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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]

Background: Bovine tuberculosis (TB) control programs generally rely on the tuberculin skin test (TST) for ante-mortem detection of Mycobacterium bovis-infected cattle. Results: Present findings demonstrate that a rapid antibody test based on Dual-Path Platform (DPP®) technology, when applied 1-3 weeks after TST, detected 9 of 11 and 34 of 52 TST non-reactive yet M. bovis-infected cattle from the US and GB, respectively. The specificity of the assay ranged from 98.9% (n = 92, US) to 96.0% (n = 50, GB) with samples from TB-free herds. Multi-antigen print immunoassay (MAPIA) revealed the presence of antibodies to multiple antigens of M. bovis in sera from TST non-reactors diagnosed with TB. Conclusions: Thus, use of serologic assays in series with TST can identify a significant number of TST non-reactive tuberculous cattle for more efficient removal from TB-affected herds. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis is an important animal pathogen worldwide that parasitizes wild and domesticated vertebrate livestock as well as humans. A comparison of the five M. bovis complete genomes from the United Kingdom, South Korea, Brazil, and theUnited States revealed four novel large-scale structural variations of at least 2,000 bp. A comparative phylogenomic study including 2,483 core genes of 38 taxa from eight countries showed conflicting phylogenetic signal among sites. By minimizing this effect, we obtained a tree that better agreeswith sampling locality. Results supported a relatively basal position of African strains (all isolated from Homo sapiens), confirming that Africa was an important region for early diversification and that humans were one of the earliest hosts. Selection analyses revealed that functional categories such as "Lipid transport and metabolism," "Cell cycle control, cell division, chromosome partitioning" and "Cell motility" were significant for the evolution of the group, besides other categories previously described, showing importance of genes associated with virulence and cholesterolmetabolism in the evolution of M. bovis. PE/PPE genes, many of which are known to be associatedwith virulence, were major targets for large-scale polymorphisms, homologous recombination, and positive selection, evincing for the first time a plethora of evolutionary forces possibly contributing to differential adaptability in M. bovis. By assuming different priors, US strains originated and started to diversify around 150-5,210 ya. By further analyzing the largest set of US genomes to date (76 in total), obtained from 14 host species, we detected that hosts were not clustered in clades (except for a few cases), with some faster-evolving strains being detected, suggesting fast and ongoing reinfections across host species, and therefore, the possibility of new bovine tuberculosis outbreaks. [ABSTRACT FROM AUTHOR]

Central memory T cell (Tcm) and polyfunctional CD4 T cell responses contribute to vaccine-elicited protection with both human and bovine tuberculosis (TB); however, their combined role in protective immunity to TB is unclear. To address this question, we evaluated polyfunctional cytokine responses by CD4 T cell effector/memory populations from bacille Calmette-Guerin (BCG) vaccinated and non-vaccinated calves by flow cytometry prior to and after aerosol challenge with virulent Mycobacterium bovis. Polyfunctional cytokine expression patterns in the response by Tcm, effector memory, and effector T cell subsets were similar between BCG-vaccinated and M. bovis-infected calves, only differing in magnitude (i.e., infected > vaccinated). BCG vaccination, however, did alter the kinetics of the ensuing response to virulent M. bovis infection. Early after challenge (3 weeks post-infection), non-vaccinates had greater antigen-specific interferon-γ (IFN-γ)/tumor necrosis factor-α (TNF-α) and lesser IFN-γ/TNF-α/IL-2 responses by Tcm cells than did vaccinated animals. Importantly, these differences were also associated with mycobacterial burden upon necropsy. Polyfunctional responses to ESAT-6:CFP10 (antigens not synthesized by BCG strains) were detected in memory subsets, as well as in effector cells, as early as 3 weeks after challenge. These findings suggest that cell fate divergence may occur early after antigen priming in the response to bovine TB and that memory and effector T cells may expand concurrently during the initial phase of the immune response. In summary, robust IFN-γ/TNF-α response by Tcm cells is associated with greater mycobacterial burden, while IFN-γ/TNF-α/IL-2 response by Tcm cells are indicative of a protective response to bovine TB. [ABSTRACT FROM AUTHOR]

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]

The high-resolution WGS analyses of MTBC strains have provided useful insight for determining sources of infection for animal tuberculosis. In Spain, tuberculosis in livestock is caused by Mycobacterium bovis and Mycobacterium caprae, where wildlife reservoirs play an important role. We analyzed a set of 125 M. bovis isolates obtained from livestock and wildlife from Catalonia to investigate strain diversity and identify possible sources and/or causes of infection. Whole-genome SNP profiles were used for phylogenetic reconstruction and pairwise SNP distance analysis. Additionally, SNPs were investigated to identify virulence and antimicrobial resistance factors to investigate clade-specific associations. Putative transmission clusters (≤12 SNPs) were identified, and associated epidemiological metadata were used to determine possible explanatory factors for transmission. M. bovis distribution was heterogeneous, with 7 major clades and 21 putative transmission clusters. In order of importance, the explanatory factors associated were proximity and neighborhood, residual infection, livestock-wildlife interaction, shared pasture, and movement. Genes related to lipid transport and metabolism showed the highest number of SNPs. All isolates were pyrazinamide resistant, and five were additionally resistant to isoniazid, but no clade-specific associations could be determined. Our findings highlight the importance of high-resolution molecular surveillance to monitor bovine tuberculosis dynamics in a low-prevalence setting. [ABSTRACT FROM AUTHOR]

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]