The survey on preparation and evaluation of killed E. coli K99 antigen in laboratory experiment.

Diarrhea caused by Escherichia coli K99 in calves, especially in the first days after birth, due to casualties and economic losses resulting from is important. In this study the prevalence of E.coli K99 disease in Fars Province was investigated. And the other after separation current strains with strains submissions from Razi Institute attempted purification of the antigen. During 2009-2010 the rectal swab calves 1-30 days were collected. For separation the samples were cultured on the Mac Conkey, Eosin methylene blue and minca agar and to identify Escherichia coli K99 slide agglutination method was used. The K99 positive samples were pure using techniques sonication, Bead, and heat K99. Antigen levels obtained in each case was determined with Lowry method. Purity of protein bands seen for SDS-Page technique was used. Finally, using the column chromatography antigen obtained was pure as possible. Results were shown of 280 E. coli isolates 16 cases (5.7%) harbored K99 antigen. the same number was negative with monovalent O8 and O20 in the agglutination test.But anti O9 and O101 serum showed a positive reaction. Purification results with different methods of heating, Bead and sonication indicated temperature of 60 degrees for 20 minutes was obtained pure antigen than other methods. Therefore chromatography was used was very fruitful. Affinity chromatography columns, thus gel sepharose 4 B activated with cyanogens bromide was obtained only one band to the K99 antigen. Antigen levels obtained between 1.5 to 2.3 mg /ml respectively. Although many studies related to bacterial infection and pathogenesis of E.coli K99 has been done, but other than prevalence in a few study areas since the review by O serogroup common in Iran there has been little studied. With purified antigen also found 43 no case in Iran. Considering that the preparation of this antigen to produce the vaccine field has provided hope in the near future it may be fulfilled. Key words: Escherichia coli K99, antigen, Fars