Bernat Pérez de Val

Background: Trained immunity refers to the stimuli-induced epigenetic and metabolic reprogramming of innate immune cells, enabling enhanced non-specific responses upon secondary stimulation. Alveolar macrophages (AMs) constitute the primary immune barrier in the lung and represent a key target in enhancing protection against pulmonary pathogens. Methods: In this study, an in vitro priming-resting-re-stimulation protocol was used to characterise the ability of Mycobacterium fortuitum (MF) to induce functional features of trained immunity in caprine AMs in vitro . Pro-inflammatory cytokines (TNFα, IL-1β, IL-6) and nitric oxide synthase (iNOS)-positive cells were quantified by multiplex assay and flow cytometry, respectively. Phagosomal acidification was detected by flow cytometry and live-cell fluorescent microscopy after MF-challenge using pH-sensitive labelled bacteria. Results: MF-trained AMs displayed significantly elevated TNFα and IL-1β production and a higher frequency of inducible iNOS-producing cells compared to non-stimulated controls, consistent with a trained immunity-associated pro-inflammatory and antimicrobial phenotype. Phagosomal acidification was enhanced and sustained in MF-trained AMs following homologous challenge with MF, suggesting enhanced phagocytic activity against mycobacteria. Conclusion: These findings provide evidence that M. fortuitum induces trained immunity-associated functional responses in caprine AMs and support further trained immunity studies in the caprine lung mucosa against pulmonary infections.

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis , remains one of the major causes of death from infection worldwide, with over a million associated deaths each year. The study of biomarkers for TB is critical for advancing our understanding and management of the disease. Biomarkers, defined as measurable indicators of biological states or conditions, are invaluable for the diagnosis, prognosis and treatment monitoring of TB. Clinical studies have provided critical knowledge on the matter but are also notoriously constrained by economical, ethical and sampling limitations. The use of animal models provides a simpler, more controllable, cost-effective setting with great potential for translation to humans. They also allow the evaluation of biomarkers within the respiratory compartment, when available, which is of particular interest due to the nature of TB pathogenesis. This Review focuses on the current landscape of TB biomarker discovery in several animal models, from invertebrates to large mammals. Here we summarize the basics of host–pathogen immune interaction, describe the main methodological approaches used and highlight the most substantial findings for each animal model studied. Furthermore, we discuss the advantages, challenges and limitations associated with species-specific differences in animal models. We conclude that integrating the data obtained from animal models and human studies is absolutely required to advance the TB field to accelerate the management of this disease.

Mycobacterial diseases, including tuberculosis (TB), remain the major health and economic challenges in livestock, underscoring the need to characterise the innate immune mechanisms involved in early bacterial containment. Alveolar macrophages (AMs) are the first line of defence against inhaled mycobacteria, yet the functional links between activation, polarisation, and phagocytic capacity in caprine AMs remain poorly defined. In this study, we compared a pH-dependent live-cell fluorescence assay with a culture-based method to evaluate phagocytosis and clearance of Mycobacterium fortuitum under different immunostimulatory conditions. AMs were stimulated in vitro with LPS or heat-inactivated Mycobacterium bovis (HIMB), and phagocytosis was assessed alongside activation and pro-inflammatory markers. Both approaches showed that LPS stimulation significantly enhanced mycobacterial clearance, despite reduced initial bacterial uptake. Moreover, this improved phagocytic capacity was associated with increased expression of the inducible Nitric Oxide Synthase (iNOS), MHCII, CD80, and CD86, as well as an elevated production of some pro-inflammatory cytokines. In contrast, HIMB induced cytokine secretion but failed to enhance activation markers or bacterial clearance. Collectively, these results establish the first association between pro-inflammatory activation and functional mycobacterial phagocytosis in caprine AMs and validate a robust methodological framework for studying innate immune responses relevant to TB and vaccine development in goats.

Early immune containment of mycobacteria at the infection site is key to tuberculosis (TB) vaccine development. Intranasal delivery strategies offer a promising alternative to parenteral BCG vaccination, particularly for pulmonary TB, the predominant clinical form in humans and livestock. This study evaluated the immunogenicity of intranasal BCG and heat-inactivated M. bovis (HIMB) with or without adjuvant, as well as prime-boost strategies combining parenteral BCG or HIMB followed by intranasal HIMB in young goats. Intranasal BCG elicited systemic antigen-specific IFNγ production, with enhanced expansion of CD4 ⁺ IFNγ ⁺ and CD8 ⁺ IFNγ ⁺ T-cells, comparable to prime-boost regimens. Intranasal BCG and prime-boosted groups also induced higher local proinflammatory responses at the lung mucosa, including proinflammatory cytokine production, expansion of antigen-specific T-cells, and polarization of alveolar macrophages toward activated proinflammatory phenotype. The results underscore the potential of respiratory mucosal BCG delivery to enhance early immune responses against TB infection and support further investigation into its protective efficacy.

Bovine tuberculosis (TB), primarily caused by Mycobacterium bovis , is a chronic infectious disease of cattle with significant public health and economic implications due to its zoonotic potential and impact on livestock productivity. The control of the disease is hindered by complex epidemiological dynamics and the chronic, and often slow‐progressing nature of the disease. The recurrent outbreaks of bovine TB in endemic areas are common and threaten the success of eradication programs. To address this issue, long‐term reemergent outbreaks in Catalonia (Northeastern Spain) were retrospectively investigated in depth. In 2009, an outbreak caused by M. bovis spoligotype SB0120 was detected in four extensively managed cattle herds. Following intensive eradication measures, all herds recovered the officially TB‐free status by 2012. In 2021, 9 years later, a new outbreak involving the same spoligotype was detected in three herds located in the same area, one of which had been affected in the previous outbreak. Extensive sampling of TB‐positive slaughtered cattle and hunted wild ungulates was conducted. Whole genome sequencing (WGS) of M. bovis isolates from cattle affected in both outbreaks, as well as from two culture‐positive wild boars was performed. Epidemiological and phylogenetic analyses were conducted to elucidate the origin and transmission dynamics of the outbreaks. The results revealed a long‐term residual infection in the cattle herd that linked the first and second outbreaks. This herd was also the most likely source of transmission of M. bovis to wild boars. Since residual infections can jeopardize the final stages of the eradication in low‐prevalence settings, thorough investigation of reemerging strains is essential for risk assessment and for guiding TB control decisions.

Wild small rodents are considered the natural reservoirs of Mycobacterium microti, a member of the Mycobacterium tuberculosis complex (MTBC) that can cause tuberculosis (TB) in humans and animals, as well as interfere with current tuberculosis eradication plans in livestock. A cross-sectional study was carried out in the Catalan Pyrenees (Iberian Peninsula) in an area where M. microti was previously isolated from wild boars, to evaluate the role of micromammals in the epidemiology of this outbreak. A total of 350 wild rodents were necropsied (306 Murinae and 44 Arvicolinae) in spring and autumn during two consecutive natural years. Tissues were analyzed by histopathology to look for TB-like lesions and by qPCR and culture to detect MTBC. Sera were analyzed by MTBC-specific ELISA. No evidence of TB infection in wild rodents was confirmed. Results suggest that small rodents did not play a role in the epidemiology of M. microti in the area. The source of this mycobacterium remains unknown, but previous detections of M. microti in various species in southern France suggest the movements of wild boars across the French Pyrenees as the most likely origin of the outbreak detected in the Iberian Peninsula.

Background: Goats are natural hosts of tuberculosis (TB) and are a valid animal model to test new vaccines and treatments to control this disease. In this study, a new experimental model of TB in goats based on the intranasal nebulization of Mycobacterium caprae was assessed in comparison with the endobronchial route of infection.Methods: Fourteen animals were divided into two groups of seven and challenged through the endobronchial (EB) and intranasal (IN) routes, respectively. Clinical signs, rectal temperature, body weight, and immunological responses from blood samples were followed up throughout the experiment. All goats were euthanized at 9 weeks post-challenge. Gross pathological examination, analysis of lung lesions using computed tomography, and bacterial load quantification in pulmonary lymph nodes (LNs) by qPCR were carried out.Results: The IN-challenged group showed a slower progression of the infection: delayed clinical signs (body weight gain reduction, peak of temperature, and apparition of other TB signs) and delayed immunological responses (IFN-γ peak response and seroconversion). At the end of the experiment, the IN group also showed significantly lower severity and dissemination of lung lesions, lower mycobacterial DNA load and volume of lesions in pulmonary LN, and higher involvement of the nasopharyngeal cavity and volume of the lesions in the retropharyngeal LN.Conclusion: The results indicated that the IN challenge with M. caprae induced pathological features of natural TB in the lungs, respiratory LN, and extrapulmonary organs but extremely exaggerating the nasopharyngeal TB pathological features. On the other hand, the EB route oversized and accelerated the pulmonary TB lesion progression. Our results highlight the need to refine the inoculation routes in the interest of faithfully reproducing the natural TB infection when evaluating new vaccines or treatments against the disease.