Feeding beneficial bacteria: A natural solution for increasing efficiency and decreasing pathogens in animal agriculture1

©2009 Poultry Science Association, Inc.

Feeding beneficial bacteria: A natural solution for increasing efficiency and decreasing pathogens in animal agriculture1 J. F. Flint and M. R. Garner2 MicroBios inc., ithaca, nY 14850

SUMMARY Consumer demand for natural and organic foods has risen steadily. Correspondingly, the demand for technologies to enhance animal performance through natural and organic solutions continues to increase. One such technology receiving considerable attention is the feedstuff application of live, beneficial bacteria, commonly called probiotics, but properly referred to as direct-fed microbials (DFM). The use of DFM in animal agriculture has grown continually over the last decade, yet the commercial success of DFM in poultry production to date has been limited. Overviewed here are the history, commercial applications, and research trials of DFM technology in animal agriculture as a means to gain some foresight into the future commercial use of DFM in poultry. We discuss successful probiotic trials in animal agriculture and emphasize those in poultry production that demonstrate 1) improved BW gain, 2) decreased morbidity, 3) decreased incidence of bird and human pathogens, and 4) increased economic profitability. Last, we present current and future obstacles and basic microbiological concepts that are essential for functional probiotic technologies. Key words: probiotic, direct fed microbial, organic, natural, poultry, antibiotic alternative 2009 J. Appl. Poult. Res. 18:367–378 doi:10.3382/japr.2008-00133

INTRODUCTION With increased worldwide demand for food products, intense pressure is being placed on livestock-rearing operations to enhance animal productivity while maintaining high levels of product quality and safety. Numerous advances in animal productivity have been made through the use of nutritional supplements and the incorporation of antimicrobial compounds into livestock feed. Although undeniably effective 1

in increasing animal productivity and reducing food costs, the future application of antibiotics to animal feed may be reduced because of academic and public scrutiny. In fact, the European Union has passed legislation that prohibits the subtherapeutic application of antibiotics in animal agriculture [1]. The food supply within the United States is considered the safest in the world, yet millions of food-borne illnesses occur every year from microbially contaminated food [2]. Whereas

Papers from the Current and Future Prospects for Natural and Organic Poultry Symposium were presented at the Poultry Science Association’s 97th Annual Meeting in Niagara Falls, Ontario, Canada. 2 Corresponding author: [email protected]

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Primary Audience: Researchers, Nutritionists, Poultry Producers, Veterinarians, Feed Manufacturers

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Antibiotic Use in Animal Feed As the demand of consumers for organic and naturally produced livestock increases, antibiotic use in animal feed will decrease and will be limited to therapeutic treatment of diseases, or will be eliminated outright in evolving animal production systems. To date, antibiotic use has become an essential tool to increase animal productivity in concentrated animal-feeding and animal-rearing operations. Remarkable increases in improved profitability are seen with the adoption of antibiotic application at subtherapeutic levels (i.e., used for animal growth promotion rather than specifically to treat disease symptoms). It is estimated that 11.2 million kg of antibiotics are used annually as growth promoters in livestock [6]. Consequently, it is hypothesized that widespread use of antimicrobial agents has led to increases in antimicrobial-resistant pathogens. As an example, in 2001, 19% (75/384) of

human Campylobacter isolates were resistant to ciprofloxacin, whereas from 1989 to 1990, no ciprofloxacin-resistant strains were identified [6]. The emergence of ciprofloxacin-resistant Campylobacter infections within the United States coincided with the US Food and Drug Administration approval of 2 fluoroquinolones for use in poultry production in 1995 and 1996. Concern over the emergence of antibioticresistant pathogens from animals fed antibiotics has resulted in legislative initiatives in Europe to eliminate their prophylactic use in animal feed [1]. Since their prohibition, the occurrence of antibiotic-resistant bacteria harbored in animal systems has reduced dramatically [1, 7]. The percentage of vancomycin-resistant Enterococcus isolated from broiler feces declined from 75% in 1995 to 5% in 2001 [7]. However, antibiotic withdrawal has not been without negative consequences. Swine morbidity and mortality have increased in Denmark, and overall animal productivity has decreased, according to Casewell et al. [1]. These authors also noted that similar reductions in performance have been observed in poultry-rearing operations in Denmark and France. Whereas the inclusion of antibiotics in livestock feed is aimed at eliminating or reducing specific or general bacterial populations in a preventive manner, the addition of beneficial bacteria to feedstuffs may be a viable substitute to increase the profitability of animal agriculture. Function of Beneficial Bacterial in Animals Bacteria are essential to a functional gastrointestinal tract and immune system in animals. Vertebrate gastrointestinal systems harbor an abundant assemblage of microorganisms that serve many functional roles for the animal, including degradation of ingesta, pathogen exclusion, production of short-chain fatty acids (SCFA), compound detoxification, vitamin supplementation, and immunodevelopment [8–10]. It is estimated that more than 400 bacterial species make up the intestinal community in humans [11]. Molecular analyses indicate that some 240 species are recognized residents of the chicken intestinal tract [12]. Neonate gastrointestinal tracts are sterile at birth, but microorganisms located in or near the vagina and anus of the mother rapidly establish residency in the neonate after birth [7]. Successive populations

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postslaughter measures such as antimicrobial dips and preservatives are effective, novel research in preslaughter prevention technologies is actively being sought [3]. One method receiving considerable recent attention as a natural alternative to enhancing animal productivity and improving product safety is the feeding of viable microorganisms [4, 5]. The practice of applying beneficial bacteria to animal feedstuffs is commonly referred to as probiotics; however, it is more correctly known as direct-fed microbials (DFM). The advantages of DFM consumption in humans have been recognized for centuries; however, their application and efficacy in livestock operations have only recently been pursued. As consumers begin to look for minimally processed, organic, and “naturally” raised products, alternative technologies are required to maintain livestock productivity. Additional questions have begun to emerge concerning these future endeavors. Can the feeding of beneficial bacteria improve livestock performance? Can the incorporation of microorganisms into animal feed positively influence animal rearing in a profitable manner? In this review, we survey current perspectives of the use and benefits associated with microbial feed additives for livestock applications and discuss the future potential and pitfalls of DFM application.

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DFM It has long been observed that the consumption of certain fermented foods and certain bacteria can elicit beneficial effects in the host.

Yogurt consumption has continually increased during the last decade because of claims of its health-promoting benefits [21, 22]. Additionally, the ensilage processes of forage materials, in which microorganisms convert free carbohydrates and protein components to acid, preserves the feed from microbial spoilage and provides a unique nutritive profile [23, 24]. As certain industries try to distance themselves from subtherapeutic antibiotic applications, new technologies have emerged as possible solutions to enhance the productivity of animal rearing. One such technology increasing in popularity in animal agriculture is DFM. For example, the last 15 yr have seen a steady >15% per annum increase in the application of DFM to feedstuffs for cattle [25]. The beneficial microorganisms fed are commonly referred to as probiotics. Several definitions that describe probiotics have been used, but one commonly accepted by the World Health Organization is that “live microorganisms, when administered in adequate amounts, confer a beneficial effect upon the host” [26]. Some definitions, however, consider feed ingredients other than bacteria, such as biologically derived extracts, including dead yeasts, essential oils, enzymes, and even seaweed, to be probiotics. Because of this lack of distinction, the US Food and Drug Administration and the Association of American Feed Control Officials mandated the term “direct-fed microbials” for the use of live microorganisms provided as a feed ingredient. Currently approved microorganisms for animal feed application can be found in Table 1. Modes of Action The mechanisms by which DFM elicit their beneficial effects on the host can vary widely. Some effects result from direct interactions with the host epithelia and immune system, whereas others mediate their effects via modulation of resident intestinal flora and prevention of pathogen establishment. For the purposes of this review, we classify the modes of action of DFM into 3 main categories: chemical inhibition, competitive exclusion, and microbially mediated immunodevelopment. Chemical Inhibition. The production of antagonistic chemicals such as organic acids, small inhibitory chemicals (reuterin), hydrogen per-

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establish themselves over time and eventually result in a climax community. Mature intestinal microbial populations are generally stable [13]; however, dietary changes or the administration of antibiotics can alter the composition of microbial populations and can lead to the proliferation of pathogenic organisms [14, 15]. Intestinal microflora have a profound impact on the digestion and metabolism of feedstuffs. Microbial metabolism in the intestine results in the production of fermentation products, including SCFA, which are also known as volatile fatty acids, predominantly formed as acetate, propionate, and butyrate [16, 17]. Microbially derived SCFA are absorbed or transported by the host epithelia, and then utilized for cellular energy sources and aid in colonocyte formation and differentiation [16, 18]. Some animals, particularly ruminants, receive a considerable portion of their daily energy requirements from microbially derived SCFA [10]. Ruminants rely on microbial fermentation of feedstuffs in a specialized voluminous chamber of the stomach called the rumen. The rumen, accounting for up to 20% of the total body mass, is dedicated to microbial degradation and fermentation of consumed feed and SCFA absorption, and contains microbial concentrations greater than 1010 microorganisms per gram of ruminal contents [19]. Intestinal microflora also play a crucial role in gastrointestinal function and metabolism in nonruminants. In one study, conventionally raised mice (with indigenous microbiota) contained 42% more body fat compared with mice raised under germ-free conditions [20]. However, colonization of germ-free mice with cecal microflora from conventionally raised mice resulted in a dramatic 57% increase in total body fat within 2 wk, irrespective of diet intake. Intestinal colonization of germ-free mice resulted in enhanced carbohydrate uptake, intestinal SCFA production, and altered host expression of fasting-induced adipose factor, a lipoprotein lipase suppressor that is responsible for triglyceridederived fatty acid uptake and storage by adipocytes [20].

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370 Table 1. Currently approved microorganisms for livestock feed application1

Kluyveromyces marxianus Lactobacillus acidophilus Lactobacillus brevis Lactobacillus buchneri (cattle only) Lactobacillus bulgaricus Lactobacillus casei Lactobacillus cellobiosus Lactobacillus curvatus Lactobacillus delbrueckii Lactobacillus farciminis (swine only) Lactobacillus fermentum Lactobacillus helveticus Lactobacillus lactis Lactobacillus plantarum Lactobacillus reuterii Leuconostoc mesenteroides Pediococcus acidilacticii Pediococcus cerevisiae (damnosus) Pediococcus pentosaceus Propionibacterium freudenreichii Propionibacterium acidipropionici (cattle only) Propionibacterium shermanii Saccharomyces cerevisiae

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Adapted with permission from [94]. An asterisk (*) indicates formerly cataloged as Streptococcus.

oxide, and antimicrobial peptides (bacteriocins) by intestinal organisms have been implicated in the control of intestinal populations [27]. These chemicals can kill pathogens directly or can generate localized “microenvironments” unfavorable for pathogen establishment. Short-chain fatty acids, the principal luminal anions of the intestine, are end products of microbial fermentation. Short-chain fatty acids are known to possess pH-dependent antimicrobial properties, of which the antimicrobial potential varies between the SCFA because of different proton association constants [e.g., lactate is a stronger acid than acetate because of its lower pKa (−log10 of the acid dissociation constant)]. At neutral pH, dissociated SCFA predominate. As pH declines, nondissociated forms become prevalent and are able to freely pass the cellular membrane. On encountering the elevated intracellular pH, protons dissociate from the SCFA, leading to an accumulation of charged anions and protons. The accretion of these ions leads to the disruption of the proton motive force and a diminution of intracellular pH, thus preventing adenosine triphosphate generation and eventually resulting in cell death. Other small inhibitory metabolites produced by some lactic acid bacteria (LAB)

include hydrogen peroxide, benzoic acid, diacetyl, mevalonolactone, and reuterin (β-hydroxypropionaldehyde) [4, 28]. Reuterin is broad-spectrum antimicrobial synthesized by Lactobacillus reuterii as a by-product of glycerol metabolism and is believed to prohibit growth as an inhibitor of ribonucleotide reductase [29, 30]. Hydrogen peroxide can be generated by some LAB through a reaction of molecular oxygen with flavoproteins. Although hydrogen peroxide production by lactobacilli is considered important in vaginal ecosystems, its contribution to limiting pathogenic populations within intestinal systems is unknown [31]. Another form of chemical inhibition is through compounds called bacteriocins. Bacteriocins are small ribosomally synthesized peptides that have a narrow spectrum of antimicrobial activity. The inhibitory action of most bacteriocins produced by gram-positive bacteria is mediated by their insertion and pore formation within the cell membrane, resulting in the dissipation of the proton motive force [32]. In vivo experiments have demonstrated the protective potential of several bacteriocin-producing strains to reduce or eliminate pathogenic populations in mice [33, 34]. More information on this topic is reviewed elsewhere.

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Aspergillus niger Aspergillus oryzae Bacillus coagulans Bacillus lentus Bacillus licheniformis Bacillus pumilus Bacillus subtilis Bacteroides amylophilus Bacteroides capillosus Bacteroides ruminicola Bacteroides suis Bifidobacterium adolescentis Bifidobacterium animalis Bifidobacterium bifidum Bifidobacterium infantis Bifidobacterium longum Bifidobacterium thermophilum Enterococcus cremoris* Enterococcus diacetylactis* Enterococcus faecium* Enterococcus intermedius* Enterococcus lactis* Enterococcus thermophilus*

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tures significantly reduced chick colonization by 55.6% and decreased cecal Salmonella counts by an average of 2.97 log10 cfu/g of material [43]. These results suggest that competition for mucosal binding sites limits Salmonella infection in chickens. Interestingly, these same CE cultures have a limited ability to prevent Campylobacter colonization. Although some CE cultures are undoubtedly effective, it has been suggested that complex cultures of “undefined” or uncharacterized populations collected from intestines are better able to elicit protective effects [4, 44, 45]. Some suggest that studies demonstrating little to no benefit in animal performance after CE culture application may be the result of “defined” cultures (those prepared from identified axenic cultures) [4, 44]. Microbially Mediated Immunodevelopment. Intestinal bacteria have a profound effect on the immunodevelopment of the gastrointestinal system and are a major source of antigenic material that stimulate the development of gut associated lymphoid tissue [46, 47] and Peyer’s patches [48–51], the production of antimicrobial peptides [8, 52, 53], and the production of protective IgA molecules [46, 54, 55]. The immunomodulating properties associated with DFM application in poultry are well known. A study performed by Haghighi et al. [56] showed significant increases in natural serum IgA, IgG, and IgM antibodies specific for tetanus toxoid after a single intragastric application of a 3-strain DFM mixture in immunized chickens. Likewise, Koenen et al. [57] reported increases in total IgG and IgM titers in chickens receiving L. plantarum, L. paracasei, or a combination of the 2 strains, and also observed increased phagocytotic activity of gut-associated immunity cells toward S. enteritidis (currently recognized as S. enterica ssp. enterica) [37]. Direct-fed microbial bacteria have also been described as increasing heterophil oxidative burst and degranulation in chickens [58]. For DFM using Bacillus species, the majority of evidence suggests immunostimulation as the primary mode of action. Bacillus spp. as DFM Traditionally, the use of LAB such as Lactobacillus has dominated DFM products. Recently, however, the use of Bacillus spp. as

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Competitive Exclusion. “Beneficial” intestinal microflora can protect the host from intestinal pathogens, termed a “barrier effect” or “competitive exclusion” (CE) [26]. This refers to the process in which epithelial associated or bound microorganisms preclude contact between pathogens and host epithelial cells [35, 36]. It is generally accepted that a key property when selecting potential DFM strains is the ability to bind to host epithelia. Attachment allows for longer residence within the intestinal system and more intimate interaction with potential pathogens at the host-pathogen interface. In vitro experiments have shown that attachment and invasion of enteric pathogens into intestinal epithelial cells are impaired by coinfection or preincubation with LAB. Lactobacillus acidophilus LA1 decreases adhesion of diarrheagenic Escherichia coli to Caco-2 cells by 85% and prevents invasion of the same cells by E. coli (95%), Yersinia pseudotuberculosis (64%), and Salmonella enterica serovar Typhimurium (37%; currently recognized as S. enterica ssp. enterica) [37, 38]. Ingrassia et al. [39] demonstrated the inhibitory effects of Lactobacillus casei DN-114 001 on adherent-invasive E. coli (AIEC) in intestinally derived tissue culture cells. There it was demonstrated that attachment of AIEC to Caco-2 and Intestine-407 cells was reduced by 47 and 62%, respectively. The authors also showed a depression of cellular invasion by AIEC with preincubation of L. casei DN-114 by 98.7% in Intestine-407 cells and 89% in Caco-2 cells. Others have shown the capacity for Lactobacillus rhamnosus GG to prevent E. coli O157:H7-induced lesions in Caco-2 cells [40]. The principle of CE is well established in poultry, as was first proposed for chicken protection by Nurmi and Rantala [41, 42]. The authors reported only 23% (103 cell inoculum) and 31% (106 cell inoculum) infection with Salmonella infantis in chicks receiving adult cecal contents, whereas all chicks in the control groups were colonized [41]. Since then, CE culture research and products have been well investigated. It has been found that CE cultures derived from mucosal scrapings provide better protection against Salmonella colonization than those from luminal preparations. Cecal-derived cultures reduced cecal Salmonella populations by 0.25 log10 cfu/g of cecal material, whereas mucosal-derived cul-

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DFM APPLICATIONS IN POULTRY The ultimate goal of commercial application of DFM is to increase economic profitability modulated by 3 expectant results: 1) a demonstrable increase in animal performance; 2) reduction in morbidity and mortality in the animals; and 3) reduction in human pathogenic bacterial populations. These goals are not mutually exclusive and beneficial outcomes are, in fact, tightly interwoven with one another, as discussed below.

Reduction of Food-Borne Pathogens Poultry are considered a major reservoir for pathogenic population of Salmonella and are a serious contributor to the estimated 1.4 million cases of salmonellosis contracted each year in the United States [66]. Decreasing the number of viable Salmonella on chicken carcasses will likely decrease downstream contamination and illness. Use of DFM products to reduce pathogen populations has been suggested as a possible solution to decreasing Salmonella incidence. Some research has focused on early colonization of chicks by pathogens. It has been shown that administration of microbial cultures derived from chicken cecal contents not only decreased Salmonella isolated from chick cecae from an average of 106 cfu/g to an average of 102 cfu/g, but also reduced the number of chicks colonized by the pathogen when challenged with 104 Salmonella Typhimurium (currently recognized as S. enterica ssp. enterica) [37, 67]. Further studies with this culture demonstrated that a defined culture of 9 different strains isolated from the original undefined inoculum provided similar efficacy in reducing Salmonella in challenged chicks [68]. Interestingly, these studies revealed that reductions in Salmonella cecal populations had a direct relationship with increases in cecal propionic acid concentrations. It has been shown that early application of DFM reduces chick colonization of Salmonella by 70% [69]. Here, increasing levels of efficacy corresponded with application doses of as few as 104 viable cells and up to 108 viable cells per chick. Doses of greater than 108 cells showed no increase in performance, and heat-killed (dead) cells were ineffective. Corrier et al. [70] also found a dosage-dependent response with a CE product, with higher doses of 107 and 108 cfu showing greater efficacy in preventing chick colonization by Salmonella Typhimurium (currently recognized as S. enterica ssp. enterica) [37]. Vicente et al. [71] evaluated an 11-strain DFM mixture for its ability to reduce Salmonella colonization of chicks. After 24 h, 5% of treated chicks tested positive for Salmonella compared with 70% of the control group. At 72 h after challenge, 25% of the treated chicks tested positive for Salmonella, whereas 65% of the control group was colonized [71]. A complex CE product containing 29

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DFM organisms is becoming more prevalent in livestock applications, especially in the poultry industry [59]. The Bacillus species are appealing DFM candidates because of the ability of members within this genus to form endospores that are resistant to extreme temperatures and processing environments of food pelletization, and to result in DFM products with extended, if not indefinite, shelf lives [59, 60]. Before considering Bacillus as a DFM, it is important to consider carefully how the endospore-based product interacts with the intestinal ecological system. One point of particular concern is whether Bacillus endospores germinate and propagate in animal intestines because Bacillus is not typically considered autochthonous to intestinal systems. Bacillus subtilis spores do germinate at very low frequencies in mice (approximately 1% of ingested spores) [61, 62], and very similar results have been observed for spores applied to 1-d-old chicks [60]. Endospore preparations may not be appropriate for all livestock operations, particularly with animals that have short intestinal transit times. Cartman et al. [60] detected spore germination 12 h after application in chicks, but at least 20 h was required for the highest levels of germination, which subsequently diminished to undetectable levels within 48 h of ingestion. Thus, given the few vegetative cells present and their short intestinal duration, the beneficial effects of Bacillus spores are more likely due to immunostimulatory effects (e.g., enhanced IgA concentrations, increased γ-interferon, and IL-4 production) of the spores themselves or the relatively small percentage of vegetative cells, and not through bacterial antagonism or competitive exclusion [63–65].

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ineffective in reducing Salmonella cecal colonization. Failure to prevent S. infantis colonization by Lactobacillus cultures was also reported by Adler and DaMassa [79]. Increase BW Gain and Increased FE It is important to consider the economic implications of DFM application. It will be difficult, if not impossible, to convince poultryraising facilities to adopt DFM technologies if increased costs are incurred that diminish profits. Increasing animal BW gain and improving FCR are measures that can indicate increased profitability for the producer. The inclusion of DFM may positively affect these measures in poultry and are discussed below. Measurements of animal BW gain in poultry receiving DFM treatment have been met with variable results. Complex cultures of multiple organisms tend to demonstrate greater efficacy in augmenting BW gain than specified single or few strains. Jin et al. [80] also noted a dosedependent response with DFM application. The authors described reduced mortality in chickens receiving 0.10% (wt/wt) Lactobacillus culture, from 8.2% in control birds to 3.2% in treated animals. These animals also demonstrated superior FCR. However, higher doses (0.15% wt/wt) resulted in lower productivity, near that of the control group receiving no DFM. Chickens receiving the low 0.05% (wt/wt) DFM dose demonstrated bird performance values between the 0.10 and 0.15% dosed birds. Reduced mortality rates and improved FCR were also observed by Timmerman et al. [81] when a multiple-strain DFM product derived from chicken ceca was applied. Here, the authors found that efficacy of the DFM was reduced under high-production conditions. Large increases in broiler performance were demonstrated by Khan et al. [82] in birds receiving intragastrically administered Lactobacillus, although statistical analyses could not be applied because of the small sample size. Other studies have effectively demonstrated that DFM organisms can provide bird performance similar to that of control groups receiving performanceenhancing antibiotic supplementation [83]. Notable observations from DFM studies in agriculture showed that DFM demonstrated greater potential in lower performing animal-rearing

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different bacterial isolates significantly reduced the horizontal spread of Salmonella Gallinarum from infected chicks to treated chicks and also resulted in significantly decreased mortality among treated chicks [72]. Similarly, a collection of 28 isolated strains provided chick colonization protection similar to that of undefined fecal cultures. However, the protective ability of this combination diminished over time with subsequent culture transfers [73]. Stavric [45] also reported that large combinations of 50 different defined organisms were protective against Salmonella colonization of chicks by several orders of magnitude. Although early prevention of colonization can help limit pathogen populations, other DFM products can provide long-term colonization benefits. One-day-old chicks challenged with Salmonella Enteritidis (currently recognized as S. enterica ssp. enterica [37]) showed a 70% colonization rate 21 d after challenge, whereas none of the chicks also receiving Lactobacillus salivarius CTC2197 with the pathogen challenge were Salmonella positive at 21 d of rearing [74]. A large study with 68 different flocks performed by Palmu and Camelin [75] showed that only 6% of flocks of chicks treated with a commercial product tested positive for Salmonella after 45 d of animal rearing, compared with the 42% contamination rate seen in control flocks. It was also demonstrated that administration of L. acidophilus and Streptococcus faecium to chicks reduced fecal shedding frequency of Campylobacter jejuni by 70% [76]. Lactobacillus johnsonii FI9785 did not significantly reduce colonization of chicks by E. coli O78:K80 or Salmonella Enteritidis (currently recognized as S. enterica ssp. enterica [37]) over a period of 36 d, but reduced Clostridium perfringens fecal shedding to nondetectable levels 15 d after challenge, even though shedding was still prevalent in the control group at least 36 d after challenge [77]. Additionally, significantly fewer tissue samples tested positive for C. perfringens when treated with L. johnsonii FI9785 than in nontreated individuals. However, it is important to note that poultry DFM products have not always provided efficacious results when applied in poultry-rearing operations. Hinton and Mead [78] reported that cultures of Lactobacillus and Enterococcus were

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CONSIDERATIONS ABOUT DFM Labeling Inaccuracies Many commercially available DFM products contain inaccurately labeled organisms for both

the type of organism and the concentration of viable organisms. Whether this is due to misidentification or inadvertent inclusion is likely case dependent. Yeung et al. [88] found that 26 of 58 strains collected from commercial products and bacteria manufacturers were incorrectly identified. Within that study, 48% (14 of 29) of the commercial products analyzed possessed label discrepancies, and 6 products contained bacteria not disclosed in the product. Similar results were obtained in a recent study that found 28% of commercial DFM cultures were incorrectly identified strains [89]. Additionally, DFM product evaluations performed in our laboratory have determined that many commercial agriculture DFM products do not contain the concentration of organisms described on the product label. However, with the increasing popularity of DFM use, federal agencies will undoubtedly require more stringent labeling practices to prevent fraudulent marketing and to ensure animal and public safety. DFM Viability and Detection An important aspect of DFM application to animal feeds that is often overlooked or improperly evaluated is the dosage of bacteria provided to an animal. The DFM bacteria need to be presented in sufficiently viable numbers to elicit beneficial effects on the host. Nonviable bacterial cells may have little impact on the health of the host [69], especially where an altered intestinal fermentation profile, the production of microbial metabolites, or bacterial antagonism are the suggested modes of action. The DFM organisms are usually applied to animal feed in a preserved or dormant state, most commonly in freeze-dried or spore form.  Consideration must be given to product (microbial) viability during production, storage, and application of DFM. Viable bacteria are lost, frequently by several orders of magnitude, during feed application until the time of ingestion. Many environmental factors adversely react with preserved bacteria, including ambient temperature, humidity, and feed temperature. Although endospores are stable under most conditions encountered in the field, to evaluate DFM product viability properly, methods must be used to enumerate viable populations accurately, preferably at the bacterial strain (target bacterium) level.

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facilities than in those with near optimal animal performance [84]. This could, in part, explain some of the lack of, or minimal, benefits often seen in organized field trials. Greater attention is provided to the animals, and those performed in university institutions require prior approval from animal care committees and follow strict Institutional Animal Care and Use Committee standards. These conditions are rarely seen in large animal production environments. Zulkifli et al. [85] found that DFM organisms were able to provide poutry production benefits under temperature-stressed conditions. There, the authors found the DFM cultures not only provided enhanced BW gain, but also improved FCR to chickens reared under the stressful environments. Torres-Rodriguez et al. [84] evaluated the animal performance of turkeys raised under a variety of conditions and found that turkeys demonstrated greater performance increases under suboptimal conditions. However, when all conditions were considered, significant differences in FCR and bird BW gain were not found. It was determined that an additional $0.10 per turkey was received (including cost of DFM) with product use when the results from all studies were considered [84]. However, it is important to note that poultry DFM products do not always result in enhanced bird productivity when applied in poultry-rearing operations. O’dea et al. [44] found that using 2 commercially available DFM products in chickens did not result in significant differences with respect to BW gain, feed conversion, or chick quality compared with control groups. The lack of positive effect on BW or FCR in chickens fed DFM has also been reported elsewhere [86, 87]. This lack of improved production results could be due to a multitude of factors, including use of a product that that did not deliver sufficient numbers of viable organisms to the animals, use of a product that was improperly manufactured or that contained inappropriate organisms, or impairment of bird performance by infectious agents that were not affected by the DFM product applied.

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determine the correct application methods, and establish the efficacy of any DFM technology. Negative Consequences The detriment of widespread antibiotic use is only now becoming apparent. As with antibiotics, any technology can bring negative consequences as a side effect of the positive benefits. Correspondingly, what concerns should be given with scientific evidence supporting the efficacy of DFM in animal agriculture? Bacteria are highly adaptable, and resistant strains can be selected rapidly under stressful conditions. As with antibiotics, certain bacterial populations may become resistant to the modulating properties associated with DFM organisms, and the efficacy of that product may wane. Continued monitoring of animal performance, DFM, and pathogenic populations during field trials and commercial use will help elucidate if resistance to DFM is a concern. The establishment of large bacterial collections and databases through proper sampling and monitoring will help prevent this from arising.

CONCLUSIONS AND APPLICATIONS







1. Direct-fed microbials are a developing technology that may support the profitability and safety of poultry production and offer meaningful alternatives for natural and organic production. 2. Research indicates that the beneficial application of viable microorganisms to livestock feed can improve animal performance and reduce human and animal pathogen populations under certain conditions. 3. Further research is needed to 1) define the precise modes of action, 2) identify the appropriate bacterial strains for specific applications, and 3) identify the appropriate DFM dosage for specific applications. 4. Viable cell detection should be incorporated into future research, field applications, and manufactured product audits as a final validation of the amount of viable organisms delivered.

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Several requirements exist for implementation of optimal bacterial detection strategies, including the ability to obtain results rapidly, detect few false positives, accurately quantify viable target bacteria, detect target bacteria in the presence of high background populations, detect target bacteria in complex matrices (e.g., feed, manure, digesta, soil), be relatively inexpensive, and be easy to implement in field settings. Classic cultivation techniques have been used [90, 91], making it difficult to enumerate a specific strain accurately among the large background of indigenous, and potentially related, bacteria present in most environmental samples (i.e., animal feed or feces). Recent technological advances in nucleic acid detection now allow for rapid detection of specific microorganisms. Although enabling for more precise and accurate detection, these methods cannot differentiate between viable and nonviable organisms. Few reports are available that have monitored DFM viability using DNAbased detection methods. Fujimoto et al. [92] developed a quantitative polymerase chain reaction method to enumerate L. casei strain Shirota, a common strain used in human DFM products, which targeted a genomic fragment identified in randomly amplified polymorphic DNA analyses. With this target sequence, L. casei strain Shirota could be enumerated in fecal samples containing as few as 104 cells per gram. Detection of Viable DFM. The authors have found a large discrepancy between PCR detection and cultivation enumeration methods, and they acknowledge the presence of nonviable cell detection (false positives) via PCR-based enumerations. Flint and Angert [93] developed a rapid strain-specific detection method that accurately enumerated viable DFM populations from 106 to as few as 102 viable cells applied to cattle feed. This method has been implemented in the field and used for the strain-specific detection of bacteria in livestock feed, manure, and digesta. It has provided a useful tool to monitor the ability of the product to survive feed processing and to assess accurate and precise application of DFM to feedstuffs, and it is easily extended to poultry feed and gastrointestinal monitoring of probiotic organisms of interest. Detection and enumeration of viable DFM bacteria will be an essential component to understand the modes of action,

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REFERENCES AND NOTES

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