Optimization of a bovine whole-blood cytokine recall assay for the detection of interferon-gamma and interleukin 10 following stimulation with <i>Coxiella burnetii</i> antigens

The intracellular bacterium, <i>Coxiella burnetii</i>, causes Q fever in humans and coxiellosis in animals. Cell-mediated immunity (CMI) has been shown to be important in Q fever; however, few studies have investigated the role of CMI in coxiellosis in cattle. Therefore, we aimed to optimize a whole-blood cytokine recall assay (CRA) and to evaluate <i>C. burnetii</i>-specific interferon-gamma (IFNγ) and interleukin 10 (IL10) responses in naturally infected cattle. Duplicate blood samples were collected from cows in an endemically infected dairy herd (as determined by serologic and molecular testing as well as through microbiologic culture) and subsequently transported at ambient temperature or on ice before stimulation with phase 1 and 2 <i>C. burnetii</i> antigen at various concentrations for 24 or 48 h; IFNγ and IL10 were measured in supernatant by ELISA. Using the optimized assay, the cytokine response was measured at calving and ~8 wk post-calving. The effect was assessed using generalized linear models and Spearman correlation coefficient (ρ). The greatest mean cytokine responses were elicited when blood samples were transported at ambient temperature and stimulated for 48 h with the highest <i>C. burnetii</i> antigen concentration. There was a significantly greater mean IFNγ (2.04 times) and IL10 (1.86 times) response at 8 wk post-calving compared to calving. At 8 wk post-calving, the correlation between cytokine response to phase 1 and 2 <i>C. burnetii</i> was ρ = 0.84 for IFNγ and ρ = 0.91 for IL10. Ultimately, our optimization study will guide informed use of the CRA in cattle research, allowing effective investigation of CMI in coxiellosis.