Ferrets as a model for tuberculosis transmission

Abstract

Even with the COVID-19 pandemic, tuberculosis remains a leading cause of human death due to a single infectious agent. Until successfully treated, infected individuals may continue to transmit Mycobacterium tuberculosis bacilli to contacts. As with other respiratory pathogens, such as SARS-CoV-2, modeling the process of person-to-person transmission will inform efforts to develop vaccines and therapies that specifically impede disease transmission. The ferret (Mustela furo), a relatively inexpensive, small animal has been successfully employed to model transmissibility, pathogenicity, and tropism of influenza and other respiratory disease agents. Ferrets can become naturally infected with Mycobacterium bovis and are closely related to badgers, well known in Great Britain and elsewhere as a natural transmission vehicle for bovine tuberculosis. Herein, we report results of a study demonstrating that within 7 weeks of intratracheal infection with a high dose (>5 x 10³ CFU) of M. tuberculosis bacilli, ferrets develop clinical signs and pathological features similar to acute disease reported in larger animals, and ferrets infected with very-high doses (>5 x 10⁴ CFU) develop severe signs within two to four weeks, with loss of body weight as high as 30%. Natural transmission of this pathogen was also examined. Acutely-infected ferrets transmitted M. tuberculosis bacilli to co-housed naïve sentinels; most of the sentinels tested positive for M. tuberculosis in nasal washes, while several developed variable disease symptomologies similar to those reported for humans exposed to an active tuberculosis patient in a closed setting. Transmission was more efficient when the transmitting animal had a well-established acute infection. The findings support further assessment of this model system for tuberculosis transmission including the testing of prevention measures and vaccine efficacy.

Keywords: animal model; ferret; mycobacterium; transmission; tuberculosis.

Figure 1

Infection study: Ferrets infected with M. tuberculosis strain Erdman generate a strong delayed-type hypersensitivity (DTH) response to PPD. The flanks of the infection-study ferrets were shaved and different areas injected intradermally with 1 or 2 µg PPD or 1,000 IU Old Tuberculin 7 weeks after intratracheal infection with the indicated M. tuberculosis dose. The diameter of the induration (mm) was measured at 24 hours after injection. The black dots mark the ends of the induration. (A) Data represent mean ± SEM from groups of 3 infected ferrets and 1 uninfected ferret, and (B) arrows indicate typical areas of induration observed in ferrets given PPD.

Figure 2

Infection study: Bacterial loads in infected ferret tissues. Mycobacterial CFU in homogenates of infection study ferret lungs (upper row), spleen (middle row), and mediastinal lymph nodes (MLN) (lower row) were obtained at (A) 4 weeks and (B) 7 weeks post infection with low, medium, or high dose of M. tuberculosis bacilli. Data represent mean ± SEM from 3 ferrets in each group at each time point, except the mediastinal lymph node (MLN) from the low dose-infected group (n = 1), since we could not isolate them cleanly during necropsy. Data were analyzed by One-way ANOVA and Kruskal-Wallis comparison test, *p < 0.05.

Figure 3

Infection study: Histopathology of the ferret lung post-challenge. Representative hematoxylin and eosin (H&E)-stained lung sections from ferrets 4 weeks (A-C) and 7 weeks (D-F) after infection with a high, medium, or low dose of M. tuberculosis (G) Percent of examined lung area affected and number of granulomas per examined lung area 4 weeks and 7 weeks after infection with high, medium, or low-dose M. tuberculosis. Data represent n = 3 ferrets for low-and medium-dose infections and n = 2 for the high-dose group. Data analyzed by One-way ANOVA and Bonferroni’s multiple comparison test, *p < 0.05, **p <0.01, ***p < 0.001. (H) Representative H&E-stained mediastinal lymph node, spleen, and liver sections from ferrets 7 weeks after infection with high-dose M. tuberculosis. 400X magnification. Red arrows on high-dose, week-7 sections (D, H) identify caseous necrotic lesions.

Figure 4

Infection study: Ferret lung cytokine and serum antibody levels. (A) Expression of the indicated cytokines/chemokines in the lungs was measured by qRT-PCR. Pooled RNA from lung tissue of infection study ferrets at 4 and 7 weeks post infection with high, medium, or low doses of M. tuberculosis was examined. Data represent fold changes relative to pre-infection levels and 18S as house-keeping gene. (B) Sera from ferrets at week 2,4 and 7 weeks after intratracheal infection with the various doses of M. tuberculosis bacilli were assayed for antibodies against M. tuberculosis whole cell lysate by ELISA. The y-axis indicates absorbance at wavelength 405 nm. The x-axis indicates the serum dilution. Serum samples were measured in duplicate. Data represent mean ± SEM from 3 groups at various time points. The groups were analyzed by One-way ANOVA for non-parametric samples followed by Kruskal-Wallis and Dunn’s multiple comparison; *p < 0.05.

Figure 5

Transmission study using medium-high dose-infected transmitters: DTH reaction to PPD. Sustained DTH to 2 µg PPD in M. tuberculosis-infected transmitter (TR) ferrets was observed by week 8 post infection. One sentinel, SE5 (red boxes) co-housed with TR4 in week 17 of the study, tested positive beginning 5 weeks later. Data represent mean ± SEM of n = 4 transmitters and n = 7 sentinels.

Figure 6

Transmission study using medium-high dose-infected transmitters: IS6110 PCR of ferret nasal wash samples. Shown is a gel image of IS6110 PCR products from transmitter (TR), sentinel (SE), and uninfected control (UI) ferrets resolved on a 2% agarose gel. The arrow indicates the expected band size corresponding to the 106 bp IS6110 amplicon. Lane 1 = 100 bp ladder (NEB), 2 = TR1, 3 = TR2, 4 = TR3, 5 = Tr4, 6 = SE1, 7 = SE2, 8 =SE3, 9 = SE5, 10 = SE6, 11 = SE7, 12 = UI (F8), 13 = UI (F9), 14 = positive control amplified with M. tuberculosis genomic DNA template, and 15 = negative control DNA. The samples were collected at study termination.

Figure 7

Transmission study using medium-high dose infected transmitters: Mycobacterial loads in ferret lungs. At the termination of the transmission study (week 27 post-infection), lung homogenates of the transmitter and sentinel ferrets were processed for M. tuberculosis CFU. Data represent mean ± SEM; n = 4 transmitters and n = 7 sentinels of which only 4 had measurable M. tuberculosis CFU detected.

Figure 8

Transmission study using medium-high dose infected transmitters: Histopathology of direct-contact transmission study ferret lungs. Percent of examined lung area affected 27 weeks post-TR infection and numbers of granulomas per examined lung area are shown. Data represent mean ± SEM from groups of 4 ferret transmitters and 7 sentinels.

Figure 9

Transmission study using medium-high dose-infected transmitters: Ferret lung cytokine gene expression and splenocyte and antibody responses to M. tuberculosis antigens. (A) Expression of genes encoding IFN-g and TNF-a was measured by qRT-PCR of RNA extracted from lung homogenates of uninfected controls and transmitter and sentinel ferrets 27 weeks after initial transmitter infection. Data represent mean ± SEM of gene expression change in four transmitter or six sentinel ferrets relative to uninfected controls. Animals not represented in figure did not have gene expression levels greater than controls. SE4 lung homogenates were not assessed in this experiment. (B) Splenocytes harvested and cryopreserved from transmitter and sentinel animals were thawed, washed, and 2X106 cells were cultured in duplicate with M. tuberculosis whole cell lysate (WCL). Culture supernatants were assayed at day 1 and day 6 post stimulation for IFN-g release by ELISA. Positive controls were stimulated with PHA or PMA + ionomycin. Cells in medium only served as the negative control. Statistical analysis was performed using a Mann-Whitney test for four transmitter or six sentinel ferrets relative to uninfected controls. SE4 splenocytes were not assessed in this experiment. (C) Sera were collected from uninfected controls, and transmitter and sentinel ferrets 27 weeks after initial transmitter infection and were assayed for antibodies against M. tuberculosis whole cell lysate by ELISA. The y-axis indicates absorbance at a wavelength of 405 nm. The x-axis indicates the serum dilution. Serum samples were diluted and measured in duplicate. No antibodies were detected in SE6 and SE7. Data analyzed by One-way ANOVA and Bonferroni’s multiple comparison test, *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 10

Transmission study using very-high dose-infected transmitters: Bacterial loads in transmitter ferret tissues. Mycobacterial CFU in homogenates of the lungs, mediastinal lymph nodes (MLN), spleen, liver, and trachea were obtained from transmitter ferrets after euthanasia. Data represent mean ± SEM from 6 transmitter ferrets.

Figure 11

Transmission study using very-high dose-infected transmitters: ferret lung cytokine and chemokine levels. Expression of the indicated cytokine genes was measured by qRT-PCR of pooled RNA from lung tissue from transmitter ferrets at the time of humane endpoint or at study termination 10 weeks post infection. Data represent mean ± SEM of gene expression changes in ferrets relative to n = 3 uninfected control tissues. (A) n = 6 transmitters (TRA1- TR-A6), (B) n = 3 direct-sentinels (SE-D1 -SE-D3), and (C) n = 6 aerosol-sentinels (SE-A1-SE-A6).

Figure 12

Transmission study using very-high dose-infected transmitters: Humoral antibody response against M. tuberculosis whole cell lysate. Sera were collected from (A) uninfected control and sentinel ferrets, and (B) transmitter ferrets at time of euthannasia. Data represent mean ± SEM of antibody titers against M. tubercculosis whole cell lysate by ELISA. The y-axis indicates absorbance at a wavelength of 405 nm. The x-axis indicates the serum dilution. Serum samples were measured in duplicate.