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-␥) by bovine T cells in whole-blood culture (IFN-␥ assay). We have analyzed various parameters of the in vitro IFN-␥ assay, ranging from blood sampling to execution of the IFN-␥ test, in view of potential simplifications of the assay. Here, we show that IFN-␥ 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-␥ 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-␥ 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-␥ is stable under various routine laboratory temperature scenarios. These findings demonstrate opportunities for improvements in the bovine IFN-␥ test platform and flexibilities in test application.Bovine tuberculosis (TB), caused by Mycobacterium bovis, has an important and adverse effect on socioeconomic conditions, public health, and trade of animals and animal products (2). Eradication of bovine TB in cattle is based on detection and slaughter of infected animals or whole herds. The standard antemortem screening test for detection of TB is the intradermal tuberculin skin test (i.e., intradermal injection of tuberculin eliciting a cell-mediated immune response [CMI] at the site, which in turn leads to skin thickening). As an alternative, the CMI can be measured in vitro by stimulating blood cells with tuberculin, which in turn leads to production of gamma interferon (IFN-␥), which can then be quantified by an enzyme-linked immunosorbent assay (ELISA; Bovigam IFN-␥ assay) (15).The Bovigam assay constitutes a laboratory-based TB test and is widely used complementarily to the tuberculin skin test (4, 11), as it offers national TB control programs and industry an additional tool for curtailing the spread of TB in cattle and other Bovidae. The assay critically depends on the sample quality, culture conditions, and quality control of stimulation reagents. The CMI, both in vivo and in vitro, may be negatively affected by stress or corticosteroid application (5). Thus, parallel stimulation of blood leukocytes with mitogen or superantigen in the IFN-␥ assay is commonly used as an indicator of sample quality and potential for underlying CMI suppression, thereby re...
