Reactivation of latent tuberculosis: variations on the Cornell murine model

Abstract

Mycobacterium tuberculosis causes active tuberculosis in only a small percentage of infected persons. In most cases, the infection is clinically latent, although immunosuppression can cause reactivation of a latent M. tuberculosis infection. Surprisingly little is known about the biology of the bacterium or the host during latency, and experimental studies on latent tuberculosis suffer from a lack of appropriate animal models. The Cornell model is a historical murine model of latent tuberculosis, in which mice infected with M. tuberculosis are treated with antibiotics (isoniazid and pyrazinamide), resulting in no detectable bacilli by organ culture. Reactivation of infection during this culture-negative state occurred spontaneously and following immunosuppression. In the present study, three variants of the Cornell model were evaluated for their utility in studies of latent and reactivated tuberculosis. The antibiotic regimen, inoculating dose, and antibiotic-free rest period prior to immunosuppression were varied. A variety of immunosuppressive agents, based on immunologic factors known to be important to control of acute infection, were used in attempts to reactivate the infection. Although reactivation of latent infection was observed in all three variants, these models were associated with characteristics that limit their experimental utility, including spontaneous reactivation, difficulties in inducing reactivation, and the generation of altered bacilli. The results from these studies demonstrate that the outcome of Cornell model-based studies depends critically upon the parameters used to establish the model.

FIG. 1

Drug-induced model of latent tuberculosis (variant A of the Cornell model). Mice were infected i.v. with 5 × 10³ CFU of M. tuberculosis for 4 weeks, and the infection was then treated with a 4-week course of the antimycobacterial drugs INH (0.1 g/liter) and PZA (15 g/liter) delivered in drinking water. After the treatment period, mice were maintained on plain water. Numbers of viable bacilli were determined at the indicated times by plating homogenates of spleen (squares) and lungs (circles) and counting colonies 21 days later. The dashed line denotes the limit of detection for the assay (80 CFU). Each point represents the mean for two to four mice; error bars show standard errors.

FIG. 2

Reactivation of infection in lungs of mice by immunosuppression in variant A of the Cornell model. Mice were infected with 5 × 10³ CFU of M. tuberculosis for 4 weeks and then treated for 4 weeks with INH-PZA. (A) Following the antibiotic regimen, IFN-γ was neutralized in vivo by using monoclonal anti-murine IFN-γ (squares) while control mice (diamonds) received normal rat IgG₁. (B) Following the antibiotic regimen, mice were treated with dexamethasone (squares) or with PBS (diamonds). For both panels, mice were sacrificed at various time points, and the number of viable bacilli in the lungs was determined by plating serial dilutions of lung homogenates onto 7H10 agar and counting colonies after 21 days at 37°C. The dashed line denotes the limit of detection for the assay (80 CFU). Each point represents the mean for three to four mice; error bars show standard errors. ∗, P ≤ 0.05 by Wilcoxon rank sum test.

FIG. 3

L-NIL inhibits nitric oxide production in vitro and exacerbates acute M. tuberculosis infection. (A) IFN-γ-primed J774 cells were treated with dilutions of L-NIL (diamonds), aminoguanidine (squares), or α-t-butoxycarbonyl–lysine, the precursor for L-NIL synthesis (circles), for 4 h prior to activation with lipopolysaccharide (1 μg/ml). Nitrite levels in the medium were determined 24 h later by Griess assay. (B) Mice were infected i.v. with 5.5 × 10⁵ CFU of M. tuberculosis and then given 2.5% (wt/vol) aminoguanidine (solid bars), 4 mM L-NIL (stippled bars), or 9 mM L-NIL (hatched bars) ad libitum in drinking water or plain water (open bars). Mice were sacrificed 21 days later, and the numbers of viable bacilli were determined by plating serial dilutions of organ and counting colonies after 21 days at 37°C. Each bar represents the mean for three mice; error bars show standard errors.

FIG. 4

In vivo neutralization of TNF-α with MP6-XT22 exacerbates acute murine tuberculosis. Mice were infected i.v. with 2 × 10⁵ CFU of M. tuberculosis 1 day after being injected i.p. with MP6-XT22 or normal rat IgG₁. After 17 days of infection, numbers of viable bacteria were determined in lungs of mice that received 0.5 mg of MP6-XT22 i.p. twice weekly (shaded bar), 1.0 mg of MP6-XT22 i.p. weekly (solid bar), or 0.5 mg of normal rat IgG₁ i.p. twice weekly (open bar). Each bar represents the mean for three to four mice; error bars show standard errors.