Isolation And Identification Of Mycoplasma Synoviae And Establishment Of MS-H Identification Methods Of Isolates And Vaccine Strains

Mycoplasma synoviae（MS）infection can cause symptoms such as synovitis,tenosynovitis,and egg tip syndrome in chickens and turkeys.After mixed infection with other pathogenic microorganisms,the pathogenicity of pathogenic microorganisms is exacerbated,resulting in an increase in the mortality rate of flocks,a decrease in the feed utilization rate of broilers,a degradation of carcasses,a decrease in egg production of laying hens,and a poor eggshell quality,which has caused great economic losses to the poultry breeding industry.At present,the disease is endemic in China.In this study,a fluorescence detection method for Mycoplasma synoviae was established.The etiology of MS infection was detected in broiler flocks of a broiler enterprise in Fujian Province.Mycoplasma synoviae was isolated and identified from broilers infected with MS to detect its pathogenicity and drug sensitivity.A multiplex PCR reaction system for rapid differentiation of clinical isolates from vaccine strain MS-H was established,laying a foundation for the prevention and control of MS infection.1.First,MS universal primers were designed according to 16S rRNA in the conserved region of MS gene,and after verifying the primer specificity,a fluorescence detection method for MS was established.Positive plasmids were constructed to verify that the assay was specific for MS.Plasmid concentration was determined and its copy number was calculated to be 2.18×10¹⁰copies·μL^-1based on the copy number formula.After 10-fold dilution,the lowest detection concentration was 2.18×10¹copies·μL^-1.Different flocks were sampled to detect the positive rate of MS.The results showed that the average MS positive rate of the sampled flocks was 28.0%,the MS positive rate of 1-21 flocks was in the range of0.9%-25%,the average positive rate of 22-40 flocks was in the range of33.3%-73.6%,and the average positive rate of 22-40 flocks was 56.9%.It is highly prevalent in January,March,April,and November of the year and is speculated to be caused by climatic factors such as months exchanged between autumn and winter and winter and spring,and large diurnal temperature differences.2.Six strains of Mycoplasma synoviae were isolated and identified from broilers suspected of MS infection,which were identified by observing colony morphology,hemadsorption test,bacterial L-form identification,and hemagglutination test,all of which were in accordance with the biological characteristics of Mycoplasma synoviae.Specific primers were designed for the Vlh A gene,and their PCR amplification products were sequenced and analyzed for homology with MS sequences uploaded on Gen Bank.The results showed that the homology of Vlh A gene sequence of the isolates with MS reference strains in the database was greater than 95%,indicating that the isolates in this study were all MS;homology analysis showed that the isolates and MS strains such as HN01 and 5-9 in China were in the same clade and had distant homology with MS-H of the vaccine strains,indicating that the isolates in this study were all wild strains.3.7 SPF chickens were infected with 0.2 ml of MS bacterial solution at a concentration of 10⁸CCU/ml through the footpad.At 7 d and 14 d after challenge,blood was collected to separate serum,and the pathological changes of tissues and organs were dissected and observed.Liver,kidney,spleen,trachea and other tissues were taken and fixed with formaldehyde for pathological section;the levels of IL-1β,IL-6,INF-γand TNF-αcytokines in liver,spleen and kidney,as well as biochemical parameters such as alkaline phosphatase,creatine kinase,and alanine aminotransferase were measured.Pathological examination of the lesions showed:SPF chickens infected with MS test had pathological phenomena such as splenic hemorrhage,renal enlargement,and hepatomegaly;the detection results of cytokine levels showed that the levels of the four cytokines in serum and kidney,spleen,and liver tissues were higher than those in the blank control group,of which the levels of the four cytokines in liver tissues were significantly（P>0.05）higher than those in the blank control group,indicating that MS proliferated in vivo after challenge,causing an inflammatory response in the body,and this inflammatory response may appear as early as in the liver;the serum biochemical results showed that the alkaline phosphatase level decreased and the creatinine level increased in the challenged experimental group compared with the blank control group,combined with the results of pathological section of the kidney tissues,indicating that MS proliferation caused damage to the liver in vivo;the alanine aminotransferase level increased in the challenged experimental group,combined with the up-regulation of pathological section of the liver and cytokine expression level.4.In order to establish a rapid and accurate method for differentiating MS field isolates from live vaccines MS-H.According to the two base mutation sites of ppm and deo D genes of Mycoplasma synoviae,two pairs of specific primers were designed by mismatch amplification mutation analysis（MAMA）PCR.Multiplex PCR amplification system was used to amplify the target gene.Through the optimization of reaction conditions,a multiplex PCR detection method that can identify the two by one PCR amplification and gel electrophoresis was established,and the specificity and sensitivity of this method were detected.The results showed that the method established in this study could specifically amplify the specific target bands of live Mycoplasma synoviae vaccine MS-H strain and MS isolate,and there was no specific amplification of several other common avian pathogens,with high specificity;the lowest sensitivity to MS-H strain and MS isolate was4.21×10⁵and 6.47×10⁵copies·μL^-1,respectively,the lowest detection concentration of the detection method to universal primers was 2.18×10³copies·μL^-1,the lower limit of detection concentration to bacterial solution of live vaccine MS-H strain and Mycoplasma synoviae isolate was 10⁵CCU/ml,and the lowest concentration of bacterial solution detected by universal primers was 10³CCU/ml.The establishment of this method provides a reliable and rapid diagnostic technique for the differentiation of live vaccine MS-H strains and MS isolates.