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Bovine tuberculosis (bTB), a global zoonotic disease, causes negative effects on human and animal health. PhoP protein is a key regulator of pathogenic phenotypes in members of the Mycobacterium tuberculosis complex, which includes the causative agent of bTB. Despite extensive research on this protein focused in deciphering its regulatory role, little was explored about it as a diagnostic antigen. In humans, a novel role of anti-PhoP antibodies as a possible marker for the diagnosis of TB was demonstrated. However, this issue was not addressed in bovines. In this study, antigenic properties of the PhoP protein were evaluated in naturally Mycobacterium bovis (M. bovis) infected bovines. A high homology of PhoP (≥ 75 %) was observed in environmental mycobacterial species and other genera such as Salmonella and Pasteurella. Using the IFN-gamma release assay (IGRA), we detected cell-mediated immune response against PhoP in cattle from infected herds (25 %; IC 95 % 3.2-65.1), although it was significantly lower than that evoked by the reference antigens, ESAT-6/CFP-10/Rv3615c (75 %; IC95 % 34.9-96.8), and the purified protein derivative (87.5 %; IC 95 % 47.4-99.7) (p < 0.05)). Animals from a bTB free area showed no response against PhoP when analyzed by IGRA. Although, the humoral response detected 62.5 % (CI95% 24.5-91.5) of naturally infected animals, there was 100 % cross-reactivity among TB-free cattle. These results suggest that the PhoP protein is not a promising candidate for bTB diagnosis, due to it had relatively low levels of test sensitivity in the IGRA test, and very low specificity in a humoral antibody western blot assay., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

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.

Ante-mortem diagnosis of bovine tuberculosis (bTB) is based mainly on the tuberculin skin test (TST) and the ɣ-IFN release assay (IGRA). Some infected animals escape screening tests, thus, limit herd sanitation. Previous reports have suggested a predominant pattern of multi-organ lesions attributable to Mycobacterium bovis (the causative agent of bTB) bacteraemia. A case-control study was conducted to investigate blood PCR as an alternative tool for improving ante-mortem detection of TST false-negative bovines. Cases comprised 70 TST false-negative bovines (cases), which were serology positive, and controls included 81 TST positive bovines; all of them confirmed as infected with M. bovis . Detection of the IS 6110 target through touchdown blood-PCR (IS 6110 TD-PCR) was performed. The positivity of the blood-PCR was 27.2% in the control group. This performance was similar to the 15% obtained among cases ( p  = 0.134). Most cases identified by the IS 6110 TD-PCR exhibited focalized lesions ( p  = 0.002). Results demonstrated that blood-PCR could detect TST false-negative cattle, even if they are negative for IGRA. Considering that cases exhibited humoral response to M. bovis , further studies conducted in a pre-serological stage could provide evidence about the real contribution of the technique in herds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Encinas, Ferrara Muñiz, Sammarruco, Ruiz Menna, Garro, Delgado, Macías, Magnano, Zumárraga, Garbaccio and Eirin.)