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Bovine tuberculosis, caused by Mycobacterium tuberculosis var. bovis (M. bovis), is an important enzootic disease affecting mainly cattle, worldwide. Despite the implementation of national campaigns to eliminate the disease, bovine tuberculosis remains recalcitrant to eradication in several countries. Characterizing the host response to M. bovis infection is crucial for understanding the immunopathogenesis of the disease and for developing better control strategies. To profile the host responses to M. bovis infection, we analyzed the transcriptome of whole blood cells collected from experimentally infected calves with a virulent strain of M. bovis using RNA transcriptome sequencing (RNAseq). Comparative analysis of calf transcriptomes at early (8 weeks) versus late (20 weeks) aerosol infection with M. bovis revealed a divergent and unique profile for each stage of infection. Notably, at the early time point, transcriptional upregulation was observed among several of the top-ranking canonical pathways involved in T-cell chemotaxis. At the late time point, enrichment in the cell mediated cytotoxicity (e.g., Granzyme B) was the predominant host response. These results showed significant change in bovine transcriptional profiles and identified networks of chemokine receptors and monocyte chemoattractant protein (CCL) coregulated genes that underline the host-mycobacterial interactions during progression of bovine tuberculosis in cattle. Further analysis of the transcriptomic profiles identified potential biomarker targets for early and late phases of tuberculosis in cattle. Overall, the identified profiles better characterized identified novel immunomodulatory mechanisms and provided a list of targets for further development of potential diagnostics for tuberculosis in cattle.

While Mycobacterium tuberculosis is the primary cause of tuberculosis in people, multiple other mycobacteria are capable of doing so. With the World Health Organization's goal of a 90% reduction in tuberculosis by 2035, all tuberculous mycobacteria need to be addressed. Understanding not only the similarities, but importantly the differences between the different species is crucial if eradication is ever to be achieved. Mycobacterium bovis, while typically thought of as a disease of cattle, remains a possible source of human infection worldwide. Although this species' genome differs from Mycobacterium tuberculosis by only 0.05%, significant differences are present, creating unique challenges to address. This review focuses on features which distinguish this bacterium from Mycobacterium tuberculosis, including differences in origin, structure, environmental persistence, host preferences, infection and disease, host immune response, diagnostics and treatment., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Unlabelled: An emerging Mycobacterium tuberculosis complex (MTC) pathogen, M. mungi, infects wild banded mongooses (Mungos mungo) in Northern Botswana, causing significant mortality. This MTC pathogen did not appear to be transmitted through a primary aerosol or oral route. We utilized histopathology, spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR), quantitative PCR (qPCR), and molecular markers (regions of difference [RDs] from various MTC members, including region of difference 1 [RD1] from M. bovis BCG [RD1(BCG)], M. microti [RD1(mic)], and M. pinnipedii [RD1(seal)], genes Rv1510 [RD4], Rv1970 [RD7], Rv3877/8 [RD1], and Rv3120 [RD12], insertion element IS1561, the 16S RNA gene, and gene Rv0577 [cfp32]), including the newly characterized mongoose-specific deletion in RD1 (RD1(mon)), in order to demonstrate the presence of M. mungi DNA in infected mongooses and investigate pathogen invasion and exposure mechanisms. M. mungi DNA was identified in 29% of nasal planum samples (n = 52), 56% of nasal rinses and swabs (n = 9), 53% of oral swabs (n = 19), 22% of urine samples (n = 23), 33% of anal gland tissue (n = 18), and 39% of anal gland secretions (n = 44). The occurrence of extremely low cycle threshold values obtained with qPCR in anal gland and nasal planum samples indicates that high levels of M. mungi can be found in these tissue types. Histological data were consistent with these results, suggesting that pathogen invasion occurs through breaks in the nasal planum and/or skin of the mongoose host, which are in frequent contact with anal gland secretions and urine during olfactory communication behavior. Lesions in the lung, when present, occurred only with disseminated disease. No environmental sources of M. mungi DNA could be found. We report primary environmental transmission of an MTC pathogen that occurs in association with social communication behavior., Importance: Organisms causing infectious disease evolve modes of transmission that exploit environmental and host conditions favoring pathogen spread and persistence. We report a novel mode of environmental infectious disease transmission that occurs in association with olfactory secretions (e.g., urine and anal gland secretions), allowing pathogen exposure to occur within and between social groups through intricate social communication behaviors of the banded mongoose host. The presence of M. mungi in these environmentally deposited secretions would effectively circumvent natural social barriers (e.g., territoriality), facilitating between-group pathogen transmission in the absence of direct physical contact, a rare occurrence in this highly territorial species. This work identifies an important potential mechanism of pathogen transmission of epidemiological significance in social species. We also provide evidence of a novel mechanism of pathogen transmission for the MTC complex, where pathogen movement in the environment and host exposure dynamics are driven by social behavior., (Copyright © 2016 Alexander et al.)

Background: Early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis) involved in phagolysosome escape of the bacillus and, potentially, in the efficient induction of granulomas. Upon tuberculosis infection, multi-nucleate giant cells are elicited, likely as a response aimed at containing mycobacteria. In tissue culture models, signal regulatory protein (SIRP)alpha (also referred to as macrophage fusion receptor or CD172a) is essential for multi-nucleate giant cell formation., Methodology/principal Findings: In the present study, ESAT-6/CFP-10 complex and SIRPalpha interactions were evaluated with samples obtained from calves experimentally infected with M. bovis. Peripheral blood CD172a(+) (SIRPalpha-expressing) cells from M. bovis-infected calves proliferated upon in vitro stimulation with ESAT-6/CFP-10 (either as a fusion protein or a peptide cocktail), but not with cells from animals receiving M. bovis strains lacking ESAT-6/CFP-10 (i.e, M. bovis BCG or M. bovis DeltaRD1). Sorted CD172a(+) cells from these cultures had a dendritic cell/macrophage morphology, bound fluorescently-tagged rESAT-6:CFP-10, bound and phagocytosed live M. bovis BCG, and co-expressed CD11c, DEC-205, CD44, MHC II, CD80/86 (a subset also co-expressed CD11b or CD8alpha). Intradermal administration of rESAT-6:CFP-10 into tuberculous calves elicited a delayed type hypersensitive response consisting of CD11c(+), CD172a(+), and CD3(+) cells, including CD172a-expressing multi-nucleated giant cells., Conclusions/significance: These findings demonstrate the ability of ESAT-6/CFP-10 to specifically expand CD172a(+) cells, bind to CD172a(+) cells, and induce multi-nucleated giant cells expressing CD172a.

An attenuated Mycobacterium tuberculosis RD1 knockout and pantothenate auxotroph (mc(2)6030) vaccine administered at 2 weeks of age failed to protect calves from low dose, aerosol M. bovis challenge at 2.5 months of age. In contrast, M. bovis bacille Calmette Guerin (BCG)-vaccinates had reduced tuberculosis-associated pathology as compared to non- and mc(2)6030-vaccinates. Mycobacterial colonization was not impacted by vaccination. Positive prognostic indicators associated with reduced pathology in the BCG-vaccinated group were decreased antigen induced IFN-gamma, iNOS, IL-4, and MIP1-alpha responses, increased antigen induced FoxP3 expression, and a diminished activation phenotype (i.e., downward arrow CD25+ and CD44+ cells and upward arrow CD62L+ cells) in mycobacterial-stimulated mononuclear cell cultures. The calf sensitization and challenge model provides an informative screen for candidate tuberculosis vaccines before their evaluation in costly non-human, primates.

Wild banded mongooses (Mungos mungo) in northeastern Botswana and northwest Zimbabwe are infected with a novel Mycobacterium tuberculosis complex (MTC) pathogen, Mycobacterium mungi. We evaluated gross and histologic lesions in 62 infected mongooses (1999–2017). Many tissues contained multifocal irregular, lymphohistiocytic to granulomatous infiltrates and/or multifocal or coalescing noncaseating to caseating granulomas with variable numbers of intralesional acid-fast bacilli. Over one-third of nasal turbinates examined had submucosal lymphohistiocytic to granulomatous infiltrates, erosion and ulceration of the nasal mucosa, bony remodeling, and nasal distortion. Similar inflammatory cell infiltrates expanded the dermis of the nasal planum with frequent ulceration. However, even in cases with intact epidermis, acid-fast bacilli were present in variable numbers among dermal infiltrates and on the epidermal surface among desquamated cells and debris, most commonly in small crevices or folds. In general, tissue involvement varied among cases but was highest in lymph nodes (50/54, 93%), liver (39/53, 74%), spleen (37/51, 73%), and anal glands/sacs (6/8, 75%). Pulmonary lesions were present in 67% of sampled mongooses (35/52) but only in advanced disseminated disease. The pathological presentation of M. mungi in the banded mongoose is consistent with pathogen shedding occurring through scent-marking behaviors (urine and anal gland secretions) with new infections arising from contact with these contaminated olfactory secretions and percutaneous movement of the pathogen through breaks in the skin, nasal planum, and/or skin of the snout. Given the character and distribution of lesions and the presence of intracellular acid-fast bacilli, we hypothesize that pathogen spread occurs within the body through a hematogenous and/or lymphatic route. Features of prototypical granulomas such as multinucleated giant cells and peripheral fibrosis were rarely present in affected mongooses. Acid-fast bacilli were consistently found intracellularly, even in regions of necrosis. The mongoose genome has a unique deletion (RD1mon) that includes part of the encoding region for PPE68 (Rv3873), a gene co-operonic with PE35. These proteins can influence the host’s cellular immune response to mycobacterial infections, and it remains uncertain how this deletion might contribute to observed patterns of pathology. M. mungi infection in banded mongooses is characterized by both a unique transmission and exposure route, as well as accompanying pathological features, providing an opportunity to increase our understanding of MTC pathogenesis across host-pathogen systems. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis is a zoonotic disease of global public health concern. Development of diagnostic tools to improve test accuracy and efficiency in domestic livestock and enable surveillance of wildlife reservoirs would improve disease management and eradication efforts. Use of volatile organic compound analysis in breath and fecal samples is being developed and optimized as a means to detect disease in humans and animals. In this study we demonstrate that VOCs present in fecal samples can be used to discriminate between non-vaccinated and BCG-vaccinated cattle prior to and after Mycobacterium bovis challenge. [ABSTRACT FROM AUTHOR]

The hallmark lesion of tuberculosis in humans and animals is the granuloma. The granuloma represents a distinct host cellular immune response composed of epithelioid macrophages, lymphocytes, and multinucleated giant cells, often surrounding a caseous necrotic core. Within the granuloma, host-pathogen interactions determine disease outcome. Factors within the granulomas such as cytokines and chemokines drive cell recruitment, activity, function and ultimately the success or failure of the host’s ability to control infection. Hence, an understanding of the granuloma-level cytokine response is necessary to understand tuberculosis pathogenesis. In-situ cytokine expression patterns were measured using a novel in situ hybridization assay, known as RNAScope® in granulomas of the lungs, tracheobronchial lymph nodes and caudal mediastinal lymph nodes of cattle experimentally infected with Mycobacterium bovis via aerosol exposure. In spite of microscopic morphological similarities, significant differences were seen between late stage granulomas of the lung compared to those of the tracheobronchial lymph nodes for IL-17A, IFN-γ, TGF-β, IL10 and IL-22 but not for TNF-α. Additionally, significant differences were noted between granulomas from two different thoracic lymph nodes that both receive afferent lymphatics from the lungs (i.e., tracheobronchial and caudal mediastinal lymph nodes) for TNF-α, IL-17A, IFN-γ, TGF-β and IL-10 but not for IL-22. These findings show that granuloma morphology alone is not a reliable indicator of granuloma function as granulomas of similar morphologies can have disparate cytokine expression patterns. Moreover, anatomically distinct lymph nodes (tracheobronchial vs caudal mediastinal) differ in cytokine expression patterns even when both receive afferent lymphatics from a lung containing tuberculoid granulomas. These findings show that selection of tissue and anatomic location are critical factors in assessing host immune response to M. bovis and should be considered carefully. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis and M. bovis share >99% genetic identity and induce similar host responses and disease profiles upon infection. There is a rich history of codiscovery in the development of control measures applicable to both human and bovine tuberculosis (TB) including skin-testing procedures, M. bovis BCG vaccination, and interferon-γ release assays. The calf TB infection model offers several opportunities to further our understanding of TB immunopathogenesis. Recent observations include correlation of central memory immune responses with TB vaccine efficacy, association of SIRPα+ cells in ESAT-6:CFP10- elicited multinucleate giant cell formation, early γδ T cell responses to TB, antimycobacterial activity of memory CD4+ T cells via granulysin production, association of specific antibody with antigen burden, and suppression of innate immune gene expression in infected animals. Partnerships teaming researchers with veterinary and medical perspectives will continue to provide mutual benefit to TB research in man and animals. [ABSTRACT FROM AUTHOR]

Background: Early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis) involved in phagolysosome escape of the bacillus and, potentially, in the efficient induction of granulomas. Upon tuberculosis infection, multi-nucleate giant cells are elicited, likely as a response aimed at containing mycobacteria. In tissue culture models, signal regulatory protein (SIRP)a (also referred to as macrophage fusion receptor or CD172a) is essential for multi-nucleate giant cell formation. Methodology/Principal Findings: In the present study, ESAT-6/CFP-10 complex and SIRPα interactions were evaluated with samples obtained from calves experimentally infected with M. bovis. Peripheral blood CD172a+ (SIRPα-expressing) cells from M. bovis-infected calves proliferated upon in vitro stimulation with ESAT-6/CFP-10 (either as a fusion protein or a peptide cocktail), but not with cells from animals receiving M. bovis strains lacking ESAT-6/CFP-10 (i.e, M. bovis BCG or M. bovis ΔRD1). Sorted CD172a+ cells from these cultures had a dendritic cell/macrophage morphology, bound fluorescently-tagged rESAT-6:CFP-10, bound and phagocytosed live M. bovis BCG, and co-expressed CD11c, DEC-205, CD44, MHC II, CD80/86 (a subset also co-expressed CD11b or CD8α). Intradermal administration of rESAT-6:CFP-10 into tuberculous calves elicited a delayed type hypersensitive response consisting of CD11c+, CD172a+, and CD3+ cells, including CD172a-expressing multinucleated giant cells. Conclusions/Significance: These findings demonstrate the ability of ESAT-6/CFP-10 to specifically expand CD172a+ cells, bind to CD172a+ cells, and induce multi-nucleated giant cells expressing CD172a. [ABSTRACT FROM AUTHOR]

Abstract: The mainstay of tuberculosis diagnosis in cattle and deer has been the tuberculin skin test. Recent advances have allowed the incorporation of blood based assays to the diagnostic arsenal for both cattle and deer. Use of defined and specific antigens has allowed for improved specificity of cell mediated assays in both cattle and deer and advances in antibody tests for tuberculosis have potential for use in free-ranging and captive cervid populations. Combined use of blood-based assays with skin testing will require further understanding of the effect of skin testing on the accuracy of blood based assays. Models of experimental infection of cattle have allowed for increased understanding of natural disease pathogenesis. Differences likely exist; however, between cattle and deer in both disease distribution and primary route of inoculation in naturally infected animals. [Copyright &y& Elsevier]

The Mycobacterium tuberculosis complex (MTBC) is a group of bacteria that cause tuberculosis (TB) in diverse hosts, including captive and free-ranging wildlife species. There is significant research interest in developing immunodiagnostic tests for TB that are both rapid and reliable, to underpin disease surveillance and control. The aim of this study was to carry out an updated review of diagnostics for TB in non-bovid species with a focus predominantly on those based on measurement of immunity. A search was carried out to identify relevant papers meeting a pre-defined set of inclusion criteria. Forty-one papers were identified from this search, from which only twenty papers contained data to measure and compare diagnostic performance using diagnostic odds ratio. The diagnostic tests from each study were ranked based on sensitivity, specificity, and diagnostic odds ratio to define high performing tests. High sensitivity and specificity values across a range of species were reported for a new antigenic target, P22 complex, demonstrating it to be a reliable and accurate antigenic target. Since the last review of this kind was undertaken, the immunodiagnosis of TB in meerkats and African wild dogs was reported for the first time. Suid species showed the most consistent immunological responses and highlight a potential dichotomy between humoral and cellular immune responses. [ABSTRACT FROM AUTHOR]

Abstract: Pioneer studies on infectious disease and immunology by Jenner, Pasteur, Koch, Von Behring, Nocard, Roux, and Ehrlich forged a path for the dual-purpose with dual benefit approach, demonstrating a profound relevance of veterinary studies for biomedical applications. Tuberculosis (TB), primarily due to Mycobacterium tuberculosis in humans and Mycobacterium bovis in cattle, is an exemplary model for the demonstration of this concept. Early studies with cattle were instrumental in the development of the use of Koch's tuberculin as an in vivo measure of cell-mediated immunity for diagnostic purposes. Calmette and Guerin demonstrated the efficacy of an attenuated M. bovis strain (BCG) in cattle prior to use of this vaccine in humans. The interferon-γ release assay, now widely used for TB diagnosis in humans, was developed circa 1990 for use in the Australian bovine TB eradication program. More recently, M. bovis infection and vaccine efficacy studies with cattle have demonstrated a correlation of vaccine-elicited T cell central memory (TCM) responses to vaccine efficacy, correlation of specific antibody to mycobacterial burden and lesion severity, and detection of antigen-specific IL-17 responses to vaccination and infection. Additionally, positive prognostic indicators of bovine TB vaccine efficacy (i.e., responses measured after infection) include: reduced antigen-specific IFN-γ, iNOS, IL-4, and MIP1-α responses; reduced antigen-specific expansion of CD4+ T cells; and a diminished activation profile on T cells within antigen stimulated cultures. Delayed type hypersensitivity and IFN-γ responses correlate with infection but do not necessarily correlate with lesion severity whereas antibody responses generally correlate with lesion severity. Recently, serologic tests have emerged for the detection of tuberculous animals, particularly elephants, captive cervids, and camelids. B cell aggregates are consistently detected within tuberculous lesions of humans, cattle, mice and various other species, suggesting a role for B cells in the immunopathogenesis of TB. Comparative immunology studies including partnerships of researchers with veterinary and medical perspectives will continue to provide mutual benefit to TB research in both man and animals. [Copyright &y& Elsevier]