真鲷弧菌病病原的研究

福建省某水产养殖场养殖的种用真鲷(Pagrosomus major)于1989年3月发生了一种以鳞片脱落、鳍基部出血为主要症状的疾病,从病死真鲷体内分离到一株弧菌,其生化特性、培养特性、致病性与鳗弧菌基本一致,抗原性与鳗弧菌相同,因此认为这次真鲷疾病是由鳗弧菌引起的真鲷弧菌病。     

海洋水产动物弧菌病的生物防治

弧菌病是由弧菌属(vibriosis)细菌引起的一类细菌性疾病,该病在全世界范围内广泛发生,其发病率高、危害大,不仅给对虾还给鱼类、贝类、其它甲壳类等海水养殖生物造成巨大的影响。因此,该病的防治一直受到国内外研究工作者的密切关注,是海水养殖动物病害研究的主要领域之一[1,2]。对于弧菌病的防治,以往一直采用抗菌素和消毒剂等化学药物治疗,然而使用化学药物具有药物残留和抗药性的问题,造成环境污染,给人类带来危害。近年来人们探索用中药制剂、生物方法和免疫技术进行弧菌防治,取得一些进展。本文仅就生物防治方法进行综述与分析,以…     

山西省虹鳟鱼弧菌病病原菌的分离与鉴定

1985年6～9月间,山西省虹鳟鱼实验场鱼种爆发了一起细菌性疾病,从病鱼的肝、脾、肾中分离到细菌,用此菌人工感染虹鳟能得到与天然发病病鱼相似的症状,并从被感染鱼的体内重新分离到这种菌,证实为该病病原菌。经鉴定为鳗弧菌生物变种Ⅲ(V.anguillarum biovarⅢ,biovar nov)隶属于弧菌Ⅰ组中的淡水亚组弧菌。     

中国对虾病原菌(鳗弧菌)的研究

1987年7～10月福建省平谭县对虾养殖场发生了一起严重的流行病。病虾活动力减弱,食欲下降,个体消瘦,头胸甲心区附近呈白色或浅桔红色,血淋巴液稀薄、混浊、不能凝固。因病虾步足、游泳肢及尾扇呈鲜红色,被称为“红腿病”。死亡率可高达90％以上。从五只垂死病虾的心脏血淋巴液中分离到细菌作纯焙养,经肌肉注射和浸泡的人工感染方法,得到了与虾池中患病虾相同的症状。菌原菌是革兰氏阴性短杆鼠。以极生单鞭毛运动。发酵葡萄糖,产酸不产气。氧化酶、过氧化氢酶阳性。能还原硝酸盐成亚硝酸盐。精氨酸脱羧;鸟氨酸、赖氨酸不脱羧。V.P.阳性.在42℃中不能生长。不能在无盐和含有8％NaCl 的胨水中生长,但在含有3％和6％NaCl的胨水中生长良好。对弧菌抑制剂0/129敏感。经鉴定为鳗弧菌(Vibrio anguillarum Bergman)。     

鳗弧菌、溶藻胶弧菌外膜蛋白的分离及特性

分别用SDS、Sarkosyl及PMSF法分离制备鳗弧菌（Vibrio anguillarum）、溶藻胶弧菌（Vibrio alginolyticus）主要外膜蛋白（MOMP），通过SDS-PAGE分析比较2种弧菌主要外膜蛋白的组成结构。结果表明，SDS、Sarkosyl和PMSF法分离提取的鳗弧菌和溶藻胶弧菌外膜蛋白中，SDS-PAGE电泳图谱不同，鳗弧菌外膜蛋白分子量为27-138kD；溶藻胶弧菌外膜蛋白分子量为27-104kD，其中，45kD、30kD和27kD外膜蛋白为鳗弧菌和溶藻胶弧菌所共有。经比较，Sarkosyl法对2种弧菌外膜蛋白的提取效果较好。对3种方法提取的2种弧菌的主要外膜蛋白进行SDS PAGE后，分别与吸附后的兔抗鳗弧菌、抗溶藻胶弧菌血清的Western-blotting印迹显示，兔抗鳗弧菌血清与其菌株的外膜蛋白主要有3条免疫反应带，其分子量分别为97kD、51kD和30kD，说明其可能与鳗弧菌抗原的特异性有关；兔抗溶藻胶弧菌血清与其菌株的外膜蛋白主要有2条免疫反应带，其分子量分别为51kD和27kD，说明可能与溶藻胶弧菌抗原的特异性有关，而51kD外膜蛋白可能是2种弧菌共有的特异性抗原。     

文蛤增养殖技术讲座 第七讲 文蛤弗尼斯弧菌病的研究与防治技术

第七讲文蛤弗尼斯弧菌病的研究与防治技术随着文蛤养殖集约化程度的提高，海洋富营养化程度的日趋加重，文蛤的病害也跟着发生，发病的情况越来越重，受灾的面积越来越大，损失惨重，仅1989年，江苏南部沿海文蛤死亡数量高达6000多吨，造成的直接经济损失达数千万元人民币。通过广泛调查，实地考察，数百次室内的对比试验，证实：江苏南部沿海多年来文蛤发生大批死亡的主要致病菌是一种海洋弧菌，学名叫弗尼斯弧菌（V凡rnjksii）。文蛤患上弗尼斯弧菌病，一般的症状为：钻出滩面，闭壳肌松驰，出水管喷水无力，贝壳光泽暗淡，不吃食、瘦弱…     

文蛤病原菌(溶藻弧菌)的分离与性状及病文蛤组织的电镜观察

近年来,江苏南部沿海在8～10月份常发生文蛤大批死亡,从病文蛤体内分离到病原菌,经人工感染试验得到证实。病原菌为革兰氏阴性短杆菌,以极生单鞭毛运动,在TCBS琼脂平板上形成黄色大菌落,在固体培养基上能游动。发酵葡萄糖产酸不产气,精氨酸—碱反应阴性,赖氨酸、鸟氨酸脱羧阳性。在无盐蛋白胨水中不能生长,在10％NaCl 蛋白胨水中生长良好。在43℃下能正常繁殖。对弧菌抑制剂0/129(150μg)和新生霉素(5μg)敏感,被鉴定为溶藻弧菌(Vibrio alginolyticus Sakazaki)。病文蛤超薄切片的电镜观察表明,肠上皮及肝组织被细菌侵袭。病原菌在肠上皮细胞质中增生,形成上百个细菌的集群。上皮细胞核变形,被挤向一侧,线粒体内嵴模糊,部分上皮细胞微绒毛的结构被严重破坏,细菌周围的组织被腐蚀成空斑。     

养殖大黄鱼低温弧菌病病原的研究

浙江沿海深水网箱养殖大黄鱼冬天发生大量死亡，从病鱼体内分离出致病力较强的细菌GYC0227。人工感染试验证实这株菌为大黄鱼的病原菌。对细菌进行了形态、生理生化特性的测定，并进行了16S rRNA分子鉴定。PCR扩增获得了GYC0227菌株大小约1．5kb的16S rDNA部分基因片段，产物经回收纯化，克隆到pMD18-T载体，转化大肠杆菌TG1，对阳性克隆子进行酶切及PCR鉴定，并测序，将测定的序列递交NCBI进行BLAET同源序列比对，与溶藻弧菌的标准菌株的16S rDNA具有96％的同源性。结合菌株的生理生化特性，GYCD227菌株属于溶藻弧菌。该菌适应于低温生长，生长温度范围为7—25℃，并测定了其对14种药物的敏感性。     

中国对虾弧菌病病原菌分离与鉴定

1991～1992年间,从唐山沿海四县7个对虾养殖场采集弧菌综合症病虾81尾,并将虾体主要患病部位进行弧菌分高培养,虾体弧菌检出率为95.1％(71/81),主要患病部位弧菌阳性分离率为86.8％(146/168),对其中64株弧菌进行生化反应鉴定,结果为麦氏弧菌8株(12.5％),哈佛弧菌6株(9.4％),弯曲弧菌7株(10.9％),溶藻弧菌21株(32.8％),河弧菌10株(15.6％),副溶血弧菌1株(1.6％),霍利斯弧菌11株(17.2％)。     

弧菌疫苗对斑节对虾和日本对虾免疫预防的作用

尝试使用免疫学的方法来防治对虾弧菌病。将病原副溶血弧菌制成福尔马林灭活疫苗,在实验条件下分别以浸泡、口服和注射等方式接种于斑节对虾和日本对虾,结果发现,在一定的范围内,对虾的免疫保护率随疫苗浓度的增加而升高。在注射接种的方式中,斑节对虾在１９＾９、１０＾８、１０＾７ｃｅｌｌｓ．ｍＬ＾－１的疫苗剂量下的保护力分别为４０％、５０％和４０％,浸泡组（疫苗浓度分别为１０＾８ｃｅｌｌｓ．ｍＬ＾－１、１０＾７     

Growth and performance of Atlantic salmon, Salmo salar L., following administration of a rhabdovirus DNA vaccine alone or concurrently with an oil-adjuvanted, polyvalent vaccine

This research demonstrates for the first time an absence of growth-related side effects in Atlantic salmon, Salmo salar L., following the injection of a DNA vaccine alone or concurrently with a commercially available, polyvalent, oil-adjuvanted vaccine. Using weight and specific growth rate measurements, individually tagged Atlantic salmon were monitored for 2028 degree days (dd) post-vaccination. During this time, DNA-vaccinated fish did not differ in weight, length, condition factor or specific growth rate compared to unvaccinated control fish. While differences in weight were observed between unvaccinated control and concurrently vaccinated fish, there were no significant differences in weight, length, condition factor or specific growth rate between concurrently vaccinated fish and adjuvant-vaccinated fish, suggesting that only adjuvant vaccination affected growth. To further determine if concurrent injection of a DNA vaccine and a polyvalent, oil-adjuvanted vaccine had a physiological impact on the Atlantic salmon, swimming performance tests were performed at 106 dd post-vaccination with U _crit,1, U _crit,2, the U _crit recovery ratio (RR) and the normalized RR being similar to values obtained from unvaccinated control fish. In summary, this study shows that concurrent injection of a DNA vaccine and a polyvalent, oil-adjuvanted vaccine does not negatively influence the growth or swimming performance of Atlantic salmon compared to adjuvant vaccination alone.     

基因工程疫苗概述

疫苗在动物疫病的防控中起着巨大作用,但传统疫苗(灭活苗、弱毒苗)已不能跟上当前畜牧业发展的的步伐。基因工程疫苗作为分子生物技术发展的新兴产物克服了传统疫苗的生产成本高、免疫途径局限、安全性较低等缺点,成为动物疫苗发展的新方向。文章概述了几种常用基因工程疫苗的特点及研究进展,为进一步研究提供文献参考。     

Development,distribution and expression of a DNA vaccine against nodavirus in Asian Seabass,Lates calcarifier (Bloch, 1790)

Fish nodavirus (betanodavirus), a viral pathogen responsible for viral nervous necrosis (VNN) was isolated from infected Asian sea bass (Lates calcarifer). The distribution, clearance and expression of nodavirus vaccine, on the basis of DNA vaccine (pFNCPE42 DNA‐pcDNA3.1) construction, were analysed in tissues of the Asian seabass by PCR, RT‐PCR, ELISA and Immunohistochemistry. Fish immunized with a single intramuscular injection of 20 μg of the pFNCPE42‐DNA vaccine showed a significant increase in the serum antibody level in the 3rd week after vaccination, compared to control eukaryotic expression vector pcDNA3.1 vaccinated fish. Results from PCR studies indicated that the vaccine‐containing plasmids were distributed in heart, intestine, gill, muscle and liver 10 days after vaccination. Clearance of pFNCPE42‐DNA vaccine was studied at 10, 25, 50, 75 and 100 days of post vaccination (d p.v). At 100 days p.v. pFNCPE42‐DNA was cleared from muscle of vaccinated sea bass. In vitro and in vivo expression of fish nodavirus capsid protein gene (FNCP) was determined by fluorescent microscopy. Asian seabass was immunized with pFNCPE42‐DNA vaccine at a dose of 20 μg per fish and were challenged with betanodavirus by intramuscular injection. The vaccinated seabass was protected from nodaviral infection and 77.33% of relative percent survival (RPS) was recorded.     

Randomized clinical field trial of a bacterial kidney disease vaccine in Atlantic salmon,Salmo salar L.

A randomized, blinded clinical trial was performed to assess the relative effectiveness of five commercial and one experimental vaccine in a population of farmed fish experiencing a bacterial kidney disease (BKD) outbreak that occurred in one study cage that was part of a larger clinical field trial. A total of 6000 uniquely identified Atlantic salmon S1 presmolts were randomly assigned to vaccine groups in the hatchery and transferred to a commercial marine aquaculture site. Repeated sampling events to evaluate growth, inherent physical conditions and health status were carried out over the entire production cycle. During the second summer at sea, the study cage developed an outbreak of BKD that lasted approximately 240 days. The effectiveness of the selected vaccines was evaluated using survival analysis methods. The sole vaccine group offering protection for BKD was found to significantly decrease the hazard of dying (hazard ratio, HR = 0.68, P = 0.018) during the outbreak, compared to the industry standard, vaccine group. Additionally, during the outbreak, fish with a shortened operculum had a significantly decreased hazard (HR = 0.38, P = 0.033) compared to those fish with a normal operculum, while fish with jaw deformities had a significantly increased hazard (HR = 2.55, P = 0.001) compared to fish with normal jaw status.     

An experimental vaccine against Aeromonas hydrophila can induce protection in rainbow trout,Oncorhynchus mykiss (Walbaum)

A candidate vaccine against Aeromonas hydrophila in rainbow trout, Oncorhynchus mykiss, was developed using a bacterial lysate. To test the strength of protection, A. hydrophila challenge models were compared using injection into both the intraperitoneal (IP) cavity and the dorsal sinus (DS) with selected doses of live bacteria washed in saline or left untreated. Unlike the IP route, injection into the DS with either saline washed or unwashed cells resulted in consistent cumulative mortality and a dose response that could be used to establish a standard challenge having an LD₅₀ of approximately 3 × 10⁷ colony forming units per fish. Survivors of the challenge suffered significantly lower mortality upon re‐challenge than naïve fish, suggesting a high level of acquired resistance was elicited by infection. Passive immunization using serum from hyper‐immunized fish also resulted in significantly reduced mortality indicating protection can be transferred and that some portion of resistance may be antibody mediated. Vaccination of groups of rainbow trout with A. hydrophila lysate resulted in significant protection against a high challenge dose but only when injected along with Freund’s complete adjuvant. At a low challenge dose, mortality in all groups was low, but the bacterial lysate alone appeared to offer some protection.     

草鱼“四联”疫苗在草鱼池塘养殖生产中的应用

利用4口池塘,每公顷放养75～100g/尾的草鱼种31 612尾～36 486尾。其中2口池草鱼种放养时接种草鱼"四联"疫苗,另2口池设为对照。经6个月的饲养试验,结果表明:接种疫苗的试验池草鱼与未接种疫苗的对照池相比,发病率比对照池低10.7%,成活率与平均产量比对照池分别提高12.8%和20.8%。试验初步证明,接种草鱼四联疫苗能有效地增强草鱼对"四病"的抵御能力,提高草鱼养殖成活率,是有效防治草鱼"四病"的有效措施之一。     

灭活菌苗免疫的南方鲇外周血液细胞免疫指标的变化

以嗜水气单胞菌(Aeromonashydrophila)灭活菌苗为免疫原,以平均体重为86 20g人工饲养的健康南方(Silu rusmeridionalis)为实验对象,通过腹腔注射免疫原(浓度为1 0×108/mL)免疫,剂量为0 2mL/尾,并分别在免疫后的第1、2、4、7、14、21、28天对实验鱼尾静脉采血,进行血液生理指标和血液中吞噬细胞活性的测定。结果显示,嗜水气单胞菌灭活菌苗(FKC)可以增加南方外周血液白细胞数量并引起不同种类白细胞组成比例的变化,提高吞噬细胞的吞噬活性。在免疫后第1、2、4、7、14、21、28天,南方外周血中白细胞数量呈明显上升趋势,其中嗜中性粒细胞、单核细胞、淋巴细胞变化趋势更为明显;而且吞噬细胞活性大为提高,吞噬百分比较对照组平均升高了17 66%,吞噬指数比对照组平均升高了1 16,在免疫后第4天吞噬百分比和吞噬指数均达到最高值,吞噬百分比为48 40%,吞噬指数为4 82;免疫后第21天,吞噬百分比和吞噬指数开始下降,但仍高于对照组。由此表明,使用疫苗后,鱼类非特异细胞免疫在初期(尤其是在第2～7天)明显增强,这是鱼用疫苗在免疫早期保护作用的主要机制之一。     

哈维氏弧菌外膜蛋白OmpW基因克隆、表达及DNA疫苗的免疫效果

参照GeneBank上登录的哈维氏弧菌外膜蛋白OmpW基因序列,设计引物扩增OmpW的开放阅读框,序列分析结果显示该基因全长645 bp,编码214个氨基酸。将其定向克隆到原核表达载体pET-32a( ),在大肠杆菌BL21中成功表达出带His-tag的融合蛋白,融合蛋白分子量约为43.8 ku,且主要以包涵体形式存在。优化的表达条件为温度37 ℃,IPTG浓度0.1 mmol/L,诱导时间5 h。将纯化的融合蛋白免疫SPF昆明小鼠制备多克隆抗体,ELISA方法检测抗体效价达1:30,000,Western-blot结果表明鼠抗OmpW血清能与诱导后的重组蛋白发生特异反应,提示OmpW可能是哈维氏弧菌的一种重要保护性抗原。为了进一步研究哈维氏弧菌外膜蛋白OmpW基因DNA疫苗对红笛鲷的免疫保护效果,构建了真核表达重组质粒pCDNA-OmpW,然后将真核质粒免疫接种红笛鲷。PCR结果显示,免疫接种第7、28 d,在红笛鲷的肌肉、头肾、肝脏和脾脏组织均存在质粒分布;RT-PCR结果显示,免疫接种第7、28 d,红笛鲷不同组织均有目的基因的表达。ELISA结果表明,红笛鲷体内血清产生了抗OmpW蛋白的高效价抗体。Western- blot分析表明,DNA疫苗免疫后红笛鲷体内表达了相应的目的蛋白,并诱导产生了相应抗体。攻毒试验结果表明,免疫保护率高达60 %,可见pCDNA-OmpW可作为红笛鲷预防哈氏弧菌感染的有效候选疫苗之一。