Isolation And Identification Of Mycoplasma Pneumoniae LY-1 Strain And Cloning And Sequence Analysis Of HSP70 And P30 Genes

Sheep contagious pleuropneumonia is a highly contagious disease caused by Mycoplasma,and Mycoplasma ovipneumoniae is one of the most important pathogens.In this study,1 strains of mycoplasma pneumonia were successfully isolated from the diseased material of the diseased sheep,and the biological characteristics and immunogenicity genes were studied systematically:1.In November 2016,the scale of Lushi County in Henan province of sheep field suspected contagious ovine pleuropneumonia cases.Lambs and adult sheep may occur,but lamb symptoms are more severe.Lamb mortality was about 30%,and adult sheep did not die.The sheep disease fever,depression,anorexia,cough,dyspnea and sudden death.Necropsy revealed the chest,pleural and lung serous and fibrinous inflammation is the main pathological characteristics,the specific performance of pleural and pericardial effusion with pale yellow,pleural thickened and rough,yellowish white with cellulose deposition,part of sheep,pericardial and pleural pleural adhesions;lung surface bleeding point.Lung interlobular tissue becomes wide,darkening the typical lesions of lung.Based on the clinical symptoms and pathological examination,the disease was identified as contagious pleuropneumonia.2.I suspected strains of Mycoplasma were isolated from the diseased material of the diseased sheep,named LY-1 strain.The culture is difficult,but in PPLO solid medium,the 37 C static culture 5-7,can be grown into a round,transparent,like dew tip size of the colony;culture of 14-16,can be grown into a round,dark,transparent,center around diameter 2-3mm "fried Poached Egg" colony.The results of Gram staining showed that the bacteria were spherical,bipolar,rod-shaped,etc..The results of biochemical tests showed that it could ferment glucose,but could not use arginine and urea.The culture characteristics,morphology and biochemical characteristics of Mycoplasma were all in accordance with the characteristics of mycoplasma.The results of drug sensitivity test showed that the strain was highly sensitive to azithromycin,ofloxacin and ciprofloxacin.It was sensitive to tetracycline and erythromycin,gentamicin and kanamycin.3.The genome was extracted as the template,and the Mycoplasma PCR was used to identify the bacteria.The bacteria were identified as Mycoplasma,and the specific primers PCR were used to identify the strain of Mycoplasma ovipneumoniae(361 bp).PCR was used to amplify the 16 S rRNA gene(1469 bp)and sequencing and BLAST analysis,16 S rRNA gene sequence and homology were NCBI database published mycoplama,the homology was more than 99%.The phylogenetic tree of 16 S rRNA gene was further constructed,which indicated that the LY-1 strain had a close relationship with FJ-FZ strain and FJ-SM strain.The results showed that the LY-1isolate was Mycoplasma ovipneumonia.4.Heat shock protein HSP70 and adhesion factor protein P30 are important protective antigens of mycoplasma.In the LY-1 genome as the template,the P30 gene were amplified by the size of HSP70 gene and 786 bp 738 bp,the BLAST results showed that,HSP70 gene and NCBI reported Mycoplasma hyopneumoniae CP003131.1 sequence homology was 77%.The homology between P30 gene and NCBI has been reported to be 80% CP003131.1 of Mycoplasma pneumoniae.Its gene and protein characteristics are worthy of further study...