Recovery of Salmonella serovar Enteritidis from inoculated broiler hatching eggs using shell rinse and shell crush sampling methods 1

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1 Recovery of Salmonella serovar Enteritidis from inoculated broiler hatching eggs using shell rinse and shell crush sampling methods 1 M. L. Webb,* J. L. Spickler, 2 D. V. Bourassa, N. A. Cox, J. L. Wilson, and R. J. Buhr 3 * Biology Department, Claflin University, Orangeburg, SC 29115; Poultry Microbiological Safety Research Unit, Richard B. Russell Agricultural Research Center, USDA, Agricultural Research Service, Athens, GA 30605; and Department of Poultry Science, The University of Georgia, Athens ABSTRACT This study compared the recovery of Salmonella from hatching eggs using 3 sampling methods (eggshell rinsing, eggshell crush following a previous rinse, and eggshell crush without previous rinse). Eggshells were drop-inoculated with approximately 10 1, 10 2, or 10 3 cfu/eggshell of Salmonella Enteritidis and allowed to dry at room temperature for 1 or 24 h. For the shell rinse groups, each inoculated egg was rinsed with buffered peptone water. These rinsed eggs were used for the shell crush with previous rinse groups, and each egg was aseptically cracked, the contents discarded, and the eggshell and membranes crushed with buffered peptone water. This same crush procedure was used for the shell crush without previous shell rinse eggs. The recovery of Salmonella 1 h after inoculation for shell rinse sampled eggs was 16% positive at 10 1, 49% at 10 2, and 93% at 10 3 cfu/eggshell challenge. For the shell crush with previous shell rinse, sampled egg recovery was 0% positive at 10 1, 3% at 10 2, and 17% at 10 3 cfu/eggshell. For the shell crush, sampled eggs had recovery of 23% positive at 10 1, 69% at 10 2, and 96% at 10 3 cfu/eggshell challenge. The recovery of Salmonella 24 h after inoculation for the shell rinse eggs was 3% positive at 10 1, 12% at 10 2, and 22% at 10 3 cfu/eggshell challenge; recovery for shell crush with previous shell rinse sampling was 2% positive at 10 1, 8% at 10 2, and 5% at 10 3 cfu/eggshell challenge; and for the shell crush sampling recovery was 2% at 10 1, 32% at 10 2, and 42% at 10 3 cfu/eggshell challenge. Eggshell crush was a more sensitive (~10 percentage points) sampling method than eggshell rinse at both 1 and 24 h, but both methods were equally optimal when the inoculum was at 10 3 and samples were collected after 1 h. Waiting 24 h after inoculation to sample significantly lowered the recovery for both the shell rinse and shell crush sampling methods by ~40 percentage points. Key words: broiler hatching egg, shell crush, eggshell Salmonella, shell rinse, Salmonella inoculation 2014 Poultry Science 93 : INTRODUCTION 2014 Poultry Science Association Inc. Received February 27, Accepted May 6, Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal-opportunity provider and employer. 2 Present address: Sam D. Hamilton Noxubee National Wildlife Refuge, Brooksville, MS Corresponding author: jeff.buhr@ars.usda.gov Salmonella contamination of broiler hatching eggs continues to be a serious concern for the poultry industry because regulations concerning consumer food safety are continuously lowering acceptable prevalence on fully processed carcasses. Salmonella is considered to be the most important human enteropathogen associated with shell eggs (Baker and Bruce, 1995). Although all serotypes of Salmonella are classified as potential human pathogens, table eggs contaminated by Salmonella Enteritidis and Heidelberg have been identified as the main source in egg-transmitted human illness (Gast et al., 2005). More than 80% of salmonellosis outbreaks in the United States from 1998 to 2008 associated with Salmonella serotypes Enteritidis, Heidelberg, and Hadar were attributed to eggs or poultry meat (Jackson et al., 2013). Regarding hatching eggs, reducing or eliminating the contamination on eggshells could potentially help decrease the prevalence of contaminated chicks (Christensen et al., 1997). The presence of Salmonella on eggshells, contamination of chicks in the hatchery, and the transfer of Salmonella-contaminated chicks to the growout farm can potentially affect the food safety of broiler raw meat products (Lahellec and Collin, 1985; Christensen et al., 1997). The shell of broiler hatching eggs can be contaminated in many ways in the laying house. Hens often bring fecal material into the nest box that contains many different types of bacteria (Cox et al., 2000b). Breeder flocks and commercial hatcheries can be early 2117

2 2118 Webb et al. opportunities for Salmonella contamination of hatching eggs (Cox et al., 2007) because Salmonella can be found in on-farm egg holding rooms, farm to hatchery egg transport trucks, and throughout the hatchery environment (Cox et al., 2000b). Although the cuticle of the eggshell provides protection and is considered a defense mechanism against bacteria, a small percentage of eggs may be laid without sufficient cuticle thickness or shell coverage (Board and Halls, 1973). The penetration of Salmonella Typhimurium through the cuticle, shell, and shell membrane can occur in as little as 10 min (Padron, 1990). Therefore, bacteria, especially Salmonella, can easily and quickly penetrate the eggshell. Bacteria penetration of the eggshell is enhanced when there is moisture present during the temperature differential period immediately following lay (Berrang et al., 1999). A temperature differential occurs because the egg is warmer than the environment when it is first laid, and as it cools the contents of the eggs contract during air cell formation (Berrang et al., 1999). This contraction forms a negative pressure within the egg, which can cause bacteria to be pulled deep into the egg through the eggshell pores (Lock et al., 1992). In freshly laid eggs, the cuticle quickly dries within 3 min but remains hydrated with gradual compositional changes to 6 h postlay. At 24 h, fissures and thinning of the cuticle occur (Rodríguez-Navarro et al., 2013), but the cuticle has rehydration (to 2 h postlay values) with moisture loss from the egg albumen through the shell. Fertile eggs that were inoculated with high levels of Salmonella (Typhimurium) still had an 86% hatching rate, indicating they can likely contaminate the other chicks while in the hatching cabinet or on the growout farm (Cason et al., 1994). Therefore, an efficient method for detection of low levels of Salmonella on eggshells is needed. Some of the reported methods for recovery of Salmonella from eggshells are rinsing, swabbing, and crushing shells (Kawasaki et al., 2008). Eggshell rinsing and crushing sampling methods have also been used to assess hatching egg sanitization and table egg Salmonella (Musgrove et al., 2005; Spickler et al., 2011; Chousalkar and Roberts, 2012). The objectives of this study were to determine 1) if shell rinsing or shell crushing sampling methods equally recovered Salmonella when inoculated with cfu/egg, and 2) if the time that the inoculated Salmonella remained in contact with the shell before sampling (1 vs. 24 h) affected the recovery percentage. MATERIALS AND METHODS Sample Preparation For 5 trial days, 120 day-of-lay nest-clean broiler hatching eggs were collected and held without refrigeration. All eggs were evaluated and any eggshells with visible dirt, wrinkles, light spots, dark speckling, or rough surfaces were culled. The selected 90 eggs were placed horizontally on plastic 36-egg flats and arranged into 6 groups of 15 eggs, with 3 flats representing shell crush without a previous shell rinse groups, and the other 3 flats representing both the shell rinse and the shell crush after shell rinse groups. The flats of eggs were warmed in an incubator at 42 C for 16 h to create a temperature differential at the time of inoculation. One flat of 15 eggs, in sequence, was removed and received inoculation levels of 10 1, 10 2, or 10 3 cfu/eggshell of a nalidixic-acid-resistant strain of Salmonella Enteritidis. The number of cfu in each 10 µl drop was monitored and averaged for the stock solution, which was diluted to concentrations of 10 1, 10 2, or 10 3 cfu/10 µl drop for inoculation. Warm eggs were individually drop (10 µl) inoculated near the equator of the egg using a pipette to deliver the cell suspension of Salmonella in 0.85% saline at room temperature. The inoculum was spread over the upper surface of the eggshell with a sterile plastic loop in a circle approximately 3 cm in diameter, avoiding the sides of the eggshell or contact with the egg flat. The inoculum was allowed to dry for 1 h on the eggshells on the laboratory bench before proceeding with the sampling methods for all 5 trials. Following trial 1, for the 4 subsequent trial days an additional 120 eggs were selected and inoculated with the same dilutions, but these eggs were sampled after leaving the inoculated Salmonella on the eggshells for 24 h. Following inoculation, the egg flats were placed into an incubator set to 24 C and approximately 45% RH for sampling the following day. The same 3 sampling methods were used after 24 h as are described below for 1 h. Microbiological Sampling Eggs were sampled by 3 methods: 1) shell rinse, 2) shell crush only, and 3) shell crush following shell rinse. The shell crush procedure was done in a manner similar to that described by Musgrove et al. (2005). For the shell rinse group, each egg was placed in a plastic bag containing 30 ml of 1% buffered peptone water (BPW, Acumedia, Lansing, MI) and massaged by hand for 1 min before being aseptically returned to the flat in the same position to be used for the shell crush with previous shell rinse group. The plastic egg flats (Jamesway 36-egg plastic, Indian Trail, NC) have 4 tabs ( cm) extending above the square egg hole that suspend the egg horizontally restricting the surface contact between the eggshell and the egg flat. For the eggshell crush with previous shell rinse group, with a new glove each egg was aseptically cracked on a sterile surface, the contents discarded (albumen and yolk), and the shell and membranes complex gently crushed in a gloved hand and forced into a 50 ml centrifuge tube containing 30 ml of 1% BPW. This same procedure was followed for the eggs from the shell crush only group that had not been previously rinsed. The shell crush following shell rinsing sampling was conducted to determine if Salmonella not recovered in the

3 SALMONELLA RECOVERY FROM INOCULATED HATCHING EGGS 2119 Table 1. Salmonella recovery (%) 1 from inoculated broiler hatching eggshells sampled by shell rinse, shell crush with previous shell rinse, and shell crush only methods 1 and 24 h after inoculation Inoculum level Treatment Time (h) Average Shell rinse Shell crush with previous shell rinse Shell crush 1 16 c (12/75) 2 49 b (37/75) 93 a (70/75) b # (2/60) 3 12 ab # (7/60) 22 a # (13/60) b * (0/75) 3 b * (1/75) 17 a * (13/75) (1/60) 8 (5/60) 5*# (3/60) c (17/75) 69 b * (52/75) 96 a (72/75) b # (1/60) 32 a *# (19/60) 42 a *# (25/60) 25 a c Percentages within a sampling method and time (in the same row) with different superscripts differ significantly (P < 0.05). 1 Number positive Salmonella recovery/number of eggshells sampled. 2 Total for 5 replications, 15 eggshells per replication (n = 75). 3 Total for 4 replications, 15 eggshells per replication (n = 60). *Significantly different from the shell rinse sampling values (P < 0.05) within a sampling method and time. #Significantly lower recovery at 24 h from samples at 1 h using the same sampling method (P < 0.05). shell rinsate could be recovered after enrichment of the crushed eggshell in PBW for 24 h. Once collected, all eggshell and shell rinse samples were incubated at 37 C for 18 to 24 h before plating. To plate the samples, 2 loops (a total of 20 µl) of enriched rinsate were streaked onto Brilliant Green Sulfa (Acumedia, Lansing, MI) agar containing 200 mg of nalidixic acid/l (Sigma Aldrich, St. Louis, MO). Plates were incubated for 24 h at 37 C, after which the plates were evaluated, separated, and recorded as positive (agar color turned from brown to pink and displays smooth, round colonies that are translucent gray) or negative (plate remained brown in color and no evidence of colony growth) reaction. A few positive colonies from each eggshell sampling method group were confirmed as serogroup D (Salmonella Enteritidis) with latex and Poly O/Poly H agglutination reactions (Becton Dickinson, Sparks, MD). Statistical Analysis Fisher s exact test (SAS Institute Inc., Cary, NC) was used to identify differences in Salmonella prevalence recovery among the 3 eggshell sampling methods (shell rinse, shell crush with a previous shell rinse, and shell crush only) across the 3 inoculum levels (10 1, 10 2, or 10 3 cfu/eggshell), and between samples collected at 1 or 24 h. For all analyses, significance was determined at P < RESULTS AND DISCUSSION Eggshells that were rinse sampled 1 h after inoculation exhibited a 16% recovery of Salmonella for inoculation level at 10 1, 49% at 10 2, and 93% at 10 3 cfu/ eggshell (Table 1). With shell crush after shell rinse at the inoculation level of 10 1 cfu/eggshell, there was 0% recovery, at 10 2 there were 3% positive, and at 10 3 there were 17% positive. Crushed eggshells (without prior shell rinse) were 23% positive for inoculation level at 10 1, 69% positive at 10 2, and 96% positive at 10 3 cfu/eggshell. After holding inoculated eggs for 24 h at 24 C, rinse sampling eggshells resulted in 3% positive at 10 1, 12% positive at 10 2, and 22% positive at 10 3 cfu/eggshell (Table 1). Shell crush after previous rinsing recovery was lowest with 2% positive at 10 1, 8% positive at 10 2, and only 5% positive at 10 3 cfu/eggshell. Crushing the eggshell (without prior shell rinse) resulted in 2% positive at 10 1, 32% positive at 10 2, and 42% positive at 10 3 cfu/eggshell. In this study crushing eggshells after a previous eggshell rinse significantly reduces the recovery of Salmonella in comparison with both shell rinse and shell crush only sampling methods at both 1 and 24 h following inoculation. It has been previously demonstrated that shell crush after previous shell rinse yielded significantly lower (0.5 log cfu/ml) aerobic plate counts compared with shell rinse and shell crush only sampling methods (Spickler et al., 2011). Salmonella recovery was significantly higher for shell crush only than for shell rinse at 1 h postinoculation with 10 2 cfu/eggshell and at 24 h postinoculation with 10 2 and 10 3 cfu/eggshell. The lack of a significant difference in Salmonella recovery between shell rinse and shell crush sampling at 1 h postinoculation with 10 3 cfu/eggshell or at 24 h postinoculation at 10 1 cfu/eggshell may be attributed to the corresponding very high or very low recoveries observed. Salmonella recovery from rinsing eggshells was comparable with crushing eggshells at inoculation levels of 10 1 and 10 3 cfu/eggshell when sampled at 1 h postinoculation. However, at 10 2 cfu/eggshell, eggshell crushing resulted in 20% more positive samples, indicating that when sampling to recover Salmonella, it is recommended to use shell crush as a sampling method. Crushing eggshells after rinsing was a significantly less sensitive recovery method, with a maximum of 17% recovery obtained at inoculation levels of 10 3 cfu/eggshell

4 2120 Webb et al. and 8% at 10 2 cfu/eggshell. Similar trends in shell rinse versus shell crush sampling methods were observed with shell eggs (Musgrove et al., 2005). Generally shell crush sampling was more effective in detecting Salmonella than shell rinse but not for every farm sampled. Only 2 eggshells sampled 24 h after inoculation (one for each 10 1 and10 2 cfu/egg) had the eggshell rinses negative and the subsequent eggshell crush positive for Salmonella. This suggests that the significantly lower recovery for eggs sampled 24 h after inoculation by eggshell rinse was not attributed to firmer attachment of Salmonella to the eggshell, compared with sampling at 1 h. However, Salmonella that were firmly attached would be recovered with incubation of the eggshell for 24 h in BPW before plating. If adhesion of Salmonella to the eggshell had contributed to lower recovery by eggshell rinse at 24 h, then the subsequent eggshell crush samples would have resulted in significantly higher recovery, which did not occur. Furthermore, Kraft et al. (1958) demonstrated that the internal shell membranes can restrict bacteria penetration through the eggshell to the albumen for 1 to 24 h postinoculation but that the membranes were not bactericidal. Williams et al. (1968) found that eggshells are penetrated almost immediately when challenged with moist Salmonella-contaminated chicken feces. The cuticle layer may serve as the first line (external) of defense of the egg while the internal shell membranes are but a temporary barrier to bacterial movement, then shell structure assumes a critical role as a physical impediment of bacteria penetration (Nascimento et al., 1992). The cuticle is a complex entity on the shell surface with water resistant and antibacterial properties consisting of the inner vesicular cuticle layer and the outer nonvesicular cuticle layer, both layers varying in thickness (Sparks and Board, 1984; Fraser et al., 1999). The cuticular vesicles contain crystalline/granular material rich in the phosphoric mineral hydroxyapatite (Fink et al., 1993) that may be involved in the termination of calcite crystal growth of the eggshell (Dennis et al., 1996). Lipoprotein and protein-lipid complexes are also major components of the eggshell cuticle but were not considered bactericidal (Sparks, 1994). However, Wellman-Labadie et al. (2008, 2010) demonstrated that eggshell surface constituents (cuticle proteins and lipid components) possess antimicrobial activity in vitro when extracted from the cuticle and outer eggshell. Rose-Martel et al. (2012) recovered additional antimicrobial proteins from the cuticle in eggshell rinses (lysozyme C, ovotransferrin, ovocalyxin-32, cystation, and ovinhibitor). Eggshell proteins within the cuticle and on the outer eggshell are in contact with the external environment and represent the first line of defense for the avian embryo. Gram-positive bacteria dominate the flora on the outside of eggs, possibly because of their tolerance of dry conditions, whereas gram-negative bacteria dominate the interior of rotten eggs (Board and Tranter, 1986). It is likely that the antimicrobial activities present within the cuticle influence the selection of gram-negative bacteria that occurs during the process of infection of the egg contents (Seviour and Board, 1972). An egg holding room RH of above 95% was required to grow pseudomonads on the eggshell surface for eggs incubated at 30 C (Board et al., 1979). Messens et al. (2005) reported only 38.7% of the hen eggshells were penetrated by Salmonella Enteritidis when dip inoculated (2.59 log cfu inoculum) and subsequently stored at 20 C and 60% RH. Most shell penetration occurred on d 1 with 95% of those shells penetrated by d 6 of storage, and as hens aged and shell characteristics changed there were no significant correlations with the shell penetration. Musgrove et al. (2010) used 3 methods of eggshell bacterial inoculation (egg immersion, fecal smear, or drop) and concluded that inoculating eggs by immersion into a solution of bacteria creates the greatest challenge to sanitizer chemicals and none of those chemicals tested were able to completely eliminate the inoculated Salmonella. In contrast, inoculating eggs using a single drop of Salmonella in suspension resulted in eggshells that could be sanitized and thereby enabled the efficacy of sanitizing chemicals to be compared. In the breeder hen nestbox, eggshell contact with feces or litter results in a localized region of the shell coming in contact and contamination by Salmonella. Immersing eggs in Salmonella solutions for inoculation has been commonly used for evaluating long-term egg storage (Jones et al., 2004; 1 to 5 wk), inoculum ph from 5.0 to 9.5 (Sauter et al., 1979), or extreme sanitization protocols such as vacuum immersion (Cox et al., 2000a; 0.4 bar) or irradiation (Serrano et al., 1997; 0.5 kgy) with recoveries greater than 95% after drying for 24 h in nontreated positive control eggshells. Both the level of inoculum and sampling time significantly affected Salmonella recovery in this study. At both 1 and 24 h postinoculation, eggshells inoculated with 10 3 cfu of Salmonella had the highest recovery, eggshells inoculated with 10 2 cfu had medium recovery, and eggshells inoculated with 10 1 cfu had the lowest recovery. After 24 h the recovery of Salmonella decreased for all sampling methods across all inoculation levels. Garcia et al. (2011) reported only 34% Salmonellapositive eggshell rinses (a composite of 6 eggs) from a Salmonella Enteritidis-positive cage layer flock at 27 wk of age and they were unable to recover Salmonella from the egg contents. The eggs had been shipped and held under refrigeration before sampling on the day following lay. Kretzschmar-McCluskey et al. (2009) evaluated eggs from commercial layer strains and recovered only 1 Salmonella positive shell rinsate (Salmonella Infantis) at 20 wk and 1 shell and membranes rinsate (Salmonella Ohio) at 40 wk. They used pooled samples of 9 eggs, resulting in a Salmonella recovery level of 0.83% from 120 egg sets. These eggs had been collected the day before sampling and held at 4 C prior and both the eggshell and the eggshell plus membranes rinse were enriched by incubation for 24 h at 37 C before plating. Gast and Beard (1992) challenged hens with Salmonella

5 SALMONELLA RECOVERY FROM INOCULATED HATCHING EGGS Enteritidis and sampled egg contents on the day of lay and after 7 d for eggs held at 7.5 or 25 C. On the day of lay, 3% (4+/132 eggs) of the egg contents were Salmonella-positive, which did not change after 7 d when eggs were held at 7.5 C (3.7%, 5+/134 eggs) but significantly increased to 19% (22/138 eggs) for eggs held at room temperature of 25 C. In the present study, egg contents were not sampled, so it is not known whether Salmonella penetration into the contents occurred during the 24 h holding period. These results reaffirm recommendations that eggshell sampling for Salmonella should occur as soon as possible after the egg has been laid on the farm to detect low levels ( cfu) of Salmonella. Sampling eggshells by crushing the shell and membranes is a more sensitive method than rinsing the eggshell when low levels of Salmonella ( cfu/eggshell) are present. However, at higher levels of Salmonella (10 3 cfu/eggshell) the nondestructive eggshell rinsing method was just as sensitive as eggshell crushing. The significantly lower recovery from eggshells that were rinsed before sampling by eggshell crush indicates that sampling should occur before all sanitizing, rinsing, or washing of the eggs. Future research should evaluate Salmonella recovery from eggshells for eggs sampled on the day of lay and compared with the recovery of those eggs held at room or the recommended refrigerated temperatures (7.2 C for table eggs and 20 C for hatching eggs) and sampled the following day. ACKNOWLEDGMENTS The authors are grateful for the assistance provided by Jeromey S. Jackson and Luanne L. Rigsby of the USDA-ARS Richard B. Russell Research Center for media preparation and plating, and Beverly L. McLendon of The University of Georgia for arranging egg collection and pickup on the day of lay. REFERENCES Baker, R. C., and C. 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