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Clinical and Vaccine Immunology, August 2009, p. 1196-1202, Vol. 16, No. 8
1071-412X/09/$08.00+0     doi:10.1128/CVI.00150-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Optimization of a Whole-Blood Gamma Interferon Assay for Detection of Mycobacterium bovis-Infected Cattle{triangledown}

Irene Schiller,1 W. Ray Waters,2 H. Martin Vordermeier,3 Brian Nonnecke,2 Michael Welsh,4 Nicolas Keck,5 Adam Whelan,3 Teresa Sigafoose,6 Christoph Stamm,1 Mitchell Palmer,2 Tyler Thacker,2 Roland Hardegger,1 Beatrice Marg-Haufe,1 Alex Raeber,1 and Bruno Oesch1*

Prionics AG, Schlieren, Switzerland,1 National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa,2 Veterinary Laboratory Agency, Addlestone, Great Britain,3 AFBI, Veterinary Sciences Division, Stormont, Great Britain,4 Laboratoire Départemental Vétérinaire de l'Hérault, Montpellier, France,5 National Veterinary Services Laboratories, APHIS, U.S. Department of Agriculture, Ames, Iowa6

Received 3 April 2009/ Returned for modification 11 May 2009/ Accepted 19 June 2009

Antigens of Mycobacterium bovis elicit a cell-mediated immune response upon intradermal injection in cattle. In vitro, such antigens stimulate the production of gamma interferon (IFN-{gamma}) by bovine T cells in whole-blood culture (IFN-{gamma} assay). We have analyzed various parameters of the in vitro IFN-{gamma} assay, ranging from blood sampling to execution of the IFN-{gamma} test, in view of potential simplifications of the assay. Here, we show that IFN-{gamma} responses may be reduced under certain animal handling/holding conditions and that a delayed time from blood collection to culture may lead to a reduced in vitro IFN-{gamma} response. Delayed initiation of culture in a purified-protein-derivative-based assay (24 h compared to 8 h after blood collection), however, resulted in a significant improvement of specificity (97% compared to 85%), whereas there was only a modest reduction of sensitivity (from 96% to 90%), which was statistically not significant. Furthermore, we show that the stimulation temperature needs to be 33°C or higher; that carbon dioxide is not required for stimulation; and that various plate formats, ranging from 24 to 96 wells per plate, can be utilized. The produced IFN-{gamma} is stable at 4°C for 28 days as well as after repeated freeze-thaw cycles. Thus, stimulation of samples may be initiated in the field without the need for a carbon dioxide source, and bovine IFN-{gamma} is stable under various routine laboratory temperature scenarios. These findings demonstrate opportunities for improvements in the bovine IFN-{gamma} test platform and flexibilities in test application.

* Corresponding author. Mailing address: Prionics AG, Wagistrasse 27A, 8952 Schlieren, Switzerland. Phone: 41 44 200 2014. Fax: 41 44 200 2010. E-mail: Bruno.oesch{at}prionics.com

{triangledown} Published ahead of print on 1 July 2009.

Clinical and Vaccine Immunology, August 2009, p. 1196-1202, Vol. 16, No. 8
1071-412X/09/$08.00+0     doi:10.1128/CVI.00150-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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