REVIEW
ONTOGENY OF GUT ASSOCIATED IMMUNE COMPETENCE IN THE
CHICK
Bar-Shira E., and Friedman A.*
Section of Immunology, Department of Animal Sciences, Faculty of Agricultural,
Nutritional and Environmental Sciences, Hebrew University of Jerusalem, Rehovot, Israel
Address for correspondence: Aharon Friedman, PhD Department of Animal Sciences Faculty of
Agriculture
POB 12 Rehovot 76100, Israel.
Phone: 972-8-9489027 Fax: 972-8-9489869 Email: friedman@agri.huji.ac.il
Summary
To accommodate the rapid transition to external nutrients, the chick’s gastrointestinal tract undergoes dramatic changes within the first few days of life.
These include a rapid increase in mass, villus number and length, enterocyte number, crypt depth and proliferating cells. A rapid development of the gut associated lymphoid tissue (GALT) occurs concomitantly with the development of digestive structures and functions. This lymphoid system functions within and in concert with digestive tract parenchyma, however, there is little information describing the normal development and immunological function of the avian GALT in the immediate post-hatch period. The purpose of this review is to summarize current knowledge on the structure and function of the avian GALT during the early post-hatch period. At hatch, the gut is poorly populated by both innate immune leukocytes and lymphocytes. The basal numbers of lymphocytes are the result of early waves of cells migrating in embryo from the thymus and bursa of
Fabricius. Further waves of lymphocyte migration occur after 4 days of life and continue intermittently with time. Adaptive immunity develops in concert with this pattern of lymphocyte population. Hence, the gut of the hatchling is unprotected against colonizing microorganisms by adaptive immunity during the first few days of life. Protection during this critical period might be the result of maternal antibody activity or that of the innate immune system. This system appears to be functional at this time, though much work is needed to establish this possibility.
Upon maturity of the immune system, most of the immunological activity within the chick GALT is concentrated in the hind gut, specifically in the caecal tonsils and bursa of Fabricius. Once immune responses have become established the relevant cells disseminate systemically and to other areas of the small intestines.
Finally, observations on the beneficial effects of early feeding on development of gut and GALT are discussed with reference to management of hatchlings.
Keywords:

Gut associated lymphoid tissue (GALT), immune system, mucosal immune response, digestive tract, chick, ontogeny, post-hatch.
Preface
Mucosal membranes are the animal's largest interface with the outer world and are major entry sites for foreign antigens some of which might be harmful. To protect the animal against invasion of harmful antigens/pathogens, an elaborate defense mechanism has developed throughout the mucosae. This defense mechanism is comprised of physical and cellular barriers including the epithelial lining and its associated mucus layer, and the mucosal associated lymphoid tissues (MALT). The general histological features of the mucosal barriers are identical within various mucosal tissues; however local specialties as required by the tissue's anatomical location and physiological function exist. On this basis the immunological barrier of
MALT was divided into a number of subdivisions: GALT - the gut associated lymphoid tissue, BALT - the bronchus associated lymphoid tissue, NALT - nasopharyngeal lymphoid tissue as well as the salivary and thegenitourinary lymphoid tissues. In the chicken MALT also includes the head associated lymphoid tissues of the Harderian gland and the conjuctiva. GALT, a major component of MALT, is comprised of an intricate infrastructure of organs and immune cells residing within the epithelial layer and the underlying lamina propria. GALT is comprised of several types of cells including specialized inducer, immunoregulatory, and effector cell types distinct from their counterparts in the systemic immune system (1). In the chicken, which is devoid of mammalian type lymphoid nodes, GALT and the spleen are the major sites for generation and induction of immune responses. Therefore investigation of GALT development and its functional maturation are fundamental in understanding immunological phenomena exclusive to the avian species such as immune responses to oral soluble protein antigens or induction of tolerance at young ages (2-4). The knowledge obtained by such studies may be utilized for vaccine development and improvement of animal health and welfare.
In contrast to the vast knowledge of avian GALT responses to intestinal pathogens and infections, there is a paucity of information describing the normal development of immunological function in the avian GALT during the immediate post-hatch period.
The purpose of this review is to summarize current knowledge on the integration between development and maturation of GALT in the developing intestinal tissue during the early post-hatch period
Gross anatomy and histology of the gastrointestinal tube (GIT) and GALT in chicks
GIT - Longitudinal Organization
The digestive tract of the chick includes the esophagus (gullet) that conveys food from the mouth to the stomach, the crop, an expansion of the esophagus, located in the lower neck area, the glandular stomach (proventriculus), and the muscular stomach (gizzard). The small intestines are comprised of a duodenal loop enclosing the pancreas and a jejunum and ileum. The end of the jejunum is defined by the
Meckel's diverticulum (MD), a remnant of the attachment of the yolk stalk. At the end

of the ileum muscular ileo-cecal valves are present at the entrance to two prominent ceca which discharge into a short large intestine which empties into the cloaca.
GALT- Longitudinal organization
The GALT, in contrast to other immune systems associated with lumens, is confronted with two types of antigenic molecules: a) innocuous antigens, namely those that are basically nutrients and as such should not evoke immune responses. b)
Antigens derived from intestinal or external pathogens that should evoke protective immune responses. Hence, the balance between response and tolerance in the gut is finely tuned and depends to a great deal on the interaction between immune cells and those of the gut parenchyma. In a broad sense it might be argued that any antigenic molecule that is absorbed via enterocytes (intracellular transcytosis) is tolerogenic, whereas any antigenic moiety that penetrates the intestinal lining, either via transcellular pathways or via phagocytic lining cells (i.e. M cells) is immunogenic (5,
6).
The chicken GALT is organized as scattered immune cells located in the epithelial layer of the GIT and the underlying lamina propria (7, 8); additional lymphoid aggregates and structures are located at several locations along the alimentary tract.
The upper segment of the intestinal tract leading to the gizzard is poor in lymphoid structures except for the esophageal tonsil located at the junction of the esophagus and proventriculus (9) and lymphoid aggregates located in the proventricular lamina propria (10). Scattered Peyer's patches were reported to appear distally in the gizzard towards the ileocecal junction(11, 12) as well as lymphoid accumulations at the MD
(13). Lymphoid follicles are abundant in the ceca; besides the major lymphoid follicles, known as cecal tonsils (CT) located at the proximal region of the ceca ,
Kitagawa et al reported the presence of numerous lymphoid nodules along the ceca, the majority of which were located in the apical region(14, 15). These authors also found numerous lymphoid nodules at thececal apex a finding which was supported by others (16). The colon is devoid of lymphoid follicles, but these become abundant again in the canal leading to the cloacal bursa, a primary and secondary lymphoid organ located in the proctodeal region of the cloaca . The mucosal and submucosal regions of the bursal canal are heavily populated by lymphoid follicles (17). Solitary lymphoid nodules are also found at the proctodeum and urodeum (12)
The distribution pattern of lymphoid tissue along the chicken gut is not surprising.
In the chicken, most contact with microflora occurs in the distal intestine. This is due to the fermentative nature of the ceca (14) and to the influx of bacteria via the cloaca by means of retrograde peristalsis (18). The retrograde movement of the cloaca and colon has been traditionally explained as a means to extend water reabsorption from kidney secretions excreted to/via the cloaca. However, we believe that this movement also serves two immunologically relevant functions: a) it is a means to absorb antibodies secreted via the bursal canal and that originate from the bursa of Fabricius and bursal canal lymphoid follicles, and b) it serves to sample external bacteria via the rectum.
In accordance with this pattern, development of lympoid follicles in the avian gut was shown to be associated with gut colonization by microflora (19, 20). Thus, the chicken’s foregut is relatively poor in lymphoid tissue organized as follicles (12).
Upon transition to the hindgut, at the ileal-cecal junction, numerous lymphoid follicles appear. The bursal canal has the structure of the alimentary canal: columnar epithelium underlain by lamina propria, submucosa (containing exocrine glands), and most importantly - muscularis propria the function of which is to propel bursal

secretions towards the cloaca (Adlerstein and Friedman, unpublished observations).
The mucosal and submucosal regions are heavily populated by lymphoid follicles.
Intraepithelial lymphocytes and lymphoid cells of the lamina propria are abundant throughout the gut as well as innate leukocytes (7, 8).
The convergence of the bursal canal with the spaces between the bursal manifolds extends the conceptual function of the bursa of Fabricius. While the bursa has been conventionally regarded as a differentiation organ for B lymphocytes, compelling evidence from our research and that of others shows the bursa to function also as a peripheral lymph node.
GIT and GALT- Cross-Sectional organization
The luminal intestinal lining has a villus-like structure with differing dimensions according to gut section. The villus lining is covered by simple columnar absorptive brush border epithelium, and secretory goblet cells. This lining has two compartments: a) the villus proper, the lining of which is heavily populated by intraepithelial leukocytes, and b) the crypt region - which is a region for maturation and differention of enterocyte, goblet, enteroendocrine and Paneth cells (not yet described in the chicken (21, 22)). The lining is protected externally by mucosal secretions and from within by penetrating IEL. The IEL of the chicken are a diverse population of lymphocytes including NK (23), TcRgd and TcRab(7) as well as Bu-1 bearing cells (though not classical B cells) (8) and may also include heterophils as is observed inthe ceca (Bar-Shira and Friedman unpublished observations ). The IEL major T cell populations are further divided according to the T cell co-receptors CD4 and CD8. CD8 subset is the major population in the chicken IEL, while CD4 subset is considered a minor population (24). Collectively this is mostly an innate population that affects immunity by immediate release of cytokines following activation; the antigen specificity of these cells in the chicken has yet to be determined. The crypt region in the mammal contains Paneth cells capable of secreting lysozyme, defensins and other anti-bacterial substances (25, 26). While epithelial defensins have been described in avian species including the chicken (27, 28), the cell type responsible for their secretion is presently unknown, as Paneth cells appear to be scarce in birds (29).
The main source for antibacterial substances in chicks might be macrophages or heterophils rather than typical mammalian-type Paneth cells (27, 30, 31). The lamina propria, as in mammals, contains a mixture of immune cells of all types, including plasma cells, effector T lymphocytes and memory lymphocytes, macrophages and granulocytes (24, 32, 33).
Peyer’s patches are not a hallmark of the chicken GALT. A few have been described along the small intestine; however, most lymphoid follicles can be found along the ceca, urodeum, proctodeum (12) and bursal canal (Adlerstein and Friedman unpublished observations). Though smaller in size, their structure is reminiscent of the mammalian Peyer’s patch: specialized lympho-epithelium containing M cells, underlying macrophages and dendritic cells (34-36), follicular structure-lymphocyte rich T- and B- niches in which the cells undergo division and differentiation. The marginal zones of the follicles contain macrophages and effector lymphocytes of all types (32).
Post-hatch Development of the Intestinal Tract in Chicks
GIT- Development
At hatch endogenous yolk is the only nutrient source in chicks and poults. After clearing the shell birds start pecking and learn to associate pecking with ingestion and

feeding by day 3 post-hatch (37). Efficient utilization of exogenous feed is subject to the development of the required intestinal structures and functions. In the immediate post-hatch period the intestines are subjected to vast morphological and functional changes which result in an increased intestinal absorptive surface and relative mass
(38-43).
It appears that the avian intestines grow in direct proportion to the age-related increases in metabolic rates. Furthermore, in avian species, patterns of intestinal growth appear to be correlated with patterns of whole-body growth rates. King et al suggested that in these species, including the chicken, rapid intestinal hyperplasia is a prerequisite for sustained rapid posthatch growth (44).
In the immediate posthatch period dramatic changes occur in the chick gastrointestinal tract. Small intestines and ceca increase in weight more rapidly than the whole body mass and this rapid relative growth was maximal at 6-10 d in the chick (45-47). The preferential early growth of the intestines occurs both in thepresence and absence of feed although in the absence of exogenous feed both absolute and relative growth is lower (48). Temporal increases in intestinal weight and length are not identical in the different segments of the small intestine (49) .
At hatch the small intestinal villi are small and crypts are not detectable in the intervillar spaces. In the first hours post-hatch crypts begin to form and become well defined by 2-3 d. The number of crypts increases rapidly after hatching and this reaches a plateau after 48-72 h post-hatch. The crypts increase in size as estimated by the number of cells per crypt in the 48 h post-hatch in the fed chick and growth rate plateaus after 48 h (50). Immediately post-hatch enterocytes increase rapidly in length and develop a pronounced polarity (41). Changes occur in absorptive surface in small and large intestine (51) reaching a plateau within 2 weeks post hatch (50). Patterns of development differ between intestinal segments; the duodenum and jejunum continue to develop after the ileum has reached a constant number of crypts per villus (50).
Changes are also observed in enterocyte differentiation, proliferation rate, the proportion of proliferating cells within the villi as well as dynamics along the cryptvillus exis (41, 50, 52).
Thus the extensive changes in the morphological development close to hatch include the basic differentiation of enterocytes and crypt definition as well as a multifold increase in the intestinal absorptive surface area. These intensive changes are stimulated by nutrient supply and microflora (39, 53)
Intestine functional maturation
Uptake of nutrients by the small intestine occurs after hydrolysis of macromolecules initially by gastric, then pancreatic and finally brush border hydrolases. Initiation of gastric and pancreatic secretions occurs before hatch and increases with feed intake post-hatch. In contrast, brush border enzymes appear to have different temporal patterns of development before and immediately post-hatch. Thus lipids are well absorbed close to hatch whereas uptake of glucose and methionine increases after hatch and is stimulated by intake of feed (39). In addition it is proposed that functional development of the intestine as a digestive and absorptive organ is closely related to its development as a major lymphoid organ (54, 55).
Post-Hatch Development of GALT
Development of the avian GALT before and after hatch has not been studied

extensively, with the exception of B lymphocyte development in the bursa of
Fabricius (56, 57). Histologically, gut sections in hatchlings appear to be poor in lymphoid content - both innate and acquired elements (Bar-Shira and Friedman, unpublished observations). More sensitive probes such as expression of B and T cell receptor genes indicate the gut to contain basal levels of lymphocytes at hatch (58).
These basal levels may coincide with early reported waves of T emigration from the thymus (59) and peripheral B lymphocyte appearance (60, 61). Functionally these lymphocytes appear to be in non-activated forms as cytokine (IL-2 and IFNg) expression in thisperiod is very low (58). A major wave (the “second” wave) of lymphoid population occurs after day 4 of life with similar dynamics for both T and B lymphocytes (58). This wave coincides and slightly follows the development of intestinal parenchyme. The rate of lymphoid population of the gut was similar though slightly earlier in the cecal tonsils. A dramatic increase of cytokine expression (IL-2 and IFNg, indicative of activation and effector functions respectively) followed the second wave of population, indicating this wave to contain fully mature lymphocytes undergoing stimulation in response to activation (58). The increase of lymphoid function in the chick intestine is dependent on the presence of bacteria and coincides with enterocyte and villus development (62). Bar-Shira et al., demonstrated that functional maturation of the chick large intestine (cecum and colon) precedes that of the small intestine and is sensitive to feed deprivation (58, 62). Hence, taken together these observations indicate that functional maturity of the gut is linked to the maturation of the local immune system. Interestingly this presents a new issue, which is to explain the chick’s capacity to defend itself against intestinal pathogen insult during the first week of life. Two possibilities might account for this: a) secretion of maternal antibodies present in yolk directly into the gut lumen, and b) immune defense during the first week is provided by the innate immune system.
The important function of maternal antibodies in the prevention of disease in hatchlings is long known (63). While the specific sites for this protection have not been investigated, it is possible that these antibodies function in sites other than the peripheral blood. For example, maternal antibodies have been shown to protect hatchlings against colonization of the gut by Campylobacter jejuni (64), and were isotyped as IgG in another study (65). As mucosal IgA and IgG are involved in protection of chicks against intestinal Campylobacter jejuni (65), maternally-derived
IgG transferred to the mucosa could participate in this protection. In contrast, maternal antibodies are less successful in protection against bacteria such as
Sallmonella spp. that parasitize cells as a strategy for their dissemination (66).
The involvement of the innate system in defense during the first week of the chick’s life has not been extensively studied out of context with pathogenicity of enteric bacteria. Thus, chemotaxis of phagocytes towards site of infection and ensuing phagocytosis was demonstrated following oral infection of day 1 chicks by
Salmonella (67). The function of macrophages may be indicative of an active innate immune system in the gut following hatch. Recent work from our laboratory examined the protective potential of innate GALT in newly hatched chicks. We found basal mRNA expression of proinflammatory cytokines (IL1b) and chemokines (IL8 and K203) at hatch which was followed by rapid increase in expression concomitant with exposure to environmental antigens. The rapid increase in expression of these genes demonstrates the capability of the enteric immune system to respond rapidly to inflammatory stimuli of external origin (Bar-Shira and Friedman, submitted). We also examined the dynamics of polymorphonuclear cells in newly hatched chicks using

light microscopy. We foundan increase in polymorphonuclear cells in all parts of the intestine particularly in the cecum, during the first two weeks post hatch® Presenilin
1 which is important in fate decision processes involving Notch was recently found in avian cells (68). In addition Notch was recently implicated as an important factor in myeloid cell differentiation (69). Using both microscopical examination and
Presenilin 1 or b defensin expression we found that polymorphonuclear cells complete their post-hatch maturation in the intestine (Bar-Shira and Friedman, submitted).
GALT function in the immunologically mature chicken
Protection of the gut is achieved by both innate and adaptive pre-emptive means.
Conceptually, the best strategy to avoid infection is to prevent binding between pathogen and enterocyte (5). This may be achieved by secreting anti-bacterial substances from innate cells of the epithelial lining (Paneth-like cells of the crypt) or more specifically by secreting neutralizing antibodies into the intestinal lumen. Thus, innate nonspecific and specific immunological barriers serve to prevent binding and absorption of pathogenic moieties (5). The use of specific antibodies is preferred for it ensures action against dedicated bacteria, while the general anti-bacterial arsenal might be counterproductive in the sense that symbiotic bacteria might be eliminated too. Furthermore, many bacteria, be they pathogenic or not, share antigenic determinants and as a result the neutralizing antibodies are probably cross reactive.
Hence neutralization (i.e. prevent binding of bacteria to epithelial cells) rather than elimination is more beneficial because it allows survival of antibody-bound bacteria in the gut lumen. Neutralizing antibodies are either of IgG, monomeric IgA or dimeric
IgA isotypes (2, 70). IgG and monomeric IgA are secreted into the gut via bile in the foregut or via the bursal canal in the hindgut (71, and Adlerstein and Friedman, unpublished observations). Plasma cells secreting IgG or dimeric IgA reside in the intestinal wall as well as in other anatomical sites in the chicken (bone marrow, spleen) (72-75), and dimeric IgA is secreted via enterocytes (70). In this case dimeric
IgA, secreted by local plasma cells, is taken up by a putative polymeric Ig receptor
(still to be shown in birds) located in the basal membrane of enterocytes. IgA is then transported to the apical membrane, and secreted into the intestinal lumen (70, 76).
The possibility that immune prevention is accomplished in the lumen rather than in the lamina propria is supported by paucity of lymphoid infiltrates, the low number of heterophils (or neutrophils in the mammal), and the un-congested lumens of lymph vessels.
Bacteria or other infectious agents capable of breaching the epithelial lining and lumen protective measures are confronted by IEL. As mentioned above, IEL in the chicken are composed by several cell types including NK, TcRab and TcRgd lymphocytes (7). The innate leukocytes, gd T and NK cells are preferentially located in the epithelium (77), and as far as is presently known these cells share many developmental and functional similarities with those of mammals (7, 32). In the chicken, gd T lymphocytes are of thymic origin (59, 78), while the possibility of an intestinalsource for these cells, as demonstrated in mice (79), has not been investigated. Functionally, gd and NK cells are capable of response immediately upon activation, which is strictly controlled for it requires exogenous cytokines as well as antigen (80, 81). Cytokines may be derived from activated CD4 or CD8 lymphocyte during an established immune responses (80), and possibly from stressed enterocytes during the initial phases of inflammation (82, 83). Thus, activated innate cells in the intestinal lining contribute to protection and concomitant activation of lamina propria residing cells.

The hallmark of the developing adaptive immune response is the lymphoid follicle.
This unique structure contains naïve lymphocytes, both T and B, undergoing differentiation and division in the process of generating effector lymphocytes.
Dividing lymphocytes are selected by merit of antigen binding as presented by follicular dendritic cells. Selected cells then differentiate into effectors or memory cells, both of which may migrate to tissues. As mentioned above, primary follicles of this nature are scarce in the small intestines of the chick (12). This is not to say that immune responses cannot be generated in the chick foregut, only that this is not a common feature of the chick immune system. Most primary responses are probably generated in the chick hindgut, bursal canal, bursa of Fabricius and spleen. Recent a study by Mehr et al, demonstrated the importance of the cloacal bursa in establishing a wide primary repertoire (84). Hence, most immunological activities observed in the chick foregut are probably of secondary nature, and these appear either in the lamina propria or lumen (from either bile or enterocyte transcytosis). The main sources for antigen against which primary responses are generated are probably derived from the hind gut as described above. These primary responses become systemic insofar as locally produced antibody is found in the plasma and effector cells migrate to other parts of gut or chick (spleen, bone marrow etc.). Hence, protection of the gut is achieved by generating immune responses in the hind gut and bursal canal followed by systemic dissemination of these responses throughout the gut and bird.
A key issue in the function of the hindgut in generation of immune responses lies in its ability to sample antigen material from the exterior. Evidence supporting this notion is indirect and is drawn from studies demonstrating retrograde contractions of the intestinal duct for other purposes. Thus retrograde peristalsis of the colon has been attributed to the need to improve water reabsorption from urine, and that of the ceca as a means to take up cellulose and nitrogenous compounds (18, 85, 86). Previous studies in hatchlings and mature birds have indicated reverse peristalsis of digesta from mid-jejunum to duodenum and even gizzard (hatchlings and mature birds) (17).
Together these studies indicate an avenue for rectally-derived external material to the immunologically active ceca, and importantly, a pathway for bursa-derived antibodies right up to the small intestines (Adlerstein and Friedman, unpublished observations).
The notion that the bursa actively contributes to immune responses in the GALT is not new, though most investigators have emphasized bursal function in the differentiation of immature Blymphocytes (61, 87). Our contention is that the bursa and bursal canal have a significant role in the generation of gut-protective immune responses. Antigen has been demonstrated to be actively transported via the bursal canal into the bursal lumen (88-90). Furthermore, antigen has been shown to be bound by follicular associated epithelium (FAE) (91). This antigen would then induce immune responses in both the canal wall and bursal tissue in which both mature T and
B lymphocytes have been previously demonstrated (92). Effector plasma cells may be observed in connective tissue proximal to FAE, and in the lamina propria of the bursal canal lining (93; Adlerstein and Friedman, unpublished observations). The epithelium also contains goblet cells and larger mucoid- like enclosures, which could contribute to antibody secretion into the bursal lumen (94). The bursa is enclosed in a capsule containing smooth muscle and is transversed by trabeculae containing smooth muscle and connective tissue. Contraction of smooth muscle could then propel secreta towards the bursal canal, and from there into the cloaca. Indeed, we have recently isolated anti-BSA specific antibodies from bursal secretions collected directly from the bursal canal (Adlerstein and Friedman, unpublished observations). The secreta

would be then transported by the colon via retrograde peristalsis, and distributed to more proximal gut segments as indicated above.
As stated above the GALT is confronted with both noxious and innocuous antigens, which lead to either immune responses, ignorance or tolerance respectively. This dichotomy has been demonstrated in several mammalian species where oral protein antigens induced tolerance (oral tolerance) (1). Surprisingly, in the chicken, however, oral protein antigens delivered in aqueous solutions induced potent immune responses and not oral tolerance (2, 71, 95). Interestingly, the same protein antigens were immunologically ignored if supplied as powder in the ration (71, 96). As these observations were initially made in adult chickens, similar experiments were conducted in hatchlings up to day 10 of life. The results of these experiments showed a brief 3-4 day period in which tolerance may be induced by oral antigen (71). The functional difference in the 4 day old intestine and GALT that allows induction of tolerance rather than response has yet to be determined, but it is interesting to note that tolerance induction precedes the second wave of lymphoid colonization described above. Thus, as the chick immediately begins to forage, the intestinal immune system is geared towards tolerance, while the subsequent colonization by flora is met with an immunologicaly mature GALT programmed for response. Interestingly, maintenance of antigen specific tolerance induced during the 4 day window is dependent upon reexposure to the tolerizing antigen; if such an antigen is denied for 4-6 weeks — tolerance is replaced by oral responses (Klipper and Friedman, unpublished).
Presently, it is not known whether oral tolerance is induced in the gut, peripheral or central immune organs. For example, if proteins are absorbed intact from the hatchling gut, one could present an argument that blood borne absorbed antigen induced central tolerance upon arrival at the thymus. However, in this case one would expect tolerance to be permanent following clonal deletion while in fact it is not.
Summary: Management and GALT Development
While development and functional maturation of enterocytes is driven by feeding, the development of functional GALT is more influenced by exposure to microflora. Thus the functional maturation of the hind gut GALT precedes that of the small intestine which is not heavily populated with bacteria (58, 62). This pattern was different with data showing long lasting effects of delayed feeding on feed consumption and body weights. Furthermore, while delayed feeding had long-lasting effects on growth and weight gain, the comparable delay in development of immuneity was overcome by two weeks of age.
In light of the above suggestions concerning management aspects are appropriate.
One is that access to feed should be provided as soon as possible, as several advantages are attained, including better tolerance, earlier responses, better protection, and a better developed intestinal tract. This is in addition to the desired promotion of muscle growth. In addition, we have indicated that early exposure to the environment in absence of a mature lymphoid system could well compromise health. This may be partially alleviated by an adequate supply of maternal antibodies, which emphasizes the importance of vaccination of layers. Finally, early foraging on litter might promote immunity by increasing access of the hindgut to environmental microflora.
Acknowledgement s
Our investigations mentioned herein were supported by grants from the Israeli
Ministry of Agriculture and Rural Development, and from the Israeli Poultry

Marketing Board.
Bibliography
1. Friedman A, al-Sabbagh A, Santos LM, Fishman-Lobell J, Polanski M, Das MP,
Khoury SJ, Weiner HL. Oral tolerance: a biologically relevant pathway to generate peripheral tolerance against external and self antigens. Chem Immunol. 58: 259-90,
1994.
2. Klipper E, Sklan D, Friedman A. Response, tolerance and ignorance following oral exposure to a single protein antigen in gallus domesticus. Vaccine 19: 2890-7, 2001.
3. Klipper E, Sklan D, Friedman A. Maternal antibodies block induction of oral tolerance in newly hatched chicks. Vaccine 22: 493-502, 2004.
4. Solomon JB. Ontogeny of defined immunity in birds. Amsterdam-London: North
Holland Publishing Company, 1971.
5. Sanderson IR, Walker WA. Mucosal barrier: an overview. London: Academic
Press. 5-18 pp, 1999.
6. McGhee JR, Mestecky J, Dertzbaugh MT, Eldridge JH, Hirasawa M, Kiyono H.
The mucosal immune system: from fundamental concepts to vaccine development.
Vaccine 10: 75-88, 1992.
7. Lillehoj HS, Chung KS. Postnatal development of T-lymphocyte subpopulations in the intestinal intraepithelium and lamina propria in chickens. Vet Immunol
Immunopathol 31: 347-60, 1992.
8. Vervelde L, Jeurissen SH. Postnatal development of intra-epithelial leukocytes in the chicken digestive tract: phenotypical characterization in situ. Cell Tiss Res 274:
295-301, 1993.
9. Olah I, Nagy N, Magyar A, Palya V. Esophageal tonsil: a novel gut-associated lymphoid organ. Poult Sci 82: 767-70, 2003.
10. Matsumoto R, Hashimoto Y. Distribution and developmental change of lymphoid tissues in the chicken proventriculus. J Vet Med Sci 62: 161-7, 2000.
11. Kajiwara E, Shigeta A, Horiuchi H, Matsuda H, Furusawa S. Development of
Peyer's Patch and Cecal Tonsil in Gut-Associated Lymphoid Tissues in the Chicken
Embryo. J Vet Med Sci 65: 607-14, 2003.
12. Befus AD, Johnston N, Leslie GA, Bienenstock J. Gut associated lymphoid tissue in the chicken. I. morphology, ontogeny, and some functional characteristics of peyer's patches. J Immunol 125: 2626-32, 1980.
13. Olah I, Glick B, Taylor RL, Jr. Meckel's diverticulum. II. A novel lymphoepithelial organ in the chicken. Anat Rec 208: 253-63, 1984.
14. Kitagawa H, Imagawa T, Uehara M. The apical caecal diverticulum of the chicken identified as a lymphoid organ. J Anat 189: 667-72, 1996.
15. Kitagawa H, Hiratsuka Y, Imagawa T, Uehara M. Distribution of lymphoid tissue in the caecal mucosa of chickens. J Anat 192 (Pt 2): 293-8, 1998.
16. del Cacho E, Gallego M, Sanz A, Zapata A. Characterization of distal lymphoid nodules in the chicken caecum. Anat Rec 237: 512-7, 1993.
17. Friedman A, Bar Shira E, Sklan D. Ontogeny of gut associated immune competence in the chick. World's Poultry Sci J 59: 209-19, 2003.
18. Clench MH. The Avian Cecum: Uptade and Motility Review. J Exp Zool 283:
441-7, 1999.
19. Barnes E. The intestinal microflora of poultry and game birds during life and after storage. J App Bacteriol 46: 407-19, 1979.
20. Honjo K, Hagiwara T, Itoh K, Takahashi E, Hirota Y. Immunohistochemical analysis of tissue distribution of B and T cells in germfree and conventional chickens.

J Vet Med Sci 55: 1031-4, 1993.
21. Porter EM, Bevins CL, Ghosh D, Ganz T. The multifaceted Paneth cell. Cell Mol
Life Sci 59: 156-70, 2002.
22. Nile CJ, Townes CL, Michailidis G, Hirst BH, Hall J. Identification of chicken lysozyme g2 and its expression in the intestine. Cell Mol Life Sci 61: 2760-6, 2004.
23. Gobel TW, Kaspers B, Stangassinger M. NK and T cells constitute two major, functionally distinct intestinal epithelial lymphocyte subsets in the chicken. Int
Immunol 13: 757-62, 2001.
24. Lillehoj HS, Min W, Dalloul RA. Recent progress on the cytokine regulation of intestinal immune responses to Eimeria. Poult Sci 83: 611-23, 2004.
25. Lehrer RI, Ganz T. Endogenous vertebrate antibiotics. Defensins, protegrins, and other cysteine-rich antimicrobial peptides. Ann N Y Acad Sci 797: 228-39, 1996.
26. Lehrer RI, Ganz T. Defensins of vertebrate animals. Curr Opin Immunol 14: 96-
102, 2002.
27. Sugiarto H, Yu P-L. Avian antimicrobial peptides: the defense role of [beta]defensins. Biochem Biophys Res Comm 323: 721-7, 2004.
28. Zhao C, Nguyen T, Liu L, Sacco RE, Brogden KA, Lehrer RI. Gallinacin-3, an inducible epithelial beta-defensin in the chicken. Infect Immun 69: 2684-91, 2001.
29. Bezuidenhout AJ, Van Aswegen G. A light microscopic and immunocytochemical study of the gastrointestinal tract of the ostrich (Struthio camelus L.). Onderstepoort J
Vet Res. 57: 37-48, 1990.
30. Evans EW, Beach GG, Wunderlich J, Harmon BG. Isolation of antimicrobial peptides from avian heterophils. J Leukoc Biol 56: 661-5, 1994.
31. Brockus CW, Jackwood MW, Harmon BG. Characterization of beta-defensin prepropeptide mRNA from chicken and turkey bone marrow. Animal Gen 29: 283-9,
1998.
32. Lillehoj HS. Avian gut-associated immune system: implication in coccidial vaccine development. Poult Sci 72: 1306-11, 1993.
33. Harmon BG. Avian heterophils in inflammation and disease resistance. Poult Sci
77: 972-7, 1998.
34. Burns RB, Maxwell MH. Ultrastructure of Peyer's patches in the domestic fowl and turkey. J Anat 147: 235-43, 1986.
35. Gallego M, Del Cacho E, Zapata A, Bascuas JA. Ultrastructural identification of the splenic follicular dendritic cells in the chicken. Anat Rec 242: 220-4, 1995.
36. Jeurissen SH, Wagenaar F, Janse EM. Further characterization of M cells in gutassociated lymphoid tissues of the chicken. Poult Sci 78: 965-72, 1999.
37. Hogan JA. Pecking and feeding in chicks. Learn Motiv 15: 360-76, 1984.
38. Dror Y, Nir I, Nitsan Z. The relative growth of internal organs in light and heavy breeds. Br Poult Sci 18: 493-6, 1977.
39. Sklan D. Development of the digestive tract of poultry. World's Poult Sci J 57:
415-28, 2001.
40. Uni Z, Noy Y, Sklan D. Development of the small intestine in heavy and light strain chicks before and after hatching. Br Poult Sci 37: 63-71, 1996.
41. Uni Z, Platin R, Sklan D. Cell proliferation in chicken intestinal epithelium occurs both in the crypt and along the villus. J Comp Physiol 168: 241-7, 1998.
42. Mitjans M, Barniol G, Ferrer R. Mucosal surface area in chicken small intestine during development. Cell Tiss Res 290: 71-8, 1997.
43. Planas JM, Villa MC, Ferrer R, Moret M. Hexose transport by chicken cecum during development. Pflugers Arch Eur J Physiol 407: 216-20, 1986.
44. King DE, Asem EK, Adeola O. Ontogenetic Development of Intestinal Digestive

Functions in White Pekin Ducks1. J Nutr 130: 57-62, 2000.
45. Noy Y, Sklan D. Yolk utilisation in the newly hatched poult. Br Poult Sci 39: 446-
51, 1998.
46. Akiba Y, Murakami, H. Partitioning of energy and protein during early growth of broiler chicks and contribution of vitelline residue. Presented at 10 th European
Symposium on Poultry Nutrition, Antalia, Turkey, 1995.
47. Katanbaf MND, E.A., Siegel, P.B. Allomorphic relationships from hatching to 56 days in parental lines and F1 crosses of chickens selected over 27 generations for high or low body weight. Growth Develop Age 52: 11-22, 1988.
48. Noy Y, Sklan D. Energy utilization in newly hatched chicks. Poult Sci 78: 1750-6,
1999.
49. Uni Z, Noy Y, Sklan D. Posthatch development of small intestinal function in the poult. Poult Sci 78: 215-22, 1999.
50. Geyra A, Uni Z, Sklan D. Enterocyte dynamics and mucosal development in the posthatch chick. Poult Sci 80: 776-82, 2001.
51. Ferrer R, Planas JM, Moreto M. Cell apical surface area in enterocytes from chicken small and large intestine during development. Poult Sci 74: 1995-2002, 1995.
52. Uni Z, Smirnov A, Sklan D. Pre- and posthatch development of goblet cells in the broiler small intestine: effect of delayed access to feed. Poult Sci 82: 320-7, 2003.
53. Yan F, Polk DB. Commensal bacteria in the gut: learning who our friends are.
Curr Opin Gastroenterol 20: 565-71, 2004.
54. Thompson FM, Catto-Smith AG, Moore D, Davidson G, Cummins AG. Epithelial growth of the small intestine in human infants. J Pediatr Gastroenterol Nutr 26: 506-
12, 1998.
55. Thompson FM, Mayrhofer G, Cummins AG. Dependence of epithelial growth of the small intestine on T-cell activation during weaning in the rat. Gastroenterol 111:
37-44, 1996.
56. Masteller EL, Thompson CB. B cell development in the chicken. Poult Sci 73:
998-1011, 1994.
57. Masteller EL, Pharr GT, Funk PE, Thompson CB. Avian B cell development. Int
Rev Immunol 15: 185-206, 1997.
58. Bar-Shira E, Sklan D, Friedman A. Establishment of immune competence in the avian GALT during the immediate post-hatch period. DevelopComp Immunol 27:
147-57, 2003.
59. Dunon D, Courtois D, Vainio O, Six A, Chen CH, Cooper MD, Dangy JP, Imhof
BA. Ontogeny of the immune system: gamma/delta and alpha/beta T cells migrate from thymus to the periphery in alternating waves. J Exp Med 186: 977-88, 1997.
60. Lawrence EC, Arnaud-Battandier F, Grayson J, Koski IR, Dooley NJ, Muchmore
AV, Blaese RM. Ontogeny of humoral immune function in normal chickens: a comparison of immunoglobulin-secreting cells in bone marrow, spleen, lungs and intestine. Clin Exp Immunol 43: 450-7, 1981.
61. Yamamoto H, Watanabe H, Mikami T. Distribution of immunoglobulin and secretory component containing cells in chickens. Am J Vet Res 38: 1227-30, 1977.
62. Bar-Shira E, Sklan D, Friedman A. Impaired immune responses in broiler hatchling hindgut following delayed access to feed. Vet Immuno Immunopathol 105:
33-45, 2005.
63. Malkinson M. The transmission of passive immunity to Escherichia coli from mother to young in the domestic fowl (Gallus domesticus). Immunol 9: 311-7, 1965.
64. Sahin O, Zhang Q, Meitzler JC, Harr BS, Morishita TY, Mohan R. Prevalence, antigenic specificity, and bactericidal activity of poultry anti-Campylobacter maternal

antibodies. Appl Environ Microbiol 67: 3951-7, 2001.
65. Cawthraw S, Ayling R, Nuijten P, Wassenaar T, Newell DG. Isotype, specificity, and kinetics of systemic and mucosal antibodies to Campylobacter jejuni antigens, including flagellin, during experimental oral infections of chickens. Avian Dis 38:
341-9, 1994.
66. Popiel I, Turnbull PC. Passage of Salmonella enteritidis and Salmonella thompson through chick ileocecal mucosa. Infect Immun 47: 786-92, 1985.
67. Henderson SC, Bounous DI, Lee MD. Early events in the pathogenesis of avian salmonellosis. Infect Immun 67: 3580-6, 1999.
68. Mirinics ZK, Calafat J, Udby L, Lovelock J, Kjeldsen L, Rothermund K, Sisodia
SS, Borregaard N, Corey SJ. Identification of the presenilins in hematopoietic cells with localization of presenilin 1 to neutrophil and platelet granules. Blood Cells Mol
Dis 28: 28-38, 2002.
69. Schroeder T, Kohlhof H, Rieber N, Just U. Notch Signaling Induces Multilineage
Myeloid Differentiation and Up-Regulates PU.1 Expression. J Immunol 170: 5538-
48, 2003.
70. Mestecky J, Moro I, Underdown BJ. Mucosal Immunoglobulins. In Mucosal
Immunology, ed. PL Ogra, J Mestecky, ME Lamm, W Strober, J Bienenstock, JR
McGhee, pp. 133-52. London: Academic Press, 1999.
71. Klipper E, Sklan D, Friedman A. Immune response of chickens to dietary protein antigens. Vet Immunol Immunopathol 74: 209-23, 2000.
72. Fagerland JA, Arp LH. Distribution and quantitation of plasma cells, T lymphocyte subsets, and B lymphocytes in bronchus-associated lymphoid tissue of chickens: age-related differences. Reg Immunoly 5: 28-36, 1993.
73. Muir WI, Bryden WL, Husband AJ. Immunity, vaccination and the avian intestinal tract. Dev Comp Immunol 24: 325-42, 2000.
74. Scott TR, Savage ML, Olah I. Plasma cells of the chicken Harderian gland. Poult
Sci 72: 1273-9, 1993.
75. Sharma JM. Overview of the avian immune system. Vet Immunol Immunopathol
30: 13-7, 1991.
76. Muir WI, Bryden WL, Husband AJ. Investigation of the site of precursors for
IgA-producing cells in the chicken intestine. Immunol Cell Biol 78: 294-6, 2000.
77. Bucy RP, Chen CL, Cihak J, Losch U, Cooper MD. Avian T cells expressing gamma delta receptors localize in the splenic sinusoids and the intestinal epithelium. J
Immunol 141: 2200-5, 1988.
78. Dunon D, Cooper MD, Imhof BA. Thymic origin of embryonic intestinal gamma/delta T cells. J Exp Med 177: 257-63, 1993.
79. Suzuki K, Oida T, Hamada H, Hitotsumatsu O, Watanabe M, Hibi T, Yamamoto
H, Kubota E, Kaminogawa S, Ishikawa H. Gut cryptopatches: direct evidence of extrathymic anatomical sites for intestinal T lymphopoiesis. Immunity 13: 691-702,
2000.
80. Arstila TP, Toivanen P, Lassila O. Helper activity of CD4+ alpha beta T cells is required for the avian gamma delta T cell response. Eu J Immunol 23: 2034-7, 1993.
81. Kasahara Y, Chen CH, Cooper MD. Growth requirements for avian gamma delta
T cells include exogenous cytokines, receptor ligation and in vivo priming. Eur J
Immunol 23: 2230-6, 1993.
82. Berin MC, McKay DM, Perdue MH. Immune-epithelial interactions in host defense. Am J Trop Med Hyg 60: 16-25, 1999.
83. Berin MC, Yang PC, Ciok L, Waserman S, Perdue MH. Role for IL-4 in macromolecular transport across human intestinal epithelium. Am J Physiol 276:

C1046-C52, 1999.
84. Mehr R, Edelman H, Sehgal D, R. M. Analysis of Mutational Lineage Trees from
Sites of Primary and Secondary Ig Gene Diversification in Rabbits and Chickens. J
Immunol 172: 4790-6, 2004.
85. Brummermann M, Braun EJ. Effect of salt and water balance on colonic motility of white leghorn roosters. Am J Physiol 268: R690-8. 1995.
86. Lai HC, Duke GE. Colonic motility in domestic turkeys. Am J Dig Dis 23: 673-
81, 1978.
87. Mansikka A, Veromaa T, Vainio O, Toivanen P. B-cell differentiation in the chicken: expression of immunoglobulin genes in the bursal and peripheral lymphocytes. Scan J Immunoly 29: 325-31, 1989.
88. Sorvari R, Naukkarinen A, Sorvari TE. Anal sucking-like movements in the chicken and chick embryo followed by the transportation of environmental material to the bursa of Fabricius, caeca and caecal tonsils. Poult Sci 56: 1426-9, 1977.
89. Sorvari R, Sorvari TE. Bursal fabricii as a peripheral lymphoid organ. Transport of various materials from the anal lips to the bursal lymphoid follicles with reference to its immunological importance. Immunol 32: 499-505, 1978.