To identify the resistance phenotype against Mycobacterium bovis in cattle, we used a bactericidal assay that has been considered a marker of this trait. Three of 24 cows (12.5%) were phenotyped as resistant and 21 as susceptible. Resistance of bovine macrophages (MΦ) to BCG challenge was evaluated for its association with SLC11A1 GT microsatellite polymorphisms within 3′UTR region. Twenty-three cows (95.8%) had a GT13 genotype, reported as resistant, consequently the SLC11A1polymorphism was not in agreement with our bactericidal assay results. MΦ of cows with resistant or susceptible phenotype were challenged in vitro with virulent M. bovis field strain or BCG, and nitric oxide production, bacterial killing and apoptosis induction were measured in resting and LPS-primed states. M. bovis field strain induced more apoptosis than BCG, although the difference was not significant. Resistant MΦ controlled better the replication of M. bovis (P<0.01), produced more nitric oxide (P<0.05) and were slightly more prone to undergo apoptosis than susceptible cells. LPS pretreatment of MΦ enhanced all the functional parameters analyzed. Inhibition of nitric oxide production with n G-monomethyl-L-arginine monoacetate enhanced replication of M. bovis but did not modify apoptosis rates in both resistant and susceptible MΦ. We conclude that nitric oxide production not apoptosis is a major determinant of macrophage resistance to M. bovis infection in cattle and that the influence of SLC11A1 gene 3′UTR polymorphism is not associated with this event.

Mycobacterium bovis, the causative agent of bovine tuberculosis encodes different virulence mechanisms to survive inside of host cells. One of the possible outcomes in this host–pathogen interaction is cell death. Previous results from our group showed that M. bovis induces a caspase-independent apoptosis in bovine macrophages with the possible participation of apoptosis inducing factor mitochondria associated 1 (AIFM1/AIF), a flavoprotein that functions as a cell-death regulator. However, contribution of other caspase-independent cell death mediators in M. bovis-infected macrophages is not known. In this study, we aimed to further characterize M. bovis-induced apoptosis, addressing Endonuclease G (Endo G) and Poly (ADP-ribose) polymerase 1 (PARP-1). In order to accomplish our objective, we infected bovine macrophages with M. bovis AN5 (MOI 10:1). Analysis of M. bovis-infected nuclear protein extracts by immunoblot, identified a 15- and 43-fold increase in concentration of mitochondrial proteins AIF and Endo G respectively. Interestingly, pretreatment of M. bovis-infected macrophages with cyclosporine A, a mitochondrial permeability transition pore inhibitor, abolished AIF and Endo G nuclear translocation. In addition, it also decreased macrophage DNA fragmentation to baseline and caused a 26.2% increase in bacterial viability. We also demonstrated that PARP-1 protein expression in macrophages did not change during M. bovis infection. Furthermore, pretreatment of M. bovis-infected bovine macrophages with 3-aminobenzamide, a PARP-1 inhibitor, did not change the proportion of macrophage DNA fragmentation. Our results suggest participation of Endo G, but not PARP-1, in M. bovis-induced macrophage apoptosis. To the best of our knowledge this is the first report associating Endo G with caspase-independent apoptosis induced by a member of the Mycobacterium tuberculosis complex.Electronic supplementary materialThe online version of this article (10.1186/s13567-018-0567-1) contains supplementary material, which is available to authorized users.

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Molecular typing of bacterial isolates provides a powerful approach for distinguishing Mycobacterium bovis (M. bovis) genotypes. It is known that M. bovis strain virulence plays a role in prevalence and spread of the disease, suggesting that strain virulence and prevailing genotypes are associated. However, it is not well understood whether strain virulence correlates with particular genotypes. In this study, we assessed the in vitro intracellular growth of 18 M. bovis isolates in bovine macrophages as an indicator of bacterial virulence and sought a relationship with the genotype identified by spoligotyping. We found 14 different spoligotypes—11 were already known and three spoligotypes had never been reported before. We identified 2 clusters that were phylogenetically related, containing 10 and 6 strains, respectively, and 2 orphan strains. Intracellular growth and phagocytic rates of 18 M. bovis strains were heterogeneous. Our results suggest that M. bovis intracellular growth and phagocytosis are independent of the bacterial lineage identified by spoligotyping.

Mycobacterium bovis is the main cause of bovine tuberculosis (BTB) in cattle and can also infect humans. Zebu cattle are considered more resistant to some infectious diseases compared with Holstein-Friesian (HF) cattle, including BTB. However, epidemiological studies may not take into account usage differences of the two types of cattle. HF cattle may suffer greater metabolic stress due to their more or less How to cite this article: Alcaraz-López OA, Flores-Villalva S, Cortéz-Hernández O, et al. Association of immune responses of Zebu and Holstein-Friesian cattle and resistance to mycobacteria in a BCG challenge model.