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Faeces, FACS, and functional assays – preparation of Isospora suis oocyst antigen and representative controls for immunoassays

Published online by Cambridge University Press:  26 May 2010

H. L. WORLICZEK ,
B. RUTTKOWSKI ,
A. JOACHIM ,
A. SAALMÜLLER  and
W. GERNER
H. L. WORLICZEK*
Affiliation:
Institute of Parasitology, University of Veterinary Medicine Vienna, Department of Pathobiology, Veterinaerplatz 1, 1210 Vienna, Austria
B. RUTTKOWSKI
Affiliation:
Institute of Parasitology, University of Veterinary Medicine Vienna, Department of Pathobiology, Veterinaerplatz 1, 1210 Vienna, Austria
A. JOACHIM
Affiliation:
Institute of Parasitology, University of Veterinary Medicine Vienna, Department of Pathobiology, Veterinaerplatz 1, 1210 Vienna, Austria
A. SAALMÜLLER
Affiliation:
Institute of Immunology, University of Veterinary Medicine Vienna, Department of Pathobiology, Veterinaerplatz 1, 1210 Vienna, Austria
W. GERNER
Affiliation:
Institute of Immunology, University of Veterinary Medicine Vienna, Department of Pathobiology, Veterinaerplatz 1, 1210 Vienna, Austria
*
*Corresponding author: University of Veterinary Medicine Vienna, Department of Pathobiology, Institute of Parasitology, Veterinaerplatz 1, 1210 Vienna, Austria. Tel: +43 1 25077 2227. Fax: +43 1 25077 2290. E-mail: [email protected]
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Summary

Highly purified antigen and appropriate controls are essential for antigen-specific immunoassays. In the case of Isospora suis, the causative agent of neonatal porcine coccidiosis, the only current source of antigen is oocysts isolated from faeces. The aim of this study was to develop a procedure for high-grade purification of I. suis oocysts from piglet faeces to obtain both antigen and representative controls suitable for in vitro re-stimulation of lymphocytes. This was achieved by use of filtration, density-gradient centrifugation and fluorescence-activated cell sorting (FACS). The feasibility for immunological studies was demonstrated with IFN-γ ELISPOT assays after in vitro re-stimulation of lymphocytes from previously infected swine using the obtained antigen. The developed method allowed the production of highly purified antigen and representative controls from faeces with an oocyst recovery rate of 14%. Regarding the application of the obtained material it could be shown that lymphocytes from I. suis-infected pigs react in an antigen-specific manner in terms of an in vitro recall response by the production of IFN-γ. This demonstrates the suitability of the developed method for the production of antigen and controls for sensitive immunological readout systems. Moreover, the detected specific IFN-γ response encourages further functional studies on the cellular immune response to I. suis.

Keywords

Isospora suisrecall responseantigen preparationFACSELISPOTcellular immune responsepig
Type
Research Article
Information
Parasitology , Volume 137 , Issue 11 , September 2010 , pp. 1637 - 1643
Copyright
Copyright © Cambridge University Press 2010

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