Characteristics Of Extracellular Protease Of Stenotrophomonas Maltophilia From Ictalurus Punctatus And Diagnosing Of The Bacteria

Infectious intussusception of channel catfish(IICC) is a bacterial infectious acute and lethal disease to the breed Ictalurus Punetatus in recent. The characteristics of the desease were high morbility, high death rate and quick infecting. The characteristic pathological changes contained circular, oval or unsteady color fading spots, ascites, enteritis and intussusception of hindgut. The desease caused serious economic lost to breed channel catfish of our country. The research was conducted to study the extracellular protease(ECPase) of the pathogen Stenotrophomonas maltophilia, including the purification, characterization, chemical modification and pathogenicity. The results showed the basic imformation to the pathopoiesisng mechanism of the bacterium through detecting whether the protease was the main causative agent of Stenotrophomonas maltophilia. PCR and in situ PCR diagnostic method were established and were effective in detecting the bacteria and diagnosisng the disease.The effects of culture conditions on the production of extracellular protease by Stenotrophomonas maltophilia was studied by madding TSB as the basic medium. It was found that the production of extracellular protease was affected by carbon sources, nitrogen sources, temprature, and the initial pH of the medium. When Stenotrophomonas maltophilia was cultured at 20℃for 72 hours on a shaker with a speed of 120r/min, the optimal culture condition as follows: sucrose as carbon source, yeast extract as nitrogen source, the initial pH 7.0. The bacteria could secrete maximum dose protease when S.maltophilia cultured in the optimal condition.An extracellular protease was partially purified from the culture solution of S.maltophilia by ammonium sulfate, DEAE Sephadex A-50 fast flow. The result showed that the molecular weight of the purified protease from channel catfish by SDS-PAGE was 45.7 KDa. The purified protease caused a great deal of death of mice and channel catfish by injecting intraperitoneally. The LD₅₀ of extracellular protease to mice and channel catfish were 4.33μg/g and 3.491μg/g respectively. It showed that the optimal temperature was 20℃and the optimal pH was 9.0. PMSF did not affect the activity, but EDTA, Ca²⁺, Hg²⁺, Cu²⁺ reduced the activity of protease significantly. Co²⁺ increased the activity. It demonstrated that it was a alkaline metalloproteinase.The injected mice showed polypnea, energy languor, and a mucous stool on its anus. Serious stomach and intestines inflammation and flatulence were seen after dissection. The stomach lining and intestinal wall became very thin. It was full of yellowy phlegm. The spleen was tumescence and hemorrhage. The intestinal mucosa showed necrosis and fell off by microscope. Cardiac muscle cell was granular degenemouseion and the hepatic cell had degenemouseion and necrosis. In the same time, the purified protease was injected to channel catfish intraperitoneally. It also showed different death. Fish had a degenemouseion and necrosis in hepar and kidney, splenorrhagia and the gill dwelling. There were not serious inflammation in stomach and intestines. Adding the purified protease to Vero cell culturing in 96-pore plate after tenfold diluting, the Vero cell had an obvious degenemouseion, necrosis, felling off and having different void spot vary in size.The chemical modification of extracellular protease of S.maltophilia was studied with phenylmethanesulfonyl fluoride (PMSF), p-chloromercuribenzoate (PCMB), N-acetylimidazole (N-AI), chloramine-T (Ch-T), N-bromosuccinimide (NBS), 2-mercaptoethanol (2-ME) as the modification reagents. The results indicated that the protease was not affected by N-AI, PMSF and PCMB, but Ch-T, NBS and 2-ME reduced the activity significantly. It implied that Ser, Tyr and sulfhydryl residues were not activity dependent groups, while Met, Trp and disulfide linkage groups played an important role in the activity of extracellular protease. Meanwhile, EDTA could reduce the activity significantly, which showed that the enzyme was a meta-protease.A PCR method for diagnosing the disease of Stenotrophomonas maltophilia with a pair of primers designed and synthesized based on the 16SrDNA gene of the bacterium, and the sensitivity, specificity were carried out. The optimal PCR system in 50μl consisted 4pmol primers, 150pmol MgCl₂, 10pmol dNTP, 3.33U Taq polymerase and the annealing temperature was 50℃for 30s, the extending temperatue was 72℃for 10min. The PCR method could detect as low as 144.6ng template DNA after at least 25 cycles. The sensitity test showed that the PCR assay did not cross-react with Aeromonas hydrophila, Pseudomonas aeruginosa, Aeromonas caviae, Aeromonas sobria, and it could be applied in the diagnosing of disease conducted by Stenotrophomonas maltiphila in channel catfish.The Ictalurus punctatus was artificial infected Stenotrophomonas maltophilia by intraperitioneal injection. Then dissected five fishes in different time to get the heart, liver, spleen, kidney, stomach, bowel, brain, gill, skin and muscle for routine paraffin imbedding, and cut sheet. Subsequently, specific primers were used to detect the bacteria by using in situ PCR and in situ hybridization. The Positive signals were amethyst spots and it appeared in hepar after 4 hours after injection, others organs were not be detected in this time. After 8 hours, positive signals were detected in the kidney and liver which main distribute to hepatic and renal interstitium. Lots of positive spots were detected in many tissues and organs including liver, spleen, kidney 16 hours later. After 24 hours, major tissues of the fish were found positive spots including liver, kidney, spleen, intestines, stomach and brain. After 48 hours, positive spots could also be detected in the hearts and gills. But swim bladders, gonads, skin and eyes never found positive spots all the time.