| Mycoplasma synoviae(MS) infection of chickens and turkeys is an acute and chronic infectious disease, with the main symptoms of joint or footpad swelling, synovial capsule and tendon sheath inflammation. The MS can also cause respiratory disease, airsacculitis or serious systemic infection to chickens when it co-infected with Newcastle disease virus, avian infectious bronchitis virus, Escherichia coli or other pathogenic microorganisms. The MS infection is worldwide epidemical, and its epidemiological surveillance of it showed there was a high prevalence of MS in commercial chickens in many countries. In our country, the MS infection is more and more common in recently years, and it becomes quite serious in some areas or seasons. MS infection can cause lameness, growth retardation, ketones downgrade and decreased egg production to chicken, which seriously affect the production performance and caused huge economic losses to the world poultry industry.This study isolate Mycoplasma isolation on the sick chickens from a chicken farm in Jiangsu Province, and obtained a Mycoplasma-like isolate. By colony observation, serological test and 16 S r RNA sequencing with universal primer, the Mycoplasma-like isolate was identified as a strain of Mycoplasma synoviae. Then we identified it with the standard strain of MS WVU1853 in our lab, the results showed that it was different from the MS WVU1853, thus we named it as MS JS1.This study conducted a preliminary exploration on attack model of Mycoplasma synoviae in SPF chickens. The 21-day-old SPF chickens were challenged with MS JS1 through 4 different ways, including joint attack, intramuscular attack, foot pad attack, and nasal cavity attack. Each group contained ten SPF chickens, and the infective dose was 109 CCU. The results showed that the different attack ways caused different incidence rates. The joint attack group was with the highest incidence rate, which was 9/10, the second highest was foot pad attack group with 8/10, the nasal cavity attack was with 4/10, and the intramuscular attack group was with 0/10. Except the chickens of intramuscular attack group were with no obvious symptom, those of the other 3 attack groups were with vary degrees of characteristic lesions, such as joint and footpad swelling, chest subcutaneous cyst, the lameness and pale comb. The Mycoplasma synoviae bacteria could also be isolated from the lesions of joints, footpads and lungs.In order to develop a multiplex PCR for simultaneous detection of the Mycoplasma synoviae, Mycoplasma gallisepticum(MG), Escherichia coli(EC) and Salmonella(SA) in chickens, primers were designed for the specific amplification of the pdh A gene of MS, the gap A gene of MG, the pho A gene of EC, and the inv A gene of SA. By screening primer combinations and optimizing the system conditions, the optimal primers combination and reaction system were determined. Specificity tests showed that the PCR method could amplify all the four genes from MS, MG, EC and SA, without any PCR products from other avian pathogens. Sensitivity tests showed that the PCR sensitivity for MS and MG was both up to 1 ×102 CCU/reaction, and 1×102 CFU/reaction for EC, 1×103 CFU/reaction for SA. Clinical infection tissue samples showed that multiple PCR detection results were consistent with the results of isolation and identification, the detection rate of up to 83.8%.In this study, we separated and identified the JS1 strain of Mycoplasma synoviae. Then we explored the challenge model for Mycoplasma synoviae in SPF chickens preliminarily. In addition, the multiplex PCR detection method of SA, MG, EC and MS for the simultaneous detection of common pathogen in chickens was successfully established. This study is useful for epidemiological investigation of Mycoplasma synoviae infection and will be helpful for the development of the Mycoplasma synoviae vaccine. |
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