In order to isolate Mycoplasma hyopneumoniae(Mhp)and select virulent strains with high growth titer from the isolates to acclimate at high temperature to obtain attenuated strains.Forty lung and trachea tissues were aseptically collected from pigs suspected of Mhp in several farms in Nanyang City.A total of 6 strains of Mhp were isolated through pathogen isolation,colony observation,PCR identification and sequencing.For each isolate,round colonies with neat edges,dewdrop like particles and ridges in the center were observed on solid medium.PCR could amplify the target gene bands of each isolated P36.After sequencing,the amplified products were compared with the gene sequences of ATCC 25934 reference strain(J strain)P36 registered in NCBI,and the homology was more than 99％.The growth titer of TH-1 strain measured by CCU experiment could reach 1011 CCU/mL,which showed a high growth titer.TH-1 strain was selected for continu-ous passage at high temperature(40 ℃)in vitro to obtain different passages(P50,P1 00,P1 50).TH-1 P150 still had a higher growth titer(1010 CCU/mL)at 40 ℃ growth temperature.TH-1 P1,P50,P100 and P150 were injected into the trachea to infect 25-day-old weaned piglets,and the re-sults of virulence evaluation showed that 4 pigs(4/5)in the P1 infection group had typical"meat or shrimp meat"lesions,and serious monocyte and lymphocyte infiltration were found in the lung HE pathological sections,and the cilia were clutter-tangled,shortened and exfoliated.P150 infec-tion group did not show typical Mhp characteristic lesions,lung HE and tracheal scan sections did not show microscopic pathological changes,indicating that TH-1 was a virulent strain,but its viru-lence was continuously decreased after continuous in vitro passage,and TH-1 P150 was a low-viru-lence strain.TH-1 P150 strain,as a attenuated vaccine strain,has good immunogenicity,and the av-erage reduction rate of pneumonia disease after immune challenge was 83.3％.In conclusion,a low-virulence strain with high growth titer(TH-1 P150)was obtained by continuous passage at high temperature in vitro for the first time,and its immunogenicity was good,which laid the foundation for further research and development of Mhp attenuated vaccine.