黄沙鳖源嗜水气单胞菌的致病力与毒力基因型相关性

Studies on the correlation with pathogenicity and virulence genes of Aeromonas hydrophila by Truogx sinensis

为探明黄沙鳖(Truogx sinensis)源嗜水气单胞菌(Aeromonas hydrophila)所携带毒力基因种类对其致病力的影响,以从广西各地患病黄沙鳖中分离得到的嗜水气单胞菌为材料,通过对黄沙鳖和小鼠进行人工感染测定试验菌株的致病力,PCR检测供试菌株的hly、Aer、Alt、Act、ahal和ahp 6种毒力基因。结果表明,47株黄沙鳖源嗜水气单胞菌共包含9种毒力基因型,48.94%的菌株携带全部6种毒力基因,基因型为hly+Aer+Alt+Act+ahal+ahp+,是主要的毒力基因型;95.79%的菌株携带hly基因,均为有毒株;嗜水气单胞菌对黄沙鳖的致病力是多个毒力基因共同作用的结果,hly和Act是重要的毒力基因,Alt和ahal基因对菌株的致病力与其他毒力基因之间存在协同作用,ahp基因缺失对菌株的致病力无影响;将对黄沙鳖的致死率达到和超过50%的菌株判定为强毒株,致死率不足50%判定为弱毒株时,强毒株至少携带4种毒力基因,并同时携带有hly和Act基因,弱毒株只携带包括hly在内的2种或3种毒力基因,本研究首次发现只携带Alt和ahal 2种毒力基因的菌株对黄沙鳖和小鼠均无致病力;在所检测的6种毒力基因中,较易缺失的基因主要有Alt、ahal和ahp;在有毒株中,不同基因型的菌株对黄沙鳖和对小鼠的平均致死率差异均为极显著(P<0.01),当存在基因缺失现象时,同一基因型的菌株对黄沙鳖的平均致死率也极显著高于对小鼠的致死率(P<0.01),表明黄沙鳖源嗜水气单胞菌对黄沙鳖的致病力显著强于对小鼠的致病力。根据本研究结果,可将hly基因作为嗜水气单胞菌有毒株的鉴定标准,携带hly和Act基因并同时携带Aer、Alt、ahal、ahp中的2种或2种以上毒力基因作为强毒株的鉴定标准。

This study aims to investigate the correlation of the pathogenicity and virulence genes of Aeromonas hydro-phila isolated from diseased Huangsha soft shelled turtle Truogx sinensis in different areas of Guangxi Zhuang Autonomous Region. An artificial infection test in Huangsha soft shelled turtle T. sinensis and in mice Mus musculus was measured on the pathogenicity of the isolated A. hydrophila strains, including a PCR assay of detecting the bacte-rial strains of 6 kinds of virulence genes including hly, Aer, Alt, Act, ahal and ahp. The results were as follows. Forty seven strains of pathogenic A. hydrophila isolated from diseased Huangsha soft shelled turtle T. sinensis contained 9 kinds of bacterium virulence genotypes in all, including hly＋ Aer＋ Alt＋Act＋ ahal＋ ahp＋, hly＋ Aer＋ Alt– Act＋ ahal＋ ahp–, hly＋ Aer＋ Alt＋ ahal＋ Act– ahp＋, hly＋ Aer＋ Alt＋ Act– ahal– ahp–, hly– Aer– Alt＋ Act– ahal＋ ahp–, hly＋ Aer– Alt＋ Act＋ahal–ahp＋, hly＋ Aer– Alt– Act＋ ahal– ahp＋, hly＋ Aer– Alt＋ Act– ahal– ahp＋ and hly＋ Aer– Alt– Act＋ ahal– ahp–. Around 48.94 percent of the A. hydrophila strains carried all the 6 kinds of virulence gene detected, and the genotype of hly＋ Aer＋ Alt＋ ahal＋ ahp＋Act＋ was the main virulence genotype. About 95.79 percent of the A. hydrophila strains carried gene hly, and all of them were the virulence strains. Our results also showed that pathogenicity of A. hydrophila to Huangsha soft shelled turtle T. sinensis was the outcome of combined action of multiple virulence genes, among which gene hly and Act were the key virulence genes, gene Alt and ahal of the isolated strains had correlated action with other virulence genes, whereas the absence of gene ahp had no effect on the pathogenicity of the isolated A. hydrophila strains to Huangsha soft shelled turtle T. sinensis or to mice M. musculus based on the artificial infection test. Strain which caused a fatality rate at or above 50 percent in Huangsha soft shelled turtle T. sinensis was determined to be a particularly virulent strain, whereas the one caused a fatality rate below 50 percent was determined to be a low virulent strain. Particularly virulent strains carried at least 4 kinds of virulence genes according to our study, and at the same time hly and Act genes were both pre-sent. Low virulent strains carried only two or three virulence genes besides gene hly. The study found for the first time that strains which carried only Alt and ahal genes have no pathogenicity to mice M. musculus or to Huangsha soft shelled turtle T. sinensis. In the detection of the 6 kinds of virulence genes, genes Alt, ahal and ahp were prone to miss-ing. Among all the virulent strains, the strains with different genotypes varied significantly in causing fatalities rates in Huangsha soft shelled turtle T. sinensis and in mice M. musculus （P〈0.01）. When any virulence gene is absent, the pathogenicity of the strains with the same genotype contributing to Huangsha soft shelled turtle T. sinensis was signifi-cantly stronger than to mice M. musculus （P〈0.01）, which indicates that pathogenic A. hydrophila isolated from dis-eased Huangsha soft shelled turtle T. sinensis contributes significantly stronger pathogenicity to Huangsha soft shelled turtle T. sinensis than to mice M. musculus. According to the results of this study, a PCR detection of the virulence gene hly could be recommended to infer whether a strain is virulent or not, and the co-existence of virulence genes hly and Act, as well as two or more virulence genes among Aer, Alt, ahal together with ahp, could be considered as a reasonable predictor of the particularly virulent strains.