This study reports two novel selective differential media. A first differential medium can be applied in methods for the isolation of non-O157 Shiga

Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan^® Environmental Master Mix 2.0; UMM: TaqMan^® Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material.

Aims: The aim of the study was to assess the impact of Salmonella present on the slaughter line before processing on broiler carcass contamination during processing. Methods and Results: Three Belgian broiler slaughterhouses were each visited twice. Samples were taken from the slaughter line after the cleaning and the disinfection process and before slaughter of the first flock. During the slaughter of the first flock, feathers and neck skins were collected at various points of the slaughter process. Swab samples were also taken from the crates in which the birds were transported. In two slaughterhouses, the slaughter line was contaminated with Salmonella before the onset of slaughter, especially the shackles, conveyer belt and the plucking machine in the dirty zone. During slaughter, the carcasses of the first Salmonella-free flock became contaminated with the same strains as isolated previously from the slaughter line. Conclusion: Contamination of the slaughter line with Salmonella leads to carcass contamination. Significance and Impact of the Study: Implementation of logistic slaughter is only successful when the cleaning and disinfection process completely eliminates the Salmonella contamination of the slaughter line. Only if this is achieved, will the slaughter of Salmonella-free flocks result in the absence of Salmonella on the carcasses after slaughter.

There is paucity of information regarding the epidemiology of Escherichia coli O157: H7 in developing countries. In this study, we investigated the occurrence of E. coli O157: H7 associated with beef cattle at processing plants and at retail shops in Ethiopia. Various samples were collected from beef cattle at slaughter/processing plants, carcass at retail shops and humans at health centers. E. coli O157: H7 was isolated, identified and characterized for antimicrobial resistance, using standard microbiological methods. At the processing plants E. coli O157: H7 was detected in 1.89% of fecal, 0.81% of intestinal mucosal swab, 0.54% of skin swab and 0.54% of carcass internal swab samples. At retail shops it was detected in 0.8% of carcass and 0.8% of cutting board swab samples, while all samples from utensils, hands from workers, and fecal and stool samples were negative. All isolates were resistant to Amoxicillin, moderately resistant to Cefoxitine and Nitrofurantoins but susceptible to other antimicrobials tested. E. coli O157: H7 occurs at low prevalence in beef cattle, and the current sanitary dressing procedures in the processing plants and storage conditions in the retail shops are effective against E. coli O157: H7.

Understanding the potential drivers of microbial meat contamination along the entire meat supply chain is needed to identify targets for interventions to reduce the number of meatborne bacterial outbreaks. We assessed the hygienic practices in cattle slaughterhouses (28 employees) and retail shops (127 employees) through face-to-face interviews and direct personal observations. At the slaughterhouses, stunning, de-hiding and evisceration in vertical position, carcass washing and separate storage of offal were the identified good practices. Lack of hot water baths, absence of a chilling room, infrequent hand washing, insufficiently trained staff and irregular medical check-up were practices that lead to unhygienic handling of carcasses. At the retail shops, cleaning equipment using soap and hot water (81%), storing unsold meat in refrigerators (92%), concrete floors and white painted walls and ceilings were good practices. Adjacently displaying offal and meat (39%), lack of a cold chain, wrapping meat with plastic bags and newspapers, using a plastic or wooden cutting board (57%), infrequent washing of equipment and floors, and inadequately trained employees were practices that could result in unhygienic handling of beef. Our study identified unhygienic practices both at the slaughterhouses and retail shops that can predispose the public to meatborne infections, which could be improved through training and implementation of quality control systems.