Abstract

Forty layer farms from 2 states participated in a study to examine the risk factors and incidence of Salmonella Enteritidis from multiple samples, including environmental drag swabs from the bird areas, feed, water, flies, rodents, live rodent traps, and environmental swabs from areas occupied by other livestock. Twenty-four of these farms had between 3,000 and 31,000 bird flocks (medium-sized flocks) and 16 had less than 3,000 birds (small-sized flocks). All were housed in cage-free production systems. Twenty-two farms included outside pasture areas for the birds. Most of the participants had just come under the FDA Egg Rule and had not yet tested their flocks (flocks under 3,000 birds are exempt) for Salmonella Enteritidis. Many, however, obtained their pullets from commercial Salmonella Enteritidis-clean breeder sources hatched in National Poultry Improvement Plan hatcheries. Vaccination against Salmonella Enteritidis was performed on 21 of the 40 farms (combination of live and killed vaccines). Salmonella Enteritidis was detected on 7 out of the 40 farms, primarily in rodents, their feces, or from swabs taken inside live traps. Of these 7 Salmonella Enteritidis-positive farms, 3 farms that had vaccinated their pullets with live Salmonella Typhimurium vaccine and killed-Salmonella Enteritidis vaccine; no Salmonella Enteritidis was isolated from the environmental drag swabs taken from the bird area or from the eggs on these farms. However, on the farms that had not vaccinated for Salmonella Enteritidis, the organism was isolated from 4 environmental drag swabs and 3 egg pools. The last 4 farms had flocks under 3,000 birds. No Salmonella Enteritidis was isolated from any of the samples of feed, flies, water, or swabs taken from other livestock areas. Based on the initial findings in this study, we suggest the 2 most important risk factors for Salmonella Enteritidis contamination inside the bird area and in the eggs in these small- and medium-sized flocks are the presence of infected rodents and the absence of an Salmonella Enteritidis vaccination program.

Primary Audience: Egg Industry Personnel, Food Scientists, Veterinarians, Poultry Scientists

DESCRIPTION OF PROBLEM

Salmonella Enteritidis is a leading food-borne pathogen in the United States, with many outbreaks in humans traced back to shell eggs [1]. As a result, the implementation of effective strategies to prevent Salmonella Enteritidis infection in table egg-producing layer flocks is an essential step in reducing Salmonella Enteritidis in humans caused by ingestion of infected eggs. Several states subsequently developed Egg Quality Assurance Programs to reduce the incidence of Salmonella Enteritidis in shell eggs. Pennsylvania was the first to develop such a program based on hazard analysis critical control points principles [2]. On July 9, 2010, the FDA first implemented national regulations to reduce Salmonella Enteritidis contamination of shell eggs by requiring producers to adopt measures such as egg refrigeration, rodent and fly control, on-farm biosecurity, and periodic testing for Salmonella Enteritidis in the bird environment. These regulations were required by all shell egg producers with flocks of 50,000 or more hens (large flocks). However, it was not until July 9, 2012, that similar regulations were required by shell egg producers for flock sizes between 3,000 and 50,000 hens (medium-sized flocks). Whereas considerable information describing Salmonella Enteritidis incidence and risk factors in large flocks exists, including the role of infected rodents, flies, and other biosecurity issues [3–7], it is not known whether the risk factors for Salmonella Enteritidis are similar in small- and medium-sized flocks. Vaccination against Salmonella Enteritidis has shown to be a useful tool to reduce shedding and contamination of this organism in laying hens and their eggs in conventional flocks [8]. However, its usefulness in medium and small flocks has not been well studied. In fact, little is known concerning the overall incidence of Salmonella Enteritidis in medium and small flocks in the United States. Management and housing are quite different in floor houses, and outside access of hens is common, especially in farms identified as organic or pasture-reared. Exposure to wild birds, rodents, and predators is not unusual. Even less is known concerning the incidence of Salmonella Enteritidis in flocks smaller than 3,000 hens (small flocks). Currently, this group is exempt from the FDA rules and regulations for Salmonella Enteritidis monitoring and prevention. However, a growing trend has been observed for direct retail of eggs to consumers from small flocks via home businesses, farmers’ markets, and the surge of domestic chickens kept for eggs in urban settings. Any information on Salmonella Enteritidis status in this group would be useful from a food safety standpoint.

Some comparisons of Salmonella Enteritidis incidence and risk factors have been studied between large, conventionally raised layer operations and smaller, floor-reared flocks, but the results of such studies are often difficult to interpret. Multiple factors come into play, such as flock size, multiple ages, cage production versus deep litter housing, wet versus dry manure, prevalence of disease vectors, and bird stress levels [9–11]. Comparisons are often difficult between such different types of production systems. The objectives of the current study were to determine the incidence of Salmonella Enteritidis infection on egg farms in flocks in the medium- and small-size categories and the significant risk factors for infection in these settings.

MATERIALS AND METHODS

Recruitment of Participating Poultry Farms

Recruiting farms to the study was accomplished by contacting egg farmers and companies with ties to layer flocks with less than 50,000 hens, such as egg processors, feed companies, vaccine or management service providers, and cooperatives in primarily 2 major shell egg-producing states. Three training workshops were held for producers that covered the obligations of the FDA Egg Safety Rule in 2012 and 2013, which included speakers from cooperative extension and the FDA. At these meetings, an informational sheet was shared that described the goals and an invitation to participate in the present study. In exchange for their participation, the project would provide to the farmer (1) all FDA-required Salmonella testing performed by certified testers or project investigators at no cost for 2 yr of the study (including travel and laboratory testing). Sample collection and laboratory testing would be performed according to FDA protocols [12, 13] (2) and flock owners would be given assistance and training with development of their FDA Salmonella Enteritidis plan, including opportunities to participate in workshops on the FDA Egg Safety Rule; (3) in addition, participants would be given 12 rodent live traps for monitoring mouse activity in the egg rooms, coolers, and in the bird area; and lastly, (4) participants or sample collectors would be given a $100 honorarium for their cooperation and access to the farms and flocks.

Three groups of participating farmers were obtained for the study, including those with flocks from 50,000 to 3,000 hens (medium-sized flocks), those with flocks of less than 3,000 hens (small sized flocks), and the pullet farmers rearing these birds. Forty farms were recruited the first year of the study. The majority of these farms were composed of cage-free flocks with some access to outside, fenced pasture. The bulk of the farms also had other animals, including dogs, cats, and other livestock, such as horses, dairy or beef cattle, sheep, goats, deer, pigs, and rabbits. A few also had other poultry, including turkeys, guinea fowl, peafowl, ducks, geese, quail, and miscellaneous types of chickens. Several farms engaged in other on site businesses, including the production and sales of produce, green house plants, construction materials, and eggs.

An on-farm interview gathered participant information, including the name of the pullet or layer farm grower, their phone number, email, and farm address. Hatchery source, hatch date, pullet strain, number of pullets, and date the pullets would be housed in the hen house were recorded as part of the flock history. The destination hen house producers and owners name, address, phone number, and email were recorded as well. Next, a description of the pullet or lay house, dimensions, map, floor plan and description of the lighting program, type of equipment, spacing, ventilation, cage equipment versus floor-reared, and bird density were taken. A description of the watering system (well, spring, city), drinkers, feeders, brooders, or egg-gathering equipment was recorded along with the feed supplier, their address, phone, email, diet phases (nutrients and bird ages), and details about the beak trim and vaccination program, specifically Salmonella live and killed products. Specifics about manure management, storage, mortality management, egg gathering, egg storage (cooler size, temperature, and percent RH), egg price payment, pick up schedule, and the name of the egg processor, address, phone, and email address were recorded. Finally, a map of the surrounding farm structures, barns, pastures with other livestock, and proximity to streams and other surface water was drawn and notations on wild birds, wild life, predators, and any other observations were recorded. A survey questionnaire gathered information on their biosecurity plan and practices, lighting program, vaccination plan (Salmonella Enteritidis-killed bacteria in the leg or breast, age, or live attenuated Salmonella Typhimurium in spray or water), bird health program, water sanitation program, rodent, fly and beetle control program, records kept, equipment borrowed versus owned, labor force (number) and division of labor, egg processing (off-line or in-line), and procedures such as washing temperatures, rinse, packaging materials, egg cooler and refrigeration (temperature and percent RH), and frequency of egg pick-up by the processor.

Cooperator pullet and layer farms were visited at 14 to 16 wk of age as well as 29 to 31, 40 to 45, and 4 to 6 wk postmolt (when applicable) to take environmental samples utilizing the standard procedures of the Pennsylvania Egg Quality Assurance Program for both floor and caged houses [14]. For most houses this meant 6 swabs of the hen or pullet house. Pullet growers were also asked to send in 5 papers used to line the chick boxes from the hatchery to test for the presence of Salmonella on the voided meconium. Additional samples included 1 feed sample from the bin per visit, 1 water sample from the primary source per visit, and culture swabs of any other areas with signs of rodent activity. Rodent live traps in and around the poultry house and egg room were opened and swabbed for Salmonella Enteritidis using a standard culture transport swab with Amies media [15]. If rodents were recovered from the traps they were taken for Salmonella Enteritidis culture as well. A fly sample was captured from the house or egg room using a sticky tape or fly index cards. Additional environmental swabs of any paddocks, coops, pens, or stalls with additional livestock or poultry were taken. All environmental and rodent live trap swabs, and fly tape samples were placed in sterile Whirl-Pak bags [16] and approximately 5 mL of evaporated canned milk was added to each bag, sealed, and stored in a cooler with ice packs until delivery to the laboratory for Salmonella culture. If any of the environmental drag swabs or rodents tested positive for Salmonella Enteritidis, the study participants were asked to submit up to 1,000 eggs every 2 wk for 8 wk total for Salmonella culture. The number of eggs submitted was based on

  • Flocks with 200 hens: 50 eggs per week;

  • Flocks with 500 hens: 100 eggs per week;

  • Flocks with 1000 hens: 200 eggs per week;

  • Flocks with 3,000 hens: 300 eggs per week;

  • Flocks with 5000 hens: 500 eggs per week;

  • Flocks with 10,000 hens: 500 eggs per week;

  • Flocks with 20,000 hens: 800 eggs per week;

  • Flocks with 30,000 hens: 900 per week;

  • Flocks with 40,000 hens: 1,000 eggs per week;

  • Flocks with 50,000 hens: 1,000 eggs per week.

The study participants were reimbursed for the submitted eggs at market value plus 10%.

Salmonella Culture and Identification

All samples except shell eggs were transported to the laboratory in insulated boxes containing ice packs. Samples including shell eggs were kept refrigerated until they were processed. Poultry house drag swabs and shell eggs were processed according to the Bacteriological Analytical Manual Method of FDA [12, 13]. The other sample types were processed according to the protocols used at the Animal Diagnostic Laboratory, Pennsylvania State University, with slight modifications as described in detail below. Agar media [except bismuth sulfite (BS) agar] were purchased as ready-to-use-media from Remel [17] and all other media were purchased from BD Diagnostics [15].

Poultry House Drag Swabs and Other Types of Swabs.

The outside surface of the Whirl-Pak bags containing the environmental gauze pad was disinfected with 70% alcohol, and 100 mL of buffered peptone water were added to the bag. After mixing the contents thoroughly, the bags were incubated for 24 ± 2 h at 35°C. One-milliliter and 0.1-mL volumes of the preenriched sample were transferred into 10 mL of tetrathionate (TT) and 10 mL of Rappaport-Vassiliadis (RV) broths, respectively. The inoculated TT broth medium was incubated at 43 ± 0.2°C for 24 ± 2 h, whereas inoculated RV medium was incubated at 42 ± 0.2°C for 24 ± 2 h in a circulating water bath. A loopful (10 μL) of each broth media was inoculated onto brilliant-green agar with novobiocin (BGN) and xylose lysine tergitol 4 (XLT4) agar plates. The RV medium was incubated for additional 24 h and streaked again onto BGN and XLT4 agar plates. After 24 ± 2 h of incubation at 35°C, the plates were examined for Salmonella-like colonies. On BGN plates, the typical Salmonella colonies appear as pink to white opaque colonies surrounded by red zones in the medium. On XLT4 plates, Salmonella colonies appear black or black-centered with a yellow peripheral area after 18 to 24 h of incubation, and the colonies become entirely black or pink to red with black centers upon continued incubation. Colonies of H2S-negative Salmonella strains appear pinkish yellow on XLT4 plates.

At least 5 colonies of presumptive Salmonella from each plate were inoculated onto triple sugar iron agar and lysine iron agar slants and incubated at 35°C for 24 ± 2 h. Isolates fitting the biochemical profile for Salmonella (alkaline slant and acid butt with or without production of H2S in triple sugar iron and alkaline butt in lysine iron agar) were transferred to trypticase soy agar slants and incubated at 35 ± 0.2°C for 24 ± 2 h. Identification was confirmed using serogroup D1 antiserum. Positive isolates for D1 antiserum were further serotyped as Salmonella Enteritidis at the National Veterinary Services Laboratory [18].

Shell Eggs.

Eggs were gently placed in a rack and the surface was disinfected by spraying with a disinfectant solution consisting of 3 parts 70% ethyl alcohol and one part iodine and potassium iodide. The iodine and potassium iodide solution contained 50 g of iodine and 100 g of potassium iodide per liter and was made immediately before use. Eggs were air-dried and aseptically cracked into a sterile 64 ounce Whirl-Pak bag, pooling 20 eggs into 1 bag. The outside of the bag was thoroughly massaged to ensure that all yolk material was completely blended with the albumen. Bags were incubated at room temperature (20–24°C) for 96 ± 2 h. A 25-mL volume from each bag was preenriched in 225 mL of trypticase soy broth supplemented with ferrous sulfate (35 mg of ferrous sulfate added to 1,000 mL of trypticase soy broth) and incubated at room temperature for 60 ± 5 h. After mixing the contents in the bags thoroughly, the pH of the contents was adjusted to 6.8 ± 0.2 with NaOH or HCl. The bags were incubated for an additional 24 ± 2 h at 35°C. Volumes of 0.1 mL and 1 mL from each bag were transferred into tubes containing 10 mL of RV broth and 10 mL of TT broth, respectively. The RV tubes were incubated at 42 ± 0.2°C for 24 ± 2 h, whereas the TT tubes were incubated at 35 ± 2°C for 24 ± 2 h. The tubes were mixed well and streaked onto xylose lysine desoxycholate agar, freshly prepared BS agar Hektoen enteric agar. The selective or differential agar plates were incubated at 35°C for 24 ± 2 h. The BS agar plates were incubated for an additional 24 h (48 ± 2 h total) and read at 48 h if no typical colonies were present at 24 h. Presumptive colonies of Salmonella (xylose lysine desoxycholate agar: pink colonies with or without black centers or completely black colonies; BS agar: brown, gray or black colonies, may have a metallic sheen; Hektoen enteric agar, blue-green colonies with or without black centers) were inoculated onto triple sugar iron agar and lysine iron agar slants. Presumptive Salmonella were confirmed using serogroup D1 antiserum and serotyped at National Veterinary Services Laboratory.

Mice.

Mice were taken from Whirl-Pak bags and placed in a quaternary ammonium disinfectant (Roccal II) to disinfect surface fur. The mouse carcasses were eviscerated aseptically by opening along the ventral midline with sterile instruments. Viscera from up to 6 mice were pooled together and ground up in a sterile blender system. The contents were transferred into a sterile Whirl-Pak bag containing TT broth and incubated at 35 ± 2°C for 18 to 24 h. A loopful (10 μL) of preenriched broth was inoculated onto BGN and XLT4 plates and incubated at 35°C for 24 ± 2 h. Presumptive colonies of Salmonella were selected, confirmed, and serotyped according to the previously mentioned procedure.

Feed.

A 100- to 125-mL volume of buffered peptone water was added to a Whirl-Pak bag containing 50 g of feed. The contents in the bag were mixed well and incubated at 35 ± 2°C for 2 h. A 500-mL volume of TT broth was added to the bag and incubated at 35 ± 2°C for 18 to 24 h. A 10-µL aliquot from the bag was inoculated onto BGN and XLT4 plates and incubated at 35°C for 24 ± 2 h. Presumptive colonies of Salmonella were selected, confirmed, and serotyped according to the previously mentioned procedure.

Water.

A 10-mg tablet of sodium thiosulfate [19] was added to 200 to 300 mL of water to neutralize any preexisting treatment with chlorine. The sample was filtered through a 250-mL filter assembly with a 0.45-µm pore size [20]. The membrane filter was then removed aseptically from the filter assembly, placed in 100 mL of TT broth, and incubated at 35°C for 24 ± 2 h. A 10-µL aliquot of preenriched sample was inoculated onto BGN and XLT4 plates and incubated at 35°C for 24 ± 2 h. Presumptive colonies of Salmonella were selected, confirmed, and serotyped according to the previously procedure.

Flies and Fly Cards or Tapes.

Flies, fly tapes, and fly cards collected from a single poultry house were pooled and enriched in 100 mL of TT broth as indicated for other sample types. After incubation at 35°C for 24 ± 2 h, presumptive colonies of Salmonella were selected, confirmed, and serotyped according to the previously mentioned procedure.

RESULTS AND DISCUSSION

Between the 2 states participating in the study, a total of 40 farms were tested for Salmonella Enteritidis in the first year of the project. Of these, 24 farms had 3,000 to 31,000 bird flocks and 16 had less than 3,000 birds per flock. All were cage-free production systems. Four were farms housing purebred chickens for hatching egg production for a commercial hatchery. Vaccination against Salmonella Enteritidis was performed on 21 of the 40 farms (combination of live and killed vaccines).

A total of 103 chick box papers, 769 environmental drag swabs, 81 samples of flies, 105 water samples, 114 feed samples, 98 live trap boxes or rodents or their pellets, and 105 manure and environmental drag swabs were collected from other areas on the farms where livestock were housed or wild birds such as pigeons were roosting. None of the feed, water, flies, or other samples from livestock areas were positive for Salmonella Enteritidis (Tables 1 and 2).

Table 1.

Salmonella Enteritidis-positive farms

Farm Hens (no.) Age (wk) Salmonella Enteritidis vaccination status Salmonella Enteritidis status Animals on farm 
9,000 45 Yes, Samonella Typhimurium live vaccine in hatchery at 2 wk; killed-Salmonella Enteritidis injection at 12 wk Positive rodent culture; negative drag swabs, negative eggs Horses, cats, dogs 
7,288 30 Yes, Salmonella Typhimurium live vaccine in hatchery at 2 wk; killed-Salmonella Enteritidis injection at 12 wk Positive rodent culture; negative drag swabs, negative eggs Silkie chickens 
5,500 15 Yes, Salmonella Typhimurium live vaccine in hatchery at 2 wk Positive rodent culture; negative drag swabs Horses, dairy cattle, goats, pigs 
2,999 14 No Positive rodent trap swab, positive drag swabs Horses, beef cattle, pigs, cats, dog 
1,800 15 No Positive drag swab, positive eggs Horses 
  30 No   
1,500 15 No Positive drag swab, positive eggs Horses 
  30 No   
1,000 45 No Positive drag swab, positive eggs Mixed backyard chickens 
Farm Hens (no.) Age (wk) Salmonella Enteritidis vaccination status Salmonella Enteritidis status Animals on farm 
9,000 45 Yes, Samonella Typhimurium live vaccine in hatchery at 2 wk; killed-Salmonella Enteritidis injection at 12 wk Positive rodent culture; negative drag swabs, negative eggs Horses, cats, dogs 
7,288 30 Yes, Salmonella Typhimurium live vaccine in hatchery at 2 wk; killed-Salmonella Enteritidis injection at 12 wk Positive rodent culture; negative drag swabs, negative eggs Silkie chickens 
5,500 15 Yes, Salmonella Typhimurium live vaccine in hatchery at 2 wk Positive rodent culture; negative drag swabs Horses, dairy cattle, goats, pigs 
2,999 14 No Positive rodent trap swab, positive drag swabs Horses, beef cattle, pigs, cats, dog 
1,800 15 No Positive drag swab, positive eggs Horses 
  30 No   
1,500 15 No Positive drag swab, positive eggs Horses 
  30 No   
1,000 45 No Positive drag swab, positive eggs Mixed backyard chickens 
Table 2.

Summary of Salmonella Enteritidis culture results

Culture location Total sampled Salmonella Enteritidis-positive (no.) 
Farms tested 40 7 (17.5%) 
Rodents, feces or live trap swab 98 4 (4%) 
Environmental drag swabs inside poultry houses 769 4 (0.5%) 
Egg pools from Salmonella Enteritidis-positive farms 3 (60%) 
Feed samples 114 0 (0%) 
Pullet chick box papers 103 0 (0%) 
Water samples 105 0 (0%) 
Fly samples 81 0 (0%) 
Culture location Total sampled Salmonella Enteritidis-positive (no.) 
Farms tested 40 7 (17.5%) 
Rodents, feces or live trap swab 98 4 (4%) 
Environmental drag swabs inside poultry houses 769 4 (0.5%) 
Egg pools from Salmonella Enteritidis-positive farms 3 (60%) 
Feed samples 114 0 (0%) 
Pullet chick box papers 103 0 (0%) 
Water samples 105 0 (0%) 
Fly samples 81 0 (0%) 

Salmonella Enteritidis was detected on 7 of the 40 farms participating in the present study (Tables 1 and 2). Three of these farms had greater than 3,000 bird flocks (medium-sized flock category), 4 had under 3,000 bird flocks (small flock category). Of these Salmonella Enteritidis-positive farms, 4 of the 40 were associated with positive rodents (10%) and another 4 had at least 1 positive drag swab (10%). Two of the Salmonella Enteritidis-positive farms also had backyard poultry present. One of the farms had a small collection of mixed chicken breeds obtained from a livestock auction. The second farm had a small flock of silkies.

Three of the 7 positive farms had used some type of vaccination program for Salmonella Enteritidis prevention. Two of the rodent Salmonella Enteritidis-positive layer farms had negative drag swabs and negative eggs. Two of the vaccinated pullet flocks also had Salmonella Enteritidis negative environmental swabs. These farms had vaccinated 3 times against Salmonella Enteritidis, using the live attenuated Salmonella Typhimurium vaccine twice in the chicks (at day of age and at 2 wk of age) followed by an injection with the killed-Salmonella Enteritidis vaccine in the leg muscles at approximately 12 to 13 wk. Of the 3 layer farms and 2 pullet flocks with Salmonella Enteritidis-positive environmental swabs and positive eggs, all were small flocks with less than 3,000 birds. Two of these farms (E and F) had purebred chickens for hatching egg production.

CONCLUSIONS AND APPLICATIONS

  1. Because data from this study are preliminary and the project will continue for an additional year, definitive conclusions cannot be made. However, based on these preliminary observations, we suggest the most important risk factor for Salmonella Enteritidis in small- to medium-sized flocks is the presence of Salmonella Enteritidis-infected rodents.

  2. The importance of infected mice as vectors of Salmonella Enteritidis is significant in small- and medium-sized flocks. The presence of Salmonella Enteritidis-infected rodents appears to correlate with increased incidence of Salmonella Enteritidis-positive environmental swabs and positive eggs, especially in chickens not vaccinated against Salmonella Enteritidis.

  3. Vaccination appears to be a useful tool for cage-free flocks to reduce Salmonella Enteritidis in the environment and the eggs.

  4. Most of the farms had buildings with access to the outdoors, exposure to wild birds, and multiple species of livestock. Outside human traffic onto the farms, especially on those premises with multiple businesses, was common, although actual visitors inside the bird area were not observed. Whereas these are certainly biosecurity risks for many poultry diseases, they did not seem to correlate with increased incidence of Salmonella Enteritidis in the study flocks.

  5. Fly cultures were also negative, even though flies may be vectors for Salmonella Enteritidis based on early studies on the epidemiology of Salmonella Enteritidis in caged layer houses. The reason for the negative findings is unclear.

  6. The incidence of Salmonella Enteritidis infection in eggs from medium to small cage-free flocks is not well known. In studies that have attempted to compare Salmonella Enteritidis incidence in large caged flocks versus smaller cage-free flocks, many variables appear to play a role, including flock size, stocking density, wet manure, cleaning and disinfection between flocks, rodent numbers, multiple flocks and ages on a farm, seasonal variation, and bird stress.

  7. As the number of cage-free egg production farms increases, it is important to objectively understand what potential food safety risks are present and the optimal procedures to reduce these risks.

1
Presented as a part of the symposium “Reducing Salmonella Enteritidis Contamination of Shell Eggs with an Integrated Research and Outreach Approach” at the Poultry Science Association’s annual meeting in San Diego, California, July 22–25, 2013.

The authors greatly appreciate funding from the USDA-National Integrated Food Safety Initiative that supported this study.

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