青鱼肠道出血病病原菌的分离鉴定与疫苗的初步研究
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摘要
嗜水气单胞菌感染引起的水生动物细菌性出血病,是危害水生动物种类最多、影响区域最广、流行季节最长、发病率和死亡率高、造成经济损失最严重的一种传染性疾病。
2009年7月,湖北省荆州市某青鱼养殖示范区域暴发严重的肠道出血病。笔者从患典型肠道出血病的青鱼肝脏中分离到优势菌1株BCB01,人工腹腔注射感染试验可复制出与自然发病相似的症状。单克隆菌落的Biolog系统鉴定结果表明,菌株BCB01为嗜水气单胞菌。细菌16S rRNA基因测序与系统进化树分析结果表明,菌株BCB01与嗜水气单胞菌(EF645799)的亲缘关系最近,其相似性为99%。药物敏感性试验结果显示,该菌株对大多数试验药物敏感,其中对复达欣、头孢西丁、先锋必等11种药物的高度敏感。
将福尔马林灭活的嗜水气单胞菌(F-AH)、菌体外膜蛋白(OMP)和菌体脂多糖(LPS)作为免疫原,分别免疫健康青鱼。在免疫1、2、4、7、14、21、28d后分别从每组中随机各取5尾试验鱼,尾静脉采血,进行外周血的血细胞计数和白细胞分类计数,测定细胞吞噬活性和抗体效价。结果表明：3种免疫原均可诱导红细胞和白细胞数量增加,并引起各种白细胞分类百分比变化,提高吞噬活性和抗体效价；免疫应答早期(第1周)主要是红细胞、单核细胞和中性粒细胞数量明显增加,吞噬细胞的吞噬活性迅速提高,吞噬百分比(PP)和吞噬指数(PI)第4天达峰值：F-AH组、OMP组和LPS组的PP、PI值依次分别为39.84%、46.53%、41.59%；3.94、4.26、3.77；随后则是淋巴细胞大量增殖,第21天淋巴细胞和抗体效价达峰值：F-AH组、OMP组和LPS组的抗体效价分别为1：426.67、1：341.33和1：213.33。免疫28d后活菌攻毒的结果表明,OMP组的免疫保护率为75%；LPS组为67.8%,均明显优于F-AH组(50%)。可见3种免疫原均能通过促进青鱼血细胞增殖、提高吞噬细胞的吞噬活性、产生特异性抗体等方式增强机体的免疫保护力。
Aquatic bacterial hemorrhage caused by Aeromonas hydrophila induced high incidence and mortality of disease in aquatic animals each year. It endangered the most aquatic species and different stage of fish and farming areas. As a severe infectious disease, aquatic bacterial hemorrhage lead to huge economic loss.
In July 2009, a serious enteric hemorrhage disease outbroke in Jingzhou, Hubei province in a Black carp farming demonstration area. The dominant bacterial strain, designated as BCBOl, was isolated from the kidney of Black carp, Mylopharyngodon piceus suffering with a severe enteric hemorrhage. Artificial intraperitoneal injection with the bacterial suspensions to healthy black carp could reproduce the typical diseased symptoms as occurred in nature. Identification of the pathogen by Biolog automatic identification system showed that the isolated causative pathogen BCBOl was Aeromonas hydrophila. According to its 16SrRNA gene sequence and molecular phylogenetic dendrogram, the strain BCBOl was most close to Aeromonas hydrophila with a similarity of 99%. The susceptibility test to antibiotics demonstrated that the isolated strain BCBOl was susceptible to most of the selected antibiotics, specifically it was highly susceptible to 11 kinds of antibiotics, such as fortum, cefoxitin, cefobid,etc.
Black carp were immunized with three vaccines (formalin killed Aeromonas hydrophila:F-AH, outer membrane protein: OMP and lipopolysaccharide:LPS, respectively. Then, the blood samples were collected from immunized fish through caudal vein in all groups on the Day 1,2,4,7,14,21 and 28 post immunization for the study of the changes of immune parameters, such as haematocyte number, differential leucocyte count(DLC), serum antibody titer and phagocytic activity of phagocytes. The challenge tests with live Aeromonas hydrophila were conducted for evaluation of the relative percent survival. The results showed:the number of erythrocytes and leucocytes in peripheral blood of immunized fish by three vaccines all increased during the 4 days post immunization and peaked on 4th day, then gradually decreased to the level of control group. The differential leucocyte count (DLC) of the immunized fish with three vaccines all increased and were significantly higher than that of control group for monocytes and neutrophils from day 4 to day 7, for lymphocytes from the 14th day to 28th day and peaked on 21th day. The phagocytic percentage(PP) and phagocitic index(PI) of phagocytes in immunized fish with three vaccines were significantly higher than those of the non-immunized fish between day 2 and day 7, and reached the maximum value on day 4. The PP value of F-AH group, OMP group and LPS group were 39.84%,46.53%, 41.59% and PI value were 3.94,4.26,3.77, respectively. The serum antibody titers gradually increased in immunized fish with three vaccines during the 21 days post immunization and reached the highest value on day 21, then gradually dropped off. The serum antibody titers of F-AH group, OMP group and LPS group were 1:426.67, 1:341.33 and 1:213.33, respectively. The result of challenge test with live Aeromonas hydrophila showed that black carp immunized with three vaccines had good relative percent survival(RPS). OMP had the highest RPS, which was 75%, followed by LPS(67.8%) and F-AH(50%). All the results stated above suggested that three vaccines could enhance black carp immunity against the bacterial pathogen infection.
2009年7月,湖北省荆州市某青鱼养殖示范区域暴发严重的肠道出血病。笔者从患典型肠道出血病的青鱼肝脏中分离到优势菌1株BCB01,人工腹腔注射感染试验可复制出与自然发病相似的症状。单克隆菌落的Biolog系统鉴定结果表明,菌株BCB01为嗜水气单胞菌。细菌16S rRNA基因测序与系统进化树分析结果表明,菌株BCB01与嗜水气单胞菌(EF645799)的亲缘关系最近,其相似性为99%。药物敏感性试验结果显示,该菌株对大多数试验药物敏感,其中对复达欣、头孢西丁、先锋必等11种药物的高度敏感。
将福尔马林灭活的嗜水气单胞菌(F-AH)、菌体外膜蛋白(OMP)和菌体脂多糖(LPS)作为免疫原,分别免疫健康青鱼。在免疫1、2、4、7、14、21、28d后分别从每组中随机各取5尾试验鱼,尾静脉采血,进行外周血的血细胞计数和白细胞分类计数,测定细胞吞噬活性和抗体效价。结果表明：3种免疫原均可诱导红细胞和白细胞数量增加,并引起各种白细胞分类百分比变化,提高吞噬活性和抗体效价；免疫应答早期(第1周)主要是红细胞、单核细胞和中性粒细胞数量明显增加,吞噬细胞的吞噬活性迅速提高,吞噬百分比(PP)和吞噬指数(PI)第4天达峰值：F-AH组、OMP组和LPS组的PP、PI值依次分别为39.84%、46.53%、41.59%；3.94、4.26、3.77；随后则是淋巴细胞大量增殖,第21天淋巴细胞和抗体效价达峰值：F-AH组、OMP组和LPS组的抗体效价分别为1：426.67、1：341.33和1：213.33。免疫28d后活菌攻毒的结果表明,OMP组的免疫保护率为75%；LPS组为67.8%,均明显优于F-AH组(50%)。可见3种免疫原均能通过促进青鱼血细胞增殖、提高吞噬细胞的吞噬活性、产生特异性抗体等方式增强机体的免疫保护力。
Aquatic bacterial hemorrhage caused by Aeromonas hydrophila induced high incidence and mortality of disease in aquatic animals each year. It endangered the most aquatic species and different stage of fish and farming areas. As a severe infectious disease, aquatic bacterial hemorrhage lead to huge economic loss.
In July 2009, a serious enteric hemorrhage disease outbroke in Jingzhou, Hubei province in a Black carp farming demonstration area. The dominant bacterial strain, designated as BCBOl, was isolated from the kidney of Black carp, Mylopharyngodon piceus suffering with a severe enteric hemorrhage. Artificial intraperitoneal injection with the bacterial suspensions to healthy black carp could reproduce the typical diseased symptoms as occurred in nature. Identification of the pathogen by Biolog automatic identification system showed that the isolated causative pathogen BCBOl was Aeromonas hydrophila. According to its 16SrRNA gene sequence and molecular phylogenetic dendrogram, the strain BCBOl was most close to Aeromonas hydrophila with a similarity of 99%. The susceptibility test to antibiotics demonstrated that the isolated strain BCBOl was susceptible to most of the selected antibiotics, specifically it was highly susceptible to 11 kinds of antibiotics, such as fortum, cefoxitin, cefobid,etc.
Black carp were immunized with three vaccines (formalin killed Aeromonas hydrophila:F-AH, outer membrane protein: OMP and lipopolysaccharide:LPS, respectively. Then, the blood samples were collected from immunized fish through caudal vein in all groups on the Day 1,2,4,7,14,21 and 28 post immunization for the study of the changes of immune parameters, such as haematocyte number, differential leucocyte count(DLC), serum antibody titer and phagocytic activity of phagocytes. The challenge tests with live Aeromonas hydrophila were conducted for evaluation of the relative percent survival. The results showed:the number of erythrocytes and leucocytes in peripheral blood of immunized fish by three vaccines all increased during the 4 days post immunization and peaked on 4th day, then gradually decreased to the level of control group. The differential leucocyte count (DLC) of the immunized fish with three vaccines all increased and were significantly higher than that of control group for monocytes and neutrophils from day 4 to day 7, for lymphocytes from the 14th day to 28th day and peaked on 21th day. The phagocytic percentage(PP) and phagocitic index(PI) of phagocytes in immunized fish with three vaccines were significantly higher than those of the non-immunized fish between day 2 and day 7, and reached the maximum value on day 4. The PP value of F-AH group, OMP group and LPS group were 39.84%,46.53%, 41.59% and PI value were 3.94,4.26,3.77, respectively. The serum antibody titers gradually increased in immunized fish with three vaccines during the 21 days post immunization and reached the highest value on day 21, then gradually dropped off. The serum antibody titers of F-AH group, OMP group and LPS group were 1:426.67, 1:341.33 and 1:213.33, respectively. The result of challenge test with live Aeromonas hydrophila showed that black carp immunized with three vaccines had good relative percent survival(RPS). OMP had the highest RPS, which was 75%, followed by LPS(67.8%) and F-AH(50%). All the results stated above suggested that three vaccines could enhance black carp immunity against the bacterial pathogen infection.
引文
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