Evaluation of an Avian-Specific Probiotic and Salmonella typhimurium-specific Antibodies on the Colonization of Salmonella typhimurium in Broilers

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1 176 Journal of Food Protection, Vol. 61, No.2, 1998, Pages Copyright, International Association of Milk, Food and Environmental Sanitarians Evaluation of an Avian-Specific Probiotic and Salmonella typhimurium-specific Antibodies on the Colonization of Salmonella typhimurium in Broilers B. PROMSOPONE,l T. Y. MORISHITA,l* P. P. AYE,l C. W. COBB,z A. VELDKAMP,z AND J. R. CLIFFORD3 ldepartment of Veterinary Preventive Medicine, The Ohio State University, 1900 Coffey Road, Columbus, Ohio 43210; 2Loveland 1ndustries, 1nc., P.O. Box 1289, Greele)\ Colorado 80632; and 3United Staes Department of Agriculture, Animal and Plant Health 1nspection Service, Veterinary Services, Stonecreek Drive, Pickerington, Ohio 43147, USA MS 97-92: Received 5 May 1997/Accepted 14 July 1997 ABSTRACT Salmonella typhimurium colonizes the intestinal tract of poultry and causes food-borne illness in humans. Reduction of S. typhimurium colonization in the intestinal tract of poultry reduces potential carcass contamination during slaughter. The purpose of this study was to determine the effect of an avian-specific pro biotic and S. typhimurium-specific antibodies on the colonization of S. typhimurium in broilers and on body weights. Broiler chicks were spray-vaccinated at the hatchery with the commercial product, Avian Pac Plus, which contains Lactobacillus acidophilus, Streptococcus faecium, and S. typhimurium-specific antibodies. At placement, these chicks were administered Avian Pac Plus in the water. Six hours postplacement, chicks were orally challenged with 1.8 X 10 7 CFU of S. typhimurium. Chicks were administered Avian Pac Plus for two additional days postchallenge. Chicks were evaluated for S. typhimurium colonization and shedding every 3 to 4 days for the first 2 weeks and every 7 days for 6 weeks. The mean cecal and colonic concentration of S. typhimurium from the Avian Pac Plus-treated group was significantly lower at day 31 (P = ), day 38 (P = ), and day 43 (P = ) than the nontreated control group. These results indicated that a combination of Lactobacillus acidophilus, Streptococcus faecium, and S. typhimurium-specific antibodies have a beneficial effect in reducing the colonization of S. typhimurium in market-aged broilers. Salmonellae, including Salmonella typhimurium, have been isolated from up to 70% of broiler carcasses in the United States (18). Control programs have been initiated to reduce this potential food-borne pathogen in dressed carcasses. Much of the effort to control S. typhimurium in poultry has been concentrated in the processing area (1). Preventing S. typhimurium infection and colonization in poultry is a major concern for both the poultry and food processing industries as well as for the consumer (29, 40). Bailey (1) stated that minimizing cross-contamination of the dressed carcasses with intestinal contents will improve the microbiological quality of processed chickens. The intestinal colonization of S. typhimurium plays a significant role in carcass contamination during processing. Therefore, reducing intestinal colonization of S. typhimurium during the grow-out period is crucial to improving carcass microbiological quality during processing. Competitive exclusion (CE) is one mechanism that has been used to prevent Salmonella intestinal colonization of chickens (25). Numerous researchers have confirmed the protective effect of indigenous intestinal flora against Salmo- *Author for correspondence. Tel: ; Fax: ; nella colonization (2, 3, 10, 11, 23, 25, 27, 30, 33, 36). Competitive exclusion has been documented to be an effective means of controlling Salmonella colonization (1). Giving 1- to 2-day-old chicks CE microflora prior to oral infection with Salmonella infantis resulted in reduced cecal colonization and these chicks were subsequently free of infection 1 to 2 weeks later (25, 28). A variety of bacteria sources for CE microflora (crop and lower intestinal contents, cultured cecal microflora, and feces from healthy adult chickens) have been given to young birds and have protective properties that inhibited the establishment and growth of invading enteric pathogens (16, 19,25,26,27,31,33,37). The crop and cecum are the major sites of Salmonella colonization in the chicken (6, 34), and these sites are responsible for the movement of Salmonella spp. into internal tissues (22). However, the preferred site of Salmonella colonization is in the caudal intestinal region (38, 39) where Salmonella spp. have a predilection to establish chronic infections in the ceca (12). Cecal contents and cecal tonsils of chickens were found to be superior sites for Salmonella isolation when compared to other areas of the intestinal tract (5). In chickens that were orally infected with Salmonella spp. the organisms persisted in the ceca, whereas in birds exposed through egg infection the organisms were found almost exclusively in the jejunum (14).

2 J. Food Prot., Vol. 61, No.2 REDUCTION OF SALMONELLA COLONIZATION 177 Avian lactobacilli competitively exclude S. typhimurium from adhering to the crop mucosa (13, 35). However, it is unknown if avian lactobacilli can reduce Salmonella cecal colonization. In addition, the use of S. typhimurium-specific antibodies to prevent the establishment of Salmonella cecal colonization has not been determined. Thus, the purpose of this project was to determine the effectiveness of a combination of Lactobacillus acidophilus, Streptococcus faecium, and S. typhimurium-specific antibodies in reducing Salmonella cecal and colonic colonization in market-aged broilers. Moreover, the effect of this combination of probiotics and antibodies on the body weight was determined. MATERIALS AND METHODS Birds. Four hundred day-old chicks were obtained from a commercial hatchery. These chicks were processed at the hatchery similar to other chicks that were going into field production. All chicks were vaccinated at the hatchery with Marek's disease virus vaccine at day O. Test article and route of administration. Avian Pac Plus was provided by Loveland Industries, Inc. (Greeley, CO). Avian Pac Plus contains Lactobacillus acidophilus and Streptococcus faecium, which were isolated from avian sources and have been adapted to avian intestinal conditions. In addition, Avian Pac Plus contained S. typhimurium-specific antibodies which were produced by immunizing hens with killed S. typhimurium and subsequently harvesting their egg yolk products for antibodies. Birds were given Avian Pac Plus at the manufacturer's recommended dosage (150 grams per 120 gallons of drinking water for every 10,000 birds) via spray at the hatchery and via drinking water from day 1 to day 3. This route was chosen to closely approximate field conditions for use of the product. Avian Pac Plus was prepared by mixing lyophilized product with the appropriate amount of double-distilled water. Control (nontreated) birds were given double-distilled water containing no probiotic or antibodies. Bacteria used for challenge. A pathogenic isolate of S. typhimurium obtained from T. Y. Morishita (Ohio State University) from a field case of salmonellosis in broilers was used as the challenge bacterium in this study. This S. typhimurium was susceptible to the following drugs: amikacin (:58.00 /lg/ml), ampicillin (1.00 /lg/ml), ceftiofur (0.50 /lg/ml), neomycin (:52.00 /lglml), streptomycin (moderate susceptibility; 32.0 /lg/ml), and TMP/Sulfa (trimethoprim, :50.12 /lg/ml; sulfamethoxazole, :52.38 /lg/ml). This isolate of S. typhimurium was resistant to apramycin (:54.0 /lg/ml), cephalothin (:54.0 /lg/ml), clindamycin (>4.00 /lg/ml), gentamicin (8.00 /lg/ml), erythromycin (>8.0 /lg/ml), nitrofurazone (32 /lg/ml), oxacillin (>4.00 /lg/ml), penicillin (8.00 /lg/ml), spectinomycin (> 16.0 /lg/ml), sulfisoxazole (> /lg/ml), tetracycline (16.0 /lg/ml), and tylosin (>8.0 /lg/ml). Five randomly chosen colonies from a 24-h plate of S. typhimurium grown on 5% sheep blood agar (Becton Dickinson Microbiology Systems, Cockeysville, MD) were inoculated into brain heart infusion broth. This suspension was incubated for 0.5 h at 37 C in a shaking water bath (Lab-Line Aquabath Model 3545, Lab-Line Instruments, Inc., Melrose Park, IL). At 0.5 h, this suspension was removed from the water bath and was used to challenge the chickens. The viable population of the challenge inoculum was determined by serial dilutions and plating on Hektoen enteric (HE) agar. Housing conditions. Chicks were reared in floor-pens with litter shavings as the flooring substrate. Supplemental heat was provided with temperatures set at 30 to 32 C when measured outside the brooder canopy and 5 cm above the top of the litter. This supplemental heat was reduced at a rate of 5 C per week until the chicks were 4 weeks of age. Food and water were provided ad lib. Samples of the feed, water, and litter were cultured for S. typhimurium prior to chick placement. Cultural methods included delayed secondary enrichment of primary enrichment broths to increase isolation of salmonellae from these environmental samples. In this method, a 0.25-ml aliquot of the originally incubated tetrathionate broth was transferred to 10 ml of fresh tetrathionate broth and incubated at 42 C for 24 h and then plated on HE agar. No S. typhimurium cells were isolated from these samples of feed, water, and litter. Experimental design. Four hundred broiler chickens were weighed and randomly allocated into one of two groups (control and treatment group including 1 replicate) containing 100 chicks each. The treatment group was defined as chicks spray-vaccinated with Avian Pac Plus at the hatchery and given Avian Pac Plus for the first 3 days after placement. The control group was defined as chicks not given any Avian Pac Plus at the hatchery or at placement. At 1 day of age, the mean body weight of the nontreated (control) birds and treated (Avian Pac Plus treatment) birds was g and g, respectively. There was no significant difference among the groups in regard to their mean bqdy weights. Water was withheld for 2 h in both groups prior to treatments to ensure that the birds orally ingested either the treated (Avian Pac Plus) or untreated (no Avian Pac Plus) double-distilled water. Six hours after oral administration of the probiotic suspension (treatment group) or water (control group), the chicks were challenged with S. typhimurium. The chicks were orally inoculated with 0.5 ml of S. typhimurium which contained 3.6 X 10 7 CFU/ml suspended in brain heart infusion (BHI) broth. Control birds were given 0.5 ml of BHI alone. Every 3 to 4 days for the first 2 weeks and every 7 days until 6 weeks of age, 10 birds were weighed and sacrificed from each group, and their lower intestinal (cecum and colon) contents were cultured for S. typhimurium. Collection periods were on day 0,1,3, 7,10,17,24,31,38, and 43. Isolation of S. typhimurium. At each collection period (day 0, 1,3,7, 10, 17,24,31,38, and 43), the cecal and colonic contents were aseptically collected in a 50-ml centrifuge tube (Coming Incorporated, Coming, NY). To determine the concentration of S. typhimurium in the cecum, the cecal contents of each bird were serially diluted at 1/10, 1/100, 1/1,000, 1/10,000, and 1/100,000 dilutions in BHI broth. A I-ml portion of each dilution was plated on an HE agar plate using the spread plate technique. The plates were aerobically incubated for 24 h at 37 C and the number of S. typhimurium colonies were counted. Typical Salmonella colonies were biochemically confirmed by using triple sugar iron agar, lysine iron agar, urea agar slant, sulfide indole motility (SIM) agar, dulcitol, maltose, mannitol, dextrose, and malonate broth. Morever, these salmonellae were confirmed to have the same antibiotic profile as the challenge isolate. In addition, other bacterial growth on the HE agar plates were identified with further biochemical tests, including triple sugar iron agar, urea agar slant, Simmons citrate agar, and SIM agar. A replicate was performed for each sample to obtain a mean CFU/g for each sample. Statistical analysis. Data were collected and subsequently analyzed by a chi-square test to determine if there was a statisti-

3 178 PROMSOPONE ET AL. J. Food Prot., Vol. 61, No.2 cally significant difference between the treatment and control groups at each collection period. RESULTS Concentration of S. typhimurium in intestinal contents. There was a significant difference in the isolation of S. typhimurium from the cecum and colon when probiotictreated and non-probiotic-treated groups were compared. The pro biotic-treated group had a significantly lower concentration of S. typhimurium colonization at day 31 (P = ), day 38 (P = ), and day 43 (P = ) when compared to the nontreated, control group (Table 1). Other bacterial growth found on the Hektoen enteric agar were predominately E. coli isolates. Mean body weight. There was no significant difference in the mean body weight between the two experimental groups at each collection period, except at day 10. At day 10, the probiotic-treated group had a significantly higher mean body weight compared to the control group (P = ) (Table 2). DISCUSSION This study demonstrated that the administration of Lactobacillus acidophilus, Streptococcus faecium, and S. typhimurium-specific antibodies when administered via spray to broiler chicks at 1 day of age and administered orally for the first 3 days after placement significantly reduced S. typhimurium colonization of the cecum and colon in marketaged broilers. It is crucial that probiotic and antibodies be administered as early as 1 day of age as previous studies on the ecological succession of the intestinal microbial flora of poultry demonstrated that Salmonella colonization can occur as early as 1 day of age (21). Unlike other researchers (9, 24) who have challenged chickens with dosages of 10 4 to 10 5 CFUs, we explored the effect of Avian Pac Plus at a challenge dosage of 10 7 CFUs. Our results indicate that TABLE 1. The effect of Avian Pac Plus (Loveland Industries, Inc.) on the mean concentration of Salmonella typhimurium from the cecum and colon in broiler chickens Age Day 0 Day 1 Day 3 Day 7 Day 10 Day 17 Day 24 Day 31 Day 38 Day 43 log CFU S. typhimurium per g of cecal contents (n = 10; mean:!: SD) Avian Pac Plus-treated group O:±:O 3.3 X 10 8 :±:7.8 X X 10 7 :±: 1.3 X X 10 6 :±: 1.6 X X 10 6 :±:5.4 X X 10 4 :±:9.0 X X 10 4 :±:4.0 X X 10 4 :±:6.3 X 10 4 A" 6.0 X 10 3 :±:4.8 X 10 3 A 1.5 X 10 3 :±:9.0 X 10 2 A Control group O:±:O 6.0 X 10 8 :±:1.3 X X 10 7 :±:8.9 X X 10 6 :±:2.4 X X 10 6 :±: 4.7 X X 10 5 :±:2.8 X X 10 5 :±:1.0 X X 10 5 :±:2.3 X 10 5 B 4.3 X 10 4 :±:4.4 X 10 4 B 1.0 X 104 :±:5.9 X 10 3 B a Values in a row followed by different letters differ significantly (P < 0.05). TABLE 2. Comparisonof mean body weights (g) betweenprobiotictreated (Avian Pac Plus-treated and non-probiotic-treated broiler chickens Age Day 0 Day 7 Day 10 Day 17 Day 24 Day 31 Day 38 Day 43 Body weight, g (n = 10; mean:!: Avian Pac Plus-treated 42.46:±: :±: :±:33.18A" :±: :±: :±: :±: :±: group SD) Control group :±: :±: :±:32.57B :±: :±: :±: :±: :±: a Values in a row followed by different letters differ significantly (P < 0.005). Avian Pac Plus has beneficial effects even when broiler chicks are exposed to the worst-case scenario, a challenge dose. The Avian Pac Plus-treated group had significantly lower numbers of S. typhimurium in the ceca when compared to the control group. However, it was noted that there is a similar rate of decline in the population of S. typhimurium in the ceca of both groups, and this may represent the normal microbiological succession of S. typhimurium in the ceca of broiler chicks. Although there were still 10 3 CFUs and 10 4 CFUs for the treated and control groups, respectively, this may be a reflection of the initially high challenge dose given to the day-old chicks. Lactic acid-producing bacteria, such as Lactobacillus spp. are among the first bacteria to colonize the digestive tract in young chicks (32). Lactic acid produced from the utilization of lactose is probably responsible for the decreased cecal ph (7, 8, 16, 17). Acetic and propionic acids produced from lactic acid in the ceca of young chicks by anaerobic cultures of normal intestinal microflora from adult chickens can reduce S. typhimurium cecal colonization of young chicks (4, 17). Soerjadi et al. (34) demonstrated that avian lactobacilli could competitively exclude S. typhimurium from colonizing the crop mucosa. Hinton et al. (15) demonstrated that Streptococcus CA331 inhibited enteropathogens like S. typhimurium and E. coli 0157:H7 in the cecum. Hence, the combination of Lactobacillus acidophilus and Streptococcus faecium and S. typhimurium-specific antibodies can be effective in inhibiting Salmonella colonization in the ceca and colon of market-aged chickens. Unlike other studies, this study followed orally S. typhimurium-challenged chicks up to market age at 42 days. It is important to determine the colonization during this period as these birds are going to slaughter and intestinal colonization can playa role in carcass contamination during processing. This study demonstrated that Avian Pac Plus significantly reduced S. typhimurium colonization in marketaged broilers and thus has the beneficial effect of potentially improving the microbial quality of processed carcasses. Earlier studies in our laboratory also demonstrated that the

4 bacteria Lactobacillus acidophilus and Streptococcus faecium when administered alone or in combination with S. typhimurium-specific antibodies can reduce the E. coli loads in the liver (20). To date, there has been no studies to determine the effect of S. typhimurium-specific antibodies on Salmonella colonization in the intestinal tract of poultry. More work is needed to determine if S. typhimurium-specific antibodies alone have a beneficial effect to reduce S. typhimurium colonization. In conclusion, Avian Pac Plus has beneficial effects in reducing S. typhimurium colonization in market-aged broilers when administered at the hatchery and at the farm from day I to day 3. ACKNOWLEDGMENTS We would like to thank our fellow colleagues at The Ohio State University Elizabeth Angrick, Brian S. Harr, and Amanda Porter for their technical and laboratory assistance and Sakol Teeravarunyou and Wichai Hannitinun for their secretarial support with this project. 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