Isolation And Detection Of Common Pathogenic Bacteria And Associated Pathogenic Research On Laboratory Zebrafish

Zebrafish （Danio rerio, Zebrafish）, also known as the blue fish, HuaTiao fish, etc, which is a common kind of tropical fish that’s native to South Asia. In recent years, with the deepening of the modem life science and technology research, zebrafish has become a new vertebrate model organism and is widely applied in the research of life science related field,such as developmental genetics, environmental toxicology, human disease models and the new drug screening.But in the quality control of the bacteria, there is still a large gap Compared with the laws and regulations standardized experimental animals such as mice.Under the condition of laboratory artificial breeding, pathogenic bacteria is an important pathogen which interfere the health status of zebrafish. In order to prevent and control bacterial pathogens interfere the zebrafish’s health effectively and to ensure its quality for scientific experiments, how to establish a fast and accurate detection method of pathogenic bacteria has become one of the important topics in experimental study and application of the zebrafish.In this study, select the laboratory （Shanghai laboratory animal research center zebrafish lab） naturally infected with typical symptoms zebrafish as the sampling object, make the laboratory health zebrafish and select from Shanghai southern health research center and Shanghai bird and flower market health adultfish as experimental control.pathogenic bacteria isolated from the sick fish’s lesion tissue and Normal control group’s same tissue and pure culture, received a total of8dominant strains, which have adopted the methods of morphological observation, biochemical identification and selection of PCR and gives the analysis of the result, and then draw a conclusions that number1-8bacteria respectively correspond to:Pseudomonas sp., Vibrio,Aeromonas sobria, Aeromonas hydrophila, Plesiomonas shigelloides,Aeromonas sobria, Pantoea and Budvicia aquatica.Further to make use of the8kinds of bacteria carry out the artificial infection pathogenicity pre-experiment on the healthy zebrafish,and found that number3,4and6strains showed high pathogenicity, which infected zebrafish appeared abdominal swelling,tail decay and other more serious pathological symptoms, and the zebrafish of high concentration test group have all died within24hours. These3kinds of bacteria all belong to the genus Aeromonas, this shows that the Aeromonas is the main pathogenic bacteria which trigger this laboratory zebrafish’s disease.In order to further research about the pathogenicity that Aeromonas infection the zebrafish,choose the number4Aeromonas hydrophila which isolated from the own laboratory’s sick fish as the main research object, take the cataloged as CGMCC1.2017Aeromonas hydrophila which bought from Chinese common microbe preservation management center （CGMCC） as the positive control strain. On account of Aeromonas hydrophila aerolysin gene AHA₀438, extracellular serine protease gene ASP, Aeromonas hydrophila conserved gene gyrB and16S rDNA, and Aeromonas specific gene designed primers and proceed PCR amplification, the position of electrophoresis lane was consistent with the expected results.Choose the conserved gene gyrB and16S rDNA and sequencing those PCR amplification products,through to the NCBI BLAST software conduct the homology analysis for the sample sequencing results,found the homology Compared with other Aeromonas hydrophila login the GenBank is above94%, use MEGA5.2software to construct a phylogenetic tree and then can further confirmed the bacteria strain4typing position in Aeromonas from the molecular level.In addition, make use of number4strain and the positive control bacteria strain respectively carry out the artificial infection on the healthy zebrafish, to ascertain the pathogenicity capability of two strains, and through make the tissue sections of infected zebrafish, observed and analyzed the histopathological changes of which zebrafish infected of Aeromonas hydrophila. In conclusion, it’s an important part of which the implementation of standardization bacteria quality control for laboratory animal to ensure scientific research quality.this study conducted on samples taken from three different fish breeding center in Shanghai Shanghai area, and make the natural incidence zebrafish of the Shanghai laboratory animal research center as the main research object, constructs the detection method of common Pathogenic bacteria of zebrafish, and focuses on the Pathogenic research that Aeromonas hydrophila infect the zebrafish which isolated from the own Laboratory’s sick fish, and that will provide scientific basis and reference in experimental fish bacterial dsease control.