PROCESSING, PRODUCTS, AND FOOD SAFETY

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1 PROCESSING, PRODUCTS, AND FOOD SAFETY Salmonella Populations and Prevalence in Layer Feces from Commercial High-Rise Houses and Characterization of the Salmonella Isolates by Serotyping, Antibiotic Resistance Analysis, and Pulsed Field Gel Electrophoresis X. Li,* J. B. Payne, F. B. Santos, J. F. Levine,* K. E. Anderson, and B. W. Sheldon 1 *Department of Population Health and Pathobiology, and Department of Poultry Science, North Carolina State University, Raleigh ABSTRACT Salmonella species are recognized as a major wk (second peak of production cycle)]. Bird ages and cause of foodborne illnesses that are closely associated with the consumption of contaminated poultry and egg molting practice did not significantly affect (P > 0.05) Salmonella populations with an average of 1.25, 1.27, 1.20, products. The objectives of this study were to evaluate and 1.14 log most probable number/g for the 18-, 25- to the Salmonella populations and prevalence in layer feces 28-, 66- to 74-, and 75- to 7-wk birds, respectively. However, the 18-wk birds had the highest prevalence of Salmo- during the laying cycle and molting of the hen and to nella (55.6%), followed by the 25- to 28-wk birds (41.7%), characterize the layer fecal Salmonella isolates by serotyping, antibiotic resistance analysis, and pulsed field gel 75- to 78-wk birds (16.7%), and 66- to 74-wk birds (5.5%). Of the 45 Salmonella isolates characterized, the most predominant serovar was Salmonella Kentucky (62%). Thirty- electrophoresis. Fecal samples were collected from a commercial layer complex consisting of 12 houses. Composite five percent of the Salmonella isolates were resistant to fecal samples across each row were collected as a function at least 1 antibiotic. As expected, considerable genetic of bird age [18 wk (at placement), 25 to 28 wk (first peak of production cycle), 66 to 74 wk (molting), and 75 to 78 diversity was observed within and across the different serovars. Key words: Salmonella, layer feces, population, prevalence, characterization 2007 Poultry Science 86: INTRODUCTION The Centers for Disease Control and Prevention (CDC) estimate that approximately 40,000 cases of salmonellosis are diagnosed annually in the United States, resulting in approximately 600 deaths (Mead et al., 1999). Although Salmonella are generally inactivated during standard cooking practices and consumer education efforts advocate careful handling and preparation of meat products, contaminated eggs and animal meat products continue to contribute to the number of human salmonellosis cases. Raw meat and poultry are recognized as the primary sources for transmitting Salmonella species to humans, with 40% of the clinical cases attributed to the consumption of egg and poultry products (Sanchez et al., 2002). Modern livestock industries, including poultry production, are frequently unprofitable unless a significant economy of scale can be achieved (Bossman, 2005). To achieve this economy of scale, poultry are generally reared under 2007 Poultry Science Association Inc. Received August 19, Accepted December 2, Corresponding author: brian_sheldon@ncsu.edu confinement in large populations. High-rise poultry housing consists of an arrangement of cages for laying hens in which manure generated in each cage is dropped through an open space beneath the cages and stored below on the lower poultry house floor. Typically, a 10,000- bird commercial layer flock can generate more than 100 tons of manure per year on a wet-weight basis (Patterson and Lorenz, 1996; Lorimor and Xin, 1999). In general, high-rise houses only require manure removal once a year during the molting period or when flocks are changed. After removal, manure is generally used as fertilizer and applied to fields for growing agricultural commodities (US Poultry and Egg Association, 1998). The large volume of manure produced in concentrated poultry production areas can contribute to the contamination of ground and surface waters (Mallin and Cahoon, 2003). Most environmental concerns over land application of animal manure have focused on either the effect of applied nutrients, especially N and P; on surrounding water quality; or have emphasized odor problems and air quality issues. However, the presence of human pathogens such as Salmonella in agricultural soils amended with manure may also pose a public health risk. Salmonella spp. have frequently been found in broiler (Payne et al., 2005) 591

2 592 LI ET AL. and turkey (Santos et al., 2005) feces. For laying hens, factors such as the egg production cycle may affect Salmonella shedding; however, their specific role in the dynamics of Salmonella ecology in commercial egg production facilities is not clearly defined. These factors may influence the prevalence and populations of Salmonella in layer feces. Accordingly, we measured the prevalence and populations of Salmonella in feces collected from a commercial laying hen complex during different phases of the production cycle: at pullet replacement, peak of first egg production cycle, molting, and peak of the second egg production cycle. Salmonella isolates obtained were also characterized by serotyping, antibiotic resistance analysis, and pulsed field gel electrophoresis (PFGE). Although the focus of this study was not specifically directed at quantifying the actual safety risks associated with land application of layer wastes, it does provide an initial assessment of populations, serotypes, and antibiotic resistance and PFGE profiles of Salmonella species found in layer wastes, which would benefit future risk assessment studies directed at these waste management practices. MATERIALS AND METHODS Study Site and Sample Collection This study was conducted in a commercial layer facility containing 12 high-rise houses. Each house measured ft ( m) and housed approximately 77,000 egg-laying White Leghorn hens in cages arranged across 6 rows. Temperatures in the layer houses were maintained from 20 to 28 C. Pullets were placed at 18 wk of age. Each flock replacement per house took 2 to 3 d. All flocks were molted from 66 to 74 wk, resulting in 2 egg production cycles. Birds were fed a low-protein energy basal diet during the first 2 wk of the molting period, a lowenergy Ca diet during the third through sixth weeks, and a postmolt diet during the seventh and eighth weeks. Approximately 300 g of fresh feces was collected under the cages across the entire length of each row, mixed, and stored in a sterile Whirl-Pak bag (Fisher Scientific Int., Bohemia, NY). The samples were kept in coolers containing blue ice and analyzed for Salmonella on the same day of sample collection. Salmonella Enumeration and Detection A most probable number (MPN) procedure was used to estimate Salmonella populations in layer feces. Upon arrival at the laboratory, the fresh fecal samples were thoroughly mixed by hand while contained in the Whirl- Pak bags. Twenty-five grams of each composite sample was placed into a sterile stomacher filter bag containing 50 ml of buffered peptone water (BPW; Oxoid, Ogdensburg, NY). Individual bags were stomached for 1 min. For preenrichment, the mixtures were serially diluted in BPW, incubated at 37 C for 18 to 24 h (Morinigo et al., 1986; Tate and Miller, 1990; Tate et al., 1992), and then 0.1 ml of the appropriate dilutions from each tube were transferred to 3 tubes containing 10 ml of Rappaport- Vassiliadis (RV) broth (Oxoid). All RV tubes were incubated at 42 C for 18 to 24 h for selective enrichment of Salmonella spp. Following incubation, 1 loopful (approximately 10 L) from each RV tube was streaked onto modified Lys-Fe agar (Oxoid), selective medium for Salmonella, and incubated at 37 C for 18 to 24 h. Suspect black colonies on modified Lys-Fe agar plates were picked and confirmed for Salmonella by inoculation onto triple-sugar Fe slants (Oxoid) and agglutination using Salmonella poly- O antiserum (Difco Laboratories, Detroit, MI). Populations of Salmonella spp. were calculated using Thomas approximation of MPN/g = P/(NT) 1/2, where P = the number of positive tubes; N = the total quantity of sample (g) in all negative tubes; and T = the total quantity of sample (g) in all tubes (Swanson et al., 2001). The minimum detection limit of this method was 10 organisms/ g of excreta (1 log). The prevalence of Salmonella in the excreta was also tested as follows. Each 25-g sample was placed into a sterile stomacher filter bag containing 100 ml of BPW. The bags were stomached for 1 min, and then an additional 125 ml of BPW was added, mixed thoroughly, and incubated at 37 C for 18 to 24 h. One milliliter from each bag was added to a bottle containing 100 ml of RV broth and incubated at 42 C for 18 to 24 h. The remaining procedures for isolating and identifying Salmonella were as described above. Serotyping Forty-five Salmonella isolates (approximately 50% of total isolates) collected from laying hen feces were selected for serotyping (18, 17, 7, and 3 isolates taken from the 18-, 25- to 28-, 66- to 74-, and 75- to 76-wk-old layer samples, respectively). For serotyping, the Salmonella isolates were transferred onto tryptic soy agar (Difco Laboratories) slants, grown overnight at 37 C, and shipped by overnight courier to the USDA National Veterinary Service Laboratories in Ames, Iowa. Antibiotic Resistance Analysis The antimicrobial susceptibility of the 45 Salmonella isolates submitted for serotyping was determined for 15 antimicrobials listed in Table 1 using the disk diffusion method described by the National Committee for Clinical Laboratory Standards (2000). Antimicrobials selected for these assays reflect the recommendations of the National Committee for Clinical Laboratory Standards. The susceptibility tests were conducted using the Sensititre susceptibility system (Sensititre, Trek Diagnostic Systems Inc., Cleveland, OH). The system encompasses a microtiter plate that is dosed with 15 individual antimicrobial agents at specified concentrations. A standard protocol and resistance break point developed under the National Antimicrobial Resistance Monitoring System was followed to study the susceptibility of the Salmonella isolates. Each Salmonella isolate was cultured on brain heart infu-

3 SALMONELLA POPULATION AND PREVALENCE IN LAYER FECES 593 Table 1. Antibiotics, their concentrations in the microtiter plate, and minimum inhibitory concentration (MIC) break point MIC break point ( g/ml) Concentration Antibiotic (abbreviation) ( g/ml) Resistant Sensitive Amikacin (Ami) 0.5 to Ampicillin (Amp) 1 to Amoxicillin-clavulanic acid (Amo) 1/0.5 to 32/16 32/16 8/4 Ceftriaxone (Ceftri) 0.25 to Chloramphenicol (Chlor) 2 to Ciprofloxacin (Cip) to Trimethoprim-Sulfamethoxazole (Sulfa) 0.12/2.38 to 4/76 4/76 1/19 Cefoxitin (Cefox) 0.5 to Gentamicin (Gen) 0.25 to Kanamycin (Kan) 8 to Nalidixic acid (Nal) 0.5 to Sulfisoxazole (Sulfi) 16 to Tetracycline (Tet) 4 to Streptomycin (Strep) 32 to Ceftiofur (Ceftio) 0.12 to sion agar plates (Oxoid) at 37 C for 18 to 24 h. A nephelometer (Promega, Madison, WI) was calibrated using a 0.5 McFarland BaSO 4 turbidity standard (Sensititre, Trek Diagnostic Systems Inc.). One to 2 colonies from the brain heart infusion agar plate were transferred to 5 ml of sterile saline (0.9% NaCl) and adjusted to a 0.5 McFarland reading using the nephelometer. Seventy-five microliters of the saline cell suspension was then transferred to 10 ml of Mueller Hinton broth (Oxoid). After transfer of 50 L of each Mueller Hinton broth cell suspension into separate wells of a microtiter plate containing 15 antimicrobials as listed in Table 1, the plate was sealed with an adhesive seal and incubated at 37 C for 18 to 24 h. The contents of the wells were manually read for bacterial growth under a fluorescent lamp. Salmonella Typhimurium DT104 (ATCC ) was used as the quality control strain for this assay. PFGE The CDC PulseNet 1-d standardized laboratory protocol for molecular subtyping of Escherichia coli O157H7, nontyphoidal Salmonella serotypes, and Shigella sonnei by PFGE was used for extraction of Salmonella genomic DNA and for establishing the analysis conditions (CDC, 2004). After electrophoresis, the gels were stained for 20 min in 0.01% ethidium bromide solution and then destained 3 times for 30 min each in distilled water. Gel images were digitally captured using an Alphaimager (model 3300, Alpha Innotech Corp., San Leandro, CA). Gels were visually analyzed with PFGE profiles differing in 1 or more bands considered different. Statistical Analysis The composite fresh fecal sample collected under each row of cages served as an independent trial replicate. There were 18, 24, 18, and 18 replicates evaluated for the 18-wk (pullet placement), 25- to 28-wk (peak of first production cycle), 66- to 74-wk (molting), and 75- to 76- wk-old (peak of second production cycle) birds, respectively. The independent variable was bird ages. The Salmonella population data were analyzed by the GLM procedure for ANOVA. The residual replicate age MS was used for testing the main effects (age, replicates; SAS Institute, 2000). When a significant effect was observed, means were compared using the PDIFF option of SAS. Moreover, the χ 2 test was used to determine if Salmonella prevalence differences were significant among the 4 ages of birds. Model and parameter adequacy were considered significant at P 0.05 unless otherwise noted. RESULTS AND DISCUSSION Salmonella Populations and Prevalence Salmonella populations and prevalence for the 18, 25- to 28-, 66- to 74-, and 75- to 76-wk-old birds are shown in Table 2. No significant difference (P > 0.05) in Salmonella populations was detected among the 4 age groups (overall mean of log 1.21 MPN/g). This finding may be due in part to the large SD and relatively low population of Salmonella recovered from the feces (mean of <20 MPN/ g). In contrast, there were significant differences (χ 2 value of ) in the prevalence of Salmonella among these groups. The 18-wk-old hens had the highest prevalence of Salmonella (55.6%), followed by the 25- to 28-wk-old hens, who had a prevalence of 41.7%. Salmonella prevalence (5.5%) was lowest in the molted hen groups. Critics of the shell egg industry have reported that layers are physiologically stressed when reared in close confinement in caged systems and during egg production and molting. Induced molting is used to rejuvenate the reproductive system of laying hens and to improve eggshell quality and egg production (Landers et al., 2005). An induced molt is generally accomplished by either fasting the flock or by limiting critical nutrients such as protein, Ca, and Na until or beyond the time egg production ceases. The findings from the present study indicate that the molting program used by this commercial producer

4 594 LI ET AL. Table 2. Populations and prevalence of Salmonella spp. in layer feces Population (log MPN/g) Bird age 1 (wk) Prevalence Mean ± SD 2 Population range (10/18) 1.25 ± 0.10 <1.00 to to (10/24) 1.27 ± 0.11 <1.00 to to (1/18) 1.20 ± 0.15 <1.00 to to (3/18) 1.14 ± 0.07 <1.00 to 1.89 Across all ages 30.8 (24/78) 1.21 ± 0.11 <1.00 to A total of 18, 24, 18, and 18 independent replicates were used for testing prevalence and most probable number (MPN) for 18-, 25- to 28-, 66- to 74-, and 75- to 76-wk-old birds, respectively. 2 Detection limit for MPN method was 1 log MPN/g. did not exacerbate Salmonella shedding problems. During the molting period, the hens were not fasted but received a modified molting diet. Holt (2003) also found that the use of low-energy, low-ca diets instead of complete feed withdrawal dramatically decreased Salmonella shedding during the molting process. However, laying hens that have been experimentally infected with 10 6 Salmonella Enteritidis organisms yielded the highest level of fecal shedding during molting (Holt and Porter, 1992, 1993; Holt, 1993; Holt et al., 1994). These previous findings have also shown that under experimental and control conditions, induced molting by feed removal significantly depressed the cell immune response of layers and increased the susceptibility of the hen to Salmonella contamination (Holt, 1992, 1999). The potential sources of Salmonella contamination on poultry farms are numerous and include pullets, air, feed, water, flies, rodents, and humans (Hoover et al., 1997; Craven et al., 2000; Heyndrickx et al., 2002; Liljebjelke et al., 2005). For the commercial farm evaluated in this study, we observed numerous flies outside the layer houses but few inside. Periodically we also observed a few dead rodents inside the houses. Characterization of Salmonella Isolates Eight Salmonella serotypes were identified from the processed fecal samples (Table 3): Salmonella Kentucky (62% of isolates), Montevideo (11%), Typhimurium (var. 5-, 4%), Heidelberg (4%), Senftenberg (2%), 8,(20): Nonmotile (2%), 8,(20):-:z6 (2%), and an untypeable serovar (11%). Surveillance studies of Salmonella serotypes conducted by the CDC identified Salmonella enterica Typhimurium and Enteritidis as the 2 most commonly reported serovars associated with human illness (CDC, 2003); Salmonella Heidelberg, Montevideo, and Senftenberg were also listed among the top 20 serovars identified in the 2003 report (CDC, 2003). The USDA (1999) found that although Salmonella Kentucky was not among the most common serovars isolated from human sources, approximately 50% of the isolates from chicken and turkey sources were Salmonella Kentucky. In the present study, Salmonella Kentucky was the most prevalent serotype (62%) identified across 3 of the 4 age categories (18, 25 to 28, and 75 to 76 wk); Salmonella Enteritidis, an egg-associated serovar, was not detected. According to the USDA (1999) report, Salmonella serovars routinely isolated from broilers and turkeys have been markedly different than those isolated from human clinical patients. The factors for why Salmonella Kentucky is commonly found in poultry but not in human clinical isolates is not clear. Proposed explanations include host-specific differences, varying dose responses, and the varied cultural methods used by different laboratories (Juven et al., 1984; Chalker and Blaser, 1988; Mohammed and Hinton, 1993; Sarwari et al., 2001). These 45 Salmonella isolates with known serovars were further characterized by PFGE and antibiotic resistance profiling. Thirty-five percent (16 of 45) of the Salmonella isolates were resistant to at least 1 antibiotic (Table 4). There were 10 antibiotic resistance patterns detected including Tet, Strep, Strep/Tet, Amp/Ceftio, Amp/Tet, Amp/Tet/Ceftio, Amp/Cefox/Ceftio, Amp/Strep/Tet, Amp/Strep/Tet/Ceftio, and Amp/Strep/Tet/Cefox/ Ceftio. A high percentage of the serotyped Salmonella iso- Table 3. Serotypes of Salmonella isolates Distribution by bird age (wk) Serotype Isolates (n) Percentage to to to 76 Kentucky Salmonella untypeable Montevideo Typhimurium (var. 5-) Heidelberg Senftenberg ,(20): Nonmotile ,(20):-:z Total

5 SALMONELLA POPULATION AND PREVALENCE IN LAYER FECES 595 Table 4. Antibiotic resistance and pulsed field gel electrophoresis (PFGE) profiles by serotype for Salmonella strains isolated from layer feces Isolates Resistant Resistance PFGE Resistance Serotype (n) isolates (n) rate (%) type profile 1 Kentucky D, F, G, H Tet Strep Strep/Tet Amp/Tet/Ceftio Amp/Strep/Tet/Ceftio Untypeable E Amp/Ceftio Amp/Cefox/Ceftio Amp/Strep/Tet/Cefox/Ceftio Montevideo A, I Amp/Tet Typhimurium (var. 5-) C Amp/Strep/Tet Heidelberg D NR 3 Senftenberg B NR 8,(20): Nonmotile Untypeable NR 8,(20):-:z Untypeable NR Total Antibiotics are defined in Table 1. 2 A total of 45 Salmonella isolates were serotyped and tested for antimicrobial susceptibility and PFGE types. 3 NR = no antibiotic resistance detected. lates were resistant to tetracycline, ampicillin, streptomycin, and ceftiofur, which are widely used in the treatment of human systemic salmonellosis (D Aoust et al., 1992). No detectable resistance to gentamicin, kanamycin, and nalidixic acid was observed. Livestock and poultry producers rely on antibiotics to treat a host of diseases and infections as well as use low doses of antibiotics as growth promoters. Such treatments help promote the health and well-being of the animal. However, there are concerns about the potential influence of antibiotic use on the development of bacterial-resistant factors that limit antimicrobial efficacy (Levy, 1987). Among the 8 identified serotypes, Salmonella Kentucky, untypeable, Typhimurium (var. 5-), and Montevideo were antibiotic resistant, whereas Salmonella Heidelberg and Senftenberg, 8,(20): Nonmotile and 8,(20):-:z6 were susceptible to all of the antibiotics tested. There were 5, 3, 1, and 1 antibioticresistant patterns observed for Salmonella Kentucky, un- typeable, Montevideo, and Typhimurium (var. 5-), respectively. Among the 16 Salmonella isolates that were resistant to antibiotics, 8, 5, 1, and 2 of the isolates were associated with Salmonella Kentucky, untypeable, Montevideo, and Typhimurium, respectively. When expressed as percentage of resistance, 29 (8 of 28), 100 (5 of 5), 20 (1 of 5), and 100% (2 of 2) of the Salmonella Kentucky, untypeable, Montevideo, and Typhimurium samples exhibited antibiotic resistance, respectively. Concerning layer age, 17 (3 of 18), 47 (8 of 17), 43 (3 of 7), and 67% (2 of 3) of the antibiotic-resistant Salmonella serotypes fell within the 18-wk, 25- to 28-wk, 66- to 74-wk, and 75- to 76-wk-old layers, respectively (Table 5). Among the Salmonella isolates typed using PFGE, there were 9 different genotypes identified: A, B, C, D, E, F, G, H, and I (Table 4). Isolates were considered genetically indistinguishable when their restriction patterns had the same number of bands and the corresponding bands were Table 5. Antibiotic resistance profiles of Salmonella isolates by bird age Bird age Isolates tested Resistant Resistance Resistance (wk) (n) isolates (n) rate (%) profile Tet Amp/Strep/Tet 25 to Tet Strep Strep/Tet Amp/Ceftio Amp/Tet/Ceftio Amp/Cefox/Ceftio 66 to Amp/Tet Amp/Strep/Tet/Ceftio Amp/Strep/Tet/Cefox/Ceftio 75 to Strep/Tet Amp/Cefox/Ceftio Total Antibiotics are defined in Table 1. 2 A total of 45 Salmonella isolates were tested for antimicrobial susceptibility.

6 596 LI ET AL. of the same size (Tenover et al., 1995). The Salmonella Kentucky serotype possessed 4 different PFGE types: D, F, G, and H. One of the Salmonella Kentucky strains also shared the same PFGE type D profile with the Salmonella Heidelberg strain. Salmonella strains with similar genotype fingerprint patterns can represent different serotypes, because one or more nucleotide repeating units can alter the transcription signals or change the reading frame of genes encoding for cell surface molecules used for differentiating serotypes. The findings from this study show that in this particular commercial layer complex, multiple Salmonella serovars were isolated of varying antibiotic-resistance patterns and PFGE genotypes; layer life stages also affected the prevalence of Salmonella recovered from the feces. Moreover, the modified diet molting program used on this farm did not increase the incidence of shedding of Salmonella into the feces. Among the 8 Salmonella serovars identified, Salmonella Kentucky was the most common serotype (62%) recovered from the fecal samples. The high prevalence rate of ampicillin, tetracycline, and streptomycinresistant Salmonella strains isolated from layer feces may be a potential public health concern if these organisms were to contaminate surface waters or crops intended for human consumption. Based on the findings of this study, further investigation into the actual safety risks involving land application of Salmonella-infected manures is warranted. ACKNOWLEDGMENTS This work was supported by a USDA Initiative for Future Agricultural and Food Systems grant. We are grateful to the owners of the commercial layer complex for their cooperation on this project. REFERENCES Bossman, D Saving American agriculture and building the rural economy by solving the manure problem. Pages 9 11 in Proc. Dev. Altern. Technol. Process. Use Anim. Waste. North Carolina State Univ., Raleigh. 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