副溶血弧菌hemoD敲除菌株构建及致病力评估

Vibrio parahaemolyticus hemoD knockouted and pathogenicity evaluation

摘要:

通过同源重组技术，构建了hemoD基因缺失的副溶血弧菌敲除菌株ΔhemoD，对ΔhemoD菌株的毒力和感染四膜虫及凡纳滨对虾（Penaeus vannamei）的能力进行了评估。相比野生型菌株，ΔhemoD菌株溶血活性下降了45.7%，生物膜形成能力下降了36.2%，生长无明显变化。相比野生型菌株，ΔhemoD菌株对四膜虫的毒力降低，四膜虫对ΔhemoD菌株具有更强的摄食能力，增殖能力也更强。针对凡纳滨对虾的攻毒实验，发现ΔhemoD菌株半数致死量为1.3×10⁸ cfu/mL，相较于野生型菌株提高了5.06倍，ΔhemoD菌株定殖凡纳滨对虾肠道能力明显下降。病理切片结果显示，ΔhemoD菌株对肝胰腺的损伤能力相较于野生菌株也有所下降，肝胰腺细胞溶解及空泡化现象较轻微。这些研究结果表明，ΔhemoD菌株的毒性明显减弱，对四膜虫和凡纳滨对虾的致病力明显减弱，是一株减毒的副溶血弧菌菌株，为后续进一步开发副溶血弧菌减毒疫苗奠定基础。

Abstract:

Vibrio parahaemolyticus is a Gram-negative rod-shaped bacterium with flagella， widely distributed in estuaries and oceans， and is one of the major pathogenic bacteria in aquaculture. For future development of attenuated vaccine of V. parahaemolyticus， in this study we knocked out hemoD gene in V. parahaemolyticus using homologous recombination. Compared with wild type of V. parahaemolyticus， ΔhemoD strain showed a decrease in hemolytic activity by 45.7%， a decrease in the ability of biofilm formation by 36.2%， and no significant change in growth rate. When co-cultured with Tetrahymena thermophila， the injury of Tetrahymena by ΔhemoD strain was less with more Tetrahymena survived， and hence engulfed more cells of ΔhemoD strain than the wild strain of V. parahaemolyticus. The whiteleg shrimp Penaeus vannamei was further used for pathogenicity evaluation. The P. vannamei werechallenged with strain ΔhemoD and wild type strain of V. anguillarum through administration， respectively， the LD₅₀ of ΔhemoD strain was 5.06 times higher than that of the wild type strain. Compared with wild type of V. parahaemolyticus， the colonization ability of ΔhemoD strain in the intestinal tract of P. vannamei was obviously weakened， and the damage ability to hepatopancreas was reduced， and the phenomena of hepatopancreatic cytolysis and vacuolisation were less serious. These results indicate that the ΔhemoD strain has a reduced pathogenicity to P. vannamei. Onthe whole， we constructed an attenuated strain of V. parahaemolyticus， which will be developed as a potential attenuated vaccine.