688 Journal of Food Protection, Vol. 59, No.7, 1996, Pages 688-fi93 Copyright, International Association of Milk, Food and Environmental Sanitarians Reduction of Crop and Cecal Colonization by a Characterized Competitive Exclusion Culture in Broilers during Grow-Out t MICHAEL E. HUME,* DONALD E. CORRIER, DAVID J. NISBET, and JOHN R. DELOACH u.s. Department of Agriculture, Agricultural Research Service, Food Animal Protection Research Laboratory, 2881 F &B Road, College Station, Texas 77845, USA (MS# 95-217: Received 21 August 1995/Accepted 20 December 1995) ABSTRACT Broiler chicks were inoculated by gavage on the day of hatch with a characterized continuous-flow (CF3) competitive-exclusion culture that contained 29 different bacterial isolates to determine the effects on cecal and crop colonization during grow-out. Chicks at 3 days old were challenged by gavage with 10 4 typhimurium. Propionic acid significantly increased (P:s 0.001) in the ceca of 3-day-old CF3-treated chicks compared to control chicks. Ceca from market-age control chickens in two trials contained log 2.6 and log 1.4 CFU/g of cecal contents, respectively, while log 0.4 CFU/g of cecal contents were detected in both trials in ceca from CF3-treated chickens. Percentages of culture-positive ceca in the two trials, respectively, were 80% and 60% in controls and 27% in treated chickens in both trials. Crops from market-age control chickens in the two trials averaged log 0.7 CFU/g of cecal contents, while crops from treated chickens averaged log 0.4 CFU/g of cecal contents. In trial 1, 60% of control chick crops and 27% of treated chick crops tested culture positive. Litter contamination by spp. at 5 weeks was reduced significantly (P:S 0.01) in pens ofcf3-treated groups compared to litter from control pens. Results indicate that CF3 reduced cecal and crop colonization by S. typhimurium during grow-out, which may reduce the number of cells entering the processing plant and decrease the potential for carcass contamination during processing. Key words: chicken crop, ceca, competitive exclusion, continuous flow, Numerous studies have focused on the reduction of colonization in poultry and the prevention of carcass contamination during processing (7, 12, 13, 16, 26). Reductions in the total numbers of cells colonizing chickens as well as reductions in the numbers of infected t 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. * Author for correspondence. Tel: 409-260-9404; Fax: 409-260-9377; Email: hume@usda.tamu.edu. chickens entering the processing plant would likely result in decreased carcass cross contamination during processing. Cross contamination during processing frequently results from the rupture of digestive organs during evisceration and the subsequent immersion of carcasses in a common chill bath (14). Crops and ceca are major sites for colonization in poultry (11). Carcass contamination resulting from crop colonization may playa more significant role than cecal colonization. In a recent study by Hargis et al. (7) only 0.3% of the ceca ruptured during evisceration of broiler carcasses, while 25.7 % of the crops were ruptured by the mechanical cropper. In the same study, 14.6% of the ceca were culture positive and 52% of the crops were culture positive, indicating that crops may be a greater source for cross contamination than ceca. Treatment of young chicks with intestinal bacteria from the digestive tracts of adult chickens reduces cecal (8, 19, 25) and crop (2, 3, 19) colonization. Newly hatched chicks lack a digestive microflora protective against colonization, but become less susceptible to colonization with age (6, 15, 16). Chicks traditionally obtain inocula of protective bacteria from the egg outer surface, from the hatchery and shipping containers, and through contact with fecal droppings from adult chickens (3). Contemporary rearing practices minimize contact with sources of protective microflora and led to the development of the Nurmi concept for treating young chicks with competitive-exclusion (CE) bacteria (19). A characterized culture consisting of 29 different isolates of anaerobic cecal bacteria has been developed in our laboratory, maintained in continuous-flow (CF) culture, and subsequently designated CF3 (4). Previous studies have focused on the effects of CF3 on cecal colonization in lo-day-old chicks. Because cells in the crop may be an important source of contamination in the processing plant (7), there is clearly a need for prophylactic treatment that effectively reduces colonization in both the crop and ceca. Additionally, colonized chicks excrete cells into the environment through fecal shedding. Reductions in colonization in chicks
GROW-OUT CECAL AND CROP SALMONELLA 689 would probably result in decreased litter contamination and a decreased potential for reintroduction of spp. into chicks from successfully treated flocks. The purpose of the present study was (i) to evaluate the effect of the characterized CF3 culture on crop and cecal colonization during grow-out through market age, and (ii) to determine the effect of CF3 treatment on contamination in floor pen litter at the end of grow-out. MATERIALS AND METHODS Experimental design Male broiler chicks were obtained on the day of hatch from a local hatchery and maintained on new pine-shavings litter in floor pens under continuous lighting conditions. Chicks were provided an unmedicated corn and soybean meal diet that contained or exceeded critical nutrient requirements recommended by the National Research Council (17) and tap water, ad libitum. The liners from the chick shipping cartons and feed (25 g) were each incubated successively in buffered peptone water, selenite cystine broth, and on brilliant green agar (BGA) (Oxoid, Unipath Ltd., Basingstoke, Hampshire, England) plates as previously described (1) and examined for colonies characteristic of. spp. were not detected in the liners or feed. In trial I, chicks were randomly assigned to two groups (70 chicks each) and were either (i) not treated (controls) or (ii) treated with collected effluent of CF3 by oral gavage. Fifteen randomly selected chicks from each group at each time point (see below; except at 3 days old) were placed in partitioned sections of corresponding rearing pens and feed was withdrawn for 12 h prior to sacrifice. The experimental design was repeated in a separate trial (trial 2) using newly hatched chicks after completion of trial I. A primary poultry isolate of typhimurium obtained from the National Veterinary Services Laboratory, Ames, Iowa was selected for resistance to naladixic acid (NA) and novobiocin (NO) in the authors' laboratory and maintained in media containing 20 Ilg/ml NA and 25 Ilg/ml NO. On day 3, 2 days after treatment with CF3, all chicks were challenged by oral gavage with 2 X 10 4 CFU/0.5 ml of S. typhimurium from an overnight culture that was previously transferred three times in Trypticase soy broth (Difco Laboratories, Detroit, MI) and serially diluted in sterile phosphatebuffered saline to a concentration of 4 X 10 4 CFU/ml. The viable cell concentration of the challenge inoculum was confirmed by colony counts on BGA plates containing NA and NO. Characterized competitive exclusion culture Development of the characterized CF3 culture using continuous-flow culture systems was described previously (4). The characterized culture was determined to be composed of 15 facultative anaerobes and 14 obligate anaerobic bacteria representing 10 different genera (4). Cecal and crop ph, lactic acid and volatile fatty acid concentrations Ten chicks per group were sacrificed by cervical dislocation and one cecum was excised from each on day 3, prior to challenge of the remaining chicks. A portion (0.2 g) of the contents was added to a sterile tube containing 1.8 ml of sterile distilled H 2 0 (sdh 2 0) followed by vigorous shaking. The contents of the tubes were used to determine the levels of lactic acid and volatile fatty acids (VFA) as indicators of the establishment of CF3 (4). The remaining chicks were challenged with 10 4 S. typhimurium. When the challenged chicks were 1.5, 3, 4, and 5 weeks of age, a cecum was removed from each of 15 sacrificed chicks per group and 0.2 g of contents was added to a sterile tube containing 1.8 ml of sdh 2 0; after vigorous shaking, the mixture was used to determine ph (Cole-Parmer Instrument Co., Niles, IL) and the concentrations of lactic acid and VFA. Lactic acid concentrations were determined by an enzymatic method (9) and VFA concentrations were determined by gas-liquid chromatography as described previously (10). Crops were excised aseptically from 15 chicks (see above) in each group at 1.5, 3, 4, and 5 weeks of age. Crop contents was released as each crop was aseptically cut into several pieces into individual stomacher bags (Tekmar Corp., Cincinnati, OH) containing 10 ml of sdh 2 0 and blended (Stomacher 80 Lab Blender; Seward Medical, London SEI IPP England) for 30 s. A portion (2 m!) of the blended sample was removed to determine ph and the concentration of lactic acid. Cecal and crop A portion (0.2 g) of cecal contents from the excised cecum (see above) from each of 15 chicks at 1.5, 3,4, and 5 weeks of age was added to 1.8 ml of sdh 2 0 and shaken vigorously. A sample (I ml) was removed and serially diluted through 2 tubes containing 9 ml each of sterile Butterfield's buffer (I: 100 and I: 1,000 dilutions). A portion (0.1 ml) was removed from the undiluted sample and each dilution tube and spread onto BGA plates containing NA and NO for determination of CFU/g of cecal contents at I: 100, I: 1000, and I: 10,000 dilutions. The remaining cecum from each chick was excised aseptically, minced with scissors into a blender bag containing 30 ml of tetrathionate broth (Difco), and incubated for 24 h at 37 C. After incubation, the broth was streaked onto BGA plates containing NA and NO, incubated for an additional 24 h at 37 C and examined for typical colonies. Typical colonies were confirmed by biochemical tests on triple sugar iron agar (Difco) and lysine iron agar (Difco) and serologically tested with 0 Antiserum Group B, Factors 1,4,5, and 12 (Difco). crop colonization was determined by removing I ml from each blender bag (see above) for serial dilution through 2 tubes containing 9 ml each of:sterile Butterfield's buffer (1:100 and 1:1,000 dilutions). A portion (0.1 ml) was removed from undiluted sample and each dilution tube and spread onto BGA-NA-NO plates for determination of CFU at I: 100, I: 1000, and I: 10,000 dilutions. Tetrathionate broth was added to the remainder of the homogenate in the blender bag, agitated, and then incubated overnight at 37 C. The broth was streaked onto BGA-NA-NO plates and then incubated overnight at 37 C and examined for typical colonies. S. typhimurium colonies were confirmed biochemically and serologically as described above. in floor pen litter Samples of litter were collected from the floor pens at the end of the grow-out period. Litter (10 g) samples from 10 predetermined locations in each pen were added individually to 90 ml of Butterfield's buffer and I ml from each sample was serially diluted through 3 tubes containing 9 ml of Butterfield's buffer. A portion (0.1 ml) from the undiluted sample and each dilution tube was spread onto BGA-NA-NO plates, incubated for 24 h at 37 C, and examined for CFU/g of litter at 1:100, 1:1,000,
690 HUME, CaRRIER, NISBET, AND DELOACH I: 10,000, and I: 100,000 dilutions. The remammg sample was added to 100 ml of tetrathionate broth and incubated for 24 h at 37 C. After incubation, the broth was streaked onto BGA-NA-NO plates, incubated an additional 24 h, and examined for typical colonies. Statistical analysis Students' t test, conducted using Microsoft Excel@) version 4.0a software (Microsoft Corp., Redmond, WA), was used to determine the significance of differences (P < 0.05 or as indicated) between treatment groups. Chi-square analysis was used to determine the significance of differences (P < 0.05 or as indicated) in crop and cecal colonization rates between treatment groups (27). All data were analyzed by individual trial. RESULTS Cecal and crop ph, lactic acid, and VFA The ph of cecal (5.6 to 6.7) and crop (5.7 to 6.3) contents fluctuated among the control and CF3-treated groups during both trials without significant treatment effect (data not shown). The concentrations of acetic, butyric, and lactic acids in the cecal contents fluctuated during both trials I and 2 without consistent treatment effect (data not shown). Propionic acid increased significantly (P ::5 0.05) in CF3-treated chicks compared to control chicks in each trial and at each time (Table 1). Propionic acid concentrations in the ceca of TABLE I. Effects of treatment with a characterized culture of cecal bacteria on the concentrations of cecal propionic acid and total volatile fatty acids during grow-out of broiler chickens fllllole/gof cecal contents" (mean ± SD, n = 15) Trial 1 Trial 2 Chick age b (week) Treatment C Propionic Total VFA Propionic Total VFA 0.4 Control 0.3::':: 0.9 15.3 ::'::2.3 2.3 ::'::1.3 28.1 ::'::7.4 CF3 11.0 ::'::6.3*** 27.0::':: 8.6*** 24.6::':: 17.1** 61.0 ::'::21.4*** 1.5 Control 3.3::':: 0.5 44.3 ::'::13.5 5.1 ::'::1.5 100.8::':: 29.8 CF3 22.6::':: 7.2*** 61.3 ::'::15.6* 41.4 ::'::11.3*** 101.8 ::'::19.1 3 Control 11.5 ::'::4.0 107.2::':: 32.1 9.5 ::'::1.6 93.4 ::'::14.1 CF3 19.4 ::'::3.7*** 81.7::':: 12.8* 14.4 ::'::3.0*** 78.3::':: 13.9* 4 Control 9.7 ::'::1.6 100.4 ::'::18.2 8.4 ::'::2.5 80.7::':: 18.3 CF3 17.0::':: 3.4*** 77.4 ::'::15.7** 13.8 ::'::4.9*** 75.2::':: 26.2 5 Control 16.6 ::'::5.9 102.6::':: 43.9 8.2::':: 2.2 93.6::':: 32.4 CF3 20.8 ::'::6.4* 104.7::':: 26.8 20.0 ::'::4.8*** 103.1 ::'::18.8 a At 0.4 wk (3 days of age) n = 10. Values followed by asterisks are significantly time: *P:S 0.05; **P:S 0.01; ***P:S 0.001. different from controls within trials and within the same b Chicks at 3 days of age were challenged by gavage with 10 4 S. typhimurium. Feed was withdrawn 12 h prior to sacrifice. C Chicks at I day of age were given the continuous-flow culture (CF3) by gavage; control, no treatment. TABLE 2. Effect of treatment with a characterized culture of cecal bacteria on CFU/g of cecal contents and ceca culture-positive during grow-out of broiler chicks colony-forming units a Trial! Trial 2 Chick age b log CFU/g Positive/total Positive/total (week) Treatment C (mean ± SD) (%) log CFU/g (%) 1.5 Control 6.2::':: 0.5 15/15 (100) 5.9 ::'::1.1 15/15 (100) CF3 3.0::':: 2.6*** 12/15 (80) 1.9 ::'::1.7*** 10/15 (67)** 3 Control 6.2::':: 0.3 15/15 (100) 3.3 ::'::1.6 14/15 (93) CF3 1.5 ::'::1.5*** 10/15 (67)** 0.2::':: 1.1*** 4/15 (27)*** 4 Control 3.4 ::'::1.1 15/15 (100) 1.9 ::'::1.9 10/15 (67) CF3 0.3::':: 0.6*** 3/15 (20)*** 0.3::':: 0.6** 3/15 (20)** 5 Control 2.6 ::'::1.6 12/15 (80) 1.4 ::'::1.7 9/15 (60) CF3 0.4 ::'::0.7*** 4/15 (27)** 0.6::':: 1.3* 4/15 (27) a Mean::':: SD, n = 15. Values followed by asterisks are significantly different from controls within trials and within the same time: *p :S 0.05; **P:S 0.01; ***P:S 0.001. b Chicks at 3 days of age were challenged by gavage with 10 4 S. typhimurium. Feed was withdrawn 12 h prior to sacrifice. C Chicks at 1 day of age were given the continuous-flow culture (CF3) by gavage; control, no treatment.
GROW-OUT CECAL AND CROP SALMONELLA 691 TABLE 3. Effects of treatment with a characterized culture of cecal bacteria on CFU/ml of crop contents and crops culture-positive during grow-out of broiler chicks colony-forming units" Trial I Trial 2 Chick age b logcpu/ml Positive/total log CFU/ml Positive/total (week) Treatment C (mean ± SD) (%) (mean ± SD) (%) 1.5 Control 0.4 ± 0.7 4/15 (27) 0.9 ± 0.7 10/15 (67) CF3 0.2 ± 0.5 2/15 (13) 0.3 ± 0.8 7/15 (47) 3 Control 1.5±1.1 11/15 (73) 0.3 ± 0.5 2/15 (13) CF3 0.2 ± 0.6** 1/15 (7)*** 0.0 ± 0.0 0/15 (0) 4 Control 1.0 ± 1.1 8/15 (53) 0.5 ± 0.9 4/15 (27) CF3 0.0 ± 0.0** 0/15 (0)*** 0.4 ± 1.5 1/15 (7) 5 Control 1.4 ± 1.3 9/15 (60) 0.0 ± 0.0 0/15 (0) CF3 0.7 ± 1.4 4/15 (27) 0.1 ± 0.4 1/15 (7) a Mean ± SD, n = 15. Values followed by asterisks are significantly different from controls within trials and within the same time: **p ~ 0.01; ***p ~ 0.001. b Chicks at 3 days of age were challenged by gavage with 10 4 S. typhimurium. Feed was withdrawn 12 h prior to sacrifice. C Chicks at 1 day of age were given the continuous-flow culture (CF3) by gavage; control, no treatment. control chicks increased (P:::; 0.05) during grow-out, but continued to be lower (P :::; 0.05) at 5 weeks than those detected in the ceca of treated chicks. Total VFA (acetic, propionic, butyric, isobutyric, valeric, and isovaleric) concentrations in the cecal contents from control and CF3-treated chicks fluctuated during both trials, but without consistent treatment effect. Crop lactic acid concentrations increased (P < 0.05) during grow-out, but no consistent treatment effect was detected in the two trials (data not shown). Cecal and crop Cecal CFU during grow-out in both trials and at all times were consistently lower (P:::; 0.05) in CF3-treated chicks when compared to cecal CFU from control chicks (Table 2). Percentages of culturepositive ceca were consistently lower to 5 weeks in both TABLE 4. Effects of treatment with a characterized culture of cecal bacteria on floor pen litter CFU/g of litter and culture-positive samples at the end of a 5-week grow-out of broiler chicks colony-fornting units" Trial I Trial 2 log CPU/ml Positive/total Positive/total Treatment (mean ± SD) (%) logcfu/ml (%) Control 3.2 ± 0.5 10/10 (100) 1.6 ± 0.8 9/10 (90) CF3 1.2 ± 0.8*** 7110 (70) 0.6 ± 0.8** 4/10 (40)* a Mean ± SD, n = 10. Values followed by asterisks are significantly different from controls within trials: *p ~ 0.05; **p ~ 0.01; ***p ~ 0.001. b Chicks at 1 day of age were given the continuous-flow culture (CF3) by gavage; control, no treatment. Chicks at 3 days of age were challenged by gavage with 10 4 S. typhimurium. Feed was withdrawn 12 h prior to sacrifice. trials in CF3-treated chicks than in ceca from control chicks. Additionally, there was significant (P:::; 0.05) treatment effect in the number of culture-positive ceca from 3 to 5 weeks in trial 1 and from 1.5 to 4 weeks in trial 2. There was no significant treatment effect in culture-positive ceca at 5 weeks in trial 2, but there was a 56% reduction in the numbers of -positive ceca for CF3-treated chicks. There were consistently fewer CFU in crops of CF3-treated chicks than in crops from untreated chicks; one exception was at 5 weeks in trial 2, when crops from untreated chicks had no detectable CFU (Table 3). By 5 weeks in trial 1 the reduction in crop CFU in treated chicks corresponded with 56% fewer crops culture-positive than in control chicks. The low numbers of CFU from 3 to 5 weeks in crops from untreated chicks in trial 2 were accompanied by low percentages of crops culture-positive. in floor pen litter Fewer (P:::; 0.01) CFU were detected in both trials at 5 weeks in floor pen litter of CF3-treated chicks compared to floor pen litter of untreated chicks (Table 4). In trial 1, the number of culture-positive litter samples was diminished (P :::; 0.06) in the treatment chicks compared to controls and significantly decreased (P :::; 0.05) in the treated chicks at 5 weeks in trial 2. DISCUSSION Treatment of young chicks with CF3 resulted in reductions in colonization in the ceca (in two trials) and crops (in one of two trials) in broiler chickens at 5 wk and decreased contamination in floor pen litter. The expected increase (P:::; 0.001) in cecal propionic acid concentration detected in 3-day-old treated chicks is a characteristic indicator of the establishment of CF3 in the digestive tract (4, 5, 18). Additional indicators of the
692 HUME, CORRIER, NISBET, AND DELOACH establishment of CF3 and previously developed CF cultures are the colonization of sites on cecal mucosal epithelial cells (5) and increases, by at least 1DO-fold, in the numbers of cecal CFU (18). Reductions in crop colonization in control chicks were likely the result of the early establishment of beneficial bacteria gained as inocula from the environment (2, 11, 23, 24). Provisioning of chicks with CE bacteria may be considered prophylactic treatment against colonization and aids in the establishment of a protective barrier in the absence of naturally acquired antisalmonellae bacteria (20). crop colonization was low in both trials compared to cecal colonization. crop colonization during trial 1 was significantly decreased at 3, 4, and 5 weeks of age in the CF3-treated chicks compared to controls. In contrast, significant differences were not detected in trial 2 in crop colonization between control and treated chicks. The results suggested that the control chicks in trial 2 had acquired protective bacteria from the environment. Undefined bacteria acquired from the environment will eventually confer some protection to chicks against colonization. However, chicks are most vulnerable to colonization during the earliest period after hatching and well before the development of a protective microflora. Importantly, during the early period of vulnerability the protective effect of CF3 culture was indicated in both trials at 1.5 weeks by the decrease (P 'S 0.001) in the numbers of in the cecal contents and by the decrease in the number of cecal culture-positive treated chicks at 3, 4, and 5 weeks of age. Reductions in crop and cecal colonization following treatment with CF3 were accompanied by reductions (P 'S 0.01) in litter CFU and the numbers of culture-positive samples. Chickens colonized by spp. continue to shed throughout grow-out (21). Additionally, market-age broilers are transported to the processing plant in crates and are likely to continue to shed during transport, resulting in the contamination of the feet and feathers of all chickens in the crates (22). Reductions in the number of -colonized chickens and in the numbers of cells shed may result in fewer spp. entering the processing plant and lower the potential for cross contamination during processing (7, 13, 14). Previous studies indicated that treatment of young chicks with CF3 resulted in significant reductions in cecal colonization in 10-day-old chicks (4). Data from the present study further indicates that treatment with CF3 significantly reduces crop and cecal colonization during grow-out and at market age. Importantly, litter contamination was decreased in the CF3-treated chickens. 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