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Informal Nutrition Symposium |
Novus International Inc., 20 Research Park Dr., Missouri Research Park, St. Charles, MO 63304
Correspondence: 2 Corresponding author: Julia.Dibner{at}novusint.com
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SUMMARY |
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 TOP SUMMARY DESCRIPTION OF PROBLEMEMBRYONIC DEVELOPMENT OF THE...EARLY POSTNATAL DEVELOPMENT OF...MICROBIOTA EFFECTS ON HOST...MICROBIOTA AND THE DEVELOPMENT...HEALTH AND PERFORMANCE EFFECTSCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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Key Words: gastrointestinal microflora • gut-associated immune system • imprinting • poultry
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DESCRIPTION OF PROBLEM |
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TOPSUMMARYDESCRIPTION OF PROBLEMEMBRYONIC DEVELOPMENT OF THE...EARLY POSTNATAL DEVELOPMENT OF...MICROBIOTA EFFECTS ON HOST...MICROBIOTA AND THE DEVELOPMENT...HEALTH AND PERFORMANCE EFFECTSCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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The concept of "metabolic imprinting" was proposed by Waterland and Garza [1] to describe adaptive metabolic responses to the influence of early nutrition that are then carried forward into the remainder of the life span, even after the initiating influence is gone. The authors listed 4 characteristics that can be used to identify imprinted adaptive responses: 1) a limited and early ontogenetic window of opportunity, 2) persistence of the effect through adulthood, 3) a specific and measurable outcome(s), and 4) a dose response or threshold relationship between the initiating factor and the outcome. Biological imprinting itself was first defined by Lorenz [2] to describe the memorization of filial relationships by neonatal birds. Lorenz proposed 2 criteria to identify imprinted influences: a critical period for 1) the exposure and 2) the persistance of the behavior. The additional constraints proposed by Waterland and Garza [1] serve to permit research into potential mechanisms associated with such an effect. On a molecular level, imprinted adaptation results in changes in gene expression associated with changes in DNA methylation, histone modification, gene conversion, and gene rearrangement [3–5]. The mechanisms that allow these changes to be inherited by daughter cells over the life span of the animal appear to be species and tissue related [6–9] and have not yet been fully described [10]. Of course, the possibility of transmitting such adaptive changes to the germ line and then to subsequent offspring has been the source of interesting debate for hundreds of years [11, 12] but is beyond the scope of this review.
To describe changes that may be the result of adaptive imprinting, it is necessary to know the state of the organism, tissue, or cell before the exposure to the conditions that effect changes in gene expression. Fortunately, in describing the impact of the microflora on the host, this prior state is simply the state of the GI system at birth, at hatch, or before conventionalization of a young, germ-free bird.
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EMBRYONIC DEVELOPMENT OF THE GI SYSTEM |
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TOPSUMMARYDESCRIPTION OF PROBLEMEMBRYONIC DEVELOPMENT OF THE...EARLY POSTNATAL DEVELOPMENT OF...MICROBIOTA EFFECTS ON HOST...MICROBIOTA AND THE DEVELOPMENT...HEALTH AND PERFORMANCE EFFECTSCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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Digestive Tract
The general direction of digestive tract development is anterior to posterior, with the fore-gut being the most differentiated at the time of hatch [17]. Although the bird has not ingested feed at the time of hatch, intestinal and pancreatic enzymes as well as nutrient transport capabilities are present [18]. Nutrient digestibility, however, is not fully mature at this time [19]. Ontogenetic changes occur, both before and after hatch, that include increased levels of pancreatic and intestinal enzymes [20–22], increases in overall GI tract surface area for absorption [23–25], and changes in nutrient transporters [26, 27].
Hatchling poultry undergo a fundamental change upon emergence from the eggshell. Simply put, the neonatal animal loses the protection of an isolated environment. During embryonic incubation, sterile yolk nutrients supply the needs of the bird and are delivered from the yolk sac via the bloodstream. It is clear that some of the biochemical changes associated with digestion and absorption occur before significant oral exposure to macromolecular nutrients. Indeed, expression of mRNA of brush border enzymes and transporters for carbohydrate digestion begins during incubation and peaks at incubation d 19 [27]. As hatch approaches, the residual yolk sac is internalized within the body wall [17]. Evidence has confirmed that near or at hatch, some of the residual yolk makes its way into the intestine via the yolk stalk, and thus provides digestible nutrients that may stimulate maturation of the digestive and absorptive functions of the intestine [28, 29]. Thus, all of this development is independent of contact with the gut microflora.
Gut-Associated Immune System
At the time of hatch, the primary immune organs, the thymus and bursa, are both present and populated by lymphoid cells. The migration of lymphocytes to the thymus occurs in several waves, beginning at d 6 of embryogenesis. The thymocytes are CD3+ (avian homolog) and develop CD4 or CD8 antigens during embryogenesis [30]. As in mammals, the development of the T-cell receptor specificity occurs primarily during embryological development and is therefore not influenced by the gut microflora. The bursa is populated by committed B lymphocytes that have already undergone heavy- and light-chain V, D, and J gene rearrangements to form the immature B-cell receptor complex [31, 32]. These committed but prediversified B cells migrate to the bursal mesenchymal anlage, and populations of those with productive V(D)J recombination are selectively expanded [32, 33]. The specificity of the prediversified receptor is not a factor in this selection, but the surface expression of an sIg receptor is required [32]. Thus, there is no requirement for the recognition of antigen—microbial or otherwise—in the development of avian embryonic B cells. At this time, the diversification of the variable region occurs by gene conversion [34] and a diverse, albeit antigen-naïve, population of B lymphocytes develops.
The effect of thymectomy or bursectomy on the development of the immune response is one indicator of its functional status at hatch. Neonatal thymectomy does not result in severe impairment of cell-mediated responses or the development of T-cell diversity, indicating a fairly high degree of development during embryogenesis [35, 36]. Bursectomy of the neonate results in an impaired adaptive response, particularly in the areas of isotype differentiation and development of antibody diversity [37]. Bursectomy as late as d 18 of incubation can result in loss of circulating IgG and IgA, leaving a primary IgM response of very limited diversity as the only humoral immune capability [38]. As the bird ages, bursectomy has less influence, indicating the existence of an ontogenetic window for bursa-dependent events.
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EARLY POSTNATAL DEVELOPMENT OF THE GI SYSTEM |
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TOPSUMMARYDESCRIPTION OF PROBLEMEMBRYONIC DEVELOPMENT OF THE...EARLY POSTNATAL DEVELOPMENT OF...MICROBIOTA EFFECTS ON HOST...MICROBIOTA AND THE DEVELOPMENT...HEALTH AND PERFORMANCE EFFECTSCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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GI Microbiota
Although the gut of birds is theoretically sterile at hatch [41], colonization proceeds immediately. Under normal circumstances, the GI lumen becomes populated rapidly by bacteria from hatchery waste or other hatchery-related microbial populations [42, 43]. Therefore, in modern production situations, birds are already populated by microorganisms before arriving at the production house. Provision of nutrients, particularly carbohydrates, is necessary to encourage colonization by saccharolytic organisms, rather than protein-fermenting putrefaction organisms [42]. The provision of an acid supplement early in life encourages expansion of acid-producing bacteria such as lactobacilli and can reduce the shedding of Salmonella [44].
The resident GI microbiota consists of approximately 400 known species, of which 20 to 40% can be cultured [15]. The availability of molecular techniques for identifying nonculturable species has opened new avenues of research into the factors that affect this complex mixture of organisms, as reviewed recently by Richards et al. [45]. Its development occurs through a series of colonization steps that are similar across species [46]. Generally, the aerobic and facultative organisms, such as coliforms, lactobacilli and streptococci, colonize first [46–48]. These organisms are thought to lower the oxidation reduction potential in the intestine, which allows the subsequent colonization by anaerobes such as bifidobacteria [46]. Both longitudinal and horizontal variation in host niches is available for colonization [46]. The longitudinal organization is based on differences in lumen pH and nutrient availability, with lower pH and higher nutrient availability in the apical compared with the distal segments. In the horizontal organization, there are 4 microenvironments: the intestinal lumen, the apical unstirred mucus layer, the deep mucus found in association with the crypts, and the epithelial cell surface itself [46]. Recently, some interesting work on the reciprocal colonization of gut microbiota of zebrafish and mice indicates that the foreign microbiota is tolerated by the host, but that the host niches determine the relative abundance of each species [49].
In poultry, enterococci and lactobacilli are the dominant species in the crop, duodenum, and ileum during the first week of life, whereas coliforms, enterococci, and lactobacilli are present in high numbers in the ceca [50–53]. Subsequently, a highly complex group of mostly obligate anaerobes begins to take over the ceca, whereas lactobacilli take over the crop, duodenum, and ileum [54]. After 2 to 3 wk, the intestinal microflora is established and stable [53, 55]. The obligate anaerobes of a stable climax hindgut microflora consist of bifidobacteria, Clostridium, and Bacteroides, living in high population densities in the cecal pouches and colon, where low nutrient concentration controls their rate of growth [42, 56, 57]. This diverse population, which still includes lactobacilli and such facultative organisms as Escherichia coli and streptococci, can be very effective in excluding newcomers, whether pathological or innocuous. However, the equilibrium of the microbiota is dynamic, responding to its environment, including changes in availability of gut secretions and dietary composition [58–60].
Gut-Associated Immune System
In contrast to the digestive tract, the gut-associated immune system develops first in the distal intestine [61, 62]. At the time of hatch, some of the immature B cells, expressing surface IgM, migrate to peripheral organs such as the cecal tonsils and Meckel’s diverticulum, where immature T cells are also present [63]. At this stage, when the animal first experiences oral exposure to feed and microbial antigens, conditions in the gut favor the development of tolerance more than immunity [64]. Interestingly, tolerance cannot be induced for any antigen to which a maternal antibody is directed [65], a condition that ensures that pathogen specificities in the nascent microbiota are not tolerogenic to the neonate.
There is a second wave of lymphocyte migration to the gut at 4 d posthatch [66], which consists of cells capable of cytokine expression and effector function. Conditions now favor the development of immunity upon exposure to microbial or soluble protein antigens [64]. The fact that the responding immune tissue is in the hindgut means that most soluble protein antigens would be digested at this point and are thus no longer antigenic. Microbial antigens, however, are abundant in the hindgut. It is at this time that the interaction of environmental antigens with enteric and immune systems begins to become essential for further maturation. The ontogeny of gut-associated immune competence has been the subject of a recent review [61].
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MICROBIOTA EFFECTS ON HOST CELL FUNCTION |
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Digestive System
Digestive Tract.
There is abundant evidence that a commensal microflora affects the structure and function of the digestive tract. Much of this evidence is from germ-free animals, including chickens. Germ-free animals have been demonstrated to have a reduction in gut size, including thinner intestinal villi and a thinner total gut wall [67]. This may be due, in part, to the reduction of trophic luminal short-chain fatty acids that are the product of microbial fermentation [68, 69]. Villi tend to be thinner and shorter but, despite that, absorption and efficiency tend to be greater [70]. The reductions in gut wall thickness and villus lamina propria density as well as the reduction in basal metabolic rate have been used to explain the enhanced nutrient digestibility seen with germ-free conditions (reviewed by Visek [71] and Furuse and Okumura [72]). Upon introduction of microflora, measurable increases occur in gut weight, villus height, and cell turnover [73].
Enteric Cells.
The fact that the enteric epithelium has a shorter half-life in the presence of a commensal microflora is well established [70]. Whether all cellular components of the enteric epithelium are equally affected is unknown. As previously described, the enteric epithelium is generated as functionally immature daughter cells of proliferative crypt stem cells. Among the factors that influence the direction their differentiation takes are the microorganisms they encounter as they migrate into the intestinal milieu. One well-documented response is that of mucin secretion, which has recently been reviewed by Deplancke and Gaskins [74] and by Liévin-Le Moal and Servin [75].
Mucin plays multiple roles in the microflora-host relationship [75, 76]. It serves to protect the epithelial cells from attachment by pathogens, to provide sites of attachment by commensal bacteria and probiotics, and to provide a readily available nutrient source to mucolytic organisms (recently reviewed by Deplancke and Gaskins [74]). Both host and microbiota influence the rate and nature of mucin synthesis, and this can be readily demonstrated by using germ-free animal models [74]. For example, when lipopolysaccharide was administered to germ-free rats, an increase in neutral mucin secretion by goblet cells of the colon was triggered [77]. This was followed by enhanced attachment of a commensal strain of E. coli in the small intestine. Another example of the role of mucin in microbial ecology is that observed by Bry et al. [78], who also used a germ-free animal model. They observed that exposure of germ-free mice to a pure culture of Bacteroides thetaiotaomicron was followed by enteric cell production of fucosylated glyco-conjugates, a readily digested substrate for B. thetaiotaomicron’s own metabolism. In research using chickens, Smirnov et al. [79] observed that the inoculation of a dietary probiotic consisting of lactobacilli and bifidobacteria increased the expression of mucin mRNA by 160% in the jejunum. Other research using in vitro systems has also demonstrated the induction of mucin gene expression by probiotic Lactobacilli [80, 81].
Another approach to identify effects of the microbiota on the host was reported by Hooper et al. [82]. This group colonized germ-free mice with the commensal B. thetaiotaomicron and used DNA microarrays to identify the global transcriptional response to its colonization in the gut. Several of the mRNA transcripts that were increased by 2-fold or more were related to lipid metabolism in the host, whereas others were associated with trace mineral uptake. The increased mineral and lipid uptake provides a mechanism that may explain the observation that colonization with B. thetaiotaomicron improves host nutrient absorption and use [83]. Another study using the same microorganism to colonize germ-free mice found that the microbial colonization induced angiogenesis in intestinal villi, and that this was mediated by Paneth cells in host intestinal crypts [84]. The effects of microbiota and Paneth cells were additive, with full villus vascular development requiring both components of the system.
Finally, the introduction of microorganisms is associated with the synthesis and secretion of a variety of products characteristic of the innate and adaptive immune responses. For example, activation of enteric cell Toll-like receptors and nucleotide-binding oligomerization domain proteins by bacterial components such as lipopolysaccharide and muramyl dipeptide results in secretion of interleukin (IL)-8 by epithelial cells and antimicrobial proteins (AMP) by Paneth cells [85–87]. Interestingly, one class of AMP proteins, cryptdins, stimulates further IL-8 secretion by an intestinal cell line in vitro by a paracrine mechanism that may provide a positive feedback loop for controlling bacteria that manage to penetrate the mucin layer and advance into the crypt [88]. Interleukin-8 is a proinflammatory cytokine that results in leukocyte infiltration. Both IL-8 and AMP are a part of the initial innate response of the host GI system to potential pathogens. This response provides nonspecific control of bacterial replication and invasion until the more specific and less energetically costly [89, 90] adaptive immune response can be mounted [91, 92]. In another report, Farnell et al. [93] described an increase in the degranulation of chicken heterophils by exposure to probiotic bacteria both in vitro and in vivo. Finally, the introduction of an isolated culture of segmented filamentous bacteria, normal commensal bacteria in mice, induces the cell-surface expression of major histocompatibility complex class II molecules on ileal epithelial cells [94]. In the same study, monoassociation of the germ-free animals with Clostridium did not trigger major histocompatibility complex expression. Thus, the microbiota stimulates host defense mechanisms whose function is to control their location and their numbers.
These examples clearly show that the intestinal microbial community affects host cell gene expression and that this can occur immediately after hatch. It is unlikely, however, that this could be described as an imprinting process, because it does not appear to be limited to a window in early postnatal time and many of these responses may be reversible. In addition, it is widely accepted that the differentiated gut epithelial cells are nonproliferative and would therefore not have the opportunity to pass the information on to daughter cells.
Secondary Gut-Associated Immune Tissue
Immune Tissue.
The essentiality of the microflora for the development of the gut-associated lymphoid tissue (GALT) in the chicken was revealed many years ago by studies in germ-free animal models and in bursectomized or bursal duct-ligated chickens [95–97]. Germ-free animals, avians and mammals alike, exhibit delayed lymphocyte and other immune cell development in the lamina propria and fewer IgA-producing cells when compared with conventionally reared animals [76, 94, 98–101]. It should be noted that in germ-free chickens, bursa, thymus, and spleen weights do not differ from those of conventional animals [96, 97]. The primary difference noted in germ-free chickens is a reduction in the amount of tissue in the cecal tonsils [96] and the low number of germinal centers and plasma cells seen in the lymphoid tissues of the ileocecal junction [97]. Lymphoid follicles of the cecal tonsils in germ-free chickens were reported to contain no germinal centers or IgA- or IgG-positive cells [102]. The importance of this observation rests in the fact that additional immune diversity is generated during the proliferation associated with class-switch recombination in germinal centers of the peripheral immune system [103]. Both gene conversion and hypermutation have been detected in the germinal centers of secondary immune tissue in chickens [104].This additional diversity, following selection by antigen binding and affinity maturation, yields memory B cells. Therefore, the mechanism for final affinity maturation appears to resemble that seen in mammals [7–9].
Immune Cells.
The consequences of a germ-free existence include effects on the primary immune effector cells of the gut-associated adaptive immune system, T and B lymphocytes. For T lymphocytes, the numbers of 
β T cells are markedly reduced in germ-free mice, but this effect is completely reversible when the mice are subsequently contaminated with normal GI microflora [100]. Interestingly, 
T-cell numbers are not affected by germ-free conditions [100]. For the humoral immune system, particularly to orally administered antigen, development of isotype switching, antibody diversity, and affinity maturation in poultry is in some way inhibited by germ-free growth conditions [37, 105, 106]. In mammals, introduction of even a single species of commensal bacteria into germ-free mammals can stimulate the development of the secretory IgA system [76, 94, 107]. Indeed, the IgA secreted across the intestinal mucosa represents approximately 70% of the total antibody production [108]. Typical adult humans secrete more than 5 g of IgA per day, most of which binds to intestinal microbes and to dietary antigens [109, 110]. These observations have led to decades of research intended to clarify the nature of the microbial-host interaction that results in full mucosal immune maturation.
The effects of the commensal microflora on the host humoral immune system are not inconsistent with the requirements of imprinting proposed by Waterland and Garza [1]. For induction of tolerance in chickens (the subject of another paper in this symposium and not discussed in detail here), a restricted window of opportunity has been demonstrated, the effect is responsive to antigen form and dose, and the changes are long term and measurable [64, 65]. The effect may not be permanent, however, because there are data showing that eventual loss of tolerance can occur [61, 65].
Regarding the development of immune sensitization in the gut of the bird, the cellular biology in birds is not yet completely understood but is under very active investigation. Recall that surface expression of an Ig receptor appears to be required for selective expansion of preimmune bursal lymphocytes, but the signal does not appear to require antigen binding [32]. During this expansion in birds, diversity is created through gene conversion, a DNA recombination process which involves intrachromosomal, non-reciprocal transfer of nucleotide sequence blocks [111, 112] through an activation-induced cytidine deaminase-dependent mechanism [113, 114]. The characteristics of avian B cells and generation of diversity by gene conversion are reviewed by McCormack et al. [34].
In birds and mammals alike, the preimmune diversity expressed during embryonic development does not appear to require contact with microorganisms. In birds, changes in bursal structure at the time of hatch appear to favor the contact between the gut microflora and the bursal lymphocytes [95]. Specifically, the bursal epithelium differentiates into cells that resemble the follicle-associated epithelium of Peyer’s patches [63], and cells resembling M cells have been observed to trap antigen delivered to the bursa via the bursal duct or the cloaca [115, 116] and to transport it to the medullary area of bursal follicles [32]. The resulting antigen-bursa interaction can be disrupted by bursectomy (chemical, hormonal, or surgical), bursal duct ligation, or bursal ectopic grafting. All of these methods have been used to study the role of contact between microbial antigens and the bursa in the development of the immune system.
Thompson and Cooper [117] reported that ectopic grafts of bursal rudiments into the abdominal wall become normally populated with lymphocytes during embryogenesis but involute following hatch, and they postulated that proximity to the contents of the intestinal lumen is essential for bursal function. Birds undergoing bursal duct ligation in ovo (embryonic d 18) were observed to have reduced antibody diversity, fewer IgA- or IgG-positive plasma cells [118], and an undetectable antibody affinity maturation following repeated vaccination [119]. Similarly, birds in which bursal development was arrested by testosterone propionate also fail to develop IgG [120]. In these and other studies, the amount of IgM was not affected by bursal ablation or by germ-free conditions but IgG, IgA, or both were affected [35, 94, 98–101, 121, 122]. Interestingly, later work in which Ig diversification was measured in primary cells and cell lines, and using 2-dimensional gel electrophoresis rather than antigen binding per se, has indicated that light-chain molecules are highly diversified in ovo [123, 124]. Because this diversification occurred during embryonic development, and was not affected by bursal duct ligation, it presumably was independent of antigen presence-, specificity-, or affinity-driven maturation. When the bursa was repopulated after cyclo-phosphamide treatment, follicular cells derived from single B-cell precursors were able to produce a broad spectrum of light-chain diversity, again as assessed by 2-dimensional gel electrophoresis, suggesting that a few bursal stem cells can generate as much potential antigen-binding diversity as the entire bursa of a nontreated bird [123]. It should be noted, however, that exposure of the bursa to antigens derived from in ovo oral vaccination [125] or in the neonate from feed, its associated microflora [44, 126], or intracloacal vaccination [127] are all associated with more rapid development of the peripheral or mucosal immune systems compared with the respective antigen-free controls. This neonatal developmental difference may be related to the reduction in B-cell proliferation associated with embryonic vitelline duct ligation [128] or bursal duct ligation [129], because embryonic development of antibody diversity in bursal cells is also dependent on B-cell division and the associated gene conversion [34].
As has been described previously [130], the progressive transition from IgM to IgA in the germinal centers of the cecal tonsils during the early postnatal period suggests that these are important secondary immune organs for the final maturation of IgA committed B cells in chickens. Support for this hypothesis can be found in the work of Yasuda et al. [131] comparing the microscopic characteristics of chicken and calf secondary immune tissue. Their studies suggest that the ileal germinal centers of the calf appear to be analogous to the bursa, with no indication of endogenous Ig class switching to IgG, whereas the germinal centers of the cecal tonsils in the chicken appear to be analogous to the jejunal Peyer’s patches of the calf. In the latter tissues, the proximity of CD4+T cells would facilitate Ig class switching to IgA, which has not been observed to occur in perinatal bursal follicles [132]. Although the occurrence of bursal lymphocytes stained for both IgG and IgM in the perinatal period was reported by Kincade and Cooper [133], subsequent work demonstrated that the IgG in such cells was not synthesized by those lymphocytes but represented Ig from the intestinal lumen. This IgG may represent immune complexes of antigen and maternal antibodies, and may play a role in preventing the development of immune tolerance to pathogen-associated antigens [65].
As the bird ages, bursectomy has less and less effect on the ability of the bird to mount a humoral response [134]. The ontogeny of this phenomenon was studied by Toivanen and Toivanen [135], who described the ability of bursal B cells to restore immune function of birds treated with cycophosphamide. They characterized the bursal stem cell as a B cell able to restore bursal morphology but incapable of further maturation without the bursal microenvironment. Furthermore, they demonstrated that the influence of the bursal microenvironment required cell-to-cell contact and would not occur if the bursal mesenchyme was enclosed in a diffusion chamber. However, once this step has occurred and the postbursal stem cells have migrated and populated the peripheral immune tissue, the bursa is no longer essential for further B-cell development [136]. Essentially, as the role of the bursa in B-cell differentiation diminishes, it becomes and remains a secondary immune organ, in which class switching and protective Ig synthesis do occur [61]. This change in function, which is gradual and age dependent, may be the cause of some conflicting results in which secretion of Ig by bursal lymphocytes has been observed by researchers using 4- to 6-wk-old chickens [137], but not by others using 1-wk-old chickens [138].
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MICROBIOTA AND THE DEVELOPMENT OF GUT IMMUNE ECOLOGY |
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TOPSUMMARYDESCRIPTION OF PROBLEMEMBRYONIC DEVELOPMENT OF THE...EARLY POSTNATAL DEVELOPMENT OF...MICROBIOTA EFFECTS ON HOST...MICROBIOTA AND THE DEVELOPMENT...HEALTH AND PERFORMANCE EFFECTSCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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The final diversification and affinity maturation of chicken B cells in germinal centers was studied by Arakawa et al. [103], who reported that, following stimulation by antigen, postbursal B cells are able to generate somatic variants in splenic germinal centers. The size of these germinal centers was maximized by d 7 of the primary response and had begun to wane by 14 d. When individual germinal centers were isolated and the nucleotide sequences of the Ig L-chain analyzed [104], the early germinal centers (d 7) contained both gene conversion and point mutation-generated diversity, whereas the late germinal centers (d 11) showed that further diversification occurred primarily through point mutations, and that novel gene-conversion events were very rare.
Taken together, these data suggest that the wide primary repertoire is generated by gene conversion in the bursa, during selective proliferation for productive V(D)J recombination [33]. This may be influenced by antigen exposure, for example, as described by Rhee et al. [142] for germ-free-appendix rabbits or as described by Weill and Reynaud [143] for GALT B cells. Once the B lymphocytes have migrated to the secondary immune tissues of the gut, B-cell proliferation in germinal centers drives further diversification, class switch recombination, and affinity maturation. This would require antigen presentation to B cells by dendritic cells at some point [31, 104].
The work described above suggests that immune maturation is influenced by the microbiota but does not prove that this process is restricted in time, one of the key criteria for imprinted effects [1, 2]. A missing experiment that would be needed to confirm the presence of an ontogenetic window of opportunity would be the demonstration that full maturation of the gut-associated immune system after exposure of a germ-free bird to gut microflora is an age-restricted phenomenon. Thus, if exposure of an adult germ-free bird to microflora did not restore IgG or IgA levels, full BCR-binding diversity, or germinal center activity in secondary immune tissue to those of conventional birds, then the role of the microbiota in these maturation processes could be shown to be restricted in time, which is currently a missing piece of data. Experimentally, it would be particularly interesting to test monoassociation by using individual bacterial species such as Bacillus subtilis, Bacteroides fragilis, B. thetaiotaomicron, or segmented filamentous bacteria, all of which have been shown to have the potential to restore full GALT development in previously germ-free mammals [15, 82, 94, 142, 144]. This would be an important step in clarifying the role of the gut microflora on avian gut immune development.
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HEALTH AND PERFORMANCE EFFECTS |
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TOPSUMMARYDESCRIPTION OF PROBLEMEMBRYONIC DEVELOPMENT OF THE...EARLY POSTNATAL DEVELOPMENT OF...MICROBIOTA EFFECTS ON HOST...MICROBIOTA AND THE DEVELOPMENT...HEALTH AND PERFORMANCE EFFECTSCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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In murine models of Crohn’s disease, development of gut inflammation is eliminated by raising the animals in germ-free conditions [157–159]. For example, IL-10 gene-deficient mice develop chronic enterocolitis unless they are raised under germ-free conditions. Neonates from this genetic line show altered GI microbial colonization within 24 h of birth [160]. Interestingly, the development of inflammation can be eliminated by treating these neonates with a lactobacillus probiotic [160]. In addition, the treatment of IL-10 gene-deficient neonates with antibiotics prevented the development of colitis for up to 12 wk after the therapy ended [161], suggesting that treatment during a period early in life may affect the later development of inflammatory disease in genetically susceptible individuals.
In poultry, the observation that a stable colonized microflora can reduce the effects of pathogens introduced later in life is well recognized [162]. Establishment of the stable or climax microflora is thought to benefit health by reducing the likelihood of growth of nonnative species, originating either from the environment or from other intestinal niches within the same animal [46, 58, 59]. For example, the presence of a lactic acid-producing microflora in the small intestine reduces the growth of acid-intolerant species normally found in the cecum. One strategy for encouraging rapid colonization of lactic acid-producing organisms is the provision of organic acids in the feed or water of neonatal poultry. There is evidence that the acid needs to be present during the establishment of the microbial population (i.e., during the first 2 wk posthatch) to achieve optimum control of pathogens such as Salmonella [163].
In poultry, enteric disease has become one of the most significant sources of production inefficiency, morbidity, and mortality [164, 165]. Diseases range from relatively benign conditions such as feed passage, to more severe problems such as runting and stunting, and finally to fatal clostridial diseases such as necrotic enteritis (NE), caused by Clostridium perfringens. Although clostridia are normally found in the gut microflora of poultry, they are most common in the lower gut, particularly in the ceca, where nutrient type and concentration control their rate of growth [56, 57]. However, the equilibrium of the microbiota is dynamic, responding to many internal environmental factors, including nutrition [59, 60].
Because retrograde peristalsis is essentially continuous in poultry [166–168], clostridia can be carried from the colon, cecum, or ileum into the jejunum at any time and enter a more rapid rate of growth in response to the nutrients available there. One factor that prevents this from occurring under normal circumstances is the presence of a dense, stable, lactic acid-producing gut microflora in the jejunum. This keeps the pH below neutral, which reduces the likelihood of dominance by acid-intolerant species such as Clostridium. However, an unstable microflora or the presence of subclinical enteritis can disrupt the ecosystem, raising the pH and increasing the rate of mucin production by goblet cells, which would favor an overgrowth of Clostridium [58, 59, 76]. Other factors that can lead to clostridial overgrowth include a rapid rate of digesta passage through the upper small intestine, particularly if the bird is consuming a high-protein diet. The increase in amino acid availability in the lower gut could favor clostridial growth [58, 59].
The effect of providing antimicrobial organic acids during the colonization and challenge period (1 to 21 d) on NE incidence and severity has been reported by Hofacre [169]. The model system used in this experiment combined coccidial (d 14) and clostridia (d 18, 19, and 20) challenge to generate NE in broilers. Among the challenged treatments, the presence of an organic acid blend (ACTIVATE WD) [170] gave lesion scores and performance significantly better than those of the untreated birds. In addition, animals given the organic acid blend had NE lesion scores that were not significantly different from those of the nonchallenged control birds. It is possible that providing an exogenous source of acid was a factor discouraging overgrowth of Clostridium in the small intestine, but it may also have simply favored the establishment and maintenance of the normal acid-tolerant microbial flora in the crop, gizzard, and upper small intestine. Thus, the use of probiotics, acids, or other gut environment modifiers can play a role not only in the rapid establishment of a stable microflora, but also in returning the gut ecosystem to a more stable balance after a challenge, restoring its ability to maintain the complex immune and microflora relationships without invoking an inappropriate inflammatory response.
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CONCLUSIONS AND APPLICATIONS |
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TOPSUMMARYDESCRIPTION OF PROBLEMEMBRYONIC DEVELOPMENT OF THE...EARLY POSTNATAL DEVELOPMENT OF...MICROBIOTA EFFECTS ON HOST...MICROBIOTA AND THE DEVELOPMENT...HEALTH AND PERFORMANCE EFFECTSCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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FOOTNOTES |
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REFERENCES AND NOTES |
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TOPSUMMARYDESCRIPTION OF PROBLEMEMBRYONIC DEVELOPMENT OF THE...EARLY POSTNATAL DEVELOPMENT OF...MICROBIOTA EFFECTS ON HOST...MICROBIOTA AND THE DEVELOPMENT...HEALTH AND PERFORMANCE EFFECTSCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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