Supplementary Material

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Abstract Investigations into the role of vitamin D in the immune response of cattle are limited. The objectives of this study were to investigate the association between circulating vitD concentration, TB vaccination and Mycobacterium bovis (M. bovis) infection outcomes in dairy calves. The study design incorporated two phases: vaccination (experimental week 0-52) and experimental infection phase (week 52-65). Twenty-four cattle less than 8 weeks old were housed throughout the study and fed a bodyweight-based allowance. Vaccinated calves (n=12) received a subcutaneous injection of a live-attenuated TB strain at week 0, whereas unvaccinated ones (n=12) were injected with saline. All animals were infected with 7,600 CFU of M. bovis 52 weeks post-vaccination and were euthanized on week 65 when lymph nodes and lungs were removed and assessed for pathology. Blood samples were taken throughout weeks 0-65. Serum 25-hydroxyvitamin D (25D) concentrations were measured at intervals using a commercial ELISA. A mixed-effects linear regression model revealed significantly higher 25D concentrations in the vaccinates post-infection (week 65) compared to the non-vaccinates. Linear regression analysis between 25D concentration and the level of M. bovis-driven pathology revealed a negative linear relationship i.e. higher concentrations were associated with lower pathology scores, irrespective of vaccination status. No correlation was detected between interferon-γ cytokine production and vitD concentration. Overall, the results support a significant role for vitD in the development of effective immunity of cattle against M. bovis. Gaining insight into the interaction between TB vaccination, M. bovis infection and vitD could potentially guide the optimization of vaccination protocols and TB control strategies going forward.

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