Polyvalent and monovalent antisera

All Shigella strains were previously cultured in trypticase soy broth (Oxoid, England) for 6 to 8 hours; Subsequently, each strain was plotted onto Salmonella–Shigella agar (Oxoid, England) and incubated at 37°C from 18 to 24 h. Presumptive identification was carried out using biochemical tests consisting of triple sugar iron agar, lysine iron agar, mobility indole ornithine agar and Simmons citrate agar (Oxoid, England). The species and serotype were determined using commercially available polyvalent and monovalent antisera (Denka Seiken Co., Ltd, Japan).

Each pre-enriched homogenate (1 ml) was aseptically added to 10 ml of Rappaport-Vassiliadis (RV) broth and incubated at 42 °C for 24 h. Then, the broths were subcultured on xylose-lysine-desoxycholate (XLD) agar (Oxoid) and incubated at 37 °C for 24 h. Next, the presumptive colonies were picked and subjected to standard biochemical methods (urea hydrolysis, H2S production on triple sugar iron agar, lysine decarboxylation, indole, methyl red test, Voges-Proskauer test and citrate utilization test). Typical Salmonella isolates were serotyped by slide agglutination test based on O and H antigens using polyvalent and monovalent antisera (DENKA SEIKEN Co., Japan) following the White-Kauffmann- Le Minor scheme [37 ].

All Shigella strains were previously cultured in trypticase soy broth (Oxoid, England) for 6 to 8 hours; Subsequently, each strain was plotted onto Salmonella–Shigella agar (Oxoid, England) and incubated at 37°C from 18 to 24 h. Presumptive identification was carried out using biochemical tests consisting of triple sugar iron agar, lysine iron agar, mobility indole ornithine agar and Simmons citrate agar (Oxoid, England). The species and serotype were determined using commercially available polyvalent and monovalent antisera (Denka Seiken Co., Ltd, Japan).