Probiotic and Prebiotic Application in Chickens J. Stan Bailey USDA, ARS, BEAR Athens, GA stan.bailey@ars.usda.gov Why the interest in pro- and prebiotics use in chickens? Pathogen control (particularly Salmonella) Improved health and associated production performance enhancement Pressure to reduce antibiotic use in animal agriculture
Broiler Production 101 Hatchery Days 0-21 Transport & Processing Days 42-56 Growout Days 42-56 Consumption 15,000 to 40,000 per house http://www.jefo.ca/pdf/intestinal_health.pdf
Gastrointestinal Tract Largest immune organ in the body (Abreu-Targan, 1996; Mayer, 2000) Underdeveloped in poultry at hatch (Uni et al.,1995; Noy and Sklan, 1997) Microflora aid in many aspects of maturation: assembly of GALT, increases integrity of mucosal barrier, educates immune system (Cebra et al., 1999; MacPherson et al., 2000; Kelly et al., 2004) Intestinal Ecosystem is Complex and Dynamic Chicken microflora determined by sequencing of 1,230 clones from a 16S rdna community DNA library. From Jiangrang et al., 2003
Human salmonellosis Estimated that 1.4 million people contract salmonellosis in the U.S. annually $3,000,000,000 estimated annual costs (WHO 2006) In 2004 Salmonella was associated with 42% of all laboratory confirmed food borne illnesses (FoodNet 2005) Poultry products serve as vehicle for human salmonellosis (Bean & Griffin, 1990; Persson & Jendteg 1992) Pathogen control Chickens are most susceptible to colonization by Salmonella when they are newly hatched or after times of stress (immature and/or disrupted intestinal microflora) CD-50 Day 1 10 0 Day 3 10 2-3 Day 7 10 6-7 Stress Temperature Subclinical disease Transport Molting
Improved health and performance Improved weight gain and feed conversion Help prevent and overcome stress during production Improve birds ability to immunologically respond to challenges Reduced antibiotic use Antibiotics are used either therapeutically to treat a disease condition or subtherapeutically to enable the chickens to reach their full growth potential by reducing subclinical disease and/or selection of more productive gut microflora Reduced subtherapeutic use is leading to increased necrotic enteritis (often caused by Clostridium perfringens) Pre- and probiotics may be an alternative
DENMARK Increases in Therapeutic Use of Antimicrobials Since the Bans on AGPs 120000 100000 80000 60000 40000 48000 57000 57300 61900 80600 91602 112500 112650 102500 96960 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 Subtherapeutic ban Source: DANMAP 1996-2005 Probiotic. A direct fed microbial which alters the microflora of the host and exert beneficial effects to the host Viable includes both defined and undefined Beneficial includes both health benefit to the animal and indirect benefit to human health such as reduction of pathogenic bacteria in/on the animal
Characteristics of ideal Probiotics Patterson and Burkholder, 2003 adapted from Simmering and Blaut, 2001 Be of host origin Non-pathogenic Withstand processing and storage Resist gastric acid and bile Adhere to epithelium or mucus Persist in the intestinal tract Produce inhibitory compounds Modulate immune response Alter microbial activities Mode of Action of Probiotics Jin et al., 1997, World Poultry Science Maintain normal intestinal microflora by competitive exclusion and antagonism Alter metabolism by increasing digestive enzyme activity and decreasing bacterial enzyme activity and ammonia production Improve feed intake and digestion Neutralize enterotoxins and stimulate the immune system
Prebiotic A nondigestible food ingredient that affects the host by selectively stimulating the growth and/or activity of beneficial bacteria in the intestinal tract. Beneficial bacteria could provide both a health benefit to the animal and indirect benefit to human health such as reduction of pathogenic bacteria in/on the animal Characteristics of ideal Prebiotics Patterson and Burkholder, 2003 adapted from Simmering and Blaut, 2001 Be neither hydrolyzed nor absorbed by mammalian enzymes or tissues Selectively enrich for one or a limited number of beneficial bacteria Beneficially alter the intestinal microbiota and their activities Beneficially alter luminal or systemic aspects of the host defense system
Probiotics Probiotics - Defined Known species of flora Lactic acid bacteria (LAB): Lactobacillus spp, Lactobacillus reuteri, Weisiella spp, Bifidobacterium spp, Enterococcus spp, Lactococcus spp, Pediococcus spp; Streptococcus thermophilus, (or various combinatios) non-lab group: >Bacillus cereus and B. licheniformis >Saccharomyces spp.
Probiotics - Undefined Normal intestinal microflora from healthy adult chicken (animal) Wider diversity of microorganisms Appears to be more effective than defined organisms Broilact,, Aviguard,, Mucosal Stater Culture Early Probiotic Work Early colonization with normal microflora improved resistance to bacterial pathogens (Nurmi (Nurmi et al.,, 1972) Increased productivity of poultry (>75 studies) Effects are dose-related, isolate specific, and require viable bacteria
1972-1995 1995 Emphasis on Prophylaxis Nurmi, E.V. and Rantala M. 1973. Prevention of the growth of Salmonella infantis in chicks by the flora of the alimentary tract of chickens. Brit. Poult. Sci. 14(6): 627-630. 630. Barrow, P.A. and J.F. Tucker. 1986. Inhibition of colonization of the chicken caecum with Salmonella typhimurium by pre- treatment with strains of Escherichia coli.. Journal of Hygiene, Camb. 96:161-169. 169. Blankenship, L.C., J.S. Bailey, N.A. Cox, N.J. Stern, R. Brewer, and O. Williams. 1993. Two-step mucosal competitive exclusion flora treatment to diminish salmonellae in commercial broiler chickens. Poultry Science. 72:1667-1672. 1672. Corrier, D.E., D.J. Nisbet, C.M. Scanlan, A.G. Hollister, and J.R. DeLoach. 1995. Control of Salmonella typhimurium colonization in broiler chicks with a continuous-flow characterized mixed culture of cecal bacteria. Poultry Science. 74:916-924 924 Recent (10 year) Probiotic Work An large number of scientific publications on Lactic Acid Bacteria and human health model studies Reports of benefits for enteric bacterial & viral diseases, reduced effects of mycotoxins, cancer intervention, increased absorption of macrominerals and other nutrients. Reid and Friendship. Animal Biotech: 13:97-112, 112, 2002. Excellent review of probiotics use in animals including poultry.
Example References- Immune Responses Fukushima Y. et al. (1998) Int. J. Food Microbiol.42:39 42:39-44. 44. Tejada-Simon MV et al. (1999) J. Dairy Sci. 82:649-660. 660. Fang H et al. (2000) FEMS Immunol. Med. Microbiol.. 29:47-52. Cukrowska B. et al. (2002) Scand. J. Immunol. 55:204-209 209. Reported Effects of Probiotics on Laying Hens Increasing egg production (Nahashon et al. 1996) Increasing egg shell quality (Mohan, B. et al. 1995) Improved Feed Conversion (Yeo and Kim, 1997) Decreasing Mortality (Kumprecht and Zobac, 1998) Decreasing Cost of Production (Jin, L. et al. 1999)
Growth Performance and Probiotics L. acidophilus added to drinking water increased weight gain by 10% (Tortuero, 1973) B. coagulans in the feed d 1-491 increased mean body weight (Cavazzoni et. al., 1998) 2 Lactobacilli in feed increased body weights by approximately 10% (d30, 35, and 40) (Lan et al., 2003) Immune Stimulation by Probiotics Strains differ in ability to change cytokine profiles (Perdigon et al., 2002; Maasen et al., 2000; Dalloul et al., 2005; Vinderola et al., 2004) Strains differ in ability to increase immunoglobulins (Yurong et al., 2005; Dalloul et al., 2005; Vinderola et al., 2004) Coincubation of 2 individual LAB with or without ST inhibit IL-8, but don t t inhibit binding of ST to cell culture (O Hara et al., 2006)
Prebiotics Prebiotics - Characteristics Non-digestible by the host Selective growth stimulation Modulates bacterial population in the GIT Brings beneficial health effects to the host Particularly for poultry production: low dietary inclusion rates!
Prebiotics - Types Carbohydrates Resistant starch Dietary fibre (non-starch polysaccharides) pectin, cellulose, hemicellulose, xilan, and guar gum Oligosaccharides lactose, lactulose, raffinose, stachyose, fructo-,, gluco-,, and mannan-oligosaccharides* Some protein and aminoacids endogenous and bacterial secretions Why lactose as prebiotic? Fermented preferentially by LAB selective Non digested by poultry species No presence of endogenous lactase considered (Denbow, 2000 ) Versatile product: feed or drinking water
Lactose Studies in Poultry Focused mainly on control of pathogens Few studies focused on performance The main problem: High doses: 2 % and up Prebiotic inclusion rates in feed: < 1 % USDA, ARS Stan Bailey prebiotic, probiotic research FOS (fructooligosaccharide) Yeast (Saccharomyces boulardii) Competitive Exclusion
Effect of FOS on Salmonella colonization of chickens (Bailey( et al., 1991) As prebiotic gave limited (12%) improve- ment in Salmonella colonization rates Improved efficacy of CE culture (3 fold) When chickens were stressed the FOS reduced Salmonella colonization from 92% to 25% Saccharomyces boulardii (Line, Bailey et al., 1998) 70 % Salmonella (+) 60 50 40 30 20 10 Positive 1X Yeast 100 X Yeast 0 Treatment
Competitive Exlusion 1973 - Dr. Esko Nurmi and co-workers in Finland were the first to use the competitive exclusion approach to control salmonellae in broiler chicks. Likely Factors in the Exclusion Process Competition for receptor sites Bacterial volatile fatty acids Substrate competition Low redox potential Bacteriocins
Summary of CE 1. Newly hatched chicks can be infected by a single cell of Salmonella. 2. Older birds are far more resistant to colonization because of normal gut microflora. 3. The introduction of flora from an adult bird into newly hatched chicks speeds gut maturation and increases resistance to salmonella colonization. Mucosal Starter Culture (MSC) Mucosal Competitive Exclusion flora (MCE), the basic material for the commercial product Mucosal Starter Culture (MSC) is derived from the mucosal scrapings of ceca from healthy adult chickens which are then propagated under anaerobic conditions.
Field Trial Testing of Mucosal Starter Culture TM Results: Competitive Exclusion Puerto Rico On Farm: 11 % to 2 % Processing Plant: 41 % to 10 % Georgia On Farm: 2 % to 0 % Processing Plan: 9 % to 4.5 % Turkey s s At 6 Weeks: 40 % to 3 %
Pre-Chill and Post-Chill Salmonella Data Pooled across all locations (3 trials) Location Pre- Chill Post- Chill Control 23+/180 9+/180 MSC Treated 12+/180 0+/180 Chi Square P=0.05 P=0.02 Advantages of CE Treatment Easily applied Low cost Non-specific protection Rapid host response Compatible with other measures
Antibiotic Resistance Risk / Benefit Potential for direct transfer of resistance from bacteria in the cultures Potential for genetic transfer between bacteria in the CE mixtures CE has been shown to significantly reduce C. perfringens induced necrotic enteritis Likely that the use of CE will allow the significant reduction of subtherapeutic antibiotics without loss of performance Why not wide acceptance of Probiotics? Many products contain few viable organisms Some products contain strains selected for growth in dairy products or at different temperatures Antimicrobial or disinfectant interference Necessary to deliver high dosage (>10 6 CFU) of viable organisms in a short time
How to Improve Probiotic Performance? Genetic engineering GMO controversy Continuous screening for probiotic isolates Laborious but so far effective Better understanding of the GIT ecology: Improving conditions that favor probiotic bacteria activity / replication / establishment Nutrition of bugs prebiotics