health in commercial broiler operations

• Enterotoxigenic organisms produce toxins that kill cells (e.g. α-toxin from Clostridium perfringens) or upset cell function (e.g. shiga toxins from Escherichia coli).

• Enteroinvasive organisms like E. coli, some Salmonella species and Listeria monocytogenes are able to transgress the flora/host barrier by stimulating endocytosis by epithelial cells, initiation of inflammatory cytokine release and causing cell death.

• Enteropathogenic E. coli causes typical attaching and effacing (A/E) lesions characterized by adhesion, microvillus destruction and gross cytoskeleton reorganization (Frankel et al., 1998; Donnenberg et al., 1997; Donnenberg, 2000).

Intervention strategies

Widespread acceptance of Koch’s Postulates by scientists in the 1900s led to the development and implementation of disease control strategies directed at eliminating the causal agent. These defense-oriented intervention strategies, including in-feed antimicrobial use, have allowed the intensive poultry industry to evolve to its current state. This approach to gastrointestinal disease control is however fundamentally flawed since it ignores the intricacies of gut flora ecology. Destabilization of the gut ecology, recognized in the EU as dysbacteriosis, and the emergence of antibiotic resistant bacteria are just two possible consequences of such an approach. To be sustainable, a growth-promoting gut health program needs to be holistic and include intervention at host, agent and environmental levels.

The task of managing the gut environment is virtually neglected because the ecology of the gastrointestinal lumen is complex and poorly understood. This is rather ironic since the efficiency of nutrient assimilation and hence feed efficiency is dependent on the integrity of the absorptive membrane, which is in turn dependent on the status of the gut lumen environment. Modern molecular techniques have helped elucidate details of the gastrointestinal environment that are revolutionizing understanding. 16S ribosomal RNA-based techniques have enhanced the accuracy of gut flora profiling (highlighting the inadequacy of culture techniques) and are making the detailed study of gut ecology feasible (Amann et al., 1995; Vaughan et al., 2000; Lu et al., 2003). Gene sequencing of purported commensals and host cascade reaction modulation studies (such as proand anti-inflammatory pathways) have already opened the door to the vast host/ flora cellular communication process that maintains immune-microbe homeostasis (Xu and Gordon, 2003; Xu et al., 2003).


IDENTIFYING OPPORTUNITY

HACCP (Hazard Analysis Critical Control Points) is a very thorough quality control system designed to build safety into food product manufacture and provides a useful means of critically evaluating a live production system. Hazard analysis begins with production system evaluation to identify potential pathogen entry points and or intervention opportunities. In food safety assurance programs bacterial contamination is a defined hazard, but in a gut health program seeding of the gut with favorable flora is a positive while contamination with unfavorable flora is a hazard or gut-health risk. Colonization of the gut with bacterial species that are able to modulate expression of genes in the host gut epithelia to create favorable conditions for a favorable community provides a natural form of defense against pathogen challenge (Guarner and Malagelada, 2003).


NATURAL SEEDING OF THE GUT

Although bacterial contamination of the egg can occur, the developing alimentary canal of the embryo and hence the alimentary tract of the chick at hatch should be sterile (Baxter-Jones, 1991; Barrow, 1994). Once the hatch process is initiated and the protective layers of the shell and its membranes are compromised, then colonization of the alimentary tract begins. The first organisms to gain access to the receptive environment of the hatchling gut originate from the shell and hatchery flora. In contrast to a natural setting, the commercial hatchling pips in an artificially clean environment. Under such conditions low doses of beneficial bacteria can significantly improve resistance to pathogen colonization (Fernandez et al., 2002).

Egg shells become contaminated with parent gut flora during and after oviposition (Humphrey, 1994). Shell contamination rates range from 103 to 108 CFU/egg; and although internal contamination of the egg is a possible outcome, this is not necessary in order for these organisms to colonize the hatchling gut (Bruce and Drysdale, 1994). Egg sanitation is inadequate as a means of eliminating shell contaminants from entering the hatchery (Hutchison et al., 2004). Bacterial survival on egg shells depends on the relative humidity or surface moisture and temperature. In this respect the prevailing conditions in the modern hatchery are very forgiving and it is likely that most hatchery organisms are introduced via the egg. In one study there was a poor correlation between specific serotypes found at the hatchery and the supplying breeder farm, suggesting that bacteria survive well in the hatchery environment and once introduced have the potential to form an endemic population (Bailey et al., 2002).

Steps to control gut health in broilers should ideally start at the parent flock level. Vertical transmission of gut flora provides the first seeding of the hatchling gut. Although these individual organisms may not persist throughout the life of the flock they at the very least create conditions that shape development of the climax flora (Dawson, 2005). Manipulating parent gut flora can have a beneficial effect on offspring resistance to pathogen colonization (Fernandez et al., 2002).


ARTIFICIAL SEEDING OF THE GUT: COMPETITIVE EXCLUSION CULTURES AND PROBIOTICS

The term competitive exclusion (CE) is used to describe the inability of one population of microorganisms to colonize the gut because of the presence of another population of microorganisms and was first described as a method of preventing pathogen colonization of the avian gastrointestinal tract in 1973 (Nurmi and Rantala, 1973). While a single Salmonella organism can potentially colonize the gut of a newly hatched chick, the infective dose for an adult bird increases to 103-106 organisms, primarily because the adult bird has an established population of competing organisms referred to as ‘normal flora’ (Pivnick and Nurmi, 1982). The gut flora has been extensively studied using culture techniques and more recently molecular techniques. The cecum contains a wide variety of bacterial species and while it is important to realize that the population is constantly evolving, the anaerobic organisms of the climax flora were found to be most crucial to the protective properties of the CE culture (Goren et al., 1984). Modern production systems target high levels of hygiene, a practice that could increase susceptibility to pathogen colonization by limiting opportunity for early seeding and colonization with normal flora (Baxter-Jones, 1991).

Resistance to pathogen infection is enhanced by dosing day-old chicks with flora from healthy adult birds, provided adequate colonization occurs prior to pathogen challenge (Hofacre et al., 2002). Early pathogen challenge accounts for variable field response to the use of competitive exclusion strategies. The process of gut colonization with a favorable flora requires at least 4 hrs to form an effective (reduce infection from 95% to <5%) barrier to low grade (<104 CFU) pathogen challenge (Hume et al., 1998; Methner et al., 1997).

Although the precise mechanisms by which a healthy microflora prevents pathogen colonization have not been fully elucidated, the creation of unfavorable conditions (pH, volatile fatty acids and bacteriocin synthesis/ accumulation) and competition for space (epithelial attachment sites) and nutrients play important roles (Scanlan, 1997).

Avian competitive exclusion products are undefined, quality-controlled pathogen-free preparations of live obligate and facultative anaerobic bacteria originating from the gut of normal healthy adult individuals of the avian species. In many countries including the US, only defined cultures (probiotics or direct-fed microbials) are accepted for registration. Defined cultures are composed of identified bacteria that have been shown to be beneficial and are individually isolated and cultured from a primary culture before being mixed (Scanlan, 1997). In contrast to competitive exclusion products, probiotics generally need to be fed continuously in order to be effective. Administration of Enterococcus faecium (Owings et al., 1990), Lactobacillus reuteri (Edens et al., 1997), a combination of Lactobacillus spp., B. bifidum, and Aspergillus oryzae (Mohan et al., 1996), or a combination of L. acidophilus, Bacillus subtilis and E. faecium (Chaing and Hseih, 1995) significantly improved broiler performance under research conditions.

Some companies have successfully utilized continuously -fed probiotics to enhance gut health and feed efficiency on a commercial scale. L. johnsonii (La Ragione et al., 2004) and a mixture of L. acidophilus and Streptococcus faecium have shown promise in reducing the impact of low grade necrotic enteritis on performance when used as probiotics (Fukata et al., 1991), but the only published reports in which a probiotic matches an antibiotic in protecting against necrotic enteritis induced mortality are those of Hofacre (Hofacre et al., 2003a, 2003b). In this study a single dose of a direct-fed microbial was shown to be comparable to bacitracin- MD (50 g per ton). The study also suggests that the degree of protection elicited by a direct-fed microbial is strain-specific since the two primary organisms used (L. acidophilus and E. faecium) were the same as those in an earlier less successful study (Fukata et al., 1991).

In addition to adherence to and colonization of the gut mucosa, certain strains of L. acidophilus aggregate against E. coli (Spencer and Chesson, 1994). It would appear that by selecting specific species as probiotic candidates it is possible to create a gut environment that accelerates the establishment of a favorable and stable climax flora. With this strategy the emphasis subtly shifts from working against, to working with, the natural ecology of the gut.


GUT ENVIRONMENT MANAGEMENT

Acidification

While acidification of feed and drinking water is a well researched and accepted practice in pig production, the same cannot be said for poultry. The poultry industry is becoming increasingly interested in this practice as a means of relinquishing dependence on antibiotics. The pronutrient potential of commercial acid preparations is thought to arise from the antibacterial effects of their ionization properties. Organic acids diffuse across the bacterial cell membrane rapidly when in the undissociated form (Cherrington et al., 1991). Once internalized the neutral pH of the cytoplasm causes dissociation, thus raising the intracellular concentration of both protons and anions. Bacterial proton-motive forces are exhausted in pursuance of homeostasis and the resultant rise in cytoplasm pH interferes with bacterial cell physiology. At low concentrations organic acids have a bacteriostatic effect but at high concentrations they become bactericidal (when acid concentration causes internal pH to rise to the point where denaturation of bacterial protein and DNA occurs) (Davidson, 2001; Ricke, 2003).

Acid ionization varies considerably according to type, concentration and mix of acids used and is further modified by the pH, buffering capacity and water activity of the feed, water and gut content. Since their activity in the gastrointestinal tract is so variable, systematic studies of the effects of a variety of acidifiers are not available. It is not possible to determine the overall responses to complex acidification strategies or to compare these with other supplementation strategies using objective analytical process (Rosen, 2003).


Nutrient balance: intake, absorption and excretion

Years of breeding and selection to improve growth rate and feed efficiency have also increased broiler appetite and hence daily feed intake. Using feed transit time in leghorns as a standard, it would appear that although feed retention time in the proventriculus and gizzard has decreased considerably, the time ingesta spends in the small intestine has remained fairly constant (Washburn, 1991; Denbow, 2000). Feed passage studies using insoluble or soluble markers indicate that average retention time is around 5-9 hrs (Branch and Cummings, 1978, Uden et al., 1980; Ferrando et al., 1987).

Although feed markers first appear 1.6 to 2.6 hrs after ingestion, the time taken for complete clearance appears to have been poorly documented. This is unfortunate since although feed transit time is inversely proportional to intake in broilers, retroperistalsis has been shown to be an important part of avian digestive physiology, particularly after withholding feed (Clench et al., 1989; Almirall and Esteve-Garcia, 1994). Any stimulus that suppresses feed intake may affect passage time, digestion efficiency and nutrient throughput. Normal dark periods could cause sufficient feed withdrawal to stimulate retroperistalsis, therefore experimental feed passage studies may be confounded by lighting programs and may not accurately reflect reality. Since feed transit time is inversely proportional to intake in broilers, it may be possible to improve digestion efficiency by restriction feeding (Almirall and Esteve-Garcia, 1994). Extended feed withdrawal, as would occur with fever response, would increase the amount of retroperistalsis and could raise the pH of the small intestine, thus upsetting normal ecology (Clench et al., 1989). If liver nutrient reserves were to be sufficiently depleted by feed withdrawal to increased tissue protein turnover, the increased urinary nitrogen (negative nitrogen balance) could further upset cecal flora (Denbow, 2000).

Several feed ingredient characteristics also affect passage time including viscosity, particle size, digestibility (starch), and lipid or protein content. In pursuance of gut health the nutritionist should consider ingredient blend in addition to nutrient specifications (Bedford, 1996). Some ingredient characteristics such as water content, viscosity and non-digestible nutrient composition can be enhanced with concomitant enzyme and/or osmolyte usage (Bedford, 2000). Water soluble non-starch polysaccharides (NSP) adversely affect digestibility by stimulating mucus production and increasing ingesta viscosity (Choct and Annison, 1992; Iji, 1999). Grains such as wheat, rye and barley are rich in water soluble NSPs and there is ample research to demonstrate that the use of exogenous enzymes improves digestibility (Rosen, 2000). The growthenhancing effect of dietary enzymes is comparable to that of the antibiotics when tested in controlled experimental conditions, suggesting a common mechanism of action, i.e. manipulation of the gut ecology. This may be one of the explanations for individual and ingredient variation in response to enzyme supplementation (Kocher et al., 1997). Apart from the direct feed efficiency implications of reduced digestion and absorption, the passage of undigested nutrients affects downstream gut ecology. Potentially toxic compounds such as ammonia, amines, phenols and indoles are generated by the proteolytic and ureolytic activity of the cecal flora on non-digested nutrients that make their way through to the cecal pouches (Gidenne, 1997). These toxic compounds affect gut ecology in the rabbit and the same is likely true for the broiler.

The morphology of the ileocecal junction is such that only fluid or very small (dissolved or suspended) particles enter the cecal pouch when intraluminal pressure increases during convergence of rectal retroperistaltic and ileal peristaltic contractions. Since the retroperistaltic contractions of the colon/rectum are almost continuous, 87-97% of the cecal fluid originates from the urine (Duke, 1982). Urine-derived uric acid reaching the ceca by retroperistalsis and undigested protein from the upper gastrointestinal tract provide a source of nitrogen for microbial amino acid synthesis. Subsequent degradation of cecal microbial protein by cecal proteases may provide an additional source of amino acids thus accounting for the capacity of the ceca for amino acid absorption (Bjornhag and Sperber, 1977).

The flora of the lower gastrointestinal tract spend the majority of their existence in stationary phase because intense competition for a limited source of nutrients causes near starvation (Zinser and Kolter, 2004). Stationary phase evolution occurs very rapidly and continuously through mutation, selection and takeover.

Selection favors gene expression over function and in the case of E. coli, enhanced amino acid scavenging capacity emerges. Rapid expression of new and advantageous metabolic pathways may redefine the organism’s niche and mean the loss of functionally beneficial genes. Any factor that reduces digestion efficiency in the upper gastrointestinal tract changes the nutrient supply to the lower tract and will likely favor specific stationary phase mutants (Zambrano et al., 1993).

The amount of nitrogen reaching the ceca is influenced by the amount of protein in the diet, the efficiency of protein digestion/absorption in the upper gastrointestinal tract and the state of nitrogen balance. Exogenous enzymes added to the diet to promote protein digestion affect cecal flora communities by reducing the amount of protein nitrogen reaching the ceca, especially if nutrient credits are allocated to the enzyme. Any physiological perturbation that negatively affects nutrient assimilation (intake, digestion and absorption) will increase cecal nitrogen either by increasing nutrient through flow or body protein turnover rate (nitrogen excretion via the urine). Volatile fatty acids (VFA) are by-products of uric acid degradation by cecal flora and despite passive absorption, cecal VFA concentrations (acetate>propionate>butyrate) are very high (125 nM) (Annison et al., 1968). Since these weak organic acids have antibacterial activity they likely play an important role in balancing the cecal ecology (Davidson, 2001).


Antimicrobials

Antibiotics have been an integral part of poultry feed for the past 50 years. Decades of research and field use have established the efficiency of antibiotics as growth promoters and in-feed antibiotics have been shown to subtly change the composition of the normal flora (Anderson et al., 2000). PCR-denaturant gradient gel electrophoresis (DGGE) studies indicate that while antibiotics definitely change the gut flora profile, microbial populations become more homogeneous (Collier et al., 2003). DGGE studies are useful for tracking changes in gut flora profiles but give no detail on actual composition. The use of 16S clone libraries have made it possible to study specific organism profile changes in the gut. It would appear that lactobacilli and clostridia are the two groups most significantly affected by antibiotics (Knarreborg et al., 2002).

Antibiotic growth promotion strategies have focused on manipulation of the gut ecology of the small intestine to improve feed efficiency but the impact on cecal flora, house flora and seeding of the hatchling gut of the next placement has been ignored. The gut flora changes elicited by a growth promotant are dependent on its antibacterial properties, rate of absorption, method of inactivation/metabolism, route of excretion, the degree of luminal (microorganism) enzyme inactivation or adsorption to ingesta (Sullivan et al., 2001). Many antibiotics are excreted via the urine in an active form, whether metabolized or not (Anadon et al., 1995).

Parenteral antimicrobials or those that are absorbed from the gastrointestinal tract after oral administration are concentrated in the urine and subsequently transported back to the ceca as illustrated by the high concentration of antibiotic in the cecal wall (Knoll et al., 1999). Orally administered antimicrobials that are not absorbed also reach the ceca when the intraluminal pressure at the ileocecal junction increases and liquid is forced into the cecal pouches (Duke, 1982).

The extensive reviews on in-feed antibiotic use, and those covering the various alternatives, have reported research investigating the response to first-time use of growth promoter strategies in controlled trials under carefully monitored experimental conditions. Broiler production is, in contrast, a continuous system. Broiler gut flora determines the composition of the litter/house flora, which in turn acts as the seedstock for the gut flora of the next placement. While the small intestinal ecology influences the efficiency of digestion and absorption, it is the cecal/colon/rectal flora that give rise to the house flora. Although there are literally thousands of growth-promotant trials demonstrating their efficacy (or lack thereof), the literature is devoid of data showing the long-term effect of such programs. While the use of a growth promoter can alter the gut flora within a couple of weeks, it takes several growout cycles to change the house flora (Schildknect et al., 2003). This is by no means a new concept, both rotation and shuttle programs have been used for decades to avoid the lack of response to growth promoting antibiotics after persistent use.

Virtually all of the major species of pathogenic bacteria have strains that are resistant to at least one of the antibiotics used in their control, and the emergence of multidrug-resistance (MDR) is related to quantity and method of antibiotic use (Levy, 2002). Bacteria evade antibiotic activity by target site alteration, antibiotic inactivation, altering cell wall permeability or antibiotic efflux (Wise, 1999). Acquisition of resistance genes occurs primarily through transformation (mainly Gram(+)) or conjugation (mainly Gram(-)) but is also possible through transduction (Woo et al., 2003).

Bacteria have evolved in the presence of natural antibiotics (bacteriocins) and since the background mutation rate is so high, there are a plethora of resistance genes in a non-medicated population. Antibiotic usage provides an accelerated source of resistance genes, facilitates lateral transfer (transformation) of these genes and amplifies the resistant population by providing selection pressure. Mathematical models have been developed to predict the population dynamics of resistance for primary pathogens that are normally cleared from the host by the immune system, with or without the help of antibiotic therapy. In contrast, population dynamics of resistance for commensals or opportunistic pathogens is less well defined. These organisms colonize the host asymptomatically and are not usually cleared by the immune system even with antibiotic therapy. With long-term antibiotic administration the cecal pouch provides an ideal environment for natural selection of antibiotic resistance since systemic antibiotics or their metabolites concentrate in the ceca and the indigenous inhabitants are not targeted by the immune system. Even low levels of antibiotic can cause sufficient cell wall damage to induce E. coli, at room temperature to absorb foreign plasmid DNA at a rate of 5 x 105 transformations per mg of plasmid DNA (Woo et al., 2003; Turkov et al., 1990; Massad et al., 1993).

Despite the capacity of some of the gut organisms to become opportunistically pathogenic, they pose a threat to flock health because their acquired resistance genes can be transferred to primary pathogens (Bonhoeffer et al., 1997). The selection index against antibiotic resistance in the absence of antibiotics varies from 50% to 1% and is dependent on the organism, environment, antibiotic and resistance mechanism. Once the resistance gene has become widely distributed within the gut and house flora, cessation of antibiotic therapy does not necessarily mean return to sensitivity (Schrag et al., 1997). The fitness cost of antibiotic resistance (selective advantage of sensitives in competition with resistants) declines rapidly in subsequent evolution because the rate of mutation exceeds that of reversion (Levin et al., 2000). Being so rapid, adaptation through mutation soon precludes reversion of resistant communities to sensitivity. Although the compensatory evolution hypothesis is supported by experimentation and mathematical modeling, a decline in frequency of resistance has been observed in some countries after antibiotic withdrawal (DANMAP, 2002). To rehabilitate resistant flora communities, additional selection pressure could perhaps be applied, to increase the cost of resistance in the period immediately after antibiotic withdrawal.

Just like penicillin, many of the mycotoxins that commonly contaminate poultry feed likely have antimicrobial properties. Mycotoxin research has focused on host toxicity, but it is possible that gut flora destabilization and feed efficiency are affected long before symptoms of toxicity appear (Kubena et al., 2001). Glucomannan (Mycosorb®, Alltech Inc.) is particularly useful in preventing the immune suppressive effects of feed mycotoxin contamination (Swamy et al., 2002). Mycosorb® has a very high affinity for the mycotoxin molecules, so dietary inclusion rates are low and it rapidly binds a broad spectrum of mycotoxins, so the toxic effects on intestinal epithelia and cecal flora are reduced/avoided. Molecular profiling using 16SrRNA library studies will likely provide more insight into the effect that mycotoxin contamination of feed has on gut flora and help explain how these novel strategies help to control gut health.

Although in-feed antibiotics are perceived by the poultry product consumer as a threat to human health, the organisms of the gut flora already use ‘natural’ antibiotics (bacteriocins) to compete with other microbes (Tahara et al., 1997). Manipulation of the gut flora communities to harness the power of these bacteriocins is an acceptable means of gut health enhancement.


Selective exclusion

Pathogen attachment to the intestinal epithelium is a pivotal first step in the colonization of the gut and depends on, amongst other things, pathogen type, flagella, type 1 fimbriae and pilus receptors for specific host cell docking sites (Sharon and Lis, 1993). Adherence has also been associated with mannoseresistant hemagglutinins. Scanning electron microscope studies of the cecal epithelium have shown that the organisms of the gut flora form a tightly adherent mat over the gut surface. These organisms are attached to each other and the epithelia by a series of fibrils, which effectively prevent pathogenic organisms from gaining access to epithelial receptors. The adhesive flagella of enteropathogenic E. coli (EPEC) have been shown to be induced by animal cells (Giron et al., 2002).

While competitive exclusion relies on the ability of live organisms to compete for nutrient supplies and attachment sites, it is also possible to block attachment with ‘decoy’ molecules and change gut flora communities (Parks et al., 2001). Mannan residues exposed on glycoproteins protruding from the gut epithelial cell surface form important docking sites for several unfavorable organisms. Mannan itself is relatively inefficient at blocking type 1 fimbriae attachment sites because the presentation of this active moiety does not simulate that which occurs on the enterocyte cell surface. Yeast cell wall derived mannoproteins are, in contrast, potentially very effective at blocking type 1 fimbriae docking sites.


Immune response

Any immune response bears a production cost. An appropriate immune response, adequate to contain infectious disease and minimize its effect on productivity, is the cost of health. An inappropriate, excessive or inadequate immune response will depress performance unnecessarily. Therefore, in a performance-driven broiler industry the gut health program should include an immune modulation strategy (Kelly, 2004).

The gastrointestinal environment is loaded with a plethora of antigens of feed and microorganism origin, the majority of which pose no threat of infectious disease. An inappropriate adaptive immune response to nonpathogen derived antigens is prevented by the innate immune system (Medzhitov and Janeway, 1997). Firstly the barrier function of a healthy gut lining prevents exposure to these antigens, and secondly, activation of T and B lymphocytes requires co-stimulation from antigen presenting cells of the innate immune system. Since cells of the innate immune system have the ability to recognize both pathogens (by highly conserved pathogen-associated molecular patterns) and self (by cell surface molecular markers expressed on the surface on normal uninfected cells), initiation of an inappropriate immune response is normally prevented. Leukocyte synthesis in response to antigen stimulation carries a barely detectable nutritional cost (<0.5% of broiler body mass) but the ramifications of the acute phase (fever) response range from negligible to dramatic.

This systemic component of the inflammatory response begins with acute phase protein synthesis in the liver and is followed by several behavioral, hormonal and metabolic responses. Feed intake declines, protein turnover accelerates and birds rapidly shift into negative nitrogen balance. With systemic challenge, most (70%) of the negative influence on growth rate and feed efficiency is attributed to reduced feed intake, while the inefficiencies of catabolism and nutrient absorption account for the rest (30%). Low level antigen recognition at the gut/ingesta interface probably seldom stimulates systemic/fever response, but proinflammatory mediators released in response to antigen stimulation of this nature can damage host tissue, thereby causing localized inflammation and reduced feed efficiency (Klasing, 1998).

Antigen-induced inflammation of the gut cytoskeleton stimulates an increase in mucus secretion, paracellular permeability, and feed passage (peristalsis). The cascade of events that follows is self-perpetuating. Increased paracellular permeability negatively affects cytoskeleton barrier function allowing toxin and antigen penetration and further inflammation. Excess mucus production allows mucolytic species to dominate the gut flora thus providing an opportunity for pathogenic organisms such as C. perfringens to proliferate and produce proinflammatory cytotoxins. Inflammation of the gut stimulates increased peristalsis and fluid secretion (an attempt to clear the gut of pro-inflammatory components), which reduces digestion and absorption efficiency and provides additional advantage to organisms such as C. perfringens that are capable of rapid multiplication. Both endogenous and exogenous anti-inflammatory agents modulate the immune response and help in the maintenance of gut health by preserving the integrity of the host enteron/environment interface and reducing the systemic (fever) response (Collier et al., 2003).

Local immune response is more pronounced in the lower gastrointestinal tract and high molecular weight proteins are more immunogenic. Therefore, apart from the obvious inefficiencies of nutrient wastage arising from poor digestibility or rapid feed passage, undigested proteins reaching the ceca are strongly inflammatory and thus further reduce feed efficiency (Lillehoj and Trout, 1996). High protein diets, essential to attain broiler muscle tissue accretion rates, increase the risk of downstream gut health challenges by increasing the chance of protein through-flow. Peptic digestion is already marginal because selection for growth rate has reduced feed retention time in the crop and gizzard, thus reducing enzyme/nutrient contact time (Denbow, 2000). Reduced enzyme-nutrient contact is a problem especially with soluble protein because liquids pass through the digestive tract 15% faster than solids (Sklan, 1980).

The nature and extent of the inflammatory response is influenced by several nutritional factors. Dietary polyunsaturated fatty acids (PUFA), for example, provide the building blocks for cell membrane synthesis and indirectly determine the type of immune response that follows cell damage, since the cell membrane lipids provide the substrate for immune system communication molecule synthesis (Kelly, 2004). Cereal grains are high in linoleic acid (n-6 PUFA precursor for arachidonic acid), which generates prostaglandins, leukotrienes and thromboxanes, whereas fish oil is high in n-3 PUFA which generates interleukin-1 and prostaglandin-E (Korver and Klasing, 1997).

The antigen receptors of the innate immune system are ancient, highly conserved and inherited (germ-line encoded) whereas those of the adaptive immune system are a somatically generated random repertoire (Medzhitov and Janeway, 1998). The backbone of the broiler hatchling gastrointestinal immune defense is the innate component, but it also receives a crucial reserve of adaptive immunity in the form of maternal antibodies from the hen. During the first 3 days after hatch, the chick immune system learns to tolerate innocuous gut antigens because intact antigens are able to transgress the immature gut cytoskeleton, enter the circulation and make contact with developing lymphocytes in the thymus, bursa and spleen (Brandtzaeg, 1989). The degree of immune tolerance induced during this time is proportional to the dose and frequency of antigen exposure and inversely proportional to antigen solubility (Lillehoj and Trout, 1996). Gut barrier function matures by day 3 and oral exposure to 'new' innocuous antigens after this will lead to an inappropriate immune response, which will likely reduce feed efficiency. Pre-starter rations must contain the correct balance of feed ingredient antigens to prepare the gut to tolerate feed antigen exposure after maturation of gut barrier function (day 3).

Maternally-derived antibodies play a relatively minor protective role at the enteric interface but they are crucial in guiding the development of the hatchling immune system along the fine line between tolerance and response. Maternal antibodies to hazardous/pathogen antigens block development of tolerance in the same way that they interfere with early vaccination (Klipper et al., 2004). This enables the developing immune system to differentiate potentially hazardous/pathogen antigens from innocuous feed/commensal antigens and initiate response or tolerance. The common practice of hyper-immunizing parent flocks to protect offspring against early exposure to harmful antigens likely generates additional gut health benefits. By manipulating the level and extent of maternal antibody transfer it is possible to shape the balance between tolerance and response in the broiler for life. Broiler gut health management needs to start at the parent flock level.

The practice of maximizing disease resistance (the capacity to resist disease and exclude pathogens depends on a variety of anatomical and physiological systems, including the immune system) has proven to be too costly for the modern broiler industry. Although immune stimulation has and still is used to enhance disease resistance, such intervention in a commercial broiler flock needs careful evaluation. The cost of an exaggerated or inappropriate immune response is counterproductive in terms of feed efficiency. Feed is the single largest input cost and as feed margin has declined, the cost-driven broiler industry has shifted emphasis from disease resistance to disease resilience (the capacity to maintain productivity during infectious challenge) (Klasing, 1998).

Just as the cost of an excessive or inappropriate immune response negatively affects performance, so too does an inadequate immune response. An inadequate immune response is usually recognized as an increase in flock mortality but has a negative economic impact long before flock mortality rises. Specific infectious diseases (e.g. Infectious Bursal Disease, Chick Anemia Virus, Marek’s Disease), nutritional deficiencies (e.g. vitamin E/selenium, zinc deficiency), toxicity (mycotoxins) and stress are all factors that can induce sufficient immune suppression to cause an inadequate response.

Stress only measurably affects performance once the aggregate of each individual stress exceeds the host’s coping mechanisms (Ferket and Quershi, 2001). The degree to which an adverse stimulus or stress will affect bird performance is directly proportional to the existing stress load; so good animal husbandry, nutrition and biosecurity are prerequisites to maintaining gut health. Any intervention that reduces stress will potentially enhance flock performance if there are birds within the flock that have reached or exceeded stress threshold. Immune modulation can be used to carefully manage the balance between disease resistance and tolerance in order to maintain productivity. An additive such as Bio- Mos®, which enhances the protective antibody response to enhance disease resistance while at the same time suppressing the acute phase (fever) response is unique and particularly useful in this regard.


Program design

ON-FARM RISK ANALYSIS

The success of a gut health program hinges on the ability to identify and then address risk. Aggregate risk is the sum of each individual risk of adverse health effects in an exposed population. The spread and consequence of infection is influenced by several factors, collectively termed disease determinants. It is these disease determinants that require special attention during risk analysis.

Risk assessment involves determining the probability of exposure to a given disease determinant, the probability of that exposure resulting in disease and spread of the disease, and the consequence of the disease outbreak. While the chi-square test can be used to test statistically whether a specific factor or process is a risk (correlated with disease), it gives no indication of the degree of risk. The strength of association or degree of risk is determined by the ratio of association.

For disease control purposes it is appropriate to evaluate each part of the production process in terms of the probability or chance of the process causing infection and the frequency with which that event occurs.

The chance of infection occurring is dependent on the resistance of the host and the challenge dose and virulence of the organism.

Control measures need to be focused on reducing the frequency of challenge, enhancing host resistance, reducing organism virulence and reducing the challenge dose.

On-farm evaluation is an essential part of gut health program design. Each event that carries a potential risk should be assessed in terms of the chance and frequency of affecting gut health. Water intake is a good example since it carries a relatively high risk despite the fact that potable water per se should pose little threat of disease. Firstly, water intake creates opportunity for infectious agent challenge and secondly the physical characteristics (e.g. pH and mineral content) of the water can adversely affect the gut environment. While the chance of either of these factors causing disease might be low, the bird consumes a large volume of water (1.8-2x feed intake) so frequency is high, thus raising the level of risk. Once each on-farm process has been ranked according to its relative risk, a logical strategy can be implemented to minimize input and maximize response.


MEASURING PROGRESS TO MEET LONG AND SHORT-TERM GOALS

The logistics and cost of long-term experimentation have forced researchers to neglect testing the long-term effects of intervention strategies aimed at enhancing gut health. It would be naive to expect any one intervention to have the same effect in a myriad of different field situations, so field evaluation is essential. Objective field evaluation is however complicated by a multitude of confounding factors. Stress, for example, is precipitated by several factors that vary from farm to farm and the response to an intervention strategy is dependent on (hence confounded by) the existing stress level.

In the high volume/low margin broiler industry, economic benefit is usually reached long before statistical significance, so field trial response criteria need to emphasize profit and repeatability, not necessarily performance and statistical significance. Besides, within and between flock variance usually exceeds the expected response (2% improvement, 74% of the time) and since it is usually impractical to include a negative control, statistical analysis under field conditions is virtually impossible (Rosen, 2001).

Careful and critical evaluation of the response to any intervention to improve gut health is crucial but difficult to accomplish. Flock productivity data usually only become available for scrutiny six weeks after initiation (completion of the first grow-out) and it takes 3-5 cycles to change the gut/house flora ecology. Real time, macroscopic and microscopic analysis of gut health is essential to ensure that timely decisions can be made. Unfortunately without productivity data (profitability), gut health is rather a nebulous term. There is a need to establish definite correlations between macroscopic gut characteristics and gut health, disease and performance to bring the exciting discoveries of modern molecular science to life on the farm.


Conclusions

Strategies to improve gut health in commercial operations need to be cost effective, sustainable, farm specific and holistic. Intervention/product selection needs to be science-based but practical and each intervention must address the specific objective for its inclusion. There are several opportunities for intervention to enhance gut health and productivity:


1. Seeding of the hatchling gut begins with vertical transmission of parent gut flora but is effectively modified with early administration of effective probiotics or competitive exclusion products. To be successful they must initiate the development of a primary flora that will rapidly evolve into a stable and favorable climax flora by creating suitable gut conditions and excluding unfavorable organisms.


2. Preparing the gut environment (pH, redox potential) for early transition from primary to climax flora through water/feed acidification. Candidates need to be weak acids that are buffered to withstand the neutralizing effect of minerals dissolved in the drinking water and have dissociation characteristics that make them active in the small intestine.


3. Excluding pathogens from colonizing the gut by competitive and selective exclusion. It is important that the selective exclusion product is compatible with (does not exclude) the organisms used for competitive exclusion or as a probiotic.


4. Enhancing resilience by stimulating protective immune response while suppressing the acute phase or fever response.


5. Decreasing nutrient through flow by enhancing nutrient digestion and absorption (exogenous enzyme addition and nutrient spec modification, feeding and lighting programs, careful use of antibiotics) to avoid cecal flora upset.
On-farm risk analysis is necessary to determine which interventions are most appropriate and establish the most profitable strategy for improving gut health.

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