Microbial ecology and pathogens

In many respects the microbial ecology within the gut can be compared with the ecology of the macrobiological world; the same selective pressures that emphasize survival fitness occur in all environments, including microbial ones. The intestinal tract is very diverse and dense, with over 2000 known species and a population of >10¹⁰ cells g⁻¹ digesta (Hungate, Reference Hungate1966; Drasar and Barrow, Reference Drasar, Barrow, Drasar and Barrow1985). Because of the extremely high intestinal population densities the competition for nutrients and environmental niches is more intense than that found in the macrobiological jungle (Steer et al., Reference Steer, Carpenter, Tuohy and Gibson2000). The scale of the intestinal environment means that dietary changes and other stressors on the animal cause environmental shifts to occur more rapidly in the microbial world, necessitating an enhanced adaptability to opportunities and challenges by the bacterial inhabitants.

The synergistic relationship between the host animal and its gastrointestinal microbial ecosystem is critical to the health and well being of the animal and to efficient production (Jayne-Williams and Fuller, Reference Jayne-Williams, Fuller, Bell and Freeman1971). Recent studies have demonstrated that certain members of the microbial population of the gut (in humans, at least) can impact obesity and can cause conditions such as autism (Ley et al., Reference Ley, Turnbaugh, Klein and Gordon2006; DiBaise et al., Reference DiBaise, Zhang, Crowell, Krajmalnik-Brown, Decker and Rittmann2008; Finegold, Reference Finegold2008). The benefits of the microbial population in the gut of animals are due largely to the fermentation of dietary substrates to produce volatile fatty acids (VFAs) and B vitamins that are absorbed by the host animal (Branner and Roth-Maier, Reference Branner and Roth-Maier2006), but the native intestinal microbial population also stimulates the immune system (Koenen et al., Reference Koenen, Kramer, Van Der Hulst, Heres, Jeurissen and Boersma2004; Schierack et al., Reference Schierack, Wieler, Taras, Herwig, Tachu, Hlinak, Schmidt and Scharek2007; Walsh et al., Reference Walsh, Gardiner, Hart, Lawlor, Daly, Lynch, Richert, Radcliffe, Giblin, Hill, Fitzgerald, Stanton and Ross2008) which can reduce colonization by pathogens and subsequent illnesses.

Competitive enhancement techniques that have been developed include: (1) introduction of a normal microbial population to the gastrointestinal tract (CE); (2) addition of a microbial supplement called a probiotic or a direct-fed-microbial (DFM), that improves gastrointestinal health and the diversity of the intestinal microbial ecology (Collins and Gibson, Reference Collins and Gibson1999); and (3) providing a specific limiting nutrient (a ‘prebiotic’) that allows an existing commensal microbial species or population to expand its current niche or to occupy a new niche in the gastrointestinal tract. No matter which of these strategies is employed, the overall goal is to fill all ecological niches within the gut, thereby preventing colonization by a pathogen or displacing an established pathogenic bacterial population in the gut. Competitive enhancement products have had somewhat limited applications commercially as pathogen-reduction strategies, in part due to the availability of cheap antibiotics which can counteract the effectiveness of competitive enhancement strategies (Steer et al., Reference Steer, Carpenter, Tuohy and Gibson2000). Many probiotics have been used simply as methods to improve gain or enhance milk production (Stavric and Kornegay, Reference Stavric, Kornegay, Wallace and Chesson1995; Bomba et al., Reference Bomba, Nemcová, Mudronová and Guba2002), with little or no understanding of their modes of action or reasons for inconsistencies in experimental trials (Wiemann, Reference Wiemann2003). Given increasing fears over the dissemination of antimicrobial resistance, it is expected that prophylactic antibiotic usage in food animals will become more closely regulated and expensive, causing probiotic/competitive enhancement strategies to become more economically feasible and widely used in disease prevention.

Probiotic studies in food animals have been characterized by inconsistency, primarily due to a lack of understanding of the microbial ecology of the gastrointestinal tract, and conditions that promote the growth of pathogens and the probiotic organisms utilized. Some probiotic species chosen for use in animals were isolated from other sources and were thus not ecologically suited for life in the anaerobic gut. Additionally, variations between studies can be attributed to antagonistic interactions between some probiotic species, as well as quality control issues. Some of this variation can also be attributed to the fact that mature animals contain a stable, relatively individualized intestinal microbial population that the probiotic must come to equilibrium with, but when probiotics are applied to neonates (or newly hatched chicks) with a sterile (or nearly so) intestinal tract, results tend to be more consistent. All of these factors have produced results that are in many cases unreplicatable. However, the advent of molecular methodologies has allowed more precise definitions of each probiotic and has allowed researchers to follow the specific changes caused by individual probiotic cultures and a greater understanding of the ‘normal’ gut flora and degree of individualization of the intestinal microbial ecosystem which can lead to the development of highly tailored probiotic products for use in specific animals or production environments.

CE

The bacterial species best adapted to occupy a particular niche within the intestinal tract will become the most successful and will eventually dominate the niche. An established, mature, gastrointestinal microbial population fills all available environmental niches making an animal more resistant to colonization by opportunistic bacteria, especially pathogens (Fuller, Reference Fuller1989). This natural antipathogen activity has been described as ‘bacterial antagonism’, ‘bacterial interference’, or ‘CE’ (Nurmi and Rantala, Reference Nurmi and Rantala1973; Lloyd et al., Reference Lloyd, Cumming and Kent1974).

CE technology involves the addition of a non-pathogenic bacterial culture of a single strain or multiple strains, to the intestinal tract of food animals in order to reduce colonization or decrease populations of pathogenic bacteria in the gastrointestinal tract (Fuller, Reference Fuller1989; Nurmi et al., Reference Nurmi, Nuotio and Schncitz1992; Nisbet et al., Reference Nisbet, Corrier, Scanlan, Hollister, Beier and DeLoach1993b; Steer et al., Reference Steer, Carpenter, Tuohy and Gibson2000). Typically, CE cultures are isolated from the animal species that will be treated in order to take advantage of a synergistic interaction between the host animal and its native microbial ecosystem that developed during millions of years of co-evolution. The introduction of a stable, mixed microbial consortium to the naïve gut of a neonate can aid in the early establishment of a normal microbial population that can competitively prevent the establishment of a pathogenic bacterial population (Nurmi et al., Reference Nurmi, Nuotio and Schncitz1992; Crittenden, Reference Crittenden and Tannock1999; Steer et al., Reference Steer, Carpenter, Tuohy and Gibson2000). This is especially critical in the poultry industry as chicks can be quickly colonized by pathogens after hatching (Cox et al., Reference Cox, Bailey, Mauldin and Blankenship1990). However, when CE is used in older animals, it must often compete against an established pathogen population that must be displaced. Therefore, the mixture of bacteria chosen for use as a CE culture must be specific for the animal, production stage, and scenario in which it will be utilized.

CE has been suggested to have several modes of action that eliminate pathogenic bacteria, including: (1) direct and indirect competition for nutrients, (2) competition for physical attachment sites, (3) production of antimicrobial compounds (including VFAs), (4) enhancement of host immune system activity, and (5) a synergistic interaction of two or more of the above activities. If bacteria (including pathogens) cannot grow at least as fast as the passage rate of their environment, then the constant flow of digesta will ‘wash out’ the pathogen. After a CE culture (or the natural flora) is established within the gut, bacteria bind to the surface of the intestinal epithelium (Lloyd et al., Reference Lloyd, Cumming and Kent1974) preventing opportunistic pathogens from attaching and thus obtaining a colonization foothold (Collins and Gibson, Reference Collins and Gibson1999). VFAs produced by normal microbial fermentation in the gut are toxic to some pathogenic bacteria, and may reduce the competitive fitness of these bacteria in the gut environment (Wolin, Reference Wolin1969; Barnes et al., Reference Barnes, Impey and Stevens1979; Prohaszka and Baron, Reference Prohaszka and Baron1983). Additionally, some bacteria produce antimicrobial protein compounds, such as traditional antibiotics and bacteriocins or colicins that can inhibit or eliminate species competing within the same niche (Jack et al., Reference Jack, Tagg and Ray1995; Stahl et al., Reference Stahl, Callaway, Lincoln, Lonergan and Genovese2004; Al-Qumber and Tagg, Reference Al-Qumber and Tagg2006).

In food animals, most CE research has focused on poultry (Nava et al., Reference Nava, Bielke, Callaway and Castaneda2005). This can be attributed to the need to control Salmonella colonization and production diseases in poultry, as well as the ease of application to hatched chicks with naïve gastrointestinal tracts. This has prompted CE cultures to be used widely (Stavric, Reference Stavric1992; Stavric and D'Aoust, Reference Stavric and D'Aoust1993; Bielke et al., Reference Bielke, Elwood, Donoghue, Donoghue, Newberry, Neighbor and Hargis2003) in many countries (Weinack et al., Reference Weinack, Snoeyenbos, Smyser and Soerjadi1982; Fuller, Reference Fuller1989; Nurmi et al., Reference Nurmi, Nuotio and Schncitz1992). In the US, a mixed, defined commercial CE product, comprised of several defined species of bacteria (Preempt®, MS BioScience, Dundee, IL) was developed and used to reduce Salmonella colonization of chicks (Nisbet et al., Reference Nisbet, Corrier and DeLoach1993a, Reference Nisbet, Corrier, Ricke, Hume, Byrd and DeLoach1996). Future trade regulations are expected to increase the use of CE as a green method to reduce Salmonella on eggs and in chicks shipped into the EU.

In swine, the addition of an Enterococcus faecium culture reduced intestinal colonization by enterotoxigenic Escherichia coli (ETEC) and reduced diarrhea and mortality in experimentally infected gnotobiotic pigs (Underdahl et al., Reference Underdahl, Torres-Medina and Doster1982). Ushe and Nagy (Reference Ushe and Nagy1985) tested a different strain of E. faecium in ETEC-infected cesarean-derived colostrum-deprived newborn pigs and observed a reduction in colonization of the ileum and in weight loss associated with the administration of E. faecium. In vitro co-cultures of the E. faecium and ETEC resulted in a lowered pH and reduced growth of the ETEC. Other researchers have found that a CE culture derived from the mucosa of healthy pigs reduced Salmonella populations in the gut of young pigs (Fedorka-Cray et al., Reference Fedorka-Cray, Bailey, Stern, Cox, Ladely and Musgrove1999). A swine CE culture derived from the cecal contents of healthy pigs was reported to reduce the incidence of Salmonella choleraesuis (Anderson et al., Reference Anderson, Stanker, Young, Buckley, Genovese, Harvey, Deloach, Keith and Nisbet1999; Genovese et al., Reference Genovese, Anderson, Harvey, Callaway, Poole, Edrington, Fedorka-Cray and Nisbet2003). This CE culture also reduced post-weaning diarrhea, morbidity and mortality caused by ETEC, an economically important infection for the swine industry (Genovese et al., Reference Genovese, Anderson, Harvey, Callaway, Poole, Edrington, Fedorka-Cray and Nisbet2003; Harvey et al., Reference Harvey, Ebert, Schmitt, Andrews, Genovese, Anderson, Scott, Callaway and Nisbet2003, Reference Harvey, Anderson, Genovese, Calloway and Nisbet2005). Similarly, spores of a non-toxigenic strain of Clostridium difficile given orally to piglets reduced colonization, diarrhea and growth depression from a toxigenic C. difficile (Songer et al., Reference Songer, Jones, Anderson, Barbara, Post and Trinh2007).

Because many pathogenic bacteria are inhibited or killed by high concentrations of VFAs, it was long thought that pathogenic E. coli would have limited opportunities to colonize cattle intestinal tracts (Hollowell and Wolin, Reference Hollowell and Wolin1965; Wolin, Reference Wolin1969). However, the carriage of pathogenic E. coli O157:H7 primarily in the lower intestine of cattle has negated this hypothesis. Because of the complex microbial population of the intestinal tract, researchers have sought to utilize this environmental resource by developing CE as a strategy to eliminate E. coli O157:H7 and Salmonella from cattle (Brashears et al., Reference Brashears, Jaroni and Trimbl2003b; Moxley et al., Reference Moxley, Smith, Klopfenstein, Erickson, Folmer, Macken, Hinkley, Potter and Finlay2003; Zhao et al., Reference Zhao, Tkalcic, Doyle, Harmon, Brown and Zhao2003). Researchers isolated and defined multiple non-O157:H7 E. coli strains from cattle, and found this generic E. coli CE culture could displace an established E. coli O157:H7 population from the gastrointestinal tract of calves (Zhao et al., Reference Zhao, Doyle, Harmon, Brown, Mueller and Parks1998). While this is the only true CE culture for cattle that has been shown to reduce E. coli O157:H7, there are other probiotic cultures that target this pathogen.

Probiotics

Probiotics are a general category of dietary products that can be included in animal rations to enhance performance and/or reduce pathogenic bacteria (Fuller, Reference Fuller1989; Collins and Gibson, Reference Collins and Gibson1999). A probiotic was initially defined as a ‘live microbial feed supplement which beneficially affects the host animal by improving intestinal microbial balance’ (Fuller, Reference Fuller1989). In recent years, the technical definition of probiotics has been broadened to include products containing microbes or their end products (i.e. fermented dairy products, etc.) and the effect is not necessarily restricted to a change in colonization per se. A proposed definition for probiotics is ‘a preparation or a product containing viable, defined micro-organisms in sufficient numbers, which alter the micro-flora (by implantation or colonization) in a compartment of the host and by that exert beneficial health effects in this host’ (Schrezenmeir and De Vrese, Reference Schrezenmeir and de Vrese2001). Indeed, some of the probiotic products used have been shown to affect immune parameters and increase CD8 production, as well as IgG and IgM concentrations in the serum and gut of swine (Duncker et al., Reference Duncker, Lorentz, Schroeder, Breves and Bischoff2006, Walsh et al., Reference Walsh, Gardiner, Hart, Lawlor, Daly, Lynch, Richert, Radcliffe, Giblin, Hill, Fitzgerald, Stanton and Ross2008, Zhang et al., Reference Zhang, Azevedo, Gonzalez, Saif, Van Nguyen, Wen, Yousef and Yuan2008).

Probiotic preparations for use in animals are typically comprised of individual species or mixtures of lactic acid bacteria (LAB), yeasts, or their end products and are not species-specific, or even necessarily originally isolated from animals (Wiemann, Reference Wiemann2003). Probiotics often fall into the categories of: (1) live cultures of yeast or bacteria, (2) heat-treated (or otherwise inactivated) cultures of yeast or bacteria, or (3) fermentation end products from incubation of yeast or bacteria. Many probiotics are currently marketed in North America for use in both humans and animals; many of these are sold as DFM. Regulations in this field have allowed a wide variety of claims to be made about the improvements in growth efficiency and other potential benefits, and the consistency of results in the field has not always been demonstrated (LeJeune et al., Reference LeJeune, Kauffman, Amstutz and Ward2006; Barroga et al., Reference Barroga, Salas, Martin, Besa and Santos2007). However, the most commonly used probiotic bacterial strains remain Bifidobacteria and Lactobacillus, and many studies demonstrate that these types of products can enhance production efficiency (Gomes and Malcata, Reference Gomes and Malcata1999; Barroga et al., Reference Barroga, Salas, Martin, Besa and Santos2007; Midilli et al., Reference Midilli, Alp, Kocabagli, Muglali, Turan, Yilmaz and Cakir2008).

Some probiotics have been reported to reduce food borne pathogens and bacteria that affect growth and production in food animals (Ohya et al., Reference Ohya, Marubashi and Ito2000; Tkalcic et al., Reference Tkalcic, Zhao, Harmon, Doyle, Brown and Zhao2003; Stephens et al., Reference Stephens, Loneragan, Karunasena and Brashears2007). In order to prevent post-weaning E. coli diarrhea in pigs (Bertschinger, Reference Bertschinger, Straw, D'Allaire, Mengeling and Taylor1999), early weaning procedures and antibiotics are often used (Alexander et al., Reference Alexander, Thornton, Boon, Lysons and Gush1980; Fedorka-Cray and Harris, Reference Fedorka-Cray and Harris1995, Fedorka-Cray et al., Reference Fedorka-Cray, Harris and Whipp1997); yet this disease is still a significant problem for the swine industry. Therefore, researchers have investigated several probiotic/DFM approaches to reducing this important production problem. Bomba et al. (Reference Bomba, Nemcova, Gancarcikova, Herich, Kastel, Paul and Francis1999) found that a Lactobacillus casei culture had no impact on adherence of an O8:K88 ETEC to the jejunum of experimentally infected conventional and gnotobiotic pigs. However, a mixture of the L. casei culture and maltodextrins resulted in a reduction of approximately 1 log₁₀ in adherence in the gnotobiotic pigs and of about 2.5 log units in the conventional pigs. The significance of these data is difficult to determine, as the concentrations of ETEC in the intestine were much lower than those usually associated with natural or experimental ETEC infections. Kyriakis et al. (Reference Kyriakis, Tsiloyiannis, Vlemmas, Sarris, Tsinas, Alexopoulos and Jansegers2001) reported that spores of Bacillus licheniformis and Bacillus toyoi were each effective in reducing diarrhea, mortality and weight loss in a field study involving 256 weaned pigs.

Supplementation of the diet of neonatal pigs with a Lactobacillus plantarum DFM resulted in an increase in total gut populations of lactobacilli in weaned pigs (Takahashi et al., Reference Takahashi, Egawa, Simojo, Tsukahara and Ushida2007). Another L. plantarum DFM product containing maltodextrin and/or fructooligosaccharides (FOS) reduced counts of E. coli O8:K88 in the jejunum and colon of piglets, and was associated with increased acetate concentrations in the ileum and colon (Nemcova et al., Reference Nemcova, Bomba, Gancarcikova, Reiffova, Guba, Koscova, Jonecova, Scirankova and Bugarsky2007). Daily oral administration of E. coli strain Nissle 1917 for 10 days was reported to abolish hypersecretion by the intestine associated with experimental infection of weaned pigs with an O149:K88 strain of ETEC (Schroeder et al., Reference Schroeder, Duncker, Barth, Bauerfeind, Gruber, Deppenmeier and Breves2006). Daily dosing of pigs with an E. faecium culture from birth to weaning was reported to result in a reduction in frequency of diarrhea and in improved weight gain in weaned pigs; the cause of the diarrhea was not determined (Zeyner and Boldt, Reference Zeyner and Boldt2006). Another E. faecium DFM was found to reduce populations of Enterococcus faecalis in the colon of weaned pigs which are responsible for the onset of some cases of post-weaning diarrhea (Vahjen et al., Reference Vahjen, Taras and Simon2007). A recent publication reported that inclusion of a Bacillus subtilis DFM in the feed resulted in a reduction in scours 24 h after challenge of weaned pigs with a K88-positive ETEC (Bhandari et al., Reference Bhandari, Xu, Nyachoti, Giesting and Krause2008). Probiotics comprised of Bifidobacter lactis and Lactobacillus rhamnosus individually reduced adherence of Salmonella, E. coli and Clostridum spp. to the intestinal mucosa in swine; together the two organisms were more effective and reduced each other's adherence (Collado et al., Reference Collado, Grzeskowiak and Salminen2007). Reduced mucosal adhesion by pathogens is presumed to lead to reduced severity of clinical disease.

The cattle industry has used various types of probiotics for many years (Dawson et al., Reference Dawson, Newman and Boling1990; Gomez-Alarcon et al., Reference Gomez-Alarcon, Huber, Higginbotham, Wiersma, Ammon and Taylor1991). These probiotics have been primarily utilized to increase growth rate, milk production, or production efficiency (Lehloenya et al., Reference Lehloenya, Stein, Allen, Selk, Jones, Aleman, Rehberger, Mertz and Spicer2008); however, recent years have seen the development of probiotic preparations to address other cattle health concerns. Initially, researchers compared several commercial probiotics and found that feeding probiotics provided neither benefit nor detriment in regard to food-borne pathogen levels in cattle (Keen and Elder, Reference Keen and Elder2000). Another study demonstrated that a probiotic did reduce fecal shedding of E. coli O157:H7 in sheep (Lema et al., Reference Lema, Williams and Rao2001). Since then there has been an explosion in probiotic research specifically aimed at reducing E. coli O157:H7 in cattle. A Lactobacillus acidophilus culture (a DFM) reduced E. coli O157:H7 shedding by more than 50% in finishing cattle (Brashears et al., Reference Brashears, Galyean, Loneragan, Mann and Killinger-Mann2003a, Reference Brashears, Jaroni and Trimblb). In an independent evaluation, this DFM reduced fecal shedding of E. coli O157:H7 in cattle from 46% of animals to 13% (Ransom et al., Reference Ransom, Belk, Sofos, Scanga, Rossman, Smith and Tatum2003). Further studies found that the feeding of this DFM reduced the isolation of E. coli O157:H7 from the hides of cattle by up to 75%, and the highest dose reduced Salmonella shedding in the feces by 50% (Younts-Dahl et al., Reference Younts-Dahl, Galyean, Loneragan, Elam and Brashears2004; Stephens et al., Reference Stephens, Loneragan, Karunasena and Brashears2007). This product is currently in use in many feedlots across the US and Canada because the increase in growth efficiency economically balances the cost of its inclusion in cattle rations, thus making a food safety enhancement pay for itself. In a study involving 138 feedlot steers, another DFM also reduced fecal shedding of E. coli O157:H7; shedding of Salmonella was not reduced but new Salmonella infections were reduced (Tabe et al., Reference Tabe, Oloya, Doetkott, Bauer, Gibbs and Khaitsa2008).

Further uses as disease preventatives in cattle have shown that the addition of bovine vaginal LAB (primarily) as a probiotic preparation inhibited the growth of metritis-causing organisms in dairy cattle (Otero et al., Reference Otero, Morelli and Nader-Macias2006). Other research on probiotic preparations found that the bacteria living on the surface of a healthy udder could inhibit the in vitro growth of mastitis-causing organisms, including Arcanobacteriun pyogenes (Al-Qumber and Tagg, Reference Al-Qumber and Tagg2006). Although these are not market-ready products to date, they emphasize the fact that probiotic approaches have a wide application in preventing animal diseases.

Prebiotics

Prebiotics are organic compounds that are unavailable to, or indigestible by the host animal, but are available to a specific proportion of the microbial population and are often described as ‘functional foods’ or ‘nutraceuticals’ (Schrezenmeir and de Vrese, Reference Schrezenmeir and de Vrese2001). Prebiotics have been used as dietary supplements in humans to promote intestinal health and well-being (Crittenden, Reference Crittenden and Tannock1999). Some carbohydrates (and other organic compounds) are not enzymatically degraded in the stomach or intestine and can be passed down to the cecum and colon to become ‘colonic food’ (Houdijk et al., Reference Houdijk, Bosch, Verstegen and Berenpas1998, Kontula, Reference Kontula1999). Prebiotics provide limiting nutrients to the intestinal mucosa, as well as substrates for intestinal bacteria to ferment, yielding enhanced B vitamin production (Collins and Gibson, Reference Collins and Gibson1999; Crittenden, Reference Crittenden and Tannock1999; Branner and Roth-Maier, Reference Branner and Roth-Maier2006). Some prebiotics provide a competitive advantage to specific members of the native microflora (e.g. Bifidobacteria and Butyrivibrio) (Willard et al., Reference Willard, Simpson, Cohen and Clancy2000) that can act as a natural CE culture against pathogens. Coupling the use of CE with prebiotics is a technique known as ‘synbiotics’; and this can induce a synergistic reduction of pathogens and disease (Collins and Gibson, Reference Collins and Gibson1999; Schrezenmeir and de Vrese, Reference Schrezenmeir and de Vrese2001). Recent research has indicated that the use of prebiotics, such as inulin and oligofructans, can modulate activity of the immune system directly (Seifert and Watz, Reference Seifert and Watz2007). Further studies found that the use of prebiotics can increase the resistance of animals to infection and the incidence of atopic dermatitis (Meyer, Reference Meyer2008). A role for prebiotics has also been described in reducing inflammatory bowel disease and colitis in humans (Leenen and Dieleman, Reference Leenen and Dieleman2007; Winkler et al., Reference Winkler, Butler and Symonds2007).

While much of the research into prebiotics has focused on the use in humans, prebiotics have been used in the animal feed industry to improve the health and well-being of poultry, swine, horses and dogs; however, prebiotics remain relatively expensive for use in commercial animals (Mosenthin and Bauer, Reference Mosenthin and Bauer2000; Willard et al., Reference Willard, Simpson, Cohen and Clancy2000; Torres-Rodriguez et al., Reference Torres-Rodriguez, Higgins, Vicente, Wolfenden, Gaona-Ramirez, Barton, Tellez, Donoghue and Hargis2007; Respondek et al., Reference Respondek, Goachet and Julliand2008). The use of maltodextrins and FOS in combination with L. plantarum has been shown to reduce adherence of E. coli O8:K88 to the jejunum and colon of weaned pigs (Nemcova et al., Reference Nemcova, Bomba, Gancarcikova, Reiffova, Guba, Koscova, Jonecova, Scirankova and Bugarsky2007). Galactooligosaccharides have also been demonstrated to have anti-adhesive activity, reducing the adherence of a human EPEC to the human cell lines HEp-2 and Caco-2 in a dose-dependent manner (Shoaf et al., Reference Shoaf, Mulvey, Armstrong and Hutkins2006). While the use of prebiotics in cattle has been limited due to the expense as well as the density and diversity of the ruminal microbial population and its ability to degrade most prebiotics, enhancements in rumen-protective technologies may allow these compounds to be used in feedlot and dairy cattle.