Escherichia coli constructs expressing human or porcine enterotoxins induce identical diarrheal diseases in a piglet infection model

Abstract

To develop a piglet model for studying diarrheal disease and developing vaccines, we challenged gnotobiotic piglets with isogenic Escherichia coli strains constructed to express porcine 987P(F6) fimbriae and a heat-labile or a heat-stable enterotoxin to examine clinical outcomes. Piglets developed identical diarrheal diseases when inoculated with constructs expressing human or porcine enterotoxins.

FIG. 1.

GM1 ELISA to detect expression of LT proteins from hLT and pLT constructs. Pellets from an overnight-grown culture were used for total protein preparation using B-PER. Each sample was assayed in triplicate. Anti-CT serum (1:5,000) was used as the primary antibody, and goat anti-rabbit HRP-conjugated IgG (1:5,000) was used as the secondary antibody. Optical densities were measured at a wavelength of 405 nm. PBS, phosphate-buffered saline.

FIG. 2.

STa competitive ELISA to detect expression of STa proteins from hSTa and pSTa constructs. Supernatants from overnight-grown culture (4AA medium) were used as protein samples for ELISA. Microtiter plates were coated with STa-ovalbumin conjugate, and anti-STa serum (1:10,000) was used as the primary antibody. Goat anti-rabbit HRP-conjugated IgG (1:10,000) was used as the secondary antibody. Optical densities were measured at a wavelength of 405 nm. Blocking of STa ELISA was calculated based on the standard reference curve (using a series of synthetic STa peptide samples) generated in the same assay.

FIG. 3.

cGMP ELISA to detect biological activity of STa proteins expressed from the hSTa and pSTa constructs through stimulation of cGMP in T-84 cells. The effect of stimulation on increasing cGMP levels was measured with a direct cGMP EIA kit (Correlate EIA; Assay Designs).

FIG. 4.

Porcine ligated gut loop assay to determine biological activity of the LT and STa toxins from the pSTa, hSTa, pLT, and hLT constructs. Overnight-grown culture (2 × 10⁹ CFU) from each strain was injected into a ligated loop. After 8 h of incubation, the fluid accumulated in each loop was measured in grams per centimeter of loop length.