Model of differential susceptibility to mucosal Burkholderia pseudomallei infection

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with protean clinical manifestations. The major route of infection is thought to be through subcutaneous inoculation of contaminated soil and water, although ingestion and inhalation of contaminated aerosols are also possible. This study examines infection through the intranasal route in a murine model to mimic infection through inhalation. Two strains of mice, C57BL/6 and BALB/c, exhibit differential susceptibilities to the infection, with the C57BL/6 mice being considerably more resistant. To examine host factors that could contribute to this difference, bacterial loads and cytokine profiles in the two strains of mice were compared. We found that infected BALB/c mice exhibited higher bacterial loads in the lung and spleen and that they produced significantly higher levels of gamma interferon (IFN-gamma) in the serum than C57BL/6 mice. Although tumor necrosis factor alpha and interleukin-1 could be detected in the nasal washes and sera of both strains of mice, the production in serum was transient and much lower than that of IFN-gamma. C57BL/6 mice also exhibited memory responses to bacteria upon reinfection, with the production of serum immunoglobulin G (IgG) and mucosal IgA antibodies. Thus, it is possible that the production of systemic and mucosal antibodies is important for protection against disease in C57BL/6 mice.

FIG. 1.

Bacterial loads in BALB/c and C57BL/6 mice. The bacterial loads in the lungs (A and D), livers (B and E), and spleens (C and F) of BALB/c (A, B, and C) and C57BL/6 (D, E, and F) mice at different time points after intranasal infection with virulent B. pseudomallei are shown. The logarithm of the number of CFU was plotted against time. Each symbol represents one mouse. The control mice are not represented, since no colonies were found in any of their organs. Filled symbols represent mice from one experiment, and open symbols represent animals from another experiment. Results from the two experiments performed under the same conditions are superimposed.

FIG. 2.

Expression of IFN-γ mRNA in the lungs (A and D), livers (B and E), and spleens (C and F) of BALB/c (A, B, and C) and C57BL/6 (D, E, and F) mice as determined by RT-PCR. In each set of gels, the top panel depicts IFN-γ and the bottom panel depicts the corresponding GAPDH. Lanes M, 100-bp molecular size marker; lanes N, no-template control; lanes 1 to 3, three infected mice at 24 h postinfection; lanes 4, control mock-infected mouse at 24 h postinfection; lanes 5 to 7, three infected mice at 48 h postinfection; lanes 8, control mock-infected mouse at 48 h postinfection; lanes 9 to 11, three infected mice at 72 h postinfection; lanes 12, control mock-infected mouse at 72 h postinfection.

FIG. 3.

Concentrations of IFN-γ (A and D), IL-1β (B and E), and TNF-α (C and F) in sera (A, B, and C) and nasal washes (D, E, and F) of BALB/c (white bars) and C57BL/6 (black bars) mice after intranasal infection with 50 CFU of virulent B. pseudomallei. Each bar represents pooled sera or nasal washes from three mice. The optical densities in the ELISA are expressed as picograms per milliliter corresponding to the linear portion of the standard curves.

FIG. 4.

Serum IgG (A) and mucosal IgA (B) responses in C57BL/6 mice after intranasal inoculation with 50 CFU of virulent B. pseudomallei (•), 50,000 CFU of heat-killed B. pseudomallei (▪), or PBS (▴). Each symbol represents a 1:10 dilution of serum at the indicated days after the first inoculation at day 0, the second inoculation at day 14, and reinfection with 10,000 CFU of live bacteria at day 28 (A), or represents undiluted nasal wash at the indicated days after reinfection with 10,000 CFU live bacteria at day 28 (B), from three mice pooled together. Results represent means ± standard deviations of triplicates. OD, optical density.