Feed additives company Phytobiotics has invited a panel of renowned experts from around the globe to share their knowledge and expertise into the interplay between the immune system, inflammation, the gut microbiota on the one hand, and animal health and performance on the other. In this article, the fourth in the series of Hot Talks, the specifics of the avian immune system will be discussed.
Anja Pastor: Good morning, Prof. Kaspers. In this interview we will be touching upon a fascinating topic: the avian immune system. To dive right into the topic: What is the main difference between the avian immune system and the mammalian immune system?
Bernd Kaspers: Two features of the avian immune system stand out. First, birds lack highly organised lymph nodes as described in mammals. However, rudimentary lymph nodes develop in the lymphatics but do not interrupt the lymph flow. They are located in the wall of deep lymphatics and are therefore referred to as mural lymph nodes. Their functional relevance is largely unknown. The second significant difference is the presence of a unique lymphoid organ, the bursa of Fabricius, only found in birds. The bursa is the site of B-cell development and its anlage appears around embryonic day 8-10 in chickens. At hatch, mature B-cells start to migrate from the bursa to secondary lymphoid tissues. Interestingly, the bursa involutes at sexual maturity. It is still not known if and where B-cells mature after bursal involution. B-cell development in the bursa has been studied intensively. In addition, numerous other structural and functional differences between birds and mammals are described, such as the small and simple major histocompatibility complex, the reduced numbers of cytokines and chemokines and the morphological difference of germinal centres.
Birds mount highly effective adaptive immune responses to infection or vaccination comparable to mammals. It is well described that germinal centres are formed in mucosa-associated lymphoid tissues (e.g. Harderian gland, bronchus-associated lymphoid tissue, caecal tonsils) and the spleen in response to immunisation, leading to the formation of high-affinity antibodies and class switching from IgM to IgY (IgG) immunoglobulins. It is therefore assumed that these structures compensate for the lack of lymph nodes.
While the B-cell response to infection or vaccination is easy to quantify through antibody ELISA assays, specific T-cell responses are difficult to analyse. Current methods require a cell culture lab and availability of fresh organ samples. Under routine circumstances in poultry medicine, these requirements are rarely met. A major challenge is to develop test systems, which permit sampling in the field and shipment to routine laboratories for qualitative and/or quantitative analysis of T-cell responses. In addition, we need a better understanding of antigen uptake, processing and presentation in chickens in order to target vaccines and to develop new vaccination strategies.
If one looks into the literature, a lot of immunological research is done in laying hens and some in broilers while there is not so much immunological research done in other poultry. Can you elaborate on the reasons for this?
The community of avian immunologist is small and has largely focused on the chicken. Historically, research in chickens contributed to a number of breakthroughs in immunology. This came along with the development of specific reagents including monoclonal antibodies, recombinant cytokines and assays to quantify immune responses. Most of these reagents do not cross-react with other avian species and consequently new and species-specific tools must be developed from scratch. Limited work has been done in ducks due to the interest in anseriformes (such as geese, ducks, and swans) as reservoirs for avian influenza viruses. Consequently, research in these species is limited.
Broilers are slaughtered at quite a young age. When does a chicken have a fully developed/mature immune system? What are the consequences of this for modern broiler production?
The innate immune system matures during embryonic development and is largely functional at hatch. In contrast, development of the adaptive immune system takes a few weeks after hatch. We know that a first wave of T-cells migrates from the thymus to secondary lymphoid organs a few days before hatch but the vast majority is released after hatch. B-cells start to leave the bursa of Fabricius at hatch, but colonisation of peripheral lymphoid tissues but a significant number of B-cells is not observed prior to the end of the first week after hatch. Likewise, newly formed antibodies (initially of the IgM isotype) appear between day 3 and 7 after hatch at low concentrations followed by IgY and IgA a few days later. It is difficult to define a time when the system is fully developed but it takes at least 2-3 weeks for lymphocytes to increase in numbers in secondary lymphoid organs and mucosal tissues and up to 4 weeks to fully develop organised lymphoid structures such as germinal centres. Up to this time-point vaccination may not be fully efficient. During this period, the innate immune system and maternal antibodies provide protection from pathogenic challenge.
If chicks would stay with the hen after hatching, the chicks would take up maternal microbiota. This is not possible in modern production systems. How does this have an impact on the development of the immune system?
Microbial colonisation of the gut is critical for the early and proper development of the immune system, both in the gut and systemically. Chickens raised under sterile conditions (germ free chickens) lack B-cells in the gut tissue and germinal centre development in caecal tonsils. Consequently, plasma immunoglobulin levels are very low and no IgA antibodies are formed. Colonisation with selected probiotic bacteria can induce partial maturation of the system but even combinations of up to nine bacteria do not induce full maturation if compared with maternally derived bacterial consortia provided at hatch. Future work will have to identify those strains effectively driving the development of the adaptive immune system after hatch for application in the field.
Sometimes chicks are transported for a long distance from the hatchery to the farm. Does this kind of feed deprivation have an influence on the immune system?
Immunodeficiencies are generally seen during pronounced malnutrition. Therefore, feed deprivation for a couple of hours has a minor impact on the immune system. However, stress responses associated with transport may affect animal health in several ways. Short-term stress responses lead to activation of the physiological stress axis and a higher state of immune system activation (such as increase in heterophils) which may help to control infections due to gut leakage.
You and your workgroup discovered that chPTX3 an acute-phase protein can be used to assess inflammatory conditions in laying hens. Can chPTX3 also be used in other poultry and when should it be assessed?
Acute phase proteins (APPs) are routinely used as markers of inflammation in humans. They are quantified in blood samples where they may increase up to 1000-fold under such conditions. PTX3 is just one of numerous APPs and it has still to be investigated which of these proteins would be best as an early marker. A major disadvantage is the necessity to collect blood from several birds for diagnostics, which may not be practical under most circumstances. More work is required to develop good makers for inflammation and efforts are underway to identify such markers indicative of gut inflammation in faeces. We have not investigated PTX3 in other species but the high sequence homology with PTX3 between frogs and man suggests that PTX3 from other birds is highly similar to chicken PTX3.
The chicken is still a fascinating model organismal for research into embryonic development and the immune system. What we need is to identify the most critical issues for the poultry industry and established joint programs, which would support the entire spectrum from basic research to application in the field, which may very well include other avian species of interest. For example, minimising chronic stress responses and the associated immune-suppression under field conditions would be just one of these areas and would help to improve animal health and welfare.
Stay tuned for our upcoming interview where Prof. Schuberth will discuss the development of the immune system in calves.
Aspects of antigen delivery for mucosal vaccines
Mucosal vaccination techniques using mammal-specific delivery systems could prove to be just as successful in birds. It is commonly known that in mammals, the role of intestinal villi’s lamina propria lymphoid tissues and Peyers patches in the gut immune system are completely different (45). Essentially, Peyers patches initiate antigen-specific intestinal immune responses to luminal substances, while the actual immune reactions (e.g. g. , IgA production) take place in the intestinal villi (45). Thus, DCs that prime mature naïve T cells by antigen presentation are often found in Peyers patches; in mammals, however, despite the lack of lymphoid follicular structures like Peyers patches, DCs are also widely distributed in the lamina propria (LP) of the gut intestinal villi (46). The absence of specific antibodies has made it difficult to demonstrate the presence of tissue DCs in birds. A first step was demonstrating the existence of cells in tissues, such as the spleen and bursa, that express the C type lectin receptor DEC205 (47) The distinct composition and capabilities of the avian immune system are reflected in the expression of chicken DEC205 (47) By extending their dendrites to control inflammation and immunological tolerance, a subset of LP DCs that are monocyte-derived and express CX3CR1 (a receptor for CX3CL1) can enter the intestinal lumen in mammals and directly sample luminal microorganisms (48). A recent study revealed that goblet cells, whose main job is to cover the intestinal epithelial surface with mucus, can also transfer luminal antigens to a different subset of LP DCs called CD103, which express ?E integrin and have differentiated from regular myeloid DC precursors. Among the two DC populations (i. e. CD103 DCs migrate into the mesenteric lymph nodes that drain the gastrointestinal tract to prime mature T cells for the initiation of antigen-specific mucosal immune responses (50). CX3CR1 DCs and CD103 DCs are found in the LP of the gut. Therefore, one possible method to improve the effectiveness of mucosal vaccinations is to devise a strategy that can transfer the vaccine antigen to CD103 DCs in the LP (50). However, it should be mentioned that chickens lack mesenteric lymph nodes. Thus, chickens have additional pathways for the delivery of antigens. It’s interesting to note that phage display technology was used to identify a 12-mer peptide that has broad targeting specificity for human and mouse DCs (51). Furthermore, it has been verified that lactic acid bacteria (LAB) taken orally and expressing both the DC-specific peptide and the vaccine antigen are effective (52). In particular, it has been demonstrated that oral delivery of LAB expressing DC-specific peptides can efficiently trigger antigen-specific immune responses in the gastrointestinal tract when it reaches intestinal DCs (52). It is crucial to remember that the mucosal tissues are both covered in a thick layer of mucus and lined by a tight epithelial barrier (53) Furthermore, the mucosal lumen where the vaccine antigens are delivered is still distant from the CD103 DCs found in the LP, which are found in intestinal tissues 46 Therefore, mucosal vaccines need to cross the physiological barrier (e. g. layers of mucus and epithelium) to connect with CD103 DCs and trigger intestinal immune responses. It is reasonable to assume that similar gut antigen-presenting cells exist in birds, despite the fact that markers like CD103 are still absent from these species.
Another possible vaccine delivery system is with liposomes. Over half a century ago, British biophysicist Alec Bangham used negatively stained dry phospholipid samples to test a newly acquired electron microscope at his research facility. This is how he discovered spherical lipid bilayer structures, or liposomes (54) Phospholipids, which consist of a hydrophilic head group connected to a hydrophobic tail by a glycerol backbone, are essentially what form liposomes (55) Liposomes range in size from tiny (nanoscale) to enormous (micro-scale) (55) Since liposomes have amphiphilic properties, they can encapsulate a variety of biomaterials, including protein antigens and nucleic adjuvants, regardless of their solubility. This biological property of liposomes is well-known in the field of vaccine development (55) Chemically altering the structure’s surface allows for the free modulation of liposome activity. When polyethylene glycol is applied to liposomes, for instance, the retention effect in blood is increased as opposed to when the liposomes are left bare because the coating prevents the liposomes from being captured by the reticuloendothelial system in the liver, spleen, etc. Giving liposomes tropism through the conjugation of cell-specific antibodies or prospective ligand molecules that bind to particular receptors expressed by the target tissues or cells is another possible modification (56). Furthermore, recent research has been successful in creating liposomes that are pH, heat, light, and enzyme-dependent as delivery vehicles that react to specific stimuli in vivo (57). The development of mucosal vaccines has also made use of these liposomes (55) When administered through the mucosal route, cationic liposomes derived from cationic lipids, such as dimethyldioctadecylammonium bromide (58), 3?-[N-(N?,N?-dimethylaminoethane)-carbamoyl], and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (59), for instance, can be retained in the mucosal epithelium. Consequently, the vaccine antigens and/or adjuvants contained in cationic liposomes are effectively released in the mucosal tissues, causing DCs to process them right away and trigger efficient immune responses. Amphiphilic nanoscale gels, also referred to as nanogels, are potent biomaterials that have applications in both drug delivery and vaccine development (60). Pullulan, a polymer made of glucose units that repeat regularly, is known as ?(1-4)Glu-?(1-4)Glu-?(1-6)Glu. It was initially used to create self-assembling nanogels by incorporating multihydrophobic domains made of 1 6 cholesteryl groups per 100 glucose units (61). The ability of cholesterol-bearing pullulan (CHP) nanogels to trap proteins in their highly water-filled nanoscale matrix is one of their most appealing properties (62, 63). Thus far, encapsulating several bioactive proteins in CHP nanogels has proven successful while preserving their activity, including insulin (64), ?-chymotrypsin (65), bovine serum albumin (62), and myoglobin (63). Like liposomes, CHP nanogels’ properties, like their electrical charge, are freely modifiable through chemical modification (66). Recent research using cationic CHP nanogels encapsulating various vaccine prototypes showed that the antigen was efficiently sampled by DCs in the nasal mucosa and that, when given intranasally, antigen-specific mucosal immune responses were successfully induced in both mice and non-human primates (6769). A mucosal adjuvant does not need to be administered in tandem with the cationic CHP nanogel-based nasal vaccine due to its high potency (6769). Notably, cationic CHP nanogels and encapsulated vaccine antigens when administered nasally do not build up in the brain or olfactory bulb (67). This implies that a nasal vaccine development strategy utilizing cationic CHP nanogels would be risk-free and would not result in the development of undesirable side effects like Bells palsy.
The mucosal immune system in mammals and birds
The first line of defense against pathogens entering the body through the mucosal surfaces lining the respiratory, digestive, and reproductive tracts is provided by the mucosa-associated lymphoid tissues, which are lymphoid structures within the mucosal tissues (Figure ). The mucosal immune system has fundamentally evolved to respond rapidly and efficiently to pathogenic challenges while tolerating commensal microbes. The avian mucosal immune response has distinct characteristics, despite the fact that the mucosal immune systems of mammals and birds share many characteristics that are essential to the operation of mucosa-associated lymphoid tissues. The absence of encapsulated lymph nodes and the presence of diffuse lymphoid tissue in birds distinguish birds fundamentally from mammals.
When particles and pathogens are inhaled, the mucosal tissue of the nose is the first to come into contact with them. One of the main features of the nasal-associated lymphoid tissue (NALT) in chickens is the development of distinct B cell areas with caps made of CD4 T cells. In chickens, immunoglobulin-producing (Ig) B cells are mostly IgY and are located both inside NALT structures and throughout the epithelium (1). Mammals exhibit immunoglobulin class switching from IgM to IgA in the NALT, and the nasal cavity is teeming with IgA-producing plasma cells. The head-associated lymphoid tissues in chickens, which include the conjunctiva-associated lymphoid tissue (CALT) and the Harderian gland, are closely related to the mucosal tissue of the nose. The Harderian gland is situated in the orbit of the eye and resembles a typical secondary lymphoid organ in that it contains germinal centers, a large number of B and plasma cells, T cell-dependent interfollicular regions, and a dispersed population of macrophages and T cells (2, 3). When avian pathogens are exposed to the eyes, it has a significant impact on the adaptive mucosal immune response (4, 5). Chickens that are one week old can be observed to have CALT on the inner surfaces of their eyelids (6). The composition of CALT lymphocytes, the expansion of polymeric immunoglobulin receptors, the induction of antigen-specific IgA antibody-secreting cells after ocular exposure, and the production of IFN? by lacrimal fluids all point to the importance of CALT in the avian mucosal immune response (7). It should be noted that most mammals (e. g. , cats, dogs, and humans) also develop CALT. According to a recent study, mice’s tear duct-associated lymphoid tissue, a similar lymphoid structure in the lacrimal sac, is crucial for the ocular immunosurveillance system’s induction of an immune response (8).
The respiratory systems of chickens and mammals differ greatly. For instance, the human lung has bidirectional airflow, whereas the avian lung has unidirectional airflow (9) Moreover, because birds lack a diaphragm, the lung of a bird is ventilated through air sacs. As a result of unidirectional airflow, particles are mostly deposited in the lung’s caudal regions (10), which are the areas where bronchus-associated lymphoid tissue (BALT) is found. The first description of these highly ordered lymphoid structures and their widely dispersed cells was published in 1973 (11). Bird BALT structures are visible 2-4 weeks after hatching, and in some cases, they are fully developed by 6 weeks (12). Both age and environmental stimuli influence BALT development (13). Furthermore, when pathogenic microorganisms are present, the number of BALT nodules rises dramatically (14) One other distinction between the lung tissue of mammals and chickens is the absence of alveolar macrophages on the exterior of the chickens’ air capillaries (14) Interestingly, the mucosa of the larger airways, the linings of the parabronchi (15), and the connective tissue (9) all contain a vast network of macrophages and dendritic cells (DCs). In order to remove inhaled particles from the air before it reaches the delicate and thin air capillaries, phagocytic cells are positioned strategically at the beginning of the gas-exchange areas. The location where phagocytic cells present the particles to the immune system is unknown because chickens do not have draining lymph nodes. Particle presentation can happen locally in the spleen, the BALT, or the interstitial follicles between parabronchi.
Gut-associated lymphoid tissues (GALTs) are well developed in birds (16). It is made up of lymphoid cells found in the lamina propria and epithelial lining, as well as specialized lymphoid structures like cecal tonsils and Peyers patches. When chickens are two weeks old, their peyers are clearly visible, and as they get older, they get more numerous. They appear to be made up of specialized epithelium with M cells that cover organized follicles with distinct T and B cell areas, just like in mammals (17). The cecal tonsils share structural similarities with Peyers patches and are situated in the neck region of each ceca (18). The GALT structures collectively are crucial for the induction of immunological responses (19).
Mucosal vaccination in birds
In the poultry business, vaccination of the mucosa itself is widely employed as a cost-effective, dependable, and efficient way to immunize a big number of birds. On the other hand, both systemic and local immune responses are necessary for a mucosal vaccine to be effective (20, 21). Live attenuated viruses or inactivated viruses combined with an appropriate adjuvant are the two types of viruses used in poultry vaccinations against viral infections. The majority of live vaccines are sprayed or applied mucosally through the oculo-nasal route, which allows the vaccine to enter the respiratory tract or be absorbed by the lymphoid tissues associated with the head, where antigen-presenting cells recognize and absorb it. When immunizing birds against respiratory viruses, such as the virus that causes infectious bronchitis (22), the virus that causes Newcastle disease, and the avian metapneumovirus, spray vaccination is the recommended approach. Nevertheless, even though beads are typically used to investigate deposition patterns following aerosol or spray vaccination, the size of the beads, the size of the droplets in the bead solution, and the age of the chickens all affect the deposition pattern. Larger beads (>3. Smaller beads are dispersed throughout the respiratory tract, with the majority of 7 ?M) being deposited in the upper respiratory tract (2325). Similar to what is seen after avian influenza virus (AIV) spray vaccination (10), the greatest accumulation of beads is found at the bifurcations of primary to secondary bronchi (24), indicating that particulate antigens are also taken up in the respiratory tract at these junctions. Particles are presented to the immune system after being taken up by antigen-presenting cells (26) and entering the respiratory tract.
In addition to spray vaccination, drinking water can also be used to administer vaccines. Vaccines administered by drinking water enter the mouth and quickly travel to the esophagus and digestive system. Here, antigens will be absorbed by GALT cells and exposed to the immune system. While some reports have linked oral vaccination to protection against Salmonella and a decrease in necrotic enteritis lesions (27), other reports have shown less encouraging outcomes (28) This could be connected to the pathogen, the kind of vaccination, or the birds’ ages.
Adjuvants are necessary in addition to inactivated vaccines because they frequently have low immunogenicity and do not induce a protective immune response (29) These vaccines are not appropriate for spray vaccination because they are designed with a high antigenic mass of bacterial or viral origin delivered in an appropriate adjuvant. Therefore, different approachessuch as customized delivery systems or adjuvants with mucoadhesive qualitiesare required for the mucosal administration of inactivated vaccines. Numerous mucosal adjuvants have been used in chickens, and they fall into two categories according to how they work: by stimulating the immune system or by effectively delivering vaccine materials. The adjuvants based on toll-like receptors (TLRs) are a significant class of possible immune stimulators (30). Pattern recognition receptors, or TLRs, are a class of receptors found on immune cells that identify pathogens’ conserved molecular structures, or “microbe-associated molecular patterns.” The immune system is instantly activated upon pathogen recognition by TLRs (31) There have been reports of CpG oligodeoxynucleotides (CpG ODNs), the ligand of chicken TLR21, being used as possible adjuvants for vaccines in chickens. For instance, vaccination with CpG and NDV induced particular immune responses and protection (32), and CpG ODNs administered in vivo by themselves inhibited IBV replication in the chicken embryo (33) Additionally, reports of improved protection following CpG ODN administration have been made for infections with Salmonella enterica (35), Escherichia coli (36), and the Mareks disease virus (34). Other possible immune stimulants include the immune potentiator CVCVA5, which stimulates enhanced immune responses and protection against AIV upon vaccination, and oligopeptides complexed with an agonistic anti-chicken CD40 monoclonal antibody (37) (38, 39).
It has been proposed that mucoadhesive adjuvants, like chitosan, lengthen the mucosal residence period, thereby increasing antigen uptake and presentation (40). When Rauw and colleagues examined the impact of chitosan on the mucosal delivery of NDV vaccines in birds that were one day old, they discovered that the spleen had increased cell-mediated immunity (41). Furthermore, through improved antigen processing, particulate delivery systems like polylactic-co-glycolic acid (PLGA) nanoparticles activate mechanisms that affect vaccine immunogenicity (42). It’s interesting to note that vaccinating chickens with PLGA particles encapsulated with inactivated AIV vaccine adjuvanted with CpG ODNs reduced virus shedding and increased antibody responses (43). Additionally, hens raised without specific pathogens showed improved humoral and cellular immune responses upon intranasal administration of NDV DNA vaccine-encapsulated nanoparticles, as well as protection against challenge with a highly virulent NDV strain (44)