The Ictalurus Channel Maltophilia Oligotrophic An Indirect Elisa For The Detection Of Aeromonas Disease

Recently,the Stenotrophomonas maltophilia isolated from Channel Catfish was detected as the pathogen of an acute epidemic among channel catfish(Ictalurus Punctatus) in China.Many typical characteristics of affected fish were presented,such as many irregular,variably sized areas of bianch in the body surface,distension of abdomen,enteritis and intussusceptions etc.In order to diagnose this disease rapidly and accurately in the early stage,we have developed a rapid detection indirect enzyme-linked immunosorbent assay(ELISA) with high specificity for Stenotrophomonas maltophilia.This experiment used the Stenotrophomonas maltophilia isolated from Channel Catfish as the antigen.The antiserum against Stenotrophomonas maltophilia was obtained by immunizing rabbits.The pure IgG with high activity were obtained from the rabbits anti Stenotrophomnas maltophilia IgG serum antibody,which was purified by saturated ammonium sulfate salting-out and hydroxyapatite chromatography typeⅡ,so as to develop the indirect ELISA.Through repeatedly tests,the optimum test conditions were confirmed:the coating buffer was 0.05mol/L pH9.6,the coating antigen was dried at 60℃,the coating antigen concentration was 10~6cfu/ml,the blocking buffer was 5%skimmed milk-PBST for 90min at 37℃,the concentration of antisera was 2μg/ml for 60min at 37℃,the optimum dilution of HRP-labelled goat anti-rabbit IgG was 1:10000 for 60min at 37℃,the time for substrate reaction was 20min.The cutoff optical density(OD) of blood serum,liver,kidney,spleen,brain and gill of at 450(nm)were 0.313,0.159,0.324,0.232,0.170,0.300,respectively.The results of reproducibility by the indirect ELISA showed that coefficient of variation was between 5.21-6.15%,which were less than 10%.This indirect ELISA was specific,for it did not cross react with the Aeromonas hydrophila,Edwardsiella ictaluri,Aeromonas veronii,Yersinia Rucker,Aeromonas caviae.The lowest detectable limit of Stenotrophomonas maltophilia suspension detected limited in laboratory was 10~5cfu/ml,namely was 10~4cfu per well.The method was developed and applied to detect the blood serum,liver,kidney, spleen,brain and gill of channel catfish,which was artifically infected with Stenotrophomonas maltophilia after 24,48,72 and 96h.The result showed that the positive rates in spleen was 100%,which was the highest,and the positive rates in kidney,liver,gill and blood serum were 95%,90%,80%,70%,respectively,but only 5%in brain.Therefore,spleen and liver could be chosen as the materials for detection of Stenotrophomonas maltophilia by indirect ELISA.