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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, 1210Vienna, Austria
B. RUTTKOWSKI
Affiliation:
Institute of Parasitology, University of Veterinary Medicine Vienna, Department of Pathobiology, Veterinaerplatz 1, 1210Vienna, Austria
A. JOACHIM
Affiliation:
Institute of Parasitology, University of Veterinary Medicine Vienna, Department of Pathobiology, Veterinaerplatz 1, 1210Vienna, Austria
A. SAALMÜLLER
Affiliation:
Institute of Immunology, University of Veterinary Medicine Vienna, Department of Pathobiology, Veterinaerplatz 1, 1210Vienna, Austria
W. GERNER
Affiliation:
Institute of Immunology, University of Veterinary Medicine Vienna, Department of Pathobiology, Veterinaerplatz 1, 1210Vienna, Austria
*
*Corresponding author: University of Veterinary Medicine Vienna, Department of Pathobiology, Institute of Parasitology, Veterinaerplatz 1, 1210Vienna, Austria. Tel: +43 1 25077 2227. Fax: +43 1 25077 2290. E-mail: hanna.worliczek@vetmeduni.ac.at
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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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References

REFERENCES

Breed, D. G., Schetters, T. P., Verhoeven, N. A., Boot-Groenink, A., Dorrestein, J. and Vermeulen, A. N. (1999). Vaccination against Eimeria tenella infection using a fraction of E. tenella sporozoites selected by the capacity to activate T cells. International Journal for Parasitology 29, 1231–1240.CrossRefGoogle ScholarPubMed
Daugschies, A., Bialek, R., Joachim, A. and Mundt, H. C. (2001). Autofluorescence microscopy for the detection of nematode eggs and protozoa, in particular Isospora suis, in swine faeces. Parasitology Research 87, 409–412. DOI: 10.1007/s004360100378CrossRefGoogle ScholarPubMed
Everson, W. V., Ware, M. W., Dubey, J. P. and Lindquist, H. D. (2002). Isolation of purified oocyst walls and sporocysts from Toxoplasma gondii. The Journal of Eukaryotic Microbiology 49, 344–349. DOI: 10/1111/j.1550-7408.2002.tb00381.xCrossRefGoogle ScholarPubMed
Fang, Q., Sun, Y. Y., Cai, W., Dodge, G. R., Lotke, P. A. and Williams, W. V. (2000). Cartilage-reactive T cells in rheumatoid synovium. International Immunology 12, 659–669.CrossRefGoogle ScholarPubMed
Ferrari, B. C., Vesey, G., Davis, K. A., Gauci, M. and Veal, D. (2000). A novel two-color flow cytometric assay for the detection of Cryptosporidium in environmental water samples. Cytometry 41, 216–222. DOI: 10.1002/1097-0320(20001101)41:3<216::AID-CYTO9>3.0.CO;2-R3.0.CO;2-R>CrossRefGoogle ScholarPubMed
Fuller, A. L. and McDougald, L. R. (1989). Analysis of coccidian oocyst populations by means of flow cytometry. The Journal of Protozoology 36, 143–146. DOI: 10.1111/j.1550-7408.1989.tb01061.xCrossRefGoogle ScholarPubMed
Hermosilla, C., Barbisch, B., Heise, A., Kowalik, S. and Zahner, H. (2002). Development of Eimeria bovis in vitro: suitability of several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal, Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells. Parasitology Research 88, 301–307. DOI: 10.1007/s00436-001-0531-1CrossRefGoogle ScholarPubMed
Hermosilla, C., Zahner, H. and Taubert, A. (2006). Eimeria bovis modulates adhesion molecule gene transcription in and PMN adhesion to infected bovine endothelial cells. International Journal for Parasitology 36, 423–431. DOI:10.1016/j.ijpara.2006.01.001CrossRefGoogle ScholarPubMed
Lindsay, D. S., Blagburn, B. L. and Powe, T. A. (1992). Enteric coccidial infections and coccidiosis in swine. Compendium on Continuing Education for the Practicing Veterinarian 14, 698–702.Google Scholar
Mundt, H. C., Joachim, A., Becka, M. and Daugschies, A. (2006). Isospora suis: an experimental model for mammalian intestinal coccidiosis. Parasitology Research 98, 167–175. DOI: 10.1007/s00436-005-0030-xCrossRefGoogle ScholarPubMed
Pakandl, M., Hlaskova, L., Poplstein, M., Chroma, V., Vodicka, T., Salat, J. and Mucksova, J. (2008). Dependence of the immune response to coccidiosis on the age of rabbit suckling. Parasitology Research 103, 1265–1271. DOI: 10.1007/s00436-008-1123-0.CrossRefGoogle ScholarPubMed
Pintaric, M., Gerner, W. and Saalmüller, A. (2008). Synergistic effects of IL-2, IL-12 and IL-18 on cytolytic activity, perforin expression and IFN-gamma production of porcine natural killer cells. Veterinary Immunology and Immunopathology 121, 68–82. DOI: 10.1016/j.vetimm.2007.08.009.CrossRefGoogle ScholarPubMed
Ruttkowski, B., Joachim, A. and Daugschies, A. (2001). PCR-based differentiation of three porcine Eimeria species and Isospora suis. Veterinary Parasitology 95, 17–23. DOI: 10.1016/S0304-4017(00)00408-8CrossRefGoogle ScholarPubMed
Scala, A., Demontis, F., Varcasia, A., Pipia, A. P., Poglayen, G., Ferrari, N. and Genchi, M. (2009). Toltrazuril and sulphonamide treatment against naturally Isospora suis infected suckling piglets: is there an actual profit? Veterinary Parasitology 163, 362–365. doi: 10.1016/j.vetpar.2009.04.028.CrossRefGoogle ScholarPubMed
Schmatz, D. M., Crane, M. S. and Murray, P. K. (1984). Purification of Eimeria sporozoites by DE-52 anion exchange chromatography. The Journal of Protozoology 31, 181–183.CrossRefGoogle ScholarPubMed
Schulze, F. (1978). The quantitative composition of the gastrointestinal flora in piglets during the first weeks of life. 1. The development of the gastrointestinal flora in naturally reared piglets. Archiv für Experimentelle Veterinärmedizin 32, 155–170.Google ScholarPubMed
Taylor, J. R. (1984). Immune Response of Pigs to Isospora suis (Apicomplexa, Eimeriidae). Dissertation, Auburn University, Auburn, Alabama, USA.Google Scholar
Worliczek, H. L., Mundt, H. C., Ruttkowski, B. and Joachim, A. (2009). Age, not infection dose, determines the outcome of Isospora suis infections in suckling piglets. Parasitology Research 105 (Suppl. 1), S157–S162. doi: 10.1007/s00436-009-1507-9.CrossRefGoogle Scholar
Worliczek, H. L., Buggelsheim, M., Alexandrowicz, R., Witter, K., Schmidt, P., Gerner, W., Saalmüller, A. and Joachim, A. (2010). Changes in lymphocyte populations in suckling piglets during primary infections with Isospora suis. Parasite Immunology 32, 232–244. doi: 10.1111/j.1365-3024.2009.01184.x.CrossRefGoogle ScholarPubMed