A modified scoring system to describe gross pathology in the rabbit model of tuberculosis.

BACKGROUND: The rabbit model is an ideal means to study the pathogenesis of tuberculosis due to its semblance to the disease in humans. We have previously described the results using a bronchoscopic route of infection with live bacilli as a reliable means of generating lung cavities in sensitized rabbits. The role of sensitization in the development of disease outcomes has been well established in several animal models. We have described here the varying gross pathology that result from lack of sensitization with heat-killed M. bovis prior to high-dose bronchoscopic infection with live bacilli. RESULTS: Rabbits lacking sensitization did not generate lung cavities, but instead formed solely a tuberculoid pneumonia that replaced the normal lung parenchyma in the area of infection. Extrapulmonary dissemination was seen in approximately equal frequency and distribution in both rabbit populations. Notable differences include the lack of intestinal lesions in non-sensitized rabbits likely due to the lack of ingestion of expectorated bacilli from cavitary lesions. The experiment also employed a modified scoring system developed initially in the primate model of tuberculosis to allow for the quantification of findings observed at necropsy. CONCLUSIONS: To date, no such scoring system has been employed in the rabbit model to describe gross pathology. The quantitative methodology would allow for rapid comparative analyses and standardization of thoracic and extrapulmonary pathology that could be evaluated for statistical significance. The aim is to use such a scoring system as the foundation for all future rabbit studies describing gross pathology at all stages in TB pathogenesis.

13[C]-urea breath test as a novel point-of-care biomarker for tuberculosis treatment and diagnosis.

BACKGROUND: Pathogen-specific metabolic pathways may be detected by breath tests based on introduction of stable isotopically-labeled substrates and detection of labeled products in exhaled breath using portable infrared spectrometers. METHODOLOGY/PRINCIPAL FINDINGS: We tested whether mycobacterial urease activity could be utilized in such a breath test format as the basis of a novel biomarker and diagnostic for pulmonary TB. Sensitized New-Zealand White Rabbits underwent bronchoscopic infection with either Mycobacterium bovis or Mycobacterium tuberculosis. Rabbits were treated with 25 mg/kg of isoniazid (INH) approximately 2 months after infection when significant cavitary lung pathology was present. [(13)C] urea was instilled directly into the lungs of intubated rabbits at selected time points, exhaled air samples analyzed, and the kinetics of delta(13)CO(2) formation were determined. Samples obtained prior to inoculation served as control samples for background (13)CO(2) conversion in the rabbit model. (13)CO(2), from metabolic conversion of [(13)C]-urea by mycobacterial urease activity, was readily detectable in the exhaled breath of infected rabbits within 15 minutes of administration. Analyses showed a rapid increase in the rate of (13)CO(2) formation both early in disease and prior to treatment with INH. Following INH treatment, all evaluable rabbits showed a decrease in the rate of (13)CO(2) formation. CONCLUSIONS/SIGNIFICANCE: Urea breath testing may provide a useful diagnostic and biomarker assay for tuberculosis and for treatment response. Future work will test specificity for M. tuberculosis using lung-targeted dry powder inhalation formulations, combined with co-administering oral urease inhibitors together with a saturating oral dose of unlabeled urea, which would prevent the delta(13)CO(2) signal from urease-positive gastrointestinal organisms.

Extrapulmonary dissemination of Mycobacterium bovis but not Mycobacterium tuberculosis in a bronchoscopic rabbit model of cavitary tuberculosis.

The rabbit model of tuberculosis is attractive because of its pathophysiologic resemblance to the disease in humans. Rabbits are naturally resistant to infection but may manifest cavitary lung lesions. We describe here a novel approach that utilizes presensitization and bronchoscopic inoculation to reliably produce cavities in the rabbit model. With a fixed inoculum of bacilli, we were able to reproducibly generate cavities by using Mycobacterium bovis Ravenel, M. bovis AF2122, M. bovis BCG, M. tuberculosis H37Rv, M. tuberculosis CDC1551, and the M. tuberculosis CDC1551 DeltasigC mutant. M. bovis infections generated cavitary CFU counts of 10(6) to 10(9) bacilli, while non-M. bovis species and BCG yielded CFU counts that ranged from 10(4) to 10(8) bacilli. Extrapulmonary dissemination was almost exclusively noted among rabbits infected with M. bovis Ravenel and AF2122. Though all of the species yielded secondary lesions at intrapulmonary sites, M. bovis infections led to the most apparent gross pathology. Using the M. tuberculosis icl and dosR gene expression patterns as molecular sentinels, we demonstrated that both the cavity wall and cavity lumen are microenvironments associated with hypoxia, upregulation of the bacterial dormancy program, and the use of host lipids for bacterial catabolism. This unique cavitary model provides a reliable animal model to study cavity pathogenesis and extrapulmonary dissemination.