Necrosis plays a role in the concentration of mycobacterial antigens in granulomas from Mycobacterium bovis naturally infected cattle.

The dynamics that develop between cells and molecules in the host against infection by Mycobacterium bovis, leads to the formation of granulomas mainly present in the lungs and regional lymph nodes in cattle. Cell death is one of the main features in granuloma organization, however, it has not been characterized in granulomatous lesions caused by M. bovis. In this study we aimed to identify the profiles of cell death in the granuloma stages and its relationship with the accumulation of bacteria. We identified necrosis, activated caspase-3, LC3B/p62 using immunohistochemistry and digital pathology analysis on 484 granulomatous lesions in mediastinal lymph nodes from 23 naturally infected cattle. Conclusions: greater amounts of mycobacterial antigens were identified in granulomas from calves compared with adult cattle. The highest percentage of necrosis and quantity of mycobacterial antigens were identified in granuloma stages (III/IV) from adults. The LC3B/p62 profile was heterogeneous in granulomas between adults and calves. Our data suggest that necrosis is associated with a higher amount of mycobacterial antigens in the late stages of granuloma and the development of autophagy appears to play an heterogeneous effector response against infection in adults and calves. These results represent one of the first approaches in the identification of cell death in the four stages of granulomas in bovine tuberculosis.

Necrotic Cell Death and Inflammasome NLRP3 Activity in Mycobacterium bovis-Infected Bovine Macrophages.

Mycobacterium bovis is a facultative intracellular bacterium that produces cellular necrosis in granulomatous lesions in bovines. Although M. bovis-induced inflammation actively participates in granuloma development, its role in necrotic cell death and in bovine macrophages has not been fully explored. In this study, we evaluate the effect of M. bovis AN5 and its culture filtrate protein extract (CFPE) on inflammasome activation in bovine macrophages and its consequences on cell death. Our results show that both stimuli induce necrotic cell death starting 4 h after incubation. CFPE treatment and M. bovis infection also induce the maturation of IL-1ß (>3000 pg/mL), oligomerization of ASC (apoptosis-associated speck-like protein containing CARD), and activation of caspase-1, following the canonical activation pathway of the NLRP3 inflammasome. Inhibiting the oligomerization of NLRP3 and caspase-1 decreases necrosis among the infected or CFPE-stimulated macrophages. Furthermore, histological lymph node sections of bovines naturally infected with M. bovis contained cleaved gasdermin D, mainly in macrophages and giant cells within the granulomas. Finally, the induction of cell death (apoptosis and pyroptosis) decreased the intracellular bacteria count in the infected bovine macrophages, suggesting that cell death helps to control the intracellular growth of the mycobacteria. Our results indicate that M. bovis induces pyroptosis-like cell death that is partially related to the NLRP3 inflammasome activation and that the cell death process could control bacterial growth.

Mycobacterium bovis naturally infected calves present a higher bacterial load and proinflammatory response than adult cattle.

Granulomas are characteristic bovine tuberculosis lesions; studying this structure has improved our understanding of tuberculosis pathogenesis. However, the immune response that develops in granulomas of young cattle naturally infected with Mycobacterium bovis (M. bovis) has not been fully studied. Our previous work described an atypical pattern in granulomatous lesions of cattle younger than 4 months (calves) naturally infected previously M. bovis that did not correspond to the histological classification previously proposed. Histologically, granulomas from calves lack a connective tissue capsule and have fewer multinucleated giant cells (MGCs) and more acid-fast bacilli (AFB) than the classic tuberculosis lesions found in cattle older than 1 year (adults); this suggests a deficient immune response against M. bovis infection in young animals. Therefore, we used IHC and digital pathology analysis to characterize the in situ immune response of granulomas from young and adult cattle. The immunolabeling quantification showed that granulomas from calves had more mycobacteria, CD3+ cells, IFN-γ, TNF-α, and inducible nitric oxide synthase (iNOS) than those of adult cattle. Furthermore, calf granulomas showed lower immunolabeling of MAC387+, CD79+, and WC1+ cells without connective tissue surrounding the lesion and were associated with less vimentin, Alpha Smooth Muscle Actin (α-SMA), and TGF-ß compared with granulomas from adult cattle. Our results suggest that the immune responses in granulomas of cattle naturally infected with M. bovis may be age dependent. This implies that an exacerbated proinflammatory response may be associated with active tuberculosis, producing more necrosis and a lower microbicidal capacity in the granulomas of calves naturally infected with M. bovis.

Ocular tuberculosis in a calf.

BACKGROUND: Bovine tuberculosis is a chronic inflammatory disease that causes granuloma formation mainly in retropharyngeal, tracheobronchial, mediastinal lymph nodes and lungs of bovines. The presence of these lesions in other tissues such as the eyeball is very rare and difficult to diagnose. This study describes macroscopic and microscopic pathological findings in a calf with ocular and meningeal tuberculosis. CASE PRESENTATION: March 2019, an eight-month-old Holstein Friesian calf was identified in a dairy farm located in central Mexico with a clinical cough, anorexia, incoordination, corneal opacity and vision loss. At necropsy, pneumonia, lymphadenitis, meningitis, and granulomatous iridocyclitis were observed. The histopathological examination revealed granulomatous lesions in lung tissue, lymph nodes, meninges and eyes with the presence of acid-fast bacilli associated with Mycobacterium spp. CONCLUSION: To the best of our knowledge, this is the first report that describes macroscopic and microscopic pathological findings of ocular tuberculosis in cattle. This report highlights the importance of considering bovine tuberculosis in the differential diagnosis of corneal opacity and loss of vision in cattle.

Association of immune responses of Zebu and Holstein-Friesian cattle and resistance to mycobacteria in a BCG challenge model.

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 exclusive dairy use, whereas Zebu cattle are mainly used for beef production. In experiments conducted so far, the number of animals has been too small to draw statistically robust conclusions on the resistance differences between these cattle breeds. Here, we used a BCG challenge model to compare the ability of naïve and vaccinated Zebu and HF cattle to control/kill mycobacteria. Young cattle of both breeds with similar ages were housed in the same accommodation for the duration of the experiment. After correcting for multiple comparisons, we found no difference between naïve HF and Zebu (ρ = 0.862) cattle. However, there was a trend for vaccinated HF cattle to have lower cfu numbers than non-vaccinated HF cattle (ρ = 0.057); no such trend was observed between vaccinated and non-vaccinated Zebu cattle (ρ = 0.560). Evaluation of antigen-specific IFNγ secretion by PBMC indicated that Zebu and HF cattle differed in their response to mycobacteria. Thus, whilst there may be difference in immune responses, our data indicate that with the number of animals included in the study and under the conditions used in this work, we were unable to measure any differences between Zebu and HF cattle in the overall control of mycobacteria. Whilst determination of different susceptibilities between Zebu and HF cattle using the BCG challenge model will require larger numbers of animals than the number of animals used in this experiment, these data should inform future experiments.

Atypical granuloma formation in Mycobacterium bovis-infected calves.

Bovine tuberculosis is a chronic inflammatory disease that causes granuloma formation. Characterization of granulomatous lesions of Mycobacterium bovis (M. bovis) experimentally infected cattle has helped to better understand the pathogenesis of this disease. However, few studies have described granulomas found in M. bovis naturally infected cattle. The aim of this work was to examine granulomas from Holstein-Friesian cattle naturally infected with M. bovis from a dairy basin located in the central region of Mexico. Tissue samples from thirty-two cattle with lesions suggestive of tuberculosis were collected post-mortem. Fifteen of the 32 sampled animals (46.8%) were 4 months of age or younger (calves), whereas the rest (53.2%, 17/32) were over one year old (adults). Macroscopic lesions suggestive of tuberculosis were found in the mediastinal lymph node chain of all animals (32/32). From the 1,143 granulomatous lesions that were microscopically analyzed, 34.6% (396/1143) were collected from adult animals and subsequently classified according to the nomenclature suggested by Wangoo et al., 2005. Surprisingly, lesions from calf tissues showed an atypical pattern which could not be fitted into the established developmental stages of this classification. Granulomatous lesions found in calves covered most of the affected organ, histologically showed large necrotic areas with central calcification, absence of a connective tissue capsule, and few giant cells. Also, there was a higher percentage of lesions with acid-fast bacilli (AFB) when compared to studied granulomas in adults. Growth of Mycobacterium spp was detected in 11 bacteriological tissue cultures. Genotypic identification of M. bovis was performed by DNA extraction from bacterial isolates, formalin-fixed and paraffin-embedded (FFPE) tissues and samples without bacterial isolation. M. bovis was detected by PCR in 84.3% (27/32) of the studied cases; whereas other AFB were observed in tissues of the remaining sampled animals (5/32). Our results describe atypical granuloma formation in calves 4 months of age or younger, naturally infected with M. bovis. These findings contribute to better understanding the physiopathology of M. bovis infection in cattle.