Rapid detection of Salmonella spp. and indicators of fecal contamination in animal feed.

摘要

Salmonellosis is a cyclic problem in the food industry, of which animal feed is a major link. Current cultural methods of Salmonella spp. detection require 96 h for confirmation. A 96-h hold represents significant storage problems. Four feed ingredients, 2 monogastric diets, 6 avian diets, and 2 ruminant diets were chosen to provide a range of protein and fat concentrations, and particle sizes. Several DNA extraction methods involving guanidine isothiocyanate, isopropanol, and polyethylene glycol, a commercial polymerase chain reaction (PCR) assay, and a combined PCR - hybridization assay were evaluated to determine a rapid method of detection of Salmonella spp. in animal feed.; The feed samples contained a diverse microflora, with total aerobic populations ranging between log 2.8 and log 6.5 CFU/g feed. Five out of 9 fresh feeds and 7 out of 8 stored feeds contained RNA somatic phages, and all feeds contained male specific and somatic coliphages. When DNA was extracted from feed microflora, a procedure with polyethylene glycol 8000 (PEG) was favored. Using PEG, DNA from samples of feed could be amplified from between 55 and 75% of samples analyzed. The commercially available assay, "BAX(TM) for Screening/ Salmonella" (BAX), was also investigated for animal feed. The results of BAX agreed with cultural results in 16 out of 18 samples spiked with 1200 colony forming units/10 g feed and in 13 out of 18 samples spiked with 40 CFU/10 g feed. Indigenous Salmonella spp. that were detected in 5 out of 8 samples of poultry diets using conventional methods could be detected in 2 samples after 13 h of enrichment and 4 samples after 24 h of enrichment. Specific sequences of DNA from Salmonella spp. that were extracted from poultry diets could be detected using BAX. Using PCR and a hybridization sensor, amplicons associated with fecal contamination were detected in 15 different feed matrices without employing enrichment. The study demonstrates that it is possible to rapidly detect and confirm the presence of pathogenic bacteria in feed matrices by combining robust gene amplification reactions with appropriate post amplification detection systems.