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Selected Publication:

Publication type: Journal Article
Document type: Full Paper

Year: 2010

Author(s): Worliczek, HL; Ruttkowski, B; Joachim, A; Saalmüller, A; Gerner, W

Title: Faeces, FACS, and functional assays--preparation of Isospora suis oocyst antigen and representative controls for immunoassays.

Source: Parasitology. 2010; 137(11):1637-1643



Authors Vetmeduni Vienna:

Gerner Wilhelm
Joachim Anja
Saalmüller Armin
Worliczek Hanna

Vetmed Research Units
Institute of Immunology
Institute of Parasitology


Abstract:
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-gamma 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-gamma. 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-gamma response encourages further functional studies on the cellular immune response to I. suis.

Keywords Pubmed: Animals
Antigens, Protozoan*/immunology
Antigens, Protozoan*/isolation & purification
Centrifugation, Density Gradient
Enzyme-Linked Immunospot Assay*/methods
Enzyme-Linked Immunospot Assay*/standards
Feces/parasitology*
Female
Flow Cytometry*/methods
Interferon-gamma/biosynthesis
Isospora/growth & development
Isospora/immunology*
Isospora/isolation & purification
Isosporiasis/immunology
Isosporiasis/parasitology*
Lymphocyte Activation
Oocysts/immunology*
Sensitivity and Specificity
T-Lymphocytes/immunology


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