CN117925538A - 耐高温型迟钝爱德华菌噬菌体及其应用 - Google Patents
耐高温型迟钝爱德华菌噬菌体及其应用 Download PDFInfo
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Abstract
本发明提出一种耐高温型迟钝爱德华菌噬菌体,属于生物工程领域,能够解决现有迟钝爱德华菌噬菌体仅能耐受短时高温,但长期保存过程中效价会大幅度降低的问题。该噬菌体被命名为RKP‑ET‑22002,保藏编号为CGMCC No.45355,于2022年10月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心。该噬菌体能够用于制备防治由迟钝爱德华菌造成的污染或引起的疾病的生物制剂。该噬菌体具有耐高温的特性,不仅可以耐受短时高温,并且制粉后可长期保存,且效价仍保持在较高水平。
Description
技术领域
本发明属于生物工程领域,尤其涉及一种耐高温型迟钝爱德华菌噬菌体及其应用。
背景技术
迟钝爱德华氏菌(Edwardsiellatarda,ET)是一种短杆状革兰氏阴性致病菌,能造成多种淡、海水鱼类的系统性出血败血症。还是一种重要的人畜共患病原菌,是爱德华氏菌属中唯一感染人类的成员。在感染过程中,E.tarda能以胞内寄生模式定植于宿主重要器官,造成宿主表皮溃烂、脓肿、肠道组织腐败,最终爆发形成爱德华氏菌病。目前,世界各地都有鱼类感染E.tarda的病例报道,造成水产养殖业的严重经济损失。
近年来,水产养殖的规模化使E.tarda成为严重威胁淡、海水鱼类的细菌性病原,给水产养殖业造成巨大经济损失。该菌能引起多种经济鱼种的消化道疾病,其感染引发的出血性败血症被认为迟钝爱德华氏菌病的典型病症。此外,E.tarda病害的地理位置范围很广泛。我国渤海、黄海、东海沿海海域的养殖场,甚至内陆的淡水鱼、鳖等养殖场都有相关病例报道。E.tarda还具有兼性胞内寄生特性,是一种造成人、鱼共患病的致病菌,直接威胁人类健康。目前为止,由生食鱼类制品或机体创伤造成的人类感染E.tarda病例呈现逐年增长的趋势,且能诱发多种并发症。因此,如何有效防治该病原菌是迫切需要解决的问题。
目前,针对水产动物出现的迟钝爱德华菌病害,通常使用抗生素或其他有关的化学药剂进行治疗。如庆大霉素、氨卞青霉素、新霉素、链霉素及磺胺类药物等。但是众所周知的是,抗生素等药物的使用在短期内固然可以起到很明显的疗效,但它有其很大的弊端就是引起病原的抗药性,同时,抗生素的使用还会对养殖环境中其他的非病原微生物或益生菌群产生很大的破坏作用,打破了养殖环境中微生物菌群的竞争和平衡。
噬菌体是特异性感染细菌并在细菌细胞内复制的病毒。它们是地球上最丰富,最普遍的生物,在微生物、生理学、进化和治疗方面发挥着重要作用。近年来,使用噬菌体治疗人类和动物的慢性细菌性感染,具有很多成功的案例。噬菌体与抗生素相比具有更高的有效性和安全性。与抗生素相比,噬菌体具有高度的特异性,这种作用机制的优点是噬菌体只会裂解目标菌株,不会导致共生肠道菌群的破坏。同时噬菌体具有自我复制、治疗所需剂量小、无毒性、研发周期短、生产成本低等优势。噬菌体治疗作为消除病原菌的替代手段具有十分广阔、诱人的前景,值得研究者深入探索。Kwon等用迟钝爱德华菌菌体(ETG)做为口服制剂饲喂牙鲆,能有效的抵抗迟钝爱德华菌的感染。因此,对控制迟钝爱德华菌感染迟钝爱德华菌噬菌体是一个很好的选择。
但是在实践过程中发现,大多数迟钝爱德华菌噬菌体可以耐受短时高温,制粉,但是在保存过程中,经过1-3个月,效价会大幅度下滑。如想解决此问题,除了优化噬菌体保护剂之外,筛选耐高温、效价稳定,可长期保存的噬菌体至关重要。
发明内容
本发明针对现有迟钝爱德华菌噬菌体仅能耐受短时高温,但长期保存过程中效价会大幅度降低的技术问题,提出一种耐高温型迟钝爱德华菌噬菌体,该噬菌体具有耐高温、效价稳定、可长期保存的特点,能够用于制备防治病原菌造成的污染或引起的疾病的生物制剂。
为了达到上述目的,本发明采用的技术方案为:
耐高温型迟钝爱德华菌噬菌体,该噬菌体被命名为RKP-ET-22002,保藏编号为CGMCC No.45355,于2022年10月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号。
在一实施方式中,所述耐高温型迟钝爱德华菌噬菌体的全基因组序列如SEQ IDNO.1所示。
所述耐高温型迟钝爱德华菌噬菌体具有一个多面体立体对称的头部和尾部,所述头部和尾部通过颈部相连,所述头部包裹着核酸,其直径为100nm,所述尾部长度为120nm。
在一实施方式中,所述耐高温型迟钝爱德华菌噬菌体在37℃下培养4小时,其效价大于1010pfu·mL-1;该噬菌体在85℃条件下水浴10min,其效价保持在109pfu·mL-1。
本发明还列举了基于上述实施方式所述的耐高温型迟钝爱德华菌噬菌体的用途,该噬菌体用于制备防治病原菌造成的污染或引起的疾病的生物制剂。
在一实施方式中,所述病原菌为迟钝爱德华菌,所述生物制剂包括药物制剂、饲料添加剂、杀菌剂或者消毒剂。
药物制剂,为纯化后的耐高温型迟钝爱德华菌噬菌体制备得到的单一制剂或以纯化后的耐高温型迟钝爱德华菌噬菌体与其他有效成分组合使用得到的复配制剂。
饲料添加剂,其包含上述实施方式所列举的耐高温型迟钝爱德华菌噬菌体,通过将饲料添加剂添加至水产养殖动物饲料中,以防治迟钝爱德华菌引发的疾病。
消毒剂,其包含上述实施方式所列举的耐高温型迟钝爱德华菌噬菌体,通过向水产养殖动物的养殖环境中喷洒所述消毒剂,以防治迟钝爱德华菌的污染。
在一实施方式中,消毒剂的剂型为液体或冻干粉;水产养殖动物的养殖环境包括水产养殖动物体内、水产养殖动物体表、水产养殖动物养殖场所地面、水产养殖动物的养殖场所空气、水产养殖动物饲料、饮水以及养殖器具。
与现有技术相比,本发明的优点和积极效果在于:
1、本发明提出一种耐高温型迟钝爱德华菌噬菌体,该噬菌体以迟钝爱德华菌作为宿主分离得到,具有耐高温的优良特性,不仅可以耐受短时高温,并且制粉后可长期保存,效价较高;
2、本发明提出一种耐高温型迟钝爱德华菌噬菌体,噬菌体进行传代30次的条件下,仍然可以保持较高水平的效价,表明这株噬菌体具有较强的遗传稳定性。
附图说明
图1为本发明所提供的迟钝爱德华菌的菌落形态图;
图2为本发明所提供的耐高温型迟钝爱德华菌噬菌体对宿主菌的噬菌斑;
图3为本发明所提供的耐高温型迟钝爱德华菌噬菌体的电镜示意图;
图4为本发明所提供的耐高温型迟钝爱德华菌噬菌体的最佳感染复数测定结果;
图5为本发明所提供的耐高温型迟钝爱德华菌噬菌体的一步生长曲线图;
图6为本发明所提供的耐高温型迟钝爱德华菌噬菌体的生长温度测定结果;
图7为本发明所提供的耐高温型迟钝爱德华菌噬菌体的pH稳定性测定结果;
图8为本发明所提供的耐高温型迟钝爱德华菌噬菌体的遗传稳定性测定结果;
图9为本发明所提供的耐高温型迟钝爱德华菌噬菌体的耐高温试验测定结果;
图10为本发明所提供的耐高温型迟钝爱德华菌噬菌体的喷粉试验测定结果;
图11为本发明所提供的耐高温型迟钝爱德华菌噬菌体的中高温长期保存试验测定结果。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
为了更清楚详细地介绍本发明实施例所提供的耐高温型迟钝爱德华菌噬菌体及其应用,下面将结合具体实施例进行描述。
实施例1
本实施例为耐高温型迟钝爱德华菌噬菌体RKP-ET-22002的分离鉴定,具体为:
(1)迟钝爱德华菌的分离及其鉴定:
从多宝鱼养殖场采样,将样品划线接种于TSA血琼脂培养基上,置于37℃恒温培养箱培养18~24h,观察平板上的菌落形态及颜色。挑取表面光滑,湿润、半透明,具有狭窄的β型溶血环的菌落。
挑取表面光滑,湿润、半透明,具有狭窄的β型溶血环的菌落,划线接种于TSA血琼脂培养基上,传代培养纯化3~5次,直至获得形态均一的菌落(如图1所示),挑取单菌落划线接种于TSA血琼脂培养基中,挑取菌落,然后经16SrRNA基因测序技术进行鉴定;鉴定完成后,刮菌苔置于30%的甘油肉汤中,保存于-80℃。
(2)噬菌体的分离及纯化:
(2-1)养殖水样处理:从养殖场采取污泥水样,称取5g加入10mL的SM缓冲液中浸泡过夜,然后将过夜后的浸出液10000rpm离心5min,取上清液用0.22μm滤器过滤,将滤出液4℃保存备用;
(2-2)噬菌体富集:取0.1mL迟钝爱德华菌悬液和1mL滤出液加入5mLLB肉汤中,37℃、摇床培养过夜,然后10000rpm离心10min,将上清液用0.22μm滤器过滤,滤出液备用;
(2-3)噬菌体分离:采用双平板法进行噬菌体的分离,取迟钝爱德华菌菌悬液0.1mL与0.6%的LB软琼脂混合铺在LB固体平板上,待软琼脂凝固后将滤出液0.1mL点到平板上,静放至混合液吸收,放于37℃培养箱培养6-8h后,用接种环挑取透明的噬菌斑置于2mL的SM缓冲液中,4℃保存过夜,让噬菌体完全释放,即得噬菌体;
(2-4)噬菌体纯化:采用双平板法进行噬菌体的分离,取混合菌悬液滤出液0.1mL和宿主迟钝爱德华菌菌悬液0.2mL混合均匀,加入冷却至50℃左右的软琼脂混合均匀,然后铺双平板,放于37℃培养箱培养6-8h后,挑取边缘光滑的单个透明斑于1mL的SM缓冲液中,4℃过夜保存。取0.1mL过夜保存的浸出液和0.1mL菌悬液混合,加入冷却至50℃左右的软琼脂混合均匀,铺双平板,37℃培养箱培养4-6h,照此步骤纯化至噬菌斑为大小均一、边缘光滑的噬菌斑,即得纯化好的噬菌体,该噬菌体对宿主菌的噬菌斑如图2所示;
(2-5)噬菌体的制备:取0.2mL迟钝爱德华菌菌悬液和1mL纯化好的噬菌体悬液加入到50mLLB肉汤中,37℃摇床培养过夜,然后10000rpm离心10min,将上清液用0.22μm滤器过滤,噬菌体滤出液备用;
(2-6)噬菌体的保藏方式:将噬菌体悬液1∶1加入60%的甘油混合后,于-80℃中保存,命名为RKP-ET-22002。
(3)噬菌体的电镜观察:
取20μL含噬菌体粗制颗粒的液体滴于铜网上,自然沉淀15min,并用滤纸从侧面吸去多余液体,加一滴2%的磷钨酸(PTA)于铜网上对噬菌体染色10min,然后用滤纸从侧面吸去染色液,待样品干燥后用电子显微镜观察噬菌体形态,如图3所示。
由图3可知,噬菌体RKP-ET-22002有一个多面体立体对称的头部,包裹着核酸,直径约为100nm,有一个长约120nm的尾,颈部连着头部和尾部。根据国际病毒分类学组织病毒分类第九次报告,该噬菌体归类为有尾病毒目肌尾病毒科。
(4)噬菌体的全基因组测序及分析:
采用IlluminaTruSeqTMNanoDNASamplePrepKit方法构建文库,具体步骤为:
①以1μg噬菌体基因组DNA起始量建库;
②Covaris M220超声打断DNA到300-500bp;
③补平3’端加A、连接index接头(TruSeqTM Nano DNA Sample Prep Kit);
④文库富集,PCR扩增8个cycles;
⑤2%琼脂糖胶回收目的条带(Certified Low Range Ultra Agarose);
⑥TBS380(Picogreen)定量,按数据比例混合上机;
⑦cBot固相载体上进行桥式PCR扩增,生成clusters;
⑧Illumina Hiseq测序平台,进行2×150bp测序,该噬菌体的全基因组序列如SEQID NO.1所示。
实施例2
本实施例为耐高温型迟钝爱德华菌噬菌体RKP-ET-22002的效价测定,具体为:
取培养到指数期的宿主菌液(迟钝爱德华菌)100μL混入5mL离心管上下摇匀,置于LB半固体琼脂上,完整覆盖于的20mLLB固体琼脂平板(9cm直径)上,待半固晾干后分别吸取纯化好的噬菌体浓缩液100μL置于平板上,一种噬菌体铺3个板,37℃培养过夜,抠取噬菌斑于20mL的SM缓冲液中,放入4℃,次日取上清液,经0.22μm滤膜过滤,取100μL滤液经无菌水10倍梯度稀释,然后分别将菌液与噬菌液各100μL的混合于双平板,平板在37℃培养箱中过夜培养。通过以上方法对噬菌体PKP-ET-22002进行噬菌体效价的测定。选择可数平板,计数噬菌体菌斑,
计算噬菌体效价(PFU/mL)为菌斑数×稀释倍数/样品体积(mL)。
实验结果及分析:经测定,该噬菌体PKP-ET-22002效价可达到1010PFU/mL。
实施例3
本实施例为耐高温型迟钝爱德华菌噬菌体RKP-ET-22002的最佳感染复数(MOI)测定,具体为:
MOI指初始感染时噬菌体数量与宿主菌(迟钝爱德华菌)数量的比值。将培养至指数期的宿主菌菌液浓度调整为108CFU/mL。设置MOI分别为0.0001、0.001、0.01、0.1、1、10、100,将已测定效价的噬菌体液按照比例分别加入到菌液中,菌液和噬菌体液各500μL,混匀,37℃,200r/min振荡培养5h。将混合培养物10000r/min离心10min,测噬菌体效价。效价最高的感染复数即为最佳感染复数。通过以上方法对噬菌体PKP-ET-22002进行最佳感染复数的测定,测定结果如图4所示。
由图4可知,噬菌体PKP-ET-22002的感染复数为0.1时噬菌体效价最高,表明该噬菌体PKP-ET-22002的最佳感染复数为0.1,对应最高效价为1010PFU/mL。
实施例4
本实施例为耐高温型迟钝爱德华菌噬菌体RKP-ET-22002的一步生长曲线,具体为:
将噬菌体液与宿主菌(迟钝爱德华菌)液各500μL以最佳感染复数需要的比例混合,37℃孵育15min。10000r/min离心1min,将沉淀用LB液体洗涤3次。加入10mL 37℃预热的LB液体培养基,混匀后迅速置于37℃摇床中振荡培养。每隔10min取样一次,测定噬菌体的效价,做3个平行,结果取平均值,以感染时间为横坐标,感染体系中噬菌体滴度的对数值为纵坐标,绘制一步生长曲线,得到噬菌体PKP-ET-22002的潜伏期、爆发期,计算出爆发量,计算公式如下:
爆发量=噬菌体爆发末期的总个数/噬菌体初期细菌的总个数。
该噬菌体的一步生长曲线如图5所示,噬菌体PKP-ET-22002感染宿主菌60min内,噬菌体数量无增加,这段时间称为潜伏期;感染宿主菌后的60~210min,噬菌体数量急速增加,这个时期为噬菌体的爆发期,即噬菌体的爆发期约为150min,爆发量为104pfu·mL-1。
实施例5
本实施例为耐高温型迟钝爱德华菌噬菌体RKP-ET-22002的最适生长温度测定试验,具体为:
将噬菌体原液十倍梯度到适合浓度取100μL与等量菌液混合,倒双层平板,分别置于15℃、20℃、25℃、30℃、37℃、40℃、45℃和50℃下培养4小时,检测噬菌体效价。以温度为横坐标,以噬菌体效价的对数值为纵坐标,分别绘制噬菌体PKP-ET-22002的生长温度曲线(如图6所示)。
由图6可知,该噬菌体在37℃时,效价最高,其最适生长温度为37℃。
实施例6
本实施例为耐高温型迟钝爱德华菌噬菌体RKP-ET-22002的pH稳定性测定,具体为:
将LB液体培养基的pH值分别调整为3、4、5、6、7、8、9、10、11、12,各取100μL与等体积的噬菌体原液混合,置于37℃作用2h后,用双层平板法测定效价,并且每个pH设定3个重复。以pH为横坐标,以噬菌体效价的对数值为纵坐标,分别绘制噬菌体PKP-ET-22002的pH稳定性曲线。
该噬菌体pH稳定性测定结果如图7所示,该噬菌体在pH=7时效价最高,且其效价在pH=5~10的范围内都能维持在108~1010pfu·mL-1较高的水平。
实施例7
本实施例为耐高温型迟钝爱德华菌噬菌体RKP-ET-22002的稳定性遗传实验,具体为:
将该噬菌体连续传代30次,每代培养基中加入等量的噬菌体和宿主菌(迟钝爱德华菌),每代培养时间6h,然后铺双平板,将噬菌体连续传29代后,测定其效价,结果如图8所示。
由图8可知,该噬菌体PKP-ET-22002在传代过程中,效价整体处于稳定状态,表明噬菌体的生物遗传稳定性较好,是生产过程的优异选择。
实施例8
本实施例为耐高温型迟钝爱德华菌噬菌体RKP-ET-22002的裂解谱系分析,具体为:
采用双层平板法点板法,测定本发明的噬菌体RKP-ET-22002对菌株库保存的20株迟钝爱德华菌的裂解效果,所用的菌株均为致病性毒株,其中10株菌为海洋来源,10株菌为淡水来源。
制备不同的迟钝爱德华菌菌液,取200μL培养6h的待测菌菌液加入到0.6%的LB半固体培养基中混匀,倒入平皿中,静置凝固。取20μL噬菌体悬液滴加到软琼脂平皿上,37℃培养6~8h左右,观察有无噬菌斑出现,测定结果见表1。
表1迟钝爱德华噬菌体RKP-ET-22002的裂解谱
由表1所知,本发明噬菌体RKP-ET-22002对不同的迟钝爱德华菌毒株的裂解高达85%。由此可见,该噬菌体RKP-ET-22002对淡水与海洋来源的迟钝爱德华菌毒株都具有很好的裂解效果。
实施例9
本实施例为耐高温型迟钝爱德华菌噬菌体RKP-ET-22002的耐高温实验,具体为:
将制备好的噬菌体PKP-ET-22002(实验组)以及噬菌体PKP-ET-2022001(对照组),以每分钟10,000转的速度离心10分钟,以便沉淀寄宿主细菌,将上清液转移至新的离心管中,使用0.22微米孔径的过滤器过滤掉残余的菌体,这样就得到不含寄主细菌的噬菌体原液,然后将噬菌体原液置于85℃水浴锅中,水浴10min后,将处理液,梯度稀释,测其效价。(注:对照组用到的噬菌体PKP-ET-2022001已申请相关专利,申请日为2023年7月6日,申请号为CN202310824749.7,该噬菌体已于2022年8月31日保藏于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.45264。)
测定结果如图9所示,对照组的噬菌体PKP-ET-22001置于85℃水浴锅中,水浴10min,效价保持在107pfu·mL-1,本发明所提供的耐高温型迟钝爱德华菌噬菌体PKP-ET-22002置于85℃水浴锅中,水浴10min,效价仍可保持在109pfu·mL-1,试验表明本发明所提供的噬菌体可耐受较高的温度。
实施例10
本实施例为耐高温型迟钝爱德华菌噬菌体RKP-ET-22002的喷粉试验,具体为:
将制备好的噬菌体原液和辅料(淀粉、β-环状糊精、甘露醇)进行混合,比例为原液、淀粉、β-环状糊精、甘露醇=100∶5∶8∶3(v/m),置于喷雾干燥机中,设置进口温度为80℃,出口温度为58℃,收集噬菌体粉剂,测定其效价。
该噬菌体的粉剂效价测定结果如图10所示,该噬菌体液体经喷雾干燥机喷粉,制成噬菌体粉剂后,效价仍可保持在109pfu·g-1。
实施例11
本实施例为耐高温型迟钝爱德华菌噬菌体RKP-ET-22002的中高温长期保存实验,具体为:
将实施例10中制备好的噬菌体粉剂,放置于30℃、50℃这两个中高温环境中储存,按照1天、2天、4天、6天、8天、14天的时间段取样,测定其噬菌体效价。
噬菌体粉剂效价测定结果如图11所示,结果表明该噬菌体粉剂在30℃、50℃这两个中高温条件下,储存2周,噬菌体效价仍可维持在107pfu·g-1。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.耐高温型迟钝爱德华菌噬菌体,其特征在于,所述耐高温型迟钝爱德华菌噬菌体被命名为RKP-ET-22002,保藏编号为CGMCC No.45355,于2022年10月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心。
2.根据权利要求1所述的耐高温型迟钝爱德华菌噬菌体,其特征在于,所述耐高温型迟钝爱德华菌噬菌体的全基因组序列如SEQ ID NO.1所示。
3.根据权利要求1所述的耐高温型迟钝爱德华菌噬菌体,其特征在于,所述耐高温型迟钝爱德华菌噬菌体具有一个多面体立体对称的头部和尾部,所述头部和尾部通过颈部相连,所述头部包裹着核酸,其直径为100nm,所述尾部长度为120nm。
4.根据权利要求1所述的耐高温型迟钝爱德华菌噬菌体,其特征在于,所述耐高温型迟钝爱德华菌噬菌体在37℃下培养4小时,其效价大于1010pfu·mL-1;所述耐高温型迟钝爱德华菌噬菌体在85℃条件下水浴10min,其效价保持在109pfu·mL-1。
5.根据权利要求1~4中任意一项所述的耐高温型迟钝爱德华菌噬菌体的用途,其特征在于,该噬菌体用于制备防治病原菌造成的污染或引起的疾病的生物制剂。
6.根据权利要求5所述的用途,其特征在于,所述病原菌为迟钝爱德华菌,所述生物制剂包括药物制剂、饲料添加剂、杀菌剂或者消毒剂。
7.药物制剂,其特征在于,为纯化后的如权利要求1~3中任意一项所述的耐高温型迟钝爱德华菌噬菌体制备得到的单一制剂或以纯化后的权利要求1~4中任意一项所述的耐高温型迟钝爱德华菌噬菌体与其他有效成分组合使用得到的复配制剂。
8.饲料添加剂,其特征在于,其包含如权利要求1~4中任意一项所述的耐高温型迟钝爱德华菌噬菌体,通过将所述饲料添加剂添加至水产养殖动物饲料中,以防治迟钝爱德华菌引发的疾病。
9.消毒剂,其特征在于,其包含如权利要求1~4中任意一项所述的耐高温型迟钝爱德华菌噬菌体,通过向水产养殖动物的养殖环境中喷洒所述消毒剂,以防治迟钝爱德华菌的污染。
10.根据权利要求8所述的消毒剂,其特征在于,所述消毒剂的剂型为液体或冻干粉;所述水产养殖动物的养殖环境包括水产养殖动物体内、水产养殖动物体表、水产养殖动物养殖场所地面、水产养殖动物的养殖场所空气、水产养殖动物饲料、饮水以及养殖器具。
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