Chemotaxis And Adhesion Of Vibrio Fluvialis To The Mucus Of Paralichthys Olivaceus And The Antibacterial Protein In The Serum Of Paralichthys Olivaceus

During the intensive culture of Paralichthys olivaceus in Fujian province, Vibrio fluvialis has been associated with epizootics in cultured P. olivaceus and caused considerable losses. The chemotaxis and adhesion to fish mucus is considered to be the virulent factors of pathogens, the understanding of the characteristics in the bacterial chemotaxis and adhesion is helpful for understanding the pathogenesis and the control of the pathogens. The antibacterial proteins in the serum play an important role in the anti-infectious immunity. The aim of this paper is to obtain more knowleges in the early stage of the infection and the anti-infectious immunity of P. olivaceus by probing into the bacterial chemotaxis and adhesion as well as the antibacterial protein in the serem.The growth of V. fluvialis in skin, gill and intestinal mucus of P. olivaceus, chemotactic response and adhesion of V. fluvialis to the 3 kinds of mucus of P. olivaceus were investigated. The results showed that: significant growth of V. fluvialis were found in all of the 3 kinds of mucus, the growth characteristics exhibited Logistice growth model at prophase and Gompertz growth model at anaphase, maximum biomass yield in gill mucus were higher than those in skin and intestinal mucus; V. fluvialis exhibited strong chemotactic response towards 3 kinds of mucus, the chemotaxis to gill mucus was significantly stronger than that to skin mucus （P<0.05）, and the chemotaxis to intestinal mucus was extremely significant lower （P<0.01） than those to gill mucus and skin mucus; the bacteria adhesion to skin mucus and gill mucus were significantly higher （P<0.05） than that to intestinal mucus after incubation for 30, 60 and 90min.The influences of bacteria concentration, incubation time, and environmental factors, such as temperature, pH, salinity and carbohydrate on the bacterial chemotaxis were investigated by the method of modified capillary assay and isotope tracer. The results showed that: the bacteria number of chemotactic V. fluvialis toward skin mucus of P. olivaceus increased with the bacteria concentration and incubation time, respectively. Saturation of bacterial chemotaxis was obtained after incubating at room temperature for 60 min; the chemotactic bacteria increased with the incubation temperature from 5℃to 15℃and peaked at 15℃, optimal chemotaxis was observed at pH 8; the bacterial chemotaxis weakened with the increase of NaCl concentration from 0.8 % to 3.6 %. Among 8 kinds carbohydrate tested, mannitol, lactose and seminose promoted the bacterial chemotaxis remarkably. The influence of bacteria concentration, incubation time, incubation temperature, pH, cation and carbohydrate on the bacterial adhesion of pathogenic V. fluvialis to the skin mucus of P. olivaceus were investigated by the method of isotope tracer. The results showed that: the adhesive quantity of V. fluvialis increases with bacteria concentrations, a saturation kinetics hyperbola: y=417.89Ln（x）+691.57 （R2=0.986）,was obtained by plotting adhered bacteria against the concentration of bacteria added; the adhered bacteria increased with the incubation time, and reached saturation after incubated for 180 min; the adhered bacteria increased with the incubation temperature from 4℃to 30℃and peaked at 30℃, the bacterial adhesion quantity at 37℃was lower than that at 30℃; more bacteria adhered was found at acidic condition and optimal adhesion was found at pH 5; the bacterial adhesion enhanced with the concentration of NaCl increased from 0.5% to 4.5%. Ca²⁺ and Mg²⁺ promoted the bacterial adhesion, Ca²⁺ more efficiently; all of the 8 kinds of carbohydrate tested promoted the bacterial adhesion remarkably.P. olivaceus were infected by injecting intramuscularly of dorsal region with V. fluvialis at a density of 107cell·mL-1. The antibacterial activity of sera from infected P. olivaceus were higher than those of control P. olivaceus during 1～72h. The serum antibacterial activity of infected P. olivaceus peaked and had a significant difference （P <0.01） to the control at 24h post-injection. P. olivaceus sera of second infected by V. fluvialis were collected 24h post-injection. The sera were separated by Sephadex G-25 gel filtration. The results of OD280 showed that there were 3 main peaks of the fractions collected. The antibacterial activity of serum to V. fluvialis was determined and the results showed that the first peak contained the main antibacterial protein. The antibacterial range of the antibacterial fractions were determined by using 6 strains as indicating bacteria, the results showed that some indicator-bacteria, e.g. Escherichia coli, Bacillus subtillis were inhibited. Besides, in the range of 4～125℃, the temperature was higher, the antibacterial activity of the antibacterial fractions was better. The antibacterial fractions were separated by ion-exchange chromatography. The results of OD280 showed that there was 2 main peaks of the fractions collected. The extractions with antibactericidal activities against V. fluvialis were at the frist apex. The result of SDS-PAGE assay showed high-molecular-weight of the antibacterial protein.These results indicated that pathogenic V. fluvialis could grow and colonize in the mucus of P. olivaceus, especially in gill mucus. Pathogenic V. fluvialis had a strong chemotaxis toward and adhere to the skin mucus of P. olivaceus, which would convenient for the subsequently infection; they were influenced remarkably by environmental factors, such as temperature, pH value, salinity and carbohydrate. Furthermore, Paralichthys olivaceus would produce multi-antibacterial-component and released them into serum soon after infected by V. fluvialis; the major antibacterial protein had high-molecular-weights and inhibited some Gram-positive and Gram-negative bacteria; they were rather stable at 4～125℃.