Development and validation of a one-tube, nested real-time PCR method suitable for routine detection of Mycobacterium bovis in animal tissue.

Aims: Development and validation of a real-time PCR test for high-throughput routine screening of animal tissue for Mycobacterium bovis and other Mycobacterium tuberculosis complex (MTBC) members.

Methods And Results: A preliminary study compared the results of a combination of five tissue preparation/DNA extraction methods and nine PCR assays on a panel of 92 cattle tissue samples of known M. bovis culture status (55 positive and 37 negative).

Assessment of the frequency of Mycobacterium bovis shedding in the faeces of naturally and experimentally TB infected cattle.

Aims: To assess the prevalence of Mycobacterium bovis bacilli in faecal samples of tuberculous cattle, and to better understand the risk of environmental dissemination of bovine tuberculosis (TB) through the spreading of manure or slurry.

Methods And Results: Faecal samples were collected from 72 naturally infected cattle with visible lesions of TB that had reacted to the tuberculin skin test and 12 cattle experimentally infected with M. bovis.

Efficacy and immunogenicity of different BCG doses in BALB/c and CB6F1 mice when challenged with H37Rv or Beijing HN878.

Two strains of mice (BALB/c and CB6F1) were vaccinated with a range of Bacille Calmette-Guérin (BCG) Danish doses from 3 × 10 to 30 CFU/mouse, followed by aerosol infection with Mtb (H37Rv or West-Beijing HN878 strain). The results indicated that both strains of mice when infected with HN878 exhibited significant protection in their lungs with BCG doses at 3 × 10-3000 CFU (BALB/c) and 3 × 10-300 CFU (CB6F1). Whereas, a significant protection was seen in both strains of mice with BCG doses at 3 × 10-300 CFU when infected with H37Rv.

Airway delivery of both a BCG prime and adenoviral boost drives CD4 and CD8 T cells into the lung tissue parenchyma.

Heterologous BCG prime-boost regimens represent a promising strategy for an urgently required improved tuberculosis vaccine. Identifying the mechanisms which underpin the enhanced protection induced by such strategies is one key aim which would significantly accelerate rational vaccine development. Experimentally, airway vaccination induces greater efficacy than parenteral delivery; in both conventional vaccination and heterologous boosting of parenteral BCG immunisation.

The effect of BCG vaccination on macrophage phenotype in a mouse model of intranasal Mycobacterium bovis challenge.

In order to develop improved vaccinations against tuberculosis, it is essential to understand the effect of vaccination on the immune response, and to overcome the mechanisms by which mycobacteria regulate this immune response. In this study, we examine the effect of intradermal vaccination with Mycobacterium bovis bacille Calmette-Guèrin on macrophage phenotype following intranasal challenge with virulent Mycobacterium bovis. Preserved lung tissues used in the present study were obtained from a previous vaccination trial in BALB/c mice.

Induction and maintenance of a phenotypically heterogeneous lung tissue-resident CD4 T cell population following BCG immunisation.

Tuberculosis (TB) is the biggest cause of human mortality from an infectious disease. The only vaccine currently available, bacille Calmette-Guérin (BCG), demonstrates some protection against disseminated disease in childhood but very variable efficacy against pulmonary disease in adults. A greater understanding of protective host immune responses is required in order to aid the development of improved vaccines.

Novel Solutions for Vaccines and Diagnostics To Combat Brucellosis.

Brucellosis is diagnosed by detection of antibodies in the blood of animals and humans that are specific for two carbohydrate antigens, termed A and M, which are present concurrently in a single cell wall O-polysaccharide. Animal brucellosis vaccines contain these antigenic determinants, and consequently infected and vaccinated animals cannot be differentiated as both groups produce A and M specific antibodies. We hypothesized that chemical synthesis of a pure A vaccine would offer unique identification of infected animals by a synthetic M diagnostic antigen that would not react with antibodies generated by this vaccine.

CD1 and CD1 porcine blood dendritic cells are enriched for the orthologues of the two major mammalian conventional subsets.

Conventional dendritic cells (cDC) are professional antigen-presenting cells that induce immune activation or tolerance. Two functionally specialised populations, termed cDC1 and cDC2, have been described in humans, mice, ruminants and recently in pigs. Pigs are an important biomedical model species and a key source of animal protein; therefore further understanding of their immune system will help underpin the development of disease prevention strategies.

Parenteral adenoviral boost enhances BCG induced protection, but not long term survival in a murine model of bovine TB.

Boosting BCG using heterologous prime-boost represents a promising strategy for improved tuberculosis (TB) vaccines, and adenovirus (Ad) delivery is established as an efficacious boosting vehicle. Although studies demonstrate that intranasal administration of Ad boost to BCG offers optimal protection, this is not currently possible in cattle. Using Ad vaccine expressing the mycobacterial antigen TB10.

Boosting BCG with inert spores improves immunogenicity and induces specific IL-17 responses in a murine model of bovine tuberculosis.

Tuberculosis (TB) remains a global pandemic, in both animals and man, and novel vaccines are urgently required. Heterologous prime-boost of BCG represents a promising strategy for improved TB vaccines, with respiratory delivery the most efficacious to date. Such an approach may be an ideal vaccination strategy against bovine TB (bTB), but respiratory vaccination presents a technical challenge in cattle.

Phenotypic characterization of bovine memory cells responding to mycobacteria in IFNγ enzyme linked immunospot assays.

Bovine tuberculosis (bTB) remains a globally significant veterinary health problem. Defining correlates of protection can accelerate the development of novel vaccines against TB. As the cultured IFNγ ELISPOT (cELISPOT) assay has been shown to predict protection and duration of immunity in vaccinated cattle, we sought to characterize the phenotype of the responding T-cells.

BALB/c mice display more enhanced BCG vaccine induced Th1 and Th17 response than C57BL/6 mice but have equivalent protection.

It is generally assumed that the inbred mouse strains BALB/c (H-2(d)) and C57BL/6 (H-2(b)) respond to mycobacterial infection with distinct polarisation of T helper responses, with C57BL/6 predisposed to Th1 and BALB/c to Th2. We investigated this in a BCG-immunisation, Mycobacterium bovis challenge model. Following immunisation, lung and spleen cell cytokine responses to in vitro re-stimulation with a cocktail of seven secreted, immunogenic, recombinant mycobacterial proteins were determined.

Persistent BCG bacilli perpetuate CD4 T effector memory and optimal protection against tuberculosis.

Tuberculosis (TB) remains one of the most important infectious diseases of man and animals, and the only available vaccine (BCG) requires urgent replacement or improvement. To facilitate this, the protective mechanisms induced by BCG require further understanding. As a live attenuated vaccine, persistence of BCG bacilli in the host may be a crucial mechanism.

Development of an unbiased antigen-mining approach to identify novel vaccine antigens and diagnostic reagents for bovine tuberculosis.

Previous experiments for the identification of novel diagnostic or vaccine candidates for bovine tuberculosis have followed a targeted approach, wherein specific groups of proteins suspected to contain likely candidates are prioritized for immunological assessment (for example, with in silico approaches). However, a disadvantage of this approach is that the sets of proteins analyzed are restricted by the initial selection criteria. In this paper, we describe a series of experiments to evaluate a nonbiased approach to antigen mining by utilizing a Gateway clone set for Mycobacterium tuberculosis, which constitutes a library of clones expressing 3,294 M.

Immunogenicity comparison of the intradermal or endobronchial boosting of BCG vaccinates with Ad5-85A.

Experiments in small animal models have indicated that intranasal vaccination confers a greater degree of protection against TB than other routes such as intradermal (i.d.) or intramuscular.

The duration of antigen-stimulation significantly alters the diversity of multifunctional CD4 T cells measured by intracellular cytokine staining.

The assessment of antigen-specific T cell responses by intracellular cytokine staining (ICS) has become a routine technique in studies of vaccination and immunity. Here, we highlight how the duration of in vitro antigen pre-stimulation, combined with the cytokine accumulation period, are critical parameters of these methods. The effect of varying these parameters upon the diversity and frequency of multifunctional CD4 T cell subsets has been investigated using a murine model of TB vaccination and in cattle naturally infected with Mycobacterium bovis.

Transcriptional profiling of disease-induced host responses in bovine tuberculosis and the identification of potential diagnostic biomarkers.

Bovine tuberculosis (bTb) remains a major and economically important disease of livestock. Improved ante-mortem diagnostic tools would help to underpin novel control strategies. The definition of biomarkers correlating with disease progression could have impact on the rational design of novel diagnostic approaches for bTb.

Development of an antibody to bovine IL-2 reveals multifunctional CD4 T(EM) cells in cattle naturally infected with bovine tuberculosis.

Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2).

Systemic BCG immunization induces persistent lung mucosal multifunctional CD4 T(EM) cells which expand following virulent mycobacterial challenge.

To more closely understand the mechanisms of how BCG vaccination confers immunity would help to rationally design improved tuberculosis vaccines that are urgently required. Given the established central role of CD4 T cells in BCG induced immunity, we sought to characterise the generation of memory CD4 T cell responses to BCG vaccination and M. bovis infection in a murine challenge model.

Mycobacterium bovis-BCG vaccination induces specific pulmonary transcriptome biosignatures in mice.

Background: In the present study, we applied microarray technology to define biosignatures by microarray transcriptome analysis in lung and spleen samples after BCG vaccination and M. bovis infection of BALB/c mice. The aims were two-fold, namely to define biosignatures that could predict vaccine success before challenge, and biomarker patterns that correlated with anamnestic protective responses following exposure to virulent M.

Adjuvants induce distinct immunological phenotypes in a bovine tuberculosis vaccine model.

Tuberculosis (TB) remains one of the most important infectious diseases of humans and animals. Mycobacterium bovis BCG, the only currently available TB vaccine, demonstrates variable levels of efficacy; therefore, a replacement or supplement to BCG is required. Protein subunit vaccines have shown promise but require the use of adjuvants to enhance their immunogenicity.

Development of a Mycobacterium bovis intranasal challenge model in mice.

Tuberculosis caused by infection with Mycobacterium tuberculosis or Mycobacterium bovis remains one of the most important infectious diseases of man and animals. The current vaccine, M. bovis bacille Calmette-Guérin (BCG) demonstrates variable efficacy in humans and cattle, and so an urgent need exists for a new vaccine to replace or supplement BCG.

Screening of highly expressed mycobacterial genes identifies Rv3615c as a useful differential diagnostic antigen for the Mycobacterium tuberculosis complex.

Tuberculous infections caused by mycobacteria, especially tuberculosis of humans and cattle, are important both clinically and economically. Human populations can be vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG), and control measures for cattle involving vaccination are now being actively considered. However, diagnostic tests based on tuberculin cannot distinguish between genuine infection and vaccination with BCG.