Contamination of eggs by Salmonella Enteritidis in experimentally infected laying hens housed in conventional or enriched cages

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1 Contamination of eggs by Salmonella Enteritidis in experimentally infected laying hens housed in conventional or enriched cages Richard K. Gast,* 1 Rupa Guraya,* Deana R. Jones,* and Kenneth E. Anderson * Egg Safety and Quality Research Unit, Agricultural Research Service, USDA, Athens, GA 30605; and Department of Poultry Science, North Carolina State University, Raleigh ABSTRACT Both epidemiologic analyses and active disease surveillance confirm an ongoing strong association between human salmonellosis and the prevalence of Salmonella enterica subspecies enterica serovar Enteritidis in commercial egg flocks. The majority of human illnesses caused by this pathogen are attributed to the consumption of contaminated eggs. Animal welfare concerns have increasingly influenced commercial poultry production practices in recent years, but the food safety implications of different housing systems for egg-laying hens are not definitively understood. The present study assessed the effects of 2 different housing systems (conventional cages and colony cages enriched with perching and nesting areas) on the frequency of Salmonella Enteritidis contamination inside eggs laid by experimentally infected laying hens. In each of 2 trials, groups of laying hens housed in each cage system were orally inoculated with doses of cfu of Salmonella Enteritidis. All eggs laid between 5 and 25 d postinoculation were collected and cultured to detect internal contamination with Salmonella Enteritidis. For both trials combined, Salmonella Enteritidis was recovered from 3.97% of eggs laid by hens in conventional cages and 3.58% of eggs laid by hens in enriched cages. No significant differences (P > 0.05) in the frequency of egg contamination were observed between the 2 housing systems. Key words: Salmonella Enteritidis, chicken, egg, conventional cage, enriched cage 2014 Poultry Science 93 : INTRODUCTION During the first decade of this century, the incidence of human Salmonella infections in the United States did not change significantly, and this disease was responsible for estimated annual costs of up to $11 billion (Centers for Disease Control and Prevention, 2011; Scharff, 2012). Moreover, the incidence of Salmonella enterica subspecies enterica serovar Enteritidis infections increased by 44% during the same period (Chai et al., 2012). Salmonella Enteritidis first emerged as a significant international public health problem in the 1980s, and the majority of human illnesses caused by this pathogen have been attributed to the consumption of contaminated eggs (Braden, 2006; Greig and Ravel, 2009). Significant resources from both government and private industry have been invested in Salmonella Enteritidis testing and risk reduction programs for egg-producing poultry (Gast, 2007; US Food and Drug Administration, 2009). After a sustained commitment to these efforts over time, international progress 2014 Poultry Science Association Inc. Received September 21, Accepted November 24, Corresponding author: Richard.Gast@ars.usda.gov has been documented in regard to the incidences of both egg contamination (Esaki et al., 2013) and human infections (Mumma et al., 2004; Poirier et al., 2008; O Brien, 2013). However, both epidemiologic analyses and active disease surveillance confirm an ongoing strong association between human salmonellosis and the prevalence of Salmonella Enteritidis in commercial egg flocks (Centers for Disease Control and Prevention, 2011; Chai et al., 2012; Havelaar et al., 2013). The deposition of Salmonella Enteritidis inside the edible interior contents of eggs is a consequence of bacterial dissemination to reproductive tissues (ovary and oviduct) in systemically infected hens (Gast et al., 2004; Gantois et al., 2009). The number of Salmonella Enteritidis cells administered to hens can significantly affect both the frequency and location of resulting egg contamination (Gast et al., 2013a), but experimental infection with very large oral doses of Salmonella Enteritidis has typically only yielded low frequencies of egg contamination and small initial bacterial cell concentrations (Humphrey et al., 1991; Gast and Beard, 1992; Gast and Holt, 2000). Even lower frequencies of contamination are usually reported for commercially produced eggs, perhaps because laying flocks are sporadically exposed to environmental sources of Salmonella Enteritidis at relatively low doses (Ebel and Schlosser, 728

2 2000; DeWinter et al., 2011; Esaki et al., 2013). Although the frequency of Salmonella Enteritidis colonization of internal organs in mature chickens declines sharply during the first few weeks after oral inoculation (Gast et al., 2007, 2011a), persistent infections in individual birds might prolong the horizontal transmission of infection throughout flocks (Gast et al., 2009). Opportunities for laying hens to be exposed and infected can also be enhanced by extended bacterial survival in the housing environment, which is often sustained and amplified by rodent or insect vectors (Davies and Breslin, 2003; Dewaele et al., 2012b; Lapuz et al., 2012). The production environment may also serve as a reservoir for the periodic emergence of pathogen strains with heightened abilities to cause systemic infection and egg contamination (Henzler et al., 1998; Dewaele et al., 2012a). Housing systems for egg-laying hens have been widely discussed and analyzed in recent years in a variety of contexts, including animal welfare and food safety. However, poultry housing conditions have diverse and complex influences on Salmonella contamination and infection, so no conclusive or authoritative consensus about their food safety implications has yet emerged from the published scientific literature on the topic (Holt et al., 2011). Several studies have reported an increased frequency of environmental Salmonella isolation from laying houses containing conventional battery cages (Huneau-Salaün et al., 2009; Snow et al., 2010; Van Hoorebeke et al., 2010b), but other investigators observed lower incidences of Salmonella infection and egg contamination associated with cages than with floor systems (Kinde et al., 1996; Hannah et al., 2011). Enriched (furnished) cages have been considered as an intermediate alternative to conventional cages and cage-free systems. Although no clear or consistent pattern of differential effects on Salmonella prevalence has yet been found to distinguish the various cage systems (De Vylder et al., 2009; Van Hoorebeke et al., 2011), a significantly higher frequency of invasion to internal organs occurred among experimentally infected hens housed in conventional cages than in enriched cages (Gast et al., 2013b). The objective of the present study was to determine the effects of 2 different housing systems (conventional and enriched cages) on the frequency of Salmonella Enteritidis contamination inside eggs laid by experimentally infected laying hens. MATERIALS AND METHODS Experimental Housing of Laying Hens In each of 2 similar trials, 68 laying hens were obtained from the specific-pathogen-free flock of Single Comb White Leghorn chickens (negative for antibodies to Salmonella in periodic routine monitoring) at the Southeast Poultry Research Laboratory in Athens, Georgia. These hens (30 and 37 wk old at the beginning of the first and second trials, respectively) were HOUSING AND SALMONELLA ENTERITIDIS IN EGGS distributed into 4 separately housed groups in different rooms of a disease-containment facility containing cage systems designed to simulate commercial conditions. Hens in 2 rooms (18 per room) were housed in conventional laying cages. Each of these cages housed 6 hens and provided 648 cm 2 of floor space per bird. Hens in 2 other rooms (16 per room) were housed in enriched laying cages. Each of these cages housed 16 hens and provided 1,216 cm 2 of floor space per bird, including access to 2 perches and a single enclosed nesting area. The stocking densities in both housing systems represented two-thirds of the maximum levels recommended by the cage manufacturer. All hens were provided with water (via 2 automatic nipple-type drinkers in each conventional cage and 6 in each enriched colony cage) and feed (a pelleted, antibiotic-free layer-breeder ration) ad libitum. All protocols involving these hens were approved by the Institutional Animal Care and Use Committee of the Southeast Poultry Research Laboratory. Experimental Infection of Laying Hens with Salmonella Enteritidis In each trial, all laying hens were orally inoculated with a measured dose of Salmonella Enteritidis. Hens in trial 1 received a phage type 13a isolate, originally isolated from a contaminated egg yolk by C. Benson at the University of Pennsylvania, Kennett Square, Pennsylvania. Hens in trial 2 received a phage type 4 isolate, originally isolated from the liver of an infected chicken by D. Munro at the Scottish Salmonella Reference Laboratory, Glasgow, United Kingdom. Two different Salmonella Enteritidis phage types (both of which are epidemiologically important) were included in this study to minimize the strain specificity of results. The inoculum strains were resuscitated by transfer into tryptic soy broth (Acumedia, Neogen Corp., Lansing, MI) for 2 successive 24-h incubation cycles at 37 C. After cell numbers in each incubated culture were estimated by determining its optical density at 600 nm, further serial 10-fold dilutions in 0.85% saline produced a desired final cell concentration of approximately cfu (confirmed by subsequent plate counts). Fecal Samples 729 Immediately before inoculation, sterile cotton swabs were used to collect samples of voided feces from polystyrene trays (food-grade but not sterile) placed under each cage. A total of 30 samples were collected from each room, evenly distributed among all occupied cages. These samples were transferred to 9 ml of tetrathionate broth (Acumedia) and incubated for 24 h at 37 C. A 10-μL portion from each broth culture was then streaked onto brilliant green agar (Acumedia) supplemented with 0.02 mg/ml of novobiocin (Sigma Chemical Co., St. Louis, MO) and incubated for 24 h at 37 C. The identity of presumptive colonies of Sal-

3 730 Gast et al. Table 1. Isolation of Salmonella Enteritidis from the contents of eggs laid by experimentally infected hens in different housing systems (Salmonella Enteritidis-positive/total with percent in parentheses) 1 Item Trial 1 Trial 2 All trials Conventional cage 18/478 (3.77) a 17/403 (4.22) a 35/881 (3.97) a Enriched cage 20/529 (3.78) a 15/448 (3.35) a 35/977 (3.58) a a Values sharing no common superscripts are significantly (P < 0.05) different. 1 Eggs were collected for sampling between the d 5 and 25 after oral inoculation of hens with approximately 10 8 cfu of phage type 13a (trial 1) or phage type 4 (trial 2) Salmonella Enteritidis strains. monella was confirmed biochemically and serologically (Waltman and Gast, 2008). Egg Content Samples All eggs laid on the day before inoculation and between 5 and 25 d postinoculation were cultured to detect internal contamination with Salmonella. Eggshell surfaces were disinfected by dipping for 5 s in 70% ethanol and the shells were then broken against a sharp edge covered by sterile foil strips. The entire liquid contents of each egg were transferred to 50 ml of tryptic soy broth supplemented with 35 mg/l of ferrous sulfate (Sigma), mixed by vigorous shaking for 15 s, and incubated for 24 h at 37 C. A 1-mL portion of each incubated tryptic soy broth culture was transferred to 9 ml of Rappaport Vassiliadis broth (Acumedia) and incubated for 24 h at 37 C. A 10-μL aliquot from each of these broth cultures was then streaked onto brilliant green agar and incubated for 24 h at 37 C. After incubation of these plates for 24 h at 37 C, typical Salmonella Enteritidis colonies were subjected to biochemical and serological confirmation (Waltman and Gast, 2008). Statistical Analysis For each trial (and for both trials combined), significant differences (P < 0.05) between housing systems in the mean frequencies of Salmonella Enteritidis isolation from egg contents were determined by Fisher s exact test. Similarly, significant differences (P < 0.05) between Salmonella Enteritidis strains in their mean frequencies of recovery from eggs were determined by Fisher s exact test for each housing system (and for both systems combined). Because the 2 replicate groups of hens for each housing system did not differ significantly within either trial in Salmonella Enteritidis recovery from eggs, their results were combined for analysis and presentation. Data were analyzed with Instat biostatistics software (GraphPad Software, San Diego, CA). RESULTS AND DISCUSSION None of the fecal samples or eggs collected before inoculation in either trial were positive for Salmonella. In trial 1, Salmonella Enteritidis (phage type 13a) was recovered from 3.77% of eggs laid between 5 and 25 d after oral inoculation by hens housed in conventional cages and from 3.78% of eggs laid by hens housed in enriched cages (Table 1). In trial 2, Salmonella Enteritidis (phage type 4) was recovered from 4.22% of eggs laid between 5 and 25 d postinoculation by hens housed in conventional cages and from 3.35% of eggs laid by hens housed in enriched cages. No significant differences (P > 0.05) were observed between housing systems in the frequencies of Salmonella Enteritidis isolation from eggs in either trial or for both trials combined. Likewise, no significant differences were observed between the 2 Salmonella Enteritidis strains in their associated frequencies of egg contamination in either housing system or for both systems combined. Salmonella Enteritidis has often caused higher incidences of egg contamination than other serovars in oral inoculation experiments (Gast et al., 2005, 2007), perhaps as a consequence of stronger adherence to reproductive tract mucosa (Wales and Davies, 2011). For example, a recent study (Gast et al., 2011b) found Salmonella inside eggs at significantly different frequencies following experimental infection of hens with serovars Enteritidis (3.6%), Heidelberg (0.5%), or Hadar (0%). The deposition of Salmonella Enteritidis inside developing eggs may result from the sequential expression of bacterial virulence properties, which are relevant at different stages of infection in susceptible laying hens (Gast et al., 2002; Guard et al., 2010). The expression of very long lipopolysaccharide O-antigen enhances both reproductive tract colonization and survival in forming eggs, and may thus distinguish egg-contaminating Salmonella Enteritidis strains from other environmental salmonellae (Guard-Bouldin et al., 2004; Coward et al., 2013). The accumulation of single nucleotide changes within relevant genes can lead to divergence among isolates of the same phage type in their abilities to invade internal organs and eggs (Guard et al., 2011). Phage typing has been a useful tool for identifying the sources of outbreak-related strains, but no consistent patterns of expression of virulence properties have differentiated the various phage types of Salmonella Enteritidis (Gast and Benson, 1996; Gast and Holt, 2000; Gantois et al., 2009). Hens were infected with strains of 2 different phage types in the present study, but laid contaminated eggs at similar frequencies. The environment of poultry housing facilities can serve as an important source of Salmonella Enteritidis introduction to laying flocks (Henzler et al., 1998; Dewaele et al., 2012 a,b). Different environmental reservoirs for Salmonella persistence have been associated with the

4 various commercial housing systems (Carrique-Mas et al., 2009b). A higher environmental prevalence of Salmonella has been reported in larger flocks (Mollenhorst et al., 2005; Namata et al., 2008; Huneau-Salaün et al., 2009), older flocks (Namata et al., 2008; Pitesky et al., 2013), multiple-age flocks (Mollenhorst et al., 2005; Huneau-Salaün et al., 2009; Snow et al., 2010), and older facilities (Van Hoorebeke et al., 2010a). Diverse flock management and housing parameters can also serve as risk factors for the transmission of Salmonella Enteritidis infection and for egg contamination. High stocking densities or unsanitary conditions can increase the susceptibility of chickens to infection (Asakura et al., 2001). Some comparative studies have reported a higher frequency of horizontal transmission of Salmonella infection in cage-free housing systems than for cage systems (De Vylder et al., 2011; Hannah et al., 2011), especially if outdoor areas allow Salmonella introduction from external environmental sources (Mollenhorst et al., 2005). Other studies have found a greater risk of Salmonella infection in caged-based housing, particularly when rodents are present in high numbers (Namata et al., 2008; Carrique-Mas et al., 2009a). However, several other investigators detected no significant differences between cage and cage-free systems (Siemon et al., 2007; Jones et al., 2012) or between conventional and enriched cage systems (De Vylder et al., 2009; Van Hoorebeke et al., 2011) in the environmental prevalence of Salmonella. In one recent experiment, Salmonella Enteritidis was recovered at a significantly higher frequency from internal organs of infected hens housed in conventional cages than from hens in enriched colony cages (94 vs. 53% of spleens and 25 vs. 10% of ovaries), suggesting that parameters such as bird density or behavioral restriction might influence susceptibility to systemic dissemination of Salmonella Enteritidis (Gast et al., 2013b). Other environmentally mediated stressors, such as heat exposure, feed restriction, or water deprivation, can increase susceptibility to Salmonella infection (Humphrey, 2006). However, no comparable difference between these same 2 caging systems was observed for the incidence of egg contamination in the present study. Although systemic infection is a necessary component step in the egg contamination process, previous experiments have sometimes shown an imprecise relationship between the frequency or magnitude of organ invasion and the production of contaminated eggs (Gast et al., 2004, 2007, 2011c). Accordingly, environmental influences which affect systemic infection may not necessarily have a corresponding effect on egg contamination. Egg-borne transmission of Salmonella Enteritidis to consumers is epidemiologically linked to the presence of the pathogen in edible yolk and albumen (and the present study focused on this critical issue), but contaminated egg shells can also pose an indirect threat to food safety. Previous research has indicated that housing systems can affect the external microbiology of HOUSING AND SALMONELLA ENTERITIDIS IN EGGS eggs. 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