Summary Existing strategies for long‐term bovine tuberculosis (bTB) control/eradication campaigns are being reconsidered in many countries because of the development of new testing technologies, increased global trade, continued struggle with wildlife reservoirs of bTB, redistribution of international trading partners/agreements, and emerging financial and animal welfare constraints on herd depopulation. Changes under consideration or newly implemented include additional control measures to limit risks with imported animals, enhanced programs to mitigate wildlife reservoir risks, re‐evaluation of options to manage bTB‐affected herds/regions, modernization of regulatory framework(s) to re‐focus control efforts, and consideration of emerging testing technologies (i.e. improved or new tests) for use in bTB control/eradication programs. Traditional slaughter surveillance and test/removal strategies will likely be augmented by incorporation of new technologies and more targeted control efforts. The present review provides an overview of current and emerging bTB testing strategies/tools and a vision for incorporation of emerging technologies into the current control/eradication programs.

Previous work with small-animal laboratory models of tuberculosis has shown that vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacillus Calmette-Guérin (BCG) to prime and modified vaccinia virus Ankara strain (MVA85A) or recombinant attenuated adenoviruses (Ad85A) expressing the mycobacterial antigen Ag85A to boost may increase the protective efficacy of BCG. Here we report the first efficacy data on using these vaccines in cattle, a natural target species of tuberculous infection. Protection was determined by measuring development of disease as an end point after M. bovis challenge. Either Ad85A or MVA85A boosting resulted in protection superior to that given by BCG alone: boosting BCG with MVA85A or Ad85A induced significant reduction in pathology in four/eight parameters assessed, while BCG vaccination alone did so in only one parameter studied. Protection was particularly evident in the lungs of vaccinated animals (median lung scores for naïve and BCG-, BCG/MVA85A-, and BCG/Ad85A-vaccinated animals were 10.5, 5, 2.5, and 0, respectively). The bacterial loads in lymph node tissues were also reduced after viral boosting of BCG-vaccinated calves compared to those in BCG-only-vaccinated animals. Analysis of vaccine-induced immunity identified memory responses measured by cultured enzyme-linked immunospot assay as well as in vitro interleukin-17 production as predictors of vaccination success, as both responses, measured before challenge, correlated positively with the degree of protection. Therefore, this study provides evidence of improved protection against tuberculosis by viral booster vaccination in a natural target species and has prioritized potential correlates of vaccine efficacy for further evaluation. These findings also have implications for human tuberculosis vaccine development.More than 50 million cattle are infected with Mycobacterium bovis, resulting in economic losses of approximately $3 billion annually (34). Over the last two decades, in Great Britain, failure of the (tuberculin) test-and-slaughter strategy has resulted in a dramatic rise in the incidence of tuberculosis (TB) in cattle (19). The urgent need for new and improved cattle vaccines and diagnostic reagents has been acknowledged by the British government, and development of a cattle vaccine is a research priority. As cattle can be considered a large-animal model for human TB vaccination, experiments with cattle will

In cattle, the kidney has been the only known site for production of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] from 25-hydroxyvitamin D(3) [25(OH)D(3)] by 1alpha-hydroxylase (1alpha-OHase). Based on human studies, it was hypothesized that bovine monocytes could produce 1,25(OH)(2)D(3) upon activation and 1,25(OH)(2)D(3) would regulate expression of vitamin D-responsive genes in monocytes. First, the effects of 1,25(OH)(2)D(3) on bovine monocytes isolated from peripheral blood were tested. Treatment of nonstimulated monocytes with 1,25(OH)(2)D(3) increased expression of the gene for the vitamin D 24-hydroxylase (24-OHase) enzyme by 51+/-13 fold, but 1,25(OH)(2)D(3) induction of 24-OHase expression was blocked by lipopolysaccharide (LPS) stimulation. In addition, 1,25(OH)(2)D(3) increased the gene expression of inducible nitric oxide synthase and the chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted) in LPS-stimulated monocytes 69+/-13 and 40+/-12 fold, respectively. Next, the ability of bovine monocytes to express 1alpha-OHase and produce 1,25(OH)(2)D(3) was tested. Activation of monocytes with LPS, tripalmitoylated lipopeptide (Pam3CSK4), or peptidoglycan caused 43+/-9, 17+/-3, and 19+/-3 fold increases in 1alpha-OHase gene expression, respectively. Addition of 25(OH)D(3) to LPS-stimulated monocytes enhanced expression of inducible nitric oxide synthase and RANTES and nitric oxide production in a dose-dependent manner, giving evidence that activated monocytes convert 25(OH)D(3) to 1,25(OH)(2)D(3). In conclusion, bovine monocytes produce 1,25(OH)(2)D(3) in response to toll-like receptor signaling, and 1,25(OH)(2)D(3) production in monocytes increased the expression of genes involved in the innate immune system. Vitamin D status of cattle might be important for optimal innate immune function because 1,25(OH)(2)D(3) production in activated monocytes and subsequent upregulation of inducible nitric oxide synthase and RANTES expression was dependent on 25(OH)D(3) availability.

Throughout the natural course of human immunodeficiency virus (HIV) infection, follicular dendritic cells (FDCs) trap and retain large quantities of particle-associated HIV RNA in the follicles of secondary lymphoid tissue. We have previously found that murine FDCs in vivo could maintain trapped virus particles in an infectious state for at least 9 months. Here we sought to determine whether human FDCs serve as an HIV reservoir, based on the criteria that virus therein must be replication competent, genetically diverse, and archival in nature. We tested our hypothesis using postmortem cells and tissues obtained from three HIVinfected subjects and antemortem blood samples obtained from one of these subjects. Replication competence was determined using coculture, while genetic diversity and the archival nature of virus were established using phylogenetic and population genetics methods. We found that FDC-trapped virus was replication competent and demonstrated greater genetic diversity than that of virus found in most other tissues and cells. Antiretrovirus-resistant variants that were not present elsewhere were also detected on FDCs. Furthermore, genetic similarity was observed between FDC-trapped HIV and viral species recovered from peripheral blood mononuclear cells obtained 21 and 22 months antemortem, but was not present in samples obtained 4 and 18 months prior to the patient's death, indicating that FDCs can archive HIV. These data indicate that FDCs represent a significant reservoir of infectious and diverse HIV, thereby providing a mechanism for viral persistence for months to years.

Bovine tuberculosis persists as a costly zoonotic disease in numerous countries despite extensive eradication and control efforts. Sequential serum samples obtained from Mycobacterium bovis-infected cattle were evaluated for seroreactivity to mycobacterial antigens. Animals received M. bovis by aerosol, intratonsil, intranasal, or intratracheal inoculation. Assays included the multiantigen print immunoassay for determination of antigen recognition patterns, immunoblot analysis for sensitive kinetic studies, and the VetTB STAT-PAK test, a novel, rapid test based on lateral-flow technology. Responses to MPB83 were detected for all M. bovis-infected animals regardless of the route or strain of M. bovis used for inoculation. Other less commonly recognized antigens included ESAT-6, CFP-10, and MPB70. Responses to MPB83 were detectable as early as 4 weeks after inoculation, were boosted upon injection of purified protein derivatives for skin testing, and persisted throughout the course of each of the four challenge studies. MPB83-specific immunoglobulin M (IgM) was detected prior to MPB83-specific IgG detection; however, early IgM responses rapidly waned, suggesting a benefit of tests that detect both IgM-and IgG-specific antibodies. The VetTB STAT-PAK test detected responses in sera from 60% (15/25) of the animals by 7 weeks after challenge and detected responses in 96% (24/25) of the animals by 18 weeks. These findings demonstrate the potential for new-generation antibody-based tests for the early detection of M. bovis infection in cattle.Tuberculosis (TB) in humans may result from exposure to any one of the tubercle bacilli included within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedii, and M. microti). Mycobacterium bovis, unlike M. tuberculosis, has a wide host range, is the species most often isolated from tuberculous cattle, and has several wildlife maintenance hosts, including the Eurasian badger (Meles meles), brush-tailed possum (Trichosurus vulpecula), and white-tailed deer (Odocoileus virginianus). Wildlife reservoirs have made M. bovis eradication from national herds in several developed countries, including the United Kingdom, New Zealand, and the United States, particularly difficult (3, 4, 16). Eradication campaigns in these countries have generally relied on test and removal, slaughterhouse surveillance, movement restriction, and/or wildlife reservoir control strategies. The tests most widely used for the detection of TB in humans and cattle include the measurement of delayed-type hypersensitivity (i.e., skin testing) to purified protein derivatives (PPDs) and/or in vitro assays for gamma interferon produced in response to mycobacterial antigen stimulation (i.e., Bovigam [Prionics AG, Schlieren, Switzerland] and Quantiferon Gold [Cellestis Inc., Carnegie, Victoria, Australia]). These tests rely on early cell-mediated responses, a hallmark of TB immunopathogenesis. In contrast, the poor sensitivity of antibodybased tests has prevented the widespre...

Mycobacterium bovis, the causative agent of bovine tuberculosis, persists within granulomas. Formation of granulomas involves a complex array of immune activation and cellular migration. To examine temporal changes in granuloma development, we inoculated 32 cattle with M. bovis of deer origin. Tissues from 4 calves each were examined at 15, 28, 42, 60, 90, 180, 270, and 370 days after inoculation. Granulomas in the medial retropharyngeal lymph node were staged (I-IV) on the basis of cellular composition and the presence or absence of necrosis and peripheral fibrosis. Immunohistochemistry for inducible nitric oxide synthase (iNOS), CD68, CD4, CD8, and gamma/delta T cells was performed. Fifteen days after inoculation only stage I granulomas were seen, while between 28 and 60 days, there was a steady progression through granuloma stages such that by day 60, granulomas of all 4 stages were seen. Acid-fast bacilli were present in moderate-to-large numbers in stage I granulomas 15-60 days after inoculation. Stage IV granulomas contained large numbers of acid-fast bacteria. Abundant iNOS immunoreactivity was associated with granulomas from day 15 through day 60 but was minimal from day 90 to the termination of the experiment. The relative number of CD4+ and CD68+ cells remained constant throughout the study. In contrast, at time points >60 days, numbers of CD8+ and gamma/delta T cells diminished. Tuberculous granulomas are dynamic lesions that follow an orderly progression through disease stages. Diminished expression of iNOS and reduced numbers of CD8+ and gamma/delta T cells late in the progression of tuberculous granulomas may represent a failure of the host response to control infection.

As a consequence of continued spillover of Mycobacterium bovis into cattle from wildlife reservoirs and increased globalization of cattle trade with associated transmission risks, new approaches such as vaccination and novel testing algorithms are seriously being considered by regulatory agencies for the control of bovine tuberculosis. Serologic tests offer opportunities for identification of M. bovis-infected animals not afforded by current diagnostic techniques. The present study describes assay development and field assessment of a new commercial enzyme-linked immunosorbent assay (ELISA) that detects antibody to M. bovis antigens MPB83 and MPB70 in infected cattle. Pertinent findings include the following: specific antibody responses were detected at ϳ90 to 100 days after experimental M. bovis challenge, minimal cross-reactive responses were elicited by infection/sensitization with nontuberculous Mycobacterium spp., and the apparent sensitivity and specificity of the ELISA with naturally infected cattle were 63% and 98%, respectively, with sensitivity improving as disease severity increased. The ELISA also detected infected animals missed by the routine tuberculin skin test, and antibody was detectable in bulk tank milk samples from M. bovis-infected dairy herds. A high-throughput ELISA could be adapted as a movement, border, or slaughter surveillance test, as well as a supplemental test to tuberculin skin testing.

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.