养殖大菱鲆病原菌迟缓爱德华氏菌的分离鉴定及其疫苗研制

Isolation of Edwardsiella tarda from diseased turbot( Scophthalmus maximus) and vaccination against Edwardsiella tarda

2006年9月山东胶南某养殖场大菱鲆(Scophthalmus maximus)发生病害,从患病大菱鲆脾脏分离出优势菌株WY28,人工感染试验证实WY28菌株对大菱鲆和斑马鱼(Danio rerio)有较强的致病性,其对大菱鲆和斑马鱼的半数致死剂量(LD50)分别为39cfu/g(BW)(3.3×102cfu/ind)和2.1×104cfu/g(BW)(6.4×103cfu/ind)。综合该菌在形态与生理生化特征及16SrDNA同源性等方面的实验结果,确定WY28菌株为迟缓爱德华氏菌(Edwardsiella tarda)。该菌对先锋霉素V、庆大霉素、氟哌酸、痢特灵等抗生素敏感。WY28菌株经福尔马林灭活制成灭活疫苗,对大菱鲆进行腹腔注射免疫,免疫后第4周测得免疫鱼血清中抗迟缓爱德华氏菌的抗体效价平均为1∶1280。免疫后第6周,免疫鱼血清中抗迟缓爱德华氏菌的抗体效价达到更高水平(平均值为1∶3289.6)。攻毒试验表明,受免鱼的相对存活率明显高于对照组。由此可见,利用从病鱼体内分离的迟缓爱德华氏菌菌株制备的灭活疫苗能使大菱鲆较有效抵御迟缓爱德华氏菌强毒株的攻击。

The diseased turbot,exhibiting haemorrhaging of the basal fin and ulceration of the body surface,were taken aseptically from the hepatopancreas,blood,gall bladder,kidney,spleen and brain,and inoculated immediately onto LBN（ LB medium supplement with 2% NaCl） plates,which were incubated at 28 ℃ for 48 h. A Gram-negative, rod shaped bacterium（ designated as strain WY28） was isolated from the spleen of diseased fish. WY28 gave positive reactions for ornithine decarboxylase,lysine decarboxylase,H2S,citrate utilisation,indole and fermentation of glucose, but was negative for arginine dihydrolase,Voges-Proskauer reaction,oxidase,β-galactosidase,tryptophan deaminase, urease,gelatinase,motility and acid production from amygdalin,arabinose,inositol,sorbitol,rhamnose,melibiose, sucrose and mannitol. WY28 was similar to Edwardsiella tarda in morphological,most of the physiological,biochemical characteristics and 16S rDNA sequence except motility and Citrate utilization. WY28 was identified to be E. tarda. Antibiotic sensitivity was determined by using antibiotic discs impregnated with 30 kinds of antibiotics. WY28 was sensitive to cefazolin（ 30 μg）,gentamycin（ 10 μg）,norfloxacin（ 10 μg）,furazolidone（ 300 μg）. Pathogenicity assays revealed that WY28 was virulent to turbot and zebrafish（ Danio rerio） by intraperitoneal injection challenge. The LD50 of WY28 to turbot and zebrafish was calculated as 39 cfu/g of fish（ 3.3×10^2 cfu/fish） and 2.1×10^4 cfu/g of fish（ 6.4×10^3 cfu/fish）,respectively. In addition,WY28 was re-isolated as pure cultures from internal organs of turbot and zebrafish. The results of pathogenicity assays demonstrated that although the LD50 of WY28 for turbot and zebrafish weredifferent,zebrafish as model animals had potential use for determining pathogenicity of marine bacterial pathogens. Turbots were vaccinated with formalin-killed vaccine of WY28 via intraperitoneal injection. Controls were injected with same volumes of saline. The evident antibody titres（ average was 1∶1 280） were detected in vaccinated fish 4 weeks after immunization. The antibody titres reached a higher level（ average was 1∶3 289.6） 6 weeks after immunization. Vaccinated turbots were intraperitoneal injected with different concentrations of WY28 to detect relative percent survival（ RPS） of formalin-killed vaccine of WY28. The vaccinated fish showed higher RPS against bacteria than the unvaccinated fish. When the vaccinated fish were challenged with 107 cfu/mL of bacteria,the RPS was calculated as 0%; When the vaccinated fish were challenged with 10^6 cfu/mL of bacteria,the RPS was calculated as 16.7%. However,when the vaccinated fish were challenged with 10^5 cfu/mL of bacteria,the RPS was calculated as 66.7%. This demonstrated that the protective effects of inactivated vaccine of WY28 were affected by the concentration of challenging bacteria. The formalinEdwardsiella tardakilled vaccine of WY28 showed higher RPS when the turbots were challenged with lower concentration of pathogen, which was considered to be similar to natural culture environment. Therefore,the inactivated vaccine of WY28,to a large extent,could prevent turbot diseases caused by E. tarda.