Polymorphism Of The OLA-DRB1、DRB3 Exon 2 In The Argali And Bashibai Sheep Population And Its Association With Mycoplasma Ovipneumoniae Infection

The major histocompatibility complex(MHC) is an organized cluster of tightly-linked genes and diversity of polymorphic alleles found in most vertebrates. Sheep MHC is also called ovine lymphocyte antigen(OLA). The highly polymorphic genes of the MHC play a central role in the immune recognition of pathogens and parasites. The antigens coded by DRB at MHC-â…¡ play important roles in immune system, that the relationship between MHC and susceptibility or resistance to diseases was closely linked. Mycoplasma ovipneumoniae(M.O) is a kind of veterinary pathogen causing contagious pleuropneumonia in sheep, goats and wild sheep worldwide,and different sheep breeds of M.O susceptibility exist obvious difference. On the basis of that the first generation lamb of wild argali and Bashibai sheep hybrid confirmed outbreaks of mycoplasma pneumonia, argali and Bashibai sheep were detected by M.O-ab Elisa kit. The polymorphisms of the classâ…¡OLA-DRB1 and DRB3 exon 2 were detected by PCR-RFLP analysis in argali and Bashibai sheep. We characterized the relationship between the polymorphism of OLA-DRB1 or DRB3 exon 2 and genetic resistance to M.O infection in these two sheep breeds. Results of the present study may provide theoretical support for sheep hybridization and anti-disease breeding. The main content of the experiment and the results are as follows.1. To determine the cause of respiratory disease of argali, which was prevalent in a zoo of Xinjiang province, 5 strains of mycoplasma were isolated from epidemic materials of the argali flock infected the disease. All isolates were identified as M.O by germ culture characteristics, colonial morphology observation, biochemical experiments, PCR identification, artificial infection test and nucleotide sequencing, respectively. The diagnosis result indicated that M.O is responsible for the pleuropneumonic disease in the argali flock. It is reported for the first time in China, and for the epidemiological characteristics of M.O infection increased the new content.To detect infection of M.O of argali, we designed a pair of specific primers to amplify a 406 bp fragment of the 16 S r RNA gene of M.O. After analysis of the optimization reaction condition, e.g., the specificity and sensitivity for PCR of M.O gene, anneal temperature, concentration of Taq DNA polymerase and that of the primers. We established a rapid, sensitive and specific PCR method for detection of M.O infection. The PCR method has significant economical and practical advantages.2. The polymorphism of the OLA-DRB3 exon 2 was detected by the PCR-RFLP analysis in argali and Bashibai sheep. By comparing test genotypic and allele frequencies of OLA-DRB3 exon 2 of healthy sheep with that of M.O infectious ones, the differences were statistically analyzed. Using three restriction enzymes, i.e., Taq I, Pst I and Hae â…¢, yield 9 genotypes, 8 alleles in argali, and 24 genotypes, 11 alleles in Bashibai sheep. 122, 154, 168, 241 base positions in OLA-DRB3 exon 2 were polymorphic in the two flock. The results of x2 test of the OLA-DRB3 exon 2 revealed that Pstâ… , Haeâ…¢ restriction enzyme sites in argali and Taq I, Pst I sites in Bashibai sheep population were fit with Hardy-Weinberg equilibrium(P>0.05), the other restriction sites did not reach the gene balance(P<0.01). The statistical result of population genetic parameters shows the polymorphism of Bashibai sheep population has a high degree of genetic variation in which He, PIC, Ne is 0.541, 0.4854 and 3.2918, respectively. Argali has medium in which He, PIC, Ne is 0.300, 0.3339, 1.5037, it showed that argali has a more stable genetic structure.In OLA-DRB3 exon 2, genotype Hae â…¢-AA(P<0.01) in argali and Hae â…¢-BC(P<0.05) in Baishibai sheep may be resistant to M.O, but Hae â…¢-DD(P<0.05) genotype of Baishibai sheep may be susceptible. Pst I B(P<0.01) and Hae â…¢ C(P<0.01) were resistant to M.O, Pst I A(P<0.01) was susceptible.3. The OLA-DRB1 exon second polymorphism was determined by PCR-RFLP in argali and Bashibai sheep. Using three restriction enzymes, i.e., Sac I, Bsa HI and Hae III, yield 8 genotypes, 6 alleles in argali, and 12 genotypes, 9 alleles in Bashibai sheep. And base positions 296, 178, 173, 123, 118, 71 were polymorphic, respectively. Hardy-Weinberg equilibrium state of OLA-DRB1 exon 2 enzyme loci in the two different breeds of sheep is not consistent. In argali, Sac I site was Hardy-Weinberg equilibrium(P>0.05), Bsa HI and Hae III does not reach equilibrium, while at these sites were not fit with equilibrium in Bashibai sheep. The average heterozygosity(He), polymorphic information content(PIC) and effective number of alleles(Ne) in argali and Bashibai sheep population was 0.327, 0.3604, 1.5102 and 0.479, 0.4794, 2.8925, respectively.Comparing the frequency of genotypes and allele in M.O infection sheep with the no infection control group, genotypes and allele in OLA-DRB1 exon 2 genes of argali and Bashibai sheep were not found associated with M.O infection.In summary, the polymorphism of the OLA-DRB3 exon 2 in argali and Baishibai sheep was related to M.O resistance and susceptibility, but the polymorphism of the OLA-DRB1 exon 2 was not related.