Background and aim:
Immune response must be measured in highly controlled conditions for reproducibility. Furthermore, whereas the cellular response plays a major role, only antibody production is assessed in most cases. For the purpose of determining responsiveness in ruminants, we developed a custom 15-plex cytokine/ chemokine assay with MERCK-Millipore.

Methods:
To evaluate the bovine cellular response, two approaches were used. Whole blood samples were collected from dairy cows, and immediately stimulated during 24 hours with heat-killed bacteria, TLR ligands, or soluble anti-CD3/CD28 monoclonal antibodies. A sample without stimulation was assayed in parallel. In another experiment, the response to an intravenous injection of ultrapure LPS was measured in plasma for the 24 hours post-injection. Bovine clones were used to reduce genetic variability.

Results:
Using ANOVA and data mining methods, we showed that the protocol is able to detect differences amongst conditions. For in vitro assays, the response varies according to the bacteria species, with S. uberis being the most potent stimulus, compared to S. aureus and E. coli that were distinct but more closely related to each other. Similarly, TLR ligands could be distinguished, and Gardiquimod was significantly different from the TLR2/4-activating ligands. LPS and E. coli provided very similar profiles confirming previous data indicating that LPS signaling mediates a large part of the E. coli response. Furthermore, when LPS was injected IV, plasma concentrations of cytokines and chemokines varied in parallel with the development of clinical signs and differed according to age and genetic background.

Conclusion:
We showed that the assay is able to reliably detect production of bovine cytokines and chemokines. High variability of the responses indicates that both environmental and genetic factors control the cellular response. Cytokine profiling is achievable for evaluation of the responses to infectious diseases and immunomodulation in cattle.