Isolation, Identification And Detection, Analysis Of Virulence Gene Of Tilapia In Guangxi With Streptococcus Agala Ctiae Disease

In recent years, Stretococcus agalactiae has become an important pathogenic bacterium of tilapia’s bacteriosis, which causes tilapia industry great economic losses. Through investigation, it is found that Streptococcus iniae and Streptoccus agalactiae are the main reasons for tilapia’s streptococcicosis recently. Thirty Stretococcus agalactiae strains (Nanning7, Qinzhou City3, Beihai7, Liuzhou5, Wuzhou City1, Yulin City7) are isolated from672moribund tilapias witch collected in12counties and8cities of Guang Xi province, the rate is4.46%. And these isolates are identified by API20Srep and16S rRNA molecular biological characterization. Also there are9strains of which are not hemolytic, the remaining21strains of test hemolysis are positive. Analysising the similarities of the30test strains’16S rRNA gene sequences and counterparts of streptococcus registered in GenBank with BLAST, showed that16S rRNA of all the strains possessed high similarities with Streptococcus agalactiae (JF423947isolated from Hainan province, JQ039375and KF111315isolated from Guangdong province, JQ990156isolated from Guangxi province)(98.5%～100%), possessed relatively low similarities with streptococcus iniae (EU622514/FJ870987)(93.4%～95.8%), streptococcus dysgalactiae (AB159678/X59030)(88.9%～96.4%) and Streptococcus pyogenes (AB023575/EU660335)(95.3%～96.1%). The16S rRNA gene sequences between isolates are highly homologous (99.1%～100%).Artificial infection test shows that all these30strains are pathogenic, but their virulence is different. The fatality rate of GXYL2G、XYL4、GXYL6、 GXYL7is100%, the strongest virulence; the fatality rate of GXNN2、GXBH1、 GXBH3、GXBH4、GXBH5is lower than80%; And the fatality rate of the remaining21strains is80%to97.5%. Drug sensitivity test shows that the30 strains are sensitive to7kinds of antibiotics, that is, ampicillin, ceftriaxone, cefobid, sarafloxacin hydrochloride, sulperazon, cefradine VI, doxycycline. And the sensitive rate is above70%, up to100%. Besides, the30strains are resisting sulfadimidine, sulfamonomethoxine, sulfamethoxydiazine, compound sulfadiazine, gentamycin, streptomycin, sulfamethoxazole trimethoprim, enoxacin and neomycin. And the drug resistance rate is above80%.By using the PCR method, the study tests the30Stretococcus agalactiae’s three virulence genes cfb, cpsE, sip, and it turns out that the30strains belong to the same gene type cfb+cpsE+sip+.Based on sources, the study clones and sequences6representative strains’ cfb, cpsE, sip, which shows that the nucleotide homology of cfb, cpsE, sip genes in isolated strains is99.1%to100%,99.6%to100%,99.5%to100%respectively; and the amino acid homology is98.8%to99.6%,100%,99.1%to100%separately. According to the analysis of the three virulence genes’phylogenetic evolutionary tree, the three virulence genes each gather in the same group and the homology is up97.7%to100%compared with corresponding domestic gene reference strains JQ289564(Guangdong), HQ148672(Guangdong), HQ148671(Guangdong), JQ289567(Hainan), HQ888682(Hainan), EF694027(Shandong). Therefore, it indicates that the kinship among the strains isolated from tilapia in Guang Xi is close, and the relations with domestic reference strains are close, too.