Effect of Cecal Cultures Lyophilized in Skim Milk or Reagent 20 on Salmonella Colonization in Broiler Chicks 1

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1 Effect of Cecal Cultures Lyophilized in Skim Milk or Reagent 20 on Salmonella Colonization in Broiler Chicks 1 A. G. HOLLISTER, 2 D. E. CORRIER, D. J. NISBET, R. C. BEIER, and J. R. DeLOACH USDA, Agricultural Research Service, Food Animal Protection Research Laboratory, Route 5, Box 810, College Station, Texas ABSTRACT Mixed cultures of cecal bacteria that were grown under continuous flow anaerobic conditions were prepared as lyophilized powder in skim milk or Reagent 20 (R-20; a mixture containing sucrose and bovine serum albumin fraction V) and compared with broth cultures for reduction of Salmonella typhimurium enteric colonization. Day old broiler chicks were provided a standard corn-soybean diet with: 1) no culture, (control); 2) broth culture administered by crop gavage; 3) broth culture added to the drinking water; 4) culture lyophilized in skim milk and added to drinking water; 5) culture lyophilized in skim milk in gelatin capsules and force-fed; 6) culture lyophilized in R-20 and added to drinking water; and 7) culture lyophilized in R-20 in gelatin capsules and force-fed. All groups were challenged on Day 3 with 10 4 cfu of S. typhimurium per chick. Culture by crop gavage, culture in the drinking water, skim milk powder in capsules, and R-20 powder in the water and in capsules significantly (P <.05) reduced mean Salmonella colonyforming units in cecal contents by 3.21 to 5.26 log 10 units at 10 d of age. Likewise, the number of cecal-culture-positive chicks in the same groups was significantly less than controls with reductions of 27 to 67%. The numbers of Salmonella per gram of cecal contents and the percentage of cecal-culturepositive chicks in the skim milk powder in the drinking water group were not different from control chicks in one of two experiments. The results suggest that the culture of cecal bacteria lyophilized in R-20 was as effective as the same culture in broth form for the reduction of Salmonella colonization in broiler chicks and demonstrate a practical delivery method that could be easily incorporated into current industry practices. (Key words: Salmonella, skim milk, sucrose, lyophilization, broiler) INTRODUCTION Lyophilized, mixed, competitive exclusion (CE) cultures grown in vitro, processed as a group, and delivered directly to chicks in feed or water have only recently been reported (Hollister et Received for publication April 18, Accepted for publication June 16, Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. 2 To whom correspondence should be addressed Poultry Science 73: al, 1994; Holton and Day, 1994). Mixed defined. or undefined CE cultures are normally delivered to chicks in liquid form (broth) by crop gavage (Nurmi and Rantala, 1973; Lloyd et al, 1974, 1977; Snoeyenbos et al, 1978; Bailey et al, 1988; Corrier et al, 1990; Stavric et al, 1992), by diluting the broth with drinking water (Snoeyenbos et al, 1978; Pivnick et al, 1981; Pivnick and Nurmi, 1982; Mead and Impey, 1984; Impey et al, 1987; Wierup et al, 1988; Schneitz et al, 1990; Stavric et al, 1991), or by spraying the culture on hatchlings (Goren et al, 1988; Blankenship et al, 1991; Schneitz, 1992). Cecal homogenates collected from specific-

2 1410 HOLLISTER ET AL. pathogen-free birds have been successfully lyophilized and rehydrated before delivery (Mulder and Bolder, 1991), and lyophilized, mixed CE cultures have been "batch" grown in liquid media for 24 h or more (to increase numbers of organisms) before delivery (Pivnick et ah, 1982). Ideally, a CE culture should be designed so that the delivery method is easily incorporated into current industry practices without requiring additional time, labor, equipment, or expertise on the part of the poultry producer. The product should not only be effective, but also easy to store, handle, and transport. Lyophilized cultures readily fit these criteria because they have a longer potential shelf life (Harrison and Pelczar, 1963; Lapage and Redway, 1974; Heckly, 1978), they are much lighter than wet products (therefore easier and less expensive to handle and transport), and they are relatively unaffected by normal shipping and storage temperatures while in the dry form (Heckly, 1978). If survival of organisms is high (not requiring "batch up" to increase numbers), the culture can then be administered directly in feed, drinking water, or by spraying it on hatchlings, thus requiring minimal alterations to current industry practices. "Batch up," however, requires additional time, labor, equipment, and expertise. The potential disadvantage of lyophilized culture, when compared with fresh culture, is that viability of the microorganisms can be adversely affected if freezing, drying, packaging, and rehydration are not carried out under appropriate conditions. For example, losses due to freezing or drying have varied from 0 to 100%, depending on the species involved, its age, handling and processing conditions, and method and time of storage (Lapage and Redway, 1974). Among the numerous factors that affect the survival of microorganisms subjected to lyophilization, the composition of the suspending medium may be one of the more critical considerations for prevention of damage during the freezing and thaw- 3 Brower, Houghton, IA ing stages (Heckly, 1961, 1978; Morichi, 1970; Lapage and Redway, 1974). Cryoprotective agents in the suspending medium are thought to prevent or minimize damage to cells by: 1) controlling the amount of residual water, to prevent overdrying; 2) providing amino groups, to neutralize toxic carbonyl radicals; 3) minimizing electrolytes that may become toxic at high concentrations; and 4) providing cake-forming substances, to prevent mechanical damage and promote uniform drying (Greaves, 1964; Meryman, 1971). Skim milk is probably the most popular general purpose medium for lyophilization, followed by solutions containing sucrose (Marshall and Scott, 1970; Janssen and Busta, 1973; Heckly, 1978). Reagent 20 (R-20; a mixture of sucrose and bovine serum albumin fraction V) is the suggested solution for anaerobic bacteria preserved in culture collections (Simione and Brown, 1991). Hollister et ah (1994) found that an undefined continuous-flowgrown culture, which had been selected for Salmonella colonization control, in combination with dietary lactose was effective at reducing Salmonella colonization when lyophilized in alginate beads with skim milk and top-dressed on the feed, but was not effective when lyophilized in skim milk alone and mixed with the feed. The present study tested the effectiveness of a characterized cecal culture grown under continuous flow conditions lyophilized in skim milk or R-20 and resuspended in the drinking water for the reduction of Salmonella colonization in broiler chicks. MATERIALS AND METHODS Animals and Husbandry Day of hatch male broiler chicks (Indian River x Indian River) were purchased from a local hatchery. Twenty chicks were randomly assigned to each treatment group, housed in battery brooders 3 in a climatecontrolled room, and provided continuous fluorescent lighting. An unmedicated cornsoybean meal mash ration that was formulated to meet NRC requirements (1984) and water were provided for ad libitum con-

3 LYOPHILIZED CECAL CULTURES FOR SALMONELLA CONTROL 1411 sumption. The treatment groups were: 1) no culture, (control); 2) broth culture by crop gavage; 3) broth culture in the drinking water; 4) culture lyophilized in skim milk and added to drinking water; 5) culture lyophilized in skim milk in gelatin capsules; 6) culture lyophilized in R-20 and added to drinking water; and 7) culture lyophilized in R-20 in gelatin capsules. Delivery methods were as follows. The broth culture was provided by either crop gavage (.5 ml containing 10 8 cfu per chick) or 80 ml per pen was mixed with the first drinking water in a ratio of one to four. The lyophilized powder was provided either by mixing the equivalent of 20 ml of dry skim milkculture or R-20-culture mixture with 400 ml of the first drinking water (the same final ratio as in the broth plus water or up to 4 ml of broth per chick) or.100 g was placed in a gelatin capsule and force-fed to each chick (equivalent to.5 ml broth per chick). The drinking water used on Day 1 in these trials was chlorinated tap water from the local public water system mat had been allowed to stand 24 h in open containers to allow most of the chlorine to dissipate before use. Feed was withheld for 4 h. At the same time that the feed was introduced, any remaining culture-drinking water mixture was discarded and all chicks were switched to fresh drinking water (chlorinated tap water). The paper liners from the shipping containers in which the chicks were transported and the feed ration were determined to be Sa/moneZ/fl-culturenegative using procedures described by Andrews et al. (1978). On Day 3, all birds were challenged orally with 10 4 cfu of S. typhimurium. This challenge dose was suggested by Mead et al. (1989) and is recommended by laboratories in the United Kingdom, France, The Netherlands, Canada, and the United States to be a standard challenge dose in order to facilitate interlaboratory comparisons. On 4 Difco Laboratories, Detroit, MI American Type Culture Collection, Rockville, MD «J. T. Baker, Inc., Phillipsburg, NJ Sigma Chemical Co., St. Louis, MO FTS Systems, Inc., Stone Ridge, NY Coy Laboratory Products, Ann Arbor, MI Day 10, all birds were killed by cervical dislocation and cecal contents were aseptically collected to determine the presence of Salmonella and Salmonella colony-forming units per gram of cecal contents. Samples of cecal contents were also collected to measure cecal ph and concentrations of acetic, propionic, butyric, and lactic acid. The experimental design was repeated in Experiment 2 using newly hatched chicks. Culture Preparation The cultures utilized in the current experiments were collected from the ceca of adult broiler chickens and maintained under continuous-flow anaerobic conditions. Effluent was collected anaerobically in a 1-L flask at 4 C. Bacterial cells were concentrated by centrifugation (4,000 x g) for 20 min at 0 C. The cells were resuspended in one-fourth the original volume of sterile, chilled, 20% skim milk 4 or sterile, chilled, R-20 (ATCC5 formula; 20 g sucrose 6 and 10 g bovine serum albumin fraction V 7 /100 ml of water). The culture lyophilized in skim milk or R-20 was prepared by pipetting 10-mL volumes of the skim milk-culture mixture or R- 20-culture mixture into 40-mL glass serum bottles, capped with rubber stoppers, and placed in a -80 C freezer. After 2 h in the freezer, the bottles were placed in the freeze dryer, 8 the rubber stoppers were removed, and the samples allowed to dry for 24 h (-50 C, 40 mtorr). The chamber was subsequently flushed with a mixture of oxygen-free nitrogen, hydrogen, and carbon dioxide (approximately 85, 10, and 5%, respectively), the rubber stoppers were replaced, the bottles were sealed with aluminum seals, and stored at 4 C until used. All processing was conducted under anaerobic conditions in a chamber 9 or in sealed containers. The viable cell concentration of the chemostat culture effluent and lyophilized powder was determined by serial dilution of the cultures, or powder dissolved in PBS, and spread plating on Brain Heart Infusion agar 4 plates. Anaerobic colony-forming units per milliliter of broth culture or per gram of powder were determined by colony counts after anaerobic incubation at 37 C for 48 h.

4 1412 HOLLISTER ET AL. Salmonella Challenge A S. typhimurium primary poultry isolate provided by the National Veterinary Services Laboratory, Ames, IA 50010, was selected for resistance to novobiocinnalidixic acid (NONA) in the authors' laboratory and maintained on nutrient agar. Media used to culture the resistant isolate contained 25 /ig/ml novobiocin and 20 /ig/ml nalidixic acid to inhibit the growth of other bacteria. Inocula for challenge was prepared from overnight tryptic soy broth 4 cultures serially diluted in sterile PBS. The viable cells present were determined from colony counts on NONA brilliant green agar (BGA) plates. 10 Salmonella typhimurium Cecal Colonization Cecal contents of one cecum from each chick were serially diluted in PBS, spreadplated on NONA BGA plates to final dilutions of 1:100,1:1000, and 1:10,000, and incubated for 24 h at 37 C. Total colonyforming units of S. typhimurium per gram of cecal contents was determined on an automatic colony counter. 11 Minced tissues and contents from the other ceca were aseptically placed in 30 ml of selenite cysteine broth, 4 agitated vigorously, and incubated overnight at 37 C. The broth was streaked on NONA plates, incubated overnight, and examined for typical Salmonella colonies. Cecal contents that were negative at the 1: 100 dilution on BGA plates but positive after culturing in selenite-cysteine and plating on BGA plates were arbitrarily assigned a value of 1.5 log 10 Salmonella cfu/ g of cecal contents. Cecal contents that were negative at the 1:100 dilution on BGA plates and negative after selenite-cysteine enrichment and BGA plating were scored as 0 log 10 cfu. Salmonella colonies were confirmed by biochemical tests on triple sugar iron agar and lysine iron agar 4 and confirmed as S. typhimurium by serological tests with Salmonella O antiserum, poly A and group B factors 1, 4, 5, and "Oxoid U.S.A., Inc., Ogdensburg, NY New Brunswick Scientific Co., Inc., Edison, NJ Cecal ph and Organic Acid Concentrations Cecal contents (.2 g) were collected from each chick, suspended in.8 ml sterile glassdistilled water, and the ph measured with a glass ph electrode. The concentrations of acetic, propionic, and butyric acids in the cecal contents were measured with a gas chromatograph utilizing the method described by Hinton et al. (1990). Lactic acid concentration was measured by the enzymatic method of Hohorst (1965). Statistical Analyses Differences in cecal logio Salmonella counts among treatment groups were determined by analysis of variance using the General Linear Models procedures of PCSAS (Luginbuke and Schlotzhauer, 1987). Significant differences were further separated with Duncan's multiple range test using commercial statistical analysis software, PCSAS Release 6.02 (Luginbuke and Schlotzhauer, 1987). Chi-square analysis was used to determine significant differences between groups in Salmonella cecal colonization rates. Differences in all experiments were considered to be significant based on the.05 level of probability. RESULTS AND DISCUSSION The number of S. typhimurium in the cecal contents of chicks in all treatment groups in both experiments, except the skim milk powder in drinking water group in Experiment 1, were significantly less than in the control groups (Table 1). Mean reductions in Salmonella colonyforming units per milliliter of cecal contents in the effective groups ranged from 2.7 to 5.1 log 10 units (approximately 1,000- to 100,000-fold reduction). Similarly, the percentage of cecal-culture-positive chicks in the group provided culture in skim milk powder in the drinking water was not different from the controls, whereas there were significantly fewer Salmonella cecal-culture-positive chicks in all other treatment groups than in controls (Table 2). Mean reductions in percentage cecal-culture-positive chicks ranged from 33 to 56%. There were no differences

5 LYOPHILIZED CECAL CULTURES FOR SALMONELLA CONTROL 1413 TABLE 1. Log 10 Salmonella typhimurium per gram of cecal contents in broiler chicks provided cecal cultures by gavage, in the drinking water, or in gelatin capsules TABLE 2. Percentage Salmonella cecal-culturepositive broiler chicks provided cecal cultures by gavage, in the drinking water, or in gelatin capsules Treatment 1 Control Broth 2 by gavage Broth in water SM 2 in water R-20 2 in water SM in capsule R-20 in capsule ± 1.27* 1.35 ± 1.45c 1.06 ± 1.72' 4.52 ± 2.42*.89 ± 1.58 c 2.93 ± 2.57 bc 1.79 ± 1.95' Experiment ± 1.06 a 1.93 ± 1.68' 1.41 ± 2.00c 2.88 ± 2.69"" 3.37 ± 2.52bc 2.02 ± 2.49c 2.98 ± 2.40 bc a_c Means with no common superscript differ significantly (P <.05). 1 Broiler chicks were administered cecal cultures on Day 1 (except controls), challenged orally with 10 4 S. typhimurium on Day 3 and killed on Day 10. Treatments were: 1) no culture, (control); 2) broth culture by crop gavage; 3) broth culture in the drinking water; 4) culture in lyophilized skim milk powder added to drinking water; 5) culture in lyophilized skim milk powder in gelatin capsules; 6) culture in lyophilized Reagent 20 powder added to drinking water; and 7) culture in lyophilized Reagent 20 powder in gelatin capsules. 2 Broth = culture in liquid form; SM = culture lyophilized in 10% skim milk; R-20 = culture lyophilized in Reagent 20; a mixture of sucrose and bovine serum albumin fraction V. among the remaining treatment groups in Salmonella per gram of cecal contents or percentage cecal-culture-positive chicks, indicating that the culture tested was equally effective at reducing Salmonella colonization when provided in lyophilized or fresh broth form (with the possible exception of skim milk in drinking water, as mentioned above). Under the conditions of these experiments, no consistent differences between skim milk and R-20 were apparent, based on Salmonella colonization reduction. It has been suggested, however, that solutions containing sucrose (like R-20) may provide better long-term viability than skim milk (Staab and Ely, 1987). The relatively greater variation in response, between experiments, of the chicks in the groups provided culture lyophilized in skim milk or R-20 and added to the drinking water suggests that (assuming there was no pen effect, processing differences, or errors in counting procedures), in some cases, there may not be as many organisms surviving Treatment 1 Control Broth 3 by gavage Broth in water SM 3 in water R20 3 in water SM in capsule R20 in capsule 1 100O' 2 60>>c 40c 87ab 33= 67bc 60>* Experiment C'n) 2 100" 73* 47c 67bc 73 bc 53* 7 3 bc "-cmeans with no common superscript differ significantly (P <.05). 1 Broiler chicks were administered cecal cultures on Day 1 (except controls), challenged orally with 10 4 S. typhimurium on Day 3 and killed on Day 10. Treatments were: 1) no culture, (control); 2) broth culture by crop gavage; 3) broth culture in the drinking water; 4) culture in lyophilized skim milk powder added to drinking water; 5) culture in lyophilized skim milk powder in gelatin capsules; 6) culture in lyophilized Reagent 20 powder added to drinking water; and 7) culture in lyophilized Reagent 20 powder in gelatin capsules. Percentage of chicks Salmonella cecal-culturepositive (n = 15). 3 Broth = culture in liquid form; SM = culture lyophilized in 10% skim milk; R-20 = culture lyophilized in Reagent 20; a mixture of sucrose and bovine serum albumin fraction V. and subsequently colonizing the chicks' digestive tract. The number of viable organisms present in a CE culture does affect the level of Salmonella colonization control (Nisbet et al, 1994). Some of the variation in results may have been due to the tap water that was used to rehydrate the lyophilized cultures, because no attempt was made to standardize it (other than overnight aging to release chlorine) and nothing was added to neutralize the chlorine or other sanitizers that may have been present. It has been demonstrated previously that plain water is not the optimal rehydration solution for maximizing the number of viable organisms (Leach and Scott, 1959; Valdez et al, 1985), and many organisms die quickly in distilled water (Heckly, 1978). Various species also respond differently to lyophilization (Steel and Ross, 1963). Further studies are necessary to test

6 1414 HOLLISTER ET AL. methods of improving viability of lyophilized cultures upon rehydration. It is very likely that methodology already in use for the delivery of vaccines and other biologicals via the drinking water will also help improve the consistency of performance of water-delivered CE cultures. Anaerobic plate counts at the beginning of each experiment determined that broth culture contained 10 8 cfu/ml, whereas dry skim milk and R-20 powder both contained 10 9 cfu/g after 7 d of storage (rehydrated in PBS). The fact that the lyophilized powder in a gelatin capsule was as successful as broth and lyophilized powder in the drinking water confirms the work of HoUister et al. (1994), suggesting that mixed CE cultures can also be successfully delivered in a dry form if consumed quickly or protected in some way. The ph of cecal contents did not vary significantly among the groups (the range was 5.45 to 5.74; data not shown). Likewise, the concentration of acetic, butyric, and lactic acid in cecal contents did not consistently differ among the groups (data not shown). The concentration of propionic acid, however, was significantly higher in all treatment groups than in controls, in agreement with earlier studies (Hollister et al, 1992, 1994; Nisbet et al, 1993). Mean propionic acid concentrations were 2.76 vs /miol/g of cecal contents for controls and all others, respectively. In summary, CE cultures made up of compatible microorganisms that have been grown together can be processed, lyophilized, and delivered to young chicks as a mixed culture, eliminating the need for separate growing and processing for each species. Lyophilized cecal microorganisms, rehydrated in the drinking water, provided a practical delivery method that may be easily incorporated into current industry practices as well as Salmonella colonization reduction comparable to that achieved with broth culture delivered by oral gavage or in the drinking water. ACKNOWLEDGMENTS The authors thank Kathleen Andrews, Clayton Myers, Jim Snodgrass, Albert Blanks, and Earl Munson for their excellent technical assistance. REFERENCES Andrews, W. H., P. L. Polema, C. R. Wilson, and A. Romero, Isolation and identification of Salmonella. Pages 1-29 Chapter 6 in: Bacteriological Analytical Manual. 5th ed. Association of Official Analytical Chemists, Washington, DC. Bailey, J. S., L. C. Blankenship, N. J. Stern, N. A. Cox, and F. McHan, Effect of anticoccidial and antimicrobial feed additives on prevention of Salmonella colonization of chicks treated with anaerobic cultures of chicken feces. Avian Dis. 32: Blankenship, L. C, J. S. Bailey, N. J. Stern, and N. A. Cox, Experiences with CE treatment field trials to control Salmonella and Campylobacter in chickens. Page 18 in: Abstracts of the International Symposium "Colonization Control of Human Pathogens in Poultry." Het Spelderholt, ed. Doorwerth, The Netherlands. Corrier, D. E., A. Hinton, Jr., R. L. Ziprin, R. C. Beier, and J. R. DeLoach, Effect of dietary lactose on cecal ph, bacteriostatic volatile fatty acids, and Salmonella typhimurium colonization of broiler chicks. Avian Dis. 34: Goren, E., W. A. de Jong, P. Doornenbal, N. M. Bolder, R.W.A.W. Mulder, and A. Jansen, Reduction of Salmonella infection of broilers by spray application of intestinal microflora: a longitudinal study. Vet. Q. 10: Greaves, R.I.N., Fundamental aspects of freezedrying bacteria and living cells. Page 407 in: Aspects Theoriques et Industriels de la Lyophilisation. L. Rey, ed. Hermann, Paris, France. As reported by Lapage and Redway, Harrison, Jr., A. P., and M. J. Pelczar, Jr., Damage and survival of bacteria during freezedrying and during storage over a ten-year period. J. Gen. Microbiol. 30: Heckly, R. J., Preservation of bacteria by lyophilization. Pages 1-76 in: Advances in Applied Microbiology. Volume 3. W. W. Umbreit, ed. Academic Press, New York, NY. Heckly, R. J., Preservation of microorganisms. Pages 1-78 in: Advances in Applied Microbiology. Volume 24. Academic Press, New York, NY. Hinton, Jr., A., D. E. Corrier, G. E. Spates, J. O. Norman, R. L. Ziprin, R. C. Beier, and J. R. DeLoach, Biological control of Salmonella typhimurium in young chicks. Avian Dis. 34: Hohorst, H. J., Lactate. Pages in: Methods of Enzymatic Analysis. H. V. Bergmeyer, ed. Academic Press, New York, NY. Hollister, A. G., D. J. Nisbet, D. E. Corrier, and J. R. DeLoach, Effect of cecal microflora and dietary lactose on cecal volatile fatty acid concentrations in broiler and layer chicks and turkey poults. Poultry Sci. 71(Suppl. 1): 46.(Abstr.) Hollister, A. G., D. J. Nisbet, D. E. Corrier, and J. R. DeLoach, Effect of cultures encapsulated

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