Differential virulence and disease progression following Mycobacterium tuberculosis complex infection of the common marmoset (Callithrix jacchus)

Abstract

Existing small-animal models of tuberculosis (TB) rarely develop cavitary disease, limiting their value for assessing the biology and dynamics of this highly important feature of human disease. To develop a smaller primate model with pathology similar to that seen in humans, we experimentally infected the common marmoset (Callithrix jacchus) with diverse strains of Mycobacterium tuberculosis of various pathogenic potentials. These included recent isolates of the modern Beijing lineage, the Euro-American X lineage, and M. africanum. All three strains produced fulminant disease in this animal with a spectrum of progression rates and clinical sequelae that could be monitored in real time using 2-deoxy-2-[(18)F]fluoro-d-glucose (FDG) positron emission tomography (PET)/computed tomography (CT). Lesion pathology at sacrifice revealed the entire spectrum of lesions observed in human TB patients. The three strains produced different rates of progression to disease, various extents of extrapulmonary dissemination, and various degrees of cavitation. The majority of live births in this species are twins, and comparison of results from siblings with different infecting strains allowed us to establish that the infection was highly reproducible and that the differential virulence of strains was not simply host variation. Quantitative assessment of disease burden by FDG-PET/CT provided an accurate reflection of the pathology findings at necropsy. These results suggest that the marmoset offers an attractive small-animal model of human disease that recapitulates both the complex pathology and spectrum of disease observed in humans infected with various M. tuberculosis strain clades.

Fig 1

Marmosets infected with different M. tuberculosis complex strains had different patterns of infection-induced weight loss and time to disease progression. (A) Six marmosets were infected by aerosol exposure to each M. tuberculosis complex strain; within each group, twin animals share the same color to indicate the distribution among the infection groups. (B) All infected animals lost weight, but those infected with Beijing strain K04 lost weight more rapidly (measured by comparing the differences in slope by one-way ANOVA; P = 0.0009) with accelerated loss in the K04 high-dose group. There was similar weight loss (assessed by slope) in animals infected with the high and low doses of CDC 1551 and N0091. Open symbols indicate the high-dose groups, and closed symbols indicate the low-dose group. (C) The median number of days to disease progression for the M. tuberculosis Beijing K04 group was 36.5 days, compared to 59 and 57 days for CDC 1551- and N0091-infected groups, respectively (P = 0.0001 by Mantel-Cox test). Exposure to the high-dose aerosols decreased survival within each strain, much more so when the strain was K04.

Fig 2

Dense, diseased lung volume increased more rapidly in marmosets infected with the M. tuberculosis Beijing strain K04. (A) The total 3D lung volume of the serial CT scans was summed by binning volumes spanning 50 HU and graphed to determine the HU changes in the lung over the course of the infection. Most notably, the peak around 50 HU captures the increase in lung density as the granulomas form. (B) The increase in abnormal lung density (>−400 HU) for each group of NHP increases with time p.i. (n = 6). In the Beijing strain K04-infected group, the disease volume increases significantly more rapidly (comparison of slopes by one-way ANOVA; P < 0.01) than in the CDC 1551-infected group or M. africanum-infected group. The slope of disease volume increase was not different between the two doses within each infection group, so the data are presented in aggregate. In addition, a slight increase in abnormal lung density in the animals given the higher infectious dose of K04 was observed (data not shown). The differences in mean lesion volume were significant at 4 weeks p.i. for K04 compared to N0091 and CDC 1551 and at 6 and 8 weeks between N0091 and CDC 1551 (P < 0.05). (C) Comparison of the dense lung volume observed in marmosets 4 weeks postinfection and the time to signs of clinical illness.

Fig 3

Lesions show increased FDG-PET uptake during disease development and progression. The images show serial 3-dimensional projections of the anterior aspect of the chest of a single representative animal infected with a low dose of M. tuberculosis Beijing strain K04 (A), M. tuberculosis CDC 1551 (B), or M. africanum N0091 (C) scanned 4 or more times (labeled in days at the top). The blue signal forming the body is derived from the CT scan, with increased light blue intensity indicating tissue with a higher HU. All images were projected with a common [¹⁸F]FGD uptake scale, indicated at the bottom right. The heart (H) had variable [¹⁸F]FGD uptake and is marked in each scan where it was avid; labeling of the heart is variable even with fasted animals. G indicates the gallbladder position if [¹⁸F]FGD avid. [¹⁸F]FGD uptake in the lung was minimal prior to infection (0) and at 2 weeks, but as lesions expanded, focal areas of [¹⁸F]FGD activity appeared. [¹⁸F]FGD uptake in the lymph nodes (LN) was observed as early as 2 to 4 weeks in Beijing K04- and M. africanum-infected animals and increased over time (arrowheads in panels B and C). The FDG uptake of the CDC 1551-infected lungs showed more heterogeneity than that of the other strains, with some early lesions having very low uptake (arrows in panel B). As lesions became larger, the [¹⁸F]FGD uptake of the central part of the lesion often was lower. (D) The increase in [¹⁸F]FGD uptake, or total glycolytic activity of the lung and LNs for each animal, was determined, and the group means over time were determined. The K04-infected animals had higher glycolytic activity than either the CDC 1551- or N0091-infected animals (P = 0.0005 by one-way ANOVA). The increases in glycolytic activity for the K04 high- and low-dose groups were different and are shown separately but did not reach significance. (E) The increase in glycolytic activity of the lung correlated with the diseased lung volume (P = 0.004; r² = 0.44) as measured by CT.

Fig 4

Marmosets infected with the M. tuberculosis Beijing strain K04 had higher bacterial loads in all organs assayed. Open symbols indicate samples from the high-dose groups, and closed symbols indicate those from the low-dose groups. (A) Bacterial burden/g of the lung (right middle lobe) and grossly normal lung from marmosets infected with the M. tuberculosis Beijing strain K04 was 1.5 to 2 logs higher than those from lung harboring CDC 1551 or the M. africanum strain N0091 (P < 0.0001 by ANOVA), but there was little difference in CFU between the high and low infection doses. In excised small nodular lesions, K04-infected animals carried CFU burdens that were 1.3 and 2.1 logs higher than that of CDC 1551 or N0091, respectively (P < 0.001 by ANOVA). CDC 1551 normal lung and isolated lesions also had a significantly greater bacteria load than the samples from M. africanum (P < 0.05). (B) Dissemination of the bacteria from the lung occurred rapidly, with both liver and spleen having cultivatable M. tuberculosis within 1 month. The extrapulmonary burden increased with time, so that animals receiving the low-dose aerosol of K04 (which survived longer) had significantly higher CFU (P = 0.008 by t test); otherwise, differences in CFU related to the dose of bacteria administered were not significant. The bacterial burden of whole liver, spleen, and lymph nodes/g from marmosets infected with the M. tuberculosis Beijing strain K04 was higher than that of animals infected with CDC 1551 or M. africanum N0091 (P < 0.001 by ANOVA). Bacterial loads of the spleen and liver of marmosets infected with CDC 1551 were generally lower than those of animals infected with N0091 (P < 0.05 by ANOVA posttest). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars indicate means.

Fig 5

Histopathology of marmoset lungs infected with the 3 M. tuberculosis complex strains is similar to that seen in human disease. Both nonnecrotizing lesions (A) and necrotizing lesions (B) were observed in each infection group and were characterized by neutrophil and epithelioid macrophage aggregates, often with central necrosis surrounded by concentric layers of lymphocytes and fibrosis. (C) Lesion erosion into airways forming cavities with variable amounts of liquefactive material with leukocytic infiltration (arrow) was observed in CDC 1551-infected animals. (D) Masson's trichrome staining (D and E) revealed increased collagen (arrow, blue staining) in the lesions from CDC 1551 infections versus lower collagen deposition in granulomas in K04- and N0091-infected animals (E) at the same time p.i. (F) Staining with hypoxyprobe antibody revealed areas where in vivo-administered pimonidazole HCl was trapped in the lesion tissue, indicating areas of hypoxia (brown staining). Low-magnification images of lung sections from K04 (G)-, CDC 1551 (H)-, and N0091 (I)-infected animals are also shown. Alveolar pneumonia extending outward from granulomatous infiltrates occurred in all groups but was predominant in K04- and N0091-infected lungs. CDC 1551-infected lungs contained a more multifocal lesion distribution, but tuberculous pneumonia was also present (the arrow indicates adhesion to the lung surface). Grossly normal-appearing lung regions contained occasional small cellular lesions (arrowhead in panel I). Images shown in panels A to C and G to I were stained with hematoxylin and eosin. N, area of necrosis. Scale bars were 1 mm (G to I), 0.2 mm (A, B, and D to F), and 0.5 mm (C).

Fig 6

Intersibling comparison of infection progression demonstrates reproducibility and strain-related differences in lesion expansion. The images are 3D reconstructions of CT scans with the low-density lung shown in green and the high-density lesions depicted in red. (A and B) Twins infected with the high dose of M. tuberculosis Beijing strain K04 at 4 weeks formed very similar numbers (38 and 29) and sizes of lesions (see Table S2 in the supplemental material). (C and D) Twins infected with the same dose of different strains of M. tuberculosis (K04 [C] and CDC 1551 [D]) demonstrated similar numbers of granulomas at 4 weeks (26 and 31), but the lesions in the CDC 1551-infected twins were much smaller at this time point.