Submitted to: Veterinary Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/10/2006
Publication Date: 1/1/2007
Citation: Waters, W.R., Nonnecke, B.J., Olsen, S.C., Palmer, M.V. 2007. Effects of pre-culture holding time and temperature on interferon-gamma responses in whole blood cultures from Mycobacterium bovis-infected cattle. Veterinary Microbiology. 119(2-4):277-282. Interpretive Summary: Despite highly successful eradication efforts in several countries, tuberculosis of cattle remains a serious health concern worldwide. In addition, recent outbreaks of tuberculosis in Michigan, California, Texas, Minnesota, and New Mexico demonstrate that the disease is far from eliminated from the United States. The Bovigam™ assay is approved for use within the United States as a complimentary test for bovine tuberculosis. This assay relies on live cells for production of a measurable response. In the field, samples are subjected to various holding time and temperature combinations that may negatively impact the response. In the present study, it was determined that high temperatures and delays in set-up of the assay interfere with the response. This interference may result in false negative responses. Knowledge obtained from this study will assist in the standardization of sample handling necessary for successful diagnosis of tuberculosis-infected cattle.
Technical Abstract: The Bovigam™ assay is approved for use within the United States as a complimentary test for tuberculosis. Prior to whole blood culture and the ensuing ELISA to detect interferon- (IFN) ', samples are subjected to various holding time / temperature combinations due, in part, to practical constraints associated with shipment of samples to approved laboratories. Five-month-old Holstein calves (n = 7) received 10**3 cfu Mycobacterium bovis by aerosol. Heparinized blood was held at 4°C or 22°C for 0 hr, 8 hr or 24 hr prior to whole blood culture. A subset of samples was held for 2 hr at 37°C at the beginning of the holding period. These samples (i.e., 2 hr hold at 37°C) had diminished responses to mycobacterial antigens or pokeweed mitogen. Differences in responses of samples held at 4°C versus 22°C were minimal. Responses of samples held for 8 or 24 hr were comparable and were lower than responses of cultures prepared immediately after blood collection, regardless of holding temperature. However, samples held at 37°C for the initial 2 hr of a 24 hr pre-culture period had lower responses to all stimulants than did samples held at 37°C for the initial 2 hr of a 8 hr pre-culture period. Pre-processing conditions, particularly delays in set-up and initial high sample temperatures, may reduce IFN-gamma responses of cells from infected cattle increasing the risk of false negatives in this important assay.