Optimization of a Whole-Blood Gamma Interferon Assay for Detection of Mycobacterium bovis-Infected Cattle |
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| Authors: | Irene Schiller W. Ray Waters H. Martin Vordermeier Brian Nonnecke Michael Welsh Nicolas Keck Adam Whelan Teresa Sigafoose Christoph Stamm Mitchell Palmer Tyler Thacker Roland Hardegger Beatrice Marg-Haufe Alex Raeber Bruno Oesch |
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| Affiliation: | 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. |
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| 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-γ) by bovine T cells in whole-blood culture (IFN-γ assay). We have analyzed various parameters of the in vitro IFN-γ assay, ranging from blood sampling to execution of the IFN-γ test, in view of potential simplifications of the assay. Here, we show that IFN-γ 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-γ 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-γ 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-γ is stable under various routine laboratory temperature scenarios. These findings demonstrate opportunities for improvements in the bovine IFN-γ 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-γ), which can then be quantified by an enzyme-linked immunosorbent assay (ELISA; Bovigam IFN-γ 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-γ assay is commonly used as an indicator of sample quality and potential for underlying CMI suppression, thereby reducing the risk of false-negative test results. Conditions such as the anticoagulant used for blood collection, the temperature and time of blood storage, and the culture duration also affect IFN-γ production in whole-blood culture (8). Furthermore, delays in culture setup, initial high sample temperatures (2 h at 37°C, followed by 22 h at 22°C, prior to 24 h of culture at 37°C), or a low sample temperature (4°C) diminishes IFN-γ responses with samples from M. bovis-infected cattle (14). Thus, sample quality, as affected by pre- and postcollection parameters, affects the accuracy of the IFN-γ test. Once the sample reaches the laboratory, additional variables, such as the culture plate format, the culture conditions, the antigens used for TB-specific stimulation, the nonspecific stimulation control reagents, and the cutoff for the final test interpretation, may also influence test performance. While the ability to modify IFN-γ test parameters offers the possibility to adapt the assay more closely to the needs within a TB program, this option also provides challenges to ensure standardization of testing procedures and quality assurance. Indeed, variations in assay protocols, with both the skin test and the IFN-γ test, have resulted in disparate results in test accuracy between studies (reviewed in reference 4).We therefore analyzed the capacities of animals under various field conditions to produce IFN-γ. Furthermore, we evaluated different parameters of the in vitro assay, including the influence of time to culture initiation, culture vessel geometry, the cell culture temperature, the need for carbon dioxide (generally used in tissue culture to stabilize pH), the animal holding conditions, and the stability of the produced IFN-γ under various standard storage scenarios. All of these parameters were analyzed in view of defining a range of possible conditions and potentially simplifying the assay for use in bovine TB eradication programs. |
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