Isolation And Identification And Antigenicity Of Mycobacterium Marinum

Mycobacterium marinum was a pathogen which could infect both aquatic animals and people. There had already many reports on human infections of M. marinum, but had rare report on bacteria isolation from the aquatic animals. In this study, we isolated strains from organizations of Sebastodes fuscescens , on the basis of comparison with isolates from lesions in patients, The strain was identified to be M.marinum by physiological and biochemical characterization and molecular and high performance liquid chromatography methods. Studying on the choice of the carbon source of strain by culturing in medium with different carbon components. A rat was immunized, and immunofluorescence antibody technology(IFAT) was used to detecte the speciality of the antiserum. Extracting cell wall proteins from strains cultured in 28°C, 34°C and 37°C respectively which were used to analysis protein composition and differences of the antigenic structure by SDS-PAGE, Western-blot and two-dimensional electrophoresis methods. Phenol - chloroform -methanol method, ethanol - phenol water method and the traditional hot phenol water method were used to extract LPS of the strain, the difference of lipopolysaccharide antigen were analyzed by periodate oxidation and serological comparison. This experiment was the theoretical basis for researching pathogenicity of M.marinum and its subunit vaccine. Specific methods and results are as follows:In this study, we isolated the same strain FZ09 from the mucus, dorsal fins, gills and other parts of Sebastodes fuscescens, all concentrations of the Sebastodes fuscescens had no incidence of the phenomenons by artificial infection of M.marinum strain Rl and strain FZ09, the Sebastodes fuscescens remained healthy after a month and the mortality rate was 0. The same strains can be extracted from mucus, dorsal fin and other parts of infected Sebastodes fuscescens. The strain FZ09 was identified as same physiological and biochemical characteristics with the strain R1 by physiological and biochemical characterization and molecular and high performance liquid chromatography analysis. It was acid-fast positive, small lily-like, no flagella, branching trend, diameter 1-10μm; producing yellow pigment when exposed in light, and the phylogenetic tree showed that FZ09 clustered with M. marinum (AJ307636) and M.marinum (MMU55831) into a branch with high level of confidence, which was 85%. The above characteristics and mycolic acid patterns indicated that FZ09 was M. marinum, it was as same as strain R1 which was isolated from granulomatous lesions of patients. This also indicated that the hard spine aquatic animals carrying with M. marinum though could not be dead, they could indirectly infect the soft spines of fish and humans, and M. marinum was originated from the aquatic animals.Culturing the M.marinum FZ09 in the medium with carbon components such as Na2CO3, Tween80, Tween20 and glycerol respectively for four weeks, the colony growth result was:compared with Na2CO3, Tween80, Tween20, glycerol remained the best carbon source ingredients which could promote M.marinum growth vigorously. And Tween80 has certain advantages in fast identification of mycobacterium species, and there existed distinctions between M.marinum and M.tuberculosis at organic carbon selection.A rat was immunized with M. marinum FZ09 whole cells, it was found that strain FZ09 could effectively stimulate the rat to produce immunity response. The result of immunofluorescence antibody technology(IFAT) showed that the antiserum could specially combine to FZ09, and enzyme-linked immunosorbent assay(ELISA) found that the titer of the antiserum was 6400. IFAT was used to detect the speciality of the antiserum. It was found that the antiserum could combine to the strain FZ09 specifically, and had cross reactions with M.tuberculosis and M.kansas, but had no cross recations with bacteria not belonged to genus Mycobacterium.Extracting cell wall proteins from strains culturing in 28℃,34℃and 37℃respectively which were used to analysis protein composition and differences of the antigenic structures by SDS-PAGE, Western-blot and two-dimensional electrophoresis methods, it showed that:at 28℃,34℃culture conditions, the M.marinum FZ09 had same wall protein bands, including 97 kDa,84kDa,56kDa,50kDa,45 kDa,35 kDa,28 kDa,23 kDa,19 kDa,11 kDa proteins. But only 45 kDa protein had good antigenicity, isoelectric point distributed during the pH4.5-5.5. At 37℃the protein bands decreased obviously, the major article was 19 kDa and 11 kDa, and had no antigen protein, isoelectric point decreased partly. It indicated that in 37℃, the strain may change its cell structure and lost the antigenicity. And 45 kDa protein may be associated with antigen specificity and pathogenicity of FZ09, and it was possibly a vaccine candidate against defferent strains of Mycobacterium.Phenol-chloroform-methanol method, ethanol-phenol water method and the traditional hot phenol water method were used to extract LPS of the strain. The lipopolysaccharide antigen difference was analyzed by SDS-PAGE and Western blot periodate oxidation. and the sensitivity of serum antibody was detected by ELISA. The results showed that LPS had the highest purity and antigenicity by the method of ethanol-extracted. LPS was basically the same by three extractions, the main bands with molecular weights of 14,16 and 23 kd. Mouse serum mainly combinated with core polysaccharide part of the LPS, and formated massive structure. After treated with mild periodate, the core polysaccharide was no longer recognized, which led to a sharp decline in ELISA absorbance values. In addition, serological testing found that the Kunming mice could be infected with liver granulomas by abdominal injection, LPS was significantly higher sensitive than the bacteria, and the infected group showed significantly positive results; secondly, the value of infected group from the Kunming mice was significantly higher than rats in each group.