Differential susceptibility of inbred mouse strains to Burkholderia thailandensis aerosol infection

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis, an emerging bacterial disease that accounts for high rates of septicaemia and death in parts of Southeast Asia and Northern Australia. The closely related species Burkholderia thailandensis is considered avirulent in humans and has been used as a surrogate for B. pseudomallei in several studies. The pathogenesis of B. pseudomallei and the role of Toll-like receptors (TLRs) in host immunity to infection are not well-defined. In this study, we exposed four strains of inbred mice (BALB/c, C57BL/6, TLR4-deficient C3H/HeJ, and TLR4-competent C3H/HeN) to increasing doses of aerosolized B. thailandensis to determine strain susceptibility and the role of TLR4 during pulmonary infection. Our results indicate an increased susceptibility in the C57BL/6 and BALB/c strains, who displayed lethality, bacterial burden in organs, and pulmonary and systemic inflammation. C3H/HeJ were as resistant as C3H/HeN mice to B. thailandensis at the highest challenge dose examined, but TLR4-deficient animals exhibited a modest increase in chronic pulmonary inflammation. These results demonstrate that B. thailandensis can be used as a surrogate for experimental laboratory investigation of melioidosis in small animal models and that TLR4 may not play a prominent role during acute pneumonic melioidosis.

Fig. 1

Survival of inbred mouse strains following aerosol infection with B. thailandensis. Groups of ten (1 × 10⁴ cfu (panel A)) or six (1 × 10⁵ (panel B) and 1 × 10⁶ cfu (panel C)) mice were infected by the aerosol route and their survival monitored for up to 28 days. Mice that did not succumb within the first 4 days survived for the entire 4-week study period; therefore only the first 7 days are shown.

Fig. 2

B. thailandensis recovery from lung, liver, and spleen after low (1 × 10⁴ cfu) and high dose (1 × 10⁶ cfu) aerosol challenge. (A) Bacterial burdens in the C57BL/6 mice that succumbed to aerosol challenge with 1 × 10⁴ cfu compared to BALB/c that were asymptomatic at the time of sacrifice (72 h p.i. for both strains). (B) Bacterial burdens in the C57BL/6 and BALB/c mice that succumbed to aerosol challenge with 1 × 10⁶ cfu at 48 h and 72 h p.i., respectively. A cohort of 3 mice per strain per time point was used. Counts of B. thailandensis from each mouse are presented as solid squares (C57BL/6) or open circles (BALB/c) and are expressed as cfu/mg tissue. The median value for each strain is indicated by a bar.

Fig. 3

Pulmonary and serum cytokine levels in susceptible C57BL/6 and BALB/c strains after high dose aerosol challenge. Cytokines were measured in the lung homogenates (left columns) and the sera (right columns) of C57BL/6 (solid bars) and BALB/c (open bars) mice after succumbing to 1 × 10⁶ cfu. Data shown are mean values with error bars showing the SEM from three mice at each time point. Samples from uninfected controls had undetectable levels of cytokines and are not shown. The assays were performed in duplicate. * indicates statistical significance between strains.

Fig. 4

Pulmonary and serum cytokine levels in resistant C3H/HeN and C3H/HeJ strains after high dose aerosol challenge. Cytokines were measured in the lung homogenates (left columns) and the sera (right columns) of C3H/HeN (horizontal lines) and C3H/HeJ (cross-hatched) after 4 weeks p.i. with 1 × 10⁶ cfu. Data shown are mean values with error bars showing the SEM from three mice at each time point. Samples from uninfected controls had undetectable levels of cytokines and are not shown. The assays were performed in duplicate. * indicates statistical significance between strains.

Fig. 5

Lung pathology in susceptible (C57BL/6 and BALB/c) and resistant (C3H/HeN and C3H/HeJ) mice after high dose aerosol challenge. Moderate pneumonia observed in the lungs of C57BL/6 (A) compared to severe pneumonia in BALB/c (B) that succumbed to 1 × 10⁶ cfu at 48 h and 72 h p.i., respectively. Examination of C3H/HeJ lungs revealed moderate lymphoid hyperplasia (C) at 4 weeks p.i. compared to normal lung architecture in C3H/HeN (D).