Identification and lipopolysaccharide antigenicity analysis of Mycobacterium marinum isolated from Sebastes schlegeli

Mycobacterium marinum is an opportunistic pathogen which can infect 150 kinds of aquatic animals.In this paper,the same bacteria strain FZ09 was isolated from the mucus,dorsal fins and gills of Sebastods schlegeli,and was identified as Mycobacterium marinum by physiological,biochemical characterization and heat shock protein 65 DNA sequence analysis.Lipopolysaccharide(LPS)was extracted from strain FZ09 by phenol-chloroform-methanol method,ethanol-phenol-water method and hot phenol-water method,respectively,and the composition and antigenic differences of the extracted LPS were analyzed by SDS-PAGE,Western-blotting and ELISA.The results showed that LPS extracted by three methods shared the same compositions and the main bands had molecular weights of 14,16 and 23 ku.LPS extracted by ethanol-phenol method had the highest purity and concentration,and LPS extracted by traditional hot phenol water method had the most bands and best antigenicity.Western-blotting showed that anti-FZ09 antiserum mainly reacted with 14 ku band of the LPS,which showed a negative reaction after mild treatment with periodate except for LPS extraction by traditional hot phenol-water method that demonstrated weak positive.ELISA revealed that reactivity of anti--FZ09 antiserum and LPS decreased after periodate oxidation and OD₄₀₅ reduced significantly.