Pathogenicity and immune response measured in mice following intranasal challenge with enterotoxigenic Escherichia coli strains H10407 and B7A

Abstract

The pathogenicity and immunogenicity induced in BALB/c mice by intranasal (i.n.) inoculation of enterotoxigenic Escherichia coli (ETEC) strains H10407 (O78:H11:CFA/I:LT(+):ST(+)) and B7A (O148:H28:CS6:LT(+):ST(+)) (two ETEC strains previously used in human challenge trials) were studied. The i.n. inoculation of BALB/c mice with large doses of ETEC strains H10407 and B7A caused illness and death. The H10407 strain was found to be consistently more virulent than the B7A strain. Following i.n. challenge with nonlethal doses of H10407 and B7A, the bacteria were cleared from the lungs of the mice at a steady rate over a 2-week period. Macrophages and neutrophils were observed in the alveoli and bronchioles, and lymphocytes were observed in the septa, around vessels, and in the pleura of the lungs in mice challenged with H10407 and B7A. In mice i.n. challenged with H10407, serum immunoglobulin G (IgG) and IgM antibodies were measured at high titers to the CFA/I and O78 lipopolysaccharide (LPS) antigens. In mice i.n. challenged with B7A, low serum IgG antibody titers were detected against CS6, and low serum IgG and IgM antibody titers were detected against O148 LPS. The serum IgG and IgM antibody titers against the heat-labile enterotoxin were equivalent in the H10407- and B7A-challenged mice. The CFA/I and O78 LPS antigens gave mixed T-helper cell 1-T-helper cell 2 (Th1-Th2) responses in which the Th2 response was greater than the Th1 response (i.e., stimulated primarily an antibody response). These studies indicate that the i.n. challenge of BALB/c mice with ETEC strains may provide a useful animal model to better understand the immunogenicity and pathogenicity of ETEC and its virulence determinants. This model may also be useful in providing selection criteria for vaccine candidates for use in primate and human trials.

FIG. 1.

Kinetics of clearance following i.n. challenge with H10407 (2 × 10⁸ bacteria) and B7A (4 × 10⁸ bacteria). Three mice from each group, H10407 and B7A challenged, were euthanized each 24-h period postchallenge, and the lungs were aseptically removed. A CFU procedure was performed to determine the number of bacteria present in the lungs at a given time postchallenge. The points indicate the means of the CFU from three mice for each of the ETEC strains. The error bars represent ±1 standard deviation of the mean response.

FIG. 2.

Histopathology of lungs of mice following i.n. challenge with H10407 (2 × 10⁸ bacteria) and B7A (4 × 10⁸ bacteria). Three mice from H10407- and B7A-challenged and PBS control groups of mice were euthanized each 24-h period postchallenge, and the lungs were removed, fixed, and stained with H&E. The lungs were examined microscopically, and the number of inflammatory cells was noted. Moderate-to-severe (solid black bars) indicates that the section of lung affected was from 11 to >41% with high numbers of inflammatory cells (neutrophils, macrophages, lymphocytes, and plasma cells); minimal-to-mild (hatched bars) indicates that the section of lung affected was from 1 to 10% with low numbers of inflammatory cells; and none (solid white bars) indicates that no histopathological changes were observed. No histopathological changes were observed in the lungs of the mice administered only PBS. MP, macrophages or histiocytes; PMN, polymorphonuclear leukocytes (neutrophils); LP, lymphoid proliferation (lymphocytes and plasma cells).

FIG. 3.

Mouse lung histopathology. (A) Control mouse i.n. administered PBS at day 1 has normal alveoli and bronchioles and fine, delicate septa. (B) Mouse challenged with H10407 (2 × 10⁸ bacteria) at day 1 has pneumonia with increased macrophages and neutrophils that obscure alveoli and thicken the septa (arrows). Many macrophages contain intracellular bacilli (data not shown). H&E was used for staining. Magnification, ×200.

FIG. 4.

Mice were challenged with H10407 (2 × 10⁸ bacteria) and B7A (4 × 10⁸ bacteria), and serum was collected at days 10, 14, 21, 28, 35, and 42. Serum IgG (A), IgM (B), and IgA (C) antibody responses were measured against CFA/I, O78 LPS, and LT following i.n. challenge with H10407, and serum IgG (D) and IgM (E) antibody responses were measured against CS6, O148 LPS, and LT following i.n. challenge with B7A. No serum IgA antibody response was measured in mice challenged with B7A (data not shown). Endpoint ELISA titers were expressed as the reciprocal of the highest dilution of immune serum at which the A₄₀₅ was at least twice that of the nonimmune control serum (mice administered PBS) and the A₄₀₅ of the immune serum was at least 0.2. Data are presented as the mean titers (of four mice) of the IgG, IgM, and IgA antibodies. The error bars represent ±1 standard deviation of the mean response. At all titers P was <0.05 compared with the PBS control mice, except on day 10 for O148 LPS IgG (D) and days 10 and 14 for LT IgG for both H10407 (A)- and B7A (D)-challenged mice.

FIG. 5.

Serum IgG subclass antibody responses measured to CFA/I, CS6, LPS, and LT. Serum IgG subclass antibody responses were measured against CFA/I (A), O78 LPS (B), and LT (C) following i.n. challenge with H10407, and the serum IgG subclass antibody responses were measured against CS6 (D), O148 LPS (E), and LT (F) following i.n. challenge with B7A. Endpoint ELISA titers were expressed as the reciprocal of the highest dilution of immune serum at which the A₄₀₅ was at least twice that of the nonimmune control serum (mice administered PBS) and the A₄₀₅ of the immune serum was at least 0.2. Data are presented as the mean titers (of four mice) of the IgG subclasses. The error bars represent ±1 standard deviation of the mean response. *, P < 0.05 compared to the PBS control mice.