An assay to compare the infectivity of Mycobacterium tuberculosis isolates based on aerosol infection of guinea pigs and assessment of bacteriology.

The aim of this study was to establish an assay to compare Mycobacterium tuberculosis strains, and cells grown under different growth conditions, in terms of their ability to cause a lung infection and disseminate to the spleen. M. tuberculosis strains H37Rv, Erdman, South Indian (TMC120, SI) and H37Rv cells grown aerobically or under low oxygen/iron limitation in a chemostat were assayed for infectivity. Groups of 8 animals were challenged with 3 different doses of each strain. Lung and spleen bacteriology was assessed at 16 days post-infection for all strains. Bacteriology and lung pathology at day 56 was studied for H37Rv, Erdman and SI. Strains H37Rv and Erdman had a statistically significantly higher pathogenic potential than SI and this was confirmed by analysis of lung pathology performed at 8 weeks post-infection, although the Erdman strain caused more extensive caseation without calcification and little encapsulation. The model could discriminate between cells grown under different growth conditions; low-oxygen/iron-limited cells had a significantly higher infectivity than those grown aerobically. This study presents a quick and reliable method for comparing with statistical confidence, the pathogenic potential of M. tuberculosis strains and the impact of in vitro growth conditions on the infectivity of M. tuberculosis in vivo.

Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis.

The TB Vaccine Cluster project funded by the EU Fifth Framework programme aims to provide novel vaccines against tuberculosis that are suitable for evaluation in humans. This paper describes the studies of the protective efficacy of vaccines in a guinea pig aerosol-infection model of primary tuberculosis. The objective was to conduct comparative evaluations of vaccines that had previously demonstrated efficacy in other animal models. Groups of 6 guinea pigs were immunized with vaccines provided by the relevant EU Vaccine Cluster partners. Survival over 17 or 26 weeks was used as the principal measure of vaccine efficacy following aerosol challenge with H37Rv. Counts of mycobacteria in lungs and spleens, and histopathological changes in the lungs, were also used to provide evidence of protection. A total of 24 vaccines were evaluated in 4 experiments each of a different design. A heterologous prime-boost strategy of DNA and MVA, each expressing Ag85A and a fusion protein of ESAT-6 and Ag85B in adjuvant, protected the guinea pigs to the same extent as BCG. Genetically modified BCG vaccines and boosted BCG strategies also protected guinea pigs to the same extent as BCG but not statistically significantly better. A relatively high aerosol-challenge dose and evaluation over a protracted time post-challenge allowed superior protection over BCG to be demonstrated by BCG boosted with MVA and fowl pox vectors expressing Ag85A.

Proteolytic inactivation of the bovine spongiform encephalopathy agent.

Thermostable proteases have been investigated for their ability to provide a novel biological solution to decontamination of prion agents responsible for transmissible spongiform encephalopathies (TSEs). Proteases were identified that digested total mouse brain homogenate (MBH) protein from uninfected mice. These proteases were then evaluated for digestion of BSE (301V) infectious MBH over a range of pH and temperatures, screened for loss of anti-prion antibody 6H4 immunoreactivity and protease-treated infectious MBH assessed in mouse bioassay using VM mice. Despite a number of proteases eliminating all 6H4-immunoreactive material, only the subtilisin-enzyme Properase showed a significant extension in incubation period in mouse bioassays following a 30-min incubation at 60 degrees C and pH 12. These results demonstrate the potential of the method to provide a practical solution to the problems of TSE contamination of surgical instruments and highlight the inadequacy of using Western blot for assessment of decontamination/inactivation of TSE agents.

Experimental aerogenic Burkholderia mallei (glanders) infection in the BALB/c mouse.

The object of this study was to develop and characterize experimental Burkholderia mallei aerosol infection in BALB/c mice. Sixty-five mice were infected with 5000 [approx. 2.5 median lethal doses (MLD)] B. mallei strain ATCC 23344(T) bacteria by the aerosol route. Bacterial counts within lung, liver, spleen, brain, kidney and blood over 14 days were determined and histopathological and immunocytochemical profiles were assessed. Mortality due to B. mallei infection occurred between days 4 and 10 post-infection. Bacterial numbers were consistently higher in the lungs than in other tissues, reaching a maximum of approximately 1.0 x 10(6) c.f.u. ml(-1) at 5 days post-infection. Bacterial counts in liver and spleen tissue remained approximately equal, reaching a maximum of approximately 1.0 x 10(4) c.f.u. ml(-1) at day 4 post-infection. By day 14 post-infection, bacterial counts were in the range 1.0 x 10(3)-1.0 x 10(4) c.f.u. ml(-1) for all tissues. Infection of the lungs by B. mallei resulted in foci of acute inflammation and necrosis. As infection progressed, the inflammatory process became subacute or chronic; this was associated with the development of extensive consolidation. Lesions in liver and spleen tissue were typical of those that might be expected in bacteraemia or bacterial toxaemia. These results suggest that the BALB/c mouse is susceptible to B. mallei when delivered by the aerosol route and that this represents a model system of acute human glanders that is suitable for research into the pathogenesis of and vaccines against this disease.