Molecular Epidemiology And Host Resistant Gene Against Gastrointestinal Nematode Infection In Sheep

The infection of Gastrointestinal nematodes(GIN)in sheep is caused by a variety of nematode parasites in Gastrointestinal tract,which can hinder growth,slow weight gain,reduce feeding reward,increase cost,and seriously affect economic benefit.In addition,the widespread use of antihelmintic agents for prevention and control of GIN lead to increase of treatment costs,generation of large number of resistant strains,and drug residues in animal products,exacerbating the risk of GIN infection.Therefore,on the basis of fully understanding the epidemic characteristics and rules of GIN infection,selecting individuals with genetic resistance to GIN and breeding GIN resistant varieties can contribute to reduce GIN infection and the use of chemical drugs,slow down the emergence of drug-resistant strains,and decrease the economic losses of GIN infection.Nowadays,breeding the GIN resistant individuals or breeds is an important strategy and technical means for the prevention and control of gastrointestinal nematodes infection in sheep.Objective: The traditional fecal examination method was combined with ribosomal r DNA detection method to carry out molecular epidemiological investigation of gastrointestinal nematode(GIN)infection in various populations and individuals of Kazakh sheep,in order to establish molecular diagnosis technology of dominant parasitic species and understand the molecular epidemic characteristics of various parasitic species.At the same time,by constructing the phylogenetic tree of the main infecting species,we can learn the epidemic and evolutionary patterns of the species,providing basic data for molecular epidemiological investigation and host resistance evaluation.On this basis,the candidate gene method and Mass ARRAY(?) SNP genotyping technology was employed to analyze the genetic polymorphism of the SNP loci of the GIN-resistant candidate genes in Kazakh sheep.And further the correlation between genotype and GIN infection intensity was explored.Our study laid a foundation for further identification of GIN-resistant candidate genes in sheep,for in-depth study of the molecular mechanism of GIN-resistant candidate genes and GIN-resistant molecular breeding.Methods: The thesis consists of three parts.Part I: Epidemiological investigation on the infection of gastrointestinal nematodes in Kazakh sheep and their hybrid population.This is an epidemiological study on the gastrointestinal nematode(GIN)infections in Kazakh sheep and the F1 and F2 individuals of Kazakh × Texel sheep crosses.A total of 7599 sheep fecal samples were collected from the Zhaosu County and Nilka County in Ili Kazakh Autonomous Prefecture in the four seasons-spring,summer,autumn,and winter of 2019.The parasite causing the infection was identified by the saturated saline floating method,and the the number of eggs infected with GIN was calculated by the modified Mc Master method.SPSS19.0 was used to log(n +10)conversion for faecal egg counts(FEC)to obtain LFEC,and variance analysis the differences seasons,regions,populations and species in LFEC of GIN and to understand the epidemic characteristics of GIN infection.Part Ⅱ: : Molecular identification and phylogenetic analysis of gastrointestinal nematodes in sheep.The internal transcribed spacer 2(ITS-2)sequences of ribosomal DNA(r DNA)was used as the target sequence,and three dominant species of nematodes,Haemonchus contortus,Trichostrongylus spp.,and Teladorsagia circumcincta from the various populations of Kazakh sheep were subjected to molecular identification and phylogenetic analysis.The fecal genomic DNA and single larva genomic DNA were extracted and amplified by PCR using specific primers to determine the infection rate of the three nematode species.In addition,the PCR products were sequenced and homology analysis to construct a phylogenetic tree,the genetic evolutionary relationships among different species and regions were analyzed.In addition,the molecular detection results and egg detection results were compared and analyzed to establish an accurate and highly specific detection method.Part Ⅲ: Candidate gene association study for resistance of gastrointestinal nematodes in sheep.A total of 381 Kazakh adult female sheep were selected.The GIN infected log(n +10)conversion for fecal egg counting(LFEC)was calculated,and the genomic DNA of individual sheep was extracted and purified.According to the reported candidate genes for GIN resistance,a total of 29 single nucleotide polymorphism(SNP)loci of the 28 candidate genes related to the immune system such as infection and inflammation were screened.Further,the Mass ARRAY(?) SNP Genotyping was conducted and statistical analysis of population genetics was performed on the SNP loci.SPSS19.0 was used to carry out association analysis between SNP genotype and LFEC phenotype data,and to preliminary identify GIN-resistant candidate genes.Results:(1)The results showed that high infection rate and moderate mean infection intensity(1235.06 A/g)of GIN in the Zhaosu County and Nilka County of Ili.Among them,the average infection intensity of Zhaosu county was 1386.21 A/g,and the average infection intensity of the Nilka county was796.30 A/g,and the mean infection intensity of GIN infection in the Zhaosu area was significantly higher than that in the Nilka area(P<0.001).The infection intensity of GIN infection in the four seasons was the highest in spring,followed by in summer,then in autumn,and the lowest in winter(P<0.001).The mean infection intensity of GIN infection of Kazakh sheep was significantly higher than the F1 generation,which was then significantly higher than the F2 generation.In summer and autumn,the GIN infection rate and mean infection of Kazakh sheep were highly significantly higher than those of the F1 generation(P <0.001).In addition,nine types of sheep GIN were initially identified,with the dominant parasite species of Haemonchus contortus,Trichostrongylus spp.,and Ostertagia spp.in different populations of Kazakh,Texel × Kazakh and different seasons.(2)The results showed that all the three species had their ITS-2 specific bands amplified.The infection rates based on fecal DNA were 35.59% of H.contortus,25.55% of Trichostrongylus spp.,and11.24% of T.circumcincta respectively,and the infection rates based on larva DNA were 19.75% of H.contortus,23.54% of Trichostrongylus spp.,and 10.03% of T.circumcincta respectively.The results were consistent with the PCR results of fecal samples and larval DNA from Trichostrongylus spp.and T.circumcincta,except H.contortus.The phylogenetic tree showed that the ITS-2 sequences of the three species were on the same branch as the ITS-2 sequences of the same species in NCBI,and on different branches from those of the ITS-2 sequences of different families,genera and species,and on different branches from H.contortus and T.circumcincta in different regions(Zhaosu County and Nilka County).The same 93 fecal samples were analyzed by molecular identification and saturated saline solution floatation method respectively.The molecular identification of hatched larva from fecal samples exhibited an infection rate of 100.00% with respect to the three dominant species,higher than the infection rate of96.77% by the saturated saline solution floatation method.Therefore,the present molecular identification is more reliable in specificity and accuracy of these three species.(3)Mass ARRAY genotyping was performed on the 29 SNP loci of the 28 GIN-resistant candidate genes of Kazakh sheep,and successfully detected 27 SNP loci,of which 24 SNP loci were polymorphic.The genotype analysis of these 24 SNP loci displayed 3 genotypes for 13 SNP loci,and 2 genotypes for 11 SNP loci.And 22 SNP loci among these 24 loci had missense mutations occurred in the coding region,one SNP loci had a nonsense mutation in the coding region,and one SNP loci had a mutation in the intergenic region.Hardy-Weinberg equilibrium(HWE)analysis showed that all but four sites reached HWE.In addition,polymorphism analysis demonstrated that 9 SNP loci were moderately polymorphic(0.5 >Polymorphism Information Content(PIC)> 0.25),and the remaining 15 loci were low polymorphic(PIC< 0.25).Association analysis was performed between the genotypes of 24 SNP loci and the mean LFEC value,the loci rs426080384 of MMRN1,rs426086535 of TLR1,rs411685804 between SPPI/EGF,rs406469938 of ITGAM,and rs424271485 locus of CD163 were significantly correlated with the mean LFEC value.Bioinformatics analysis predicted that the mutations of four SNP sites would affect the structure and function of the encoded protein,thereby affecting the host’s defense response to GIN infection.Conclusion: From this study,we learned the epidemiological pattern of GIN infection in the Texel and Kazakh F1 hybrid and the progressive F2 sheep in Ili Zhaosu County and Nilka County.Regarding the four seasons,the GIN infection intensity was the highest in spring and the lowest in winter.The GIN infection intensity of Texel and Kazakh F1 hybrid sheep was significantly lower than that of Kazakh sheep,presumably related to the long-term GIN resistance breeding of Texel sheep abroad.With respect to the specific identification of GIN species,the molecular detection method based on r DNA ITS-2 sequences showed higher specificity and convenience than the conventional saturated saline flotation method.In this study,molecular diagnostic methods for the main infecting insect species H.contortus,Trichostrongylus spp.,and T.circumcincta were established.The epidemiological characteristics and pattern of GIN infection in the two counties of Ili were learned.Furthermore,five SNP loci related to GIN resistance candidate genes in Kazakh sheep were identified.The possible mechanism of SNP mutations impacting on GIN infection/resistance by affecting the function of their encoded protein was preliminarily explored,which laid a foundation for the research of GIN resistance molecular breeding and resistance mechanism in sheep.