Review: The use of direct fed microbials to mitigate ... use of direct fed... · Review: The use of direct fed microbials to mitigate pathogens and enhance production in cattle T

Review: The use of direct fed microbials to mitigatepathogens and enhance production in cattle

T. A. McAllister1, K. A. Beauchemin1, A. Y. Alazzeh1, J. Baah1, R. M. Teather1, andK. Stanford2

1Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta, Canada T1J 4B1; and2Alberta Agriculture and Rural Development, Lethbridge, Alberta, Canada T1J 4V6

(e-mail: [email protected]). Received 29 May 2010, accepted 19 January 2011.

McAllister, T. A., Beauchemin, K. A., Alazzeh, A. Y., Baah, J., Teather, R. M. and Stanford, K. 2011. Review: The use ofdirect fed microbials to mitigate pathogens and enhance production in cattle. Can. J. Anim. Sci. 91: xxx�xxx. Direct-fedmicrobials (DFM) have been employed in ruminant production for over 30 yr. Originally, DFM were used primarily inyoung ruminants to accelerate establishment of the intestinal microflora involved in feed digestion and to promote guthealth. Further advancements led to more sophisticated mixtures of DFM that are targeted at improving fiber digestionand preventing ruminal acidosis in mature cattle. Through these outcomes on fiber digestion/rumen health, second-generation DFM have also resulted in improvements in milk yield, growth and feed efficiency of cattle, but results havebeen inconsistent. More recently, there has been an emphasis on the development of DFM that exhibit activity in cattleagainst potentially zoonotic pathogens such as Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus.Regulatory requirements have limited the microbial species within DFM products to organisms that are generallyrecognized as safe, such as lactic acid-producing bacteria (e.g., Lactobacillus and Enterococcus spp.), fungi (e.g., Aspergillusoryzae), or yeast (e.g., Saccharomyces cerevisiae). Direct-fed microbials of rumen origin, involving lactate-utilizing species(e.g., Megasphaera elsdenii, Selenomonas ruminantium, Propionibacterium spp.) and plant cell wall-degrading isolates ofButyrivibrio fibrisolvens have also been explored, but have not been commercially used. Development of DFM that areefficacious over a wide range of ruminant production systems remains challenging because[0] comprehensive knowledge ofmicrobial ecology is lacking. Few studies have employed molecular techniques to study in detail the interaction of DFMwith native microbial communities or the ruminant host. Advancements in the metagenomics of microbial communitiesand the genomics of microbial�host interactions may enable DFM to be formulated to improve production and promotehealth, responses that are presently often achieved through the use of antimicrobials in cattle.

Key words: Direct fed microbials, acidosis, bacteriocins, cattle, Escherichia coli O157:H7, Lactobacillus, rumen

McAllister, T. A., Beauchemin, K. A., Alazzeh, A. Y., Baah, J., Teather, R. M. et Stanford, K. 2011. Survol de

l’administration directe d’agents microbiens aux bovins pour attenuer l’incidence des microorganismes pathogenes et accroıtrela production. Can. J. Anim. Sci. 91: xxx�xxx. On administre directement des agents microbiens aux ruminants depuis plusde trente ans. Au depart, on recourait surtout a cette pratique pour accelerer l’etablissement de la microflore intestinaleparticipant a la digestion et concourir a la sante du tube digestif chez les jeunes ruminants. Divers progres ont cependantdebouche sur des melanges plus complexes qui avaient pour but d’ameliorer la digestion des fibres et de prevenir l’acidosedu rumen chez les sujets adultes. Consecutivement aux ameliorations obtenues sur ces deux plans, des agents microbiens dedeuxieme generation ont concouru a rehausser le rendement laitier, la croissance et la valorisation des aliments chez lesbovins, en depit d’un manque d’uniformite au niveau des resultats. Plus recemment, on s’est interesse au developpementd’agents microbiens susceptibles de combattre les agents pathogenes a l’origine de certaines zoonoses, comme la soucheO157:H7 d’Escherichia coli, Salmonella spp. et Staphylococcus aureus. La reglementation limite toutefois les especespouvant etre administrees directement et qu’on estime generalement etre inoffensives comme les bacteries lactiques (asavoir, especes des genres Lactobacillus et Enterococcus), les cryptogames (par ex., Aspergillus oryzae) ou les levures (parex., Saccharomyces cerevisiae). On a aussi explore l’administration directe d’agents microbiens issus du rumen, comme lesespeces utilisant le lactate (par ex., Megasphaera elsdenii, Selenomonas ruminantium, Propionibacterium spp.) et les isolatsde Butyrivibrio fibrisolvens qui s’attaquent a la paroi cellulosique des cellules vegetales, mais ces produits n’ont pas etecommercialises. L’elaboration d’agents microbiens efficaces pour une vaste gamme de systemes d’elevage des ruminantscontinue de poser des difficultes faute d’une connaissance approfondie de l’ecologie des microorganismes. Peu d’etudes ontrecouru aux techniques moleculaires pour preciser l’interaction des agents microbiens avec la microflore naturelle ou avecle ruminant servant d’hote. Il se peut que les progres realises dans la metagenomique des populations d’unicellulaires etdans la genomique des interactions entre la bacterie et l’hote permettent la formulation de produits qui amelioreront laproduction et bonifieront la sante, reactions qui resultent souvent deja de l’administration d’antibiotiques aux bovins.

Mots cles: Administration directe d’agents microbiens, acidose, bacteriocines, bovins, E. coli O157:H7, Lactobacillus, rumen

Abbreviations: DFM, direct-fed microbials; GIT, gastro-intestinal tract; LAB, lactic acid-producing bacteria; VFA,volatile fatty acids

Can. J. Anim. Sci. (2011) 91: 1�19 doi:10.4141/CJAS10047 1

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There is a new sense of importance in the development ofeffective direct fed microbials (DFM) for use in livestockproduction as concerns over the use of antibiotics inlivestock production and the need for pathogen ex-clusion continue to grow. The terms ‘‘probiotics’’ and‘‘DFM’’ are often used interchangeably, but in fact theyare not truly synonymous. Probiotics are defined as ‘‘livemicroorganisms which when administered in adequateamounts, confer a health benefit on the host’’ (FAO-WHO 2001). Some probiotics, however, also con-tain enzymes and/or crude extracts in addition to livemicrobes (Yoon and Stern 1995). The Office of Reg-ulatory Affairs of the US Food and Drug Administra-tion and The Association of The American Feed ControlOfficials have defined DFM as feed products that con-tain only a source of live or naturally occurring micro-organisms (Brashears et al. 2005).

Direct fed microbials have been used in the cattleindustry for over 20 yr, primarily to improve growth per-formance, milk production or feed conversion effici-ency (LeJeune and Wetzel 2007). They are administereddirectly to the animal in the form of an encapsulatedbolus or mixed with the feed. A number of mechanismswhereby DFM may improve gut health and animalperformance have been proposed (Fig. 1), but few ofthese have been directly examined in experiments withruminants. The majority of studies that have attemptedto define possible modes of action have examined theability of DFM to favorably alter digestion in the rumen,through modulating ruminal acid production, promoting

the establishment of desirable rumen microbial popula-tions or enhancing ruminal fiber digestion.

Direct fed microbials may also alter microbial acti-vity in the lower digestive tract, but far fewer experi-ments have been conducted to specifically examine theirimpact on nutrient absorption or immune responses inthe small or large intestine. Where studies have beenconducted, they have primarily focused on the ability ofDFM to competitively exclude undesirable pathogenssuch as Escherichia coliO157:H7 from the intestinal tract(Brashears et al. 2003). Bacterial DFM may also affectinnate, humoral and cellular immune parameters as de-monstrated by increased serum concentration of IgA,IgG and IgM and intestinal concentration of IgG andIgM in poultry (Haghighi et al. 2006) and swine (Zhanget al. 2008), respectively. Similar studies have not beenconducted in ruminants, but an inflammatory responsehas been observed in steers fed a mixed DFM containingbacteria and yeast (Emmanuel et al. 2007). In poultry,DFM may also influence intestinal integrity by alteringtight junctions and increasing mucus production bygoblet cells (Chichlowski et al. 2007a), factors that couldultimately influence nutrient absorption. Finally, we arenot aware of studies that have examined the extent towhich DFM persist in the environment, a factor thatwould be an obvious benefit if fecal-oral transmissionoccurred and the DFM conferred health or performancebenefits to other individuals in a herd. Studies thatclearly define the extent to which DFM establish andpersist as well as the mechanisms whereby they alter

• Modulation of fermentation • Enhanced fiber digestion• Ecosystem enhancement• Antimicrobial effects

Rumen

• Competitive exclusion• Immune stimulation

Smallintestine

• Competitive exclusion

Largeintestine

DFM consumptionor administration

Fecaltransmission

Fig. 1. Proposed mode of action of direct-fed microbials (DFM) in ruminants. DFM may alter ruminal fermentation, nutrientabsorption, intestinal immune function and competitively exclude select microbes from the intestinal tract. Some DFM may alsoremain viable after passage through the intestinal tract and excretion in feces.

2 CANADIAN JOURNAL OF ANIMAL SCIENCE

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intestinal function are the key to developing the nextgeneration of effective products.

TYPES OF DIRECT FED MICROBIALS

Rumen MicrobesAlthough the rumen contains a large number ofbacteria, protozoa and fungi (Miron et al. 2001), onlya few members of this complex community have beenexplored for their potential as DFM (Table 1). Mostapproaches have focused on the use of rumen bacteriato either alter the profile of fermentation products ordetoxify plant secondary compounds such as mimosine(Jones et al. 2009). A few studies have explored theextent to which inoculation with ruminal fungi mayimprove fiber digestion (Lee et al. 2000; Sehgal et al.

2008), but the potential of rumen protozoa as a DFMhas not been investigated.

The majority of studies using rumen bacteria DFMhave been targeted at enhancing ruminal lactic acidmetabolism through inoculation with lactic acid-utilizing bacteria such as Megasphaera elsdenii (Klieveet al. 2003), Selenomonas ruminantium (Wiryawan andBrooker 1995) or Propionibacterium freudenreichii(Raeth-Knight et al. 2007).

Others have taken the approach of attempting toreduce the amount of lactic acid produced by introdu-cing rumen bacteria (Prevotella bryantii 25A) that utilizestarch, but produce fermentation end products otherthan lactic acid (Chiquette et al. 2008). The fibrolyticrumen bacteria Ruminococcus albus (Krause et al. 2001)and Ruminococcus flavefaciens (Chiquette et al. 2007)

Table 1. Summary of published work and proposed modes of action for direct fed microbials (DFM) used in ruminant studies since 1991. The cited

reference represents the latest work published on the DFM of interest in each livestock class

Bacteria Applicationzy Example references Mode of action

Lactic acid producersEnterococcus faecium C (1)

D (8)F (4)L (1)

Nocek et al. (2003)Fleige et al. (2007)Emmanuel et al. (2007)Abas et al. (2007)

� stimulation of lactic acid utilizers� Competitive exclusion� Direct antibacterial effect� Enhanced immune response

Lactobacillus plantarum C (3)D (4)F (4)L (1)

Aydin et al. (2009)Jatkauskas and Vrotniakien (2007)Nocek et al. (2002)Lema et al. (2001)

Lactobacillus casei D (3)L (1)

Yasuda et al. (2007)Lema et al. (2001)

Lactobacillus acidophilus C (1)D (1)F (3)L (1)

Al-Saiady (2010)West et al. (2005)Tabe et al. (2008)Lema et al. (2001)

Rumen bacteriaMegasphaera elsdenii D (1)

F (3)L (1)

Henning et al. (2010)Leeuw et al. (2009)Aikman et al. (2009)

� Increased propionate� Moderation of pH

Prevotella bryantii D (1) Chiquette et al. (2008)Selenomas ruminantium L (1) Wiryawan and Brooker (1995)

OtherPropionibacterium freudenreichii F (4) Vasconcelos et al. (2008) � Increased propionate

� Moderation of pHPropionibacterium jensenii C (2)

D (1)Adams et al. (2008)Francisco et al. (2002)

Propionibacterium acidipropionici C (1) Kim et al. (2000)Bifidobacterium spp. C (1) Krehbiel et al. (2003) � Lower tract functionBacillus spp. C (4)

D (1)F (2)

Aydin et al. (2009)Qiao et al. (2009)Arthur et al. (2010)

� Substrate utilization

Escherichia coli C (2) Schamberger et al. (2004) � Competitive exclusion

Yeast and FungiSaccharomyces cerevisiae C (4)

D (14)F (9)L (2)

Kalmus et al. (2009)Liou et al. (2009)Thrune et al. (2009)Chaucheyras-Durand et al. (2010)

� Rapid establishment of microbial consortia in newborn� Improved fiber digestion� Enhanced lactic acid utilization� Oxygen scavenging� Unidentified growth factors/nutrients� Source of hydrolytic enzymes

Aspergillus oryzae F (1) Miranda et al. (1996)

*C: calzC, calves; D, dairy cattle; F, feedlot cattle; L, lamb.yNumbers within parenthesis indicate number of studies reported since 1991.

MCALLISTER ET AL. * DIRECT FED MICROBIALS FOR CATTLE 3

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have also been used as a DFM in an effort to enhancefiber digestion, but with limited success. Logically, onemight hypothesize that DFM derived from the rumenmay more readily integrate into the microbial commu-nity as they are being introduced into the environmentfrom which they were derived. Such a scenario would beadvantageous if the DFM established and persistedwithin the microbial community, as it would eliminatethe need for daily administration. However, culturedrumen bacteria frequently fail to persist in the rumen(Flint et al. 1989; Krause et al. 2000, 2001) and haveoften been only successfully established in gnotobioticruminants (Fonty et al. 1983). It is widely recognizedthat with repeated culture, many rumen bacteria un-dergo morphological and metabolic changes relative totheir wild type counterparts (Groleau and Forsberg1981; Stewart et al. 1997). Cultured rumen bacteria arenot exposed to myriad selective pressures that are in-herent to the intestinal environment and consequentlymay lack the fitness required to integrate or competewithin rumen microbial communities.

Perhaps the greatest obstacle to the development ofrumen-derived DFM is that many of the candidatemicrobes are obligate anaerobes, limiting cell yield andcomplicating their culture in commercial fermenta-tion facilities. Media required for the growth of rumenmicrobes are often chemically complex, expensive andnot well-defined as clarified rumen fluid is still an im-portant ingredient of many formulations. Exclusion ofoxygen during packaging and storage also becomes anissue and oxygen sensitivity precludes the administra-tion of anaerobic DFM with feed. Consequently, ru-men-based DFM have not been commercialized to date.

Lactic Acid-producing BacteriaThe majority of DFM used in cattle production containat least one or more lactic acid- producing bacteria(LAB) with representative genera including Lactobacillusspp., Streptococcus spp., Pediococcus spp. and Entero-coccus spp. These microbes have been administered toalmost all classes of ruminant livestock (Table 1). Insuckling calves, LAB are most often administered as abolus or associated with a carrier paste, whereas in beefand dairy cattle they are more commonly administeredthrough the diet. Additionally, LAB are frequently inoc-ulated onto forages just prior to ensiling as a means ofenhancing the preservation, feed value and aerobicstability of silage (Kang et al. 2009; Schmidt et al.2009). In these cases viability of LAB during feed pro-cessing or ensiling is a key selection criterion for theirusefulness as a DFM.

Lactic acid-producing bacteria are desirable as DFMas they lend themselves to industrial culture, are envir-onmentally robust and have a number of mechanismswhereby they may alter or influence microbial commu-nities. Obviously, lactic acid is one key antimicrobialcompound produced by LAB that can disrupt the intra-ceullular pH of bacterial competitors (Servin 2004), but

these bacteria are also known to produce antimicro-bial peptides known as bacteriocins (see below). In thepresence of oxygen, LAB can also produce hydrogenperoxide, which has been shown to limit Salmonellaactivity in vitro (Pridmore et al. 2008), but the role thatthis antimicrobial plays in the intestinal tract, whereoxygen concentrations are limited, is unknown. Othercompounds such as benzoic acid, diacetyl, mevalono-lactone, methylhydantoin and reuterin can be producedby some, but not all strains of LAB (Brashears et al.2005). In many commercial DFM, LAB are adminis-tered in combination with other bacteria or yeast to geta multi-factorial response to the use of these products.Although such an approach makes sense from a pro-duction perspective, from an experimental perspectiveit makes it very difficult to attribute performance orpathogen exclusion responses to a single microbial com-ponent within the DFM. The possibility of interactiveeffects of mixed DFM on performance parameters canalso not be excluded.

Other BacteriaOther bacteria such as Bacillus spp. and Bifidobacteriumspp. have also been used as DFM, but primarily inpoultry (Flint and Garner 2009). The ability of Bacillusspp. to form thermotolerant and environmentally stableendospores has obvious advantages in ensuring theirsurvival during feed pelleting and prolonged storage.Although Bacillus spp. have been isolated from therumen (Oyeleke and Okusanmi 2008) they are oftenpresent in low numbers and may only play a minor rolein plant cell wall degradation. Strains of Bifidobacteriumspp. have also been isolated from the rumen, but thestrains used as DFM are not of rumen origin. In mono-gastrics, Bifidobacterium spp. colonize the intestinaltract shortly after birth (Lievin-Le Moal and Servin2006) and play a key role against enterovirulent micro-organisms involved in diarrhea (Servin 2004). In therumen, Bifidobacterium spp. most likely play a role instarch digestion (Stewart et al. 1997), with their role inthe metabolism of sugars in the lower intestinal tractbeing less pronounced than in monogastrics owing tothe fact that only low levels of soluble carbohydratesescape the rumen. We are aware of only one study whereBifidobacterium spp. were included as a DFM that wasadministered to feedlot calves, and as they were admini-stered along with three other microbial species, it isimpossible to discern if any of the responses observedwere due to their presence (Krehbiel et al. 2001).

Yeast CultureSaccharomyces cerevisiae has been used extensively as aDFM for ruminants with the most common use being indairy cattle (Table 1). Desnoyers et al. (2009) recentlycompleted a comprehensive meta-analysis on the influ-ence of S. cerevisiae on ruminal parameters and per-formance in dairy cows and concluded that this DFMincreased dry matter intake, rumen pH, rumen volatile


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fatty acids (VFA) and organic matter digestibility, aswell as decreasing rumen lactate concentration. Theseresponses were more pronounced in dairy cattle fedhigher levels of concentrate in the diet. Saccharomycescerevisiae can metabolize lactic acid, but as theyare aerobic, the extent to which they actively metabolizelactic acid within the anaerobic environment of therumen remains a matter of debate. Alternatively, addi-tion of S. cerevisiae may cause shifts in rumen bacterialpopulations, such as an increase in the numbers of fibro-lytic rumen bacteria, a scenario that has been offered asan explanation for the improvements in fiber digest-ibility that are occasionally observed with yeast supple-mentation. Some have proposed that these shifts inbacterial populations reflect the ability of yeast to utilizethe trace amounts of oxygen present in the rumen,thereby creating an environment that is more conducivefor the activity of anaerobic cellulolytic bacteria (Jouanyet al. 1999). Alternatively, others have proposed that theyeast culture itself contains micronutrients that simu-late the growth of rumen microbial populations therebyaltering rumen fermentation (Robinson and Erasmus2009).

Crude enzyme extracts, as opposed to whole cells,are the primary form in which Aspergillus oryzae andAspergillus niger have been added to the diets of rumin-ants. As these are extracts, in the strictest sense they arenot true DFM. These preparations are primarily tar-geted at increasing fiber or starch digestion in therumen, but may alter feed utilization in ruminants viaseveral mechanisms (McAllister et al. 2001). As theseextracts are often crude, it seems probable that some ofthese preparations contain viable fungal cells. However,as Aspergillus spp. are also aerobic, their impact on ru-men fermentation as a result of direct metabolism orgrowth is likely minimal.

DIRECT FED MICROBIAL MODES OF ACTION

Bacteriocins

General Nature of BacteriocinsBacteriocins are a heterogeneous group of ribosomallysynthesized antibacterial peptides and proteins and areperhaps the most characterized of the antimicro-bials produced by bacterial DFM. They are usuallycapable of inhibiting bacteria closely related to theproducing strain, which are presumably competing forthe same ecological niche, but they may also inhibit awider range of target organisms. In Gram-positivebacteria these substances consist primarily of cationic,amphipathic peptides which are divided into two maingroups: lantibiotic (class I), and non-lantibiotic (class II)bacteriocins (Klaenhammer 1993; Eijsink et al. 2002;Nes et al. 2007).

The bacteriocins produced by Gram-positive organ-isms also include large, enzymatically active peptides(class III) and complex peptide-containing molecules(class IV). Bacteriocins produced by Gram-negative

bacteria and archaea are similarly diverse (Riley andWertz 2002; Gillor et al. 2008). Bacteriocin productionhas been identified in all major bacterial groups.Environmental surveys have identified prevalence levelsof bacteriocin-producing isolates of 3 to 90% (Gordonand O’Brien 2006; Gillor et al. 2008), and it has beensuggested that most bacteria produce at least one bac-teriocin (Klaenhammer 1988). Consequently, bacterio-cins likely play a significant role in the mode of action ofmost bacterial DFM.

Classes, Modes of Action, Spectrum of ActivityThe bacteriocins produced by Gram-negative bacteriafall into two classes, colicins and microcins. The colicins,first described in E. coli (hence the name colicin) arelarge, heat labile peptides that generally kill their targetorganism by membrane permeabilization or by nucleicacid degradation (Riley and Wertz 2002). Microcins ap-pear to be similar in many respects to the class I and IIbacteriocins of Gram-positive bacteria, but kill througha variety of mechanisms (Duquesne et al. 2007). Theyare not as well characterized as the colicins. The bac-teriocins produced by Gram-positives bacteria, histori-cally most extensively studied among the lactic acidbacteria, comprise four distinct classes. The lantibiotics(class I) are small heat-stable peptides (20�35 aminoacids for the mature peptide). The original transla-ted peptide, or prebacteriocin, is generally significantlylarger and undergoes extensive post-translational mod-ification that includes cleavage of a leader peptide andmodification of amino acid residues to form dehydroa-lanine, lanthionine, and/or 3-methyllanthionine residues(Twomey et al. 2002). They generally act by formingpores in the cytoplasmic membrane of the target cell orby interfering with cell wall synthesis, and in some caseshave been shown to require interaction with specifictarget or docking molecules for optimal activity (Gilloret al. 2008).

The class II bacteriocins are also small heat-stablepeptides that, like the lantibiotics, are synthesized with aleader peptide. However, post-translational processingis largely limited to cleavage of the leader peptide, and inmost cases they do not contain modified amino acidresidues (Drider et al. 2006). They act by forming poresin the cytoplasmic membrane of the target cell (Eijsinket al. 2002; Gillor et al. 2008).

The class III bacteriocins are large proteins thatpossess bactericidal enzyme activity (Nilsen et al.2003), while class IV bacteriocins have lipid or carbohy-drate moieties that are required for activity (Vermeirenet al. 2006). Less is known about the distribution orsignificance of these latter groups.

Production of Bacteriocins by RumenMicroorganismsThe first report of bacteriocin-like activity in a rumenisolate was by Iverson and Millis (1976), who described


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antimicrobial activity by isolates of Streptococcus bovis.Since that time there have been reports of production ofantimicrobials by many rumen bacteria, and in manycases these have been confirmed as bacteriocins of classesI, II, or III. For example, a survey of 50 ruminal Butyri-vibrio isolates demonstrated a high prevalence of anti-microbial production. Twenty-six of the 50 isolatesexhibited activity against other strains of Butyrivibrio(Kalmokoff et al. 1996). These antimicrobials alsoshowed activity against strains of clostridium, eubacter-ium, lachnospira, lactobacillus, ruminococcus and st-reptococcus. Two of those antimicrobials have beenpurified and characterized. One, butyrivibriocin OR79A,produced by Butyrivibrio fibrisolvens OR79, was alantibiotic (class I) similar to lacticin 481 (Kalmokoffet al. 1999), while the second, butyrivibriocin AR10,produced by B. fibrisolvens AR10, is a circular, non-lantibiotic (class II) bacteriocin (Kalmokoff et al. 2003).Among the rumen cocci, R. albus 7 produces a heat-stable protein that inhibits R. flavefaciens FD-1 (Odenyoet al. 1994) and R. albus produces a heat-labile proteinwith inhibitory activity against R. flavefaciens (Chanand Dehority 1999). A rumen Enterococcus faecalisisolate, E. faecalis II/I, was shown to produce abacteriocin identical to the heat labile class III bacter-iocin enterolysin A, which was originally identified inthe non-rumen isolate, E. faecalis LMG 2333 (Nilsenet al. 2003). Five of 33 rumen coccus isolates, 2 strep-tococci and 3 enterococci, were shown to carry homo-logues to the gene for this bacteriocin, enlA (Nigutovaet al. 2007). Ruminococcus albus 7 also produces a classIII bacteriocin, albusin 7, which is active against alltested strains of R. flavefaciens (Chen et al. 2004). Aclass I lantibiotic, bovicin HC5, is produced by S. bovisHC5 (Mantovani et al. 2002). Recent studies indicatethat this lantibiotic may be as useful as monensin inlimiting methane production and amino acid degrada-tion in the rumen (Lee et al. 2002; Lima et al. 2009).A class II bacteriocin, bovicin 255, with activity againststrains of butyrivibrio, enterococcus, and lactobacillushas been shown to be produced by a rumen isolate ofStreptococcus gallolyticus (Whitford et al. 2001a). An-other class II bacteriocin, lichenin, is produced by therumen isolate Bacillus licheniformis 26 L-10/3RA. Liche-nin shows activity against some streptococci, rumino-cocci, eubacteria, lactobacilli and strains of Butyrivibrio(Pattnaik et al. 2001).

Among the reports of antimicrobial activity pro-duction by rumen bacteria there are also a number ofreports of probable bacteriocin production where thenature of the inhibitory agent has not been confirmed.Pseudobutyrivibrio xylanivorans Mz5 produces an un-characterized antimicrobial compound active againststrains of butyrivibrio and prevotella (Cepeljnik et al.2003). Butyrivibrio fibrisolvens JL5 has been shown toproduce an antimicrobial membrane-active peptidewith a comparatively wide spectrum of activity, activeagainst strains of butyrivibrio, prevotella, clostridium,

eubacterium, peptostreptococcus, ruminococcus, andfusobacterium (Rychlik and Russell 2002). A ruminalisolate of Lactobacillus fermentum produces an unchar-acterized antimicrobial compound active against strainsof S. bovis (Wells et al. 1997), and all four isolates of anovel rumen bacterium, Lachnobacterium bovis, pro-duced an uncharacterized temperature-sensitive antimi-crobial compound (Whitford et al. 2001b). Given thewidespread presence of bacteriocins, it seems certainthat some of the changes in microbial ecology observedwith DFM of rumen origin arise from the presence ofthese antimicrobials. The consequences of resistanceby rumen microorganisms to bacteriocins producedby DFM, and the effects of bacteriocins produced byrumen bacteria on the viability of DFM, are also likelyto be significant.

Resistance to BacteriocinsResistance to bacteriocins can arise from many differentsources. In bacteriocin producers, resistance to the pro-duced bacteriocin is normally a function of a gene orgenes that are expressed at the same time as the bac-teriocin. More generalized resistance, such as the usualresistance of Gram-negative bacteria to bacteriocinsproduced by Gram-positive bacteria, may be due tobarriers that limit access to the cytoplasmic membrane.These could include factors such as the Gram-negativeouter membrane or a positively-charged surface layer, orthe lack of a specific target or docking molecule on thecell surface. Proteases may also play a role. Resistancemay also be acquired by susceptible organisms throughthe same general mechanisms as employed for otherantibiotics: exclusion, degradation, or target modifica-tion (Eijsink et al. 2002). The extent to which develop-ment of resistance to bacteriocins influences the efficacyof DFM is unknown, but given that many DFM are ad-ministered to ruminants throughout the feeding periodthe possibility of resistance development should be givenconsideration.

Studies on the Role of Bacteriocins inDFM ResponseStudies on the effect of bacteriocins introduced into thegastrointestinal environment have been limited. Onlytwo specific bacteriocins have been examined, and bothof these were effective in the rumen environment.In the first example, enterocin CCM 4231, producedby the rumen isolate Enterococcus faecium CCM4231(Laukova and Marekova 1993) was shown to inhibitthe growth of enterococci, staphylococci, listeria, andE. coli in the rumen environment (Laukova andCzikkova 1998). In vitro studies of the class I lantibioticbovicin HC5, produced by S. bovis HC5 (Mantovaniet al. 2002) revealed that it may be as useful as monensinin limiting methane production and amino acid degra-dation in the rumen (Lee et al. 2002; Lima et al. 2009).Most studies of bacteriocin effects on rumen function


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have been less well defined. Cell-free supernatant fromthe bacteriocin-producer L. plantarum 80 inhibits me-thanogenesis (Nollet et al. 1998), but the mechanism ofthis effect has not been determined. Nisin- and pediocin-producing lactic acid bacteria have been shown toreduce intestinal colonization by vancomycin-resistantenterococci (Millette et al. 2008), but direct effects bybacteriocins have not been demonstrated. It has oftenbeen shown that colicinogenic strains of E. coli areinhibitory to pathogenic strains (Jordi et al. 2001). Theprobiotic E. coli strain Nissle 1917 produces microcinsH47 and M (Patzer et al. 2003), and has been shown toreduce neonatal diarrhea in calves (von Buenau et al.2005), possibly by interfering with the invasion of epi-thelial cells by Salmonella enterica var. typhimurium.However, this response appears to arise via a secretedcomponent that is independent of microcin production(Altenhoefer et al. 2004). Again a specific role of theantimicrobial compounds in this protective effect hasnot been demonstrated.

DFM AND BIOFILMSAs in most aquatic environments, the overwhelmingmajority of microbes present in the intestinal tract ofruminants reside in complex communities known asbiofilms. Microbial biofilms form on the surface of feedparticles and play an integral role in feed digestion withsubstrate exchange occurring among community mem-bers (McAllister et al. 1994). Similarly, biofilms alsoform on the surface of intestinal tissues influencingnutrient transport and intestinal health (Cheng andMcAllister 1997). As these microbes function as a com-munity in a manner that is not unlike that of a multi-cellular organism (Nikolaev and Plakunov 2007), themechanisms whereby foreign DFM may integrate intoor alter community structure is not entirely clear.

The widespread occurrence of bacteriocin productionacross the microbial kingdom, and the high incidence ofbacteriocin production and resistance in natural micro-bial communities, suggest that these compounds playan important role in determining the competitive fitnessof microbial strains. However, the presence of non-producing, sensitive strains within the same commu-nities raises the question of what that role might be, andhow important these compounds are in determining theability of a DFM to integrate with intestinal microbialcommunities in ruminants. The cost of bacteriocinproduction to the cell can be significant. For example,in Lantibiotic plantaricin NC8, 21 genes are involved inthe regulation, synthesis, post-translational processing,immunity, and export of the bacteriocin (Navarro et al.2008). Thus, the fate of a bacteriocin-producing strain ina complex environment is a balance between the produc-tion of antimicrobials to confer a competitive advantagevs. decreased reproduction due to the metabolic cost ofbacteriocin synthesis. Expression of resistance genesalone also has a cost, though it is less than the cost ofbacteriocin production.

Theoretical models indicate that the competition at asimplistic level resembles a paper/rock/scissors gamewhere producers beat sensitive cells, resistants beat pro-ducers, and sensitives beat resistants under conditionswhere expression of resistance genes offers no competi-tive advantage (Czaran et al. 2002; Kerr et al. 2002;Riley and Wertz 2002). Biofilms, where micro-coloniesinteract at short range, tend to include bacteria thatrepresent all three types. Production of the Gram-positive class I and II bacteriocins is generally regulatedby a quorum-sensing mechanism, where high local celldensities, as occurs in biofilms, triggers bacteriocin pro-duction (Gobbetti et al. 2007; Nes et al. 2007). Quorumsensing, a proposed method of communication betweenbacterial cells by the release of small diffusible sig-nal molecules, has been observed in rumen bacteria(Mitsumori et al. 2003) and likely plays an importantrole in the development and establishment of ruminalbiofilms. At this point it is not known if introducedDFM and resident intestinal bacteria in ruminantsspeak the same quorum sensing language, although se-veral quorum sensing mechanisms have been shown tobe shared among widely differing microbial species(Dickschat 2010).

Natural biofilm populations with a high degree of di-versity are predicted to tend toward a ‘‘hyper-immunity’’state whereby many strains produce no toxins; manyothers produce only one, and only a few produce many,with all strains being resistant to most or many of thetoxins. For example, in the case of the 50 butyrivibriostrains examined by Kalmokoff et al. (1996), manystrains carrying the structural gene bviA did not expressit (Kalmokoff et al. 1999), even though the gene waswidely distributed in all the butyrivibrio groups exam-ined (unpublished observations). The presence of homo-logous genes in a variety of genera suggests that thesegenes may be widely distributed by horizontal transfer(Ochman et al. 2000). Horizontal transfer of bacteriocingenes may allow organisms to become effective com-petitors in a previously unexplored niche (Lawrence1999) and selection of DFM based on their ability toacquire genes coding for bacteriocins may be an ap-proach to increasing their competitiveness in intestinalenvironments.

Establishment of DFM in intestinal communitiesappears more straightforward when it is accomplishedthrough the production of an antimicrobial that speci-fically promotes competitive exclusion. Under theseconditions DFM must simply expulse or prevent theestablishment of the target microorganism thereby pre-venting biofilm formation. Specific integration of theDFM into biofilms would be more complex as it pre-sumably would require the DFM to contribute somelimiting or absent metabolic function to the microbialcommunity. Biofilm communities are highly structuredand the microbial consortia they contain function as ateam to accomplish degradation of complex substratessuch as starch and cellulose (McAllister et al. 1994).


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Occasionally, DFM may possess unique metabolic acti-vities that promote their establishment within the intes-tinal tract, such as the ability of Synergistes jonesii todegrade mimosine in the rumen (Jones et al. 2009).Specific integration of DFM into biofilms needed formore complex functions such as the degradation ofplant cells seems less likely, especially as most commer-cial DFM lack genes coding for the enzymes involved inthe hydrolysis of plant cell walls. In these instances theprovision of undefined nutrients or the use of secondaryfermentation products may be a more plausible expla-nation for their mode of action.

IMMUNOMODULATIONBacterial DFM may impact the host immune system viaa number of mechanisms including up-regulation of cell-mediated immunity, increased antibody production andepithelial barrier integrity, reduction of epithelial cellapoptosis, enhanced dendritic cell�T cell interactions,heightened T cell association with lymph nodes andgreater Toll-like receptor signalling (Lee et al. 2010).Stimulation of epithelial innate immunity is central tothis response and may suppress intestinal inflammationby increased production of epithelial-derived TNF-aand restoration of epithelial barrier function (Pagniniet al. 2010). The impact of DFM on cytokine and che-mokine production as well as T and B cell responsesappear to depend on several factors including microbialcomposition of the DFM, the dosage and the dura-tion of administration. For example, a mixed DFMconsisting of L. casei, L. acidophilus, Bifidobacteriumthermophilum and E. faecium increased expression ofinflammatory cytokine IL-6, but decreased expressionof the anti-inflammatory cytokine IL-10 in chicks(Chichlowski et al. 2007b). In other studies administra-tion of a LAB-based DFM repressed IFN-g and IL-12levels in the intestinal tract of chickens and reducedintestinal colonization by Salmonella enterica (Haghighiet al. 2008). In contrast a Bacillus subtilis DFM hadno impact on the expression of IFN-g, IL-3, IL-4 inchickens (Fujiwara et al. 2009). Using microarrays,Brisbin et al. (2008) found that expression of IFN-g,IFN-a, STAT2, STAT4, IL-18, MyD88 were all up-regulated in the cecal tonsil cells of chickens providedwith a L. acidophilus DFM. Although numerous studieshave measured changes in the expression of genes cod-ing for various immune factors, very few have examinedthe signalling cascades that result in these responsesbeing conferred from DFM to host.

The majority of studies that have attempted to defineimmunomodulation responses to DFM have been con-ducted within cell culture or with poultry or murinemodels (Hormannsperger and Haller 2010; Lee et al.2010). Most of these studies have also examined immuneresponses to DFM after challenge with specific intestinalpathogens with little work being conducted to define theimpact of DFM on intestinal immunity in healthy hosts.Studying immunomodulation responses in ruminants is

difficult due to their extended life cycle and the expenseassociated with getting sufficient animal numbers todraw meaningful conclusions. Direct fed microbial-mediated immune responses are likely more importantin younger ruminants where intestinal populations areless established and the intestinal tract is potentiallymore susceptible to colonization by opportunistic pa-thogens. In mature dairy cows, intramammary infusionof Lactococcus lactis DPC 3147 was shown to increaseIL-1b and IL-8 gene expression (Beecher et al. 2009), butthe ability of this potential therapy to control mastitishas not been confirmed. Only one study reported that acombination of E. faecium and S. cerevisiae increasedthe concentration of the acute phase proteins, serumamyloid A, lipopolysaccharide binding protein, andhaptoglobin in plasma (Emmanuel et al. 2007). How-ever, a linkage between alterations in intestinal functionas a result of the DFM and changes in plasma concen-trations of these acute phase proteins was not established.

Modulation of Ruminal Fermentation

Yeast culture (S. cerevisiae)Active dry yeast (S. cerevisiae) and yeast cultures thatcontain yeast plus culture medium are increasingly usedin ruminant feeding to improve animal performance(Desnoyers et al. 2009; Robinson and Erasmus 2009).Commercial products vary widely in the strains ofS. cerevisiae they contain and the number of viableyeast cells present. Desnoyers et al. (2009) summarizedthe findings from 110 research papers representing abroad range of yeast products and feeding conditionsand reported a mean increase in milk production of0.8 kg d�1 due to supplemental yeast. In a more selectivereview of research conducted using three commercialyeast products, Robinson and Erasmus (2009) reporteda milk production response of 0.9 kg d�1 to addedyeast. In both reviews, the beneficial effects of yeastwere greatest in low fiber diets, consistent with observedimprovements in fiber digestion and ruminal fermenta-tion of cattle fed yeast. The effects of added yeast cultureon rumen fermentation are complex [as reviewed byChaucheyras-Durand et al. (2008)], and not well under-stood. Live yeast has been shown to stimulate thegrowth and activities of some ruminal fiber-degradingmicroorganisms, although this work has mostly beendone in vitro (Martin and Nisbet 1992; Chaucheyras-Durand et al. 2008). Various mechanisms for theincrease in fiber degradation due to yeast supplementa-tion have been proposed. Yeast may scavenge oxygenwithin the rumen, which might stimulate the growth ofcellulolytic bacteria given that most ruminal microor-ganisms are highly sensitive to the presence of oxygen(Newbold et al. 1996). In support of this oxygen-scavenging theory, the redox potential of ruminal fluidin cows is lower in the presence of live yeasts (Mardenet al. 2008), indicating that yeast can strengthen thereducing power of ruminal fluid. It is also possible that


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yeast provide rumen bacteria with growth factors,including organic acids, B vitamins, and amino acids(Callaway and Martin 1997). In addition, yeast productsmay stimulate the growth of fibrolytic bacteria bypreventing sub-acute ruminal acidosis (Chaucheyras-Durand et al. 2008).

Sub-acute ruminal acidosis is characterized by re-peated bouts of low ruminal pH, with the pH recoveringafter each bout (Krause and Oetzel 2006). Long bouts(�4 h) of low pH (B6.0) negatively affect fiber digestion(Russell and Wilson 1996) and decrease the absorptivecapacity of the ruminal epithelium (Krehbiel et al. 1995).These bouts of low pH occur when VFA production israpid and exceeds the capacity of the rumen to maintainequilibrium. With time, the VFA are absorbed, bufferedor passed from the rumen, causing the pH to rise. As aconsequence, ruminal pH follows a cyclical pattern thatincludes periods defined as sub-acute ruminal acidosis.During sub-acute ruminal acidosis, lactic acid concen-trations remain low (B5 mM) (Nagaraja and Titgemeyer2007). In a limited number of cases, sub-acute ruminalacidosis develops into acute acidosis, during whichruminal pH drops drastically (B5.0) and fails to recoverover time (Owens et al. 1998). Acute acidosis is usuallycaused by elevated concentrations of lactic acid in therumen (�50 mM) as a result of an abrupt increase in theintake of rapidly fermentable carbohydrates (Nagarajaand Titgemeyer 2007). Callaway and Martin (1997)reported that yeast culture stimulated the growth of twolactate-utilizing bacteria, S. ruminantium and M. elsde-nii, in vitro. If yeast help prevent lactic acid from accu-mulating in the rumen, it follows that providing yeastto cows could theoretically help prevent sub-acuteacidosis from developing into acute acidosis. However,sub-acute acidosis is seldom related solely to lacticacid accumulation (Krause and Oetzel 2006); thus, thismode of action is unlikely to account for the acidosis-prevention response that is occasionally observed withyeast supplementation.

The effects of yeast on stabilizing ruminal pH maydepend on the diet or the strain of yeast. In fact, manystudies report no effect of yeast and yeast culture onrumen pH (Wiedmeier et al. 1987; Erasmus et al. 2005;Longuski et al. 2009), and in some studies, rumen pHwas actually lowered by feeding yeast culture to dairycows (Harrison et al. 1988). Despite many studies thatindicate no effect of yeast on rumen pH, there arestudies showing that yeast products elevate ruminal pH.For example, Williams et al. (1991) fed yeast culture tothree steers and nadir pH following the meal was higherwhen yeast was fed. However, caution must be usedwhen interpreting these results, because the yeast andcontrol diets were fed at different times, confounding thetreatment comparison with time. Additional evidencefor the pH stabilizing effects of yeast is given in severalrecent papers, although few cows were used in thesestudies. Marden et al. (2008) fed three lactating dairycows either no yeast or live yeast (5�1010 CFU d�1) in

a Latin square design. Feeding yeast helped elevatenadir pH following the morning meal (from 5.55 to 5.95)and the response was equal to that of providing 150g d�1 of sodium bicarbonate. It is not known whetherthe response to yeast would have occurred had sodiumbicarbonate been included in the basal diet. In anotherstudy, Bach et al. (2007) fed dairy cows either no yeastor live yeast (1�1010 CFU d�1) in a crossover design.Average rumen pH was higher when yeast was supple-mented than when no yeast was provided (6.05 vs. 5.46).The effects of yeast on rumen pH appear to depend onthe strain of yeast and the dose rate. For example, in anunpublished study in our laboratory, dairy cows werefed two different strains of yeast (1�1010 CFU d�1).Strain 1 had no effect on pH variables, whereas Strain 2substantially lowered mean pH compared with thecontrol (5.98 vs. 6.27) and increased the hours that pHwas below 5.8 (7.5 vs 2.9 h d�1). Strain 2 had beenselected based on its ability to increase the rate of fiberdigestion in vitro, which may have increased the rate ofproduction and subsequent accumulation of VFA in therumen.

There is compelling evidence to indicate that yeastproducts improve production efficiency of dairy cows.The improvement is likely due to a number of interact-ing factors. However, of primary importance is theirbeneficial effects on fiber digestibility, especially in cowsfed low fiber diets where lactic acid metabolism by DFMmay offset the low rumen pH that can depress theactivity of cellulolytic bacteria and protozoa. There isalso some limited evidence to suggest that some yeastproducts may help stabilize rumen pH, but that isnot always the case. Whether yeast products reducethe risk of acidosis in dairy cows probably depends onthe strain of yeast, the dose rate, and the presence andnature of other acidotic risk factors.

Bacterial DFM and Ruminal FermentationThere is evidence that bacterial DFM can modify therumen environment in a manner that may enhanceanimal productivity (Krehbiel et al. 2003; Beaucheminet al. 2006). In terms of their impact on ruminal fermen-tation, the main bacterial species used in DFM productscan be considered in two categories: lactic acid produ-cers and lactic acid utilizers (or propionate producers).Commercial DFM products often combine variousorganisms; thus, their modes of action are usually inter-twined (Fig. 2). The rationale for feeding lactic acidproducing bacteria, such as E. faecium and Lactobacillusspp., to ruminants is based on the contention that thesebacteria produce lactic acid in the rumen, whichpromotes the growth of lactic acid utilizing bacteria.However, small, transient increases in ruminal lacticacid concentration are almost impossible to measure invivo. Consequently, an increased concentration ofpropionate in rumen fluid has been used to signifyincreased numbers of lactic acid-producing bacteria asmost lactic acid utilizers convert lactate directly to


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propionate or indirectly through succinate to propio-nate. As such, Beauchemin et al. (2003) fed E. faeciumEF212 (EF; 6�109 CFU d�1) to feedlot finishing cattleand measured an increased concentration of propionatein rumen fluid, as well as a numerical increase in thenumbers of lactic acid-utilizing bacteria. Proliferation ofthe lactic acid-utilizing bacteria would be beneficial inthe event of any sudden increase in lactic acid concen-tration in the rumen. Numerous ruminal bacterialspecies ferment lactic acid, with M. elsdenii and S.ruminantium spp. lactilytica being predominant cultur-able lactate-fermenting organisms in grain-fed animals(Nagaraja and Titgemeyer 2007). In most animals, theinherent ruminal population of lactic acid-utilizingbacteria ensures that any lactic acid produced is rapidlymetabolized, and it therefore does not accumulate. As aconsequence of this, lactic acid concentration in therumen of most ruminants is low (B5 mM; Owens et al.1998). Lactic acid utilizers can be slow to adapt to rapidchanges in diet composition; thus, abrupt changes in dietcomposition, amount of feed consumed or feed deliverycan lead to a sudden increase in lactic acid concentra-tion. Lactic acid is a very potent acid (pKa 3.9 vs. 4.9 forVFA); therefore, accumulation of lactic acid causesruminal pH to decline rapidly (Nagaraja and Titgemeyer2007), causing subacute and even acute acidosis (Krauseand Oetzel 2006). Low ruminal pH also activates lactatedehydrogenase, the enzyme involved in convertingpyruvate to lactate, a response that exacerbates theaccumulation of lactic acid in the rumen (Owens et al.1998). Furthermore, feeding a large amount of starchcan also increase ruminal concentrations of free glucose,which increases the competitiveness of lactate-producing

bacteria such as S. bovis in the rumen (Owens et al.1998). Some bacterial DFM products are composed ofboth lactic acid producers and lactic acid utilizers in aneffort to ensure that any lactic acid produced isimmediately metabolized. These combination productshave been explored mainly for use in feedlot cattle fedhigh grain diets [as reviewed by Krehbiel et al. (2003)],with some limited work in dairy cows (Raeth-Knightet al. 2007).

Other bacterial DFM products contain only lacticacid utilizers, with Propionibacterium being the predo-minant organism (Francisco et al. 2002; Stein et al. 2006;Weiss et al. 2008). Propionibacterium are natural inhabi-tants of the rumen that utilize lactic acid and producepropionate. Ruminal populations of Propionibacterium(mainly P. acidipropionici) typically range from 103

to 104 CFU mL�1 (Davidson and Rehberger 1995).The rationale for feeding Propionibacterium is to furtherensure concentrations of lactic acid in the rumen re-main low, while increasing production of propionate(Lehloenya et al. 2008), the major precursor for gluco-neogenesis in ruminants (Huntington 1990). Feedingsupplemental Propionibacterium can help improve theenergy status of ruminants, in particular, lactating dairycows (Stein et al. 2006; Aleman et al. 2007). Useof Propionibacterium alone has increased propionateconcentrations in the rumen in some studies (Stein et al.2006; Lehloenya et al. 2008), but not all (Ghorbani et al.2002). There is little published data on the effects ofPropionibacterium on rumen pH. Stein et al. (2006)reported that dairy cows fed a high dose of Propioni-bacterium P169 (6�1011 CFU d�1) had lower pH thancows on a low dose (6�1010 CFU d�1) or the control

Protection

VFA

Integration into microbial biofilms

Substratetransfer

e– transfer

A.DFM

B.DFM

Fig. 2. Interaction of direct-fed microbials (DFM) with microbial biofilms in the intestinal tract of ruminants. (A) DFM integratedirectly into the biofilm through the production of antimicrobials such as bacteriocins and organic acids or (B) DFM may eitherutilize substrates associated with the fluid environment. Occasionally, DFM may integrate into biofilms by possible possessing aunique metabolic capability that is not present in other community members. Concept adapted from McAllister et al. (1994).


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with values of 6.65, 6.94, and 6.86, respectively. Rum-inal fluid samples were taken via intubation in thatstudy, and therefore are considerably higher than ex-pected had they been measured using indwelling pHprobes. Other studies have reported no effects of feedingPropionibacterium on rumen pH in steers (Ghorbaniet al. 2002, strain P15 at 1�109 CFU d�1; Lehloenyaet al. 2008, strain P169 at 6�1011 CFU d�1).

There has also been interest in using M. elsdenii as aDFM for cattle because it metabolizes lactic acid (Kungand Hession 1995; Klieve et al. 2003). In an early study,steers were inoculated with M. elsdenii strain YE34, andthen rapidly adapted to a grain-based diet. The YE34rapidly established in the rumen after dosing with 106

cell equivalents mL�1 of rumen fluid being measuredimmediately upon providing the DFM, with this num-ber increasing 100-fold 4 d after inoculation. When thecontrol cattle were switched to the grain diet, wild typesof M. elsdenii eventually also became predominantmembers of the microbial population, but required anadditional week to become established at a level similarto the DFM. It is thought that rapid establishment ofM. elsdenii would be protective against acidosis forruminants during the periods of diet transition. To testthis hypothesis, dairy cows were fed very high grain diets(60 and 70%, dry matter basis) with M. elsdenii dosedon days 2, 10, and 20 post-partum (Hagg 2007). Con-trary to expectations, the DFM had no effect on pHor lactic acid concentrations in the rumen. Another ru-men bacterium, P. bryantii (strain 25A), selected for itsability to grow rapidly on starch and produce propio-nate rather than lactate (Rodriguez 2003), has recentlybeen evaluated in dairy cows (Chiquette et al. 2008).When this strain was administered (2�1011 cells d�1) todairy cows through a rumen cannula from �3 to 7 wkpostpartum, there was no change in milk production,but milk fat content (3.9 vs. 3.5%) and total VFAtended to be higher in inoculated cows than in controlcows. However, rumen pH or concentrations of propio-nate and lactate were not affected by treatment.

Pathogen ExclusionAs livestock may intermittently become reservoirs ofpathogenic bacteria (Callaway et al. 2008a), DFM havealso been developed specifically to limit the shedding ofpotential food-borne pathogens. In cattle, work withDFM on pathogen exclusion has focused primarily onreducing the shedding of E. coli O157 (Elam et al. 2003;Zhao et al. 2003; Callaway et al. 2004; LeJeune andWetzel 2007), the bacterium responsible for hemorrha-gic colitis and hemolytic uremic syndrome in humans.The most extensively studied DFM for cattle is L.acidophilus strain NP51, which has been found to reduceshedding of E. coli O157:H7 by cattle by 48 to 80%when fed at 109 CFU (Brashears et al. 2003; Younts-Dahl et al. 2004, 2005; Stephens et al. 2007a, b).Recently, Tabe et al. (2008) found that an alternativestrain of L. acidophilus (BT1386) reduced fecal shedding

of E. coli O157 in feedlot steers, but had no impact onthe shedding of Salmonella sp. Reduced fecal shedd-ing of Salmonella spp. in cattle was documented byStephens et al. (2007b) in steers fed a combination of 109

CFU L. acidophilus NP51 and 109 CFU P. freudenreichiiNP24. However, unlike the work of Younts-Dahl et al.(2005) these researchers did not detect a dose-dependentreduction in the shedding of E. coli O157 with increasinglevels of L. acidophilus NP51 in the diet.

Although the ability of DFM to reduce the prevalenceof pathogens in livestock has been reported, few studieshave specifically addressed the possible mechanisms re-sponsible for this phenomenon (Fig. 3). Pathogens thathave become firmly established in the gastro-intestinaltract (GIT) through the formation of mature bio-films may pose a greater challenge for DFM-mediatedremoval than those that are transiently associated withdigesta. Cells within biofilms are known to alter theirgene expression and may become thousands of timesmore resistant to antimicrobial agents (Mah and O’Toole2001; Ito et al. 2009). Under these conditions pathogenicbacteria may become insensitive to antimicrobials (e.g.,bacteriocins, organic acids) produced by DFM. Al-though recently it has been shown that a released exo-polysaccharide (r-EPS) produced by L. acidophilus A4inhibited biofilm formation by E. coli O157:H7 (Kimet al. 2009). In the newborn, complex microbial biofilmshave yet to establish and under these conditions DFMmay be more likely to cause favorable alterations in themicrobial ecology of the digestive tract. Studies in pig-lets showed that a lactobacilli-based DFM promotedcolonization of a beneficial microbiota and reduced in-testinal colonization by Clostridium perfringens (Siggerset al. 2008). Consequently, DFM may be more effica-cious at eliminating pathogens from the intestinal tractof young as compared with mature ruminants.

Competitive exclusion is one of the predominantmechanisms whereby DFM may eliminate pathogensfrom the intestinal tract (LeJeune and Wetzel 2007;Callaway et al. 2008b). Sherman et al. (2005) demon-strated that adhesion of L. acidophilus strain R0052 andL. rhamnosus strain R0011 to intestinal epithelium cells(T84) reduced subsequent colonization by both E. coliO157:H7 and E. coli O127:H6. Similarly, Chen et al.(2007) determined that surface-layer proteins pro-duced by L. crispatus ZJ001 inhibited the adhesion ofS. typhimurium and E. coli O157:H7 to HeLa cells. Morerecently, Johnson-Henry et al. (2008) confirmed thatpre-treatment of T84 cells with L. rhamnosus limitedmorphological changes in cells by reducing the forma-tion of attaching and effacing lesions after exposure toE. coli O157:H7. These researchers proposed that thisobservation arises from the ability of L. rhamnosus toprevent redistribution of tight-junction proteins at theepithelial surface. Lactobacillus plantarum has also beenshown to stabilize tight junction proteins in intestinalepithelial cells exposed to enteropathogenic E. coli (Qinet al. 2009).


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Direct binding to the intestinal epithelium is only oneof many mechanisms whereby DFM may exclude patho-gens. In some instances DFM may alter gene expressionin targeted pathogens, a phenomenon that was recentlydemonstrated when a cell-free medium from L. acid-ophilus strain La-5 was shown to inhibit the coloniza-tion of specific-pathogen-free mice by E. coli O157:H7 (Medellin-Pena and Griffiths 2009). As binding ofE. coli O157:H7 to the GIT is also mediated by thehormones epinephrine and norepinephrine secreted bythe host (Sperandio et al. 2003), other mechanisms forcompetitive exclusion perhaps involving communicationbetween the DFM and the host also likely exist, butrequire further characterization.

Other key mechanisms resulting in competitive exclu-sion of pathogenic organisms by DFM arise from thecompetition for limiting nutrients or production ofcompounds that are toxic to the pathogens (Callawayet al. 2004). Metabolism of gluconic acid, a constituentof intestinal mucus, which stimulates in vitro growth ofE. coli O157 (Chang et al. 2004) was identified by Foxet al. (2009) as a mechanism for competitive exclusionof E. coli O157 by non-pathogenic E. coli. Inhibitorymetabolites including bacteriocins and organic acidswere proposed, but not directly evaluated by Lee et al.(2008) as a mechanism for in vitro competitive exclusionof E. coli O157:H7 by L. paracasei ATCC 25598 andL. rhamnosus GG. The presence of bacteriocins wasverified by Schamberger and Diez-Gonzalez (2004)

when these researchers isolated seven colicins from non-pathogenic strains of E. coli.

Although competition for nutrients and production ofbacteriocins has been verified as mechanisms for com-petitive exclusion of pathogenic organisms by DFM,control of pathogenic organisms by production of or-ganic acids appears less likely. Our laboratory has re-cently shown that some acid-adapted strains of E. coliO157:H7 can remain viable for extended periods of time,even at a pH of 2.5 (Yang et al. 2010). Jacobsen et al.(2009) recently confirmed this acid tolerance when theydetermined that the acidic threshold for growth was pH4.3 using a selection of E. coli O157:H7 strains chosenfor diversity from a library of 2600 isolates. Conse-quently, animal performance would likely suffer due toacidosis (Nagaraja 2007) long before acid-mediatedcompetitive exclusion of this pathogenic organism occurs.Within the bovine GIT, pH at the ileum commonlyranges between 7.4 and 7.9 (Christiansen and Webb1990), falling to 6.0 in the large intestine and feces fromhind gut fermentation of starch in cattle receiving high-concentrate diets (Berg et al. 2004), a pH range thatis conducive to both the growth and establishment ofE. coli O157:H7.

The heightened immune responses as a result of DFMdescribed above may also aide the host in eliminatingpathogenic organisms from the intestinal tract. Oetzelet al. (2007) demonstrated that a DFM containingE. faecium and S. cerevisiae reduced requirements for

Fig. 3. Mechanisms whereby direct fed microbials (DFM) may exclude microbial pathogens from the intestinal tract of ruminants.(A) Competition for nutrients that limit microbial growth (e.g., Fe�2); (B) Direct antagonism through the production ofantimicrobials (e.g., bacteriocins, organic acids); (C) Competitive exclusion through occupation of specific binding sites; (D)Stimulation of the immune response resulting in host-exclusion of the pathogen; (E) enhanced gut health through restoration ofepithelial integrity. Adapted from O’Toole and Cooney (2008).


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antibiotic treatment for second lactation dairy cows,although the mechanism(s) for this response were notidentified. Davis et al. (2007) were able to provide someinsight into the relationship between probiotics and hostimmunity by demonstrating that a DFM containingL. brevis increased the number of mucin-producing gob-let cells and altered numbers of antigen-presenting cellsand T cells within the jejunal villi of pigs. More recently,Szabo et al. (2009) demonstrated that a DFM contain-ing E. faecium NCIMB 10415 enhanced production ofspecific antibodies (serum IgM and IgA) against Sal-monella serovar Typhmurium DT104 in weanling piglets.Similarly, Lessard et al. (2009) found an increase inIgA and enhanced resistance to exterotoxigenic E. coliinfection in piglets after use of a DFM containing Pedio-coccus acidilactici and S. cerevisiae boulardii. Themechanisms by which DFM control pathogenic organ-isms are in some cases exceedingly complex and due tothe plethora of microorganisms used as constituents ofDFM, much additional study will be required to es-tablish a basic framework for improved and uniformefficacy. However, as many mechanisms by which DFMcontrol pathogenic organisms have been verified rela-tively recently, accelerated expansion of our knowledgein this area is likely. For cattle, new DFM, which arecapable of controlling a variety of pathogenic organ-isms, will be perhaps most suitable for use in youngcalves.

CONCLUSIONDirect fed microbials have the potential to reduce thecurrent reliance on antimicrobials as a tool to pro-mote health and optimize productivity in cattle. How-ever, for DFM to be adopted, positive productionresponses as a result of their administration must bepredictable and consistent. Advances in molecular bio-logy are just now providing the enabling technologiesthat allow microbial�host interactions to be examined ata level that was previously impossible using traditionalmicrobial culture or histological techniques. Metage-nomics and transcriptomics should provide new insightinto how DFM alter microbial ecology within the GItract and gene expression in the host. As these relation-ships between DFM and increased production andimproved health become defined, selection of DFMfor properties such as the exclusion of specific pathogensor optimization of host immune function will becomemore feasible. Only through a growing emphasis onmechanistic research will the true extent to which DFMimprove the microbial community as well as the rumin-ant host come to light. Learning the way DFM work isthe key to the development of more effective DFM.

ACKNOWLEDGEMENTSThe researchers thank K. Jakober and K. Munns fortheir editorial assistance and S. Torgunrud for excellentgraphical support.

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