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Title:   Author  item
                                            SCHILLER, I - PRIONICS AG, SWITZERLANDitem
                                            MARG-HAUFE, B - PRIONICS AG, SWITZERLANDitem
                                            VORDERMEIER, M - VET. LAB. AGENCY, UKitem
                                            Waters, Wadeitem
                                            WHELAN, A - VET. LAB. AGENCY, UKitem
                                            WELSH, M - VET. SCIENCES DIV. UKitem
                                            KECK, N - LAB. DEP. DE L'HERAULTitem
                                            MOYEN, J - LAB. DEPT. D'ANALYSES, FRitem
                                            Palmer, Mitchellitem
                                            Thacker, Tyleritem
                                            RAEBER, A - PRIONICS AG, SWITZERLANDitem
                                            OESCH, B - PRIONICS AG, SWITZERLAND
  Submitted to: Meeting Abstract
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 10/16/2008
                                    Publication  Date: 10/16/2008
                                    Citation: Schiller, I., Marg-Haufe, B., Vordermeier, M., Waters, W.R., Whelan, A., Welsh, M., Keck, N., Moyen, J.L., Palmer, M.V., Thacker, T.C., Raeber, A., Oesch, B. 2008. Interferon Gamma Assay for the Diagnosis of Bovine Tuberculosis [abstract]. Paper No. 25.Interpretive Summary:
                                Technical Abstract: Contact
Irene Schiller
Prionics AG
Wagistrasse 27A
CH-8952 Schlieren
Switzerland
irene.schiller@prionics.com
	 

Introduction
Bovine tuberculosis (bTB), a zoonotic disease with a major economic impact, continues to be a significant problem with a global perspective and increasing prevalence in various countries. Control and eradication of bTB are based on a test and slaughter strategy. The BOVIGAM® interferon  gamma (IFN-gamma) assay (Prionics, Switzerland) constitutes a laboratory-based test detecting the hosts's cell-mediated immune response (CMI). It is widely used complementary to the tuberculin skin test. The principle of this test has been adapted for TB diagnosis in humans (QuantiFERON®-TB). 

Aim
Environmental conditions before and during the culturing of the leucocytes influence the efficacy of in vitro IFN-gamma production. We have analyzed various parameters of the IFN-gamma test in view of optimal conditions and potential simplifications in order to streamline the assay procedure.


Results
We show that the culture temperature needs to be 33°C or higher and that there is no need to use incubation in the presence of CO2. Furthermore, various plate formats are shown to be feasible. The produced IFN-gamma was stable at 4°C for 28 days as well as after repeated freeze-thaw cycles.

The CMI is known to be impacted negatively by stress. We have stimulated fresh blood from animals with or without stress with mitogens resulting in significantly lower IFN-' production in stressed animals and therefore potentially leading to false negative results. 

Tuberculosis-specific stimulation is currently done with tuberculins. We have analyzed tuberculins at different concentrations from different sources and will report on the optimal standardized tuberculin activity. As an alternative, a cocktail of recombinant antigens for stimulation resulted in improved diagnostic sensitivity and specificity. 

Conclusion
Simplified culture conditions in combination with the high stability of natural IFN-gamma and optimized stimulation antigens will simplify the assay as well as the logistics for blood samples considerably. These recent developments in measuring the CMI represent excellent tools for control and eradication of bTB.


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