蟹源假单胞菌致病机理的研究
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摘要
应用细菌学和分子生物学方法对本实验室分离的一株蟹源致病菌(X-1株)进行了鉴定和致病机理的研究。
首先，根据细菌的形态、生理生化特性和血清学反应等常规鉴定和细菌编码鉴定，确定其为假单胞菌。
其次，对该株细菌进行了致病性测定。细菌对小鼠和河蟹均具有致病性，死亡率分别为100％(8／8)和87.5％(7／8)。对小鼠的半数致死量(LD_(50)为2.9×10~7CFU／只。同时，还研究了环境对细菌致病性的影响。结果显示，在30℃、25℃和20℃的水温下细菌对河蟹致死率分别为100％、80％和60％；水体pH值在5.0和10.0时，10h内细菌对河蟹的致死率均为100％，而pH 7.5时，致死率仅为40％。充气与不充气条件下细菌在10h内对河蟹的致死率分别为60％和100％。表明环境因素影响细菌对河蟹的致病性。此外，该菌还具有凝集鸡、小鼠、牛、鲫鱼、鲢鱼、兔、家鸽的红细胞和粘附Hela细胞的能力，表明其具有较强的粘附侵袭力。
进一步测定了细菌的内毒素和胞外产物(ECP)的致病作用。采用三氯乙酸(TCA)溶解法提取的细菌内毒素脂多糖的致病性实验结果显示，LPS可引起河蟹发病，但不能导致河蟹死亡，说明该菌内毒素毒力较弱。
在培养温度为20℃、25℃和30℃条件下细菌产生的胞外产物提取物中的蛋白质含量分别为3.107mg／mL、3.952mg／mL和3.372mg／mL。SDS-PAGE电泳图谱显示，不同温度组间的大部分蛋白条带无明显差异，只有相对分子量为54 000蛋白条带差异较明显。三组的ECP均具有溶血性，但均无细胞毒性和酪蛋白酶活性；三种温度的ECP对河蟹均有致病性，但死亡率不同，分别为60％(25℃ ECP)、20％(20℃ ECP)和20％(30℃ ECP)。
综上所述，本实验室分离的蟹源细菌X-1株为假单胞菌弱毒株，其对河蟹的致病机理主要是粘附素，内毒素和胞外产物中的溶血素等毒性蛋白的共同作用。
A strain of pathogenic bacteria (strain X-l) isolated from diseased crab was identified and the pathogenic mechanism was also studied by using bacteriological and molecular biological methods.
First,according to morphology,physiology and biochemistry,serological reaction,and numerical system,the bacterium was identified as Pseudomonas.
Second,the bacterium was pathogenic to mouse and crab in animals experiments and the mortalities were 100% and 87.5% respectively,and the bacterial LDsowas 2.9 X107 CPU per mouse. The effects of aquatic environment factors on bacterial pathogenicity were also studied,At different water temperatures (30,25 and 20),the moralities for crabs were about 100%,80% and 60% respectively. When water pHs were 5.0 and 10.0,the moralities for crabs were all about 100% within 10 hours,however,at pH7.5 it was only 40%. With supplying enough air in water and not doing it,the moralities for crabs were 60% and 100% respectively within 10 hours. In addition,the bacterium could agglutinate erythrocytes of chicken,mouse,cow,Crucian crap,Silver carp,rabbit and dove,and adhere to Hela cell strongly.
When the lipopolysaccharide (LPS) of endotoxin extracted by Trichloroacetic Acid (TCA) lysis was analyzed,the result indicated that LPS had pathogenicity,but no lethality to crab.
There were about 3.107 mg/mL,3.952mg/mL and 3.372mg/mL to the concentrations of the preliminarily extracted Extracellular Products (ECPs) which were obtained from culture mediums at different culture temperatures (20 ,25 and 30). The mortalities of ECPs were 20% (20),60% (25 ) and 20% (30) to crabs. Except for a 54 000 protein band,most protein bands were alike among ECPs by analyzing the profiles of SDS-PAGE. The ECPs had hematolytic activities,but no cell toxicities or casein enzymatic activities.
All results in this study suggests that the main pathogenic mechanism of the lowly pathogenic Pseudomonas (strain X-l) might be the cooperation of adhesin,
endotoxin and toxoproteins in ECPs to crab.
首先，根据细菌的形态、生理生化特性和血清学反应等常规鉴定和细菌编码鉴定，确定其为假单胞菌。
其次，对该株细菌进行了致病性测定。细菌对小鼠和河蟹均具有致病性，死亡率分别为100％(8／8)和87.5％(7／8)。对小鼠的半数致死量(LD_(50)为2.9×10~7CFU／只。同时，还研究了环境对细菌致病性的影响。结果显示，在30℃、25℃和20℃的水温下细菌对河蟹致死率分别为100％、80％和60％；水体pH值在5.0和10.0时，10h内细菌对河蟹的致死率均为100％，而pH 7.5时，致死率仅为40％。充气与不充气条件下细菌在10h内对河蟹的致死率分别为60％和100％。表明环境因素影响细菌对河蟹的致病性。此外，该菌还具有凝集鸡、小鼠、牛、鲫鱼、鲢鱼、兔、家鸽的红细胞和粘附Hela细胞的能力，表明其具有较强的粘附侵袭力。
进一步测定了细菌的内毒素和胞外产物(ECP)的致病作用。采用三氯乙酸(TCA)溶解法提取的细菌内毒素脂多糖的致病性实验结果显示，LPS可引起河蟹发病，但不能导致河蟹死亡，说明该菌内毒素毒力较弱。
在培养温度为20℃、25℃和30℃条件下细菌产生的胞外产物提取物中的蛋白质含量分别为3.107mg／mL、3.952mg／mL和3.372mg／mL。SDS-PAGE电泳图谱显示，不同温度组间的大部分蛋白条带无明显差异，只有相对分子量为54 000蛋白条带差异较明显。三组的ECP均具有溶血性，但均无细胞毒性和酪蛋白酶活性；三种温度的ECP对河蟹均有致病性，但死亡率不同，分别为60％(25℃ ECP)、20％(20℃ ECP)和20％(30℃ ECP)。
综上所述，本实验室分离的蟹源细菌X-1株为假单胞菌弱毒株，其对河蟹的致病机理主要是粘附素，内毒素和胞外产物中的溶血素等毒性蛋白的共同作用。
A strain of pathogenic bacteria (strain X-l) isolated from diseased crab was identified and the pathogenic mechanism was also studied by using bacteriological and molecular biological methods.
First,according to morphology,physiology and biochemistry,serological reaction,and numerical system,the bacterium was identified as Pseudomonas.
Second,the bacterium was pathogenic to mouse and crab in animals experiments and the mortalities were 100% and 87.5% respectively,and the bacterial LDsowas 2.9 X107 CPU per mouse. The effects of aquatic environment factors on bacterial pathogenicity were also studied,At different water temperatures (30,25 and 20),the moralities for crabs were about 100%,80% and 60% respectively. When water pHs were 5.0 and 10.0,the moralities for crabs were all about 100% within 10 hours,however,at pH7.5 it was only 40%. With supplying enough air in water and not doing it,the moralities for crabs were 60% and 100% respectively within 10 hours. In addition,the bacterium could agglutinate erythrocytes of chicken,mouse,cow,Crucian crap,Silver carp,rabbit and dove,and adhere to Hela cell strongly.
When the lipopolysaccharide (LPS) of endotoxin extracted by Trichloroacetic Acid (TCA) lysis was analyzed,the result indicated that LPS had pathogenicity,but no lethality to crab.
There were about 3.107 mg/mL,3.952mg/mL and 3.372mg/mL to the concentrations of the preliminarily extracted Extracellular Products (ECPs) which were obtained from culture mediums at different culture temperatures (20 ,25 and 30). The mortalities of ECPs were 20% (20),60% (25 ) and 20% (30) to crabs. Except for a 54 000 protein band,most protein bands were alike among ECPs by analyzing the profiles of SDS-PAGE. The ECPs had hematolytic activities,but no cell toxicities or casein enzymatic activities.
All results in this study suggests that the main pathogenic mechanism of the lowly pathogenic Pseudomonas (strain X-l) might be the cooperation of adhesin,
endotoxin and toxoproteins in ECPs to crab.
引文
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