Caecal Culture Enhances Performance and Prevents Salmonella Infection in Broilers

01999Applied PoultrySciencz, Inc

CAECAL CULTURE ENHANCES PERFORMANCE AND PREVENTS SALMONELLA INFECTION IN BROILERS

Primary Audience: Nutritionists, Poultry Producers, Veterinarians, Feed Formulators

however, has been strictly regulated due to DESCRIPTION OF PROBLEM the potential for antibiotic residues. Feed adModulation of the microflora population in the gastrointestinal (GI) tract of chickens can improve growth performance and feed efficiency. Bactericidal additives, including copper and antibiotics, have been used for years as growth promoters. Use of antibiotics, 1 To whom correspondence should be adressed

ditives with little or no residue potential are appealing as substitutes for antibiotics. Probiotics that contain viable organisms have the greatest potential as feed additives. Probiotics exert a beneficial effect on animals'performance through modifcation of

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BI W Department of Animal Science, National Chung-Hsing University, 250 Kuo-KuangRoad Taichung, Taiwan HAU-YANG "SEN Department of Food Science, National Chung-Hsing University, Taichung, Taiwan PETER WEN-SHYG CHIOU~ Department of Animal Science, National Chung-HsingUniversity, 250 Kuo-KuangRo& Taichung, Taiwan Phone: 88642870613 FAX: 88642860265

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MATERIALS AND METHODS PREPARATION OF CAECAL CULTURE The caecal cultures were prepared from the fresh caecal contents of healthy mature donor chickens that were not contaminated with Salmonella [13].

SOURCE O F SALMONELLA FOR CHALLENGE The source of challenge Salmonella used in this trial was Salmonella typhimunum 29E with resistance to novobiocin and nalidixic acid from Alltech, Inc. (Nicholasville, KY). The viable cell concentration of Salmonella in brilliant green agar (BGA) was determined by counting cherry red colonies [14]. CHALLENGE MODEL AND FEEDING In order to study the effects of caecal culture treatment on performance and the prevention of Salmonella infection in broilers, all broilers were challenged with Salmonella. One additional treatment without Salmonella challenge (positive control group) was included in Trial 1 to simulate practical conditions. In Trial 1, 360 day-old Harber broiler chicks from a commercial hatchery were screened and found free of Salmonella contamination. Screening was performed by randomly selecting droppings from the paper liners of the package boxes the chicks were transported in and subjecting the droppings to a BGA culture examination. These chicks were randomly allocated into 12 groups of 30 chicks, each with equal numbers of males and females. The trial was a completely randomized design with three treatments and four replicates. The three treatments were caecal culture treatment, negative control, and positive control. The negative control group received only water as a placebo; the 1-day-old chicks in the caecal culture treatment group received the caecal VL culture through drinking water upon arrival. Both groups were challenged through drinking water with 1mL of 1x 104CFU Salmonellatyphimurium at 3 days of age. The positive control group received only caecal culture at 1day of age and were not challenged with Salmonella at 3 days of age. The chickens were wing-banded and fed the same diet (without any added medication) in floor pens (2.1 x 2.0 m2) for a 6-wk

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the GI microflora [l]. Chateauet al. [2] showed that probiotics can also effectively reduce pathogenic infections. Mechanism that may inhibit the growth of pathogenic microorganisms include production of organic acid, a decrease in environmental pH, production of H202, bacteriocin [3], enhancement of animal immunity [4], and promotion of hostspecific adhering activity [5]. Fuller [l] suggested that the effectiveness of probiotics depends on the interaction between the ingested organisms and the intestinal environment. Only those organisms that tolerate low gastric pH and bile salts in the small intestine and are capable of adhering specifically to the host’s intestinal epithelium can become the dominant organisms in the host’s intestine. Salmonella-related diseases have historically been a problem in the poultry industry. The poultry industry experiences few losses due to salmonellosis at the production level. However, the potential for salmonellosis as a source of human food poisoning caused by contaminated poultry and egg consumption is a matter of concern. Stavric et al. [6] treated chickens with purified Lactobacillus and mixed strains of Lactobacillus acidophilus and Bifidobacenum bifidum, and found no inhibitory effect on Salmonella infection. On the other hand, the competitive exclusion treatment of day-old chicks with caecal cultures or gut contents of adult chickens has been shown to increase resistance to potentially pathogenic microorganisms, including Salmonella [7,8,9]. The microflora that have been effectivein competitive exclusion of Salmonella are made by anaerobicallyculturing the caecal contents or fresh feces of adult chickens.These cultures contain a large number of anaerobic and aerobic microorganisms [lo, 111, which can establish a balance of microflora that can effectively protect chicks against exogenous microbes in the intestine. Tellez et al. 1121 showed that induced resistance to Salmonella enteritidis invasion using dietary lactose was associated not only with increasing organic acid production from microflora fermentation but also with changes in the caecal mucosal morphology. This study investigatesthe caecal culture treatment of day-old chicks on performance and on the prevention of Salmonella infection in chickens.

CAECAL CULTURE FOR BROILERS

Research Report YU et al.

DETERMINATION O F CAECAL WEIGHT AND pH O F CAECAL CONTENTS Caeca weights for 20 chicks from each group were measured. The pH values of the caecal contents were directly measured using the glass electrode of a portable pH meter (Sentex TS-1, Taiwan). DETERMINATION O F LACTATE, VOLATILE FATTYACIDS (VFA) OF CAECAL CONTENT Samples of caecal contents for 20 chicks from each treatment were prepared according to the method of Marsili et al. [15] for lactic acid analysis using high pressure liquid chromatography (L-6200A, Hitachi Ltd., Japan). The column was Phenomenex 00H-0138-K0,300 x 7.8 mm, serial number 151634. Caecal content analysis was performed for an additional 20 chicks to analyze the concentration of VFA according to the procedures outlined by Parker and McMillan

WICAECAL HISTOLOGY A section 0.5 cm in length was taken from the middle of the caeca of 10 chicks from each treatment group for histological observation [12,17l.

OBSERVATION O F MICROBIAL ADHESION IN THE CAECA BY SEM Caecal samples about 1 cm2 were taken from 3-day-old and 10-day-old chicks for scanning to observe the microbial adhesion in the caecal mucosa [18,19]. The contents of the crop and caeca from 20 chicks were aseptically squeezed and serially diluted in peptone water to enumerate total aerobic and anaerobic bacteria, Salmonella, Lactobacillus, a n d coliform bacteria [9,20,21]. STATISTICALANALYSIS All data were analyzed using the general linear models procedure for statisticalanalysis systems [22]. Si&icant differences at the 0.05 level among treatments were separated using Duncan’s multiple range test.

RESULTS AND DISCUSSION EFFECT ON THE GROWTH PERFORMANCE OF CHICKENS Table 1presents the growth performance of the broiler chickens in the three treatment groups. Chickens in the positive control group gained significantly more body weight (P < .OS) during the growing period (0-3 wk). However, body weight gain during the finishing period ( 4 4 wk), body weight at 42 days old, feed intake, and feed conversion during the growing and finishing periods were not signiftcantly different among the three treatment groups. EFFECT ON CAECAL CHARACTERISTICS AND HISTOLOGY Table 2 presents the caecal weight, lumen pH, organic acids of the caecal contents, and caecal histology of the 10-day-old chickens. A significantly heavier caecal weight and higher concentration of total VFA in the caecal culture-treated group was observed, compared to the negative control group ( P e .05). The lumen pH and lactic acid concentrations in the caecal contents of chicks in both groups were not significantly different. The mucosal layer was significantly thicker in the culture-treated group than in the negative control group (P e .OS).

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feeding trial. Diets were formulated as 22.5% CP and 12.93 MJ/kg ME for growing and 19.0% CP and 13.60 MJ/kg ME for finishing broilers, respectively. Feed and water were provided ad libitum. The live weights were measured on Day 1and at 21 and 42 days of age. Feed consumption was recorded weekly. In Trial 2,200 day-old chicks were divided into culture treatment and negative control groups of 100 chicks each. They received the same challenge and feeding management as in the feeding trial. In this culture treatment trial, no positive control group was utilized. At 3 days of age, 10 chicks from each group were killed by cervical dislocation. The caeca were removed for microflora colonization examination using electronic microscope scanning (SEM) and culture counting. The remaining 180 chicks were killed and examined for caecal traits and microflora colonization in the crop and caeca of the chicks 7 days after receiving the challenge (at 10 days old).

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JAPR CAECAL CULTURE FOR BROILERS

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CAECALCULTURE TREAlMENI 3wk

NEGATIVE COrnOL

731b

6 wk

731b

1999

&3 wk

1989

69Cib

6%b

P O S m CONTROL

SEM

10.0

761a

38.0

2000

9.8

726a

1266

1251

1262

34.3

0-3 wk

946

904

973

39.7

4 4 wk

2608

2.491

2606

119.3

0-3 wk

1.27

1.27

1.26

0.03

44 wk

2.07

2.00

2.11

0.08

ITEMS

CAECAL. CULTURE

NEGATlVE CONIROL

TREATMENT Caecal weight, g Lactic acid,p mole/g

1 Total W A , p mole/g

2.1520521a 10.3320.93

1.73k052b 8.05k 1.07

88.29&4.Ua

70.62k5.29b

Lamina propria thickness, p m

4.00k0.92

5.210.77

Thickness of mucosal layer,pm

15.1421.31a

8.07k 1.32b

%ean

k

SD, n = 10 (obsemtions/bird).

a3%alues in the same row with different superscript letters are significantly different (P < .OS).

EFFECTS ON MICROBIAL COLONIZATION Figures 1 and 2 present SEM micrographs of the microbial colonization adhering to the caecal mucosa in 3- and 10-day-old chicks, respectively. The microbes shown in the SEM micrograph were the colonized or adhering microbes in the mucosal epithelium [181, since the procedure used in this trial eliminated the caecal content and the uncolonized microbes from the mucosal epithelium. The

SEM showed a slightly different histology and probable mucosal development for the undeveloped caecal mucosa in 3-day-old chicks (Figure l),as compared to the welldeveloped caecal mucosa of the 10-day-old chicks (Figure 2). Electron micrographs showed more colonized microbes in the mucosa of the treated chicks than negative control chicks at 3 and 10 days of age (Figures lB, 2B vs. lE, 2E). Micrographs also showed colonized microbes completely

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44wk

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FIGURE 1. Scanning electron micrographs of broiler caeca at 3 days after orally treated with caecal culture (A, B, C) and negative control group (D, E. F). Bars= 100pm (A&D); 20pm (B&E); 2pm (C&F). In A&B, bacteria are extensively colonized over the epithelium and crypt of mucosa (arrow). C is an enlargement of bacteria bound to the mucosal epithelium.

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(A, B, C) and negative control group (D, E. F). Bars= lOOpm (A&D); 20pm (B&E);2 p m (C&F). In A&B, bacteria are extensively colonized over the epithelium and crypt of mucosa (arrow). C is an enlargement of bacteria bound to the mucosal epithelium.

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EFFECT ON THE MICROBIAL DISTRIBUTION IN CROP AND CAECA Table 3 presents the microbial population in the crop and caeca of the chickens. The populations of Lactobacillus in the crop and caecal content of the chicks were well established at 3 days of age in both the treated and negative control groups. M e r receiving the challenge, the populations of anaerobes and coliform bacteria were significantly lower in the 10-day-old treated chicks than in the control chicks ( P c .05). The distributions of Lactobacillus in the crop and caeca were similar, with slightly higher levels in the treated group than in the negative control group. Two different cell counting procedures, with and without enrichment cultures, were used to determine the Salmonella contamination in this trial. Results showed that the num-

CROP

CAECUM

Caecal Culture

Negative Control

Caecal Culture

Negative Control

7.78k0.24

759+0.52

852k0.06

8.59k0.18

Aerobes

-

-

7.63k0.59

7.40k0.22

Anaerobes

-

-

6.81+0.3Sb

7.24k0.22a

-

4.66+0.29b

7.21+0.46a

8.67k0.23

8.47k0.26

8.44k0.49

8.3okO.36

Coliforms

Unenriched cultureD

I

Enriched culture

I

*Mean

k

NDE 4

3

I

8

I

ND ND

I

ND

I

6

SD, n = 10.

BMean k SD, n = 20.

CSalmonella culture positive at the lo-* dilution of content. DSalmonellaculture positive after selenitecystine enrichment. %=Not

detected.

a’%alues in the same row with different superscript letters are significantly different (P < .05).

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ber of chicks with Salmonella contamination in the crop and caecal contents were lower in the treated group than in the negative control group. From the enrichment procedure, 6 out of 20 chicks in the negative control group developed Salmonella infections in the caecal contents. No Salmonella were isolated from the caecal contents of the treated chicks. Salmonella were isolated from the crops of the treated chicks. These Salmonella recovered from the crop may be transients. Since the birds were kept on the floor, coprophagy or litter eating is expected to occur. In the poultry industry, supplementation of probiotics or microbes from caecal culture to maintain the health of the host animal has been applied in practical production. These applications have yielded inconsistent results in both practice and experiments. The effectiveness apparently depends on the strains of administered microbes, which can be competitive to exclude or colonized to resist other microflora. This mechanism is not a simple one but a series of complex actions. We tried to apply a mixed culture from different strains of Lactobacillus fermentum from chicks, and did not obtain our expectations for growth promotion and the reduction of Salmonella

covering the epithelial cells and adjacent crypts (Figures 1B and 2B). From the magnified SEM micrograph (5000X)of Figures 1C and 2C, cocci or rod-shaped colonized microbes are shown aggregated together. Conversely, scattered colonized microbes occur only in small numbers in the mucosa of the negative control chicks.

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suppress other bacteria due to low lumen pH from the production of lactic acid. High VFA concentration in the caeca reflects a high degree of fermentation. Under normal conditions, facultativeanaerobes were the dominant genera in the small intestine, while anaerobes were the indigenous microorganisms in the caeca [B].The VFA concentration can be an index of the anaerobic organism population [29]. Although high caecal VFA concentration was detected in the treated chicks in this trial, the population of facultative anaerobes in the caecum did not increase. A feeding trial in chickens displayed an increase in VFA concentrations that suppressed Salmonella colonization [29]. The heavier caecal weight and the higher caecal VFA concentration in the treated chickens (Table 2) reflect a high degree of fermentation. High VFA concentrations and low lumen pH may cause some structural and histological changes in the caecal mucosa (Table 2). Comparable conditions result from a high degree of fermentation in the intestines of Leghorn chickens fed a high lactose diet

[=I*

Changes in intestinal histology have been associated with animal health and performance. Nabuurs et al. [30] showed that shorter intestinal villi and deeper crypts in pigs might lead to poor nutrient absorption. Spring [31] showed that addingmannan-oligosaccharides (MOS) to a broiler diet increased villi length by alleviating the destruction of enteric pathogens (Salmonella and coliform) in the intestinal villi. Although we did not ob5efve changes in the mucosal tissues of the small intestine in this study, changes in the caecal mucosa, microorganisms, pH, and organic acid concentration indicate that a treatment of caecal culture in day-old chicks can effectively improve the intestinal environment in favor of balancing microflora, yielding enhanced growth performance in growing chickens (0-3 wk) in the positive control group. This treatment, however, did not significantly improve the broilers’ growth performance through the entire growing-finishing period.

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and E. coli colonization in this laboratory (unpublished data). This suggests that competitive exclusion for pathogenic bacteria to maintain the balance of microflora in the GI tract of animals does not result from any single factor. Application of the undefined culture of caecal contents from healthy, mature chickens may therefore facilitate the day-old chicks’ ability to establish a balanced microflora in their GI tract, and enhance the health and growth performance of the chicks. The GI tracts of newly hatched chicks are almost sterile [23]. Either from exogenous preparations or through direct contact from the environment, the microflora with Lactobacillus appeared in the crop and caeca as early as 3 days of age (Table 3). Lloyd et al. [24] suggested that a period of 3 days is time enough for microbes to establish a flora population in the digestive tract. Savage [25] classified the colonized intestinal microbial flora into two categories for the allochthonous microbial population, such as nonindigenous or transient flora that can be cultured directly from the intestinal contents, and autochthonous microbial populations such as indigenous or resident microbes that were adhering to the mucosal epithelial cells. Figures 1and 2 show many microbial flora adhering to the mucosal epithelium in the caeca of the treated chickens. This suggests that treating newly hatched chicks with exogenous preparations that can rapidly multiply and colonize in the mucosal epithelial cells and establish as an indigenous microflora, can effectively reduce the colonization of S. lyphimurium and other enteropathogens [18,26]. Data from this trial also show a reduction in the population of coliform bacteria (Table 3). Besides the competitive exclusion through the occupation of the binding sites, other environmental factors in caeca, including low pH, high concentrations of lactic acid, and VFA, can increase the inhibitory effects of pathogen invasion [12]. Data from this trial showed heavier caeca weights and higher concentrations of WA in the caeca of the treated chickens (Table 2). Fuller [21 suggested that Lactobacillus reaching a population level of lo7 CFU/g can effectively

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CONCLUSIONS AND APPLICATIONS

REFERENCES AND NOTES .. .

invasion in LRghom chicks. Poultry Sci.

1. Fuller, R , 1986. Probiotics. J. Appl. Bacteriol. 61:15-75.

-organ m636442.

2. Chateau, N., I. Castellanos, and S.M. Deschamps, 1993. Distribution of pathogen inhibition in the L&Qbacillus isolates of a commercial probiotic consortium. J. Appl. Bacteriol. 7436-40.

13. Preparation of caecal culture: 1.0 g of caecal content was inoculated into 20 mL of Viende Levoir (VL) broth containing 10 g tryptone, 5 g NaCI, 2.4 g beef extract, 5 g yeast extract, 0.4 g cystein hydrochloride, 2 5 g glucose, and 0.6 g agar per L, creating a broth of pH 7.2-7.4 [lo]. This broth was then incubated in an anaerobic chamber (9570 COh 5% Hz, Coy Laboratory Products, Grass Lake, MI) at 37°C for 24 hr. Three consecutive subcultures were prepared with a 1:39 ratio of inoculum to VL broth. The final culture was used as an inoculum for treatment. The specimens were stored at -70°C with glycerol as a stock culture.

3. Muriana, P.M. and T.R Klaenhammer, 1991. Purification and partial characteetion. of laftacin F, a bacteriocin produced by11088. Appl. Environ. Microbiol. 57114-121.4. Perdigon, G., M.E DeMacias, S. Ahrarez, M. Mcdici, G. Oliver, R De, and A.P. Holgado, 1986. Effect of a mixture of Lactobacillus rpsei and Lactobacillus andoDhllusadministered orally on the immune system in mice. J. Food Prot. 49986-989.

5. Savage, D.C., 1983.Mechanism by which indigenous microorganism colonize gastrointestinal epithelial surfaces. Prog. Fed. Nutr. Sci. 765-75. 6. Stavric, S., T.M. Gleeson, B. Buchanan, and B. Blanchfield, 1992. Experience of the use of probiotics for Salmonellacontrol in poultry. Appl. Microbiol. 14:69-71. 7. Nurmi, E and M. Rantala, 1973. New as cts of Salmonella infection in broiler production. s a t u r e 241:210-211. 8. Impey, C.S. and G.C. Mead, 1989. Fate of Salmo-

& in the alimentary tract of chicks pretreated with a

natural caecal microflora to increase colonization resistance. J. Appl. Bacteriol. 66:469-475. 9. C o d e r , D.E, D.J. Nisbet, C.M. scanlan, A.G. Hollister, and J.R Deloach, 1995. Control of Salmonella -colonization in broiler chicks with a continuous-flow characterized mixed culture of cecal bacteria. Poultly Sci 74916924.

10. Barnes, EM., C.S. Impey, and J.H. Stevens, 1979. Factors affecting the incidence and anti-salmonella activity of the anaerobic caecal flora of the young chick. J. Hygiene 8226S283. 11. Stavric, S., T.M. Gleeson, B. Blanchfield, and H. Pivnick, 1987. Role of adhering microflora in competitive exclusion of Salmonella from young chicks. J. Food Prot. 50:928-932. 12. Teller, G., C.E Dean, D.E Coder, J.RDeLoacb, L Jaeger, and B.M. Hargis, 1993. Effect of dietarylactose on cecal morphology, pH, organic acids, and Salmonella

14. Source of Salmonella for challenge: Salmonella stock cultures were consecutively subcultured three times into a tryptic soy broth for 18 hrand then diluted into the final culturewith 0.1% tonewater to obtaina population of 1 X lo4 CFU/%!. Brilliant green a ar (BGA) contained 20 pcglmL of nalidixic acid and 28pglmL of novobiocin [9].

15. MarsUI, RT., H. Ostapenko, RE Simmons,and D.E Green, 1983. High performance liquid chromatogra hic determination of organic acid. J. Food Prot. 46:!2-57. 16. Parker, D.S. and RT. McMillan, 1976. The determination of volatile fatty acids in the caecum of the conscious rabbit. Br. J. Nutr. 35:365-371.

17. Caecal preparation for histology observation: The samples were rinsed through a 0.4 M KCI solution first and were then laced into a 10% neutral formalin buffer solution @H ?2-7.4). All specimens were embedded in paraffin and samples were then prepared by sectioning at 6 p m and stained with hematoxylin and eosin. Intestinal histologywasobserved according t o T e l l e z a d . [12].The measurement of each sample was taken by averaging 10 intact villi. A lamina propria was measured using the distance from the base membrane to the muscularis mucosa while the thickness of the mucosal layer w a s measured usin8 the distance from muscularis mucosa to the tip of the vllli. 18. Droleskey, R E , D.E Coder, DJ. Nisbet, and J.R Dehach, 1995. Colonization of cecal mucosal epithelium in chicks treated with a continuous flow culture of 29 characterized bacteria: Confirmation by scanning electron microscopy. J. Food Prot. 588374342.

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1. Use of caecal culture as competitive exclusion microflora in day-old broilers influenced caecal weight, pH, organic acids production, and number of adherent microbes in the caecal mucosa of treated chickens. This treatment can establish a beneficially balanced microflora environment. 2. Although positive caecal culture treatment did not signiticantly improve performance throughout the whole growing period, the positive control group with caecal culture treatment without Salmonella challenge displayed significantly improved growth performance at the critical growth period (0-3 wk) compared with both the caecal culture and negative control groups. A caecal culture preparation can be provided to broilers as a probiotic additive to eliminate pathogenic microorganism contamination and promote growth in broilers.

204 19.The specimens of caeca for observation of microbial adhesive by SEM: A Fmtive solution containing 3% glutaraldehyde, 50 mM phosphate buffer, pH 7.4,50 mM sucrose, and 12% picric acid was injected into the lumen of each caeca in addition to the ligature caeca that was taken from 3-day-old and lOday-old chicks [18]. Once pressurized with fkative, the samples were immersed in an additional fixative for 60 min at room temperature. Postfuration was performed using osmium tetroxide. The samples were then dehydrated, critical point dried, mounted on aluminum stubs, coatedwith gold, and placed in the SEM (Bausch & Lomb Ltd., Nonolab 2100).

Salmon& dilution on BGA plater and after enrichment and were scored as 0 CFU or (-). Colony counts that were negative at the 1:100 dilution on BGA plates but sitive after enrichment were arbitrarily assigned 1.5&/g or (+).

24. Lloyd, AB., RB. Cinumhg, and R D . Kent, 1977. Prevention of M. ' ' infection in poultry by pretreatment of- c oultry with intestinal extracts. Australian Vet. J. 53:82A!7.

25.Savage,D.C.,1977.Microbial ecologyof the gastrointestinal tract. Ann. Rev. Microbiol. 31:107-133. 26. Soerjadi, AS., S.M. Stehman, G.H. Snoeyenbos, O.M. Weinack, and C.F. Smyser, 1981. Some measurements of protection against -Sdpn@la and Epli by competitive exclusion in chickens. Avian Dis. 25:76712.

27. Fulkr, R, 197. The importance of Lactob acil1.1 in maintaining normal microbial balance in the crop. Br. Poultry Sci. 1885-94.

28. Salanitro, J.P., I.G. Blake, P.A. Muirhead, M. Maglio, and J.R Goodman, 1978.Bacteria isolated from the duodenum, ileum, and cecum of young chicks. Appl. Environ. Microbiol. 35:782-790. 29.Barnes, EM.,C.S.Impey, and D.M. Cooper, 1980. Manipulation of the crop and intestinal flora of the newly hatched chick. Amer. J. Clin. Nutr. 332426-2433.

30. Nabuurs, MJA, A. Hoogendoom, EJ. Van der Molcu, and ALM. Van Osta, 1993. Villus height and crypt depth in weaned pigs reared under various circumstances in The Netherlands. Res. Vet. Sci. 55:78-84.

21.Tortuero, F., J. Rioperez, E Fernandez, andM.L Rodriguez, 1995.Response of piglets to oral administration of lactic acid bacteria. J. Food Prot. 58:1359-1374.

31.Spring, P.,1996. Effects of mannanoligosaccharide on different cecal parameters and on cecal concentration of enteric pathogens in poultry. Ph.D. Thesis. Swiss Fed. Inst. Tech., Zurich, Switzerland.

22.SAS Institute, 1985.SAS Users' Guide: Statistics. Version 5 Edition. SAS Institute, Inc., Cary,NC.

ACKNOWLEDGEMENT

23. EwLng, W.N. and DJ.A. Cole, 1994. The Living Gut - An Introduction to Microorganisms in Nutrition. Context Publication, Northern Ireland.

The authors thank the National Science Council in Taiwan for the financial support of this project (NSCSS-2321-BOOS-071).

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20.The microflora distribution in the crop and the caeca: Aliquots of 0.1mL of diluted sampleswere spreadplated onto different nutrient media: plate count agar (Difco), reinforced clostridial agar (Oxoid), rogma SL agar (Difco), and chromocult agars (Merck). These media were used to enumerate total aerobes, anaerobes, Jactobacillu&, and coliform bacteria, respectively. Reinforced clostridial agar and R o g m agarwere incubated at 37°C for 48 hr under anaerobic conditions. Plate count agarand chromocult agar were incubated at 37°C for 24 hr in air. Salmonella counts for the crop and the caecal contents were detected at the BGA that contained nalidixic acid and novobiocin with or without selenite-

CAECAL CULTURE FOR BROILERS