Immunoglobulins, also called antibodies or Igs, are proteins associated with health maintenance of poultry and mammals. They are secretory products of B-cells originating in the bursa of Fabricius, a small outpocket of the avian cloaca located just anterior to the vent. Surgical removal of bursa from young chicks is associated with a reduced capacity to produce antibodies to salmonella. Humans who have inherited defects in the ability to produce antibodies are prone to certain bacterial diseases. Together these observations provide evidence for the importance of antibodies in the control of infection.
Igs have been shown to be a complex class of proteins differing both in structure and function. In the early phases of infection the IgM class of antibody is active. IgM is thought to be the largest among the Ig molecular classes. IgM acts early, often activating complement, an enzymatic cascade with lytic properties. Later smaller molecules of the IgG class become involved in infection control. These last longer and are more important during recurrences of infection.
IgA occurs in all mammals and in avian species such as chickens, turkeys, and ducks. It is a special class of antibody of particular importance in diseases involving tissues lined by mucous membranes such as the gut or respiratory organs. Since diseases involving these systems are prominent in poultry, IgA deserves special attention.
Structure of IgA
Ig monomers are comprised of four polypeptides combined into a tetrameric structure containing two heavy and two light chains. The heavy chains, known as μ, γ or α and a light chain type called λ, determine Ig classification as IgM, IgG, and IgA. Each class can bind antigens derived from the structural parts of pathogens and in the process cause their neutralization or inactivation. Ig binding properties are unique to individual molecules, each combining with pathogens in a highly specific manner. Often Igs show little ability to combine with pathogens other than the original inducers but sometimes cross-reactions can occur.
IgA is structurally more diverse than IgG and in mammals it predominates in mucous secretions. In chickens, IgG is secreted as well, while turkeys secrete both IgG and IgM (Cotter, 2000a). Part of IgA diversity arises as a result of maturation occurring as it passes through epithelial cells lining avian mucosal tissues. Monomeric IgA undergoes dimerization, the production of paired molecules joined by a small polypeptide called the J-chain.
Now polymerized IgA (pIgA) attaches to the basolateral surface of epithelial cells by a receptor, the poly-Ig-R (pIgR). This receptor is found in chickens and shares properties with pIgRs of mammals (Wieland, 2004). pIgA becomes internalized, and after passage through the epithelial cell cytoplasm (transcytosis) emerges on its luminal surface. Surface pIgA is released from the cell by enzyme action thereby becoming the secreted form, sIgA, retaining a piece of the pIgR, the secretory component (SC). The details of events occurring subsequent to membrane release are now a subject of interest.
Secretory IgA is known to occur in multiple forms suggesting that it undergoes additional differentiation. The existence of multiple forms suggests that IgA is functionally diverse.
Thus it is the purpose here to illustrate some of the peculiarities of avian IgA revealed by classic gel diffusion and related techniques, simple but powerful methods. Particular attention will be given to how IgA may be influenced by diet and nutritional status.
Some of differences between the chicken and turkey IgA will be illustrated.
IgA in bile
Bile, a concentrated exocrine secretion of liver, is a complex fluid containing water, electrolytes, and organic molecules including Igs. Bile acids, which are critical for digestion and absorption of fats and fat-soluble vitamins in the small intestine, are also present. Many waste products are secreted into bile and eliminated in feces. Therefore it seems that bile has multiple functions both in nutrition and immunity and it is not surprising that either may influence its composition.
HETEROGENEITY OF BILE IMMUNOGLOBULIN A REVEALED BY DOUBLE DIFFUSION
In classic Ouchterlony, double diffusion techniques gels are used in which combination of antigens with antibodies produces visible precipitation arcs. Simple measurements of length, shape and other arc characteristics can reveal enormous information about both IgA quantity and quality (Cotter, 2000b).
An illustration of principles is shown in Figures 1a and 1b. An antibody raised in goats immunized with chicken IgA was placed in the central well in Figure 1a. Bile samples obtained from six chickens were placed in peripheral wells. After diffusion was completed, precipitate arcs were formed by four samples indicating each contained IgA. In two other samples IgA could not be detected. It is clear however that the four positive precipitates differ in curvature and arc location. Clearly IgA precipitates are heterogeneous. Arc curvatures and staining intensity differences suggest that precipitates differ both in molecular weight and composition.
After substituting a plant lectin (BTB) in place of the original antibody, the same samples were retested. A strikingly different picture emerged (Figure 1b). Sample 1, located at the top in both figures, is negative when tested for IgA using antibody but is positive using the lectin. Sample 3 remains negative by both tests.
Figure 2 indicates how precipitates formed by the goat antibody and lectin reagents are related. Three bile samples were placed in the gel at peripheral positions 2, 4, and 6.
Figure 1a (left) and b (right). IgAA and IgAB precipitates respectively from chicken bile samples. Sample No.1 is negative by antibody precipitation but positive by BTB. Sample 3 is negative by both reagents (Cotter, 2000a).
The lectin BTB was placed in alternating wells marked ‘B’. The central well contained the same antibody used in Figure 1a. Simultaneously two distinct types of precipitates have formed; the edge of one touches a part of the other. These differ in length and staining intensity. Small spurs are also seen indicating that the material precipitated by both reagents is similar, but not identical. If both precipitates contain IgA, then two forms exist, each differentiated by the precipitating reagent.
Figure 2. Spur production illustrating partial immunologic identity of IgAA and IgAB precipitates in chicken bile (from Cotter, 2000a).
In studies using another lectin, a third type of chicken IgA was identified. The lectin, called JFE (Jacalin) because it comes from seeds of the jackfruit tree, was known to be able to bind differentially with one of the two IgA forms found in humans (Kondoh et al., 1986). A preliminary report indicating JFE precipitates might represent a novel form of chicken IgA was presented in 2004 (Cotter, 2004).
JFE precipitates are noticeably more curved and are in different positions than antibody or BTB precipitates (Figure 3). Both gel position and curvature indicate a higher molecular weight than the other types. Evidence supporting the claim that JFE precipitates contain IgA has been obtained recently using ‘dot-blot’ methods (Cotter and Van Eerden, 2006).
Figure 3. JFE-bile precipitates in hens illustrate variation of arc length, curvature, gel position, and stain intensity. These unique bile precipitates are called IgAJ.
IgA molecules are glycosylated by the addition of carbohydrates onto their heavy (α) chains; furthermore both the J chain and SC components are also glycosylated (Mansikka, 1992). Lectins react strongly with carbohydrates, so IgA precipitated by BTB and JFE are distinct from the type precipitated by antibody. These represent glycoforms and are now referred to as: IgAA, IgAB or IgAJ, a classification based on precipitating reagent (Cotter and Van Eerden, 2006).
NUTRITIONAL INFLUENCES ON BILE IgA
Several groups have provided immunodiffusion evidence for the existence of IgA in turkeys (Dohms et al., 1978). Both low and high molecular weight forms were found in serum, bile, gut washings and lachrymal secretions, probably corresponding to the monomeric and dimeric forms found in chickens. Very large polymeric forms were present in bile in another case (Lim and Maheswaran, 1977).
Some investigators claim that antisera that precipitate chicken IgA will cross-react with turkey IgA. However this is not always the case. Ouchterlony results (Figure 4) show that chicken α-chain specific antisera precipitated IgA from five chicken bile samples, as expected, but failed to precipitate IgA from a turkey sample (gel position ( :)). However, the same (chicken) antisera was found useful as a ‘first antibody’ for detecting turkey IgA by immuno-dot blot precipitation procedure (data not shown). The reason for the discrepancy between gel and immuno-dot results is currently unknown.
Figure 4. Ouchterlony double diffusion results indicating the inability of affinity purified α-chain specific goat antichicken IgA to precipitate turkey IgA. Gel position ( : ) contained the turkey bile IgA, precipitates formed at all other positions containing chicken samples.
Confirmation of the Ouchterlony results was obtained using a radialimmunodiffusion (Figure 5) in which the antisera was distributed throughout the gel. The resulting precipitation rings indicate the presence of both high (inner) and low (outer) MW IgAs in chicken bile, neither of which could be detected in the turkey bile sample.
Figure 5. Radialimmunodiffusion results showing the presence of both high and low MW IgAs in chicken (ck) bile, neither of which could be detected in the turkey sample (tk). The lengths of the lines indicate the extent of diffusion of the two chicken IgA types.
Rocket immunoelectrophoresis of turkey bile obtained from young toms fed diets containing yeast derived mannan oligosaccharide (Bio-Mos®, Alltech Inc.) indicated elevated levels of IgAB. Measurements of radialimmunodifusion ring diameter sizes and rocket sizes, both quantitative measures, were higher in toms fed the yeast-derived products (Table 1).
While elevated IgA is not automatically an indication of a dietderived benefit, the performance of the yeast product fed toms was also improved (Savage et al., 1996). Thus it is reasonable to conclude that the elevated IgA levels were a factor contributing to better performance. Perhaps higher IgA provided a benefit to growth by controlling detrimental gut microbes.
Table 1. Effect of Bio-Mos® and Saccharomycese boulardii on measurements (mm) of IgAB in tom turkey bile using radialimmunodiffusion (RID) or rocket immunoelectrophoresis.
adapted from Cotter, P.F. Project NE-60 Annual Report, 1995
Commercial hens were categorized as efficient or non-efficient based on their residual feed intake (Van Eerden et al., 2004a). Their immune functional capacity was measured by determining quantities of various antibodies in both types. Certain antibodies were at higher levels in non-efficient hens, suggesting that nutritional efficiency might be attained at a cost (Van Eerden et al., 2004b). If there is a genetic carryover to nutritional status, selection for improved performance might result in compromised immunity. From an energetic viewpoint the gist of the argument is that resources devoted to one physiologic process (growth) are not available to support another (immunity). Efficient animals might not possess sufficient reserves to fight infection.
As an experimental test of this idea, hens of known nutritional efficiency status were challenged by killed salmonella antigen and live Salmonella enteritidis. Antibodies representing both natural and acquired immunity were measured in both types. Serum antibody levels to the somatic and flagella antigens of salmonella were higher in efficient hens only when a live antigen was used. On the contrary, non-efficient hens produced higher salmonella antibody to killed antigen. It was suggested that the response of such hens represented immunologic inefficiency since only live antigen challenge could result in disease (Cotter and Van Eerden, 2006).
Innate immunity, represented by anti-Gal antibody (Cotter et al., 2005) was also measured in the same hens. This antibody arises as a result of a stimulus by gut microbes and is measured by the agglutination of rabbit erythrocytes that express galactose on the cell surface. No differences in anti-Gal levels were detected between hens of differing efficiency status, but hens of either type given a live salmonella challenge had higher anti-Gal levels. Thus the combined observations suggested that nutritional efficiency was not achieved at the expense of either salmonella-specific antibody or of anti-Gal.
Therefore, immune efficiency parallels dietary efficiency.
Bile IgA levels obtained from hens on the efficiency study were determined using the gel precipitation method. The three IgA glycoforms described above were found in all but one hen, which lacked IgAA. Efficiency status was not associated with differences in either IgAA, or IgAB. However IgAJ was elevated in live-challenged efficient hens and slightly lowered in non-efficient hens, suggesting that this glycoform has a functional role in maintaining health.
Investigation of bile in poultry using various precipitation methods has indicated the presence of multiple IgA forms. At least three glycoforms are found in chicken bile: IgAA, IgAB and IgAJ. Elevation of IgAJ was observed in hens challenged with live salmonella but not with killed antigen. In non-efficient hens the opposite was observed. IgAJ was slightly depressed after live challenge but neither IgAA nor IgAB levels were affected.
Turkeys, on the other hand, do not seem to possess IgAJ. However IgM, a large MW immunoglobulin, was regularly found in turkey bile, in contrast to its infrequent occurrence in chicken bile. Moreover IgAB levels of turkeys were elevated in toms fed Bio-Mos® mannan oligosaccharides. This change was accompanied by better performance.
Collectively these observations indicate that avian IgAs are influenced by nutritional factors as well as immune status. Furthermore, while IgA is found in both chickens and turkeys, clear evidence of species differences exist. It would be of interest to both nutritionists and immunologists to better define those circumstances that influence IgA glycoform levels in both species.
Cotter, P.F. 2000a. Analysis of chicken bile by gel precipitation reactions using a lectin in the place of antibody. Poult. Sci. 79:1276-1281.
Cotter, P.F. 2000b. Quantitative and qualitative heterogeneity of bile IgA in commercial laying hens. In: Current Progress in Avian Immunology Research. Proceedings of the 6th Avian Immunology Research Group Meeting (K.A. Schat, ed). Cornell University, Oct. 8-10, pp. 78-82.
Cotter, P.F. 2004. A jackfruit lectin detects a form of chicken IgA often ignored by antibody. 7th International Veterinary Immunology Symposium, July 25-30, Quebec City, Canada.
Cotter, P.F., J Ayoub and H.K. Parmentier. 2005. Directional selection for specific sheep cell antibody responses affects natural rabbit agglutinins of chickens. Poult. Sci. 84:220-225.
Cotter, P.F and E. Van Eerden. Salmonella challenge affects the antibody isotype profile of bile in hens differing in metabolic efficiency. (Accepted for publication).
Cotter, P.F. and E. Van Eerden. 2006. Natural ‘anti-gal’ and Salmonella specific antibody in bile and plasma of hens differing in diet efficiency. (In press).
Dohms, J.E., Y.M. Saif and J.E. Pitts. 1978. Isolation of turkey immunoglobulin A. Avian Dis. 22(1):151-156.
Kondoh, H.K., K. Kobayashi, K. Hagiwara and T. Kajii. 1986. Jacalin, a jackfruit lectin precipitates IgA1 but not IgA2 on gel diffusion reaction. J. Immunol. Meth. 88:171-173.
Lim, O.J and S.K. Maheswaran. 1977. Purification and identification of turkey immunoglobulin A. Avian Dis. 4:675-696.
Mansikka, A. 1992. Chicken IgA H chains. Implications concerning the evolution of H chain genes. J. Immuonol. 149:855-861.
Savage, T.F., P.F. Cotter and E.I. Zakrzewska. 1996. The effects of feeding a mannan oligosaccharide on immunoglobulins, plasma IgG and bile IgA Wrolstad MW male turkeys. Poult. Sci. 75(Suppl. 1):143.
Van Eerden, E., H. Van Den Brand, H.K. Parmentier, M.C.M. De Jong and B. Kemp. 2004a. Phenotypic selection for residual feed intake and its effect on humoral immune responses in growing layer hens. Poult. Sci. 83:1602-1609.
Van Eerden E., H. Van Den Brand, G. De Vries Reilingh, H.K. Parmentier, M.C.M. De Jong and B. Kemp. 2004b. Residual feed intake and its effect on Salmonella enteritidis infection in growing layer hens. Poult. Sci. 83:1904-1910.
Wieland, W.H., D. Orzaez, A. Lammers, H.K. Parmentier, M.W. Verstegen and A. Schots. 2004. A functional polymeric immunoglobulin receptor in chicken (Gallus gallus) indicates ancient role of secretory IgA in mucosal immunity. Biochem. J. 380:669-676.
Author: PAUL F. COTTER
Professor Emeritus, Biology Department, Framingham State College, Framingham, Massachusetts, USA