The Impact of Mouse Passaging of Mycobacterium tuberculosis Strains prior to Virulence Testing in the Mouse and Guinea Pig Aerosol Models

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.By 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.These 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

Smaller cardiac cell size and reduced extra-cellular collagen might be beneficial for hearts of Ames dwarf mice

Purpose: To test the hypothesis that cardiac morphologic differences between Ames dwarf and wild-type littermates might correlate with the increased longevity observed in the Ames dwarf mice.Methods: Hearts removed from young adult (5-7 mo) and old (24-28 mo) Ames dwarf and wild-type littermates underwent histological and morphometric analysis. Measurements of cell size, nuclear size, and collagen content were made using computerized color deconvolution and particle analysis methodology.Results: In the young mice at six months of age, mean cardiomyocyte area was 46% less in Ames dwarf than in wild-type mice (p&#60;0.0001). Cardiomyocyte size increased with age by about 52% in the wild-type mice and 44% in the Ames dwarf mice (p&#60;0.001). There was no difference in nuclear size of the cardiomyocytes between the young adult wild-type and Ames dwarf mice. There was an age-associated increase in the cardiomyocyte nuclear size by approximately 50% in both the Ames and wild-type mice (p&#60;0.001). The older Ames dwarf mice had slightly larger cardiomyocyte nuclei compared to wild-type (2%, p&#60;0.05). The collagen content of the hearts in young adult Ames dwarf mice was estimated to be 57% less compared to wild-type littermates (p&#60;0.05). Although collagen content of both Ames dwarf and wild-type mouse hearts increased with age, there was no significant difference at 24 months.Conclusions: In wild-type and Ames dwarf mice, nuclear size, cardiomyocyte size, and collagen content increased with advancing age. While cardiomyocyte size was much reduced in young and old Ames dwarf mice compared with wild-type, collagen content was reduced only in the young adult mice. Taken together, these findings suggest that Ames dwarf mice may receive some longevity benefit from the reduced cardiomyocyte cell size and a period of reduced collagen content in the heart during adulthood.</p

Differences in the Growth of Paired Ugandan Isolates of Mycobacterium tuberculosis within Human Mononuclear Phagocytes Correlate with Epidemiological Evidence of Strain Virulence

Previous studies have suggested that isolates of Mycobacterium tuberculosis responsible for tuberculosis outbreaks grow more rapidly within human mononuclear phagocytes than do other isolates. Clinical scenarios suggesting virulence of specific M. tuberculosis isolates are readily identified. Determination of appropriate “control” isolates for these studies is more problematic, but equally important for validating these assays and, ultimately, for identifying biologic differences between M. tuberculosis strains that contribute to virulence. We utilized the database from a study of Ugandan tuberculosis patients and their household (HH) contacts to identify M. tuberculosis isolates transmitted within HH and nontransmitted control isolates. Isolate pairs were evaluated from matched HH in each of three clinical scenarios: (i) coprevalent disease and no disease, (ii) incident disease and no disease, and (iii) M. tuberculosis infection (purified protein derivative [PPD] positive) and no infection (PPD negative). Intracellular growth of paired organisms was determined in a blinded fashion using two models of intracellular infection in which we have previously demonstrated correlation between intracellular growth and strain virulence, primary human monocytes (MN) and THP-1 human macrophage-like cells. In both models, transmitted isolates from coprevalent disease HH displayed more rapid growth than nontransmitted control isolates. In the THP-1 model, this was also true of transmitted isolates from HH with incident disease and their controls. Differences in production of tumor necrosis factor alpha and interleukin-10 by matched isolates showed correlation with growth patterns in the THP-1 cells but not in MN. Paired isolates characterized in this manner may be of particular interest for further investigations of the virulence of M. tuberculosis

A. Multiplication of mouse-passaged and -unpassaged <i>M. tuberculosis</i> clinical isolate in BALB/c mice.

<p>A. Growth in lungs of the isolate following intravenous infection. B. Growth in lungs following aerosol infection of mouse-passaged clinical isolate in comparison with regularly mouse-passaged H37Rv strain (JHU) in BALB/c mice. Errors are SD.</p