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Title:   Author  item
                                            Waters, Wadeitem
                                            Palmer, Mitchellitem
                                            Nonnecke, Brian
  Submitted to: American Association of Veterinary Laboratory Diagnosticians
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 7/26/2006
                                    Publication  Date: 10/12/2006
                                    Citation: Waters, W.R., Palmer, M.V., Nonnecke, B.J. 2006. Effects of pre-incubation holding time and temperature on interferon-gamma responses in whole blood cultures from Mycobacterium bovis-infected cattle [abstract]. American Association of Veterinary Laboratory Diagnosticians. p. 197.Interpretive Summary:
                                Technical Abstract: The BovigamTM assay is approved for use within the U.S. as a complementary diagnostic test for tuberculosis.  The in vitro assay detects interferon (IFN)-gamma produced in whole blood cultures stimulated with specific antigen.  Stimulants commonly used in the assay are purified protein derivatives derived from Mycobacterium avium (i.e., PPDa) and M. bovis (PPDb), M. bovis-specific proteins (e.g., rESAT-6:CFP10), and pokeweed mitogen (PWM, an indicator of functional capacity).  Prior to whole blood culture and the ensuing ELISA to detect IFN-gamma, blood samples are subjected to various holding times and temperatures due, in part, to practical constraints associated with the shipment of samples to diagnostic laboratories.  To investigate effects of holding conditions on the response, 2.5-month-old Holstein calves received 10**3 cfu M. bovis via aerosol delivery into a mask covering the mouth and nasal passages.  Two months after inoculation, blood was collected into heparinized tubes and held at 4 deg C or 22 deg C for 0, 8, or 24 h prior to set up for whole blood culture.  Pre-incubation at 37 deg C for the initial 2 h of each of the 8 and 24 h holding treatments was also evaluated.  Under each time and temperature condition, pre-incubation of the sample at 37 deg C for 2 h diminished the response to stimulation with antigen and PWM.  In general, responses to each of the stimulants were decreased in all samples held for 8 or 24 h at each of the temperature variables as compared to immediate set up; however, the response to rESAT-6:CFP10 held for 8 h at 4 deg C did not differ from that of samples set-up immediately.  Responses to PPDb, PPDa, and PWM did not differ when held at 8 h as compared to 24 h, regardless of the temperature.  Responses to rESAT-6:CFP10 decreased when held for 24 h as compared to 8 h, regardless of the temperature.  Pre-incubation of samples at 37 deg C for the initial 2 h of holding resulted in decreased responses to all stimulants held at 4 deg C or 22 deg C for the remaining 6 h (i.e., 8 h hold) as compared to responses of samples held for an additional 22 h (i.e., 24 h hold).  Conclusions include:  (1) delays in set-up of blood cultures for stimulation negatively impact the IFN-gamma response to mycobacterial antigens and mitogen, (2) pre-incubation of samples at 37 deg C for 2 h diminishes the response, and (3) increased holding periods (i.e., 8 h versus 24 h) may interfere with the response to certain antigens, particularly when samples are warmed to 37 deg C for the initial 2 h.  Sample quality is particularly crucial for the BovigamTM assay as this test relies on the maintenance of the functional capacity cells for the production of the readout parameter.


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