Abstract

There is at this moment no sensitive and specific test for evaluating the antibody response after erysipelas vaccination. The haem agglutination tests used have low sensitivity and specificity.¹ ELISA's are in general more sensitive and specific and can be used to determine IgG, IgM and IgA antibody responses. Therefore an isotype-specific ELISA with purified erysipelas antigens would be more appropriate for analysing the immune status of the animal. However anti-dolphin immunoglobulin-antisera specifically directed against dolphins IgG, IgM or IgA are necessary for such an ELISA.² Those are not commercially available.

In the present study, the cross-reaction was examined of antisera against immunoglobulins of several species with dolphin immunoglobulins.

Immunoglobulins were enriched from dolphins' sera by ammonium sulphate precipitation and were fractionated by gel filtration using ultra gel as a matrix. Fractions were subsequently examined for the presence of immunoglobulins by ELISA's and Western blotting using antisera against immunoglobulin G whole molecule, IgA (Fc) or against the gamma or the mu chain of immunoglobulins of different species. In ELISA, a strong reaction was observed with anti-dog and anti-swine total immunoglobulins and to a lesser extend, and in descending order, with anti horse, -rabbit, -bovine and -goat immunoglobulins.

In Western blotting it was shown that anti-dog IgM, anti-dog IgG and anti-horse IgG recognize specifically the heavy chains of dolphin IGM, IgG and IgG respectively.

Introduction

Anti-dolphin immunoglobulin-antisera specifically directed against dolphins IgG, IgM and/or IgA are not commercially available. The aim of the present study was to evaluate the cross reaction of commercial antisera against immunoglobulins of several species with dolphin immunoglobulins. Therefore, different immunoglobulin isotypes in serum of dolphins were separated on the basis of their molecular size using gel filtration, where after commercially available antisera against immunoglobulins of other mammals were used to determine cross-reaction.

Materials and Methods

1.  Gel filtrations of dolphins' sera
Serum samples of different dolphins were pooled. The immunoglobulins were enriched by a 50% ammonium sulphate precipitation. The obtained proteins were dissolved in PBS at a concentration of 50 mg/ml. Five milliliter of this solution was loaded on an ultra gel column (length 1 m, diameter 5 cm). The column was run with a 50 mM Tris-HCl, pH 8.6 buffer, at a flow rate of 0.2 ml/min for 46 hours. UV monitoring of the eluens at 280 mm was performed to assess the fractions which contained proteins. Eighty fractions of 6 ml each were collected.

2.  ELISA
ELISA was used for a first screening of the fractions for presence of cross reaction with antisera against immunoglobulins of several species. Hereto the fractions containing proteins, as determined with the UV monitor, were coated in different wells of a 96-wells microtitre plate. After blocking of the remaining binding sites and four washes with PBS-Tween 20, the different antisera conjugated with horseradish peroxydase were added to the wells in their appropriate dilution. Again four washes were performed, where after ABTS substrate was added. Thirty minutes later the absorbance was measured at 405 run. The absorbance was positively correlated with the intensity of cross-reaction of the antisera with proteins present in the fractions.

3.  SDS Page
Since gamma chains have a molecular weight of +/-50 kD, mu chains of +/-70 kD and lambda and kappa chains of +/-23 kD, a 12% acrylamide separating gel was chosen and samples were run under reducing conditions, during one hour, at 150 V and 120 mA.
The gels were then either directly colored with Coomassie blue or immuno blotted.

4.  Western Blotting
The gels were blotted onto a polyvinyl-fluoride membrane during one hour at 250 mA and 5OV. The membrane was then left overnight in a blocking solution of PBS supplemented with 10% (vol/vol) immunoglobulin free horse serum and 0.2% (vol/vol) Tween 20. After three washes with PBS, the chosen anti-sera, conjugated with horseradish peroxydase and appropriately diluted in the blocking solution, were added to the membrane and incubated for one hour. After three further washes binding of the antisera was revealed by adding an AEC (3-amino-9-ethylcarbazole) solution during 15 minutes at room temperature. The membrane was then further washed with PBS and aqua dest.

5.  Antisera against immunoglobulins of several species.

 swine anti-rabbit immunoglobulins (mainly IgG whole molecule)

 rabbit anti-mouse immunoglobulins (mainly IgG whole molecule)

 rabbit anti-goat immunoglobulins (mainly IgG whole molecule)

 rabbit anti-swine immunoglobulins (mainly IgG whole molecule)

 rabbit anti-bovine immunoglobulins (mainly IgG whole molecule)

 goat anti-dog/IgA(Fc)

 rabbit anti-dog/IgG(H+L)

 goat anti-dog IgG (gamma chain)

 goat anti-dog IgM (mu chain)

 goat anti-horse IgG (gamma chain)

 goat anti-horse IgM (mu chain)

Results

ELISA

ELISA results of cross-reaction between dolphin immunoglobulins and antisera against immunoglobulins of different species showed good reactions for dog, horse and swine. A weak reaction was observed for rabbit. No reaction was observed for bovine and goat.

Since cross-reaction was more pronounced for the antisera against dog and horse immunoglobulins, isotype specific antisera against the mu and gamma chain of both animals were evaluated for their cross-reaction with dolphins immunoglobulins.

Western blotting Western blotting was performed to confirm results of the ELISA. It was observed that the dog specific anti-mu or anti-gamma chain antisera specifically recognize the dolphin IgM and IgG heavy chains and no other molecules; whereas the horse specific anti-mu or anti-gamma chain antisera also recognize other proteins of other molecular weights.

It was observed that the mu chain was approximately between 67 and 84 kD, but nearer the 67 kD band. The gamma chain was approximately between 55 and 67 kD. The light chains seemed to be near 35 kD. Until now dolphin IgA could not be specifically identified in serum.

Conclusion

This study showed that anti-dog isotype specific antisera can be used to analyze specifically the immune status of a dolphin and the systemic humoral immune response against vaccines and infectious microorganisms.

 la Bourse de la Vocation, Belgium

 Hoechst, Belgium

 John G. Shedd Aquarium, USA

 Ocean Park, Hong Kong

 ZooMarine, Portugal

 Anonymous gifts

References

1.  Colgrove, G. S. 1975. A survey of Erysipelothrix insidiosa agglutinating antibody titers in vaccinated porpoises (Tursiops truncatus). Journal of Wildlife Diseases 11:234-236.

2.  Nash, D.R., and J. Mach. 1971. Immunoglobulin classes in aquatic mammals: characterization by serological cross reactivity, molecular size and binding of human free secretory component. Journal of Immunology 107:1424-1430.