length,containing 6 base mutation sites,including 2 conversion sites and 4 transposition sites.As for the 9 B.trigonocephalum isolates,the ITS sequence size was about 766 bp.The ITS1sequence had 4 genotypes,and intra-specific variation rate was 0-0.3%.The ITS2 sequence had 1 genotype,and intra-specific variation rate was 0-0.4%.The length of ITS1 fragment was about 382 bp and there was one base transposition site;the length of ITS2 fragment was231 bp and there was one base transposition site.The above results indicate that the ITS r DNA sequences of O.asperum and B.trigonocephalum from goats in Xianyang Shaanxi Province have a low degree of intra-specific variation,and no new population differentiation occurs.The results of phylogenetic tree reveal that the conspecific isolates from different areas converge on the same clade,and the correlation between the variation of ITS gene in the same species and different geographical conditions is not obvious.Despite the morphological similarity between B.trigonocephalum and Bunostomum phlebotomum,the ITS sequences of them are relatively far in evolutionary distance,which shows they belong to two different branches and they are different species.2.Based on the genus-specific primers designed in terms of ITS genes of Oesophagostomum,Bunostomum and Trichuris from sheep and goats,a single PCR detection method for three kinds of nematodes was established.The optimization results of single PCR showed that the optimum annealing temperature,amount of primer and Mg²⁺concentration of Oesophagostomum,Bunostomum and Trichuris were 57℃,10 pmol,1.5mmol/L;57℃,10 pmol,2.5 mmol/L;57℃,18 pmol and 1.5 mmol/L,respectively.The results of specificity test showed that the three single PCR detection methods had good specificity and could effectively identify the target nematodes.The results of single PCR sensitivity test showed that the minimum detectable concentrations of Oesophagostomum,Bunostomum and Trichuris were 90,3.6×10³ and 2.68×10³ copies/μL,respectively.It indicates that the three single PCR detection methods established in this study have high sensitivity.3.On the basis of the establishment of single PCR detection method,the duplex PCR detection methods were set up pairwise between Oesophagostomum,Bunostomum and Trichuris.The results of duplex PCR optimization showed that the optimum annealing temperature of Oesophagostomum and Trichuris,Bunostomum and Trichuris,Oesophagostomum and Bunostomum was 57℃.The specificity test results showed that the three duplex PCR methods demonstrated good specificity and could effectively identify the target nematodes.The sensitivity of the duplex PCR methods revealed that the minimum detectable concentrations of the duplex PCR for Oesophagostomum and Trichuris,Bunostomum and Trichuris,Oesophagostomum and Bunostomum were 9×10³ copies/μL and2.68×10⁵ copies/μL,3.6×10⁴ copies/μL and 2.68×10⁶ copies/μL,9×10⁴ copies/μL and3.6×10⁵ copies/μL,respectively,showing high sensitivity with the three methods.4.The results of microscopic examination,single PCR and duplex PCR detection of clinical fecal samples showed that there was no significant difference（P>0.05）in the positive rates of Oesophagostomum,Bunostomum and Trichuris detected by different methods,indicating that all the PCR methods established in this study can be used to detect target nematode eggs in clinical fecal samples.In conclusion,the genetic evolution of the ITS r DNA gene sequences of O.asperum and B.trigonocephalum from goats in Xianyang Shaanxi province is low.The single and duplex PCR molecular biological detection methods established in this study can be used to detect target nematode eggs in clinical fecal samples.And this study provides alternative molecular assistant diagnosis methods for the diagnosis of Oesophagostomiasis,Bunostomiasis,and Trichocerciasis in sheep and goats.