Isolation And Identification Of Chicken Mycoplasma Synoviae And Analysis Its Immunogenicity

Mycoplasma synoviae(M.synoviae)belongs to the genus Mycoplasma in the class Mollicutes,which mainly infects chickens and turkeys and causes swelling of joints and foot pads,inflammation of bursa and tendon sheaths and swelling of some parenchymal organs in poultry after infection.In recent years,M.synoviae infection has been widely prevalent in China’s chicken flocks,with rapid and widespread prevalence and high incidence,causing serious economic losses.The lack of effective vaccines and therapeutic drugs has made prevention and control difficult.Based on this,the isolation and identification of M.synoviae and immunogenicity analysis will lay the foundation for the development of a vaccine for the disease,which is of great significance for the effective prevention and control of the disease.In this study,PCR was performed to identify M.synoviae in the joint tissues of chickens with suspected infection of this disease,and the positive samples were inoculated with modified Frey’s liquid medium to obtain a strain of M.synoviae by cultivation.The morphological observation of the colonies under low magnification microscope showed that the colonies had typical mycoplasma characteristics,round"fried egg-like"colonies with diameters of about 200～400μm,and oval-shaped bacteria with diameters of 0.1～0.3μm could be observed under high power microscope by Wright’s stain.It was determined that the isolate was free from contamination by other exogenous microorganisms.The results of metabolic inhibition and growth inhibition assays showed that the isolate could be neutralized by M.synoviae-specific antiserum,and the strain was named the HLJ strain of M.synoviae.The HLJ strain was characterized by biochemical characteristics assays and hemagglutination assays,and the results showed that the HLJ strain was biochemically identical with the M.synoviae standard strain.Primers were designed according to the vlh A gene sequence of MS in Gen Bank,and the whole gene fragment was amplified by PCR and sequence analysis was performed,and the results showed that the HLJ strain had up to 100%homology with isolates from various regions in China.By phylogenetic analysis,the HLJ strain was in the same branch of phylogenetic tree as the other domestic isolates analyzed.HLJ strain was inoculated with 42-day-old SPF chickens through different injection routes for animal pathogenicity experiments,and it was found that both infection routes can cause a high proportion of SPF chickens,showing typical symptoms of M.synoviae in chickens.The characteristic pathological changes of M.synoviae capsule in chickens were all seen on autopsy,and transvenous infection was determined as the best route of infection.By q PCR test,all tissues and organs of infected chickens contained M.synoviae,with the highest bacterial counts in the joints and foot pads.The minimum infectious dose was determined to be 10~9 CCU/0.1m L after different infectious dose tests.To evaluate the immunogenicity of the HLJ strain of M.synoviae,an inactivated M.synoviae vaccine was prepared in this study,and the preparation process of the vaccine was optimized.Firstly,by measuring and analyzing the culture conditions characteristics of the isolated strain,it was determined that the optimal NAD concentration of the culture medium for in vitro culture was 0.05‰,the optimal passage time was 24 h after growing M.synoviae in passaged culture,the passage CCU was 10~8CCU/0.1 m L,the serum concentration was 15%,and the harvest time was 24 h after passage,which resulted in a live mycoplasma count content of 10~9 CCU/0.1 m L bacterial solution.By measuring the optimal inactivator concentration and inactivation time,it was determined that a formaldehyde solution with a concentration of 0.1%could be used to inactivate the bacterium solution completely at 37℃for 4 h.The inactivated vaccine was prepared with 10~9CCU/0.1 m L of the bacterial solution for immunogenicity analysis,and the minimum immune dose of the prepared inactivated vaccine was determined.The results showed that the level of hemagglutination inhibitory antibodies in the serum to be examined was proportional to the live bacterial content of the prepared vaccine,and the inactivated vaccine prepared with live bacterial content≥10~9 CCU/0.1 m L had 70%of individual chickens without M.synoviae typical clinical symptoms in each immunized dose group during the observation period after infection,and the body weight of chickens in the immunized group was significantly higher than that of the control group.The results of pathological autopsy showed that the immune protective effects of 0.2 m L and 0.4 m L immunization groups were similar and were better than those of 0.1 m L immunization group.The above results showed that the inactivated vaccine prepared by the HLJ strain of M.synoviae had good immunogenicity,and the protection rate could reach 70%at 0.2 m L/chicken with an immunization dose of 10~9 CCU/0.1 m L.The duration of immunity of the inactivated vaccine was measured,and the hemagglutination inhibitory antibody potency of the immunized chickens was maintained above 1:32 at 150 d post immunization above 1:32,which is the HI antibody level providing protection,at the 14 d and lasted to the 150 d post immunization.In summary,a field of M.synoviae was isolated and identified in this study,and the inactivated vaccine prepared with this strain had good immunogenicity and produced soils protection for chickens after immunization,laying the foundation for this strain as a candidate for inactivated vaccine against M.synoviae.