Optimization of a whole-blood gamma interferon assay for detection of Mycobacterium bovis-infected cattle.
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 degrees 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 degrees 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.
Schiller
Irene
I
Prionics AG, Schlieren, Switzerland.
Waters
W Ray
WR
Vordermeier
H Martin
HM
Nonnecke
Brian
B
Welsh
Michael
M
Keck
Nicolas
N
Whelan
Adam
A
Sigafoose
Teresa
T
Stamm
Christoph
C
Palmer
Mitchell
M
Thacker
Tyler
T
Hardegger
Roland
R
Marg-Haufe
Beatrice
B
Raeber
Alex
A
Oesch
Bruno
B
D000818:Animals; D001769:Blood / Q000276:immunology*; D002417:Cattle; D002478:Cells, Cultured; D007118:Immunoassay / Q000592:standards*; D007371:Interferon-gamma / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D008297:Male; D009163:Mycobacterium bovis / Q000276:immunology* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D013696:Temperature; D013997:Time Factors; D014376:Tuberculosis / Q000175:diagnosis / Q000662:veterinary*
D007371:Interferon-gamma