LUCRĂRI TIINłIFICE MEDICINĂ VETERINARĂ VOL. XLIII (1), 2010 TIMIOARA

PARTICULARITIES OF THE AVIAN

E. TÎRZIU, MONICA ERE

Faculty of Veterinary Medicine Timisoara, 300645, Calea Aradului No. 119, Timisoara, Romania Email: [email protected]

Summary

Because of its economic importance and consanguineous lines availability, most research on immunity in general, cellular and humoral immune response and general effectors implied in the reactivity of the body were performed on domestic ( Gallus gallus domesticus ). Direct consequence of this research is the understanding of fundamental and applied concepts of immunity in birds. Comparing to the mammalian, avian immune system appears insignificant. However, it currently provides important information regarding the fundamental immunological mechanisms. It also provided a significant number of firsts, especially in vaccinology. Relatively recent publication of genome (International Chicken Genome Sequencing Committee, 2004) allowed the orientation of research to identify new avian genes and great opportunities to develop new tools and reagents to enable immune responses analysis and immunogenetic studies. Key words: immune system, birds, lymphocytes, complement, MHC

Bursa of Fabricius and its role in B cells development is included in the group of central (primary) lymphoid organs, is present in birds and is specializes in differentiation and maturation of B lymphocyte. Bursa consists of follicles structured in cortical and medullary regions. There are about 10,000 follicles in the bursa, each of them containing about 1000 B cells and the entire compartment in the chicken is derived from about 30 000 precursors that have undergone productive rearrangement at the heavy and light chain loci. Since only one in three light chain rearrangements are in frame and only one in nine heavy chain rearrangements are productive, only about 3% of B cell precursors that have undergone rearrangement will contain both functional heavy and light chains. Therefore, from theoretical considerations, this places the minimum number of B cell precursors in the developing embryo at approximately 10 6 (21). Bursa is developing in two stages, embryonic and after hatching, after suffering involution. The bursal microenvironment is absolutely crucial for the induction of gene conversion. This has been demonstrated in chicks that have been “tailectomized” at 3 EID. Remarkably in such , relatively normal numbers of B cells develop in the periphery suggesting that in the absence of the bursa, there are alternative sites in which B cells can develop (8). However, this cells show a very restricted repertoire and their genes not contain significant levels of conversion events (17). By the time of hatching, each follicle is directly connected to the bursal epithelium which separates the lymphoid compartment from the bursal lumen and the 269 LUCRĂRI TIINłIFICE MEDICINĂ VETERINARĂ VOL. XLIII (1), 2010 TIMIOARA bursal lumen is itself connected to the gut lumen by the bursal duct. Shortly after hatching the bursa goes through a number of changes that suggest that its function may change. After hatching, the rate of growth of the bursa slows, despite the fact that B cells in the cortex of bursal follicles are rapidly dividing. It has been estimated that about 95% of newly generated bursal cells die in situ by apoptosis (21). In mammalian B cell extensive cell death results from the accumulation of nonproductive rearrangements. Cells that fail to express an Ig receptor die. This cannot be the situation in the bursa since all follicular B cells are derived from cells that express Ig. One possible explanation is that the process of gene conversion might be highly error prone, but an analysis of gene conversion events revealed an error rate of no more than 23% (26). A second possibility is that although gene conversion might generate predominantly in frame products, combinations of heavy and light chain regions could potentially be generated that fail to pair to form a functional Ig molecule. The process of forming functional B lymphocytes takes places in the bursa of Fabricius, in which cells acquire the essential membrane molecules: receptors for antigen, molecules encoded by MHC, other receptors, and adhesion molecules. Bursectomised birds synthesized few and plasmocytes disappeared quickly. Therefore busectomy does not affect too much cellmediated immune response. Bursectomised birds have a higher susceptibility to leptospirosis and salmonellosis, but not to against which cellmediated immune response is important, such as Mycobacterium avium .

Avian immunoglobulins Using immunocytochemical and genetic techniques, three classes of avian immunoglobulins have been identified as the homologues of mammalian IgM, IgA, and IgG. IgM – chicken IgM is structurally and functionally similar to its mammalian homologue. It is the predominant Bcell antigen receptor, being the first isotype expressed during embryonic development. Physical determinations mage under various conditions showed a molecular weight (MW) in the range 823954kDa, with an average value of 890 kDa. The H chain has a MW ~70 kDa and L chain 22 kDa, indicating that chicken IgM is more likely to have a tetrameric (2L2)4 rather than a pentameric (2L2)5 configuration. The MW of duck IgM is 800 kDa, with its component H and L chains that have 86kDa, respectively 22kDa; this also suggests a tetrameric structure (9). IgM is the predominant isotype produced after initial exposure to a novel antigen. As in , this humoral response is usually transient (4), although after chronic bacterial infection, such as Bordetella avium in turkeys, IgM is reported to be active for several weeks (32). IgY (IgG) – the avian homologue for mammalian IgG has similarities with both mammalian IgG and IgE. This avian isotype is the predominant form in sera, produced after IgM in the primary response and the main isotype produced in the secondary response.

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It is often referred to in the literature as IgY, as originally proposed by the first authors who have described it (14). Salt precipitation studies indicate that the avian form of Ig has different biochemical properties to mammalian IgG, hence the name IgY. Since the chicken isotype shares homology in function, Ratcliffe (24) have suggested that the term avian IgG should be retained. As a result, both IgY and IgG are used in the literature. The major difference between the chicken IgY and the mammalian homologue is the longer H chain in the chicken molecule (Table 1). Avian IgY consists of five domains (V, C1–C4), as opposed to the four that are found in mammalian IgG. Avian molecule does not possess a genetically encoded hinge, that being replaced by “switch” regions with limited flexibility at the Cυ1–Cυ2 and the Cυ3–Cυ4 domain interfaces. Table 1 Comparison of the characteristics of mammalian IgG and avian IgY after Schade et al . (27) Mammalian IgG Avian IgY Antibody sampling invasive noninvasive 200mg IgG per 50100mg IgY per (57 Antibody amount bleed (40 ml blood) per week) Amount of antibody per month 200 mg ~ 1500 mg Amount of specific antibody ~ 5% 210% ProteinA/G binding yes no Interference with mammalian IgG yes no Interference with rheumatoid factor yes no Activation of mammalian complement yes no

Although IgY is the major avian systemic antibody active in infections, detailed characterization has only been carried out in the chicken and in the duck (Figure 1). Partial characterizations of turkey and pigeon IgY show similarities with that of the chicken. Radioimmunoassay analyses have indicated antigenic similarities between avian IgY molecules and confirmed a lack of antigenic relationship with mammalian IgG or IgD. A relationship with mammalian IgA has been reported but this is now discounted (9, 10, 31).

Fig. 1. The structural differences between mammalian IgG and avian IgY

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Ducks produce two isoforms of IgY (35). The larger one is structurally analogous to chicken IgY and has been referred to as “7.8S IgG”. The smaller isoform, named “5.7S IgG”, is sometimes referred to as IgY(Fc), since it has only three H chain domains (V, C1 and C2) and resembles, structurally and antigenically, an F(ab’) 2 fragment of IgY. IgA – has been found in all mammals and, based on molecular genetic evidence from chicken and duck, it is probably present in all avian species. The phylogenetic origins of IgA are not known. There has been demonstrated the presence of a structurally and functionally homologous form of mammalian IgA in chicken secretions, especially bile (10). cDNA encoding the chicken α chain has been sequenced, and the inferred amino acid sequence confirms overall homology with mammalian α chains (16). The differences chicken α chain possesses four C domains with no genetic hinge suggesting that, similar to the relationship between avian and mammalian IgG, the primitive C H2 domain could have been condensed to give the hinge region of the mammalian molecule. Chicken IgA, extracted from secretions such as bile, is usually larger than the IgA found in mammalian secretions suggesting the avian form is a trimer (α 2L2)3 or a tetramer (α 2L2)4. Recent cloning of cDNA encoding chicken J chain and polymeric Ig receptor confirms that the origins of secretory Ig predate the evolutionary divergence of birds and mammals (33, 38). IgA has since been confirmed biochemically and antigenically in the turkey and in the pigeon. A secretory form of Ig found in the bile of ducks and geese was shown to resemble IgM, both physically and antigenically (7). Using cDNA cloning and sequencing has shown that the H chains resemble chicken α chains and studies on the expression of α chain mRNA have confirmed the delayed ontogeny of this molecule. A cDNA corresponding to the polymeric Ig receptor and J chain were also identified in duck (15). Although there were some early reports of a chicken homologue of IgD on the surface of chicken lymphocytes, it is generally accepted that there is no avian homologue for Igδ, with the majority of chicken B cells expressing IgM. Likewise no IgE isotype has been described for birds. It is possible that IgE functions are performed by IgY (6).

Avian major hystocompatibility complex (MHC) Chicken MHC is known as B locus because it was first described as a serological locus codifying BG antigen, polymorphic and highly immunogenic. This antigen BG has a high expression on blood cells and there is no equivalent in mammals. Later it was proved that locus B is the chickens’ CMH, this finding being based on the existence of a strong association with cellmediated immune functions, such as rejection of grafts, mixed lymphocyte reaction or graft versus host disease. Remarkably, and unlike mammals’ CMH, B locus of chickens is very small, covering only 92 kB comprising 19 genes, which makes it about 20 times smaller than a human’ CMH (12).

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The chicken MHC has many features that are unlike those of typical mammals, some of which may provide profound insight into the evolutionary history of the MHC. This is particularly clear now for the organization of those genes that are present, the lack of some expected genes and the presence of some unexpected genes. First, as discussed above, the existence of a single dominantlyexpressed class I molecule in chickens is due to coevolution with the closely linked TAP and tapasin genes, whereas the multigene family of highly polymorphic class I molecules in humans and most mammals is a consequence of the much larger distance to the TAP, tapasin and LMP genes. The sequence of the BF/BL region of the B12 haplotype showed that the chicken MHC is smaller, simpler and organized differently compared to the MHC of typical mammals. Long distance PCR and sequencing throughout the classical MHC has confirmed the same organization for another six haplotypes in nine chicken lines (28). Four main points emerged from this work: First, the BF/BL region is simple and compact, with only 11 genes in the 44kB of the central region spanning the class I and II B genes and only 19 genes in the sequenced 92kB. Second, some of the genes present in the MHC of typical mammals are found in this region (such as class I, II B, TAP, DM, RING3, and C4 genes) but many are absent (including class II A, LMP, DO, C2/fB, and other class III region genes). Third, the chicken genes are organized differently compared to the mammalian MHC, with the TAP genes flanked by the class I genes, the tapasin gene flanked by class II B genes, and the class III region genes represented by the complement component C4 outside of the class I and II region genes. Fourth, there are genes present in the sequenced region that would not be expected, including the Ctype animal lectin membrane protein genes, and the BG gene for which there is no mammalian orthologue). In birds there has been more controversy about the organization, particularly focusing on whether all birds have a “minimal essential MHC”. The overall organization of the quail MHC is similar to the chicken MHC, but it appears that at both ends there has been repeated tandem duplication of groups of genes, leading to multiple classes I, II B, lectinlike and BG genes. So, there seem to be at least four expressed class I genes, although only two appear to be classical and only one appears to be wellexpressed (29, 30) . The duck has five classical class I genes arranged in tandem next to the TAP genes, but like chickens the TAP genes are polymorphic and only one class I gene is expressed at a high level (19, 20). The situation is less clear for the passerine birds, with reports of multiple class I and II genes, of which several are expressed (2, 37) so it is possible that galliform and passerine birds are different. Other questions arise from the fact that there are many genes found in the mammalian MHC that are missing from the chicken MHC. The fates of many of these

273 LUCRĂRI TIINłIFICE MEDICINĂ VETERINARĂ VOL. XLIII (1), 2010 TIMIOARA genes, including the inducible proteasome components (LMP) and TNF , are unknown. Thus, the general conclusion is that genes missing from the chicken MHC have been dispersed for reasons and by mechanisms so far unknown.

Complement system particularities The avian immune system has a variety of tools at its disposal to combat infections, including the that, as an innate immune component, is immediately ready to target and eliminate virus particles and to interact with the surface of virusinfected cells (1). Complement system consists of more than 30 serum and membrane proteins present in all vertebrates, including fish, in variable quantities, according to species. In addition to blood, complement may be present in smaller proportions in other humours, the pleural and peritoneal fluid. The proteins circulate in an inactive form but, in response to the recognition of molecular components of microorganisms, they become sequentially activated, working in a cascade in which the binding of one protein promotes the binding of the next protein in the cascade. Complement activity includes an enhancing effect on phagocytosis, the ability to induce an inflammatory response, enhancing B and responses (3) and, finally, enhancing the direct killing of target cells (cytolysis). Complement activation was analyzed in detail in mammals being identified the three different mechanisms (classical pathway, alternative pathway, and lectin pathway). Complement activation in chickens is a crucial component of both innate immunity, in the forms of alternative and lectin activation pathways (11), and adaptive immunity (classical, activation) (5) acquired over time following virus infection or (34). The purpose of vaccination of chickens, as of other species, is usually to induce enhanced specific and protective immune responses such as specific antibodies or memory T cells (23) against important viral diseases at an appropriate time. In classical pathway described in mammals, C1 is the first enzyme able to cleave and activate the next two components C2 and C4. The chicken C1q molecule has been identified and has – like its human counterpart – a characteristic “bouquet of tulips” structure with six globular subunits (Figure 2). Chicken C1q has the ability to generate full C1 haemolytic activity when mixed with the human complement subcomponents C1r and C1s (40). A predicted sequence for the three chicken C1q chains was recently identified on the same chromosome (XM 417654, XM 425756, and XM 417653). In addition, the sequence for chicken C1s has been determined and mapped, whereas only a “likesequence” for chicken C1r has been found (BX 427559). The C1 molecule activates C2 and C4 by cutting a small peptide (C2a and C4a) from each via the serine protease C1. C4b then binds covalently to the pathogen surface and associates with C2b which has protease activity in a magnesiumdependent way. This C4bC2b molecule is the C3 convertase of classical pathway.

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Fig. 2. The first protein in the CP of complement activation C1, which is a complex of C1q, C1r, and C1s

In earlier studies, it was assumed that the role of C2 was fulfilled by the chicken factor B, but the predicted amino acid sequence for C2 and the partial sequence for C4 denied it. In addition, it has been shown that chicken serum protein crossreact with antihuman C4 serum (36). It therefore seems reasonable to assume that chickens possess a normal classical complement pathway. Activation of the alternative complement pathway begins when C3b or C3i binds to the cell wall and other surface components of microbes. The alternative pathway protein factor B then combines with the cellbound C3b to form C3bB in a magnesiumdependent manner. Factor D splits the bound factor B into Bb and Ba, forming C3bBb. Finally, properdin stabilizes the complex by binding to the Bb to form C3bBbP which functions as a C3 convertase. Chicken factor B is a 95 kDa glycoprotein exhibiting a genetic polymorphism with three common phenotypes. It is not known whether the polymorphism is due to single nucleotide polymorphisms or posttranslational modifications. When activated in a magnesiumdependent way, the chicken factor B is cleaved into a Ba fragment of 37 kDa and a Bb fragment of 60 kDa leading to creation of the active C3 convertase (13). Depletion of factor B abolished lytic activity indicating that factor B participates in the alternative pathway. Unlike in the human genome this gene is not linked to the chicken MHC complex. Although factor B is the only alternative pathway protein that has been characterized so far, all things considered, the results indicate that a factor B dependent alternative pathway is active in the chicken. The key component of the chicken complement system is C3, a homologue for other vertebrates’ C3 belonging to the α2macroglobulin family (18). Normally, chicken C3 is present in serum, but recently it was also found in egg yolk (25). Chicken C3 has been isolated, mapped and characterized. It has a doublechain structure with an α chain (111 kDa) and a βchain (70 kDa); both have been cloned (NM 205405). Upon

275 LUCRĂRI TIINłIFICE MEDICINĂ VETERINARĂ VOL. XLIII (1), 2010 TIMIOARA cleavage of chicken C3 by C3 convertase, two fragments are generated: the small anaphylatoxin C3a (15 kDa) and the major fragment C3b (175 kDa). The C3b active fragment is the main effector which directs complement mediated immune response (22). C3b binding covalently to the surface of pathogens, signaling their presence to the effector cells. Additionally, after binding to C3b, the C5b9 unit assembles forming membrane attack complex (MAC) (22). Regarding the function of MAC, the final stage of cleavage and activation, there are no data on the molecular basis of this lytic reaction for avian species. However, predicted amino acid sequences have recently been determined for chickens C5C9 suggesting a MAC process similar to that in mammalian species (XM 415405, XM 429140, XM 424775, XM 415563, and AI 980217) (22).

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