CN111057658B - 一株线纹海马致病菌株及表皮溃疡综合征灭活疫苗 - Google Patents
一株线纹海马致病菌株及表皮溃疡综合征灭活疫苗 Download PDFInfo
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Abstract
本发明涉及一株线纹海马致病菌株及表皮溃疡综合征灭活疫苗，其特征在于，该灭活疫苗为哈维氏弧菌的灭活菌体，灭活疫苗菌体浓度不小于109CFU/ml。本发明制备的海马表皮溃疡综合征灭活疫苗的制备工艺简单、产量稳定，成本低，可大量获得具有较高免疫效果的灭活疫苗。使用该方法制备的灭活疫苗的免疫保护率达62％，该疫苗为纯生物制剂，且具有安全和环保的特点，不会产生药物残留，不会造成环境污染，并且该方法可从根本上遏制海马表皮溃疡综合征的发生和流行，为大规模制备海马表皮溃疡综合征灭活疫苗提供了技术支持。
Description
技术领域
本发明属于微生物疫苗制备领域，具体涉及一株线纹海马致病菌株及表皮溃疡综合征灭活疫苗。
背景技术
线纹海马(Hippocampus erectus)，原产于大西洋海域，主要栖息于浅海层及珊瑚礁等区域，是具有较高的药用价值和观赏价值的珍贵物种。近年来，由于海马野生栖息地的破坏和过度捕捞等原因，使得海马的数量急剧下降。2004年，所有的海马物种都已经列入了华盛顿公约和世界自然保护联盟红色名录中。我国的海马养殖近年来有了长足的发展，但养殖过程中的高密度压力使海马极易受到环境条件致病菌的感染，除此之外，海马捕获携带大量的致病菌的桡足类和卤虫为食，因此病害问题日渐突出，严重制约了该产业的健康发展。
自2017年以来，在线纹海马的养殖过程中，频繁暴发表皮溃疡综合征，患病海马游泳力以及尾部握力减弱，皮肤大面积溃烂，且伴随出现烂尾症状，其解剖特征表现为肠道发白，肝脏及鳃部颜色暗淡，患病海马会在3d内死亡，死亡率高达80％以上。
对于线纹海马表皮溃疡综合征的治疗目前主要依赖于抗生素，导致鱼体极易产生耐药性，破坏生态平衡，威胁人体的健康和安全。因此寻找治疗该病的安全有效的途径是目前亟待解决的问题。
发明内容
本发明的目的是提供一种线纹海马表皮溃疡综合征灭活疫苗及其制备方法，即提供一种对于线纹海马所患有的表皮溃疡综合征具有预防治疗效果的灭活疫苗及其制备方法，解决使用抗生素治疗线纹海马表皮溃疡综合征时所产生的药物残留和耐药性问题。
本发明从患有表皮溃疡综合征的线纹海马组织中分离得到致病哈维氏弧菌，并于2018年03月12日保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.15442。
本发明筛选到的哈氏弧菌CGMCC No.15442用于制备灭活疫苗。
为了获得最好的防治效果，上述的灭活疫苗灭活菌体的浓度为107-1010CFU/ml
上述灭活疫苗的制备方法如下：
1)首先，将分离纯化的哈维氏弧菌接种于含有3％NaCl的LB液体培养基中，28℃震荡培养16-24小时，待菌液浓度达109CFU/ml或分光光度计检测OD600达到0.6以上时终止培养，获得菌株的扩大培养液；
2)然后按体积比5‰加入甲醛溶液，于室温灭活12-16小时，期间震荡5-10次，得到菌株的灭活菌液；
3)然后，将菌株的灭活菌液于8000g、4℃离心15分钟，弃上清液，将沉淀用无菌生理盐水洗涤2次，收集菌体，用无菌生理盐水重悬菌体，浓度约为l010CFU/mL，4℃保存备用；
4)最后取灭活疫苗100μl涂布于2216E固体培养基，28℃培养48h后无菌落长出，即为合格的灭活疫苗。
本发明制备的线纹海马表皮溃疡综合征灭活疫苗制备工艺简单、产量稳定，成本低，可大量获得具有较高免疫效果的灭活疫苗。使用该方法制备的灭活疫苗对线纹海马的免疫保护率达60％，对日本海马的免疫保护率尾42％，该灭活疫苗为纯生物制剂，且具有安全、环保的特点，不会产生药物残留，不会对环境造成污染，并且该方法可从根本上遏制海马表皮溃疡综合征的发生和流行，为大规模制备海马表皮溃疡综合征灭活疫苗提供了技术支持。
具体实施方式
实施例1本发明筛选的哈维氏弧菌CGMCC No.15442的信息
1)哈维氏弧菌的筛选
具有典型症状的患病线纹海马取自青岛清源海洋生物科技公司，将患病海马用0.01mol/L的无菌磷酸盐缓冲盐水(PBS)冲洗体表三遍，然后对患病海马进行解剖，用无菌剪刀取其溃烂肌肉及肠道组织收集于装有灭菌PBS缓冲液的离心管中，将组织剪碎、悬浮于PBS缓冲液中，取上清液进行稀释后涂布于2216E培养平板及TCBS培养平板，28℃培养箱中培养24h，挑取形态特征一致的优势菌落进行分离纯化，将纯化4次后的菌株进行常规生理生化和16S rDNA基因序列测定，确定优势菌株为哈维氏弧菌。
2)哈维氏弧菌的培养条件
将该菌涂布于2216E固体培养基平板上，28℃培养16-18小时即可长出单菌落。
3)形态特征
哈维氏弧菌接种于2216E固体培养基，28℃培养12h，观察菌落形态为圆形、菌落湿润、突起、边缘整齐，菌落呈淡黄色、半透明。哈维氏弧菌用磷钨酸负染3min，晾干后经透射电镜下观察，电镜下菌株HDM-2呈短杆状、周生鞭毛、有荚膜、无芽孢，且菌体大小大约为2.20×4.75μm。
4)生理生化特征
哈维氏弧菌为革兰氏阴性菌，可在1％～10％NaCl蛋白胨水中生长；可发酵葡萄糖、甘露糖、蔗糖、阿拉伯糖以及麦芽糖，不可发酵木糖和乳糖；脲酶、脂酶以及明胶酶阴性；甘露醇阳性，肌醇及水杨苷阴性；精氨酸脱羧酶以及赖氨酸脱羧酶阳性，鸟氨酸脱羧酶阴性；且ONPG、VP以及M.R结果均为阴性,其鉴定结果与哈维氏弧菌基本一致。
5)筛选的菌株的致病性检测
健康线纹海马由青岛清源海洋生物科技有限公司提供，养于实验室整理箱中，培养条件为盐度为30，温度为25±1℃，连续充氧，每天换水量为50％。将分离纯化的哈维氏弧菌进行注射感染，注射浓度为107CFU/mL实验组海马的死亡率为20％，注射浓度为108CFU/mL实验组海马的死亡率为100％，故LD50处于107CFU/mL与108CFU/mL之间，通过平板稀释法，确定了108CFU/mL的精确浓度为5.46×108CFU/mL。线纹海马经注射感染后，浓度为5.46×108组，线纹海马的平均死亡率达到100％，浓度为2.73×108组、1.37×108组及0.68×108组的平均死亡率分别为40％、10％以及0％，而对照组线纹海马无死亡现象，因此，LD50计算结果约为2.89×108CFU/mL。再次收集患病线纹海马的溃烂肌肉及肠道组织进行细菌再分离，得到的细菌经16SrDNA鉴定与初次分离的细菌相同。
分离得到的哈维氏弧菌具有很强的致病性，因此发明人将筛选到的该菌株制备灭活疫苗。
实施例2哈维氏弧菌CGMCC No.15442的灭活疫苗的制备
1)首先将本发明筛选到的哈维氏弧菌接种于1000ml高盐普通LB液体培养基(胰蛋白胨10g/L,酵母浸出汁5g/L,氯化钠30g/L)中，在28℃震荡培养12小时，血球计数板检测菌液浓度达109CFU/ml时终止培养，获得哈维氏弧菌的扩大培养液；
2)后按体积比0.5％加入50ml甲醛溶液，对菌株的扩大培养液于室温灭活12小时，期间震荡5次；得到菌株哈维氏弧菌的灭活菌液；
3)最后，将菌株的灭活菌液于8000g、4℃离心15分钟，弃上清液，将沉淀用无菌生理盐水洗涤2次，收集菌体，用无菌生理盐水重悬菌液，使菌液中的灭活菌体的浓度为107-1010CFU/ml。
4)取灭活疫苗0.1ml涂布于2216E固体培养基，28℃培养48h后无菌落长出表明细菌彻底灭活。将灭活疫苗以0.1ml/尾腹腔注射10尾线纹海马，对照组10尾注射同等剂量的无菌生理盐水，然后正常饲养观察一个星期，全部存活且进食及活动正常，内脏剖检未见任何异常，可认为制备的灭活疫苗安全可靠。放置在4℃条件下保存备用。
实施例3疫苗的免疫保护试验1
用制得的灭活疫苗对日本海马进行免疫效果实验。随机抽取体长2-3厘米的日本海马100尾，随机分为两个处理组，使用具体实施例2中制备的灭活疫苗进行浸泡免疫试验，第1组为对照组，第2组为免疫组，将第2组日本海马置于8％的NaC1溶液中浸泡2分钟，然后立即转移到经过1O倍稀释的灭活菌液(2×108CFU/m1)中浸泡3min，28天后对2组日本海马进行攻毒试验，对照组海马48h全部死亡，第2组疫苗处理组的海马在浸泡攻毒的33h开始死亡，48h累计死亡29尾，48h的相对免疫保护率为42％。
实施例4疫苗的免疫保护试验2
采用浸泡免疫的方法对制备的灭活疫苗的免疫效果进行检测：随机选取体长2-3cm的健康线纹海马200尾，随机分为对照组和免疫组，免疫组置于8％的NaC1溶液中浸泡2分钟，然后立即转移到经过10倍稀释的灭活菌液(2×108CFU/m1)中浸泡3min。7天后用同等方法加强免疫一次，其他饲养条件均与对照组未免疫线纹海马一致。在免疫后第20天，进行攻毒。免疫后第三天对照组线纹海马全部死亡，免疫组累计死亡20尾，免疫组的相对免疫保护率达60％。
Claims (5)
1.一株海马致病菌株，为哈维氏弧菌(Vibrio harveyi)，保藏编号为CGMCC No.15442。
2.一种海马表皮溃疡综合征的灭活疫苗，其特征在于疫苗中包含有权利要求1所述的哈维氏弧菌CGMCC No.15442的灭活菌体。
3.如权利要求2中权利要求所述的灭活疫苗，其特征在于，上述的疫苗中灭活菌体的浓度为107-1010CFU/ml。
4.如权利要求3所述的灭活疫苗，其特征在于，上述的疫苗中灭活菌体的浓度为109CFU/ml。
5.如权利要求3所述的灭活疫苗，其特征在于，所述灭活疫苗的制备方法如下：
首先，将分离纯化的哈维氏弧菌接种于含有3％NaCl的LB液体培养基中，28℃震荡培养16-24小时，待菌液浓度达109CFU/ml或分光光度计检测OD600达到0.6以上时终止培养，获得菌株的扩大培养液；
然后按体积比5‰加入甲醛溶液，于室温灭活12-16小时，期间震荡5-10次，得到菌株的灭活菌液；
然后，将菌株的灭活菌液于8000g、4℃离心15分钟，弃上清液，将沉淀用无菌生理盐水洗涤2次，收集菌体，用无菌生理盐水重悬菌体，浓度约为109CFU/mL，4℃保存备用；
最后取灭活疫苗0.1ml涂布于2216E固体培养基，28℃培养48h后无菌落长出，即为合格的灭活疫苗。
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