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Quantification of Turkey Immunoglobulins IgA, IgM and IgG in Serum and Secretions
Z. Immun.-Forsch. vol. 154, pp. 248-255 (1978) Faculty of Veterinary Medicine, Department of Immunology, Ut,recht., The Netherhnds
Quantification of Turkey Immunoglobulins IgA, IgM and IgG in Serum and Secretions J. GOUDSWAARD, A. NOORDZIJ, R. H. VAN DAM, and J. A. VAN DER DONK WITH 2 FIGURES
Received December 29, 1977 . Accepted February 28, 1978
Abstract The immunoglobulins IgA, IgM and IgG of the turkey were quantitated in individual serum samples as well as in pooled sera. The variability of Ig levels in normal, healthy birds was quite high: IgA: mean value 0.633 mg/ml (4.0 x2.5 x); IgM: mean value 4.37 mg/ml (0.5 x-l.4 x) and IgG: mean value 8.92 mg/ml (0.6 x-1.7 x). Immunoglobulin levels in egg-yolk, egg-white, bile and some intraocular tissues ,vere quantitated as well. An interesting finding was, that the forementioned variability was by far not so high with respect to IgG levels in 20 egg-yolk samples: mean value 5.1 mg/ml (0.86 ¥-1.17 x). Though IgG and IgM could be detected in pooled turkey bile, IgA predominated in this secretion. In aqueous humor, iris tissue and vitreous body only IgG could be detected.
Introduction
The immunoglobulins of a number of birds have been studied including chicken (1), turkey (2), pheasant (3), duck (4) and pigeon (5). Sera of birds have long been known to contain only two classes of immunoglobulins, IgM and IgG (6), but in the chicken (1), the turkey (2) and the pigeon (5) IgA has been recently identified too. IgA is relatively abundant in secretions such as bile and intestinal fluid, while it is found also to be present in the majority of the intestinal plasma cells of the birds studied in the mentioned papers. In spite of the large number of reports on avian immunoglobulins, there is, however, only little information on quantitative distribution of avian immunoglobulins IgA, IgG and IgM in sera and secretions. Up to now only one paper has been published (7) dealing with the quantification of the immunoglobulins of the chicken in sera and some secretions. The present investigation deals with the quantitative distribution of immunoglobulins in turkey serum and secretions. In the latter egg-yolk and egg-white are included, especially because investigations concerning the determination of antibodytiters in egg-yolk and egg-white (8, 9) of
Turkey Immunoglobulins· 249
birds have been reported. According to these investigations antibodytiters proved to be considerably higher in egg-yolk in comparison with egg-white (9). Materials and Methods Animals Turkeys, 5 months old, were used in this study.
Sera 18 individual serum samples and pooled sera (approximately n collected and stored at -20°C until use.
50) were
SeC1'etions Bile was collected post-mortem directly from the gall-bladder and pooled (approximately n = 50). Storage took place at - 20°C. Aqueous hmnor was collected from the eyes with a sterile syringe. During dissection of the eyes, the lens and the vitreous body were easy to isolate from tho eye. The iris tissue was disseoted carefully from the eyes; special oare was taken that the iris tissue was free of oontamination with other tissues. The vitreous body and the iris tissue were diluted with de-ionized cooled water to twice the original volume and homogenized. Egg-white and egg-yolk of 20 fresh eggs were separated. Beoause in 4 cases egg-white was contaminated with egg-yolk, only 16 egg-white samples were examined. The egg-yolk was diluted to twioe the original volume and 1 % Triton X 100 was added to the mixture. The samples could be used without furthor treatment. In egg-white samples, treated in the same way, the immunoglobulin levels were too low to doteot. Therefore, the samples were further dialysed against 0.1 M Na-acetate buffer, pH 4.5, to preoipitate tho muoins. The resulting precipitate was discarded and the pH adjusted to 6.8. Then, globulins were salted out with 50% ammonium sulfate, pH 6.8. The precipitated globulins wero oentrifuged at 15,000 rpm and re-dissolved in 10 ml distilled water. All samples were then concentrated 20 times in a Minicon B 15 1 ).
Anti8era The preparation of a rabbit-antiserum to egg-yolk and whole turkey serum, as well as of antisera speoifio for the heavy chains of turkoy IgM, IgA and IgG has been described (2).
Purification of immunoglob·ulins I gG: egg-yolk, diluted 1: 4 with 0.9 p~rcent N aCI and treated with dextran sulphate and CaCl 2 (10), was dialysed against 0.9 p~rcent NaCI which contained 2 gil EDTA. Then it was salted out with 45 peroent (NH4)2S04 at pH 6.8. The procipitate was taken up in a small amount of distilled water, dialysed against 0.1 M Tris HCl containing 1 M NaCI, pH 8.0, and fraotionated on Ultrogel ACA 22 2). To yield pure IgG, the first peak was ohromatographed on DEAE oellulose. IgA: bilo was collectod post-mortem fronl the gall-bladder, extensively dialysed against 2 peroent NaCl buffered, at pH 8.0, with 0.02 M Tris HCI and passed on a column of Sephadex G-75 to remove low moleoular weight pigmented material. Then, after concentration to the original voItune, the eluate was 1) Amicon Corp., Lexington, Massaohusetts, USA. 2) LKB-Produkten AB, Bromma, Sweden.
250· J. GOUDSWAARD, A. NOORDZIJ, R. H.
VAN DAMM,
and J. A.
VAN
DER DONK
dialysed against 0.01 M Tris-HCI with 1 % NaCI, pH 8.0, and chromatographed on Sepharose 4B3). The IgA-rich fraction in the second peak (not shown) was then chromatographed twice on Ultrogel ACA 22. The first peak contained pure IgA. IgJjJJ: pooled serum was treated with dextran sulphate and CaCl 2 (10) and dialysed against 0.9 percent NaCI containing 2g/1 EDTA. Then, the serum was successively precipitated with 18, 14 and 14% (w/v) of sodium sulphate. After each precipitation the precipitate was dissolved in distilled water and dialysed against 0.03 Na 3 BO s buffer, pH 8.0. After dialysis, the globulins salted out with 14% (w/v) sodium sulphate were further salted out with 9% sodium sulphate. The resultant supernatant was finally brought again to 14% sodium sulphate. The latter precipitate was redissolved in distilled water and dialysed against 0.01 M Tris-HCI containing 1 M NaCI and chromatographed on Ultrogel ACA 22. The first peak contained almost pure IgM (see under Results).
Q'U
Immunoelectrophoresis and double diffu8ion Immunoelectrophoresis and double diffusion ("Ouchterlony") were carried out according to standard procedures using 1 % agarose (Industrie Biologique Franyaise) .
Results
PU1·ification of immunoglobulin standards The preparation of IgG (2.9 mgjml) from egg-yolk and of IgA (2.3 mgjml) from bile yielded pure immunoglobulin standards, as judged by Ouchterlony analysis as well as by immunoelectrophoresis with anti-whole turkey serum (figure 1) and anti-whole egg-yolk (not shown). Upon radial immunodiffusion the IgA or IgG samples reacted only with anti-a and anti-y reagents respectively, also after being concentrated about 5 times. Although the preparation of IgM (3 mgjml) seemed to be pure, as judged immunoelectrophoretically with anti-whole turkey serum (figure 1), it still contained 0.19 mgjml IgG and 0.11 mgjml IgA, when the sample was checked with anti-y and anti-IX reagents in radial immunodiffusion. The antisera used, were specific for each immunoglobulin class, as is s,hown in figure 2. 3) Pharmacia Fine Chemicals, Uppsala, Sweden. 4) Transidyne General Corporation, Ann Arbor - Michigan.
Turkey IIllIlllmoglobulins . 251
_ lgM
-
A-Wh
-
~lgA
~
IgG
A-W. h ..r
Fig. 1: Agarose gel immunoelectrophoresis of pm'ified immunoglobulin standards IgM, IgA and IgG, developed with anti-whole turkey serum (A-Wh). Anode to the left.
Fig. 2: Agarose gel immlUloelectrophoresis of turkey serum (Se) developed with anti-whole turkey serum (A-Wh) and sp ecific anti-IgM (A-IgM), anti-lgA (A19A) and an ti-IgG (A-IgG). Anode t o the left.
252· J. GOUDSWAARD, A. NOORDZIJ, R. H. VAN DAMM, and J. A. VAN DER DONK
Quantitative distribution ot immunoglobulins in sera and secretions Immunoglobulin quantification was carried out in 18 individual serum samples and in a pool of about 50 sera. Moreover, IgG was quantitated in 20 egg-yolk samples. The results are summarized in Table 1. The IgM and IgA levels of egg-white are very low, as was expected. In 9 samples only a trace of IgM was found; the mean IgM level in the other samples was 0.024 mg(ml. The extensive treatment of the egg-white samples also did not allow to give exact values of IgA levels in individual samples: the main value, however, was 0.034 mg( ml. The IgA concentration in pooled bile (approximately n = 50) was 1. 7 mg(ml. Bile contained also IgM (0.44 mg(ml) as well as IgG (0.21 mg(ml). Table 1. Immtilloglobulin levels in 18 individual serum samples and in pooled serum (approximately n = 50). IgG levels in 20 egg-yolk samples. Immunoglobulins mg/ml. Serumnumber
IgA
IgM
IgG
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
0.52 0.33 0.66 0.55 0.62 1,48 1.60 0.39 0.53 0,46 0.52 0,43 0.74 0,41 0.26 0.62 0,44 0.84
4.80 2,40 5.80 3.08 5,48 5,46 4.12 5.80 5.12 5.80 2,48 3.58 2,40 5,46 3.80 5.88 3.28 4.00
8.2 8.2 9.0 8.2 10.8 10.0 9.2 8.6 8.0 15.2 9.0 11.2 6.8 7.2 7.2 11.0 7.6 5.2
Mean
0.633
4.37
8.92
5.1
Normal value
0,4x-2.5x
0.5x- 1.4x
0.6x-1. 7:X:
0.86x-1.17:X:
3.80
9.2
Pooled serum
0.55
Egg-yolk nunlber
IgG
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
6.0 6.0 5.6 5.2 4.6 5.6 5.6 6.0 5.2 4.2 4.6 4,4 4.6 5.2 4.6 4.6 5.0 5.0 4,4 5.6
Turkey Immunoglobulins· 253
In the pooled samples of corpus vitrium, aquaeous humor and iris (approximately n = 10) only IgG proved to be present; the concentration of this immunoglobulin class in the various eye fluids was respectively 0.24 mgjml, 0.32 mgjml and 0.24 mgjml. Discussion The only immunoglobulin class which can be quantitated in egg-yolk is IgG, for it is known (14) that neither IgA nor IglVI are present in yolk. On the other hand, IgG will not be found in egg-white. Our treatment of the yolk with Triton X 100 proved to be a successful method; this made the diffusion of the egg-yolk proteins very easy. The clear precipitation rings of the egg-yolk samples could be read within the same time as those of the serum samples. It can be concluded from the table that yolk contains large quantities of IgG (mean IgG level 5.1 mgj ml) which laying hens do loose every day. It is proved that this IgG represents the whole range of antibodies to various infectious agents (8). An interesting finding too is the small variability of the IgG levels in the different yolk samples, which is very much in contrast with the reported findings of Ig levels in chicken sera (7) and with our own findings with the turkey serum samples. This suggests a phenomenon which can not be explained by a simple transfer of the IgG to the yolk. Also, the specific passage of only IgG antibodies from hen serum to yolk and hence - if the eggs are fertilized and hatched - to the circulation of the chick via the endoderm of the yolk sac are evidence for this suggestion. The Ig levels found by us in sera of almost adult turkeys are higher as those reported by LEBAOQ-VERHEYDEN and co-workers (7) in chickens (IgA: 0.633 mgjml versus 0.377 mgjml; IgG: 8.92 mgjml versus 6.90 mgjml and IglVI: 4.37 mgjml versus 2.506 mgjml), though the variability in individual serum samples is almost comparable. This high variability of Ig levels in healthy individuals in human sera is wellknown too. The differences between our results and those of LEBAOQVERHEYDEN (7) may be explained by the differences in age of the experimental animals. Our birds were 5 months of age, while the chickens she used were only 6-8 weeks old. Paper electrophoresis of pooled turkey sera revealed 15.1 % ,(-globulin (total protein content of the serum was 44 mgjml). The values confirm our findings, because nearly all of the ,(-globulin is already part of the IgG. Though we were able to prove the IglVI and IgA content of all investigated egg-white samples, our results were not accurate enough to report about the quantitative distribution of these immunoglobulins in individual samples. There is no doubt, we loosed small quantities of Ig during the different treatments.
254· J. GOUDSWAARD, A. NOORDZU, R. H. VAN DAMM, and J. A. VAN DER DONK
The IgA content of turkey bile (1.7 mg/ml) was considerably lower as was expected. LEBACQ-VERHEYDEN and co-workers (7) found 3.15 mg/ ml. It is well-known (15) that the chicken gallbladder synthesizes and secretes IgA. There is no reason to believe that the gallbladder of other birds should behave in a different way. It is possible that the IgA in the bile collected by us underwent some enzymatic digestion, especially because it has been reported (16) that secretory IgA of chicken bile lacks secretory component. Lebacq-Verheyden and co-workers centrifuged, extensively dialysed and diluted their bile samples before use. The finding by these authors, that chicken bile contains also small amounts of IgG and IgM, was confirmed by us with pooled turkey bile. In a recently published study by SANDERS and CASE (17) chicken IgA was determined to be the only immunoglobulin class present in bile; the fact that these authors just carried out immunoelectrophoresis and immunodiffusion may be responsible for this different result. However, even with the radial immunodiffusion test we used as the more sensitive test, we were not able to detect any traces of IgM and even of IgA in pooled samples of intraocular tissues. Just low levels of IgG were detected. Therefore, we conclude that there is no direct link between these tissues and the avian Harderian gland in which mainly IgA containing immunocytes (18) can be detected. References 1. LEBAcQ-VERHEYDEN, A.-M., J.-P. VAERMAN, and J. F. HEREMANS. 1972. A possible homologue of mammalian IgA in chicken serum and secretions. Immunology 22: 165. 2. GOUDSWAARD, J., A. NOORDZIJ, R. H. VAN DAM, J. A. VAN DER DONK, and .L-P. VAERMAN. 1977. The immunoglobulins of the turkey (Meleagris gallopavol. Isolation and characterization of IgG, IgM and IgA in body fluids, eggs and intraocular tissues. Poultry Science 56: 1847. 3. LESLIE, G. A., and A. A. BENEDICT. 1969. Structural and antigenic relationships between avian inlmunoglobulins. I. The immune responses of pheasants and quail and reduchve dissociation of their immunoglobulins. J. Immunol. 103: 1356. 4. GREY, H. M. 1967. Duck immunoglobulins. II. Biologic and immunochemical studies. J. Immuno!. 98: 820. 5. GOUDSWAARD, J., J.-P. VAERMAN, and J. F. HEREMANS. 1977. Three immunoglobulin classes in the pigeon (Columbia livia). Int. Archs. Allergy app!. Immun. 53: 409. 6. LESLIE, G. A., and .L VV. CLEM. 1969. Phylogeny of immunoglobulin structure and function. III. Immunoglobulins of the chicken. J. expo Med. 130: 1337. 7. LEBAcQ-VERHEYDEN, A.-M., J.-P. VAEJIMAN, and J. F. HEREMANS. ] 974. Quantification and distribution of chicken immunoglobulins IgA, IgM and IgG in serum and secretions. Immunology 27: 683. 8. YAMAMOTO, H., H. VVATANABE, G. SATO, and T. MIKAMI. 1975. Identification of immUl1ogiobulins in chicken eggs and their antibody activity. Japn. J. Vet. Res. 23: 131.
Turkey Immunoglobulins . 255 9. KIRST, R. 1967. Spuren von Antikorpern im Hiihnerei. Zschr. Arztl. Fortbild. 61: 761. 10. BURSTEIN, M., and J. SAMAILLE. 1959. Nouvelle methode de separationet de dosage des lipoproteines de faible densiM. Ann. BioI. Clin. 17: 23. 11. MANCINI, G., A. O. CARBONARA, and J. F. HERE1I1ANS. 1965. Immunochemical quantitation of antigens by radial immunodiffusion. Immunochemistry 2 : 235. 12. HUDSON, L., and F. C. HAY. 1976. Practical Immunology. Blackwell Scient. Publ. Oxford. 13. LOWRY, O. R., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL. 1951. Protein measurement with the Folin Phenol reagent. J. BioI. Chem. 193: 265. 14. ROSE, M. E., E. ORLANS, and M. BUTTRESS. 1974. Immunoglobulin classes in the hen's egg: their segregation in yolk ad white. Eur. J. Immun. 4: 521. 15. LESLIE, G. A., R. P. STANKUS, and L. N. MARTIN. 1976. Secretory immunologic system of fowl. V. The gallbladder: an integral part of the secretory immunological system of fowl. Int. Archs. Allergy appl. Immun. 51, 2: 175. 16. WATANABE, H., and K. KOBAYASHI. 1974. Peculiar secretory IgA system identified in chickens. J. Immun. 113: 1405. 17. SANDERS, B. G., and W. L. CASE. 1977. Chicken secretory immunoglobulin: chemical ad immunological characterization of chicken IgA. Compo Biochem. Physiol. 56E: 273. 18. SURVASHE, B. D., and L D. AITKEN. 1977. Further observations on functional deletion of paraocular glands in the fowl (Gallus domesticus). Res. Vet. Sci. 23: 217. Dr. J. GOUDSWAARD, Department of Immtillology, Faculty of Veterinary Medicine, Yalelaan 1, De Uithof, Utrecht, The Netherlands.
Quantification of Turkey Immunoglobulins IgA, IgM and IgG in Serum and Secretions J. GOUDSWAARD, A. NOORDZIJ, R. H. VAN DAM, and J. A. VAN DER DONK WITH 2 FIGURES
Received December 29, 1977 . Accepted February 28, 1978
Abstract The immunoglobulins IgA, IgM and IgG of the turkey were quantitated in individual serum samples as well as in pooled sera. The variability of Ig levels in normal, healthy birds was quite high: IgA: mean value 0.633 mg/ml (4.0 x2.5 x); IgM: mean value 4.37 mg/ml (0.5 x-l.4 x) and IgG: mean value 8.92 mg/ml (0.6 x-1.7 x). Immunoglobulin levels in egg-yolk, egg-white, bile and some intraocular tissues ,vere quantitated as well. An interesting finding was, that the forementioned variability was by far not so high with respect to IgG levels in 20 egg-yolk samples: mean value 5.1 mg/ml (0.86 ¥-1.17 x). Though IgG and IgM could be detected in pooled turkey bile, IgA predominated in this secretion. In aqueous humor, iris tissue and vitreous body only IgG could be detected.
Introduction
The immunoglobulins of a number of birds have been studied including chicken (1), turkey (2), pheasant (3), duck (4) and pigeon (5). Sera of birds have long been known to contain only two classes of immunoglobulins, IgM and IgG (6), but in the chicken (1), the turkey (2) and the pigeon (5) IgA has been recently identified too. IgA is relatively abundant in secretions such as bile and intestinal fluid, while it is found also to be present in the majority of the intestinal plasma cells of the birds studied in the mentioned papers. In spite of the large number of reports on avian immunoglobulins, there is, however, only little information on quantitative distribution of avian immunoglobulins IgA, IgG and IgM in sera and secretions. Up to now only one paper has been published (7) dealing with the quantification of the immunoglobulins of the chicken in sera and some secretions. The present investigation deals with the quantitative distribution of immunoglobulins in turkey serum and secretions. In the latter egg-yolk and egg-white are included, especially because investigations concerning the determination of antibodytiters in egg-yolk and egg-white (8, 9) of
Turkey Immunoglobulins· 249
birds have been reported. According to these investigations antibodytiters proved to be considerably higher in egg-yolk in comparison with egg-white (9). Materials and Methods Animals Turkeys, 5 months old, were used in this study.
Sera 18 individual serum samples and pooled sera (approximately n collected and stored at -20°C until use.
50) were
SeC1'etions Bile was collected post-mortem directly from the gall-bladder and pooled (approximately n = 50). Storage took place at - 20°C. Aqueous hmnor was collected from the eyes with a sterile syringe. During dissection of the eyes, the lens and the vitreous body were easy to isolate from tho eye. The iris tissue was disseoted carefully from the eyes; special oare was taken that the iris tissue was free of oontamination with other tissues. The vitreous body and the iris tissue were diluted with de-ionized cooled water to twice the original volume and homogenized. Egg-white and egg-yolk of 20 fresh eggs were separated. Beoause in 4 cases egg-white was contaminated with egg-yolk, only 16 egg-white samples were examined. The egg-yolk was diluted to twioe the original volume and 1 % Triton X 100 was added to the mixture. The samples could be used without furthor treatment. In egg-white samples, treated in the same way, the immunoglobulin levels were too low to doteot. Therefore, the samples were further dialysed against 0.1 M Na-acetate buffer, pH 4.5, to preoipitate tho muoins. The resulting precipitate was discarded and the pH adjusted to 6.8. Then, globulins were salted out with 50% ammonium sulfate, pH 6.8. The precipitated globulins wero oentrifuged at 15,000 rpm and re-dissolved in 10 ml distilled water. All samples were then concentrated 20 times in a Minicon B 15 1 ).
Anti8era The preparation of a rabbit-antiserum to egg-yolk and whole turkey serum, as well as of antisera speoifio for the heavy chains of turkoy IgM, IgA and IgG has been described (2).
Purification of immunoglob·ulins I gG: egg-yolk, diluted 1: 4 with 0.9 p~rcent N aCI and treated with dextran sulphate and CaCl 2 (10), was dialysed against 0.9 p~rcent NaCI which contained 2 gil EDTA. Then it was salted out with 45 peroent (NH4)2S04 at pH 6.8. The procipitate was taken up in a small amount of distilled water, dialysed against 0.1 M Tris HCl containing 1 M NaCI, pH 8.0, and fraotionated on Ultrogel ACA 22 2). To yield pure IgG, the first peak was ohromatographed on DEAE oellulose. IgA: bilo was collectod post-mortem fronl the gall-bladder, extensively dialysed against 2 peroent NaCl buffered, at pH 8.0, with 0.02 M Tris HCI and passed on a column of Sephadex G-75 to remove low moleoular weight pigmented material. Then, after concentration to the original voItune, the eluate was 1) Amicon Corp., Lexington, Massaohusetts, USA. 2) LKB-Produkten AB, Bromma, Sweden.
250· J. GOUDSWAARD, A. NOORDZIJ, R. H.
VAN DAMM,
and J. A.
VAN
DER DONK
dialysed against 0.01 M Tris-HCI with 1 % NaCI, pH 8.0, and chromatographed on Sepharose 4B3). The IgA-rich fraction in the second peak (not shown) was then chromatographed twice on Ultrogel ACA 22. The first peak contained pure IgA. IgJjJJ: pooled serum was treated with dextran sulphate and CaCl 2 (10) and dialysed against 0.9 percent NaCI containing 2g/1 EDTA. Then, the serum was successively precipitated with 18, 14 and 14% (w/v) of sodium sulphate. After each precipitation the precipitate was dissolved in distilled water and dialysed against 0.03 Na 3 BO s buffer, pH 8.0. After dialysis, the globulins salted out with 14% (w/v) sodium sulphate were further salted out with 9% sodium sulphate. The resultant supernatant was finally brought again to 14% sodium sulphate. The latter precipitate was redissolved in distilled water and dialysed against 0.01 M Tris-HCI containing 1 M NaCI and chromatographed on Ultrogel ACA 22. The first peak contained almost pure IgM (see under Results).
Q'U
Immunoelectrophoresis and double diffu8ion Immunoelectrophoresis and double diffusion ("Ouchterlony") were carried out according to standard procedures using 1 % agarose (Industrie Biologique Franyaise) .
Results
PU1·ification of immunoglobulin standards The preparation of IgG (2.9 mgjml) from egg-yolk and of IgA (2.3 mgjml) from bile yielded pure immunoglobulin standards, as judged by Ouchterlony analysis as well as by immunoelectrophoresis with anti-whole turkey serum (figure 1) and anti-whole egg-yolk (not shown). Upon radial immunodiffusion the IgA or IgG samples reacted only with anti-a and anti-y reagents respectively, also after being concentrated about 5 times. Although the preparation of IgM (3 mgjml) seemed to be pure, as judged immunoelectrophoretically with anti-whole turkey serum (figure 1), it still contained 0.19 mgjml IgG and 0.11 mgjml IgA, when the sample was checked with anti-y and anti-IX reagents in radial immunodiffusion. The antisera used, were specific for each immunoglobulin class, as is s,hown in figure 2. 3) Pharmacia Fine Chemicals, Uppsala, Sweden. 4) Transidyne General Corporation, Ann Arbor - Michigan.
Turkey IIllIlllmoglobulins . 251
_ lgM
-
A-Wh
-
~lgA
~
IgG
A-W. h ..r
Fig. 1: Agarose gel immunoelectrophoresis of pm'ified immunoglobulin standards IgM, IgA and IgG, developed with anti-whole turkey serum (A-Wh). Anode to the left.
Fig. 2: Agarose gel immlUloelectrophoresis of turkey serum (Se) developed with anti-whole turkey serum (A-Wh) and sp ecific anti-IgM (A-IgM), anti-lgA (A19A) and an ti-IgG (A-IgG). Anode t o the left.
252· J. GOUDSWAARD, A. NOORDZIJ, R. H. VAN DAMM, and J. A. VAN DER DONK
Quantitative distribution ot immunoglobulins in sera and secretions Immunoglobulin quantification was carried out in 18 individual serum samples and in a pool of about 50 sera. Moreover, IgG was quantitated in 20 egg-yolk samples. The results are summarized in Table 1. The IgM and IgA levels of egg-white are very low, as was expected. In 9 samples only a trace of IgM was found; the mean IgM level in the other samples was 0.024 mg(ml. The extensive treatment of the egg-white samples also did not allow to give exact values of IgA levels in individual samples: the main value, however, was 0.034 mg( ml. The IgA concentration in pooled bile (approximately n = 50) was 1. 7 mg(ml. Bile contained also IgM (0.44 mg(ml) as well as IgG (0.21 mg(ml). Table 1. Immtilloglobulin levels in 18 individual serum samples and in pooled serum (approximately n = 50). IgG levels in 20 egg-yolk samples. Immunoglobulins mg/ml. Serumnumber
IgA
IgM
IgG
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
0.52 0.33 0.66 0.55 0.62 1,48 1.60 0.39 0.53 0,46 0.52 0,43 0.74 0,41 0.26 0.62 0,44 0.84
4.80 2,40 5.80 3.08 5,48 5,46 4.12 5.80 5.12 5.80 2,48 3.58 2,40 5,46 3.80 5.88 3.28 4.00
8.2 8.2 9.0 8.2 10.8 10.0 9.2 8.6 8.0 15.2 9.0 11.2 6.8 7.2 7.2 11.0 7.6 5.2
Mean
0.633
4.37
8.92
5.1
Normal value
0,4x-2.5x
0.5x- 1.4x
0.6x-1. 7:X:
0.86x-1.17:X:
3.80
9.2
Pooled serum
0.55
Egg-yolk nunlber
IgG
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
6.0 6.0 5.6 5.2 4.6 5.6 5.6 6.0 5.2 4.2 4.6 4,4 4.6 5.2 4.6 4.6 5.0 5.0 4,4 5.6
Turkey Immunoglobulins· 253
In the pooled samples of corpus vitrium, aquaeous humor and iris (approximately n = 10) only IgG proved to be present; the concentration of this immunoglobulin class in the various eye fluids was respectively 0.24 mgjml, 0.32 mgjml and 0.24 mgjml. Discussion The only immunoglobulin class which can be quantitated in egg-yolk is IgG, for it is known (14) that neither IgA nor IglVI are present in yolk. On the other hand, IgG will not be found in egg-white. Our treatment of the yolk with Triton X 100 proved to be a successful method; this made the diffusion of the egg-yolk proteins very easy. The clear precipitation rings of the egg-yolk samples could be read within the same time as those of the serum samples. It can be concluded from the table that yolk contains large quantities of IgG (mean IgG level 5.1 mgj ml) which laying hens do loose every day. It is proved that this IgG represents the whole range of antibodies to various infectious agents (8). An interesting finding too is the small variability of the IgG levels in the different yolk samples, which is very much in contrast with the reported findings of Ig levels in chicken sera (7) and with our own findings with the turkey serum samples. This suggests a phenomenon which can not be explained by a simple transfer of the IgG to the yolk. Also, the specific passage of only IgG antibodies from hen serum to yolk and hence - if the eggs are fertilized and hatched - to the circulation of the chick via the endoderm of the yolk sac are evidence for this suggestion. The Ig levels found by us in sera of almost adult turkeys are higher as those reported by LEBAOQ-VERHEYDEN and co-workers (7) in chickens (IgA: 0.633 mgjml versus 0.377 mgjml; IgG: 8.92 mgjml versus 6.90 mgjml and IglVI: 4.37 mgjml versus 2.506 mgjml), though the variability in individual serum samples is almost comparable. This high variability of Ig levels in healthy individuals in human sera is wellknown too. The differences between our results and those of LEBAOQVERHEYDEN (7) may be explained by the differences in age of the experimental animals. Our birds were 5 months of age, while the chickens she used were only 6-8 weeks old. Paper electrophoresis of pooled turkey sera revealed 15.1 % ,(-globulin (total protein content of the serum was 44 mgjml). The values confirm our findings, because nearly all of the ,(-globulin is already part of the IgG. Though we were able to prove the IglVI and IgA content of all investigated egg-white samples, our results were not accurate enough to report about the quantitative distribution of these immunoglobulins in individual samples. There is no doubt, we loosed small quantities of Ig during the different treatments.
254· J. GOUDSWAARD, A. NOORDZU, R. H. VAN DAMM, and J. A. VAN DER DONK
The IgA content of turkey bile (1.7 mg/ml) was considerably lower as was expected. LEBACQ-VERHEYDEN and co-workers (7) found 3.15 mg/ ml. It is well-known (15) that the chicken gallbladder synthesizes and secretes IgA. There is no reason to believe that the gallbladder of other birds should behave in a different way. It is possible that the IgA in the bile collected by us underwent some enzymatic digestion, especially because it has been reported (16) that secretory IgA of chicken bile lacks secretory component. Lebacq-Verheyden and co-workers centrifuged, extensively dialysed and diluted their bile samples before use. The finding by these authors, that chicken bile contains also small amounts of IgG and IgM, was confirmed by us with pooled turkey bile. In a recently published study by SANDERS and CASE (17) chicken IgA was determined to be the only immunoglobulin class present in bile; the fact that these authors just carried out immunoelectrophoresis and immunodiffusion may be responsible for this different result. However, even with the radial immunodiffusion test we used as the more sensitive test, we were not able to detect any traces of IgM and even of IgA in pooled samples of intraocular tissues. Just low levels of IgG were detected. Therefore, we conclude that there is no direct link between these tissues and the avian Harderian gland in which mainly IgA containing immunocytes (18) can be detected. References 1. LEBAcQ-VERHEYDEN, A.-M., J.-P. VAERMAN, and J. F. HEREMANS. 1972. A possible homologue of mammalian IgA in chicken serum and secretions. Immunology 22: 165. 2. GOUDSWAARD, J., A. NOORDZIJ, R. H. VAN DAM, J. A. VAN DER DONK, and .L-P. VAERMAN. 1977. The immunoglobulins of the turkey (Meleagris gallopavol. Isolation and characterization of IgG, IgM and IgA in body fluids, eggs and intraocular tissues. Poultry Science 56: 1847. 3. LESLIE, G. A., and A. A. BENEDICT. 1969. Structural and antigenic relationships between avian inlmunoglobulins. I. The immune responses of pheasants and quail and reduchve dissociation of their immunoglobulins. J. Immunol. 103: 1356. 4. GREY, H. M. 1967. Duck immunoglobulins. II. Biologic and immunochemical studies. J. Immuno!. 98: 820. 5. GOUDSWAARD, J., J.-P. VAERMAN, and J. F. HEREMANS. 1977. Three immunoglobulin classes in the pigeon (Columbia livia). Int. Archs. Allergy app!. Immun. 53: 409. 6. LESLIE, G. A., and .L VV. CLEM. 1969. Phylogeny of immunoglobulin structure and function. III. Immunoglobulins of the chicken. J. expo Med. 130: 1337. 7. LEBAcQ-VERHEYDEN, A.-M., J.-P. VAEJIMAN, and J. F. HEREMANS. ] 974. Quantification and distribution of chicken immunoglobulins IgA, IgM and IgG in serum and secretions. Immunology 27: 683. 8. YAMAMOTO, H., H. VVATANABE, G. SATO, and T. MIKAMI. 1975. Identification of immUl1ogiobulins in chicken eggs and their antibody activity. Japn. J. Vet. Res. 23: 131.
Turkey Immunoglobulins . 255 9. KIRST, R. 1967. Spuren von Antikorpern im Hiihnerei. Zschr. Arztl. Fortbild. 61: 761. 10. BURSTEIN, M., and J. SAMAILLE. 1959. Nouvelle methode de separationet de dosage des lipoproteines de faible densiM. Ann. BioI. Clin. 17: 23. 11. MANCINI, G., A. O. CARBONARA, and J. F. HERE1I1ANS. 1965. Immunochemical quantitation of antigens by radial immunodiffusion. Immunochemistry 2 : 235. 12. HUDSON, L., and F. C. HAY. 1976. Practical Immunology. Blackwell Scient. Publ. Oxford. 13. LOWRY, O. R., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL. 1951. Protein measurement with the Folin Phenol reagent. J. BioI. Chem. 193: 265. 14. ROSE, M. E., E. ORLANS, and M. BUTTRESS. 1974. Immunoglobulin classes in the hen's egg: their segregation in yolk ad white. Eur. J. Immun. 4: 521. 15. LESLIE, G. A., R. P. STANKUS, and L. N. MARTIN. 1976. Secretory immunologic system of fowl. V. The gallbladder: an integral part of the secretory immunological system of fowl. Int. Archs. Allergy appl. Immun. 51, 2: 175. 16. WATANABE, H., and K. KOBAYASHI. 1974. Peculiar secretory IgA system identified in chickens. J. Immun. 113: 1405. 17. SANDERS, B. G., and W. L. CASE. 1977. Chicken secretory immunoglobulin: chemical ad immunological characterization of chicken IgA. Compo Biochem. Physiol. 56E: 273. 18. SURVASHE, B. D., and L D. AITKEN. 1977. Further observations on functional deletion of paraocular glands in the fowl (Gallus domesticus). Res. Vet. Sci. 23: 217. Dr. J. GOUDSWAARD, Department of Immtillology, Faculty of Veterinary Medicine, Yalelaan 1, De Uithof, Utrecht, The Netherlands.