Isolation And Identification Of Mycoplasma Ovipneumoniae Intacheng, Xinjiang And Preliminary Studies On Immunogenicity Of Inactivated Oil-adjuvant Vaccine

Mycoplasma ovipneumoniae(MO) is one of the important pathogens causing interstitial pneumonia of sheep and goats. The clinic symptom in infected animals is characterized by progressive chronic pneumonia such as respiratory disorder, diarrhea, angular, growth retardation, and high mortality. It has posed a threat to the development of sheep industry. When MO was firstly isolated in 1963, the pathogen found in 1978 in China from sheep imported from abroad. Since then, the disease was widespread all over the country. Xinjiang is one of the main regions of sheep industry in China, however, the disease lead to high morbidity and mortality in lambs. So far, there is not commercial vaccine to prevent sheep from infectiion of MO. Therefore, it is of important significance to isolate and identify of epidemic strains of MO, and develop an effective inactivated vaccine in Xinjiang.In this study, the main methods and results are as follows:1. Isolation and identification of Tacheng isolates of MO25 lung samples of dead sheep suspected MO infection were collected from Tacheng Xinjiang, and the epidemic strains of MO were isolated by KM2 media 15 strains of MO were successfully isolated. All of them display typical papillary colony, and the isolates are of pleomorphic. It is dark blue in center, light color on the edge of bacteria by Dienes dying. Fourteen strains can hydrolyze glucose, adsorpt of red blood cells, while only one strain hydrolyzes urea and no adsorption ability for red blood cells, 15 isolates of mycoplasma can fade the color of meilan milk and cause hemolysis. 16 S r RNA gene sequence of isolates were amplified by PCR and sequenced. Based on the alignments of 16 S r RNA gene sequences among isolates, the identities in nucleotide between Y98 and isolates were 98.82-100%, respectively. These isolates, SC01 and Y98 strain were in the same branch of phylogenetic tree, which confirmed that 14 isolates were MO.2. Cloning, genetic variation and expression of antigen genes of MO Tacheng stains.HSP70, adhesin, haemolysin A and haemolysin C genes of MO S3 strain were amplified with specific primers by PCR, respectively, and then were cloned and sequenced. The genes of HSP70, adhesin, haemolysin A and haemolysin C are 747 bp, 3426 bp, 777 bp and 1239 bp, respectively. Compared with SC01 strain, the identities of HSP70 of S3 strain in nucleotide and amino acid was 85.24% and 83.24%, respectively. There were 64 mutations in nucleotide, leading to 24 mutations in amino acid. The identities of adhesin in nucleotide and amino acid were 93.56% and 92.19% respectively. There were 180 mutations in nucleotide, causing 62 mutations in amino acid. The identities of haemolysin A in nucleotide and amino acid were 95.24% and 93.64%, respectively. There were 32 mutations in nucleotide, causing 20 mutations in amino acid. The identities of haemolysin C in nucleotide and amino acid was 89.31% and 85.23%, respectively. There were 45 mutations in nucleotide, causing 23 mutations in amino acid. Phylogenetic tree revealed that there were relatively close relationship among tacheng isolates, SC01, M.hyopneumoniae J, 232 strains. Compared with antigen epitopes of HSP70, adhesin, hemolysin hemolysin A and C of SC01 strain, there exists some differences in antigen epitopes for S3 strain. The expressed HSP70 was confirmed to be with a molecular mass of 53 k Da by SDS-PAGE. Western blot revealed that the recombinant protein can specifically react with anti-MO polyclonal antibody. And recombinant HSP70 protein can induce specific antibodies in mice after immunization, confirming that the recombinant protein has good immunogenicity.3. Development of inactivated oil-adjuvant vaccine and it’s immunogenicityS3 strain of MO was cultured when bacteria concentration reached 109 CCU/m L. Bacterial antigen was diluted into 0.5 and 0.25 mg/m L after 20 fold concentrated. Then, bacteria were inactivated with formaldehyde in 37 ℃ for 48 h, following blending with white oil in volume ratio of 1:1 to preparate MO inactivated vaccine. The sterile and safety of vaccine was then evaluated. 27 healthy sheep of MO antibody-negative were selected and divided into three groups F1, F2, and control group, each group contains 9 sheep. Two groups of F1 and F2 were subcutaneously vaccinated with a dose of 2 m L(0.5 mg/m L and 0.25 mg/m L), respectively, while control group was injected with saline solution in the same way. All groups were immunized 2 times at interval of 15 d. Finally, blood was collected after immunization of day 15, 30, 45, 60, 75, 90 and 105 d, respectively. The antibody titers were assayed with indirect hemagglutination test. Vaccinated sheep were challenged at day 90 by S3 strain. The results showed that the specific antibody against MO of F1 and F2 group were induced in the blood. Antibody titer can reach 1:16-1:64 in day 45, while control group were negative. The results show that protection rates were 88.9%(8/9) in F1 and F2 group. The sheep in control group was sick with clinic symptoms. The furthrt analysis found that sheep with antibody titer ≥8 can be protected after challenge, which confirmed that the inactivated vaccine is of good immunogenicity.