中国七种水生动物源无乳链球菌的分子特征及其对斑马鱼的致病性

Molecular characteristics and the pathogenicity to zebrafish of Streptococcus agalactiae strains isolated from seven aquatic animals in China

为了解中国水生动物源无乳链球菌的分子流行特征,揭示其传播和流行规律,本实验对分离得到并鉴定的10株7种水生动物源无乳链球菌通过分子血清型、多位点序列分型(MLST)分型、毒力基因型和前噬菌体分型等方法进行分子分型;其次,通过斑马鱼评价7种水生动物源无乳链球菌的致病性。分子血清型分析结果表明,10株无乳链球菌可分为3种血清型,即Ⅰa、Ⅰb和Ⅲ型;MLST分型结果表明,Ⅰa型无乳链球菌均为ST7型,Ⅰb无乳链球菌均是ST261型,只有Ⅲ型无乳链球菌是ST739型。进一步分型结果表明,10株无乳链球菌可分为3种毒力基因型和4种前噬菌体基因型。根据4种分型结果可知,10株水生动物源无乳链球菌可分为4种类型,其中虎纹蛙源无乳链球菌具有独立的分子血清型、MLST型、毒力基因型和前噬菌体基因型,即Ⅲ-ST739-V1-P3;罗非鱼源无乳链球菌的基因型有3种,即Ⅰa-ST7-V2-P1、Ⅰa-ST7-V2-P2和Ⅰa-ST7-V3-P4;红尾皇冠鱼、鳙和罗非鱼源无乳链球菌的基因型相同:Ⅰa-ST7-V2-P2;卵形鲳鲹、宝石鲈和罗非鱼源无乳链球菌具有相同的基因型:Ⅰa-ST7-V2-P1;鲮和罗非鱼源无乳链球菌的基因型相同,即Ⅰb-ST261-V3-P4。致病性研究表明,7种水生动物源无乳链球菌对斑马鱼均有强致病性。研究表明,两栖类虎纹蛙源无乳链球菌和鱼源无乳链球菌的基因型明显不同,它们之间遗传变异较大,因此,无乳链球菌在两栖类和鱼类之间相互传播的可能性较小。鱼源无乳链球菌有3种基因型,且这3种基因型均在罗非鱼中流行,这表明无乳链球菌在鱼类中相互传播的可能性较大,尤其是在罗非鱼与其他鱼类之间。

The aim of this study is to obtain the molecular characteristics of Streptococcus agalactiae（Group B Streptococcus, GBS） strains isolated from seven aquatic animals in China, and then analyze the epidemiology and transmission of GBS strains in these aquatic animals. In this study, a total of 10 GBS strains were isolated from seven aquatic animals. These isolates were analyzed by four typing methods： molecular serotype, MLST（Multilocus Sequence Typing）, virulence genotype and prophage typing. In addition, the pathogenicity of GBS strains was evaluated by zebrafish. The ten GBS strains were divided into three types based on molecular serotype,namelyⅠa, Ⅰb and Ⅲ. The results of MLST indicated that serotypesⅠa, Ⅰb and Ⅲ were ST7, ST261 and ST739, respectively. The ten GBS strains were divided into three virulence genotypes（V1, V2 and V3） determined by PCR detection. The FWL1405 strain（-ST739） isolated from Hoplobatrachus chinensis carried 11 virulence genes（11/12, 91.7%）, and the a-ST7 GBS strains carried 10 virulence genes（10/12, 83.3%）, and the b-ST7 GBS strains were possessed of 7 virulence genes（7/12, 58.3%）. Prophage typing showed that the ten GBS strains were divided into four types（P1, P2, P3 and P4）, and the strains of P1 and P1 types belong to a-ST7 GBS strains. These results revealed that GBS strains from seven hosts were divided into four genotypes, and the FWL1405 strain was the unique type：-ST739-V1-P3, determined by the previous four typing methods. However, tilapia GBS strains（LT-1, WC1535, WT1451 and WC15122） were divided into three genotypes：Ⅰa-ST7-V2-P1,Ⅰa-ST7-V2-P2 andⅠa-ST7-V3-P4. More important, the GBS strains isolated from Aristichthys nobilis, Aequidens rivulatus, and Oreochromis sp., have the same genotype：Ⅰa-ST7-V2-P2. The strains isolated from Trachinotus ovatus, Scortum barcoo, and Oreochromis sp., have the same genotype：Ⅰa-ST7-V2-P1. Furthermore, the same genotype of the GBS strains were found that isolated from Cirrhinus molitorella and Oreochromis sp.. Additionally, in this study,all the GBS strains have strong pathogenicity to zebrafish（Danio rerio）. In conclusion, the genotypes of ten GBS strains were significantly different between amphibians（frog） and fishes. The possibility was little that GBS strains transmitted between amphibians and fishes. Notably, there were three genotypes of GBS strains from fishes, and the same genotype GBS strains were all found in tilapia. These results indicated that the possibility was great that GBS strains transmitted among these six species of fish, especially between tilapia and other fish species.