Prevalence and concentration of Salmonella and Campylobacter in the processing environment of small-scale pastured broiler farms

Research Note Prevalence and concentration of Salmonella and Campylobacter in the processing environment of small-scale pastured broiler farms Lisa M. Trimble,* Walid Q. Alali,*1 Kristen E. Gibson,† Steven C. Ricke,† Philip Crandall,† Divya Jaroni,‡ Mark Berrang,# and Mussie Y. Habteselassie¶ *Center for Food Safety and Department of Food Science and Technology, University of Georgia, Griffin 30223; †Center for Food Safety and Department of Food Science, University of Arkansas, Fayetteville 72701; ‡Department of Animal Science, Oklahoma State University, Stillwater 74074; #United States Department of Agriculture, Agriculture Research Service, Russell Research Center, Athens, GA 30605; and ¶Department of Crop and Soil Sciences, University of Georgia, Griffin 30223 tion (mean log10 MPN per sample weight or volume) in soil [60%, 0.97 (95% CI: 0.66 to 1.27)], compost [64%, 0.95 (95% CI: 0.66 to 1.24)], and wastewater [48%, 1.29 (95% CI: 0.87 to 1.71)] were not significantly different (P > 0.05). Although Campylobacter prevalence was not significantly different by sample type (64.3, 64.3, and 45.7% in soil, compost, and PWW, respectively), the concentration (mean log10 cfu) of this pathogen was significantly lower (P < 0.05) in wastewater [2.19 (95% CI: 0.36 to 3.03)] samples compared with soil [3.08 (95% CI: 2.23 to 3.94)], and compost [3.83 (95% CI: 2.71 to 4.95)]. These data provide insight into smallscale poultry production waste disposal practices and provides a record of data that may serve as a guide for future improvement of these practices. Further research is needed regarding the small-scale broiler production environment in relation to improving disposal of processing waste for optimum control of human pathogens.

Key words: Salmonella, Campylobacter, pastured poultry, processing environment 2013 Poultry Science 92:3060–3066 http://dx.doi.org/10.3382/ps.2013-03114

INTRODUCTION Locally produced food products have increased in popularity partially due to consumer interest in sustainable agriculture (Johnson et al., 2012). The pastured poultry production model, in which small producers raise and process an average of 1,500 broilers per year, is a growing niche in the locally grown food movement. Birds are raised on pasture, processed on the farm, and the meat is sold directly to customers (Fanatico, 2003). Floorless pens containing 50 to 90 chicks are rotated to fresh pasture on a daily basis (Salatin, 1993). Forag©2013 Poultry Science Association Inc. Received February 11, 2013. Accepted August 5, 2013. 1 Corresponding author: [email protected]

ing constitutes approximately 20 to 30% of the birds’ diet, which is supplemented with natural or organic feed and does not require the use of antibiotics (Glass, 2002). Small-scale producers and consumers are drawn to this production model based on the expectation of improved nutrition and flavor of the broiler meat, higher standards for animal welfare, increased soil fertility, sustainability of the farm environment, and community involvement (Hillmire, 2011; Fanatico, 2012). The processing method used by small-scale pastured b roiler producers depends on the availability of suitable equipment, facilities, labor, and compliance with regulations (Fanatico, 2003). Many producers choose to process their birds manually at the site of production (on-farm) in an open-air operation or an enclosed shed. Common practices include allowing processing wastewater (PWW) to empty into the surrounding soil and

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ABSTRACT A growing niche in the locally grown food movement is the small-scale production of broiler chickens using the pasture-raised poultry production model. Limited research exists that focuses on Salmonella and Campylobacter contamination in the environment associated with on-farm processing of pasture-raised broilers. The objective of this study was to establish data relative to Salmonella and Campylobacter prevalence and concentration in soil and mortality compost resulting from prior processing waste disposal in the small-scale, on-farm broiler processing environment. Salmonella and Campylobacter concentrations were determined in soil (n = 42), compost (n = 39), and processing wastewater (PWW; n = 46) samples from 4 small broiler farms using a 3-tube most probable number (MPN) method for Salmonella and direct plating method for Campylobacter. Salmonella prevalence and concentra-

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disposal in small-scale, on-farm broiler processing environments.

MATERIALS AND METHODS Over a 1-yr period, this study was conducted at 4 small-scale, independent, pasture-raised broiler farms in the southeastern United States. The farms were selected based on the farmers’ willingness to participate in the study (convenience sampling), and participation was based on the condition of privacy. All of the participating farms were exempt from federal and state inspection. Approximately 1,000 broilers were produced per year at each small-scale operation, and birds were manually processed at the site of production (on farm) as described in Trimble et al. (2013). Birds were slaughtered at 9 to 10 wk of age and were processed in a processing station located in an open-air set up or in an enclosed shed. Workers included family, friends, and customers with varying levels of experience participating in broiler processing. Farm animals such as pigs, horses, goats, cows, and herding dogs were also present on some of the farms. A total of 46 composite PWW samples, 42 composite soil samples, and 39 composite compost samples were collected during 12 on-farm visits in accordance with the farmers’ broiler processing schedules. Three visits were conducted at farm A, 2 visits were conducted at farms B and C, and 5 visits were conducted at farm D.

Sampling Scheme and Processing Farmers processed between 50 and 100 pasture-raised birds at each visit. The processing area at each farm included variations of basic equipment such as kill cones, a single-stage scalder, a batch picker, a stainless-steel table for evisceration, a water hose for spray-washing carcasses, and a container filled with ice water as a chill tank. The PWW from the scalder, picker, and runoff from the evisceration table emptied directly into the soil surrounding the processing area. Processing offal was collected and added to an on-site mortality compost pile that used the passive composting method (USDA-NRCS, 2010). Compost ingredients included a mixture of manure from farm animals (i.e., goats, pigs, cows), wood chips or straw, dead birds, and other small animals. The approximate age of the piles ranged from 6 mo to 2 yr old. Soil Samples. Samples were collected before processing began at each farm visit. Three separate areas subjected to previous wastewater disposal in the soil were chosen using the judgmental sampling approach (IAEA, 2004). In most cases, previous wastewater disposal occurred at least 1 wk before sample collection. Sampling points included the areas around the scalder discharge hose, the picker, and the evisceration table. Three 24-cm soil cores (2 cm wide) were collected at each of the 3 disposal areas using a soil auger. Samples

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the collection of processing offal for addition to an onsite mortality compost pile. Raw PWW includes fats and proteins in both particulate and dissolved forms as well as blood, feathers, viscera, and excreta (USEPA, 2002). According to US Environmental Protection Agency (USEPA), PWW from a commercial poultry plant could contain high loads (millions of cfu/100 mL) of nonpathogenic indicator bacteria such as fecal coliforms, which may indicate the presence of enteric pathogens such as Salmonella and Campylobacter (USEPA, 2002). On-farm mortality composting is used to manage animal mortality and processing waste on many small broiler farms. If managed properly, composting could minimize the transmission of pathogenic bacteria into surrounding soil, air, and water, prevent the spread of infection from diseased animals, and convert the carcass and processing waste into a constructive, nutrientrich material (Kalbasi et al., 2005). When compared with commercial composting facilities, private farms may present a challenge regarding the implementation of basic hygiene standards to reduce microbial hazards associated with mortality composting (Wilkinson, 2007). Although composting is an established method for pathogen reduction, validation of the microbial safety of mortality composting can be challenging because of diverse pile conditions and process management (Wilkinson, 2007). Land application of the PWW and finished compost may be used as a waste management strategy and as a valuable soil amendment on the farm. However, these strategies are not always congruent (Edwards and Daniel, 1992). Research has shown that repeated animal waste disposal and overapplication of animal wastes can lead to decreased crop yields (Shortall and Liebhardt, 1975; Glanville et al., 2009), soil toxicity (Weil and Kroontje, 1979), and contamination of crops (Islam et al., 2004), surface waters, and groundwater through precipitation runoff and soil leaching (Burkholder et al., 2007). Enteric pathogens such as Salmonella and Campylobacter can survive in agricultural waste composts (Gong et al., 2005; Heringa et al., 2010; Inglis et al., 2010; Singh et al., 2012) and domestic waste compost containing garden waste, fruits, and vegetables (Lemunier et al., 2005). Furthermore, survival of Salmonella and Campylobacter has been demonstrated in soil where agricultural waste has been applied (Hutchison et al., 2004, 2005; Holley et al., 2006; Ross and Donnison, 2006; You et al., 2006). Limited research exists that focuses on the prevalence and concentration of Salmonella and Campylobacter in the environment (i.e., soil and mortality compost) of small-scale, pasture-raised operations where birds are processed at the site of production. The objective of this study was to determine Salmonella and Campylobacter prevalence and concentration in soil and mortality compost resulting from prior processing waste

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Analysis for Salmonella and Campylobacter The 3-tube most probable number (MPN) method was used for Salmonella quantification according to USDA-FSIS methods (USDA-FSIS, 2008a,b). The direct plating and enrichment method was used for enumeration and detection of Campylobacter (USDA-FSIS, 2011). All samples were processed and assayed on the day of collection. Salmonella Analysis. For the preenrichment of Salmonella, each processed soil, compost, and PWW sample was added to 9 tubes containing buffered peptone water (BPW, Difco; 3 tubes each of 1 mL of 10× BPW, 9 mL of 1× BPW, and 9 mL of 1× BPW) in the

amounts of 10, 1, and 0.1 mL, respectively. Tubes were incubated at 37°C for 24 h. After incubation, 0.5 and 0.1 mL of each preenrichment tube was added to 10 mL of tetrathionate broth (TT broth, Difco) and 10 mL of Rappaport-Vassiliadis (RV broth, Difco), respectively, and incubated (24 h, 42°C). Tubes were vortexed and a 10-μL loopful from each broth was streaked onto brilliant green sulfa agar (BGS; Difco) and xylose lysine tergitol-4 agar (XLT4; Difco) plates and incubated (24 h, 37°C). Colonies typical of Salmonella were inoculated onto triple sugar iron agar (TSI; Difco) and lysine iron agar (LIA; Difco) slants and incubated (24 h, 37°C). All BGS and XLT4 plates were incubated for an additional 24 h, and colonies presumed to be Salmonella were inoculated onto additional LIA and TSI slants and incubated as previously described. Slants were examined as sets for reactions typical of Salmonella and were further tested for agglutination using Salmonella Poly O (A-I & Vi) antiserum (Difco). Colonies with a presumptive positive reaction on LIA and TSI slants that did not agglutinate were further tested using real-time PCR (Stratagene Mx 3005P, Santa Clara, CA). Total DNA was extracted from the isolates according to the method described in Anderson et al. (2010). Real-time PCR detection of Salmonella was performed as described in Bohaychuk et al. (2007) with modifications. Briefly, reactions were conducted in a total volume of 25 µL (12.5 µL of 2× Brilliant SYBR Green I qPCR Master Mix (Stratagene), 10.25 µL of nuclease-free water (Qiagen, Valencia, CA), 0.125 µL of each primer (forward: 5′-AACTTCATCGCACCGTCA-3′; reverse: 5′-TATTGTCACCGTGGTCCAG-3′; 135 nM final concentration), and 2 µL of total DNA. The reaction conditions for amplification were 95°C for 10 min, and 40 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 15 s. Colonies confirmed as Salmonella were preserved on tryptic soy agar (Difco) and stored at 4°C. The detection limit for soil and compost samples was a MPN of 3 salmonellae per 100-g sample (95% CI: <1 to 9.6) and 30 salmonellae per 1,000 mL of PWW (95% CI: 1.5 to 96). Campylobacter Analysis. Serial dilutions of the processed soil, compost, and PWW samples were prepared in PBS and were spread plated on premade modified Campy-Cefex agar plates (Hardy Diagnostics, Santa Maria, CA). For each undiluted sample, 250 μL was spread onto 4 plates and subsequent dilutions were achieved by plating 100 μL of the dilution series on duplicate plates. Plates were placed in sealable plastic bags flushed with microaerobic gas (5% O2, 10% CO2, 85% N2) and were incubated at 35°C for 48 h per the manufacturer’s recommendation. Enrichment of samples was performed using 10 mL of sample and 10 mL of Bolton Enrichment Broth (Hardy Diagnostics). After incubation, Campy-Cefex plates were examined for typical Campylobacter colonies. Confirmation of presumptive positive colonies was based on cellular morphology and motility under a phase contrast mi-

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were placed in Whirl-Pak bags (Nasco, Fort Atkinson, WI) on ice for transport to the laboratory where they were combined into 3 composite samples (approximately 100 g each). The moisture content of the soil and compost samples was not determined. Compost Samples. Three 30- to 40-g samples in each of 3 areas of the mortality compost pile were collected at each farm visit. A large sterile metal scoop was used to collect the samples at a depth of 24 to 36 cm. Samples were placed in sterile Whirl-Pak bags on ice for transport to the laboratory where they were combined into 3 composite samples (approximately 100 g each). Debris (rocks, pebbles, and bones) was removed from soil and compost samples using a sterile handheld screen. Two 10-g samples of soil and compost were each placed in a 50-mL polypropylene tube, and PBS (Difco, Sparks, MD) was added to bring the volume to 50 mL per tube. Each tube was vigorously shaken manually for 1 min and placed on a shaker at 200 rpm for 20 min at 4°C. Soil samples were centrifuged at 378 × g for 5 min at 4°C, whereas compost samples were centrifuged at 22,237 × g for 15 min at 4°C. The supernatant fluid (approximately 45 mL) was poured into a sterile tube and used for Salmonella and Campylobacter analysis. Wastewater Samples. Three composite PWW samples (1,000 mL each per 10 to 20 birds processed) were collected into sterile plastic field bottles (Nalgene, Rochester, NY) upon emptying of the scalder, picker, and during runoff from the evisceration table. Samples were placed on ice for transport to the laboratory. Feathers and debris were removed using a sterile handheld screen. Two 50-mL tubes of PWW from each composite sample were centrifuged for 20 min (22,237 × g at 4°C). The supernatant fluid (approximately 45 mL) from each tube was discarded. The pellet and the remaining 5 mL of liquid in each tube were resuspended in 20 mL of room-temperature PBS, vortexed, and used for Salmonella and Campylobacter assays. Due to conflicts in farm visit scheduling, additional samples (a total of 10 PWW, 6 soil, and 3 compost) were collected and included in the analysis over the 12 visits.

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croscope (Olympus BX40, Center Valley, PA) and a positive reaction in a latex agglutination immunoassay (Hardy Diagnostics). The detection limit was 100 Campylobacter cfu per 1,000 mL of PWW and 100 Campylobacter cfu per 100 g of soil and compost. For each sample, the dilution that contained confirmed colonies within the countable range (15 to 300 cfu per plate) was used to calculate the cfu per milliliter of sample according to the method outlined by USDA-FSIS (2011). If direct plating of the sample did not display colonies typical of Campylobacter, the Bolton Broth enrichment cultures were plated on Campy-Cefex agar and were confirmed as described previously.

gen prevalence and the sample type (soil, compost, and PWW) was assessed using a GLM with binomial error distribution, logit link function, and adjustment for dependency within farms using generalized estimated equations (GEE) in STATA software version 10.1 (Stata Corp., College Station, TX). For pathogen concentration data, the relationship between the log10 MPN or cfu per sample and sample type was assessed using the GEE model in STATA, with identity link function to adjust for dependency within farms. A P-value less than 0.05 was considered significant.

Data Analysis

The prevalence and mean log10 concentration of Salmonella and Campylobacter in soil, mortality compost, and PWW samples collected from the on-farm processing environment is shown in Table 1. The distribution of the Salmonella and Campylobacter mean log10 MPN and cfu by sample type (soil, compost, and PWW) are shown in Figures 1 and 2, respectively. The Salmonella prevalence and concentrations were not significantly different (P > 0.05) by sample type. The prevalence of Campylobacter was not significantly different by sample

The outcomes of this study were the prevalence and concentration of Salmonella and Campylobacter in soil, compost, and PWW samples associated with smallscale, pasture-raised broiler processing at the site of production. The concentration data (MPN and cfu/mL or g) were adjusted to the original sample volume or weight collected and were log10 transformed to approximate normality. The relationship between the patho-

RESULTS AND DISCUSSION

Table 1. Salmonella and Campylobacter prevalence and concentration in the small-scale broiler farm environment Salmonella Item Soil Compost Wastewater A,BValues

Prevalence 60%A 64%A 48%A

(n = 42) (n = 39) (n = 46)

Mean log10 MPN1 0.967A 0.953A 1.289A

Campylobacter 95% CI 0.660–1.273 0.664–1.243 0.868–1.710

Prevalence 64.3%A 64.3%A 45.7%A

(n = 42) (n = 39) (n = 46)

Mean log10 cfu1

95% CI

3.084A 3.827A 2.192B

2.225–3.944 2.710–4.945 0.357–3.028

in the same column with different superscripts are significantly different (P < 0.05). values for Salmonella and Campylobacter are expressed per 100 g of soil, 100 g of compost, and 1,000 mL of processing wastewater (PWW) samples in the small-scale pastured poultry farm environment. MPN = most probable number. 1Mean

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Figure 1. Percentage bar chart illustrating the log10 most probable number (MPN) of Salmonella in 100 g of soil, 100 g of compost, and 1,000 mL of processing wastewater (PWW) samples in the small-scale pastured poultry farm environment.

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type, but the concentration of this pathogen was significantly lower (P < 0.05) in PWW samples compared with soil and compost samples. The rate at which poultry waste is applied to land depends on whether the objective is to supply nutrients to the soil or to simply dispose of waste. If utilization of the fertility-related aspects of the waste is the primary focus, the application should be based on the needs of the crop and the characteristics of the waste (Edwards and Daniel, 1992). Additionally, organic poultry processing wastes should be regarded as contaminated with human pathogens unless the wastes have been treated sufficiently (Burton et al., 2004). In the current study, a common belief among the small-scale producers was that the direct application of raw PWW is innocuous and will greatly increase soil fertility (personal communication with farmers). In many cases, the farmers did not rotate the location of wastewater disposal during subsequent processing days (personal communication with farmers) and disposal occurred in areas of high foot traffic. Because most soil samples were collected at least 1 wk after application of PWW to the soil, the data suggest that between slaughter dates, both pathogens were moderately prevalent and fairly concentrated in the soil surrounding the processing area. The absence of soil management practices such as varying the location of PWW disposal, coupled with the direct application of untreated wastewater to the soil surrounding the processing area, may have contributed to the prevalence of Salmonella and Campylobacter in the collected samples. Previous research has shown that both pathogens may survive in agricultural environments. Ross and Donnison (2006) reported that 99% of Campylobacter jejuni in inoculated dairy farm effluent was retained in the top 5 cm of 4 different soil types for 25 d. Holley et al. (2006) reported that surface application of manure to soil resulted in increased survival time of Salmonella

compared with incorporation of manure into the soil. Furthermore, Dazzo et al. (1973) reported that after 3 yr of continuous exposure to manure slurry irrigation under natural field disposal or conditions, the resulting modifications to the soil environment favored the extended survival of Salmonella Enteriditis. The prevalence and concentration of Salmonella and Campylobacter in the PWW samples in this study suggest that the untreated wastewater itself represents a hazard, in addition to the soil surrounding the processing area where the PWW has been dumped. It is possible that the pathogen concentration may have been higher if sampling had occurred at the soil surface. High foot traffic, rainfall, and other animals are potential vectors in the transmission of pathogens around the processing area. The data in Figures 1 and 2 suggest that the mortality compost pile may also pose a risk for the dissemination of Salmonella and Campylobacter in the smallscale poultry production environment. In the current study, we did not survey individual farmers to collect information on practices such as compost recipes (i.e., C:N ratios), aeration, or temperature monitoring. All of the participating farms lacked enclosed, structured composting bins, and piles were located on the ground near wooded areas or piles of debris. This arrangement may have provided easy access to the compost pile for pests such as rodents, which are considered to be potential vehicles for transmission of enteric pathogens on the farm (Meerburg et al., 2006). Furthermore, the effects of precipitation and the high moisture content of carcasses and processing offal may result in runoff from the exposed pile that could contaminate the surrounding soil (Kalbasi et al., 2005). This potential for leaching of mortality compost ingredients supports the recommendation by Carter et al. (1996) to separate the compost pile from the poultry production and processing area of the farm.

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Figure 2. Percentage bar chart illustrating the log10 (cfu) of Campylobacter in 100 g of soil, 100 g of compost, and 1,000 mL of processing wastewater (PWW) samples in the small-scale pastured poultry farm environment.

RESEARCH NOTE

ACKNOWLEDGMENTS The authors thank the USDA-Southern Sustainable Agriculture Research and Education (SSARE) project #LS11-245 for providing the funding for this project. The authors also thank all participating farmers in this study. We thank Eric Adams at the USDA-Agriculture Research Services in Athens, Georgia, and Meghan Keys, Jared Smith, Nathan Flitcroft, Jarred Sturm, and David Mann at the University of Georgia Center for Food Safety.

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Recommendations for composting dead poultry include the construction of a proper composting bin that includes a roof, a foundation, and a floor (Carter et al., 1996; USDA-NRCS, 2010). Field trials reported by Shepherd et al. (2011) validated the recommendation for a cover on fresh compost; E. coli O157:H7 reduction occurred more rapidly at the surface of heaps covered with finished compost versus fresh straw or uncovered compost heaps. A temperature monitoring program that documents 3 heating and turning cycles with peak temperatures between 140 and 160°F for at least 5 d to eliminate pathogens is also recommended (USDA-NRCS, 2010). A survey of farmers conducted by Rangarajan et al. (2002) revealed that only 39% (n = 31) of compost producers consistently monitored the temperature of their compost heaps for a target peak temperature of 140°F or higher. However, research has shown that meeting time and temperature requirements does not guarantee the complete destruction of all pathogens. Pietronave et al. (2004) and Kim et al. (2009) concluded that proper storage conditions are required to prevent pathogen regrowth in finished compost. A study by Shepherd et al. (2010) concluded that the conditions on the surface (<5 cm) of poultry mortality compost heaps were suitable for pathogen survival during the first phase of composting. Additionally, the authors reported that the temperature and moisture contents of the compost heaps at participating small- and medium-sized poultry farms were often below the recommended level for pathogen inactivation, which could result in inadequate composting conditions and pathogen survival. The current study provides insight into small-scale poultry production waste disposal practices and provides a record of data that may serve as a guide for future improvement of these practices. Further research is needed regarding the small-scale broiler production environment in relation to potential disposal intervention methods.

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