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

Abstract

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...

Evaluation of gamma interferon assay for detection of bovine tuberculosis in inactive foot and mouth disease vaccine administered cattle

“…The outbreaks of this disease have been a major cause of economic losses to livestock industries worldwide [10]. The inactive FMD vaccines are widely [7,8,11,12]. Schiller et al [8] reported that the time and temperature of culture will affect the result of assay, and the stimulation temperature needs to be 33 °C or higher.…”

“…IFN-γ release in IFN-γ assay is affected by different conditions, such as the time and temperature of blood sample storage, the time of test culture, and the kind of anticoagulant used for blood collection [7,8].…”

Mycobacterium bovis antigens for the differential diagnosis of vaccinated and infected cattle

“…However, further studies suggested that this sensitivity benefit in relation to tuberculin and ESAT-6/CFP-10 could largely be realized only in freshly sampled blood, whilst it was less potent in blood that was stored overnight before the blood cultures were established (Vordermeier, unpublished data). This is was mainly due to the fact that overnight blood storage significantly improved the tuberculin specificity whilst it impacted less on sensitivity (Schiller et al, 2009). As now the routine application of the Bovigam assay in GB is based on blood stored overnight, further studies were needed to improve ESAT-6 and CFP-10 sensitivity in this testing scenario (see next paragraph).…”

“…Briefly, results in cattle have shown that the most effective vaccination strategy against bovine TB is based on priming the immune system with BCG followed by boosting with subunit vaccines containing protective antigens that are present in BCG (heterologous prime-boost strategy), DNA (Skinner et al, 2003;, protein , or viral subunit vaccines (Vordermeier et al, 2006b; Vordermeier et al, 2004;Vordermeier et al, 2009). Nevertheless, all these strategies resulted in at least a proportion of animals becoming tuberculin-test positive (both in tuberculin skin and IFN-!…”

Stimulation of Bovine Whole-Blood Samples Cultured in Media Supplemented with Recombinant Interleukin-7 (IL-7) and IL-12 Extends the Life Span of the Gamma Interferon Assay To Detect Mycobacterium bovis-Infected Cattle

“…Beyond this time limit, the IFN-␥ response diminishes and signal strength (optical density [OD]) decreases and thus reduces test sensitivity and specificity (10). This narrow time window has limited the wider application of the IFN-␥ assay in tuberculosis control programs in remote areas where long transit times between collection and laboratory analysis can lead to delays in sample processing.…”