方斑东风螺“急性死亡症”病原及其毒力基因研究

为明确引起方斑东风螺（Babylonia areolata）"急性死亡症"的病原及相关的毒力因子，对"急性死亡症"疫区的方斑东风螺进行了病原分离纯化鉴定、人工感染试验等，经鉴定为塔氏弧菌（Vibrio tubiashii）。本实验以4株分离自发病方斑东风螺且具有不同程度毒力的塔氏弧菌及东风螺组织样品为研究对象，根据半数致死剂量（LD₅₀）确定菌株毒力大小依次为:BBXP1>FBC1>FB20>FB13，针对17种弧菌毒力相关基因进行了检测分析，结果显示强毒株BBXP1、FBC1中的毒力基因检出率最高，携带12种毒力基因，弱毒株FB13毒力基因检出率最低，携带7种，表明弧菌的致病性可能与其携带毒力基因的数量具有相关性。其中，不耐热溶血毒素基因tlh、胞外碱性丝氨酸蛋白酶基因asp、金属蛋白酶基因vtmp、几丁质外切酶基因chi36、锌金属蛋白酶基因vtpA、塔氏弧菌溶血素基因vthA在4株塔氏弧菌中检测均呈阳性，且仅在患有"急性死亡症"的东风螺组织样品中检测出，在健康的组织样品中未检测出。进一步通过实时荧光定量PCR方法检测此6种毒力基因在4株不同毒力的塔氏弧菌中的mRNA表达差异，结果显示，asp、vtpA的mRNA表达量与弧菌的毒力呈正相关，随着塔氏弧菌致病性的增强，vtpA、asp的mRNA表达量呈上升趋势，且菌株的半致死剂量LD₅₀与相对表达量具有显著相关性（r2=0.933，0.948；P < 0.05）；溶血素vthA的表达量则与弧菌的致病性呈现负相关，相关性并不显著（r2=0.2401；P < 0.05），因此推测可能与其致病性关联不大；其它几种毒力基因的相对表达量与弧菌的毒力相关性不显著（P>0.05），可见同种毒力因子在不同毒力的菌株中相对表达量存在差异，推测塔氏弧菌致病株在侵染螺体的过程主要由金属蛋白酶和丝氨酸蛋白酶发挥主导作用。

Studies on the pathogenic bacteria and their virulence factors of "acute death syndrome" in Babylonia Areolata

In order to reveal the main causative agents and the related virulence factors of "acute death syndrome" in Babylonia areolata, the pathogens were isolated, purified, artificially infected.The results showed that the pathogen was Vibrio tubiashii.In this study, four strains of V.tubiashii, which had different degrees of virulence, were tested for 17 virulent-related genes.The healthy and diseased B.areolatasample were also detected these 17 genes.It showed that strain BBXP1 and strain FBC1carried 12 kinds of virulence genes while the attenuated strain FB13 took 7 kinds of virulence genes.Therefore, the pathogenicity of vibrios may be related to the number of virulence genes.Meanwhile, the results indicated that tlh (non-thermostatic hemolytic toxin), asp (extracellular alkaline serine protease), vtmp (metalloproteinase), chi36 (exochitinase), vtpA (zinc metalloproteinase)and vthA (hemolysin) were positive in all the four strains of V.tubiashii but could only be detected in the tissue samples of B.areolata with "acute death".Furthermore, the mNRA expression difference of 6 kinds of virulence genes in 4 different kinds of V.tubiashii were examined by real-time fluorescent quantitative PCR.The results displayed that the relative expression levels of asp and vtpA were positively correlated with the pathogenicity of vibrio, and the expression of asp and vtpA in virulent strains were significantly higher than that of the attenuated strain.In detail, the mRNA relative expression levels of vtpA and asp increased with the enhancement of pathogenicity of V.tubiashii, and the LD₅₀ of the strain was significantly correlated with the relative expression (r2=0.933, 0.948;P < 0.05).On the contrary, the mRNA expression of vthA was negatively correlated with the pathogenicity of vibrio(r2=0.2401;P < 0.05) and the relative expression of several other virulence genes was not significantly correlated with the virulence.It was found that the relative expression of the same virulence factor in different virulence strains was different.The metalloproteinase and serine protease from V.tubiashii played a leading role in the process of infecting in Babylonia areolata.