Characterization Of Guanidine Extraction And Extracelluar Production Of Aeromonas Hydrophila

Whole cell (WC),guanidine extracted protein (extraction) and Extracellular product (ECP) from six virulent strains of Aeromonas hydrophila,of which two isolates BSK10,TPS30 were from Carassius auratus gibelio and Mygalobrama amblycephala in,Chma,two isolates (CL999916,CL99920) were from freshwater crab (Eriocheir sinensis) in China,one isolate (T4) was from fish (Labeo rohitd) in Bangladesh and another strain (NCIMB1134) was from Rainbow Trout (Oncorhynchus mykiss] in UK,were characterized by SDS-PAGE and Western blot analysis. The bacteria were cultured in TSA,TSA with 0.1mM 2,2 dipyridyl to create an iron-depleted medium,TSA with 0.1 mM FeSO4 to create an iron-supplemented medium,AVL medium and TSA with 1% fish serum.The protein profiles of WC and extraction indicated that all the isolates on different conditions had various bands but had the same molecular weight ones. The same isolate on five conditions had the similar bands pattern.Schiff's reagent,Silver stain and Lectin staining (BS-l,ConA,UEA-l) were used to examine and characterize carbohydrate presented on extraction and ECP. The lectin staining and silver stain (proteinase k treated extraction) showed all the isolates produce different molecular weight carbohydrate-related bands,the 32 kDa band being recognized by three lectins and silver stain,34 kDa band by lectin BS-1 and UEA-1. The gels were also stained by Schiff's reagent,but only few bands appeared.1The results indicated that all the isolates produced ECP which had different molecular weights proteins but shared the same band at 35 kDa. The amount of protein produced in the ECP of the different A.hydrophila isolates on different conditions was various,ranging from approximately 1 to 6 mg protein per ml. Proteolytic activity was observed in ECP of A.hydrophila isolates using four substrates (gelatin,casein,commercial haemoglobin and fresh fish haemoglobin) incorporated into SDS-PAGE. All the strains showedthe dominant clear zone areas lysed by proteases on gelatin and casein substrate gel,but showed a fewer clear zones on haemoglobin substrate gel with different molecular weight. Silver stains were used to examine and characterize carbohydrate present on proteinase k treated ECP. The results showed all the strains produced different molecular weight carbohydrate-related bands,but had the same band around 35 kDa. The gels were also stained by Schiff's reagent,but no band appeared.Tilapia anti-A. hydrophila sera against live and killed T4 were prepared to determine the antigenicity of ECP and guanidine extraction by Western blot analysis. The results indicated that various bands of extraction of A. hydrophila could be recognized by both fish sera. However,the reaction with serum against the live bacteria was stronger than that against the killed. It appeared that all the isolates expressed a 35' kDa band in their extraction on five mediums,which was reacted with fish antibody. The results also indicated that the 3 5 kDa band ware also in their ECP from all isolates in five culture conditions,which was strong reaction with fish antibody. Thus showed that the 35kDa band may be a common antigen in all these 6 bacterial pathogens. However the 35 kDa band disappeared except in the T4 isolate,when the ECP was digested by proteinase k. This suggests that the 35 kDa band may be a protein. The antibody titer and cross-reaction of fish serum was examined by ELISA. The titers of fish antibody were 1/512,1/8192 at 2,4 weeks respectively against live T4,and 1/1024 against killed T4. Cross-reactions of fish serum against killed or live T4 with other five isolates cultured on TSA were also very strong,the titer ranged from 1/512 to 1/1024.