Background/Objectives: Vaccines may improve the control and eradication of bovine tuberculosis. However, the evaluation of experimental candidates requires the assessment of the protection, excretion, transmission and biosafety. A natural transmission trial among likely infected animals was conducted. Methods: Seventy-four male heifers were randomly distributed (five groups) and vaccinated subcutaneously with attenuated strains (M. bovis Δmce2 or M. bovis Δmce2-phoP), a recombinant M. bovis BCG Pasteur (BCGr) or M. bovis BCG Pasteur. Then, they cohoused with a naturally infected bTB cohort under field conditions exposed to the infection. Results: A 23% of transmission of wild-type strains was confirmed (non-vaccinated group). Strikingly, first vaccination did not induce immune response (caudal fold test and IFN-gamma release assay). However, after 74 days of exposure to bTB, animals were re-vaccinated. Although their sensitization increased throughout the trial, the vaccines did not confer significant protection, when compared to the non-vaccinated group, as demonstrated by pathology progression of lesions and confirmatory tools. Besides, the likelihood of acquiring the infection was similar in all groups compared to the non-vaccinated group (p > 0.076). Respiratory and digestive excretion of viable vaccine candidates was undetectable. To note, the group vaccinated with M. bovis Δmce2-phoP exhibited the highest proportion of animals without macroscopic lesions, compared to the one vaccinated with BCG, although this was not statistically supported. Conclusions: This highlights that further evaluation of these vaccines would not guarantee better protection. The limitations detected during the trial are discussed regarding the transmission rate of M. bovis wild-type, the imperfect test for studying sensitization, the need for a DIVA diagnosis and management conditions of the trials performed under routine husbandry conditions. Re-vaccination of likely infected bovines did not highlight a conclusive result, even suggesting a detrimental effect on those vaccinated with M. bovis BCG.

Bovine tuberculosis (bTB) is commonly diagnosed ante-mortem using the tuberculin skin test and interferon-gamma release assay, yet some infected cattle remain undetected. This study evaluated post-mortem confirmatory diagnostic approaches and identified the most informative tissues for infection confirmation in this population.Thirty-nine Holstein cows from endemic dairy herds underwent post-mortem examination, tissue PCR targeting Rv2807, bacteriology followed by IS6110 colony PCR (c-PCR), and histopathology. Diagnostic agreement among methods and associations between lesion distribution and confirmatory test results were assessed.Macroscopic or microscopic lesions compatible with bTB were detected in 97% of animals, with 74% showing multi-organ involvement. Tissue PCR detected Mycobacterium tuberculosis complex DNA in 64% of cattle, whereas bacteriology/c-PCR confirmed infection in 54%. Combining both methods increased confirmation to 82%. Tissue PCR and bacteriology/c-PCR frequently identified different subsets of infected cattle, supporting their complementary value. Cranial and respiratory lymph nodes provided the highest diagnostic yield, with combined detection rates of 95% and 81%, respectively. Histopathology identified more positive animals than bacteriology (p = 0.0001).These findings highlight the diagnostic gap of cell-mediated immune response-based screening tests and support an integrated post-mortem strategy combining pathology, tissue PCR, and bacteriology for bTB confirmation in endemic settings.