Overview of broiler chicken immune system and development

The immune system consists of specific cells and tissues produced or transformed from lymphoid organs that recognize and respond to invading micro-organisms, parasites, and other antigenic materials (Korver, Reference KORVER2006). Broiler chickens, like other animals, have non-specific (innate) and specific (acquired) immune systems. It is widely believed that the innate system usually recognises infectious microbes and the acquired system are subsequently activated to purge invading pathogens (Kogut and Klasing, Reference KOGUT and KLASING2009).

The structure and relevant functions of the innate and acquired immune systems in chickens have been summarized in several reviews (Korver, Reference KORVER2006; Kogut, Reference KOGUT2009; Kogut and Klasing, Reference KOGUT and KLASING2009). Briefly, the innate response, by using germ line coded receptors to recognise invading pathogens, is rapid and critical for disease resistance and susceptibility. The acquired immune system consists of cell mediated and humoral mediated reactions through T lymphocytes (T-cell) and B lymphocytes (B-cell), respectively. For broiler chickens, the primary lymphoid organs include the thymus and bursa of Fabricius (bursa). Encounter of lymphocyte cells with their respective antigens (Ag) and T-cell receptors then migrate to the secondary lymphoid tissues, including the spleen and gut-associated lymphoid tissues (GALT) (Janeway et al., Reference JANEWAY, TRAVERS, WALPORT and SHLOMCHIK2005; Sklan, Reference SKLAN2005). The GALT, a component of mucus-associated lymphoid tissue, consists of organized tissues and freely dispersed lamina propria lymphocytes (Liebler-Tenorio and Pabst, Reference LIEBLER-TENORIO and PABST2006). The organized tissues are usually made up of Peyer's patch (pp), caecal tonsils, bursa of Fabricius, Meckel's diverticulum, Harderian gland and various lymphoid aggregates located throughout the digestive tract.

The immune system of the bird is partly developed at hatch. The primary immune organs, such as the thymus and bursa, are both present and are already populated by lymphoid tissue. However, the secondary immune organs, such as the spleen, caecal tonsils, Meckel's diverticulum, Harderian gland, and the diffuse lymphoid tissue of the gut are incomplete at hatch and will take one to two weeks to fully develop, and then start by circulating IgA and IgG antibodies or begin to mature T-cells. Therefore, IgM is the dominant immunoglobulin at hatching which is derived from maternal IgM deposited in the yolk sac (Dibner et al., Reference DIBNER, KNIGHT, KITCHELL, ATWELL, DOWNS and IVEY1998). Obviously, feeding on the day of hatch may provide an early antigen stimulus and thus facilitate rapid differentiation of the humoral response or mucosal tolerance. However, this period of low immune response against antigens present in diets may be important for the development of tolerance to antigens that birds should not respond to (Maiorka et al., Reference MAIORKA, DAHLKE and MORGULIS2006).

It is well known that intestinal colonization by various bacteria commences immediately after hatch (Brisbin et al., Reference BRISBIN, GONG and SHARIF2008), although a ‘mature’ flora is not established until 14 days of age (Torok et al., Reference TOROK, HUGHES, OPHEL-KELLER, ALI and MACLPINE2009). Thus, interactions between commensal bacteria and GALT immune systems are considered to be the basic mechanism of the host against invading pathogens (Forchielli and Walker, Reference FORCHIELLI and WALKER2005), achieved by forming biofilms and by binding to the intestinal epithelium and effectively blocking the sites from pathogens (Brisbin et al., Reference BRISBIN, GONG and SHARIF2008). Invading pathogens are then trapped in the mucosa where they are inactivated by IgA or other secreted products including T-cells. The inactivated pathogens are passed out of the digestive tract later (Korver, Reference KORVER2006). Consequently, the complete development of acquired immune response in GALT will rely on commensal bacterial stimulation during 1-2 weeks post-hatch. Considering that this barrier function of the gut epithelium depends on the presence of IgA, until it is present, the hatchling is more susceptible to invading pathogens (Dibner et al., Reference DIBNER, KITCHELL, ATWELL and IVEY1996). Thus a successful GALT immune system will be assumed to permit beneficial bacteria to colonise as soon as possible and protect against pathogens by occupying available attachment sites. Obviously, before this barrier function is completely developed, mainly the maternal IgM and innate immune responses protect the host from invading pathogens (Bar-Shira and Friedman, Reference BAR-SHIRA and FRIEDMAN2005). In many cases, maternal IgM and innate immune responses are sufficient to resolve the challenge. Furthermore, the genetic selection of rapid growth for broiler chickens has resulted in little or no differences in macrophage and natural killer cell function, i.e. the innate immune responses (Rauw et al., Reference RAUW, KANIS, NOORDHUIZEN-STASSEN and GROMMERS1998). Interestingly, Bar-Shira et al. (Reference BAR-SHIRA, SKLAN and FRIEDMAN2005) demonstrated that functional maturation of chicken hindgut preceded that of the small intestine and chicken hindgut in particular caeca might play more important role in maintaining health than we assumed previously.

The gut NSP nutrition and the enteric immune system

The healthy chicken gut harbours more than 640 different species of bacteria (Apajalahti et al., Reference APAJALAHTI, KETLUNEN and GRAHAM2004), and dietary NSP are the main substrates for those organisms. Considering that the efficient GALT immune system heavily relies on beneficial bacteria competing with pathogens, NSP and exogenous NSP degrading enzymes have significant impacts on the broiler chicken immune system and hence gut health. It is becoming increasingly apparent that NSP are more complex than just being anti-nutrients or nutrient diluents (Choct, Reference CHOCT1997). In general, soluble NSP impart anti-nutritive activities due to their viscous nature and exogenous enzymes have been widely used to counter this (Choct, Reference CHOCT2001; Choct, Reference CHOCT2006; Józefiak et al., Reference JÓZEFIAK, RUTKOWSKI, JENSEN and ENGBERG2007). The current exogenous NSP degrading enzymes are able to partially depolymerise soluble NSP into smaller polymers and reduce digesta viscosity, instead of degrading them into monomers. This suggests that a considerable amount of oligosaccharides and other low-molecular weight carbohydrates is produced in situ when birds are fed diets based on viscous grains like wheat, barley, rye and triticale. Indeed, Choct (Reference CHOCT2006) speculated the effects of these carbohydrates might have on gut health and production in poultry.

However, it is not clearly understood how the degradation products of NSP in response to enzyme supplementation affects the dynamics of the gut microflora. Bedford and Apajalahti (Reference BEDFORD and APAJALAHTI2004) demonstrated that in birds fed a reasonable quality wheat-based diet, xylanase supplementation resulted in a 60% reduction in microbial numbers, which was almost comparable to that achieved by an antibiotic. Half a decade has passed since the publication of this existing piece of research and we are still not clear about the mode of action whereby enzymes act as an alternative to in-feed antibiotics. A couple of possible explanations can be put forward. Firstly, it is possible that a more complete digestion of nutrients due to the action of enzymes means that little substrate remains at the end of the intestine to sustain a large population of organisms, effectively ‘starving’ some of the microbes out of existence. Secondly, somewhat contrary to the first point but perhaps more plausible is that an increased amount of low-molecular weight carbohydrates produced from NSP by enzymes leads to the production of short chain fatty acids (SCFA) (Sakata, Reference SAKATA1987). SCFA, among other activities, can inhibit the proliferation of certain organisms, an effect that may explain results of enzymes reported by Bedford and Apajalahti (Reference BEDFORD and APAJALAHTI2004). Furthermore, low-molecular weight carbohydrates produced from NSP by enzymes indirectly stimulate the immune system by encouraging the growth of enteric lactic acid bacteria, which, through their cell wall components, evoke an immune response, especially on the intestinal mucosal surface (Santos et al., Reference SANTOS, SHELDON, SANTOS and FERKET2008). It appears to indicate that not all fermented NSP but only those fitting the criteria of pre-biotics (oligosaccharides) produce an acid pH which benefits intestinal host defence. The pre-biotics can also directly stimulate an up-regulation of regulatory (IL-10) and protective (INF-g) cytokines (Forchielli and Walker, Reference FORCHIELLI and WALKER2005).

Recently Kogut (Reference KOGUT2009) indicated that the genetic selection of today's broiler chickens for rapid growth had resulted in a diminished inflammatory response. It is reasonable to assume that the current diets for broiler chickens may not provide sufficient or suitable substrates for beneficial bacteria (endogenous probiotics) to colonise and compete with pathogens. The fact that eggs and human milk are full of oligosaccharides may confirm that newly-hatched broilers need sufficient and suitable oligosaccharides for beneficial bacteria substrates. Traditionally, the total load of intestinal micro flora has been effectively controlled by the use of in-feed antibiotics, alleviating microbial composition for nutrition with the host. Without the use of in-feed antibiotics, however, alternatives such as enzymes, prebiotics and probiotics are required to modulate gut microbial balance and maintain gut health.

Klasing (Reference KLASING2007) and Kogut (Reference KOGUT2009) have claimed that all nutrients in the diet play a fundamental role in sustaining an optimal immune response and the addition to the diet of many non-essential nutrients causes larger changes in immunity than manipulation of many essential nutrients. Therefore, insoluble NSP are not inert nutrients as previously assumed. In fact, there has been a tremendous amount of research undertaken in recent years, elucidating the effects of structural materials in feed i.e., plant materials that consist mainly of insoluble NSP, such as course oat hulls (Hetland et al., Reference HETLAND, SVIHUS and KROGDAHL2003; Choct, Reference CHOCT2006). More recently, Gonzaleź-Alvarado et al. (Reference GONZALEŹ-ALVARADO, JIMENEZ-MORENO and MATEOS2007) indicated that broiler chickens needed minimal NSP to improve their gut health due to the fact that NSP could increase gizzard weight and decrease pH value in the gizzard. It is believed that a more acidic gizzard pH may not only kill ingested pathogens but could allow beneficial bacteria in the crop to out-colonise pathogens in the lower digestive tract (Engberg et al., Reference ENGBERG, HEDEMANN, STEENFELDT and JENSEN2004).

Effect of stress on the intestinal immune system

When the immune system mounts defence functions against invading pathogens, phagocytes, as well as T-cells and B-cells release a multi-component enzyme complex which is responsible for the production of reactive oxygen species. While these radicals are killing invading pathogens, they also have the potential to damage host tissue (Costantini and Møler, Reference COSTANTINI and MØLER2009). This increased oxidative stress is a side-effect of immune responses. It is known that immune cells rely heavily on cell to cell communication, particularly via membrane-bound receptors, to work effectively. Cell membranes are rich in polyunsaturated fatty acids which, if peroxidised, can lead to a loss of membrane integrity, altered membrane fluidity and result in alterations in intracellular signalling and cell function (Hughes, Reference HUGHES1999). Thus, sufficient antioxidants may protect the host from unnecessary cell damage due to immune responses. The causes of stress in animals are varied including heat, negative energy balance, transportation, pregnancy, and mixing with unfamiliar animals. For broiler chickens, the high rate of growth may also be considered a type of stress. Various immune cells are affected when an animal is stressed. In particular, animals subjected to restraint stress fail to mount a protective immune response (Anglen et al., Reference ANGLEN, TRUCKENMILLER, SCHELL and BONNEAU2003). Therefore, antioxidants are important for the correct reaction and functionality of avian intestinal immune systems.

In addition to the commonly used dietary antioxidants, like vitamin E and organic selenium, there is increasing evidence that numerous cell wall components, such as NSP, phenolics and even phytate, have appreciable antioxidant properties. For example, cereal fructans have the ability to stabilise membranes under stress conditions, with size and species dependent differences between fructans. In addition, mixtures of fructans, as they occur in living cells, may have protective properties that differ significantly from those of the purified fractions (Hincha et al., Reference HINCHA, LIVINGSTONLII, PREMAKUMAR, ZUTHER, OBEL, CACELA and HEYER2007). Phytate, which is generally considered as an anti-nutritional factor, has recently been suggested as having antioxidative activity due to its high binding affinity for Fe²⁺ (Stodolak et al., Reference STODOLAK, STARZYNSKA, CZYSZCZON and ZYLA2007). This is because the control of free Fe is important for intestinal health maintenance due to the fact that freely available Fe diminishes or destroys normal resistance to bacterial infection (Adams, Reference ADAMS2006). In addition, phenolic acid in cereal bran has shown strong antioxidant activity (Kim et al., 2006) because it exists as ferulic acid, substitute of the arabinose side chains are substituted at the 5’ position (Hegde et al., Reference HEGDE, KAVITHA, VARADARAJ and MURALIKRISHNA2006). In an in vitro study, using exogenous multiple NSP degrading enzymes to hydrolyze the palm kernel meal and rice bran, resulted in increased total phenolics and anti-oxidative activity in palm kernel meal and rice bran (Bao, Reference BAO2009). It is further suggested that such an antioxidant may quench soluble free radicals that are continuously formed in intestinal tracts (Vitaglione et al., Reference VITAGLIONE, NAPOLITANO and FOGLIANO2008).