摘要
为研究杂交鲟“红嘴病”的病原,本研究对自然患“红嘴病”杂交鲟的肾脏、肝脏进行病原菌的分离,对分离株经形态学观察、生化特性鉴定、16S rRNA基因的PCR扩增及序列分析,并将分离菌以1.0×10^(7)cfu/mL~1.0×10^(3)cfu/mL的剂量感染健康杂交鲟,每日观察并记录鲟鱼的活力和死亡数,感染后10 d对各组发病鱼剖检并再次分离病原菌。采用SPSS 19.0的概率单位图解法计算该分离菌对杂交鲟的半数致死量(LD50)。结果显示,从患病杂交鲟肾脏中分离到一株优势革兰氏阳性菌株KS2,呈卵圆形或圆形,成双或成链状排列。生化鉴定结果显示,分离株KS2与停乳链球菌停乳亚种的生化特征一致;PCR结果显示,从分离菌中扩增到约1 500 bp的16S r RNA基因片段,其与停乳链球菌停乳亚种16S rRNA基因的同源性达100%,二者在进化树中聚为一支。以上结果表明从杂交鲟中分离到一株停乳链球菌停乳亚种KS2株。感染试验结果显示,感染组发病鲟鱼均出现嘴部充血肿胀、肛门红肿等“红嘴病”典型症状,并从濒死鲟鱼肾脏中分离的细菌与原感染菌的形态、生化特征均一致;经计算分离菌株KS2对杂交鲟的LD50为3.83×10^(4)cfu/mL。结合上述结果判定菌株KS2为杂交鲟发病的病原菌。利用0.5%甲醛制备KS2菌株灭活疫苗,经安全性检验合格后将其以两种剂量(灭活前分别为1.0×10^(8)cfu/mL及1.0×10^(7)cfu/mL的菌液)免疫杂交鲟,分别于免疫后不同时间采血,采用试剂盒测定各组鲟鱼血清中C3补体和免疫球蛋白IgM的含量,免疫后90 d每组鱼分别经腹腔注射0.5 mL(1.0×10^(7)cfu/mL)停乳链球菌攻毒,通过观察记录10 d内鲟鱼的发病和死亡情况,计算疫苗的相对保护率(RPS),评估该疫苗在实验室阶段对鲟鱼的免疫保护效果。在田间试验中利用该灭活疫苗(1.0×10^(8)cfu/mL)免疫58 637尾杂交鲟,另将66 544尾鲟鱼作为空白对照组,统计患“红嘴病”死亡鲟鱼的数量,评估该疫苗在田间试验阶段对鲟鱼的免疫保护效果。实验室免疫保护试验结果显示,免疫后60 d和90 d,免疫组鲟鱼血清中C3补体和IgM的含量均显著高于PBS对照组(P<0.05),且免疫后30 d~90 d,高剂量免疫组鲟鱼血清中上述两个指标的含量均随时间的延长而升高。攻毒结果显示:高、低剂量灭活疫苗免疫组和对照组杂交鲟的RPS分别为75%、50%。灭活疫苗田间试验结果显示,免疫组杂交鲟因“红嘴病”死亡鲟鱼1 295尾,明显低于对照组死亡的3 799尾,死亡率降低了3.5%。综上所述,本研究证实停乳链球菌停乳亚种是杂交鲟“红嘴病”的病原,对杂交鲟的致病性较强。首次制备鲟鱼源停乳链球菌灭活疫苗,通过实验室免疫保护试验和田间免疫试验证明该疫苗的免疫原性较强,能够刺激鲟鱼产生较强的体液免疫反应,对鲟鱼的免疫保护效果较好,具有很好的应用前景。本研究为鲟鱼“红嘴病”疫苗的研制提供了重要参考依据,对该病的防治具有重要意义。
To investigate the pathogen of Red mouth disease of hybrid sturgeon,the potential pathogenic bacteria were isolated from the kidneys and livers of diseased hybrid sturgeons.In order to identify the isolated bacteria and determine its pathogenicity,the biochemical and physiological analysis,16S rRNA gene analysis,and a artificial infection tests were performed.Healthy hybrid sturgeon was infected with the isolates at a dose of 1.0×10^(7) cfu/mL-1.0×10^(3) cfu/mL.The vitality and mortality rates of sturgeon were observed every day,and the pathogenic bacteria were subsequently re-isolated from the fish again 10 days post infection.Additionally,the probability unit graphical method of SPSS 19.0 was employed to calculate the half lethal concentration(LD50)of the isolated pathogenic bacteria on hybrid sturgeon.The results showed that a dominant strain KS2 was isolated from the kidney of diseased hybrid acipenser.It was a Gram-positive strain,oval or round,arranged in pairs or chains.The integrated outcomes of physiological and biochemical tests and 16S rRNA gene analysis indicated that the strain KS2 has been identified as Streptococcus dysgalactiae subsp.dysgalactiae.PCR results showed that the 16S rRNA gene fragment was amplified from the isolated strain to about 1500bp,and the homology of 16S rRNA gene between the isolated strain and Streptococcus dysgalactiae was 100%,and the two strains converged into one branch in the evolutionary tree.These results indicated that a strain KS2 of Streptococcus dysgalactiae subsp.dysgalactiae was isolated from hybrid sturgeon.The challenge test showed that this strain was lethal to hybrid sturgeons with similar clinical signs as naturally diseased fish,and the LD50 was 3.83×10^(4) cfu/mL.The above results indicated that the KS2 strain was the pathogenic bacterium responsible for the Red mouth disease of hybrid sturgeon.We further prepared an inactivated vaccine using 0.5%formaldehyde,and evaluated its immunoprotective efficacy on hybrid sturgeon at both high and low doses(1.0×10^(8) cfu/mL and 1.0×10^(7) cfu/mL bacterial solution before inactivation)via a laboratory experiment.The laboratory immune test results showed that the levels of complement C3 and IgM in the serum of hybrid sturgeon in the high-dose and low-dose vaccine groups were significantly elevated compared to the control group at both 60 days and 90 days post-vaccination(P<0.05),and the contents of the above two indexes in the serum of sturgeon in the high-dose immunization group were increased with the extension of time on days 30 to 90 after immunization.Following a 10-day infection with S.dysgalactiae,these two vaccine groups achieved immune protection rates of 75%and 50%,respectively.In pilot test,58637 hybrid sturgeons were immunized with the inactivated S.dysgalactiae vaccines,and 66544 sturgeons were selected as the control group.The number of sturgeons that died from Red mouth disease were counted to evaluate the immune protection effect of the vaccine on sturgeons during the field trial stage.The pilot test results showed that the mortality rate of sturgeon(A.gueldenstaedti)caused by Red mouth disease in the S.dysgalactiae inactivated vaccine group(1.0×10^(8) cfu/mL)decreased by 3.5%compared to the control group.In summary,this study confirmed that S.dysgalactiae subsp.Dysgalactiae was the causative agent of Red mouth disease in hybrid sturgeons and its pathogenicity was potent.This study has pioneered the development of an inactivated vaccine against S.dysgalactiae in sturgeon,demonstrating its outstanding immunogenicity and protective efficacy.These findings lay a crucial foundation for the advancement of vaccines targeting Red mouth disease in sturgeon and are of considerable significance for the prevention of this malady.
作者
胡谋
黄雷
姚嘉赟
徐洋
蔺凌云
沈锦玉
许式见
潘晓艺
HU Mou;HUANG Lei;YAO Jia-yun;XU Yang;LIN Ling-yun;SHEN Jin-yu;XU Shi-jian;PAN Xiao-yi(Zhejiang Institute of Freshwater Fisheries,Key Laboratory of Healthy Freshwater Aquaculture,Ministry of Agriculture and Rural Affairs,Key Laboratory of Fish Health and Nutrition of Zhejiang Province,Key Laboratory of Fishery Environment and Aquatic Product Quality and Safety of Huzhou City,Huzhou 313001,China;Key Laboratory of Sturgeon Genetics and Breeding,Ministry of Agriculture and Rural Affairs,Hangzhou Qiandaohu Xunlong Sci-tech Co.,Ltd.,Hangzhou 311700,China;Quzhou Sturgeon Aquatic Food Technology Development Co.,Ltd.,Quzhou 324000,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2024年第10期1072-1079,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
浙江省“尖兵领雁+X”研发攻关计划项目(2024C02005、2022C2027)
杭州市科技项目(2019S017)。
关键词
杂交鲟
停乳链球菌
细菌鉴定
灭活疫苗
免疫作用
hybrid sturgeon
Streptococcus dysgalactiae
bacterial identification
inactivated vaccine
immune effect
作者简介
共同第一作者:胡谋(1989-),男,湖南浏阳人,工程师,硕士,主要从事水产动物健康养殖研究;共同第一作者:黄雷(1992-),男,湖南道县人,助理研究员,主要从事水生动物病害防控研究;通信作者:姚嘉赟,E-mail:yaojiayun@126.com;通信作者:许式见,E-mail:xsj@kalugaqueen.com。
