Evaluation of Salmonella presence in selected United States feed mills

Abstract Salmonella is a pathogen of public health concern. Each year, Salmonella infections cost to the food industry approximately $2.3 billion and 33% of the reported cases are associated with beef, poultry, or pork. Pathogen presence in feed mills can represent one of the many potential routes for entry and transmission into the food production chain. Nevertheless, little is known about Salmonella incidence and association with these types of environments. The objective of this study was to investigate Salmonella presence in different feed mills across the United States. Eleven facilities were selected in eight states and 12 sites were sampled within each feed mill. Samples were analyzed following the FSIS guidelines for isolation and identification of Salmonella. Positive isolates were further investigated by a PCR analysis targeting the invA gene to differentiate for Salmonella enterica. The total number of environmental samples collected was 237: 66% resulted culture positive and 13.1% were PCR positive. All sampled feed mills had at least one culture positive site and following production flow the number of positive samples decreased from ingredient receiving to final product. These preliminary results demonstrate the presence of Salmonella in selected United States feed mills and suggest their potential role as vehicle for pathogen transmission and spread into the food production chain.

association with these types of environments. The objective of this study was to investigate Salmonella presence in different feed mills across the United States. Eleven facilities were selected in eight states and 12 sites were sampled within each feed mill. Samples were analyzed following the FSIS guidelines for isolation and identification of Salmonella. Positive isolates were further investigated by a PCR analysis targeting the invA gene to differentiate for Salmonella enterica. The total number of environmental samples collected was 237: 66% resulted culture positive and 13.1% were PCR positive. All sampled feed mills had at least one culture positive site and following production flow the number of positive samples decreased from ingredient receiving to final product. These preliminary results demonstrate the presence of Salmonella in selected United States feed mills and suggest their potential role as vehicle for pathogen transmission and spread into the food production chain.

K E Y W O R D S
contamination, feed, feed mills, microbial entry route, Salmonella as generally low, and historically no evidence of direct link to animal or human illness has been demonstrated in US (Burns et al., 2015;Cochrane et al., 2015;Davies, Hurd, Funk, Fedorka-Cray, & Jones, 2004;Molla et al., 2010). Nevertheless, the importance of feed as pathogen contamination source in pigs, the potential risk of transmission and survival in slaughter houses and the possible infection for consumers has been highlighted as significant and potentially high in several risk assessment models (Rönnqvist, Välttilä, Ranta, & Tuominen, 2017;Österberg, Vågsholm, Boqvist, & Sternberg Lewerin, 2006 (Li et al., 2012). These results support the importance to investigate pathogen presence and possible infection sources from feed to fork. The risk of salmonellosis from feed is difficult to quantify due to inconsistent data, sampling constrains, and lack of epidemiological information (Crump & Griffin, 2002;Jones, 2011).
Limited practices have been implemented for animal feed environments, even if these facilities have been recognized as potential source of infections in different occasions (Podolack et al., 2010;Rostagno & Callaway, 2012). Therefore, the objectives of this study were to: (a) evaluate the presence of Salmonella in selected United States commercial animal feed mills; and (b) preliminary characterize the prevalence of the pathogen in relation to sampling site and processing-associated risk factors.

| Swabbing method and sites
A diverse geographical pool of 11 feed manufacturing facilities, representative of the US swine production areas, were selected for this study. One location was identified in Colorado, Illinois, Floor dust in break or control room b Exterior of pellet mill − − − + − + n/a n/a n/a n/a n/a 33.3 16.7 Finished product bin boot samples. The sites were selected considering production flow, people traffic and dust accumulation (Table 1)

| Culture-based analysis
The for groups A through G+ iv following the manufacturer's instructions (BD Difco Salmonella O Antisera).

| Molecular-based analysis
Positive culture-based samples were further analyzed by real-time PCR. One colony from each agar plate was transferred directly and without any treatment to the PCR mixture. A protocol developed in our laboratory, that targets the invasion gene invA present in all Salmonella enterica was followed (Bai et al., 2018

| Statistical analysis
Samples exhibiting biochemical characteristics compatible with mill ID (each individual mill received an ID consisting of a number from 1 to 11), state (state where the mill is located) mill type (divided into mills producing mash or pelleted feed), and sample site (location within the mill that was analyzed). Depending on the fixed effect evaluated in the univariable and multivariable models, we incorporated a random intercept for feed mill nested within state (except when evaluating feed mill or state as fixed effect), to account for the hierarchical structure of the study. An initial univariable screen was followed by a multivariable model if more than one fixed effect was significant in the univariable screen. Mean probabilities and their 95% confidence intervals were computed and significance was indicated by p < 0.05.

| RE SULTS AND D ISCUSS I ON
For this study, both C+ and PCR+ samples were considered: results from culture-based analysis gave an indication of Salmonella genus presence (family of Enterobacteriaceae), while molecular-based analysis provided specific information about S. enterica prevalence.
Samples that were not C+ were analyzed by biochemical tests (API 20E; Biomeriux, Durham, NC). Results indicated that the majority of these isolates were either Enterobacter or Citrobacter.
Several studies have shown that Enterobacteriaceae counts tend to be higher in Salmonella positive samples and that the presence of Enterobacteriaceae can be considered as an indicator of hygiene in feed mill production systems and a tool to assess the likelihood of Salmonella incidence (Jones & Richardson, 2004). Nevertheless, since results from the Literature are conflicting, our discussion concentrated only on the presence of Salmonella (both C+ and PCR+) in feed mill environments. observed that overall the number of C+ samples decreased from the initial processing steps toward the finished product, following feed production flow. As highlighter in Figure 1 the manufacturing process within feed mill includes receiving, processing, storagepackaging, loading, and delivery. Ingredient, people and crosscontamination during production, load out, and delivery were all identified as potential hazard for microbial and viral introduction in feed mills (Cochrane et al., 2015). A biosecurity plan might offer an effective approach to reduce the likelihood of biological presence in feed mill manufacturing facilities, as well as microbial risk assessment and mitigation practices. Similar results of high pathogen presence in dust samples collected from manufacturing operations (33%-65%), storage areas (10%-27%), and worker shoes (9%-100%) were reported in a study that reviewed the practical measures to control Salmonella in animal feed (Jones & Richardson, 2004). This research highlighted the difficulty of detecting Salmonella in feed and the need to sample also dust and debris in feed manufacturing facilities to obtain a more sensitive indication of pathogen presence (Jones & Richardson, 2004). Based on these observations, in our study we selected sampling sites considering feed production flow, people traffic and dust accumulation. We also observed that the finished product bin boot had the highest number of C+ positive samples (81.1%) within the sampling sites in the production area. This equipment is in contact with the finished product before loading; therefore, it was identified as a high-risk contamination point in our research: it might represent the suitable entry point for Salmonella in the feed to fork chain.
Among the sampling sites that were not considered directly part of the production flow, worker shoes and broom had 95.2% and 63.6% C+ samples, respectively. These results highlight the high likelihood of microbial transfer and cross-contamination within the facilities based on people movements (Cochrane et al., 2015). It was also observed that overall pelleted facilities had higher percentage of microbial presence as compared to mash mills in the final products (Table 1). The unfiltered air introduced into the system to cool the feed after the pelleting step might represent the source of recontamination in this type of facilities. Similar to our observations, also another study on Salmonella contamination in US swine feed reported higher pathogen presence in pelleted commercial feed product as compared to on-farm mixed mash products (Davies et al., 2004 Most peer-reviewed studies on Salmonella presence in commercial feed manufacturing facilities focus on final product contamination, indicating the occurrence of pathogen infection, but they lack information regarding pathogen environmental presence (Jones, 2011;Li et al., 2012;Molla et al., 2010). Contaminated feed can represent a vehicle for Salmonella transmission to animals and therefore increase pathogen likelihood to be introduced into the human food (Crump & Griffin, 2002). Hence, understanding the mechanisms of contamination at preharvest level is instrumental for a more thorough hazard analysis and biosecurity plan development: the goal is to prevent and reduce pathogens contamination in animal feed and decrease the possible entrance into the human food chain (Houser et al., 2010;Li et al., 2012).
To our knowledge, this is the first study evaluating Salmonella presence in US feed mill environments. Our data showed that feed manufacturing facilities can represent a port of entry for the pathogen into the food supply chain and that effective mitigation strategies are needed to identify contamination sources and reduce risk. Future studies exploring the seasonality, genetic relatedness, as well as serotyping and antibiotic resistance profiles of Salmonella isolates are warranted to fully understand the epidemiology, ecology, and distribution of this pathogen in US feed mill environments.

ACK N OWLED G M ENTS
The authors thank the National Pork Board for the financial support

CO N FLI C T O F I NTE R E S T
The authors confirm that this article content has no conflict of interest.

DATA ACCE SS I B I LIT Y
All data are provided in full in the results section of this paper.