CN116445366A - 一株大菱鲆来源的致病性恶臭假单胞菌及其应用 - Google Patents
一株大菱鲆来源的致病性恶臭假单胞菌及其应用 Download PDFInfo
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- CN116445366A CN116445366A CN202310652472.4A CN202310652472A CN116445366A CN 116445366 A CN116445366 A CN 116445366A CN 202310652472 A CN202310652472 A CN 202310652472A CN 116445366 A CN116445366 A CN 116445366A
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- pseudomonas putida
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Abstract
本发明公开了一株大菱鲆来源的对大菱鲆具有致病性的恶臭假单胞菌PPD2及其应用，属于病原微生物筛选技术领域。其中，该恶臭假单胞菌PPD2是从腹部膨大、肛门红肿外凸、腹腔腹水、肝充血、脾肾肿胀为主要特征的患病大菱鲆的内脏中筛选获得的，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M2023180。该恶臭假单胞菌PPD2对大菱鲆具有一定的致病性，可引起养殖大菱鲆患病死亡，且该菌株对β‑内酰胺类3种、大环内酯类1种和多肽类1种抗生素具有抗性，用该恶臭假单胞菌PPD2制备灭活疫苗，可有效防控大菱鲆恶臭假单胞菌病，减少抗生素类药物滥用、保障水产品质量安全。
Description
技术领域
本发明涉及一株细菌及其应用，具体涉及一株大菱鲆来源的对大菱鲆具有致病性的恶臭假单胞菌及其应用，属于病原微生物筛选技术领域。
背景技术
大菱鲆（Scophthalmus maximus）又名多宝鱼，原产于欧洲，于1992年引进我国。因其具有生长速度快、耐低温、饲料转化率高、肉质鲜美等优点，成为我国北方沿海地区重要的海水养殖种类。随着工厂化养殖的发展，因管理不当、种质退化以及水质恶化等问题，导致病害频发，病害问题已成为制约大菱鲆养殖业健康发展的瓶颈之一。
恶臭假单胞菌（Pseudomonas putida）属假单胞菌科（Pseudomonadaceae）、假单胞菌属（Pseudomonas），系广泛分布于自然环境中的一种革兰氏阴性杆菌，典型条件致病菌，在环境不良时具有较强的感染性。动物染病后，会出现摄食减少、游动缓慢、体表溃烂、肝脏淤血肿胀和生长缓慢等症状，严重时会直接导致动物体死亡。目前，在欧洲鳗鲡（Anguilla anguilla）、条石鲷（Japanese parrotfish）、黑鲷（Sparus macrocephlus）、大黄鱼（Larimichthys crocea）及虹鳟（Oncorhynchus mykiss）等鱼类中均有不同程度感染的报道，该菌已经成为威胁水产养殖业发展的重要病原之一。
疫苗是一种绿色、高效的免疫保护方式，通过使鱼体产生特异性免疫进而达到抵抗特定病原感染的目的。有研究表明，大菱鲆来源的鳗弧菌、爱德华氏菌、杀鲑气单胞菌、海豚链球菌等制作的灭活疫苗均对大菱鲆有一定的免疫保护作用，大菱鲆专用多联疫苗也有报道。使用疫苗可有效解决抗生素使用引发的病原菌耐药、环境污染及食品安全等问题，是水产绿色、健康、高质量发展的有效有段之一。
发明内容
本发明的目的在于提供一株可感染养殖大菱鲆发病的恶臭假单胞菌及其疫苗应用。
为了实现上述目标，本发明采用如下的技术方案：
一株大菱鲆来源的致病性恶臭假单胞菌PPD2，从腹部膨大、肛门红肿外凸、腹腔腹水、肝充血、脾肾肿胀为主要特征的患病大菱鲆的内脏中筛选获得，该恶臭假单胞菌PPD2保藏于中国典型培养物保藏中心，保藏地址为中国武汉，保藏日期为：2023年02月23日，保藏编号为CCTCC NO：M2023180，分类命名为恶臭假单胞菌PPD2 Pseudomonas putida PPD2。
前述的大菱鲆来源的致病性恶臭假单胞菌PPD2在制备疫苗中的应用，前述疫苗为灭活疫苗，应用于养殖大菱鲆的疫苗接种，用于预防恶臭假单胞菌引起的以腹部膨大、肛门红肿外凸，腹腔腹水、肝充血、脾肾肿胀为主要症状的细菌性疾病。
本发明的有益之处在于：本发明公开的致病性假单胞菌是从发病的大菱鲆中筛选获得的恶臭假单胞菌PPD2，经试验证实，该恶臭假单胞菌PPD2对大菱鲆具有一定的致病性，可引起养殖大菱鲆患病死亡，且该菌株对β-内酰胺类3种、大环内酯类1种和多肽类1种抗生素具有抗性；用该恶臭假单胞菌PPD2制备灭活疫苗，可有效防控大菱鲆恶臭假单胞菌病，减少抗生素类药物滥用、保障水产品质量安全。
附图说明
图1是恶臭假单胞菌PPD2的革兰氏染色结果图。
具体实施方式
以下结合附图和具体实施例对本发明作具体的介绍。
一、菌株分离、鉴定
1、菌株分离
患病大菱鲆样品采集自山东烟台大菱鲆养殖场，个体质量在239-533g之间。患病大菱鲆病症表现为：腹部膨大、肛门红肿外凸，严重者脱肛。剖检发现：腹腔有大量腹水、肠膨大有出血点、肝充血、脾肾肿胀，严重者糜烂呈豆渣状或有白色脓液。
取具有典型症状的大菱鲆濒死个体，进行体表、鳃等组织的寄生虫检查后，无菌解剖内脏，取肝、肾和脾，无菌水冲洗、研磨后于添加2wt%NaCl的营养琼脂平板划线，28℃倒置培养48h，挑取形态一致的优势菌落进行纯化培养，直至获得纯培养物（对应的菌株记为PPD2），用终体积分数为40%的甘油保种，-80℃冰箱保存备用。
2、菌株鉴定
应用细菌16S rDNA通用引物（27F：5＇-AGAGTTTGATCMTGGCTCAG-3＇、1492R：5＇-GGTTACCTTGTTACGACTT-3＇）对菌株PPD2进行PCR扩增，得到了一个长度为1416bp的产物，该产物的基因序列见SEQ ID No.1。经NCBI在线BLAST同源性比对，该产物的基因序列与恶臭假单胞菌Pseudomonas putida一致性最高为99.86%。应用MEGA 7.0软件构建系统发育树，结果表明：菌株PPD2单独聚为一支。由此确认菌株PPD2为恶臭假单胞菌Pseudomonas putida，记为恶臭假单胞菌PPD2 （Pseudomonas putida PPD2）。
二、菌株特性检测
1、致病力验证
将保存备用的恶臭假单胞菌PPD2活化后，用PBS缓冲液洗脱培养物，获得菌悬液，采用比浊法将菌悬液浓度调整为108 CFU/mL左右，冷藏待用。
选取与患病大菱鲆个体规格相近的健康大菱鲆作为实验用鱼，暂养一周后，随机分为2组，每组20尾。其中一组腹腔注射上述菌悬液，每尾注射0.1mL，另一组作为对照组，注射同等体积的无菌PBS缓冲液。养殖水温18±1℃，全天充气，日换水30%，每天观察记录死亡数量及发病症状，取发病个体再次分离病原菌。
14d后统计感染情况，恶臭假单胞菌PPD2表现出一定的致病力，可引起80%以上的实验大菱鲆死亡，且发病死亡个体表现出与患病大菱鲆一致的临床症状，患病个体可再次分离获得恶臭假单胞菌PPD2。证实恶臭假单胞菌PPD2对大菱鲆具有致病力，可引起养殖大菱鲆死亡。
2、生理生化特性
经革兰氏染色观察，可知恶臭假单胞菌PPD2为革兰氏阴性短杆状细菌，详见图1。生理生化反应结果（表1）显示，恶臭假单胞菌PPD2具有运动性，氧化酶反应、VP反应、MR反应阴性，不产生吲哚和H2S，能利用鸟氨酸脱羧，精氨酸双水解阳性，具β-半乳糖苷酶活性，可以水解尿素和枸橼酸盐，可以还原硝酸盐，不能利用赖氨酸脱羧，不能利用苯丙氨酸脱氨，可以利用葡萄糖、麦芽糖、蔗糖产酸，不能利用阿拉伯糖、甘露醇、鼠李糖、木糖、山梨糖、半乳糖、蜜二糖、肌醇、阿拉伯醇和苦杏仁苷。
表1 恶臭假单胞菌PPD2的生理生化反应结果
3、药敏特性
按照美国临床检验标准委员会（CLSI）的纸片扩散法琼脂扩散（K-B）法对恶臭假单胞菌PPD2进行21种抗菌药物敏感性测定。药敏结果（表2）显示，恶臭假单胞菌PPD2对喹诺酮类5种、β-内酰胺类2种、氨基糖苷类2种、四环素类2种和多肽类1种敏感，对磺胺类2种、氨基糖苷类1种和利福霉素类1种中度敏感，对β-内酰胺类3种、大环内酯类1种、多肽类1种、氯霉素类1种和呋喃类1种抗生素具有抗性。
表2 恶臭假单胞菌PPD2的抗菌药物敏感性测定结果
4、全基因组序列分析
为进一步分析恶臭假单胞菌PPD2的基因组特征，对其进行了全基因组测序。对原始的测序片段进行序列组装，共得到98个重叠群，PPD2全基因序列总长度为6.32Mb，GC含量为62.0%，共预测到5878个基因。
基于原核基因COG（Clusters of Orthologous Groups）数据库注释到恶臭假单胞菌PPD2有4323个基因，结果分属于21类。其中，参与氨基酸转运和代谢的有431个、参与转录的有430个、参与无机离子转运和代谢的有270个、参与细胞壁/细胞膜/包膜合成的有216个、参与能量产生和转换的有280个。依据细菌致病因子VFDB （Virulence Factors ofPathogenic Bacteria）数据库，在恶臭假单胞菌PPD2全基因组中共预测到446个毒力基因，落入117个毒力因子条目中，主要与铁摄取系统、毒素、鞭毛调控系统、双组分系统以及转运蛋白等基因相关。应用升级版耐药基因CARD（The Comprehensive Antibiotic ResistanceDatabase）数据库分析研究恶臭假单胞菌PPD2的耐药特征，共检测出79种耐药基因，主要包括β-内酰胺酶、药物外排泵类、DNA旋转酶以及延伸因子等基因。在碳水化合物活性酶专业数据库CAZy（Carbohydrate-Active enZYmes Database），共预测到176个碳水化合物活性酶基因，包括56个糖基转移酶、42个糖类酯解酶、36个氧化还原酶、31个糖苷水解酶、7个碳水化合物结合结构域以及4个多糖裂解酶相关基因。在病原与宿主互作数据库PathogenHost Interactions Database（PHI-base）中预测到恶臭假单胞菌PPD2存在病原宿主互作基因蛋白201个。
三、制备灭活疫苗
将纯化好保存于-80℃冰箱中备用的恶臭假单胞菌PPD2活化后接种于添加2wt%NaCl的营养琼脂平板中，28℃培养48 h；挑取指数生长期的单菌落于营养肉汤中，28℃震荡培养24h，收集培养液并将培养液在4℃条件下5000rpm离心10min，将离心后的沉积菌体用pH=7.2的PBS缓冲液冲洗3遍，然后用无菌生理盐水制成浓度为109 CFU/mL的菌悬液原液；向菌悬液原液中加入质量浓度为37%的福尔马林溶液至甲醛终浓度为0.2wt%，在37℃、140rpm条件下进行灭活，分别取灭活12h和24h的菌液检测灭活情况。将灭活的菌液在4℃条件下5000rpm离心10min，将离心后的沉积菌体用pH=7.2的PBS缓冲液冲洗3遍，然后用无菌生理盐水制成浓度为109 CFU/mL的恶臭假单胞菌PPD2灭活菌悬液；将恶臭假单胞菌PPD2灭活菌悬液、天蚕素抗菌肽（华北制药福分有限公司）、弗氏不完全佐剂（SIGMA公司）按照2:1:3的体积比混合均匀，制成灭活疫苗。
四、检测灭活疫苗的免疫效果
将上述灭活疫苗浓度调整至OD600=0.1±0.01，腹腔注射免疫大菱鲆，每尾注射0.1mL，对照组注射同等剂量的无菌PBS。每组免疫10尾，设置3个重复。养殖水温18±1℃，全天充气，日换水30%；免疫30d后用恶臭假单胞菌PPD2进行人工感染毒，每尾注射浓度为3.7×1010 CFU/mL的菌液0.1mL；每天观察并记录各实验组死亡数量，计算灭活疫苗的免疫保护率。
经统计，对照组平均死亡率83.33%，而经过灭活疫苗免疫的实验组死亡率仅为26.67%。
由此可见，将恶臭假单胞菌PPD2制备成灭活疫苗进行注射免疫后，对养殖大菱鲆有一定的免疫保护作用，可有效降低恶臭假单胞菌引起的感染死亡。
五、菌种保藏
由前面的研究可知，当恶臭假单胞菌PPD2制成灭活疫苗后，可有效防控大菱鲆假单胞菌病，减少抗生素类药物滥用、保障水产品质量安全，具有非常积极的应用价值。
该菌株于2023年02月23日保藏于中国典型培养物保藏中心，保藏地址为中国武汉，保藏编号为CCTCC NO：M2023180，分类命名为恶臭假单胞菌PPD2 Pseudomonas putidaPPD2。
需要说明的是，上述实施例不以任何形式限制本发明，凡采用等同替换或等效变换的方式所获得的技术方案，均落在本发明的保护范围内。
Claims (4)
1.一株大菱鲆来源的致病性恶臭假单胞菌PPD2，其特征在于，所述恶臭假单胞菌PPD2从腹部膨大、肛门红肿外凸、腹腔腹水、肝充血、脾肾肿胀为主要特征的患病大菱鲆的内脏中筛选获得，该恶臭假单胞菌PPD2保藏于中国典型培养物保藏中心，保藏地址为中国武汉，保藏日期为2023年02月23日，保藏编号为CCTCC NO：M2023180，分类命名为恶臭假单胞菌PPD2 Pseudomonas putida PPD2。
2.权利要求1所述的大菱鲆来源的致病性恶臭假单胞菌PPD2在制备疫苗中的应用，其特征在于，所述疫苗应用于养殖大菱鲆的疫苗接种。
3.根据权利要求2所述的应用，其特征在于，所述疫苗为灭活疫苗。
4.根据权利要求2所述的应用，其特征在于，所述疫苗用于预防恶臭假单胞菌引起的以腹部膨大、肛门红肿外凸，腹腔腹水、肝充血、脾肾肿胀为主要症状的细菌性疾病。
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| CN101591638A (zh) * | 2009-06-23 | 2009-12-02 | 中国水产科学研究院黄海水产研究所 | 大菱鲆肾细胞系的构建方法 |
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| US6235882B1 (en) * | 1997-10-31 | 2001-05-22 | E. I. Du Pont De Nemours And Company | Gene encoding a putative efflux protein for solvents/antibiotics in pseudomonas mendocina |
| WO2001000835A1 (en) * | 1999-06-25 | 2001-01-04 | Smithkline Beecham Biologicals S.A. | Moraxella catarrhalis polypeptides |
| CN101016524A (zh) * | 2006-10-13 | 2007-08-15 | 北京工商大学 | 具有好氧反硝化性能的恶臭假单胞菌及其处理废水的方法 |
| CN101591638A (zh) * | 2009-06-23 | 2009-12-02 | 中国水产科学研究院黄海水产研究所 | 大菱鲆肾细胞系的构建方法 |
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