Effects of repeated comparative intradermal tuberculin testing on test results: a longitudinal study in TB-free red deer

[Background]: Diagnosing tuberculosis (TB) in farmed red deer (Cervus elaphus) is challenging and might require combining cellular and humoral diagnostic tests. Repeated skin-testing with mycobacterial purified protein derivatives (PPDs) might sensitize or desensitize the subjects to both kinds of diagnostic tools. We evaluated the effect of repeated (every 6 months) comparative tuberculin skin testing on skin test and ELISA responsiveness in farmed red deer hinds from a TB-free herd. Eighteen 8-month old hinds were inoculated with bovine and avian PPDs and the mitogen phytohaemagglutinin (PHA), as positive control and concurrently tested by ELISA for antibodies against avian (avian PPD, aPPD and protoplasmatic antigen 3, PPA3) and bovine antigens (bPPD and MPB70). Blood serum was also sampled three weeks after each skin testing round and tested for antibodies against aPPD and bPPD, in order to detect eventual antibody level boosts. Testing took place every six months from winter 2012 until winter 2015. [Results]: The skin test response to both PPDs peaked during the second and third test round, returning to standard values thereafter. Individual variability was particularly high at the first year and early second year testing rounds (first intradermal test and blood sampling; first winter). The antibody response to avian antigens increased through time, while no such increase was recorded for bovine antigens. The antibody boost three weeks after skin testing was more marked for avian PPD. However, there was no consistent trend in the boosting response through time. [Conclusion]: Repeated comparative skin testing at six month intervals did not cause progressive increments in skin test responsiveness or antibody production. Specifically, we observed no loss of the skin test response to bPPD and also no progressive loss of the boosting effect in the ELISA responses. However, we recorded increases through time in the antibody levels against avian mycobacterial antigens, possibly due to the progressive exposure to MAP or to other cross-reacting environmental mycobacteria. These findings should be taken into account in designing and interpreting TB testing schemes in farmed deer.Research funding is acknowledged to Plan Nacional grant AGL2014-56305 (MINECO, Spain and FEDER). ACA was supported by a grant from University Putra Malaysia, Malaysia, and MAR had a research contract from Junta de Comunidades de Castilla-La Mancha (JCCM), Spain.Peer Reviewe