to show significant necrosis and thus hypoxia in lung lesions [19– 21]. Therefore, the addition of a secondary animal model showing a broad range of pathological features might be necessary at later stages of TB drug development to assess drug activity against persisting bacteria in necrotic lesions. Guinea pigs infected with M. tuberculosis show greater similarities to natural infections in humans, as these animals show hypoxia, necrosis and calcification [21–25]. Recently, more attention has been brought to the importance of comparative pathology and pathogenesis in different animal models [21,26], yet little information is available regarding the actual location of the bacilli in these pulmonary lesions across animal models. There are at least two reasons that may explain the lack of this information. Many animal models showing these advanced lesion types, such as the non-human primates and the current rabbit model, show only low bacterial numbers per lesion which makes visualization in tissue sections very difficult [21,27,28]. In addition, the staining methods used by most are labor-intensive and time consuming especially when numerous organ sections from multiple animals require microscopic evaluation. To visualize M. tuberculosis either in tissues or in sputum smears, the detection method most commonly employed is the Ziehl- Neelsen (ZN) acid-fast stain [29–35]. Fluorescence is much preferred over brightfield microscopy because it provides greater sensitivity at a lower scanning magnification, yielding more consistent results with less user-fatigue [36–38]. Among the various acid-fast, fluorochrome staining reagents used in TB detection, the most common are auramine O and the auramine- rhodamine (AR) combination. A comprehensive review on the comparison of the AR and ZN staining techniques to detect M. tuberculosis in sputum smears demonstrated that both techniques showed similar specificity, but the AR technique was found to be more sensitive and facilitated easier microscopic evaluations versus the ZN stain [39]. One drawback of using fluorescent acid-fast stains is that tissue architecture is obscured due to the counterstain that darkens all but the bacilli. Only a few reports describe the use of AR in combination with other tissue stains on tissue samples [40–42]. In this study, a combination staining method was developed to not only identify M. tuberculosis bacilli, but to also navigate through tissue histology. This method enables bacilli to be accurately located within tissue sections. In an earlier study, we described that treatment of guinea pigs with the experimental drug TMC207 almost completely eradicated bacteria throughout the lesions, and that some remnants of acid fast bacilli remaining after treatment were observed to be extracellular, in the acellular rim of primary necrotic lesions by the Ziehl-Neelsen method [23]. In this study a thorough, in-depth comparison of the bacterial location in several animal models was performed during the progression of disease as well as after drug treatment using the newly developed combination staining method. To date, the activity of novel compounds in vivo is generally measured by the reduction in colony forming units (CFU) from organ homogenates of infected animals with little knowledge relating to pathology, location of the bacilli at the start and during drug treatment, and the effect of drug activity in different locations in the lung. To address these three aspects, we studied the different standard animal models used in our laboratory for evaluation of experimental compounds against tuberculosis ; a short term M. tuberculosis infection model using immunocompromised IFN-c gene knockout mice [43], the immunocompetent C57BL/6 mouse model [44] and the guinea pig model [23,45]. All M. tuberculosis infections were performed by low dose aerosol infection (using an initial inoculum of 30–100 CFU in the lungs of the animals). The ultimate goals of this study were: 1) to understand the pathogenesis Visualization of M. tuberculosis in Animal Models across the animal models in terms of lung lesion pathology and to record the location of the bacilli, 2) to gain information about drug effects on the pathology in the different animal models, and 3) to study the location of the persistent bacterial population remaining after drug treatment in the lung. A better understanding of the animal models used in TB drug development is critical in delineating the in vivo activity of an experimental compound against certain populations of M. tuberculosis bacteria based on their location. In addition, this will ensure the use of the appropriate animal model for the evaluation of new drug classes. Results Drug efficacy in immunocompetent vs. immunocompromised mice The efficacy of several clinically used TB drugs was evaluated in our standard C57BL/6 versus GKO mouse model infected via a low dose aerosol of M. tuberculosis (Fig. 1). At the start of treatment, the bacterial load in the lungs reached ,7.0 log10CFU in C57BL6 mice (at 21 days after aerosol infection) and 6.2 log10CFU in GKO mice (at 18 days after aerosol). At the completion of the study (at 28 days after aerosol infection), the bacterial load in the untreated control group remained largely unchanged for C57BL/6 mice (P.0.05), whereas the bacillary burden in GKO increased by more than 1 log10CFU (Fig. 1). The activities of the tested drugs were evaluated over 7 days with sacrifice point after 2, 5 and 7 days of treatment. INH reduced the bacterial load only slightly over 7 days in the lungs of the C57BL/6 mice (0.25 log10CFU reduction), whereas in the immunocompromised GKO mice, INH showed significant activity mainly over the first 5 days of treatment (0.85 log10CFU reduction) (P,0.05) (Fig. 1). The difference in drug activity between the two mouse strains was even more pronounced with MXF treatment at 100 mg/kg, which reduced the bacterial load in C57BL/6 mice by 0.85 log10CFU and by more than 3.5 log10CFU in GKO mice after 7 days of treatment when compared to untreated mice at the start of treatment (P,0.05). Intra- and extracellular stages of M. tuberculosis in lung lesions of GKO mice Lung sections from GKO mice were studied for the location of the bacilli after M. tuberculosis infection. The bacilli were visualized in lung sections via a modified staining method combining the auramine O and rhodamine B staining (AR) for acid fast bacteria, hematoxylin QS for staining tissue and DAPI for staining nuclei. At 18 days post aerosol infection, which is the start of treatment of our standard laboratory protocol for this model [43], the lung tissue sections mainly contained inflammatory lesions consisting primarily of macrophage (mW) and granulocyte (GC) cell accumulations arranged in an unorganized manner (Fig. 2A), as also earlier described [46–48]. Auramine-rhodamine positive (AR+) bacilli were found uniformly distributed across these inflammatory lesions (estimated at about 85–90%) (Fig. 2B), whereas a minority was found in and around airways, blood vessels and randomly dispersed throughout non-inflamed lung parenchy- ma. AR+ bacilli were predominantly intracellular during this early stage of infection within various mono- and multinucleated mW cell types (Fig. 2C, D). At a later stage (20 to 22 days post aerosol infection), there was a loss of alveolar septal architecture due to progressive inflammation and individual cell and lesion necrosis. It is at this stage that the AR+ bacillary population changes from being exclusively intracellular to becoming increasingly extracel- lular within the areas of necrosis. By 28 days post aerosol infection, which is about one week before mice would succumb to disease PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e17550