The Characteristic Of M.Hyopneumoniae ES-2 Strain And Study On The Genes Involed In Synthesis Of Capsular Polysaccharide In Streptococcus Suis Type 2

Porcine respiratory disease is one of the most important diseases that restrict the rapid development of intensive pig industry all over the world.Mycoplasma hyopneumoniae?M.hyopneumoniae?is a major pathogen causing enzootic pneumonia?EP?in pigs in various countries.As a chronic respiratory disease with high morbidity and low mortality rate,EP mainly causes chronic dry cough,slow growth and low feed utilization rate.Since EP was first reported in the 1930s,the disease has caused serious economic losses to pig farming all over the world.In practical production,pigs infected with M.hyopneumoniae are more likely to be infected with bacterial pathogens causing greater economic losses to the pig industry.Streptococcus suis type 2 is a highly pathogenic zoonotic pathogen which can infect humans and pigs,causing pneumonia and meningitis.In the current pig industry,Streptococcus suis type 2 often infect the lung tissue of infected pigs with M.hyopneumoniae.Under the mixed infection of Streptococcus suis type 2 and M.hyopneumoniae,the respiratory diseases of nursing pigs become complex and changeable.Capsular polysaccharides play an important role in the pathogenesis of Streptococcus suis type 2,and the synthesis of capsular polysaccharides requires the involvement of multiple genes.For the scientific problems of complex respiratory diseases in pigs,the isolation and culture of M.hyopneumoniae were carried out.The whole genome of the new isolate was assembled by third generation sequencing technology,and the genes specific to pathogenic M.hyopneumoniae and pathogenic genes of M.hyopneumoniae ES-2 strain were detected by comparative genomics,and animal experiments were carried out to evaluate the protective effect of the ES-2 strain as vaccine on pigs.Four genes?cps2E,cps2G,cps2J and cps2L?predicted to be involved in the synthesis of the repeating units capsular polysaccharides.The role of the four genes in the synthesis of Streptococcus suis type 2 capsular polysaccharide were detected by using gene deletion and biological characteristics study.The main results are as follows:1.Isolation,culture and pathogenicity of M.hyopneumoniae ES-2 strainIn this study,a method of multi-point sampling and continuous passage was applied.A M.hyopneumoniae strain was isolated from lung tissue of Enshi black pig in Hubei by using Friis medium and named as M.hyopneumoniae ES-2 strain.ES-2strain was cultured in the improved Friis medium for about 48 hours,and its growth titer could reach 1×10¹00 CCU/mL,which has the shape of“fried eggs”on the solid medium.ES-2 cells were round or oval ranging from 0.2 to 1.6?m in diameter,which was sensitive to kanamycinn?MIC=2?g/mL?.The results of pig infection experiments showed that M.hyopneumoniae ES-2 strain has highly pathogenic,and can cause typical pneumonia“meat changes”in the lungs,the cilia of the bronchial epithelial cells are bifurcated,shortened or even shed.Among the groups infected with differrent doses of ES-2 strain?7×10⁶ CCU,7×10⁷ CCU,7×10⁸ CCU and 7×10⁹CCU?,the highest incidence and the most serious lesions were found in the group infected with 7×10⁷ CCU of ES-2 strain.2.Characteristic of whole genome of M.hyopneumoniae ES-2 strain and the comparative genomics analysisIn this study,the whole genome of ES-2 strain was sequenced and assembled by the third generation sequencing PacBio method.The complete genome map of ES-2strain was drawn by the correction of initial loci,and ORFs were predicted by bioinformatics tools.ORFs were annotated by protein database NCBI NR and UNIPORT,and the annotated proteins were classified by COG database,and their virulence factors were extracted by comparing with VFDB database.Phylogenetic tree analysis of the complete genomes of ES-2 strain and 161 strains?collected from NCBI?isolated from different hosts showed that ES-2 strain was located in the same branch with the other 8 Swine mycoplasma,1 Mycoplasma flocculen and 2Mycoplasma dispar.M.hyopneumoniae is closely related to Mycoplasma hyorhinis and has the most distant relationship with Mycoplasma pneumoniae.In order to identify the common genes of pathogenic M.hyopneumoniae,the whole genomes of non-pathogenic and pathogenic M.hyopneumoniae were compared by BlASTP in this study.By setting different similarity?0.75,0.80,0.85,0.90,0.95 and 0.99?,a total of34 common genes of pathogenic M.hyopneumoniae were preliminarily found.Through further comparative analysis,a common gene of pathogenic M.hyopneumoniae?ES-2₀0484?was identified at amino acid and nucleotide level.3.Immune protection effect of M.hyopneumoniae ES-2 strain on pigsBased on the advantages of high growth titer and fast growth,the M.hyopneumoniae ES-2 strain was prepared into inactivated vaccine against the infections caused by M.hyopneumoniae ES-2 strain.The best immune protection dose was evaluated by immunization of piglets with different doses of inactivated M.hyopneumoniae ES-2.The results of immunological protection showed that the immune dose of 1.6×10⁹ CCU ES-2 was the best.The immunization doses of 1.6×10⁷CCU and 1.6×10⁸ CCU ES-2 could reach the same protective effect as the commercial inactivated M.hyopneumoniae vaccine?1.6×10⁸ CCU?.4.Study on the genes involved in the synthesis of capsular polysaccharide of Streptococcus suis type 2In this study,4 genes?cps2E,cps2G,cps2J and cps2L?were successfully deleted from Streptococcus suis type 2 respectively.Deletion of these genes resulted in serious loss of capsular polysaccharide on the surface of Streptococcus suis type 2,and the content of sialic acid in capsular polysaccharide was significant reduction.The interactions between the mutants?cps2E,?cps2G,?cps2J or?cps2L with the host were significantly changed,and the pathogenicity of these mutants to mice model was significantly decreased campared to the wild type starin.These results indicate that genes cps2E,cps2G,cps2J and cps2L were involved in the synthesis of capsular polysaccharides on the cell surface of Streptococcus suis type 2,and play an important role in the invasion and colonization process of Streptococcus suis type 2 to the host.