| Turbot (Scophthalmus maximus) was firstly introduced into China by Yellow Sea Fishery Research Institute in 1992. The fish was extensively cultured along the coast in north China . However, due to the high stocking density and poor management, diseases occurred frequently. Epidemiologic investigation of 2004 showed that septicemia and ulcer disease commonly occurred, they ofen caused high mortalities. The gross signs of septicemia disease fish displayed dark body and anorexia at the beginning, Several days later, the body turned to hemorrhagic, especially the fins. Dissection of the moribund fish showed that the gut was bloodshot and inflame, the stomach were empty, the liver , kidney and spleen became swell .While ulcer disease turbots displayed sluggish swimming and anorexia At the beginning, Several days later, the turbot eyes swelled, the fins and tail turned red, and the back gradually ulcerated. Dissection of the moribund fish showed that the ulcerative gills paled, the liver becamed bloodshot, the kidney and gallbladder swelled, the intestinal wall became filmy and bloodshot. The time course from appearance of disease signs to death lasted about a week.One pathogenic strain MN was isolated from turbot {Scophthalmus maximus) in an outbreak of the septicemia at fish farm. It was Gram-negative, curved or right rods shape, motile with a polar flagella and translucent cloney in TSB culture medium. It was shown from the conventional physiological and biochemical test contrast result that MN's characteristics were very similar to those of Listonella anguillarum. It's 16S rDNA sequence was analyzed and phylogenetic dendrogram was constructed, the results showed that this bacteria MN has a close phylogenetic relation to. L. anguillarum. Based on the results obtained, MN was identified as L. anguillarum. The artificial infection experiment by intramuscular injection revealed that MN was virulent to flounder (Paralichthys olivaceus), with a LD50 value of 1.49×10~5 colony forming units/fish. The results of the sensitivity test showed that Ceftriaxone Sodium , Cefotaxime Sodium, Lomefioxacin et al. were effective to inhibit the growth of the pathogenic bacterium.A dominant strain of bacteria, which was Gram-negative and short rod with single polar flagellum under electron microscope, was isolated from the ulcer turbot and designated asHI. In artificial infection test, all fish of the experimental groups died in a week after intramuscularly injected with bacterial suspension, while the control group showed no signs in 10 d post-challenge. The moribund experimental fish had similar gross signs as the natural infected fish, such as pale liver, ulcerative gills, and sma!' ulcerations on the dorsal fins and caudal fin. The bacteria re-isolated from the challenged fish also had the same characteristics as HI, which proved that the isolate HI was the pathogenic bacteria that triggered this ulcer disease. Different methods were used to identify the pathogenic bacteria. The identification result by API 20NE and API 20E system indicated that HI was Aeromonas hydrophila, with 99% reliability and 61.5% respectively. While traditional biochemical identification revealed that HI exhibited relatedness to Vibrio Harveyi. In order to confirm the different result, a 1424 bp sequence of Hi's 16S rDNA was amplified and compared with other Vibrio spp. in GenBank, homology analysis and phylogenetic study showed that HI has the highest similarity to V. harveyi, with 99% identity. According to morphological features, physiological and biological characteristics and 16S rDNA homology comparison of the bacteria, the pathogenic bacteria were V. harveyi. Drugs sensitivity test showed that the pathogenic bacteria were highly sensitive to nitrofurantoinum, chloramphenicol and ceftriaxone sodium etc. This is the first report that V. harveyi was found as the pathogenic bacteria of cultured turbot in China. The research suggests that V. harveyi should be regarded as an important pathogen of turbot and can causes ulcer disease under conditions of high temperatures. Therefore, it is necessary to prevent against ulcer disease in culture turbot in summer.Antibody titers in serum of immunized animals is a primary factor of evaluating specific immunity effects. Usually high or low level of antibody titers reflects the efficacy of specific immunization .In the study of efficacy of vaccines on Flounder, Paralichthys olivaceus, vaccine preparations were made of 0.5% rmalin-killed Listonella anguillarum.ish were vaccinated by intraperitoneal cavity injection. After fifty days since the primary vaccination, the vaccinated fish received a second vaccination by the same route. Multiple proportions serum of flounder which had been immunized for 70d was coated on 96 well plate and bound with nonradioactive DIG(digoxygenin) labeled formalin-killed vaccines of L. anguillarum in proper buffer, according to Anti-DIG-Fab-POD system, detected the specific immune response of Flounder, the antibody titers of experimental group and control group were 1:8192 and 1:32 respectively, but the antibody titers of experimental group and control group determined by agglutination were 1:128 and 1:8 respectively. When antibody data were compared, highly significant correlations were found between agglutination and DIG-labelling-antigen indirect ELISA titers. Here the results suggested that this method canbe employed as a new, sensitive and convenient way to evaluate antibody titers of serum in immunized fishes.At present time, bacteria disease is a serious problem for the fish farming industry which caused severe loss in cultured marine fish farms in the North China. Furthermore, disease caused by L.anguillarum is common and L.anguillarum is one of the most important bacterial pathogens. To control bacteria disease, vaccination is regarded as an ideal strategy. In the present study, immunological efficacy induced by vaccine preparations of formalin-killed L.anguillarum on Flounder (Paralichthys oliyaceus) was measured. Following vaccination, the efficacy of vaccine preparations on fish was studied by using nonspecific immune assays and monitoring specific antibody production (ELISA) and artificial challenge tests. The effect of vaccinations on growth was also determined. The results indicated that the vaccinated fish show higher nonspecific immune activity than the unvaccinated fish. Furthermore, the effect of vaccinations on phagocytic activity of hemocyte, bactericidal activity and lysozyme was notable, especially bactericidal activity was the most distinct. Thus, phagocytic activity of hemocyte, bactericidal activity and lysozyme could be regarded as the major indexs to value the efficacy of vaccine on fish. The ELISA assays showed the antibody levels significantly increased in the vaccinated groups and the maximal titer came to l:4096~l:8192. The challenges were performed artificially by intraperitoneal injection with L. anguillarum and the relative percentage survival (RPS) values was 76.9%. By challenging with V. parahaemolyticus, the relative percentage survival values were 40.1%. The vaccine preparations showed cross-protection against infection by V. parahaemolyticus.
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