中国南方地区罗非鱼无乳链球菌的分子流行病学研究

Molecular epidemiology of Streptococcus agalactiae isolated from tilapia in Southern China

从广东省以及海南省等地区养殖的患病罗非鱼体内分离、收集到多株致病菌,经生化分析和分子生物学鉴定,均为无乳链球菌。对这些菌株分别进行了耐药谱测定、分子分型试验以及分子血清型分析。药敏试验结果表明,2007—2010年分离到的无乳链球菌耐药谱基本相似;多位点可变数目串联重复序列分析(MLVA)试验中,选择5个高变异指数的可变数目重复位点(VNTR)进行分子分型,结果表明,所有鱼源无乳链球菌菌株为同一MLVA型,而作为对照的牛源无乳链球菌则明显不同;为了对这些菌株进一步分型,分别进行了分子血清型和表面蛋白抗原基因的检测,结果表明,鱼源无乳链球菌的分子血清型均为Ⅰa型,表面蛋白抗原均为alpha-C蛋白。这进一步说明了不同年份和不同地区的鱼源无乳链球菌在基因水平上为同一分子类型,具有相同的起源或传染源。同时也说明,我国南方地区罗非鱼无乳链球菌在这几年中未发生明显的遗传变异。这些结果为罗非鱼无乳链球菌病疫苗研制,疫病监测及药物防治的研究提供理论依据。

Streptococcus agalactiae strains were isolated and collected from moribund tilapia in Guangdong,Hainan,and Fujian Provinces,and these isolates were identified by biochemical and molecular biological characterization.In the current study,we compared the phylogenetic relationships among S.agalactiae strains.Furthermore,we compared antibiotic sensitivity,molecular classification and molecular serotype of these strains which were isolated from different years and different geographical regions.Antibiotic sensitivity assays showed that among 20 antibiotics tested,there are 10 antibiotics that had no differences,and the other 10 items showed similar sensitivity.Five most diverse loci（Variable number of tandem repeats,VNTR）were used for the construction of MLVA to evaluate the diversity of these strains.Molecular classification assays showed that all S.agalactiae strains isolated from fish were of the same molecular classification,but the S.agalactiae strain（C918）isolated from dairy cattle was different.Molecular serotype of these isolates using a multiplex PCR reaction,and the results showed that the molecular serotype of S.aglactiae was Ⅰa.However,surface protein genes assays showed that the surface protein of S.agalactiae strains is alpha-C protein except the S.agalactiae（C918）strain.Conclusion：these S.agalactiae isolates isolated from fish are of the same classification by molecular classification assays.Therefore,these results provide a theoretical basis for control of the tilapia diseases and for vaccine development of S.agalactiae in tilapia.