Rapid detection and differentiation of Salmonella species, Salmonella Typhimurium and Salmonella Enteritidis by multiplex quantitative PCR.

A multiplex quantitative PCR (qPCR) was developed and evaluated for the simultaneous detection of Salmonella spp., S. enterica serovar Typhimurium and S. enterica serovar Enteritidis in various (food) matrices. Early and fast detection of these pathogens facilitates effective intervention and prevents further distribution of contaminated food products on the market. Three primer and probe sets were designed to target the invA gene, the STM4200 gene, and the SEN1392 gene to detect and differentiate Salmonella spp., S. Typhimurium, and S. Enteritidis, respectively. The multiplex qPCR targeting these three genes was optimized for efficiency and linearity. By testing 225 Salmonella isolates and 34 non-Salmonella isolates from various sources the inclusivity and exclusivity were determined. The inclusivity of the multiplex qPCR was 100% for all Salmonella isolates, including 72 S. Typhimurium isolates, and 53 S. Enteritidis isolates. The exclusivity for Salmonella spp., S. Typhimurium, and S. Enteritidis was 100%, 94.6%, and 100%, respectively. No positive results were reported for non-Salmonella isolates. The limit of detection (LOD) for the qPCR was determined for the matrices poultry, minced meat, egg, herbs/spices, powdered milk, fish, animal feed, boot-socks with chicken feces and chicken down. LOD values for qPCR and the conventional culture methods were similar, except for the matrix boot-socks and down, for which the LOD for the conventional culture methods performed better than the qPCR method. In conclusion, the multiplex qPCR assay developed allows for rapid screening of Salmonella spp., S. Typhimurium, and S. Enteritidis in various (food) matrices.

Two outbreaks of campylobacteriosis associated with the consumption of raw cows' milk.

The present paper summarises the investigation of two different outbreaks of milk-associated Campylobacter enteritis in the Netherlands. In 2005, after a school trip to a dairy farm, 22 out of a group of 34 children developed diarrhoeal illness and Campylobacterjejuni was cultured from the stool samples of 11 of the cases. The illness was found to be epidemiologically associated with drinking raw milk during the farm visit; 86% of the cases could be explained by drinking raw milk. C.jejuni was also isolated from three of 10 faecal samples from dairy cattle collected at the farm. The human isolates and C.jejuni isolates from one of these three samples of cattle faeces revealed identical restriction patterns by both pulsed-field gel electrophoresis (PFGE) and flagellin (fla) typing by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Both epidemiological and bacteriological evidence implicated contaminated raw milk as the vehicle of transmission, though C.jejuni was not isolated from the bulk tank milk or the milk filter collected during the farm investigation. In 2007, an outbreak of enteritis was notified among people who had attended a lunch at a dairy farm where bulk tank milk was served. Of the 19 persons who had consumed raw milk, 16 (84%) had become ill. Of the persons who did not drink the raw milk, none became ill. A significant association was found between tasting the raw milk and being ill (risk difference=0.84, p=0.0011). C.jejuni was cultured from four of seven cases who had submitted a stool specimen. C. jejuni was also isolated from a sample of bulk tank milk and the isolate had an identical flaA PCR-RFLP genotype to isolates obtained from patients. Also in this outbreak both the epidemiological and bacteriological findings support raw milk as the vehicle for the enteritis. These two outbreaks highlight the health risks associated with the consumption of raw milk. As long as legislation allows the sale and distribution of untreated milk these risks will continue. Therefore, consumers need to be continuously informed about the dangers inherent in consuming unpasteurised milk or products made from raw milk. Farmers need to be strongly discouraged from serving raw milk to their visitors.