鸡滑液囊支原体ZP-1株的分离鉴定及其致病性和免疫保护效果的评估

Isolation,identification and evaluation of pathogenicity and immunoprotective effect of Mycoplasma synoviae strain ZP-1

为分离鉴定鸡滑液囊支原体(MS),并评估其致病性和免疫保护效果,本实验无菌采集南阳市多家鸡场35份疑似MS发病鸡的关节腔粘液和关节组织,通过病原分离培养后观察菌落、PCR鉴定并测序。结果显示,在改良Friis固体培养基上可见圆形、边缘整齐、隆起的“煎蛋样”菌落形态;PCR可以扩增到16S r RNA基因目的条带,扩增产物测序后与NCBI中登录的ATCC 25204标准菌株16S r RNA基因序列比对,结果显示二者的同源性达99%以上,本实验共分离到5株MS。选择ZP-1株进行生物学特性研究,通过颜色变化单位(CCU)试验测定其生长滴度、采用试剂盒测定其代谢产生的H_(2)O_(2)水平。结果显示,在培养24 h~36 h后,ZP-1株的生长滴度最高可达10^(12)CCU/mL,具有生长速度快、滴度高的特点。在甘油存在的情况下,ZP-1株产生H_(2)O_(2)的相对水平极显著高于弱毒疫苗株(P<0.001)。将ZP-1株分别通过足垫和静脉注射感染SPF鸡,评估其致病性;利用该菌株制备灭活疫苗经皮下接种鸡后攻毒,分析ZP-1株的免疫保护效果。结果显示,该分离株可复制出MS典型症状及病变;利用该菌株制备的灭活疫苗对鸡的攻毒保护率可达80%,高于商品化疫苗,其降低MS关节组织定植水平和MS抗体水平与商品化疫苗一致。本研究获得了具有较高生长滴度(10^(12)CCU/mL)的MS分离株ZP-1,发现利用该MS分离株制备的灭活疫苗具有较好的免疫保护效果,为MS疫苗的研发奠定了基础。

In order to isolate and identify Mycoplasma synoviae(MS),35 aseptic samples of joint cavity mucus and joint tissue from chickens suspected of MS infection were collected across various farms in Nanyang City.Through pathogen isolation,culture,colony observation,PCR identification,and sequencing,we successfully isolated MS strains.The results showed that the isolates were characterized by a distinct"fried egg"-like colony morphology on solid medium,and PCR analysis consistently amplified the 16S rRNA gene.Sequence alignment with the ATCC 25204 standard strain in the NCBI database revealed a high homology of over 99%,confirming the identity of MS.A total of 5 MS strains were isolated.ZP-1 strain was selected for an in-depth examination of its biological characteristics.Its growth titer was determined by Colour change unit(CCU)experiment,the level of hydrogen peroxide produced by metabolism was determined by kit,and its pathogenicity and immunogenicity were evaluated by animal experiments.The strain demonstrated a remarkable growth titer of up to 10^(12)CCU/mL within 24-36 hours,highlighting its rapid growth and high yield.The ZP-1 strain also exhibited a significantly higher relative level of hydrogen peroxide production in the presence of glycerol compared to an attenuated vaccine strain,which may be indicative of its virulence.Further animal experiments confirmed the pathogenicity of the ZP-1 strain,as it induced typical MS symptoms in SPF chickens following foot pad and intravenous inoculation.Notably,an inactivated vaccine derived from the ZP-1 strain achieved an 80%protection rate upon subcutaneous administration and subsequent challenge,a rate that rivals that of commercial vaccines.In conclusion,ZP-1 strain not only exhibits a high growth titer(10^(12)CCU/mL),but also marks the first instance where a correlation between hydrogen peroxide production and virulence has been established.The strain's demonstrated immunoprotective efficacy as an inactivated vaccine lays a robust groundwork for advancing future vaccine research and development.