The use of quantitative Real-Time PCR to estimate Salmonella shed in fecal samples from naturally infected finishing pigs

The objective of this study was to describe the shedding pattern of Salmonella in finishing pigs, as well to quantify the Salmonella load. A longitudinal study was conducted in 12 cohorts of pigs in a multi-site farrow-to-finish production system. At the beginning of each cohort, 50 pigs (10 ± 2 weeks old) were randomly selected and individually identified. Individual pig fecal samples were collected and cultured every 2 weeks for 16 weeks (8 collections). Further, quantitative real-time PCR (q-PCR) targeting the invA gene was performed in a subset of the culture negative samples (555) and in all available culture positive samples. At the time of submission, Salmonella was cultured from 397 (8.74%) of 4540 individual fecal samples. Overall incidence of Salmonella was 24.8% (149/600 pigs). The proportion of positive samples decreased over the finishing period from 16.75% (10 weeks old) to 4.30% (24-26 weeks old). At the present, all Salmonella culture negative samples subjected to q-PCR were PCR negative. Of culture positive samples, 16% were detected by q-PCR, and only 3 % of the samples were within the quantifiable range of detection (>103 colony forming units per gram of feces). Of those samples within the quantifiable range, the bacterial concentration ranged from 1.05x103 to 1.73x106 invA gene copies/g feces. The results suggest that point estimates of Salmonella prevalence may not accurately describe the Salmonella status of finishing pigs. The majority of pigs shed Salmonella at low concentrations. These preliminary data can contribute to quantitative risk assessments of the association between concentrations of Salmonella shed by pigs during the finishing phase and contributions to carcass contamination at slaughter. Introduction Salmonella is one of the major causes of bacterial foodborne disease in United States (Henao et al., 2011). Salmonella is one the most important bacteriological zoonotic hazards transmissible from pork to consumers (Boyen et al., 2008). Reduction of the Salmonella contamination of pork and pork products requires interventions at pre-harvest, harvest and post-harvest (Lo Fo Wong et al., 2002). Longitudinal studies have shown high variability in Salmonella shedding over time at both the farm and individual level (Funk et al., 2001b; Rajic et al., 2005). Therefore, there is a need to better describe Salmonella shedding in order to understand Salmonella dynamics and implement control measures to reduce Salmonella pre-harvest. Limited research has been conducted to quantify Salmonella load in naturally infected finishing pigs. Enumeration of bacterial load can be used to identify contamination pressure and to implement effective control measures to reduce contamination (Fravalo et al., 2003; Boughton et al., 2007). In addition, quantitative data is needed for quantitative microbial risk assessments (QMRA) and modeling transmission patterns of Salmonella (Bollaerts et al., 2009; Lanzas et al., 2011). The objective of this study was to describe the shedding pattern of Salmonella in finishing pigs, as well to quantify the Salmonella load. Materials and Methods A longitudinal study was conducted in three sites of a multi-site farrow-to-finish production system. At each 4-barn site (A, B, C) one barn was selected for study inclusion. For each barn selected six (sites A & B) and two (site C) consecutive cohorts of pigs were included in the study. At the beginning of each cohort, 50 pigs (10 ± 2 week old) were randomly selected and individually identified. Individual pig fecal samples (10g) were collected and cultured every 2 weeks for 16 weeks (8 total sampling periods per cohort). Fecal samples were culture using standard enrichment adapted from Davies et al. (2000) for 10 gram fecal weights (TTB, RV, XLT-4). An additional fecal sample from each pig (200mg) was kept frozen (-80°C) for later analysis using quantitative real-time PCR (q-PCR). A random selection of culture negative samples (n=555) and all the available culture positive samples (n=381) were submitted for real-time q-PCR targeting the invA gene. The