Combination of most-probable-number method with light cycler real-time PCR assay (MPN-real-time PCR) for rapid quantification of Salmonella in artificially and naturally contaminated bovine fecal samples at slaughter house

W. Ashraf*1, D. Abdunaser1, F. Almabrouk1,; S. Moez2, M. Yves2, C. Olivier2,

doi:10.33899/ijvs.2009.189563

	Combination of most-probable-number method with light cycler real-time PCR assay (MPN-real-time PCR) for rapid quantification of Salmonella in artificially and naturally contaminated bovine fecal samples at slaughter house
Iraqi Journal of Veterinary Sciences
Volume 23, Supplement 2, June 2009 PDF (702.95 K)
Document Type: Conference Paper
DOI: 10.33899/ijvs.2009.189563
Authors
D. Abdunaser1, F. Almabrouk1, W. Ashraf*1¹; M. Yves2, C. Olivier2, S. Moez2²
¹1 Department of Preventive Medicine, Faculty of Veterinary Medicine, Al Fateh University, Tripoli, Libya
²2 Epidemiology and Risk Analysis Section, National Veterinary School of Alfort, Maisons Alfort, France
Abstract
As a part of our effort in quantitative risk analysis of food-borne diseases, the objective of this study was to develop rapid and reliable protocols for detection and quantification of Salmonella in bovine fecal samples at slaughter house. First, for the detection of Salmonella in artificially and naturally contaminated fecal samples, SYBR Green I real-time PCR assay was used, where quantification of Salmonella was achieved by combining this assay with most-probable-number (MPN) method (MPN-real-time PCR). To develop the protocol for detecting and enumerating of Salmonella in artificially contaminated fecal samples, a Salmonella enterica serotype Typhimurium DT104 strain was inoculated into fecal samples at different levels of contamination. Data on artificially contaminated fecal samples indicated that both detection and quantification protocols were able to detect and enumerate as few as 1 CFU/mL of faeces after 8-h of a single non-selective pre-enrichment step in buffered peptone water. All MPN estimates corresponded well to inoculum levels. The protocol was then applied to naturally contaminate fecal samples. A total of 296 fecal and 26 environmental samples were aseptically collected from slaughterhouse located in Meaux, France weekly in February and March 2006. 9.1% (27/296), and 34.6% (9/26) fecal and environmental samples, respectively, were found Salmonella-positive, with estimated MPN values of Salmonella ranging from <1.8 - 1609 MPN/g of faeces. The mean of the log10 concentration of Salmonella is 0.62 MPN/g with standard deviations of 2.7 by using the censored regression approach. Counts were generally low, with the exception of 6 animals (>1400 MPN/g), while all the other 21 Salmonella positive animals had faeces with less than 80 MPN/g. The prevalence of Salmonella showed no significant difference (p=1) between French (8.63%, 17/197) and Belgian cattle (10%, 10/99). Furthermore, neither the animals’ area of origin (p=0.75), age (p=0.18), race (p=0.94), breed (p=0.23), or movement of the animal (p=0.89) had any impact on the prevalence of Salmonella. The results of this study demonstrate that the combination of real-time PCR assay and MPN method constitutes an effective, rapid and easy-to-perform method for quantifying low levels of Salmonella in bovine fecal samples.

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