Bacteriocins reduce Campylobacter jejuni colonization while bacteria ...

Microbial Ecology in Health and Disease. 2008; 20: 74
79

ORIGINAL ARTICLE

Bacteriocins reduce Campylobacter jejuni colonization while bacteria producing bacteriocins are ineffective

NORMAN J. STERN2, BORIS V. ERUSLANOV1, VICTOR D. POKHILENKO1, YURI N. KOVALEV1, LARISA L. VOLODINA1, VLADIMIR V. PERELYGIN1, EVGENII V. MITSEVICH1, IRINA P. MITSEVICH1, VALERY N. BORZENKOV1, VLADIMIR P. LEVCHUK1, OLGA E. SVETOCH1, YURI G. STEPANSHIN1 & EDWARD A. SVETOCH1 1

State Research Center for Applied Microbiology and Biotechnology, Obolensk, Russia and 2USDA, Agricultural Research Service, RRC, PMSRU, Athens, GA, USA

Abstract Broiler chickens are widely considered an important source of human exposure to Campylobacter jejuni because of the high numbers found colonizing the chicken gut and the consequent contamination of processed carcasses. We hoped to intervene in C. jejuni gut colonization by using a defined probiotic. Chicken intestinal contents were screened for diverse bacterial isolates that manifested C. jejuni inhibition. These antagonistic bacteria were fed directly to chickens before or after C. jejuni challenge. The prophylactic probiotic treatments were effective only when very low challenge levels of C. jejuni were used. Otherwise, probiotic treatments failed to reduce C. jejuni colonization. Regardless of treatment, as birds further aged C. jejuni numbers soon approached the levels observed in the control birds. We sought an alternative approach, as commercial broilers may be exposed to infectious levels of C. jejuni at any time during production. Two of our most promising antagonistic isolates, Lactobacillus salivarius NRRL B-30514 and Paenibacillus polymyxa NRRL B-30509, were further studied for effectiveness in reducing C. jejuni in chickens. When 250 mg of purified bacteriocins (produced by these organisms)/kg feed were fed therapeutically to chickens colonized with C. jejuni, colonization was reduced by at least one million-fold. Treatments with viable probiotic bacterial cultures were ineffective in reducing C. jejuni in chickens, while bacteriocin treatment from these corresponding bacteria substantially reduced C. jejuni colonization in the live birds.

Key words: bacteriocin, Campylobacter jejuni, colonization, probiotic, broiler chicken, competitive exclusion, Lactobacillus salivarius, Paenibacillus polymyxa

Introduction Competitive exclusion (CE) has long been known to reduce Salmonella colonization in chickens (1). The mechanism by which CE reduces Salmonella has been a matter of scientific speculation. Some explanations include: (i) the antagonizing bacteria preferentially occupy Salmonella intestinal colonization niches and/or preferentially consume required substrates; (ii) the probiotic organisms have a shorter doubling time than Salmonella and outgrow the pathogen; (iii) antagonists produce volatile fatty acids associated with Salmonella killing; (iv) probiotic organisms elicit a host immune modulation

response to clear Salmonella; or (v) the CE bacteria produce metabolites that interfere with or kill the target organism. In vivo data supporting these contentions have not definitively proven or disproven these as the central mechanism involved in demonstrable intestinal reduction of Salmonella via CE. In an attempt to use a similar practical approach, we previously observed inconsistent Campylobacter jejuni reductions by administration of CE in chickens (2). On several occasions, reduction of C. jejuni intestinal colonization was demonstrated, while at other times the same CE flora did not provide campylobacter reduction. We wanted to determine whether a more systematic selection of diverse

Correspondence: Dr Norman J. Stern, Poultry Microbiological Safety Research Unit, Russell Research Center, 950 College Station, Athens, GA 30605, USA. Tel: 1 706 546 3516. Fax: 1 706 546 3771. E-mail: [email protected]

(Received 21 August 2007; accepted 20 February 2008) ISSN 0891-060X print/ISSN 1651-2235 online # 2008 Informa UK Ltd. DOI: 10.1080/08910600802030196

Relation of bacteriocins to competitive exclusion antagonistic enteric bacteria might yet provide consistent control of broiler intestinal colonization. Fuller and Gibson (3) suggested that probiotic organisms benefit the health of the host animal by improving the balance of the intestinal microbial community. In the course of characterizing the in vitro antagonism of campylobacter by selected chicken gut flora, we gathered data to explain a mechanism by which in vivo antagonism does occur, and successfully applied these observations to dramatically reduce C. jejuni colonization in the broiler chickens.

Materials and methods Isolation of antagonistic bacteria To assure diversity of antagonistic bacteria, intestinal materials from hundreds of healthy birds, from dozens of processed Russian broiler flocks, were used as donor source material. Both broilers that were colonized with Campylobacter and non-colonized birds were used as bacterial donors. Birds were commercially processed and after evisceration ceca were aseptically excised, individually placed in sterile plastic bags, and returned on ice to the laboratory within 4 h. Three bacterial taxa were designated for further study: 1) lactic acid bacteria (LAB), 2) facultative spore-forming bacteria, and 3) Escherichia coli. Correspondingly, isolates were obtained by 1) directly streaking the cecal material onto MRS (deMan, Rogosa, Sharpe) agar and incubating at 378C for 24 h; 2) diluting cecal materials in buffer (1:10), heating at 88C for 15 min, and streaking the remaining viable organisms on either Plate Count or Starch agar and incubating at 308C for 24 and 48 h; and 3) directly streaking cecal content onto EMB (eosin methylene blue) agar and incubating plates at 378C for 24 h. Isolates were re-streaked for purity and identified using API test strip procedures. About 107 cfu of each candidate antagonistic isolate was surface plated onto agar and grown overnight on each respective agar. Subsequently,
0.5 cm3 agar plugs were aseptically excised, inverted, and transferred onto Brucella agar plates seeded with
107 cfu of C. jejuni NCTC 11168, and the cultures were incubated overnight at 428C for 36
48 h under microaerobic conditions (5% O2, 10% CO2, 85% N2). Antagonistic isolates were initially selected based on the size of the zone of inhibition. Antagonists producing larger zones were saved as most desirable. Antagonistic bacteria isolated and used for subsequent chicken treatments included Bacillus subtilis 1316, E. coli BS10, E. coli BS230, Enterococcus faecium LWP 26, Ent. faecium LWP 408, Ent. faecium 21, Ent. durans 26, Eubacteria saburreum LWP-130,

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Lactobacillus acidophilus PVD-26, Lact. acidophilus LWP-320, Lact. acidophilus VF-1, Lact. salivarius PVD-32 (NRRL 30514), Lact. salivarius PVD-279, Lact. salivarius LWP-163, Lact. salivarius LWP-287, Lact. salivarius PVD-10-5, Lact. salivarius PVD-686, Mitsuokella multiacidus LWP-92, Paenibacillus polymyxa 37, Paen. polymyxa 119, and Paen. polymyxa 602 (NRRL B-30509). Chicken handling and administration of bacteria Approval for the conduct of the following animal experiments was provided by the Institutional Animal Care and Use Committee (PMS-03-03, ‘Control of Campylobacter in Poultry Production’). Healthy dayof-hatch chicks were obtained from commercial hatcheries and returned to the laboratory facilities within 4 h of procurement. Birds were randomly distributed into groups of 5
20 chicks each. Individual birds were gavaged with selected probiotic strains of bacteria (indicated above), using inoculation volumes of 0.2
0.5 ml per bird and times of administration that ranged from day of hatch through 10 days post-hatch. Using selected strains of C. jejuni (4), mid-log cultures were grown on Campylobacter blood agar plates at 428C for 24 h, and incubated under microaerobic conditions (5% O2, 10% CO2, 85% N2). The growth was resuspended and diluted in saline buffer to provide challenges ranging from 103 to 108 cfu/chick and times from day of hatch to day 4 post-hatch. Probiotic organisms were provided both by prophylactic (before campylobacter challenge) and therapeutic (post campylobacter colonization) administrations. At designated times, chickens were sacrificed, ceca were aseptically dissected, and contents were plated onto Campylobacter selective media. Plates containing serial dilutions of cecal contents were incubated at 428C for 36
48 h, under microaerobic conditions. Representative colonies were confirmed as C. jejuni by both phase-contrast microscopy and latex agglutination tests. Enumeration was reported after log10 conversion. Mean values and standard deviations were reported for each group. Bacteriocin production and chicken treatment Bacteriocin from Paen. polymyxa B-30509 was produced and purified as described previously (5). In brief, the culture was grown for 40 h at 328C in Kugler’s broth, then cells were removed by centrifugation and filter-sterilized. The cell-free supernatant was exposed to 80% ammonium sulfate and the protein precipitate was redissolved and dialyzed before being further purified by gel filtration and ion exchange chromatography. Chicken challenge and bacteriocin treatments have been described

76

N.J. Stern et al.

elsewhere (6). In brief, the bacteriocin obtained as described above was micro-encapsulated in PVP (polyvinylpyrrolidone) and incorporated into broiler chicken feed at 250 mg bacteriocin/kg feed. Selection of Lact. salivarius B-30514, characterization and production of its bacteriocin, and use for chicken treatments are described elsewhere (7). We precisely followed these protocols for bacteriocin production and distribution into feeds. Results Providing chicks with combinations of either Lactobacillus spp. or Paen. polymyxa probiotic antagonists before C. jejuni challenge did not alter the resulting level of cecal colonization. C. jejuni colonization ranged from an average of 3 107 to 3108 per g of cecal material. This colonization occurred despite the prophylactic administration of large numbers (107
108 cfu/chick) of probiotic organisms, which had created zones of inhibition of C. jejuni in vitro. Additional, more extensive sets of multi-genera antagonistic probiotic bacterial strains, likewise, did not reduce C. jejuni colonization in chicks. Treated birds were given 108 cfu probiotics per chick, and yet C. jejuni were colonized with approximately the same numbers (
109 cfu per g) in cecal materials as found in the untreated birds. Only when chicks were gavaged daily with defined probiotic antagonists, and then only when these birds were challenged with very low levels (
103 cfu per chick) of C. jejuni, was colonization eliminated in the animals. Treated and control groups challenged with higher levels of C. jejuni were colonized at approximately 3 108 cfu per g. However, this prophylactic treatment by the probiotic bacteria was inconsistently effective, as even the treated group of chicks challenged with only
103 cfu C. jejuni per chick were subsequently colonized in excess of 109 cfu per g cecal contents. Even when a reduced level of C. jejuni colonization was noted following Lact. salivarius NRRL B-30514 treatment at 7 days of age, this significant difference (p50.017) as compared with the control birds was lost by day 10 of colonization. No reduction was seen when combinations of high levels of prophylactic probiotic bacteria were given via daily repeated feeding or by incorporating probiotics in the animal feed (Table I). Daily feeding of very high numbers of probiotic bacteria, likewise, did not reduce cecal colonization by C. jejuni. It was only after colonized chicks were fed bacteriocin on a daily basis that reduction of C. jejuni was manifested (p50.0001) among the treated birds as compared with the control group (Table II). Bacteriocins used as therapy for this study were produced by Paen. polymyxa B-30509 and Lact. salivarius

B-30514. These were bacterial isolates that were also used in the above probiotic studies. Complete elimination of C. jejuni from the 10 chick ceca was achieved among the group given 250 mg bacteriocin derived from Paen. polymyxa NRRL B-30509/kg feed. This was compared to an average of
109 cfu C. jejuni/g cecal materials among eight non-treated control chicks. The difference represented greater than a billion-fold reduction in colonization among the treated and control groups. Although total elimination was not achieved with 250 mg bacteriocin derived from Lact. salivarius NRRL B-30514/kg feed, dramatic reductions in C. jejuni numbers were observed as compared with the levels seen in the control birds. Eight of the nine treated chicks had no detectable levels of C. jejuni, while the remaining treated bird had only
103 cfu/g cecal materials. Discussion Directly fed probiotic bacterial treatments had limited influence on broiler chick colonization by C. jejuni (Table I), while purified bacteriocins provided dramatic reductions over that commensal pathogen (Table II). In 1984, Goren et al. (8) were among the first to report on Salmonella reductions in a large European field trial in which CE was administered by spray application to chicks. Intestinal carriage of Salmonella was reduced among the treated groups of chickens. The poultry industry in Europe, primarily Finland and Sweden, has continued to routinely use CE prophylactically to treat flocks deemed to be at risk for Salmonella infection (9). On the other hand, in the United States, the FDA has been reluctant to provide approval for administration of undefined microbial preparations. The FDA position has been that starting CE material would have to be comprehensively screened for all pathogens that may be present and then disseminated in such a treatment product. Consistent product efficacy would also need to be assured among CE batches. Previous studies have indicated that standard preparations of CE, even when effective against Salmonella spp., offered no resistance to colonization by Campylobacter spp. (10,11). On the other hand, Morishita et al. (12) reported reductions of C. jejuni among colonized chickens after treatment with avian-isolated cultures of Lact. acidophilus and Streptococcus faecium. Likewise, Hakkinen and Schneitz (13) reported that a non-defined CE product (Broilact) reduced colonization by C. jejuni in a chick challenge model. These approaches have not yet been reproduced and are not yet employed by the poultry industry to reduce the target organism. Chaveerach et al. (14) reported isolation of a

Relation of bacteriocins to competitive exclusion Table I. Lack of prophylactic activity of bacterial antagonists in chicks challenged at day 5 post-hatch with
105 cfu C. jejuni. Groups of chicks, experimental conditions 1. Positive control 2. Control, chicks gavaged with 109 cfu Lact. salivarius 68
6, on days 1
5 and 11 and gavaged with 107 cfu Bacillus subtilis 1316 on days 1, 4, and 8 3. Same as group 2, given feed containing
1010 cfu/kg encapsulated Lact. salivarius 68
6 and 5107 cfu/kg B. subtilis 1316; at days 1
5 and 11; free access to feed during the remaining days 4. Same as group 2, given feed containing
9 1010 cfu/kg Lact. salivarius 68
6 at days 1
5 and 11; free access to feed during the remaining days.

No. of chicks per group

Log10 mean cfu C. jejuni/g ceca

10 10

8.8590.95 8.9190.73

20

8.5790.69

10

8.5990.77

Birds were sacrificed and sampled at 13 days of age.

Lactobacillus sp. strain P93 that showed bactericidal activity against Campylobacter spp. They suggested that the bactericidal effect was due to the production of organic acids in combination with an antiCampylobacter protein. They did not, however, report on purification of the protein or on testing the efficacy in a chicken model. In the United States, colonization of the gastrointestinal (GI) tract in commercial chickens occurs in a non-predictable manner and, in part, is dependent on the chance distribution of the organism in the chicken’s environment. In the present study, Campylobacter-antagonistic probiotics were provided to alter the GI tract flora but these alterations did not reduce C. jejuni in the chicken gut. Metabolic by-products such as peroxide compounds and volatile fatty acids are recognized antimicrobials that have been attributed to the dominance of a CE flora (15
17). We had hoped to systematically employ CE to reduce C. jejuni in chicks. After constructing a diverse

77

panel of bacterial isolates antagonistic to C. jejuni, we wished to determine whether prophylactic or therapeutic administration of probiotics might provide reduction of pathogen colonization in chickens. It was only after repeated ‘failures’ in vivo that we observed the consistent benefit of administering bacteriocin produced by these isolates. Reporting a similar failure, Barnes et al. (15) had isolated Bacteroides hypermegas and Bifidobacterium sp., antagonistic to Salmonellae in vitro, but these organisms showed no reductions of Salmonella typhimurium in a chick model. The success in bacteriocin feeding reported in this manuscript corroborates results published previously (6,7). Although CE has proven effective against Salmonella spp., our results do not support the thesis that administering bacteriocin-producing bacteria would form an effective CE preparation for reduction of C. jejuni (18). Bacteriocins are known to disrupt the integrity of the cellular membrane among the target organisms by causing pore formation, cell lysis, and subsequent death (19). Bacteriocins consist of bactericidal polypeptides and the mechanism is thought to be similar to that expressed by ionophore antibiotics. The current study provided our developmental rationale for an overall approach toward bacteriocin application to reduce poultry-borne pathogens. Our completely defined bacteriocin treatment would preclude inadvertent administration of pathogens and would yield predictable and reproducible pathogen killing. CE failed to exclude C. jejuni in this study, although CE has been repeatedly demonstrated to be effective against Salmonella spp. C. jejuni reside in the crypts of Lieberkuhn, deep within the intestinal villi. Limited numbers of competing flora can be found in that niche. This is in part due to the unique nutrients exploited there (20) and the microaerobic atmosphere, to which Campylobacter spp. are adapted. Thus, competing flora were excluded from that niche and probiotic therapy appeared to have limited influence on C. jejuni colonization. However, exogenous bacteriocins, which are aqueous soluble, were able to diffuse into this unique niche and destroy Campylobacter spp. in vivo. We suggest that those intestinal niche differences manifested by Salmonella

Table II. In vivo anti-C. jejuni activity of bacteriocins produced by Paenibacillus polymyxa NRRL B-30509 and by Lact. salivarius NRRL B-30514 administered in emended feeds.

Treatment Positive control Bacteriocin from Paen. polymyxa NRRL B-30509 Lact. Salivarius NRRL B-30514

No. of birds 8 10 9

Treatment scheme None Free access to feed with bacteriocin (250 mg/kg) Free access to feed with bacteriocin (250 mg/kg)

Log10 cfu C. jejuni/g cecal content 9.0890.38 Not detected 0.3491.00

At age 2 days chicks were challenged with 6106 cfu C. jejuni. Birds were sacrificed and levels of C. jejuni/g cecal contents were monitored at 7 days post-hatch.

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N.J. Stern et al.

spp. and C. jejuni in gut colonization are likely responsible for the difference in CE outcomes. Salmonella spp. are believed to adhere to the intestinal linings (21), while C. jejuni is thought to be free swimming within the mucin layer, deep within the luminal crypts (22). In the crypts of Lieberkuhn there is a comparative lack of diversity in bacterial flora present and C. jejuni appears to be the dominant organism. Thus, our selected antagonistic probiotics did not, apparently, come in direct contact with the target organism as they do not occupy this portion of the intestine. It is likely that bacteriocins are produced in situ by bacteria in limited quantities. This quantity will effectively compete with proximally colonizing target bacteria by killing its host competition. It is unlikely that bacteriocin produced in vivo by the producer organism would be over-produced, as the anabolic costs to the organism are too great to be wasted. Because bacteriocins are aqueous soluble, the quantity we provided to the animals was adequate to reach the crypts containing the C. jejuni and killed that target. Further dose-response studies will be needed to establish requisite quantities of bacteriocins needed to kill C. jejuni in the chicken gut. These antagonistic strains were selected by virtue of our in vitro testing for zones of inhibition against C. jejuni. The zones of inhibition were due to the presence of bacteriocins (5,7). Although production of bacteriocins by intestinal bacteria has been recognized, its prominent role within gut ecology has not been appreciated. In part, this is due to the high metabolic costs expended by bacteria to elaborate and secrete these non-structural polypeptides. It is likely that bacteriocins play additional roles in regulating the intestinal flora, such as signaling within and among microbial species (23). Bacteriocins represent a novel therapeutic approach to address intestinal infections. These natural antimicrobial polypeptides have proven effective in reducing a previously rampant infection. Although bacteriocins are routinely produced by these same probiotic intestinal bacteria, those bacteriocin concentrations achieved in vivo were inadequate to kill Campylobacter spp. under in vivo conditions. The present study does not allow us to conclude that bacteriocins are the most important mechanism involved in CE, but our data do support bacteriocins as a significant contributor to the benefits derived from probiotics (24). By providing these polypeptides as a therapeutic, an alternative treatment has been offered.

Acknowledgements Funds for the conduct of this research were provided by the US State Department (ISTC #1720p), the US Department of Agriculture (CRIS 6612
32000
034
00), and the State Research Center for Applied Microbiology and Biotechnology.

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1. 2. Stern NJ. Mucosal competitive exclusion to diminish colonization of chickens by Campylobacter jejuni. Poultry Sci. 1994; 73:402
7. 3. Fuller R, Gibson GR. Modification of the intestinal microflora using probiotics and prebiotics. Scand J Gastroenterol. 1997;222:28
31. 4. Stern NJ, Bannov VA, Svetoch EA, Mitsevich IP, Volozhantsev NV, Gusev VV, et al. Distribution and characterization of Campylobacter spp. from Russian poultry. J Food Prot. 2004; 67:239
45. 5. Svetoch EA, Stern NJ, Eruslanov BV, Kovalev YN, Volodina LI, Perelygin VV, et al. Isolation of Bacillus circulans and Paenibacillus polymyxa strains inhibitory to Campylobacter jejuni and characterization of associated bacteriocins. J Food Prot. 2005;68:11
7. 6. Stern NJ, Svetoch EA, Eruslanov BV, Kovalev YN, Volodina LI, Perelygin VV, et al. Paenibacillus polymyxa purified bacteriocin to control Campylobacter jejuni in chickens. J Food Prot. 2005;68:1450
3. 7. Stern NJ, Svetoch EA, Eruslanov BV, Perelygin VV, Mitsevich EV, Mitsevich IP, et al. Isolation of a Lactobacillus salivarius and purification of its bacteriocin that is inhibitory to Campylobacter jejuni in the chicken gastrointestinal system. Antimicrob Agents Chemother. 2006;50:3111
6. 8. Goren E, de Jong WA, Doornebal P, Koopman JP, Kennis HM. Protection of chicks against Salmonella infection induced by spray application of intestinal microflora in the hatchery. Vet Q. 1984;6:73
9. 9. Mead GC. Prospects for ‘‘competitive exclusion’’ treatments to control Salmonellas and other foodborne pathogens in poultry. Vet J. 2000;159:111
23. 10. Stern NJ, Bailey JS, Blankenship LC, Cox NS, McHan F. Colonization characteristics of Campylobacter jejuni in chick ceca. Avian Dis. 1988;32:330
4. 11. Shanker S, Lee A, Sorrell TC. Horizontal transmission of Campylobacter jejuni amongst broiler chicks: experimental studies. Epidemiol Infect. 1990;104:101
10. 12. Morishita TY, Aye PP, Harr BS, Cobb CW, Clifford JR. Evaluation of an avian-specific probiotic to reduce the colonization and shedding of Campylobacter jejuni in broilers. Avian Dis. 1997;41:850
5. 13. Hakkinen M, Schneitz C. Efficacy of a commercial competitive exclusion product against Campylobacter jejuni. Br Poult Sci. 1999;40:619
21. 14. Chaveerach P, Lipman LJA, van Knapen F. Antagonistic activities of several bacteria on in vitro growth of 10 strains of Campylobacter jejuni/coli. Int J Food Microbiol. 2004;90: 43
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Relation of bacteriocins to competitive exclusion 15. Barnes EM, Impey CS, Stevens BJH. Factors affecting the incidence and anti-salmonella activity of the anaerobic caecal flora of the young chick. J Hyg (Camb). 1979;82:263
83. 16. Nisbet DJ, Corrier DE, Scanlan CM, Hollister AG, Reier RC, DeLoach JR. Effect of defined continuous-flow derived bacterial culture and dietary lactose on Salmonella typhimurium colonization in broiler chickens. Avian Dis. 1993;37: 1017
25. 17. Corrier DE, Nisbet DJ, Scanlan CM, Hollister AG, Caldwell DJ, Thomas LA, et al. Treatment of commercial broiler chickens with a characterized culture of cecal bacteria to reduce salmonellae colonization. Poultry Sci. 1995;74:1093
101. 18. Joerger RD. Alternatives to antibiotics: bacteriocins, antimicrobial peptides and bacteriophages. Poultry Sci. 2003;82: 640
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19. Tagg JR, Dajani AS, Wannamaker LW. Bacteriocins of Grampositive bacteria. Bacteriol Rev. 1976;40:722
56. 20. Hugdahl MB, Beery JT, Doyle MP. Chemotactic behavior of Campylobacter jejuni. Infect Immun. 1988;56:1560
6. 21. Finlay BB, Heffron F, Falkow S. Epithelial cell surfaces induce Salmonella proteins required for bacterial adherence and invasion. Science. 1989;243:940
3. 22. Beery JT, Hugdahl MB, Doyle MP. Colonization of gastrointestinal tracts of chicks by Campylobacter jejuni. Appl Environ Microbiol. 1988;54:2365
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Table III. Influence of selected antagonistic strains given to chicks on C. jejuni colonization.

Group 1. 2. 3. 4. 5

Control Positive control Positive control positive control

6

7

8

9

10

Antagonists None None None None Enterococcus durans 26 Ent. faecium 21 Lact. salivarius NRRL B-30514 Lact. acidophilus VF1 Enterococcus durans 26 Ent. Faecium 21 Lact. salivarius NRRL B-30514 Lact. acidophilus VF1 Ent. durans 26 Ent. faecium 21 Lact. salivarius NRRL B-30514 Lact. acidophilus VF1 Ent. durans 26 Ent. faecium 21 Lact. salivarius NRRL B-30514 Lact. acidophilus VF1 E. coli B6 & B9 Paen. polymyxa 37 and 119 Ent. durans 26 Ent. faecium 21 L. salivarius NRRL B-30514 L. acidophilus VF1 Eschcherichia coli B6 & B9 ?aenibacillus polymyxa 37 and 119 Ent. durans 26 Ent. faecium 21 L. salivarius NRRL B-30514 L. acidophilus VF1 Eschcherichia coli B6 & B9 Paenibacillus polymyxa 37 and 119

Administration of antagonists

C. jejuni challenge,
cfu

Mean log10 C. jejuni, cfu/g ceca

None None None None 2108 cfu/bird gavaged twice daily through 3 days of age


103 104 105 103

0 8.7090.45 8.2090.29 8.5090.51 Not detected

2108 cfu/bird gavaged twice daily through 3 days of age

104

8.4890.56

2108 cfu/bird gavaged twice daily through 3 days of age

105

8.6490.62

2108 cfu/bird gavaged twice daily through 3 days of age

103

0

2108 cfu/bird gavaged twice daily through 3 days of age

104

9.5390.94

2108 cfu/bird gavaged twice daily through 3 days of age

105

10.5090.88

Chicks were gavaged twice daily with the indicated probiotic bacteria from day-of-hatch through 3 days of age. Ten chicks per group were subsequently challenged with doses of
103,
104, and
105 cfu C. jejuni per bird at 4 days of age and sampled at 7 days of age. Colonization levels reported were determined by direct plating of cecal contents onto Campylobacter selective agar and incubating plates at 428C under microaerobic atmosphere for 48 h.

Table IV. Influence of selected antagonistic strains on C. jejuni colonization in chicks.

Group 1. Control 2. Positive control 3. Positive control 4

5

Antagonists None None None Ent. durans 26 Ent. faecium 21 Lact. salivarius NRRL B-30514 Lact. acidophilus VF1 Ent. durans 26 Ent. faecium 21 Lact. salivarius NRRL B-30514 Lact. acidophilus VF1

Route and interval of administration of antagonists

C. jejuni challenge dose
cfu/chick

C. jejuni, log cfu/g ceca

None None None 2 108 cfu/bird gavaged twice daily through 3 days of age


1 103 1 104 1 103

0 9.090.25 9.3090.35 9.0890.53

2 108 cfu/bird gavaged twice daily through 3 days of age

1 104

9.4790.77

Chicks were gavaged twice daily with selected bacteria from day of hatch through 3 days of age. Birds were challenged with doses of 1 103, 1104, and 1 105 cfu C. jejuni per bird at 4 days of age and sampled at 7 days of age. Colonization among 10 chicks per group was determined by direct plating of individual cecal contents on Campylobacter selective agar and incubating plates at 428C under microaerobic atmosphere for 48 h.

Table V. Day-of-hatch chicks initially provided with 2.0108 Lact. salivarius NRRL 30514/chick and
106 Lact. salivarius/g feed for 10 days.

Treatment

No. of chicks

None Lact. salivarius NRRL B-30514 None Lact. salivarius NRRL B-30514

Log10 C. jejuni cfu/g ceca
individual chicks

Day of chick sampling

7 7 7 5

7 7 10 10

9.1490.58 7.6091.36 8.3490.74 9.0290.50

Birds were challenged with
104 cfu C. jejuni at 4 days of age and sampled at 7 and 10 days of age. Enumeration of C. jejuni/g of cecum was determined by dilution plating of cecal contents onto Campylobacter selective agar and incubating at 428C under microaerobic atmosphere for 48 h.

Table VI. Lack of prophylactic activity of antagonists in chicks challenged at 5 days of age with
105 cfu C. jejuni; birds were sacrificed and sampled at 13 days of age. Groups of chicks, experimental conditions

No. of chicks per group

1. Positive control 2. Control, chicks gavaged with 109 cfu Lact. salivarius 68
6, on days 1
5 and 11 and gavaged with 107 cfu B. subtilis 1316 on days 1, 4, and 8 3. Same as group 2, given feed containing
1010 cfu/kg encapsulated Lact. Salivarius 68
6 and 5 107 cfu/kg B. subtilis 1316; at days 1
5 and 11; free access to feed during the remaining days 4. Ssame as group 2, given feed containing
9 1010 cfu/kg Lact. salivarius 68-6 at days 1
5 and 11;free access to feed during the remaining days

Log10 mean cfu C. jejuni/g ceca

10 10

8.8590.95 8.9190.73

20

8.5790.69

10

8.5990.77

Table VII. Lack of prophylactic or therapeutic activity of antagonistic Lact. salivarius isolates against C. jejuni-colonized chicks.

Treatment Positive control Lact. salivarius PVD 10-5 Lact. salivarius PVD 68-6

No. of birds 9 9 10

Treatment scheme None Daily administration of live culture;
109 cfu/chick Daily administration of live culture;
109 cfu/chick

Log10 cfu C. jejuni/g cecal content 9.0590.74 9.5590.55 9.4390.24

At 2 days of age chicks were challenged with 6 105 C. jejuni. Birds were sacrificed and levels of C. jejuni/g cecal contents were monitored at 7 days of age.

Table VIII. In vivo anti-C. jejuni activity of bacteriocins produced by Paenibacillus polymyxa NRRL B-30509 and by Lact. salivarius NRRL B-30514 administered in emended feeds.

Treatment Positive control Bacteriocin from Paen. Polymyxa NRRL B-30509 Lact. salivarius NRRL B-30514

No. of birds

Treatment scheme

Log10 cfu C. jejuni/g cecal content

8 10

None Free access to feed with bacteriocin (250 mg/kg)

9.0890.38 Not detected

9

Free access to feed with bacteriocin (250 mg/kg)

0.3491.00

At 2 days of age chicks were challenged with 6 106 cfu C. jejuni. Birds were sacrificed and levels of C. jejuni/g cecal contents were monitored at 7 days of age.