Antibiotic Alternatives to Reduce Foodborne Pathogens in Preharvest Poultry

Non-Technical Summary:<br/>
Among the foodborne pathogens transmitted through poultry products, Salmonella spp. and C. jejuni are the most common infectious agents causing disease in humans (Heres et al., 2004; Newell et al., 2010). The costs of poultry-associated cases of salmonellosis and campylobacteriosis in the U.S. range from 64 to 114.5 million dollars, and 362 to 699 million dollars, respectively (Bryan and Doyle, 1995). Recently, the USDA-FSIS announced implementation of performance standards for Salmonella and Campylobacter on broiler carcasses, in an effort to improve the microbiological safety of poultry-derived foods (USDA-FSIS, 2011). The Centers of Disease Control and Prevention (CDC) recently reported that S. Enteritidis was the most commonly isolated serotype of all Salmonella, and contribute to 27 percent of all single etiology outbreaks reported (Anonymous, 2011). Chickens are the reservoir host of S. Enteritidis and C. jejuni, with their intestinal colonization being the single most significant factor causing contamination of meat. In addition, a variety of other sources, including contaminated feed, litter, water, rodents, and insects can also play a role in the spread of Salmonella and C. jejuni in chickens. Therefore, implementation of an extensive and environmentally-friendly strategy, comprising of strict biosecurity measures at the farm and effective decontamination methods in processing plants are critical to improve the microbiological safety of poultry meat. Moreover, effective training and outreach programs targeting poultry farmers, processors and the industry are necessary to curtail microbial contamination of poultry-derived foods. A variety of approaches for reducing the colonization of S. Enteritidis and C. jejuni in chickens have been explored with limited success. Research conducted by the PD and collaborators showed that (CA) demonstrated significant antimicrobial properties against a number of bacterial pathogens in vitro and in vivo (Annamalai et al., 2004; Solis de los Santos et al., 2008, 2009). Bergsson et al. (1999) indicated that free fatty acids and monoglycerides exert their antimicrobial effect by affecting the bacterial membranes. Encapsulating these compounds may enhance their efficacy. Competitive exclusion CE is another possibility to reduce these foodborne pathogens. This principle was first applied in chickens by Nurmi and Rantala in 1973, who reported that feeding intestinal contents from adult chickens offered protection from S. Infantis infection in day-old chicks. Since then, the Nurmi concept has been demonstrated as an means for reducing but not eliminating Salmonella colonization in broilers (Heres et al., 2003). However, the protective effect of CE culture is not consistent against Campylobacter (Stern et al., 2001; Patterson and Burkholder, 2003). Our strategy for developing an effective CE culture against these pathogens is to use motility-enhanced bacteria having the marked capability to exclude these foodborne pathogens in the enteric environmental of chickens. Despite all these efforts, Salmonella and Campylobacter continue to be the most frequently isolated pathogens from poultry products.
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Approach:<br/>
Objective A. We will determine the ability of the medium chain fatty acid, caprylic acid (CA) as dietary supplement to reduce the colonization of S. Enteritidis and C. jejuni in poultry. For each pathogen chicks will be randomly divided into treatment groups receiving various amount of CA. Birds from each group will be sacrificed and pathogen populations in the cecum, small intestine, cloaca, crop, liver and spleen will be enumerated. The feed consumption and body weight of the birds will also be determined. These experiments may be repeated using a water application to determine efficacy of these treatments. Experiments will also be conducted to determine the efficacy of encapsulated plant molecules (example, trans-cinnamaldehyde) as a dietary supplement to reduce cecal S. Enteritidis and C. jejuni in chickens. The experimental design will follow a similar design as described above. Treatments will be encapsulated according to the method of Zhong and co-workers (Xiao et al., 2011), based on an emulsion-evaporation technology (McClements et al. 2007). <P>
Objective B. Ceca from approximately 100 adult chickens will be collected and frozen from at least 6 different commercial and University flocks. Cecal isolates will be tested against Campylobacter or Salmonella using the procedure of Miyamoto and coworkers (2000) with modifications. In this procedure, prospective bacterial species will be isolated from ceca by serially diluting intestinal contents and plating the dilutions onto standard DeMan Rogosa Sharp (MRS) agar and incubating plates aerobically for 24 to48 hours at 37C. Prospective cultures will be picked from standard MRS plates as single colonies and passed into fresh MRS broth, incubated 24 hours at 37C then 50 microliters of each culture was pipetted onto the center of a Tryptic Soy Agar (TSA) plate. Isolates will be grown for 24 hours; 2% soft agar will be melted and then cooled to 49C at which time a 24-48 hours culture of Campylobacter or Salmonella will be added to the soft agar at a ratio of 1:20 culture broth to soft agar. The bacterial isolates with the greatest motility will be used for the in vivo studies (Objective C).
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Objective C. To determine the efficacy of isolated collected in Objective B, will be treated with the selected isolates (described above) on the day of hatch followed by either Campylobacter or Salmonella challenge post-hatch as previously described (Bielke et al., 2003; Solis de los Santos et al., 2008). Preliminary evidence suggests it will be possible to identify candidate bacteria with efficacy against both pathogens. These individual bacteria will have to be tested in various combinations to evaluate and determine the most efficacious partnerships. For the candidate bacteria demonstrating in vivo efficacy, isolates will be sorted according to genus and species (when determined). These isolates which are capable of displacing foodborne pathogens will be subjected to determinative bacteriology (National Veterinary Services Laboratory, Ames, IA). Salmonella and Campylobacter will be determined as previously described.