黄沙鳖红底板病病原菌的分离鉴定及其毒力基因检测

Isolation,Identification and Virulence Genes Detection of Pathogenitic Bacteria from Trionyx sinensis with Red Abdominal Shell Disease

以常规方法从患典型红底板病黄沙鳖的心脏、肝脏和腹水等处分离到5株病原菌,API 20NE、16S rRNA基因序列分析进行病原菌鉴定和确定其系统发育地位,PCR检测病原菌的6种毒力基因。结果表明,5株病原菌均为嗜水气单胞菌(Aeromonas hydrophila),与Aeromonas hydrophila QDC01菌株的亲源关系最近。6种毒力基因的阳性率,hly、Aer和Act为100%,ahal、Alt和ahp为80%;毒力基因型共3种,在5株菌株中的分布情况为hly+Aer+Alt+Act+ahal+ahp+3株,hly+Aer+Alt-Act+ahal+ahp-1株,hly+Aer+Alt+Act+ahal-ahp+1株。试验所检测的5株嗜水气单胞菌的毒力基因型主要为hly+Aer+Alt+Act+ahal+ahp+,Alt、ahp和ahal基因的缺失对嗜水气单胞菌的致病力没有影响。

Five pathogens from the Trionyx sinensis infected with red abdominal shell disease were isolated from hearts、livers and ascites by routine methods.Their phylogenesises were determined by API 20NE analysis and 16Sr RNA gene PCR amplification followed by PCR assay according to 6 chosen virulence genes.Results showed 5 strains isolates are Aeromonas hydrophila.Phylogenetic tree showed 5 Aeromonas hydrophila have high similarities to Aeromonas hydrophila QDC01 bacteria.The hly Aer and Act gene could be detected in all isolates（100 ％）,also the Ahal Alt and ahp were found in 80 ％ isolates.3 genotypes were found in these isolates,showing 3 hly ＋ Aer ＋ Alt ＋ Act ＋ ahal＋ ahp＋,1 hly＋Aer＋Alt-Act＋ahal＋ahp-,1 hly＋Aer＋Alt＋Alt＋ahal-ahp＋ dominated in 5 strains.The test showed major virulence genotypes was hly＋Aer＋Alt＋Act＋ahal＋ahp＋in 5 hydrophila Aeromonas,Lack of the Alt,ahp and ahal gene had no effect to pathogenicity of Aeromonas hydrophilas.