Immunological responses following experimental endobronchial infection of badgers (Meles meles) with different doses of Mycobacterium bovis.

The Eurasian badger (Meles meles) is a wildlife reservoir for Mycobacterium bovis infection in Ireland and Great Britain and has been implicated in the transmission of tuberculosis to cattle. Vaccination of badgers is an option that could be used as part of a strategy to control the disease. In this study we used an endobronchial infection procedure to inoculate groups of badgers with three different doses (3x10(3), 2x10(2) and <10 Colony Forming Units (CFUs)) of M. bovis. After 17 weeks the disease status of each animal was determined by post-mortem pathology and culture for M. bovis. Each of the inoculum doses resulted in establishment of infection in the badgers. The cell-mediated immune (CMI) responses were measured by lymphocyte transformation assay (LTA) of peripheral blood mononuclear cells (PBMCs) cultured with bovine tuberculin (PPD-B). In each infected group the CMI responses increased with a kinetic profile corresponding to the delivered dose and the post-mortem pathology. The serological responses were measured by ELISA and a multi-antigen print immunoassay (MAPIA) in order to investigate any changes in the antigenic repertoire associated with different infective doses. In contrast to the CMI responses, the ELISA and MAPIA showed that the recognition of antigens by the badgers was intermittent and not strongly influenced by the dose of M. bovis.

TB vaccines for the World.

In September 2003, Montreal hosted the first international conference on "TB Vaccines for the World". The timing of the conference was prescient. Two major contracts for screening TB vaccines, the NIH vaccine screening contract and the European Union Fifth Framework TB Vaccine Cluster were coming to an end as was a major 5 year international initiative to develop vaccines against bovine tuberculosis. For the first time the TB vaccine community was getting a glimpse of the most promising vaccine candidates identified using a number of different animal models. Moreover, the first Phase I human trial of a new vaccine for TB based on boosting BCG with an attenuated vaccinia virus expressing Ag85A of M. tuberculosis, MVA85A, had just begun. In addition, the Aeras Global TB Vaccine Foundation had just been launched to apply an industrial pipeline approach to drive vaccine candidates through to clinical trials. The aim of this special edition of Tuberculosis is to encapsulate this defining moment in the development of TB vaccines so that it may be used it as a yardstick against which future progress may be measured. This article provides an overview of the scope of this task.

Cattle as a model for development of vaccines against human tuberculosis.

The identification of tuberculosis vaccines and vaccination strategies, which in small animal models appear to be more effective than BCG, offer some exciting possibilities for control of human tuberculosis in the future. However, some major problems remain including selecting which vaccines should go into human trials and the length of time it will take for testing these vaccines in humans. The cattle model is well suited for the secondary screening of tuberculosis vaccines as there is a strong similarity between the disease in cattle and humans and outbred animals are used. Moreover, there are many similarities in the results from field trials of BCG in both cattle and humans, with BCG often failing to protect when the trials are extended over a number of years. In addition, calves, like human infants, are immunocompetent at birth. Recent studies in calves have shown that BCG vaccination of calves within hours of birth is highly effective in protecting animals against bovine tuberculosis, but BCG revaccination at 6 weeks of age is contraindicated. Prime boost vaccination strategies using BCG and DNA vaccines have provided evidence that these combinations may give better protection in calves than either vaccine alone. Based on antigens whose genes are absent from the BCG genome, advances have also been made to develop diagnostic reagents distinguishing infected and vaccinated animals (differential diagnosis). The cattle model has been particularly useful in prioritizing such antigens for testing in humans. Finally, there is an urgent need to identify an immunological correlate of protection against tuberculosis. The cattle model can be particularly helpful in this area as it is relatively easy to collect large volumes of blood from calves at intervals following vaccination and challenge, and a large number of immunological reagents are now available for cattle.

Evaluation of variable number tandem repeat (VNTR) loci in molecular typing of Mycobacterium bovis isolates from Ireland.

Various sets of short tandem repeats such as the exact tandem repeats (ETRs), mycobacterial interspersed repetitive units (MIRUs) and variable number tandem repeat (VNTR) loci, have recently been described as effective tools in strain typing M. tuberculosis complex isolates, representative of global diversity. This study extends our previous study, evaluating the discrimination of a further 17 MIRU_VNTR loci individually and comparing the resolution of published VNTR sets and spoligotyping using a panel of 47 local M. bovis field isolates, including known epidemiologically linked isolates and 9 M. tuberculosis complex reference isolates. Individual loci differed greatly in their discrimination. The discriminatory capacity of novel combinations of the most discriminating VNTR loci was also assessed. In the panel of 47 M. bovis isolates, 17 unique profiles were resolved using VNTR set 1, whilst the MIRUs and ETRs resolved the panel into 11 and 6 profiles, respectively. A novel combination of 10 highly discriminatory VNTRs was determined, which resolved 30 unique profiles. The configuration of a multi-locus VNTR-based assay and its ability to provide a flexible, convenient and high-resolution genotyping method is discussed. We suggest a panel of VNTR markers which may be widely suitable for molecular epidemiological studies of M. bovis. However, the number and combination of informative VNTR markers selected needs to be determined empirically with reference to locally prevalent strains and will depend on the epidemiological study requirements.

Vaccination of cattle with Mycobacterium bovis BCG by a combination of systemic and oral routes.

Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine delivered to calves by the subcutaneous (s.c.) or by the oral route in a formulated lipid matrix has been previously shown to induce similar levels of protection against bovine tuberculosis. The current study was aimed at determining whether a combination of delivering BCG by s.c. and oral routes would enhance levels of protection, compared to only one route of vaccination. Forty calves were randomly divided into four groups (10/group). Calves were vaccinated with 10(6)colony forming units (CFU) of BCG Pasteur by the s.c. route or orally with 10(9)CFU BCG incorporated into a lipid formulation. One group received a combination of BCG administered by both the s.c. and oral routes and a non-vaccinated group served as a control. The two groups of calves that received s.c. BCG produced strong IFN-gamma responses in whole blood cultures stimulated with bovine purified protein derivative (PPD) 3 weeks after vaccination. Cattle vaccinated just with oral BCG in a lipid matrix produced a strong IFN-gamma response 8 weeks after vaccination, and peaking at 11 weeks after vaccination. All calves were challenged by the intratracheal route with M. bovis 15 weeks after vaccination and were euthanized and necropsied to assess protection at 17 weeks following challenge. BCG given s.c. or orally induced significant and comparable levels of protection against the virulent challenge. Vaccination of cattle by a combination of s.c./oral routes did not enhance protection beyond that achieved by s.c. or oral vaccination alone. We conclude that vaccination of cattle with BCG by a combination of routes has no beneficial additive effects, compared to a single s.c. administration of BCG or BCG given orally in a lipid formulation.

Simple objective measurement of the cutaneous delayed-type hypersensitivity reaction to tuberculin using spectrophotometry.

BACKGROUND/AIMS: A number of subjective methods have been used to quantify the extent of the cutaneous delayed-type hypersensitivity (DTH) reaction. However, because of their subjective nature, significant differences in measurements may be seen between individual observers or laboratories unless thorough training is given to each observer. METHODS: Objective measurement of the DTH reaction using a hand-held spectrophotometer is described. Guinea pigs were primed using inoculation with Mycobacterium bovis Bacille Calmette-Guerin and challenged five weeks later in the shaved flank with three doses of bovine purified protein derivative. The extent of the ensuing DTH reaction was measured 24 and 48 h later. Spectrophotometric measurement of the reaction site was compared with a control region of skin on each animal and expressed as the change within a standard colour space. Data obtained with the spectrophotometer was compared with the subjective measurement of the area of the DTH reaction by an experienced operator. RESULTS: The measurements obtained with the spectrophotometer correlated very closely with conventional measurement of the reaction area by a trained operator. The reaction size in square mm and changes along the red/green colour axis was correlated most strongly. CONCLUSION: Spectrophotometric measurement of the DTH reaction had advantages over conventional measuring techniques in terms of speed, reproducibility and reduced operator to operator variation. We conclude that the cutaneous DTH reaction may be simply and objectively quantified with the use of a hand-held spectrophotometer.

A single dose of killed Mycobacterium bovis BCG in a novel class of adjuvant (Novasome) protects guinea pigs from lethal tuberculosis.

The only vaccine currently available for the prevention of tuberculosis in man is a live attenuated vaccine, bacille Calmette-Guerin (BCG), derived from Mycobacterium bovis. Concerns over the lack of the universal efficacy and safety of BCG have resulted in efforts to develop a new generation of TB vaccines. Historically, killed whole-cell preparations of mycobacteria have been ineffective vaccines. We revisited the potential of killed whole-cell vaccines by comparing their efficacy with live BCG Pasteur in a guinea pig challenge model. BCG Pasteur was inactivated with a low concentration of formalin and showed to be non-viable in culture or severe combined immunodeficient mice. Formalin-inactivated BCG was mixed with non-phospholipid liposome adjuvants (Novasomes) and administered to guinea pigs as a single subcutaneous inoculation. All formulations were well tolerated and one conferred a significant survival advantage against lethal aerogenic challenge with M. bovis.