Abstract
BackgroundIt has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models.Methodology/Principal FindingsBy testing three M. tuberculosis H37Rv samples, one deletion mutant, and one recent clinical isolate for survival by the quantitative organ CFU counting method in mouse or guinea pig aerosol or intravenous infection models, we could discern no increase in bacterial fitness as a result of passaging of M. tuberculosis strains in mice prior to quantitative virulence testing in two animal models. Surface lipid expression as assessed by neutral red staining and thin-layer chromatography for PDIM analysis also failed to identify virulence correlates.Conclusions/SignificanceThese results indicate that animal passaging of M. tuberculosis strains prior to quantitative virulence testing in mouse or guinea pig models does not enhance or restore potency to strains that may have lost virulence due to in vitro passaging. It is critical to verify virulence of parental strains before genetic manipulations are undertaken and comparisons are made.
Highlights
Serial in vitro passaging of pathogenic bacteria and viruses is a classical method for obtaining attenuated strains that can be used either as vaccines or as safe laboratory model strains to study basic functions of the pathogen [1]
Examples in tuberculosis include the Bacille Calmette-Guerin (BCG) vaccine strain and H37Ra obtained by intentional serial culture of virulent Mycobacterium bovis and the variably virulent M. tuberculosis H37 strain, respectively [2,3]
Anti-toxin therapy, for which von Behring received the first Nobel Prize ever awarded in Medicine in 1901, was rapidly recognized to have no likelihood of success against TB as it had had against diphtheria
Summary
Serial in vitro passaging of pathogenic bacteria and viruses is a classical method for obtaining attenuated strains that can be used either as vaccines or as safe laboratory model strains to study basic functions of the pathogen [1]. Seeking a vaccine against cattle tuberculosis in order to prevent transmission of bovine bacilli through the digestive tract, which he proposed to nearly always occur in childhood and to be a prerequisite for adult pulmonary disease [6], von Behring reported in his Nobel Lecture [11] that tubercle bacilli derived from human sputum were not ‘‘unharmful’’ for cows but that they lost their virulence ‘‘through long-continued culture [6.5 years [2]] in the laboratory.’’ These bacilli could be restored to ‘‘considerable virulence in cattle’’ after passage through goats on his Marburg farm [5,11]. It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models
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