Raising Drug-Free Poultry What are the Alternatives?P. R. FerketDepartment of Poultry Science,College of Agriculture and Life Sciences,North Carolina State University,Raleigh, NC 27606-7608During the past 50 years, the livestock and poultry industries have madegreat strides in several areas including nutrition, genetics, engineering,management, and communications thereby maximizing the efficiency ofgrowth performance and meat yield. These industries are now expected tofocus more attention on how animal agriculture affects the environment andfood safety. Currently, the global paradigm is shifting from an emphasis onproductive efficiency to one of public security. Nothing demonstrates thisparadigm shift more clearly than the issues concerning the use of antibioticgrowth promoters (AGPs). For the past 4 decades, antibiotics have beenused in animal agriculture to improve the growth performance and to protectanimals from the adverse effects of pathogenic and non-pathogenic entericmicroorganisms. Recently, the use of antibiotics, have come underincreasing scrutiny because of the potential development of antibiotic-resistant human pathogenic bacteria after long use. The tide of publicopinion is forcing animal agriculture to develop alternatives to antibioticgrowth promoters. Some of these alternatives may include significantchanges in husbandry practices or the strategic use of enteric microfloraconditioners, including acidifiers, probiotics, enzymes, herbal products,microflora enhancers, and immunomodulators.The objective of this paper is to discuss the potential of non-pharmaceuticalalternatives to antibiotics. Alternatives to antibiotics promote gut health byseveral possible mechanisms including: altering gut pH, maintainingprotective gut mucins, selecting for beneficial intestinal organisms or againstpathogens, enhancing fermentation acids, enhancing nutrient uptake, andincreasing the humoral immune response. Strategic use of these alternativecompounds will help optimize growth, provided they are used in a mannerthat complements their modes of action. -------------------------------------------------------------------------------- Page 2 Enhance Pathogen Colonization ResistanceColonization of enteric pathogens is dependent upon the degree of resistanceafforded by the stability of the resident microflora and the integrity of theintestinal mucin barrier in the animal. Older animals are much lesssusceptible to the colonization of enteric pathogens than young animalsbecause they have a more stable and diverse gut microflora thatcompetitively excludes pathogen colonization. In contrast, the ability ofpathogens to colonize in the gut increases after antibiotic administrationbecause of a loss of resident microflora. The stability of resident microfloracan be enhanced by the administration of competitive exclusion cultures(probiotics) or feeding prebiotic compounds that feed the beneficialmicroflora. Hollister et al. (1999) reduced salmonella colonization in chicksby feeding a live cecal culture from salmonella-free poultry. Fedorka-Crayet al. (1999) has shown similar response to microbial cultures in youngswine. Gram-positive bacteria, including Lactobacillus, Enterococcus,Pediococcus, Bacillus, and bifidobacteria, and fungi of the Saccharomyces(yeast) genus are often fed after antibiotic therapy as a means of restoringequilibrium, by re-introducing a beneficial flora to the gut of affectedanimals. Beneficial bacteria inhibit the colonization of pathogens byproducing volatile fatty acids (VFAs) that reduce the pH of the brush-boarder microenvironment and by blocking the attachment of pathogens.Organic acids have strong antibacterial effects, especially to gram-negativepathogens.Immune Response AugmentationThe immune system is the primary defense mechanism of the animal in itsfight against infectious disease. Augmentation of humoral and cell-mediatedimmunity will increase an animal's ability to resist disease. Although thereis a small nutrient cost in the production of immunoglobulins, good antibodytiter levels indicate a far more efficient capacity to resist disease by humoralimmune responses than by an active inflammatory response (Humphrey etal., 2002). A pro-inflammatory innate immune response is associated withthe mobilization of nutrients away from growth and suppression of feedintake. Thus, dietary immunomodulators or vaccines that enhance humoralimmunity and minimize immunological stress will have a more positiveeffect on growth performance. An alternative to feeding dietary factors thatstimulate gut-associated humoral immunity, may be the feeding of specificantibodies that neutralize pathogenic organisms. To produce the specific -------------------------------------------------------------------------------- Page 3 antibodies, laying hens are exposed to particular antigens to stimulate theproduction of immunoglobulins, which are deposited in the egg. Theseimmunoglobulins are then harvested from the eggs and fed to susceptibleyoung animals. There may be some limitations to this technology, sincethese immunoproteins are sensitive to heat treatment during feed processingand the digestive processes of the animal.Diet digestibility and Enzyme supplementationGut health and enteric disease resistance is often dependent upon thedigestibility of feed components and feed formulation. Poorly digestedprotein meals cause the proliferation of putrifying bacteria in the hindgut,which increases toxic metabolites (ammonia and biogenic amines) thatcompromise gut health. Similarly, poultry fed diets containing high levels ofpoorly digested non-starch polysaccharides (NSPs) from wheat, barley orrye are more susceptible to enteric disease, such as necrotic enteritis (Riddelland Kong, 1992; Kaldhusdal and Skjerve, 1996). Langhout (1999) observedthat dietary NSPs significantly increase gut populations of pathogenicbacteria at the expense of beneficial bacteria. However, the digestibility ofwheat, barley, rye, triticale and even corn-based diets can be significantlyimproved through use of exogenous enzymes including xylanases, phytasesand ß-glucanases. In a comprehensive literature review, Rosen (2001)concluded that the effect of enzymes was nearly equivalent to the effects ofantibiotics on gain and FCR, and that in combination there was improvedperformance, although this was less than the sum of the two effects.Enzymes are perhaps the most extensively reviewed products that seem to becapable of limiting the performance losses associated with the removal ofantibiotic growth promoters (AGPs).Because supplemental enzymes mediate their beneficial effects primarily byenhancing feed digestibility and nutrient availability to the host, it must beassumed that they also influence the gut microbial ecosystem. The use ofenzymes has been shown to alter the gut microflora populations in the smallintestine and caeca (Choct et al., 1996; Hock et al., 1997; Bedford, 2000a)and reduce mortality rates (Rosen, 2001). Such a benefit is brought about bya more rapid digestion and absorption of starch, protein and fat from thesmall intestine, which effectively limits available substrate for the residentflora. In general, the improvement in nutrient digestibility achieved for thehost by the use of an appropriate enzyme is much smaller than theconcomitant loss of substrate experienced by microflora resident in the large -------------------------------------------------------------------------------- Page 4 intestinal. This starch and protein removal effect is coupled with theproduction of exogenous enzyme for fiber-derived oligomers, which serve assubstrate for specific populations of bacteria that seem to benefit the host(Bedford, 2000a).Organic Acids, Herbs, Spices, and Essential OilsMany compounds that have bacteriostatic effects can serve as alternatives toAGPs, which work in similar fashion. Organic acids have been used assalmonella-control agents in feed and water supplies for livestock andpoultry, and they are most effective in young poultry because they havelimited acid production in the proventriculus. Herbs, spices, and plantextracts may be useful because they could stimulate appetite (e.g. mentholfrom peppermint), provide anti-oxidant protection (e.g. cinnamaldehydefrom cinnamon), or suppress microbial growth (carvacol from oregano).Essential oils from Oregano is showing the greatest potential as analternative to antibiotic growth promoters. Oregano contains phenoliccompounds, such as carvacrol, that have antimicrobial activity (Akagul andKivanc, 1988). Like antibiotics, the essential oils of Oregano modify the gutmicroflora and reduce microbial load by suppressing the proliferation ofbacteria. These plant-based antimicrobials compounds, which functionfundamentally similar to antibiotic compounds produced by fungi, could beused to replace some antibiotic growth promoters. As with antibiotics,continued use of these plant-based antimicrobials may result in thedevelopment of resistance in some pathogenic bacteria. However, moreresearch is necessary to confirm this risk.OligosaccharidesOligosaccharides are promising alternatives to AGPs, because they facilitateand support the symbiotic relationship between host and microflora.Fructooligosaccharide (FOS) and mannanoligosaccharide (MOS) are twoclasses of oligosaccharides that are beneficial to enteric health, but they doso by different means.Fructooligosaccharides (FOS)Fructooligosaccharides are found in numerous plants such as the onion,Jerusalem artichoke, garlic, banana, chicory, asparagus, and wheat. Theyinfluence enteric microflora by feeding the good bacteria, whichcompetitively excludes the colonization of pathogens. Dietarysupplementation of FOS provides selective enrichment of Lactobacilli(Mitsuoka et al., 1987) and Bifidobacteria (Hidaka et al. 1991). Patterson et -------------------------------------------------------------------------------- Page 5 al. (1997) found that cecal Bifidobacteria concentrations were increased 24-fold and Lactobacilli populations increased 7-fold in young broilers fed theFOS-enriched diets. Fructooligosaccharides are well utilized by the majorityof Bifidobacteria strains (longum, brevis, and infantis) with the exception ofBifodobacterium bifidum (Hidaka and Hirayama, 1991). The Bacteroidesgroup also showed a tendency to utilize FOS as a growth source, whileLactobacillus fermentum, E. coli, and Clostridium perfringens failed toutilize FOS as a fermentative carbohydrate source. Bifidobacteria readilyferment FOS because of the innate secretion of a -fructoside enzyme.Bifidobacteria may inhibit other microbes because of its acidic surroundingsfrom the high production of VFAs or the secretion of bacteriocin-likepeptides. The improvement in gut health conditions by dietary FOSsupplementation often results in improved growth performance. Ammermanet al. (1988) demonstrated that the addition of either 0.25% or 0.50% dietaryFOS improved feed efficiency from 1 to 46 days of age and reducedmortality when fed at the higher level (0.50%). FOS-treated birds also hadless air sac lesions at day 46.Mannanoligosaccharide (MOS)Unlike FOS, MOS is not used as a substrate in microbial fermentation, but itstill exerts a significant growth-promoting effect by enhancing the animalsresistance to enteric pathogens. BioMos¡(Alltech, Nicholasville, KY) is thecommercial source of MOS that has been used in most of the publishedresearch literature. Based on the scientific literature, BioMos enhances ananimals resistance to enteric disease and promotes growth by the followingmeans: 1) Inhibits colonization of enteric pathogens by blocking bacterialadhesion to gut lining; 2) enhances immunity; 3) modifies microflorafermentation to favor nutrient availability for the host; 4) enhances the brushboarder mucin barrier; 5) reduces enterocyte turnover rate; and 6) enhancesthe integrity of the gut lining.Inhibition of pathogen colonization by MOSMannan-oligosaccharides, derived from mannans on yeast cell surfaces, actas high affinity ligands, offering a competitive binding site for a certain classof bacteria (Ofek et al., 1977). Gram-negative pathogens with the mannose-specific Type-1 fimbriae attach to the MOS instead of attaching to intestinalepithelial cells and they move through the gut without colonization. DietaryMOS in the intestinal tract removes pathogenic bacteria that could attach to -------------------------------------------------------------------------------- Page 6 the lumen of the intestine (Newman, 1994). Mannose was shown by Oyofoet al. (1989a) to inhibit the in vitro attachment of Salmonella typhimurium tointestinal cells of the day old chicken. Then Oyofo et al. (1989b) providedevidence that dietary D-mannose was successful at inhibiting the intestinalcolonization of Salmonella typhimurium in broilers. The ability of MOS tointerfere with the attachment of pathogenic bacteria in the gut raises thepossibility that it could also inhibit the binding between bacteria that isrequired for plasmid transfer via conjugation. This kind of inhibition ofplasmid transfer in the digestive tract of mice colonized with humanmicroflora has been described using lactose (Duval-Iflah, 2001). Lou (1995)demonstrated that dietary MOS supplementation decreased the proportion ofspecific groups of Gram-negative antibiotic resistant fecal bacteria in swine.Enhancement of Immune Function by MOSMOS has been shown to have a positive influence on humoral immunity andimmunoglobulin status. As mentioned above, a good humoral immuneresponse is a nutritionally more efficient means to resist disease than anactive inflammatory response (Humphrey et al., 2002). Savage et al. (1996)reported an increase in plasma IgG and bile IgA in poults fed dietssupplemented with 0.11% MOS. An increase in antibody response to MOSis expected because of the ability of the immune system to react to foreignantigenic material of microbial origin. Portions of the cell wall structure ofthe yeast organism, Saccharomyces contained in MOS has been shown toelicit powerful antigenic properties (Ballou, 1970). However, MOS mayalso enhance humoral immunity against specific pathogens by preventingtheir colonization leading to disease, yet allowing them to be presented toimmune cells as attenuated antigens. Indeed, as MOS facilitates thesecretion of IgA into the gut mucosa layer, pathogenic agents become morelabile to the phagocytic action of gut-associated lympocytes.All animals reared under commercial field conditions are subjected toimmunological stress, depending on the pathogen load in their environmentand the vaccination program. The positive growth performance effectsobserved among animals fed MOS, may be partly due to the effect of MOSon reducing acute immunological stress and associated inflammation that isdetrimental to growth and production. To test this hypothesis, Ferket (2002)induced an acute immune stress in 14-day old turkey poults byintraperitoneal injection of lipopolysaccharides (LPS) from Salmonellatyphimurium strain SL 684. The poults were fed either, 1 kg BioMos/tonne,20 g virginiamycin /tonne, or control diet from one day of age. Cloacal -------------------------------------------------------------------------------- Page 7 temperatures were measured eight hours after the LPS injection, and thenbody, liver, spleen, bursa of Fabricius, and intestinal tract weights wererecorded 24 hours post-injection. In contrast to the control and theantibiotic-fed birds, the BioMOS-fed birds showed no fever response 8 hpost-injection, even though liver and intestine weights were increased. Inother words, the MOS-fed birds retained normal body temperature afterexposure to a pro-inflammatory antigen, while the controls and thevirginiamycin (VM)-fed birds expressed elevated body temperatures. Undercommercial conditions where birds are subjected to chronic immunologicalstress, MOS may help reduce the pro-inflammatory response and associateddepression in feed intake and growth.Effect of MOS on Gut Tissue Integrity and HealthThe beneficial effects of MOS on the gut microflora, nutrient utilization, andgrowth performance may be associated with brush boarder morphology andhow it influences enteric disease resistance. To test this hypothesis, Ferket(2002) conducted an experiment to ascertain effects of MOS and VM onjejunum villi morphology. Commercial Hybridpoults were fed a corn-soycontrol diet or diets supplemented with 1 kg BioMos/tonne or 20 gvirginiamycin/tonne starting a 1 day of age. At 14 days of age, 8 birds pertreatment pen were sampled for morphometric measurements, includingvillus height, crypt depth, muscularis thickness, and goblet cell number.MOS had the greatest effect on villi morphology. Although MOS did notaffect villus height, a decrease in crypt depth approached significance andvilli height:crypt depth ratio was significantly greater than the control or VMtreatments. Iji et al. (2001) also observed an increase in jejunal villiheight:crypt depth ratio by MOS supplementation in broilers, but this wasdue to a significant increase in villi height rather than crypt depth. Theseresearchers also observed MOS to significantly increase protein/DNA ofjejunal mucosa, as well as increases in the brush boarder enzymes maltase,leucine aminopepidase and alkaline phosphatase. Turkeys receiving MOS inour experiment also exhibited a thinner muscularis layer and increased thenumber of goblet cells per mm of villus height as compared to control birds.The mucus gel layer coating the surface of the intestinal epithelium is thefirst major barrier to enteric infection. Hence, the production of mucus, asindicated by the number of goblet cells, is an important feature in theprotective scheme against pathogens. Feeding MOS resulted in an increasedproliferation of goblet cells into the surface of the villus membrane. The -------------------------------------------------------------------------------- Page 8 innate immune system recognizes key molecular structures of invadingbacteria, including lipopolysacchharides, peptidoglycans, and possibly themannose structures in the cell walls of yeasts. Oligosaccharides containingmannose have been shown to affect the immune system by stimulating liversecretion of mannose-binding protein. This protein, in turn, can bind tobacteria and trigger the complement cascade of the host immune system(Newman, 1994). Intestinal microbes might influence goblet cell dynamicsby releasing bioactive compounds or by indirect activation of the immunesystem (Bienenstock and Befus, 1980).ConclusionIn response to consumer demands and government regulations, todaysintensive animal agriculture industry must adapt to producing animals in aworld without antibiotic growth promoters. This paper presented severalalternatives to antibiotics to manage gut health. Although no singlealternative may be as effective as antibiotics, the combination of strategiesand feed additives can be used to achieve good gut health and growthperformance. The key to selecting the most cost effective approach willdepend upon the production requirements of each company, and the type ofproduction challenges they face.ReferencesAkagul, A., and M. Kivanc, 1988. Inhibitory effects of selected Turkish spices andoregano compounds on some food-borne fungi. Intl. J. Food Microbiology 6:264-268.Ammerman, E., C. Quarles, and P. Twining, 1988. Broiler response to the addition ofdietary fructooligosaccharides. Poultry Sci. 67: (Supple. 1) 46 (Abstract).Ballou, C. E., 1970. A study of the immunochemistry of three yeast mannans. J. Biol.Chem. 245: 1197-1203.Bedford, M.R., 2000a. Removal of antibiotic growth promoters from poultry diets:implications and strategies to minimise subsequent problems. Poult Sci 56, 347-365.Bedford, M.R., 2000b. Removal of antibiotic growth promoters from poultry diets:implications and strategies to minimise subsequent problems. Poult. Sci. 56:347-365.Bienenstock, J., and A. D. Befus, 1980. Mucosal Immunology: A Review. Immunology41: 249-270. -------------------------------------------------------------------------------- Page 9 Choct, M., R.J. Hughes, J. Wang, M.R. Bedford, A.J. Morgan, and G. Annison, 1996.Increased small intestinal fermentation is partly responsible for the anti-nutritiveactivity of non-starch polysaccharides in chickens. Br Poult. Sci. 37:609-621.Fedorka-Cray, P.J., J.S. Bailey, N.J. Stern, N.A. Cox, S.R. Ladely, and M.Musgrove,1999. Mucosal competitive exclusion to reduce Salmonella in swine. J. Food Prot.62:1376-1380.Ferket, P.R., 2002. Use of oligosaccharides and gut modifiers as replacements for dietaryantibiotics. Proc. 63rdMinnesota Nutrition Conference, September 17-18, Eagan,MN, pp 169-182.Hidaka, H., and M. Hirayama, 1991. Useful characteristics and commercial applicationsof fructooligosaccharides. Biochemical Society Transactions 19:561-565.Hidaka, H., M. Hirayama, and K. Yamada, 1991. Fructooligosaccharides enzymaticpreparation and biofunctions. J. Carbohydrate Chem. 10:509-522.Hock, E., I. Halle, S. Matthes, and H. Jeroch, 1997. Investigations on the composition ofthe ileal and caecal microflora of broiler chicks in consideration to dietary enzymepreparation and zinc bacitracin in wheat-based diets. Agribiol 50:85-95.Hollister, A.G., D.E. Corrier, D.J. Nisbet, and J.R. DeLaoch, 1999. Effects of chicken-derived cecal microorganisms maintained in continous culture on cecal colonizationby Slamonella typhimurium in turkey poults. Poultry Sci. 78:546-549.Humphrey, B. D., E. A. Koutsos, and K. C. Klasing, 2000. Requirements and prioritiesof the immune system for nutrients. Pp 69-77. In. Nutritional biotechnology in thefeed and food industries. Proceedings of Alltech's 18thAnnual symposium. Ed. T. P.Lyons, and K. A. Jacques.Iji, P.A., A. A. Saki, and D. R. Tivey, 2001. Intestinal structure and function of broilerchickens on diets supplemented with a mannan oligosaccharide. J. Sci. Food Agric.81:1138-1192.Kaldhusdal, M., E. Skjerve, 1996. Association between cereal contents in the diet andincidence of necrotic enteritis in broiler chickens in Norway. Prev Vet Med 28:1-16.Langhout, D. J., 1999. The role of the intestinal flora as affected by NSP in broilers.Pages: 203-212. In: Proceedings, Twelfth European Symposium on Poultry Nutrition.Veldhoven, The Netherlands, August 15-19.Lou, R., 1995. Dietary mannan-oligosaccharides as an approach for altering prevalenceof antibiotic resistance and dritribution of tetracycline resistance determinants: In:Fecal Bacteria From Swine. M.S. thesis. University of Kentucky.Mitsuoka, T., H. Hidaka, and T. Eida, 1987. Effect of fructooligosaccharides onintestinal microflora. Die Nahrung 31:5-6, 427-436.Newman, K., 1994. Mannan-oligosaccharides: Natural polymers with significant impacton the gastrointestinal microflora and the immune system. In: Biotechnology in theFeed Industry. Proceedings of Alltech's Tenth Annual Symposium. T.P. Lyons andK.A. Jacques (Eds.). Nottingham University Press, Nottingham, UK, 167-174.Ofek, I., D. Mirelman, and N. Sharon, 1977. Adherence of Escherichia coli to humanmucosal cells mediated by mannose receptors. Nature (London) 265:623-625. -------------------------------------------------------------------------------- Page 10 Oyofo, B.A., J.R. DeLoach, D.E. Corrier, J.O. Norman, R.L. Ziprin, and H.H.Mollenhauer, 1989a. Prevention of Salmonella typhimurium colonization of broilerswith D-mannose. Poultry Sci. 68:1357-1360.Oyofo, B.A., R. E. Droleskey, J.O. Norman, H.H. Hollenhauer, R.L. Ziprin, D.E. Corrier,and J.R. DeLoach, 1989b. Inhibition by mannose of in vitro colonization of chickensmall intestine by Salmonella typhimurium. Poultry Sci. 68:1351-1356.Patterson, J.A., J.I. Orban, A.L. Sutton, and G.N. Richards, 1997. Selective enrichmentof Bifidobacteria in the intestinal tract of broilers by thermally produced kestoses andeffect on broiler performance. Poultry Sci. 76:497-500.Riddell,C., X.-M. Kong, 1992. The influence of diet on necrotic enteritis in broilerchickens. Avian Dis 36: 499-503.Rosen, G. D., 2001. Multi-factorial efficacy evaluation of alternatives to antimicrobials inpronutrition. Proc. BSAS Meeting, York, UK.Savage, T.F., and E.I. Zakrzewska, 1996. The performance of male turkeys fed a starterdiet containing a mannanoligosaccharide (Bio-Mos) from day old to eight weeks ofage. Pages 47-54 in: Biotechnology in the Feed Industry: Alltechs 12thAnnualSymposium. T.P. Lyons and K.A. Jacques, eds. Nottingham University Press, UK.Savage, T.F., P.F. Cotter, and E.I. Zakrzewska. 1996. The effect of feeding a mannanoligosaccharide on immunoglobulins, plasma IgG and bile IgA of Wrolstad MW maleturkeys. Poultry Science 75 (supp. 1):Abstract S129.