Significance of heat-stable and heat-labile enterotoxins in porcine colibacillosis in an additive model for pathogenicity studies

Abstract

Although heat-stable (ST) and heat-labile (LT) enterotoxins produced by enterotoxigenic Escherichia coli (ETEC) have been documented as important factors associated with diarrheal diseases, investigations assessing the contributions of individual enterotoxins to the pathogenesis of E. coli infection have been limited. To address the individual roles of enterotoxins in the diarrheal disease caused by K88-positive ETEC in young pigs, enterotoxin-positive and -negative isogenic E. coli strains were constructed by using pBR322 to clone and express LT and STb. Four strains, K88+ astA, K88+ astA/pBR322, K88+ astA STb+, and K88+ astA LT+, were constructed and subsequently included in gnotobiotic piglet challenge studies, and their pathogenesis was assessed. The results indicated that all K88+ isogenic strains were able to colonize the small intestines of piglets exhibiting the K88 receptor. However, only LT- and STb-positive strains caused appreciable diarrhea. Piglets inoculated with the K88+ astA LT+ strain became dehydrated within 18 h, while those inoculated with the K88+ astA STb+ strain did not, although diarrhea developed in several piglets. The changes in the blood packed-cell volume and plasma total protein of gnotobiotic piglets inoculated with the LT-positive strains were significantly greater than those of pigs inoculated with the K88 astA/pBR322 strain (P = 0.012, P = 0.002). Immunochemistry image analysis also suggested that LT enhanced bacterial colonization in a gnotobiotic piglet model. This investigation suggested that LT is a major contributor to the virulence of K88+ ETEC and that isogenic constructs are a useful tool for studying the pathogenesis of ETEC infection.

FIG. 1.

Detection of LT by GM1 ELISA. Wells of 96-well microtiter plates were coated with the ganglioside GM1 as the ligand for LT. Lysates of pellets from overnight culture were tested for expression of LT with anti-CT as the primary antibodies (1:2,500) and goat anti-rabbit IgG (1:1,500) as the secondary antibodies. Reactions were visualized with peroxidase substrate, and optical densities were measured at 405 nm. Titration curves of commercialized CT (solid line), positive control strain 3030-2 (double-dotted and dashed line), strain 8035 (LT⁺) (single-dotted and dashed line), parental strain 1836-2 (circles), strain 8015 (STb⁺) (dotted line), and negative control strain 8017 (squares) are shown.

FIG. 2.

Detection of STb by ELISA. Supernatants from overnight bacterial culture were concentrated 10-fold with Centricon filters. Anti-STb rabbit serum (1:5,000) was used as the primary antibody, and goat anti-rabbit IgG conjugated with HRP (1:10,000) was used as the secondary antibody. Reactions were visualized with o-phenylenediamine and peroxide buffer, and optical densities were read at 490 nm. Student's t test indicated that the expression of STb in strains 3030-2 and 8015 was significantly different from that in strain 1836-2 (P < 0.01, P = 0.01), whereas no significantly differences were found between strain 8035 or 8017 and strain 1836-2 (P = 0.07, P = 0.39).

FIG. 3.

Ligated ileal loop assay in neonatal pigs. Individual ligated intestinal loops were inoculated with 2 ml of an overnight culture of strain 3030-2 (K88⁺ LT⁺ STb⁺ STa⁺, positive control), 8035 (K88⁺ astA LT⁺), 8015 (K88⁺ astA STb⁺), or 8017 (K88⁺ astA/pBR322, negative control). Fluid accumulation was measured at 8 h postinoculation and expressed as grams per centimeter of ligated loop. Statistical analysis indicated that the fluid accumulation in strains 3030-2 (P = 0.02), 8035 (P = 0.04), and 8015 (P = 0.048) was significantly different from that in strain 8017.

FIG. 4.

Histological sections of ileum from challenged gnotobiotic pigs were stained immunohistochemically to demonstrate bacterial colonization on the surface of the mucosal epithelium by strains 8017 (K88⁺ astA) (A), 8015 (K88⁺ astA STb⁺) (B), and 8035 (K88⁺ astA LT⁺) (C). Rabbit polyclonal antiserum against E. coli O8 somatic antigen was used as the primary antibody, and goat anti-rabbit IgG (heavy and light chain specific) was used as the secondary antibody. Avidin-biotin-alkaline phosphatase and Vector Red as the substrate were used to generate a red reaction product. Microscopic images were digitalized, and red areas were measured by the Image Processing Tool kit. Bar, 100 μm.