Rapid diagnostic e coli antisera sets

All recovered E. coli isolates were refreshed again onto nutrient agar (Oxoid, Hampshire, UK) and biochemically examined via biochemical tests such as indol, methylred, voges-proskauer, citrate utilization, and hydrogensulphide [23 ]. All E. coli isolates exhibited their characteristics from the used biochemical tests and were serologically identified according to Kok et al. [24 ] by using rapid diagnostic E. coli antisera sets (DENKA SEIKEN Co., Chuo-Ku, Tokyo, Japan) for diagnosis of the Enteropathogenic types. All the procedures were carried out according to the manufacturer’s instructions.

The isolates were serologically identified according to [25 ] by using rapid diagnostic E. coli antisera sets (DENKA SEIKEN Co., Japan) for diagnosis of the Enteropathogenic types.

In total, 116 APEC isolates recovered from 400 different samples were assayed. Samples were obtained from liver, lungs, air sacs and spleen from chicken with typical lesions of colisepticemia and were collected from 28 different broiler farms located in different geographic areas of Dakahlia Governorate in 2014 and 2015. Samples were cultured onto eosin methylene blue (EMB) agar (Oxoid, Basingstoke, UK) and incubated for 18–24 h at 37 °C. Typical colonies were subcultured onto MacConkey agar (Oxoid, Basingstoke, UK) and were biochemically confirmed using API 20E system (BioMérieux, Marcy-l’Étoile, France). Once identified, the isolates were preserved at −70 °C in brain heart infusion broth containing 20% glycerol (vol/vol) for further studies. Serotyping of E. coli isolates was performed using rapid diagnostic E. coli antisera sets (Denka Seiken Co., Japan) using 8 polyvalent, 43 monovalent somatic, and 22 flagellar antisera. Bacterial DNA for PCR analysis was prepared by boiling a bacterial culture in 300 µl of distilled water for 10 min, followed by centrifugation for 5 min at 10,000×g. A volume of 1 µl of the supernatant was used as a template for each 25 µl PCR mixture.