Shiga-toxin producing Escherichia coli (STEC) are zoonotic foodborne pathogens that are capable of causing serious human illness. Ovine ruminants are recognized as an important source of STEC and a notable contributor to contamination within the food industry. This review examined the prevalence of STEC in the ovine food production chain from farm-to-fork, reporting carriage in sheep herds, during abattoir processing, and in raw and ready-to-eat meats and meat products. Factors affecting the prevalence of STEC, including seasonality and animal age, were also examined. A relative prevalence can be obtained by calculating the mean prevalence observed over multiple surveys, weighted by sample number. A relative mean prevalence was obtained for STEC O157 and all STEC serogroups at multiple points along the ovine production chain by using suitable published surveys. A relative mean prevalence (and range) for STEC O157 was calculated: for feces 4.4% (0.2-28.1%), fleece 7.6% (0.8-12.8%), carcass 2.1% (0.2-9.8%), and raw ovine meat 1.9% (0.2-6.3%). For all STEC independent of serotype, a relative mean prevalence was calculated: for feces 33.3% (0.9-90.0%), carcass 58.7% (2.0-81.6%), and raw ovine meat 15.4% (2.7-35.5%). The prevalence of STEC in ovine fleece was reported in only one earlier survey, which recorded a prevalence of 86.2%. Animal age was reported to affect shedding in many surveys, with younger animals typically reported as having a higher prevalence of the pathogen. The prevalence of STEC decreases significantly along the ovine production chain after the application of postharvest interventions. Ovine products pose a small risk of potential STEC contamination to the food supply chain.

Molecular methods may reveal the presence of pathogens in samples through the detection of specific target gene(s) associated with microorganisms, but often, the subsequent cultural isolation of the pathogen is not possible. This discrepancy may be related to low concentration of the cells, presence of dead cells, competitive microflora, injured cells and cells in a viable but non-culturable state, free DNA and the presence of free bacteriophages which can carry the target gene causing the PCR-positive/culture-negative results. Shiga-toxigenic Escherichia coli (STEC) was used as a model for studying this phenomenon, based on the phage-encoded cytotoxins genes (Stx family) as the detection target in samples through real-time qPCR. Stx phages can be integrated in the STEC chromosome or can be isolated as free particles in the environment. In this study, a combination of PCR with culturing was used for investigating the presence of the stx1 and stx2 genes in 155 ovine recto-anal junction swab samples (method (a)-PCR). Samples which were PCR-positive and culture-negative were subjected to additional analyses including detection of dead STEC cells (method (b)-PCR-PMA dye assay), presence of Stx phages (method (c)-plaque assays) and inducible integrated phages (method (d)-phage induction). Method (a) showed that even though 121 samples gave a PCR-positive result (78%), only 68 samples yielded a culturable isolate (43.9%). Among the 53 (34.2%) PCR-positive/culture-negative samples, 21 (39.6%) samples were shown to have STEC dead cells only, eight (15.1%) had a combination of dead cells and inducible stx phage, while two samples (3.8%) had a combination of dead cells, inducible phage and free stx phage, and a further two samples had Stx1 free phages only (3.8%). It was thus possible to reduce the samples with no explanation to 20 (37.7% of 53 samples), representing a further step towards an improved understanding of the STEC PCR-positive/culture-negative phenomenon.

Shiga toxin-producing Escherichia coli (STEC) are a diverse group of pathogenic bacteria capable of causing serious human illness and serogroups O157 and O26 are frequently implicated in human disease. Ruminant hosts are the primary STEC reservoir and small ruminants are important contributors to STEC transmission. This study investigated the prevalence, serotypes and shedding dynamics of STEC, including the super-shedding of serogroups O157 and O26, in Irish sheep. Recto-anal mucosal swab samples (N=840) were collected over 24 months from two ovine slaughtering facilities. Samples were plated on selective agars and were quantitatively and qualitatively assessed via real-time PCR for Shiga-toxin prevalence and serogroup. A subset of STEC isolates (N=199) were selected for whole-genome sequencing and analysed in silico . In total, 704/840 (83.8%) swab samples were Shiga-toxin positive following RT-PCR screening, and 363/704 (51.6%) animals were subsequently culture positive for STEC. Five animals were shedding STEC O157 and three of these were identified as super-shedders. No STEC O26 was isolated. Post-hoc statistical analysis showed that younger animals are more likely to harbour STEC and STEC carriage is most prevalent during the summer months. Following sequencing, 178/199 genomes were confirmed as STEC. Thirty-five different serotypes were identified, fifteen of which were not yet reported in sheep. Serotype O91:H14 was the most frequently reported. Eight Shiga-toxin gene variants were reported, two stx 1 and six stx 2 , and three novel Shiga-toxin subunit combinations were observed. Variant stx 1c was the most prevalent, while many strains also harboured stx 2b . Importance Shiga toxin-producing Escherichia coli (STEC) are foodborne, zoonotic pathogens of significant public health concern. All STEC harbour stx , a critical virulence determinant, but it is not expressed in most serotypes. Sheep shed the pathogen via faecal excretion and are increasingly recognised as important contributors in the dissemination of STEC. In this study, we have found that there is high prevalence of STEC circulating within sheep and prevalence is related to animal age and seasonality. Further, sheep harbour a variety of non-O157 STEC, whose prevalence and contribution to human disease has been under investigated for many years. A variety of Stx variants were also observed, some of which are of high clinical importance.