Virulotyping of Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli isolated from recto-anal mucosal swabs of young diarrheic and non-diarrheic calves

Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens causing severe disease in humans worldwide, whose most important virulence factor is the production of Shiga toxins (Stx1, Stx2). Additionally, most STEC also possess the eae gene encoding for the intimin protein, which is responsible for the formation of the attaching and effacing (A/E) lesions (AE-STEC) and is also typical of enteropathogenic E. coli (EPEC). AE-STEC colonize the mucosa of the recto-anal junction of healthy adult cattle, making recto-anal mucosal swabs (RAMS) more sensitive than fecal culture to isolate them. In addition, AE-STEC also colonize the small and large intestines of young calves causing diarrhea. Conversely, the carrier state of non-diarrheic young calves is unknown. Finally, most studies address the most pathogenic O157:H7 AE-STEC serotypes, although several other non-O157:H7 AE-STEC serotypes are becoming increasingly important with regard to human outbreaks. The objective of the present study was to evaluate the colonization by and shedding of AE-STEC in young diarrheic and non-diarrheic dairy calves. RAMS from 233 young calves in 3 farms with non-O157:H7 AE-STEC in older animals were overnight enriched in Lauryl-Sulfate broth and tested with a triplex PCR targeting the eae, stx1 and stx2 genes. Positive broths were subsequently streaked on four selective agar plates (Figure 1). Up to 5 colonies per plate will be picked-up, tested by the colony hybridization assay with probes for the same genes and confirmed by the same triplex PCR.A total of 152 broths (65%) tested positive with the triplex PCR after overnight enrichment: 23 AE-STEC, 122 STEC and 7 EPEC. In farm #1, 2 RAMS tested positive for AE-STEC, 45 for STEC and 3 for EPEC. In farm #2, 3 RAMS tested positive for AE-STEC, 42 for STEC and 4 for EPEC. In farm #3, 18 RAMS tested positive for AE-STEC and 35 for STEC. All positive broths are being streaked on the selective agar plates and the 5 picked-up colonies are being prepared for the colony hybridization assay. Of the positive RAM’s the majority (80%) harbored STEC and a minority AE-STEC (15%) or EPEC (5%). This low number of EPEC-positive samples is different from previous results obtained in adult cattle in two slaughterhouses following the same methodology. In the same survey, 80% of the PCR-positive broths gave colony hybridization-positive and triplex PCR-positive colonies. Confirmed positive colonies will be O:H serotyped by PCR and all results will be associated with presence/absence of diarrhea in the calves