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Mycobacterium tuberculosis variant bovis (MBO) has one of the widest known mammalian host ranges, including humans. Despite the characterization of this pathogen in the 1800s and whole genome sequencing of a UK strain (AF2122) nearly two decades ago, the basis of its host specificity and pathogenicity remains poorly understood. Recent experimental calf infection studies show that MBO strain Ravenel (MBO Ravenel) is attenuated in the cattle host compared to other pathogenic strains of MBO. In the present study, experimental infections were performed to define attenuation. Whole genome sequencing was completed to identify regions of differences (RD) and single nucleotide polymorphisms (SNPs) to explain the observed attenuation. Comparative genomic analysis of MBO Ravenel against three pathogenic strains of MBO (strains AF2122-97, 10-7428, and 95-1315) was performed. Experimental infection studies on five calves each, with either MBO Ravenel or 95-1315, revealed no visible lesions in all five animals in the Ravenel group despite robust IFN-γ responses. Out of 486 polymorphisms in the present analysis, 173 were unique to MBO Ravenel among the strains compared. A high-confidence subset of nine unique SNPs were missense mutations in genes with annotated functions impacting two major MBO survival and virulence pathways: (1) Cell wall synthesis & transport [espH (A103T), mmpL8 (V888I), aftB (H484Y), eccC5 (T507M), rpfB (E263G)], and (2) Lipid metabolism & respiration [mycP1(T125I), pks5 (G455S), fadD29 (N231S), fadE29 (V360G)]. These substitutions likely contribute to the observed attenuation. Results from experimental calf infections and the functional attributions of polymorphic loci on the genome of MBO Ravenel provide new insights into the strain's genotype-disease phenotype associations. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis is a serious zoonotic pathogen and the cause of tuberculosis in many mammalian species, most notably, cattle. The hallmark lesion of tuberculosis is the granuloma. It is within the developing granuloma where host and pathogen interact; therefore, it is critical to understand host-pathogen interactions at the granuloma level. Cytokines and chemokines drive cell recruitment, activity, and function and ultimately determine the success or failure of the host to control infection. In calves, early lesions (ie, 15 and 30 days) after experimental aerosol infection were examined microscopically using in situ hybridization and immunohistochemistry to demonstrate early infiltrates of CD68+ macrophages within alveoli and alveolar interstitium, as well as the presence of CD4, CD8, and γδ T cells. Unlike lesions at 15 days, lesions at 30 days after infection contained small foci of necrosis among infiltrates of macrophages, lymphocytes, neutrophils, and multinucleated giant cells and extracellular acid-fast bacilli within necrotic areas. At both time points, there was abundant expression of the chemokines CXCL9, MCP-1/CCL2, and the cytokine transforming growth factor (TGF)–β. The proinflammatory cytokines tumor necrosis factor (TNF)–α and interleukin (IL)–1β, as well as the anti-inflammatory cytokine IL-10, were expressed at moderate levels at both time points, while expression of IFN-γ was limited. These findings document the early pulmonary lesions after M. bovis infection in calves and are in general agreement with the proposed pathogenesis of tuberculosis described in laboratory animal and nonhuman primate models of tuberculosis. [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]

Abstract: The paired palatine tonsils, located at the junction of the nasopharynx and oropharynx, are ideally positioned to sample antigens entering through the nasal cavity or oral cavity. Entering antigens will first contact tonsilar epithelium. To better understand the cellular composition of this important epithelial layer, palatine tonsils were collected from six, 7-month-old calves and examined by light microscopy, immunohistochemistry and electron microscopy. Morphometric analysis showed that epithelium overlying lymphoid follicles (reticular epithelium) contained more B-cells, CD4+, CD8+, CD11c+, CD172a+ and γ/δ TCR+ cells than non-reticular epithelium, with B-cells, CD4+ cells and CD11c+ cells being most numerous. Scanning and transmission electron microscopy of reticular epithelium identified an interrupted basement membrane and vascular elements within the epithelium, as well as cells with characteristics morphologically consistent with cells identified as M-cells in other species. Bovine palatine tonsilar reticular epithelium contains key immune cells, as well as potential M-cell-like cells; elements essential for antigen uptake, antigen processing and initiation of immune responses. [Copyright &y& Elsevier]

Abstract: An impediment to the development of efficacious vaccines for bovine tuberculosis has been the failure to demonstrate strong associations between immune function and protective immunity. Cytokine gene expression in response to Mycobacterium bovis (M. bovis) infection was evaluated to identify correlates of immunity. Ten Holstein calves were infected with M. bovis by intratonsillar inoculation. Five uninfected animals served as controls. At 15, 30, 60 and 85 days post-infection (dpi) peripheral blood mononuclear cells (PBMC) were isolated and stimulated with either purified protein derivative of M. bovis (PPD), a recombinant fusion protein comprised of 6kDa early secretory antigenic target and 10kDa culture filtrate antigen (rESAT6:CFP10), or PBS. After a 16h incubation period, total leukocyte RNA was isolated and gene expression evaluated using reverse transcriptase real-time PCR. In addition, gene expression adjacent to gross lesion in the retropharyngeal lymph node (LN) was analyzed. Pathology was evaluated at necropsy. Expression of IFN-γ, TNF-α, iNOS and IL-4 by PBMC increased in response to infection, whereas, IL-10 expression decreased. Differences in gene expression between PBMC from infected and uninfected animals was greatest at 30dpi. Infected animals were divided into two groups based on pathology. Animals in the low pathology group had lesions primarily in LN of the head; whereas, animals in the high pathology group also had lesions in the lungs and lung associated LN. Gene expression in PBMC and LN was compared between animals in the high and low pathology groups. Cells from animals in the high pathology group expressed more IFN-γ, TNF-α, iNOS and IL-4 than did animals in the low pathology group at early time points. IL-10 gene expression decreased with time in PBMC from animals in the high pathology group. At 85dpi, animals in the high pathology group expressed twofold less IL-10 mRNA than did animals in the low pathology group and the uninfected controls. IFN-γ and iNOS gene expression were significantly greater in tissues from infected animals compared to tissues from uninfected animals. The pathological outcome of M. bovis infection of cattle may be established early after infection since expression of both the TH1 and TH2 cytokines were differentially expressed by animals in the high and low pathology groups at early time points. In addition, more robust immunological responses were associated with increased pathology. These results suggest that early immune responses play a critical role in establishing the pathological outcome. [Copyright &y& Elsevier]

Recent studies have suggested the potential of innovative serologic tests for accurate and rapid detection of bovine tuberculosis (bTB). Dual Path Platform (DPP) technology has been used to develop rapid animal-side antibody tests for Mycobacterium bovis infection in a range of livestock and wildlife host species. The present study evaluated diagnostic performance of DPP BovidTB IgM/IgG assay designed for differential detection of bovine IgM and IgG antibodies against two chimeric antigens, DID38 and TBf2, respectively, using 662 well-characterized serum samples from M. bovis -infected and bTB-free cattle collected in the United States, Great Britain, France, and South Africa. Test sensitivity and specificity ranged from 71% to 100% and from 95% to 100%, respectively, depending on the country, with overall accuracy of 83%. No significant risk of cross-reactivity with serum samples from cattle infected with most relevant species of mycobacteria other than M. bovis was found. The DPP BovidTB IgM/IgG assay may be suitable for use in multi-test algorithms to improve current strategies for bTB surveillance. • IgM antibody detection complements IgG-based serodiagnosis of bovine tuberculosis. • Use of chimeric antigens can enhance antibody detection in bovine tuberculosis. • DPP BovidTB IgG/IgM test for bovine tuberculosis shows ∼83% diagnostic accuracy. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis (bTB) control programs can be improved by combined use of tests for humoral and cell-mediated immune responses targeting multiple biomarkers of Mycobacterium bovis. To further the diagnostic benefits of this approach, we used Dual Path Platform (DPP) technology to test sera from cattle with naturally acquired bTB in the United States (US) and Spain for the presence of M. bovis antigen, IgM and/or IgG antibodies to MPB70/MPB83 fusion antigen in conjunction with tuberculin skin tests (TST) or interferon-gamma release assays (IGRA). When TST was complemented with detection of IgM and IgG antibodies, the diagnostic sensitivity increased from 85.4% to 95.1% in the US and from 64.2% to 81.5% in Spain. Likewise, adding the DPP assays enhanced IGRA diagnostic sensitivity from 82.7% to 93.8% in Spain. Detection of circulating M. bovis antigen showed added value when used in combination with the DPP antibody assays but it was limited when analyzed in the context of TST or IGRA results. Present findings support the benefits of a multi-test approach for the ante-mortem diagnosis of bTB in cattle. • Detection of multiple biomarkers of bovine tuberculosis can enhance test sensitivity. • Use of multiple immunoassays in series can improve disease detection rates. • Diagnostic value of circulating antigen and antibody may vary between countries. [ABSTRACT FROM AUTHOR]

• Recombinant polyproteins can improve antibody tests for bovine tuberculosis. • Newly designed fusion antigens (BID109, TB1f, TB2f) offer serodiagnostic potential. • TBf2 demonstrate highest accuracy (∼84 %) of detecting infected cattle by DPP assay. Recent studies have demonstrated potential for serologic assays to improve surveillance and control programs for bovine tuberculosis. Due to the animal-to-animal variation of the individual antibody repertoires observed in bovine tuberculosis, it has been suggested that serodiagnostic sensitivity can be maximized by use of multi-antigen cocktails or genetically engineered polyproteins expressing immunodominant B-cell epitopes. In the present study, we designed three novel multiepitope polyproteins named BID109, TB1f, and TB2f, with each construct representing a unique combination of four full-length peptides of Mycobacterium bovis predominantly recognized in bovine tuberculosis. Functional performance of the fusion antigens was evaluated using multi-antigen print immunoassay (MAPIA) and Dual Path Platform (DPP) technology with panels of monoclonal and polyclonal antibodies generated against individual proteins included in the fusion constructs as well as with serum samples from M. bovis -infected and non-infected cattle, American bison, and domestic pigs. It was shown that epitopes of each individual protein were expressed in the fusion antigens and accessible for efficient binding by the respective antibodies. The three fusion antigens demonstrated stronger immunoreactivity in MAPIA than that of single protein antigens. Evaluation of the fusion antigens in DPP assay using serum samples from 125 M. bovis -infected and 57 non-infected cattle showed the best accuracy (∼84 %) for TB2f antigen composed of MPB70, MPB83, CFP10, and Rv2650c proteins. Thus, the study results suggest a potential for the multiepitope polyproteins to improve diagnostic sensitivity of serologic assays for bovine tuberculosis. [ABSTRACT FROM AUTHOR]

• Blood, serum, plasma produce consistent serology results in bovine tuberculosis. • Other than blood-derived specimen types can be used for antibody detection. • Alternative specimens reflect antigen reactivity profiles of serum antibodies. • Bronchoalveolar lavage and diaphragm extract appear suitable for antibody assays. Bovine tuberculosis (bTB) control programs can be improved by implementation of advanced ante-mortem testing algorithms. Serodiagnostic methods using traditional blood or blood-derived specimens may benefit from the use of less invasive alternative biological fluids, provided those mirror systemic antibody responses. In the present study, we used Dual Path Platform (DPP) and Multiantigen Print Immunoassay (MAPIA) to compare antibody levels in ten sample types including whole blood (fresh and hemolyzed), plasma (fresh and leftover from Bovigam testing), serum, saliva, broncho-alveolar lavage, urine, diaphragm extract, and bile collected from cattle aerosol-infected with Mycobacterium bovis. High correlation (r = 0.97−0.99) in measurements of IgG antibodies to MPB70/MPB83 fusion antigen by DPP assay was found between all blood-derived specimens, supporting matrix equivalency. Broncho-alveolar lavage and diaphragm extract yielded positive results in all the infected animals tested, showing high correlation with matching serum data (r = 0.94 and r = 0.95, respectively) and suggesting their potential use in antibody assays. Characterized by MAPIA, the antigen reactivity patterns obtained with paired sera and alternative specimens were nearly identical, with slight differences in intensity. Antibodies were also found by DPP assay in saliva, urine, and bile from some of the infected animals, but the titers were relatively low, thus reducing the diagnostic value of such specimens. The proposed approach was evaluated in a pilot field study on warthogs diagnosed with M. bovis infection. Relative levels of antibody in tissue fluid obtained from lymph nodes or lungs were consistent with those detected in sera and detectable in all infected warthogs. The findings support the diagnostic utility of non-traditional biological fluids and tissue samples when used as alternative test specimens in serologic assays for bTB. [ABSTRACT FROM AUTHOR]