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Study on Isolation, Identification and Controlling Methods of Aeromonas Hydrophila

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Tutor: LuJianFeng; YeYingWang
School: Hefei University of Technology
Course: Aquatic Products Processing and Storage Engineering
Keywords: Aeromonas hydrophila,probiotics,vaccine,relative percent survival,Carassius aura
CLC: S941
Type: Master's thesis
Year:  2012
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The main objects infected by Aeromonas hydrophila was aquatic animal food and itsposition as the causative organism has been well established with increasing fishconsumption. Now it becomes a very important concern and much attention has beendrawn to the aquatic products quality and safety. In this dissertation, isolation,identification and controlling methods of A. hydrophila were studied, and the main resultsare as follows:1) Isolation and identification of A. hydropila from freshwatr fishes. Twenty A.hydrophyla strains were isolated from these freshwatr fishes by selective media andmolecular detection. Artificial infection proved the hemorrhage disease of the fish wasinduced by this bacterium. Then, biochemical characteristics and its susceptibility of thestrains, were further studied.2) Optimization of cultural condition of A. hydrophila L1inactive vaccine. Selecting A.hydrophila L1as a starting strain for following experiment. The research progresses ofvirulence factors and control technologies in A. hydrophila were reviewed. The OD valueand growth curve of Aeromonas hydrophila L1were studied in this paper. Orthogonaldesign was employed to test the best broth components for the strain growing, and theresults showed that the best condition was peptone:10g/L, yeast extract:15g/L, sucrose:7g/L, K2HPO4:4.56g/L. The antigenicity was strongest when the conditions of makingvaccines was0.2%formaldehyde at28℃for36h, and it isqualified by sterility test andsafety inspection.The injected relative percent survival of vaccines reached90%by animalprotection test.3) Studies on influences of hematological and biochemical parameters in bacterialsepticemia Carassius auratus gibelio by formalin-killed A. hydrophila. To study theinfluences of blood and biochemical parameters on crucian carps (Carassius auratusgibelio) by A. hydrophila, the crucian carp were randomly divided into control group,infection group and immune group. Blood samples were taken from the carp’s caudal sinus.Anti-coagulate blood samples and serum samples were used to determine the8bloodsamples and13biochemical parameters respectively after treated18h and36h. To18h, compared with the control group, the ratio of aspartate aminotransferase (AST), alanineaminotransferase (ALT) and Cl-were significantly higher in the injection group, theimmune groups had no significant difference with the control group. To36h,9significantdifferent parameters (MCH, MCHC, WBC, RBC, Cl, ALT, AST, Glucose) and8differentparameters (MCV, HGB, HCT, total protein, total bilirubin, albumin, creatinine, uric acid)were observed significantly different, there was no significant difference between thecontrol group and the immune group except WBC significantly higher in the immunegroups. The results showed that formalin-killed A. hydrophila could produce a strongimmune protection, reduce the bacteria on the fish of the damage, and enhance the fish’simmune resistance.4) Selection and characteristic analysis of potential probiotic strains against A.hydrophila from the intestine of silver crucian carp, Carassius auratus gibelio andinfluences of hematological and biochemical parameters.60strains of bacteria withdifferent characters were isolated from the intestine of silver crucian carp. Using A.hydrophila as the index bacteria,12strains of bacteria were identified by dot-inoculatingmethod, and4strains of potential probiotic were selected with vary bacteriostatic abilityagainst A. hydrophila (inhibition circle to pathogens>14mm) by Oxford cup test. Accordingto their tolerances of high temperature, low pH, artificial intestinal juice, the strain YJ-9was confirmed with the best probiotic functional characteristics. Under the condition ofwater-bath of90℃for30min and pH3in Nutrient Broth medium for4h, its relativesurvival rate were1.51%and8.37%, and the number of viable cells above106CFU/mL. Itcould grow slowly in the artificial intestinal juice. In addition, it was safe for the silvercrucian carp when they were challenged by the10times experimental dose. The relativepercent survival of YJ-9reached80%by animal protection test. The results indicated thatYJ-9could be used as a new candidate probiotic strain for microbial ecological feedadditives. The results, obtained from blood parameters, showed that probiotics couldprotect fish’s liver and kidney when infecting A. hydrophila, which helps to reduce theextent of damage of the pathogens of fish, and enhance the fish’s immune resistanceforce,but its prevention is smaller than a vaccine.
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