马腺疫链球菌的分离鉴定及其灭活疫苗的研制

发布时间：2018-10-21 15:22

【摘要】：马腺疫是一种由C型链球菌马链球菌马亚种(Streptococcus equi subspecies equi,S.equi)引起的急性接触性马属动物传染病,在我国牧区发病率很高,以2岁以下幼驹易感,给牧民造成巨大的经济损失,但尚无有效的预防和控制措施。为尽快缓解马腺疫在我国牧区的流行情况,本项研究进行了马腺疫灭活疫苗制备方法的探讨,试验获得的病原菌为分离自马腺疫重症马的野毒株(HLJ2011),利用该菌株制备灭活疫苗能够为小鼠提供良好的保护力。本文从HLJ2011菌株生物学特性及马腺疫灭活疫苗小鼠免疫保护效力两个方面进行了系统的研究。结果如下:1.HLJ2011株生物学特性研究:本试验从病畜下颌破溃脓汁病料中分离出1株主要致病菌,对该菌的16S rRNA鉴定、生化鉴定、不同接种浓度生长曲线、菌落形成单位(Colony-Forming Unit,CFU)与OD值关系、马腺疫链球菌毒力测定进行了研究。结果表明,通过16S rRNA鉴定及生化鉴定该菌为S.equi,该菌不同接种浓度生长曲线表明,各浓度生长曲线基本相同,接种浓度越高,对数生长期越短;1%接种浓度培养8~10 h时CFU最大,可达1×109 CFU;用1×107 CFU腹腔攻毒8周龄Balb/c小鼠,死亡率100%,用改良寇氏法计算得出,该菌对小鼠半数致死量为5×105 CFU。2.马腺疫灭活疫苗对小鼠免疫保护效力的研究方面:通过不同温度和甲醛浓度完成HLJ2011灭活条件的筛选以及马腺疫灭活疫苗对小鼠的免疫保护效力的研究。在4℃、28℃及37℃条件下不同甲醛浓度对灭活效果的影响试验表明,温度越高或甲醛浓度越高,灭活时间越短。采用28℃、1%甲醛灭活12 h后,加入某高分子聚合物佐剂,经无菌检验后制成马腺疫灭活疫苗,对5组小鼠进行不同剂量的免疫后,分别进行不同剂量攻毒试验,结果表明小鼠经106 CFU灭活疫苗免疫后,保护率为80%。本试验完成了对分离菌株HLJ2011的生物学特征研究,以及利用该菌株制备灭活疫苗对感染小鼠保护性试验。本项研究为我国马腺疫病原学研究和疫苗研究奠定了重要基础。
[Abstract]:Adenosis is an acute contagious disease of the genus Equine caused by (Streptococcus equi subspecies equi,S.equi (Streptococcus C Maya). The incidence of the disease is very high in pastoral areas of China. It is susceptible to colt under 2 years of age and causes great economic losses to herdsmen. However, there are no effective prevention and control measures. In order to alleviate the epidemic situation of horse adenosis in pastoral areas of China as soon as possible, the preparation method of inactivated vaccine against equine adenosis was studied in this study. The pathogen of the experiment was the wild virus strain (HLJ2011) isolated from the seriously infected horse. The inactivated vaccine prepared by this strain could provide good protection for mice. In this paper, the biological characteristics of HLJ2011 strain and the immune protective effect of inactivated equine adenosis vaccine in mice were studied systematically. The results were as follows: the biological characteristics of 1.HLJ2011 strain: a main pathogenic bacteria was isolated from the material of the disease animal's mandibular burst and pus. The 16s rRNA, biochemical identification and growth curve of different inoculation concentration were obtained. The relationship between colony forming unit (Colony-Forming Unit,CFU) and OD was studied. The results showed that the strain was S. equii by 16s rRNA identification and biochemical identification. The growth curve of different inoculation concentration was basically the same, the higher the inoculation concentration, the shorter the logarithmic growth period, and the maximum CFU was when 1% inoculation concentration was cultured for 810 h. 1 脳 10 ~ 9 CFU; could be used to attack 8 week-old Balb/c mice with 1 脳 10 ~ 7 CFU intraperitoneally. The mortality rate of Balb/c mice was 100%. Calculated by modified Kou's method, the median lethal dose of this strain to mice was 5 脳 10 ~ 5 CFU.2.. The study on the immune protection efficacy of inactivated vaccine against equine adenosis: the selection of HLJ2011 inactivation conditions was completed by different temperature and formaldehyde concentration, and the immune protection effect of inactivated vaccine against equine adenosis was studied. The effects of different formaldehyde concentrations on the inactivation effect at 4 鈩，

本文编号：2285513