| Because of its fast propagation speed and high death rate, the mussel Hyriopsis cumingii plague is the most serious disease in the freshwater shellfish cultivation and pearl production. This kind of destructive disease often occurs in the pearl base areas, as well as in the new waters, and it exacerbates the water pollution, leads to sharp dropping in the high-quality freshwater shellfish resources. The 2 years old H. cumingii, that has been planted piece, is often harmed. During earlier stage, lots of mucus are secreted from the clam body. After that, H. cumingii become dull to outside stimulation. At the same time, the shells-muscle contraction ability become more weaker, and the axe foot does not stretch. The digestive gland is from dark green into brown, the wall of the the gastrointestinal tract is mildly swell, and the color of gonad is lighter like erosion. As the etiology of H. cumingii plague with bacterial and virus is not clear, great successes in pathogenesis and effective prevention of this disease are not achieved. In order to find out the pathogens of H. cumingii plague, to offer basic information for seeking effective measures of prevention and treatment. the isolation and culture of H. cumingii plague, the pathogenicity, biological characteristics, microstructure and ultrastructure, physiological and molecular identification, susceptibility testing, the cloning and expression of mainly virulence genes in the pathogen ect are studied., those results as following:1. The bacteria pathogenicity test for Hyriopsis cumingii PlagueThe strains (SJ-1?SJ-2?SJ-3) with high virulence which isolated from 15 strains of the diseased mussels were used to infect the healthy individuals. The artificially infection experiment proved that separate infection or mixed infection of the three strains could cause the death of the mussels and those symptoms are as same as naturely diseased mussels. Thereinto, acute symptoms appeared after the mix infection of the three strains and the total mortality was as high as 96%; the total mortality was 68% when infected by SJ-1 alone; while infected by SJ-2 alone, the rate was 46% and 34% by SJ-3.2. The histopathology of bacterial Hyriopsis cumingii PlagueThrough organization, dyed with Methylene blue and the microscope observation, the results as follows:Strains SJ-1 mainly infected digestive gland, gill, gonad and axe foot of sick mussels, strains SJ-2 mainly infected intestine, digestive gland, gonad and axe foot; and strains SJ-3 mainly infected kidney, axe foot, gill, and intestine. After the ultramicrostructure of diseased cells were observed and analysed. The Results showed: diseased cells were swelling,cell membrane were incomplete,their structure wree fuzzy and even broken. The nucleus of the diseased cells were changed into irregular shape and the nuclear membrane became incomplete. The electronic density in cytoplasm decreased and the number of organelles and cytoplasmic inclusions significantly reduced. Obvious pathologic changes were appeared in free surface of epithelial cells and other organelles such as mitochondria, ER, lysosome, peroxisome, golgi apparatus, centrosome and microcilli.3. The identification of pathogens and the susceptibility testing of Hyriopsis cumingii plagueThe results of gram's stain and TEM observation show that, SJ-1 strain is gram-negative coli bacteria with many flagellum ends; SJ-2 strain is gram-negative short coli bacteria with a flagellum in one of the ends; SJ-3 strain is gram-negative coli bacteria, which straight or slightly curved and obtuse in two ends, with 1-3 one-port flagellum. The result of physiological and biochemical tests show that, optimal growth salinity of SJ-1 strain is 4%; optimal pH ranges from 6.0 to 8.0, optimal growth temperature is 25-35?. Optimal growth salinity of SJ-2 strain is 3%; optimal pH is 7.0; optimal growth temperature is 28-30?, and it will not grow when the temperature is below 4?or above 45?. Optimal growth salinity of SJ-3 strain is 3%, optimal pH ranges from 6.0 to 7.0; optimal growth temperature is 25-30?, and it will stop growing when the temperature is below 4?or above 40?. Through the 16S rDNA genetic evolution and Phylogenetic Tree analysis, the following results were found, SJ-1 strain (GU294302) cluster naturally with the genus Stenotrophomonas clustering, similarity were 97%, SJ-2 strains (GU294303) cluster naturally with the genus Aeromonas, similarity 99%. SJ-3 strain (GU294304) cluster naturally with the genus Pseudomonas, similarity were 99%. Through the determination of the automatic biochemical analyzer,SJ-1 strain is similar to the rate of 95.5% with Stenotrophomonas maltophilia,SJ-2 strain is similar to the rate of 99% with Aeromonas veronii, and SJ-3 is similar to the rate of 99.9% with Pseudomonas. We synthesized the morphological characteristics of bacteria, physiological and biochemical characteristics and 16S rDNA gene evolution and phylogenetic tree analysis, the SJ-1 strain was identified as Stenotrophomonas maltophilia, SJ-2 strain was identified as Aeromonas veronii, SJ-1 strain was identified as Pseudomonas aeruginosa.The antimicrobial susceptibility tests of pathogens of H. cumingii plague were done, and some high-sensitivity drugs to pathogens were screened, they are ciprofloracin?lomefloxacin?levofloxacin?ofloxacin?norfloxaci?gatifloxacin?ceftazidime/clavulanic acid.4. The cloning and prokaryotic expression of elastase LasB gene from stenotrophomonas maltophiliaThe complete open reading frame (ORF) sequences Of elastase gene was cloned from Stenotrophomonas maltophilia. The full length of the ORF was 1497bp which encoded a protein of 498 amino acids with a predicted molecular mass of approximately 53.57 KDa. The amino acid sequence anlaysis indicated that the protein was a non-transmembrane protein with signal peptide. The homology comparison of amino acid proved that the homology between the LasB gene and other members of the family from other stains was very high. The sequences highly conserved. Phylogenetic tree analysis showed that the gene had close relationship with many elastase genes from strains. The prokaryotic expression vector with target gene was constructed, after transformation to express strains BL21, the fusion protein as the expectation was induced to expression. The study provides the foundation for research about the correlations between stenotrophomonas maltophilia and its host, the pathogenesis and the exploitation of elastase. |
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