黄河裸裂尻鱼无乳链球菌的分离鉴定与多位点序列分型

2015年10月四川省雅安市某养殖场的黄河裸裂尻鱼(Schizopygopsis pylzovi Kessler)暴发传染病,临床表现为神经症状,眼球、下颌和鳃盖等出血或无明显临床表现而突然死亡,内脏涂片见革兰氏阳性球菌。从病鱼的肝、脾和肾组织中分离到2株病原菌(ZYW151011,ZYW151012),通过人工感染、无乳链球菌种属特异性cfb基因(GBS-specific gene cfb,CAMP factor)的PCR检测和16S r RNA基因序列分析等方法对病原菌进行鉴定,发现2株病原菌均为无乳链球菌(Streptococcus agalactiae)。2株病原菌16S r RNA序列(Gen Bank登录号:KU308396和KU308397)与Gen Bank中无乳链球菌16S r RNA序列同源性达99.8%。药敏试验结果表明,2株病原菌对头孢类和喹诺酮类药物敏感,而对强力霉素、氟苯尼考和阿莫西林等耐药。进一步通过荚膜多糖血清分型和多位点序列分型技术对病原菌进行分子特征分析,结果表明2株病原菌荚膜多糖血清型均为致病性Ia型;2株病原菌多位点序列分型均被鉴定为新的序列型ZST-1,与序列型为ST-261的亲缘关系最近,此新型ZST-1在gln A位点发生变异,提交序列到MLST数据库获得新的等位基因编号81(登录ID:BIGSdb_20151110 081850_13025_35759)。

Identification of the pathogen Streptococcus agalactiae in Schizopygopsis pylzovi (Kessler) by multilocus sequence typing

In October 2015, an infectious disease characterized by hemorrhaging from the eyeball, jaw, and operculum, along with neurological symptoms or sudden death with no other clinical symptoms emerged in farms of Schizopygopsis pylzovi (Kessler) culture in Ya''an, Sichuan Province, China. A Gram-positive, chain-forming coccus was found in visceral smears. Two bacterial isolates (ZYW151011 and ZYW151012) were isolated from the liver, spleen, and kidney of the diseased S. pylzovi, and then identified using a S. pylzovi pathogenic test, PCR detection of the cfb gene (GBS-specific gene cfb, CAMP factor) from Streptococcus agalactiae, and sequence analysis of the 16S rRNA gene. Both bacterial isolates were identified as S. agalactiae. A 16S rRNA gene sequence analysis by BLAST in GenBank indicated that the two isolates (GenBank accession numbers KU308396 and KU308396) showed high levels of similarity to S. agalactiae (99.8%). Drug sensitivity test results showed that both strains were sensitive to ceftriaxone and quinolones, but were not sensitive to doxycycline, florfenicol, and amoxicillin. Serotyping and sequence typing of the two isolates was achieved using capsular polysaccharide serotyping and multilocus sequence typing (MLST), respectively. The serotype of both isolates was pathogenic type Ia. The two isolates were identified as a new sequence type (ZST-1), closely related to ST-261. The allele gene sequence has been submitted to the MLST database under the new allele number 81 (ID, BIGSdb_20151110081850_13025_35759) based on this new variation of glnA in the novel sequence type ZST-1.