| Aeromonas, an opportunistic pathogen, is widely distributed in nature. Pathogenic and non-pathogenic strains have been found. Pathogenic strains can cause sepsis in a variety of aquatic animals and diarrhea in livestock and human. Especially this pathogen has brought a serious harm in the aquaculture industry.To investigate the prevalence of Aeromonas,530samples were collected from freshwater fish farms in Jiangsu Province, and202Aeromonas strains were identified from diseased fish (n=42), healthy fish (n=120) and water (n=40), respectively.202bacterial strains were identified to species, based on the sequencing of housekeeping gene gyrB, including A. veronii (69%), A. hydrophila (10%), A. sobria (11%), A. media (4%), A. caviae (2%), A. jandaei (1%), A. salmonicida (1%), A. allosaccharophila (1%), A. bivalvium (0.5%) and A. bestiarum (0.5%). A. veronii was the most common specie. The isolation rate of A. hydrophila from diseased fish (14/20) was obviously higher than those from healthy fish (4/20) and water (2/20). Five pairs of specific primers for aer, alt, act, eprCAI, and ahp of Aeromonas, were used in PCR detection of202isolates. The result showed alt (171/202;85%) and act (175/202;87%) had highest detection rate, while the other3genes were aer (95/202;47%), eprCAI (89/202;44%) and ahp (102/202;51%), respectively. For strains from water, diseased fish and healthy fish, there was not significant difference in the distribution of5virulence factors. One virulence factor at least was detected from201of202strains, and only1strain from healthy fish was negative for5virulence factors. Among the202strains,9(19/202;9.4%) had all five virulence factors, and10of which were isolated from diseased fish, and9were identified as A. hydrophila. Using zebra fish as model, LD50was measured of the strains with all5virulence factors.The results showed that the LD50values of strains from diseased fish were significantly lower than strains from healthy fish and water. 54strains of A. hydrophila were typed using ERIC-PCR and outer membrane protein SDS-PAGE. The electrophoresis results were digitalized by means of Quantity one. All54strains showed good polymorphic fingerprinting map by both methods. Two to six bands could be amplified between600bp to4000bp by ERIC-PCR, typing54strains into4groups and8classes, while strains with same date and geographic location were inclined to1class. The result of SDS-PAGE of outer membrane protein (OMP) showed3to6bands between22kD and51kD, typing54strains into4groups and7classes. Both methods showed good typing ability, while the relevance was not significant. The results of two typing methods were relevant to the pathogenicity of strains in some way. The OMP bands were relatively less compared with the ERIC-PCR method, and OMP typing has good repeatability, stability and distinguish ability, but the process is complicated. Therefore, it can be used as reference in the laboratory; ERIC-PCR typing method is simple and fast, and requires no template sequence, which is suitable for fast typing of a large number of strains.Based on the detection of5extracellular virulence factors of A. hydrophila, including aer, alt, act, eprCAIand ahp,3strains ((NJ-35, XX-52, CS-43) with all5virulence factors and XY-16strain lack of ahp, were selected for biochemical analysis, hemolytic and proteolytic characterizations, OMP profiles and pathogenicity in zebrafish. The result showed that there were similar biochemical results, hemolytic/proteolytic ability and OMP profiles in four strains. But compared Chinese vaccine strain J-l, the OMP bands were more clear. The LD5Os of NJ-35, XX-52, CS-43and XY-16in zebrafish1.5×102CFU/mL,1.3×103CFU/mL,2.5×103CFU/mL and6.9×102CFU/mL, respectively, suggesting the four strains are highly virulent. The experiment provides the candidate strains for pathogenicity mechanism of A. hydrophila and vaccine development... |
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