Isolation, Identification And Pathogenic Mechanism Of Two Bacterial Pathogens Of Fish And Taxonomic Analysis Of Three Novel Marine Bacterial Species

In recent years, pressures on continental resources have grown with rapid increasement of population and subsequent consumption. Resources exploitation switched from continent to ocean. The marine environment is the largest habitat on the earth. Within this environment, research of marine bacteria is a growing field of interest. Marine bacteria are an important and relatively unexploied resource for novel microbial products. Some species of marine bacteria are great of benefit to human, e.g. biopharmaceuty, bioremediation, biogeochemical cycles and prevension of diseases in aquaculture (probiotics). However, some spieses of marine bacteria are harmful to human, e.g. fish and human pathogens, marine biofouling. Thus, marine bacteria resources should be exploited appropriately--taking its advantages and controlling its disadvantages. This research studied marine bacteria bilaterally, e.g. bacterial pathogens of fish and exploitation of new marine bacterial species.Development of intensive marine aquaculture, poor management practices and abuse of antibiotics, disturb the stable microbial banlance and cause diseases, especially bacterial dieases in aquculture, sometimes lead to emergence of new pathogens. In present study, two strains WY06 and WY28 were isolated form diseased tongle sole (Cynoglossus semilaevis Gunther) and turbot(Scophthalmus maximus), respectively. Pathogenicity assays revealed that WY06 was virulent to tongue sole and zebrafish (Danio rerio) by intraperitoneal injection challenge, with the LD50 being calculated as 5.5×103 cfu/g of fish (5.2×105 cfu/fish) and 1.9×103 cfu/g of fish (8.9×102 cfu/fish) respectively. WY28 was virulent to turbot and zebrafish by intraperitoneal injection challenge, with the LD50 being calculated as 39 cfu/g (3.3×102cfu/fish) and 1.9×103 cfu/g of fish 2.1×104 cfu/g (6.4×103cfu/fish) respectively. WY06 and WY28 showed 99.0% and 99.9% 16S rDNA sequences similarity to Photobacterium damselae subsp. piscicida and Edwardsiella tarda respectively. On the basis of the polyphasic taxonomic evidence presented in this study, it can be concluded that WY06 and WY28 belong to P. damselae subsp. piscicida and E. tarda respectively. P. damselae subsp. piscicida was described as fish pathogen for the first time in China.E. tarda, which had a wide range of host, caused vital economic losses in aqucuture of Ameriacan, European and Asian countries including China. In past few years, E. tarda affected the turbot cultre in China. However, the pathogenic mechanism of the organism still remained to be studied. In order to find pathogenicity related factors, caseinase, gelatinase, elastase, phospholipase, lipase, haemolytic, hydrophobic and serum resistance activities, haemagglutination, autoagglutination, siderophore and profiles of extracellular protein and outer membrane protein of virulent and avirulent strains of E. tarda were examined and compared. There are three low molecular-weight bands that are prensent in vilurent strains but not in avirulent strain. However, their identities were not verified in this study. The pathogenicity of live cells, lipopolysaccharides (LPS) and lipid A have been tested and compared with those of avirulent strains, using rainbow trout (Oncorhynchus mykiss Walbaum) as a model fish. The result revealed that LPS was one of the virulence factors of E. tarda and lipid A was a biologically active determinant of LPS as previously reported.In order to find a safe and heathy way to control fish diseases caused by E. tarda, turbots were vaccinated with formalin-killed vaccine of WY28 via intraperitoneal injection. Controls were injected with the same volumes of saline. The evident antibody titres (average was 1:1280) were detected in vaccinated fish 4 weeks after immunization. The antibody titres reached higher level (average was 1:3289) 6 weeks after immunization. Vaccinated turbots were intraperitoneal injected with different concentrations of WY28 to detect relative percent survival (RPS) of formalin-killed vaccine of WY28. The vaccinated fish showed higher RPS against bacteria than the unvaccinated fish. Therefore, the inactivated vaccine of WY28, to a large extent, could prevent turbot diseases that caused by E. tarda. Furthermore, garlic was used as immonostimulant to feed rainbow trout. Pathogenic assay revealed that fish feeded with garlic showed higher RPS against E. tarda than the control fish. Thus, garlic was a valuable candidate of disease control plants.Based on 16S rDNA sequences analysis, only 1% of marine bacteria were isolated and identified. This means most of the marine bacteria resources have not been explored. Another point of this research is to explore new species in marine environment. In this study, three strains HHS02T, WH169T and DFH11T were isolated from healthy Chinese prawn(Penaeus chinensis, O'sbeck) and superficial seawater of Yeallow Sea, respectively. Strain HHS02T comprised slightly curved rod-shaped, Gram-negative, non-endospore-forming, catalase-negative, oxidase-positive,O/129 sensitive, facultatively anaerobic cells that were motile by means of single polar flagella. Strain WH169T was a Gram-negative, non-spore-forming, catalase-and oxidase-positive, strictly aerobic, short rod-shaped bacterium with a single, polar flagellum. The cells aggregated together when incubated at 28℃for more than 3 days in marine 2216E broth. Buds and prosthecae are formed when it was grown at 20℃for 12 days on marine 2216E agar. DFH11T strain comprised Gram-negative motile, psychrophilic, nitrate reductase positive, strictly aerobic spirilli that did not produce catalase. HHS02T, WH169T and DFH11T showed 98.5%,95.1 and 97.2% 16S rDNA sequences similarity to Vibrio sp., Aestuariibacter sp. and Oleispira sp. respectively. Based on differential phenotypic properties, fatty acid profile,16S rRNA gene sequence and MLSA results, it is concluded that HHS02T, WH169T and DFH11T represents the novel species for which the name Vibrio atypicus sp. nov., Aestuariibacter aggregatus sp. nov. and Oleispira lenta sp. nov. were proposed respectively. The strains are deposited in two recognized culture collections in two different countries.