Background The natural hosts of Shigella are conventionally humans and other primates; however, the host range of Shigella has been shown to expand to many animals. Although Shigella is becoming a huge threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea. Results Fifty-four S. flexneri isolates

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... ed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, set1B and stx were 100%, 100%, 77.78%, 79.63%, 48.15%, 48.15% and 0, respectively. MLVA based on 8 VNTR loci discriminated the isolates into 39 different MTs, PFGE based on NotI digestion divided the 54 isolates into 31 PTs, and MLST based on 15 housekeeping genes differentiated the isolates into 7 STs. Conclusion Our findings of this study have enriched our knowledge of the molecular characteristics of S. flexneri collected from diarrheal calves, which will be important for addressing clinical and epidemiological issues regarding Shigellosis.