Effects of using a chicken-origin competitive exclusion culture and probiotic cultures on reducing Salmonella in broilers 1

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1 2009 Poultry Science Association, Inc. Effects of using a chicken-origin competitive exclusion culture and probiotic cultures on reducing Salmonella in broilers 1 S. F. Al-Zenki,* 2 A. Y. Al-Nasser, A. E. Al-Saffar, F. K. Abdullah, M. E. Al-Bahouh, A. S. Al-Haddad, H. Alomirah,* and M. Mashaly * Biotechnology Department, and Aridland Agriculture and Greenery Department, Kuwait Institute for Scientific Research, Safat, Kuwait 13109; and Department of Poultry Science, The Pennsylvania State University, University Park Primary Audience: Veterinarians, Nutritionists, Food Scientists SUMMARY Three commercial products, a partially defined chicken-origin competitive exclusion culture (Aviguard, 0.50 ml/chick) and 2 single-organism probiotic cultures (Saccharomyces cerevisiae, Levucell SC, 1 g/kg of feed, and Pediococcus acidilactici, Bactocell, 100 mg/kg of feed) were evaluated for their ability to reduce Salmonella in broilers and their effects on production performance. It was found that all the treatments significantly (P < 0.05) reduced Salmonella concentrations, as compared with the control, on the chicken, in the ceca, and on the chicken carcass. In addition, it was found that these treatments had no adverse effect on any of the production parameters that were measured. Finally, this study showed the importance of using these preharvest treatments as part of an integrated program to control Salmonella at the broiler farm. Key words: broiler, competitive exclusion, probiotic, production performance, Salmonella 2009 J. Appl. Poult. Res. 18 :23 29 doi: /japr DESCRIPTION OF PROBLEM The Kuwait poultry industry is considered a major component of the local food industry, successfully supplying more than 50% of poultry meat for consumption in Kuwait [1]. A major factor influencing the continued increase in poultry meat consumption in Kuwait is the microbiological safety of poultry. Contamination of poultry by foodborne pathogens is considered a major problem facing the poultry industry in Kuwait. During the last 5 yr, the poultry producers in Kuwait suffered major economic losses because of a lack of an effective pathogen reducing-monitoring program for poultry. In addition, on 2 occasions in 2006, the Kuwait Ministry of Public Health banned locally produced broilers from the market because of the high incidence of Salmonella contamination. This has created economic loss to the Kuwait poultry industry. Salmonella food poisoning associated with the consumption of poultry products is a continual problem for the local poultry industry. For 1 The use of trade names in this publication does not imply endorsement of the products mentioned or criticism of similar products not mentioned. 2 Corresponding author: szenki@safat.kisr.edu.kw

2 24 this reason, methods to control Salmonella at the farm and at the processing plant are a priority to both the industry and the public health authorities in Kuwait. Control measures are difficult because of the ubiquitous nature of Salmonella in the farm environment and its colonization of the gastrointestinal tract [2]. Therefore, it is physically and economically impossible to test every chicken carcass to ensure that they are Salmonella free before reaching the consumer [3]. A viable alternative is to reduce the concentrations of Salmonella at the farm level to the lowest possible level and implement good manufacturing practices to prevent the risk of cross contamination at processing. Novel preharvest innovative treatments have recently emerged to control the presence of this pathogen at the farm level, such as competitive exclusion [4 6] and probiotics [7, 8]. Furthermore, effects of these treatments on production performance are of great importance in determining the influence on production efficiency. Khaksefidi and Ghoorchi [9] found that feeding broiler chicks the probiotic Bacillus subtilis increased BW and improved feed conversion. In addition, Zhang et al. [10] concluded that dietary yeast components improved broiler growth performance. Therefore, the objectives of this study were to evaluate the effect of using 3 commercial products on reducing Salmonella in broilers and to study whether these treatments would have an adverse effect on broiler production performance. MATERIALS AND METHODS Bird Management One-day-old broiler chicks [11], obtained from 1 commercial hatchery, were used in the current study. This study was conducted on a commercial farm using 1 part of a house. This part was divided into 36 floor pens equipped with nipple drinkers, under continuous lighting, and wood shavings were used as floor bedding. Water and an unmedicated corn-soy-based diet that met the NRC requirements [12] were provided ad libitum. The chicks received a prestarter diet (24.4% CP, 3,029 kcal of ME/kg) from hatch until 7 d of age, a starter diet (22.5% CP, 3,036 kcal of ME/kg) from 8 until 21 d of age, and a finisher diet (21.6% CP, 3,171 kcal of ME/ kg) from 22 until 35 d of age, when the experiments ended and the birds were slaughtered. Experimental Design One hundred twenty straight-run chicks were placed randomly in each of the 36 pens mentioned above (5 m 2 per pen), thus providing m 2 /bird. The 36 pens were divided into 4 groups of 9 pens each and were used for 1 of the 4 treatments described below. This study was repeated in the summer and winter seasons to determine whether the season would have any effect on Salmonella contamination regardless of treatment used. In all, a total of 8,640 birds were used for the entire study. Treatments The 1-d-old chicks for each group, mentioned previously, received 1 of 4 treatments. These treatments included the following: the control group with no treatment and a basal diet was used (treatment 1); chicken-origin, partially defined, competitive exclusion culture [13] (Aviguard MDL; treatment 2) was sprayed on the 1-d-old chicks at a dose of 0.5 ml/bird; a probiotic Saccharomyces cerevisiae culture (Leuvcell SB20) [14] was administered in the feed throughout the 5-wk period at a concentration of 0.1% (1 kg/ton of feed; treatment 3); and a probiotic Pediococcus acidilactici culture (Bactocell PA10) [14] was also administered in the feed throughout the 5-wk period at a concentration of 0.01% (100 g/ ton of feed; treatment 4). Sample Collection and Microbiological Analysis JAPR: Research Report Samples were collected both from the farm and at the processing plant. Farm samples were collected 1) just before placement of chicks and 2) at 7, 21, and 35 d of age. The processing plant samples were collected at 1) the evisceration step to obtain the cecal samples and 2) the postchilling step to obtain the carcass samples. All of these samples were randomly collected from all 36 pens of the different treatments for each of the 2 seasons. Paper tray liners were collected from 15 transport boxes before chick placement. Each

3 Al-Zenki et al.: SALMONELLA REDUCTION IN BROILERS 25 individual paper tray liner was then placed in a large sterile bag. A 10-g representative sample was then removed from the bag and placed in a sterile Stomacher bag and diluted 1:10 in 0.1% buffered peptone water. Fifteen feed samples (10 g) and 15 water samples (10 ml) were collected in sterile Stomacher bags before chick placement and diluted 1:10 in 0.1% buffered peptone water. Air was also sampled before chick placement. A Petri dish containing brain heart infusion solid medium was placed in an Oxoid air sampler [15] set to draw 60 L in 20 s. Air samples were collected from areas containing ventilation fans as well as random areas in the house. Ethyl alcohol (70%) was applied at each sampling site to sanitize the air sampler cover. The medium was then aseptically transferred to 100 ml of 0.1% buffered peptone water. Litter samples were collected before chick placement and at 7, 21, and 35 d of age. Five litter samples (10 g) were collected from 5 different areas within each pen (near the feeders and drinker lines) to form 1 composite sample. The prevalence of Salmonella on the chicken body and in the ceca was determined before chick placement and at 7, 21, and 35 d of age. Five randomly selected chickens were removed at each sampling period from each pen from each treatment. The body of the whole bird was placed in large sterile Stomacher bags and rinsed for 2 min in 400 ml of 0.1% buffered peptone water. The whole-bird rinse solution was then poured into sterile containers. The body surface was washed with 70% ethyl alcohol and then aseptically dissected. Ceca were removed and their contents diluted 1:3 with 0.1% buffered peptone. At the processing plant, the prevalence of Salmonella on the chicken carcass and in the ceca was also determined. Numbered leg bands were placed on the chicken as a means of identification. The ceca of 5 randomly selected chickens, representing each pen from each treatment, were collected during the evisceration step. Additionally, the same posteviscerated carcasses (5 samples) were collected after air chilling. Sample preparation for the postchilled carcass and posteviscerated cecal samples was carried out as described earlier. Salmonella detection was carried out as outlined in the US Food and Drug Administration Bacteriological Analytical Manual Salmonella isolation procedure [16] with some modifications, where buffered peptone water replaced lactose broth for preenrichment. All samples were preenriched at 37 C for 24 h in 0.1% buffered peptone water. After incubation, 1 ml quantities of the preenriched samples were transferred to 9 ml of tetrathionate broth and selenite cysteine broth and incubated for 24 h at 37 C. After incubation, samples were then streaked onto xylose lysine deoxycholate and bismuth sulfite agar. Suspected Salmonella colonies were stabbed onto triple sugar iron agar and lysine iron agar slants and presumptive Salmonella were confirmed by serotyping. Production Performance Measured Twenty birds from each pen in each treatment, selected randomly each time, were weighed at hatch, 7, 21, and 35 d of age. Feed consumption for each pen was determined at 7, 21, and 35 d of age and feed consumption per bird and FCR (corrected for mortality) were calculated. Mortality was recorded daily and weekly mortality was calculated. Temperatures in the house were measured throughout the summer and winter seasons. The average temperatures throughout the summer and winter seasons were approximately 28 and 23 C, respectively. Data Analysis Data were analyzed using a 1-way ANOVA utilizing the S-Plus statistical program [17]. Treatments at each age were the main effect. Means were separated using Tukey s test, and significance was set at P < In addition, overall mean comparison between summer and winter, regardless of the treatment, was also carried out using the same S-Plus statistical program. RESULTS AND DISCUSSION Effects of Treatments on Salmonella Reduction Whole Birds, Carcasses, and Ceca. The effects of various treatments on the prevalence of Salmonella on the exterior body and in the ceca at different ages during the summer season are

4 26 JAPR: Research Report Table 1. Effects of different treatments on Salmonella reduction (%) on broiler whole body and postchill carcass at different ages in the summer and winter season 1 Age (d) a 28.9 b 22.2 b 35.6 b 35.6 a 20.0 a 13.0 a 22.2 a a 28.9 b 33.3 b 31.1 b 46.7 a 33.3 a 35.6 a 42.2 a a 22.2 b 20.0 b 22.2 b 26.7 a 4.4 b 4.4 b 6.7 b Carcass 26.7 a 2.2 b 4.4 b 6.7 b 2.2 a 2.2 a 0.0 a 0.0 a a,b Means within a season and within each age in a row with no common superscripts differ significantly (P < 0.05). shown in Tables 1 and 2, respectively. For the summer season, our results showed that all treatments applied significantly (P < 0.05) reduced Salmonella contamination on the exterior body and in the ceca of chicks at different ages when compared with the control. These results are in agreement with previous studies using different preharvest treatments to control Salmonella in poultry. Deruyttere et al. [18] reported that 24% of the control flocks were Salmonella-positive compared with none recovered from Aviguardtreated flocks. Similarly, Line et al. [8] reported a 50% reduction in yeast-treated birds compared with the positive control. It is important to note that in our study, a greater percentage reduction perhaps was not achieved partly because of the negative effect of Salmonella contamination of the 1-d-old chicks and of the farm environment. The isolation of Salmonella from the 1-d-old chicks before chick placement (body 69.2%; ceca 47.8%) may have resulted in a considerable delay in the early establishment of the competitive flora of the treatments to prevent Salmonella colonization and multiplication in the intestinal tract. In addition to Salmonella contamination of the 1-d-old chicks, the isolation of Salmonella from the paper liners (66.7%), feed (26.6%), and air (20%) before chick placement could have resulted in the exposure of chicks to Salmonella, thus jeopardizing the optimum effect of the treatments under investigation. The effect of various treatments on the prevalence of Salmonella in the ceca from posteviscerated chicken and the postchilled carcass at the processing plant during the summer season are also shown in Tables 1 and 2, respectively. For the control treatment, the percentage of Salmonella culture-positive ceca from eviscerated chickens and postchilled carcasses were 28.9 and 26.7%, respectively. A significant reduction (P < 0.05) of more than 20% was observed for Salmonella culture-positive ceca from eviscerated chickens and postchilled carcasses for all treatments compared with the control. It is noteworthy that the percentages of Salmonella on the carcass after processing represent the most important data of this study because these numbers reflect the concentrations of Salmonella present on broilers before marketing. The data shown for the control birds (26.7%) in the present study are above the 20% margin level adopted by Kuwaiti regulatory agencies and the zero Table 2. Effects of different treatments on Salmonella reduction (%) in broiler ceca at different ages in the summer and winter season 1 Age (d) a 57.8 b 44.4 b 51.1 b 35.6 a 15.6 a 22.2 a 13.3 a a 33.3 b 33.3 b 28.9 b 46.7 a 15.6 a 33.3 a 28.9 a a 8.9 b 11.1 b 13.3 b 35.6 a 6.7 b 8.9 b 2.2 b Carcass 28.9 a 6.7 b 6.7 b 6.7 b 15.6 a 4.4 a 8.9 a 6.7 a a,b Means within a season and within each age in a row with no common superscripts differ significantly (P < 0.05).

5 Al-Zenki et al.: SALMONELLA REDUCTION IN BROILERS 27 Table 3. Effects of different treatments on Salmonella reduction (%) in litter at different broiler ages in the summer and winter season 1 Age (d) a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 6.7 a 6.7 a a 37.8 ab 24.4 b 28.9 b 6.7 a 6.7 a 6.7 a 0.0 a a 35.6 b 40.0 b 26.7 b 13.3 a 0.0 b 0.0 b 0.0 b a,b Means within a season and within each age in a row with no common superscripts differ significantly (P < 0.05). tolerance for Salmonella adopted worldwide. Furthermore, an average reduction of more than 20% achieved by the treatments under investigation could contribute greatly to the safety of poultry before marketing The effects of various treatments on the prevalence of Salmonella on the exterior body and in the ceca at different ages during the winter season are also shown in Tables 1 and 2, respectively. The results show that the treatments under investigation resulted in a numerical but not significant reduction (P > 0.05) at d 7 and 21. However, the reduction was significant (P < 0.05) at d 35 (market age). These results are different from those found in the summer season, in which the reduction was significant at all ages. This could be due to the greater contamination level of Salmonella in 1-d-old chicks before chick placement during the summer season (body: 69.2%; ceca: 47.8%) compared with the winter season (body: 10%; ceca: 6.7%), which resulted in a more pronounced effect of the treatments. The effect of various treatments on the prevalence of Salmonella in the ceca from posteviscerated chicken and the postchilled carcass during the winter season are also shown in Tables 1 and 2, respectively. Contrary to the summer season, the treatments used resulted in no significant reduction (P > 0.05) on the postchilled carcass or the posteviscerated ceca of the carcass when compared with the control during the winter season (Tables 1 and 2). Salmonella cecal colonization and contamination of the exterior body are known to be the major factors in Salmonella presence at the processing plant [19]. Therefore, the insignificant reduction found in this study could be explained by seasonal variation because the overall Salmonella contamination on the body exterior was significantly (P < 0.05) less in the winter (16.8%) than in the summer (37.5%) and in the ceca was also significantly (P < 0.05) less in the winter (16.3%) than the summer (35.4%). Litter. The effect of various treatments on the prevalence of Salmonella in litter samples collected from pens of various treatments at different ages during the summer and winter seasons are shown in Table 3. In the summer season, no Salmonella were present in the litter samples from the control or the treatment groups at 7 d of age. However, a significant reduction (P < 0.05) in Salmonella-positive litter samples was observed in all treatments when compared with the control at both 21 and 35 d of age. These results show that the treatments that were effective in reducing Salmonella presence in the body and in the ceca of broilers also had a beneficial effect in reducing Salmonella in the litter. This was the case even though it had been reported that Salmonella shed by Salmonella-colonized birds remained viable in the litter from a few days to several weeks [20]. For the winter season, the treatments showed a significant (P < 0.05) Salmonella reduction at 35 d of age (Table 3). This reduction corresponds well with a similar reduction on the exterior body and in the ceca of birds found at 35 d of age (Tables 1 and 2). In addition, our results showed that overall Salmonella contamination of the litter (preplacement) was significantly (P < 0.05) less in the winter season (5.0%) than in the summer season (19.7%). This seasonal difference could be due to more stress imposed on the birds in the summer than in the winter season. Stress-associated conditions have been shown to increase shedding of Salmonella Enteritidis in chickens [21, 22]. Furthermore, McBride et al.

6 28 JAPR: Research Report Table 4. Effects of different treatments for Salmonella reduction in broilers on production performance in the summer and winter season 1 Parameter Final BW (g/bird) 1,367.9 a 1,370.3 a 1,347.6 a 1,383.6 a 1,402.0 a 1,440.3 a 1,439.6 a 1,470.8 a Total feed consumption (g/bird) 2,195.8 a 2,149.6 a 2,147.5 a 2,169.2 a 2,378.8 a 2,459.1 a 2,431.1 a 2,428.6 a FCR 1.66 a 1.62 a 1.65 a 1.61 a 1.76 a 1.76 a 1.74 a 1.70 a Total mortality (%) 4.79 a 4.74 a 4.21 a 4.12 a 3.31 a 7.35 b 3.46 a 3.93 a a,b Means for each parameter within a season in a row with no common superscripts differ significantly (P 0.05). [23] have reported greater frequency of Salmonella detection in flocks reared in the summer compared with the winter season. Production Performance. Data on production performance, including BW, feed consumption, FE, and percentage of mortality, are shown in Table 4. Our results showed that none of the treatments had any significant effects on any of the parameters measured. Our results agree with the findings of Angel et al. [24] and Mountzouris et al. [25], who found that the use of probiotic products in the feed had no significant effect on broiler BW. In addition, O Dea et al. [26] and Mountzouris et al. [25], using probiotics, and Zhang et al. [10], using yeast products, found that these products improved FE, but the differences were not significant. CONCLUSIONS AND APPLICATIONS 1. Our results suggest that the threat of the pathogen could originate from the hatchery. 2. This study has also shown a high prevalence of Salmonella on the body and in cecal contents of newly hatched chicks in Kuwait. 3. The treatments under investigation (partially defined chicken-origin competitive exclusion culture (Aviguard) and 2 single-organism probiotic cultures (S. cerevisiae, Levucell SC and P. acidilactici, Bactocell) could be used to reduce Salmonella concentrations in local broilers without adversely affecting production performance. REFERENCES AND NOTES 1. Ministry of Planning Statistics and Census Sector. Annu. Stat. Abstr. 40: Turner, A. K., M. A. Lovell, S. D. Hulme, L. Zhang- Barber, and P. A. Barrow Identification of Salmonella Typhimurium genes required for the colonization of the chicken alimentary tract and for newly hatched chicks. Infect. Immun. 66: Holleman, K. A Food Safety Fact Sheet for Poultry Producers and Processors. Oregon State University Extension Service. Oregon State University, Corvallis. 4. Nakamura, A., Y. Ota, A. Mizukami, T. Ito, Y. Ngwai, and Y. Adachi Evaluation of Aviguard, a commercial competitive exclusion product for efficiency and after Effect on the antibody response of chicks to Salmonella. Poult. Sci. 81: Stern, N., N. Cox, J. Bailey, M. Berrang, and M. Musgrove Comparison of mucosal competitive exclusion treatment to reduce Salmonella and Campylobacter spp. colonization in broiler chickens. Poult. Sci. 80: Seo, K. H., P. S. Holt, R. K. Gast, and C. L. Hofacre Elimination of early Salmonella Enteritidis infection after treatment with competitive-exclusion culture and enrofloxacin in experimentally infected chicks. Poult. Sci. 79: Johannsen, S. A., R. W. Griffith, I. V. Wesley, and C. G. Scanes Salmonella enterica serovar Typhimurium colonization of the crop in the domestic turkey: Influence of probiotic and prebiotic treatment (Lactobacillus acidophilus and lactose). Avian Dis. 48: Line, J. E., J. S. Bailey, N. A. Cox, N. J. Stern, and T. Tompkins Effect of yeast supplemented feed on Salmonella and Campylobacter populations in broilers. Poult. Sci. 77: Khaksefidi, A., and T. Ghoorchi Effect of probiotic on performance and immunocompetence in broiler chicks. Jpn. Poult. Sci. 43: Zhang, A., B. Lee, S. Lee, K. Lee, G. An, K. Song, and C. Lee Effect of yeast (Saccharomyces cervisiae) cell components on growth performance, meat quality, and lleal mucosa development of broiler chicks. Poult. Sci. 84: Al Sultan Poultry Company, Kuwait City, Kuwait. 12. NRC Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC.

7 Al-Zenki et al.: SALMONELLA REDUCTION IN BROILERS Microbial Development Ltd., Malvern Link, Worcestershire, United Kingdom. 14. Lallemand SAS, Blagnac Cedex, France. 15. Oxoid Ltd., Basingstoke, Hampshire, United Kingdom. 16. US Food and Drug Administration Bacteriological analytical manual online. Chapter 5 Salmonella. Accessed January Crawley, M. J Statistical Computing: An Introduction to Data Analysis Using S-Plus. Wiley, New York, NY. 18. Deruyttere, L., J. Klaasen, R. Froyman, and C. A. Day Field study to demonstrate the efficacy of Aviguard against intestinal Salmonella colonization in broilers. Pages in Proc. Salmonella and Salmonellosis. Zoopole Development, Institut Superieur des Productions Animales et des Industries Agro-alimentaires, Ploufragen, France. 19. Lillard, H. S Incidence and recovery of Salmonella and other bacteria from commercially processed poultry carcasses and selected pre- and post-evisceration steps. J. Food Prot. 52: Hoover, N. J., P. B. Kenney, J. D. Amick, and W. A. Hypes Pre-harvest sources of Salmonella colonization in turkey production. Poult. Sci. 76: Holt, P. S Effect of induced molting on B cell and CT4 and CT8 cell numbers in spleens and peripheral blood of White Leghorn hens. Poult. Sci. 71: Holt, P. S Effect of induced molting on the susceptibility of White Leghorn hens to a Salmonella Enteritidis infection. Avian Dis. 37: McBride, G. B., B. Brown, and B. J. Skura Effect of bird type, growers and season on the incidence of Salmonella in turkeys. J. Food Sci. 43: Angel, R., R. Dalloul, and J. Doerr Performance of broiler chickens fed diets supplemented with a direct-fed microbial. Poult. Sci. 84: Mountzouris, K., P. Tsirtsikos, E. Kalamara, S. Nitsch, G. Schatzmayr, and K. Fegero Evaluation of the efficacy of a probiotic containing Lactobacillus, Bifidobacterium, Enterococcus, and Pediococcus strains in promoting broiler performance and modulating cecal microflora composition and metabolic activities. Poult. Sci. 86: O Dea, E., G. Fasenko, G. Allison, D. Korver, G. Tannock, and L. Guan Investigating the effects of commercial probiotics on broiler chick quality and production efficiency. Poult. Sci. 85: Acknowledgments This research project was partially funded by the Kuwait Foundation for Advancement of Sciences.