Isolation And Identification Of Pathogen From Black Carp Enteric Hemorrhage And The Preliminary Study Of Vaccines

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.