The Study Of Purification And Pathogenicity Of Stenotrophomonas Maltophilia Extracellular Protease From Fish

Recently, an acute infective disease of channel catfish came out frequently. A stain of stenotrophomonas maltophilia was indentified as the pathogenic bacteria. But relvant research about the pathogenic factors were seldom reported and remained to be furthered. In order to make out the pathogenic factors and suplly the theroetic foundation for theropy, an extracellular protease was partially purified from the extracellular production of the stenotrophomonas maltophilia and the pathogenicity was studied.Base on the TSB substrate, the effects of culture conditions on the production of extracellular proteases by stenotrophomonas maltophila was conducted. It was found that the production of ECPase was effected by carbon sources, nitrogen sources, temperature, time and the initial pH. The optimal medium was as follows: Socrose as carbon source, casein tryptone as nitrogen source, and pH 7.0. In this case, when stenotrophomonas maltophila was cultured at 20℃for 72h on a shaker with a speed of 120r/min, the ECPase production could reach the maximum.Under the optimal condition of culture, an extracellular protease producted by stenotrophomonas maltophila was purified by ammonium sulfale, DEAE Sephadex A-50 fast flow. The ECPase was a single poly peptide with a molecular weight of 45.7kDa by SDS-PAGE as detemined.Take the purified protease to Vero cell culturing in 96 pore plate after twofold diluted. The Vero cell had obvious degenemouseion, necrosis, felling off and having void spot vary in size. Then injected the ECPase to mouses and channel catfish intraperitoneally, it caused a large mortality. The LD₅₀ of extracellular protease to rats and channel catfish were 4.33ug/g and 3.49ug/g respectively. The injected mousse came out energy languor and had a mucous stool on its anus. After dissection, we saw it had serious stomach and intestines inflammation and flatulence. The spleen was tumescence and hemorrhage. Under optical microscope, it showed that intestinal mucosa cell was necrosis and fell off. Cardiac muscle cell was ganular degenemouseion and the REC fulled of the spleen. The died fish's gill was tumid. There wasn't serious inflammation in its stomach and intestines. The liver, splee and kidney were tumescence. Fish had degenemouseion and necrosis in hepar and kidney. They were the target organs. Under the electrical microscope, cytopathological changed obviously. The cells of most tissues and organs were badly destroyed. The nucleus membrane expanded, chromatin dissolved and mitochomdria tumefied. Both the general pathological changes and histological pathological changes were similar to natual symptoms, though there was no intussusception. It demonstrated that ECPase of stenotrophomonas maltophalia was an important virulent factor.