Relative importance of heat-labile enterotoxin in the causation of severe diarrheal disease in the gnotobiotic piglet model by a strain of enterotoxigenic Escherichia coli that produces multiple enterotoxins

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains that produce multiple enterotoxins are important causes of severe dehydrating diarrhea in human beings and animals, but the relative importance of these enterotoxins in the pathogenesis is poorly understood. Gnotobiotic piglets were used to study the importance of heat-labile enterotoxin (LT) in infection with an ETEC strain that produces multiple enterotoxins. LT(-) (DeltaeltAB) and complemented mutants of an F4(+) LT(+) STb(+) EAST1(+) ETEC strain were constructed, and the virulence of these strains was compared in gnotobiotic piglets expressing receptors for F4(+) fimbria. Sixty percent of the piglets inoculated with the LT(-) mutant developed severe dehydrating diarrhea and septicemia compared to 100% of those inoculated with the nalidixic acid-resistant (Nal(r)) parent and 100% of those inoculated with the complemented mutant strain. Compared to piglets inoculated with the Nal(r) parent, the mean rate of weight loss (percent per hour) of those inoculated with the LT(-) mutant was 67% lower (P < 0.05) and that of those inoculated with the complemented strain was 36% higher (P < 0.001). Similarly, piglets inoculated with the LT(-) mutant had significant reductions in the mean bacterial colony count (CFU per gram) in the ileum; bacterial colonization scores (square millimeters) in the jejunum and ileum; and clinical pathology parameters of dehydration, electrolyte imbalance, and metabolic acidosis (P < 0.05). These results indicate the significance of LT to the development of severe dehydrating diarrhea and postdiarrheal septicemia in ETEC infections of swine and demonstrate that EAST1, LT, and STb may be concurrently expressed by porcine ETEC strains.

FIG. 1.

Southern hybridization of XbaI digests of genomic DNA for (A) kanamycin resistance gene (aphA) and (B) E. coli heat-labile enterotoxin (LT) genes (eltAB). Lanes: 1, molecular size marker; 2, negative control DH5α; 3, LT⁻ (ΔeltAB) mutant MUN284; 4, single-crossover mutant; 5, LT⁻ (ΔeltAB) mutant MUN285; 6, recipient strain WAM2317; 7, single-crossover mutant; 8, LT⁺ wild-type 2534-86. Horseradish peroxidase-labeled probes were used for aphA (450-bp internal PCR fragment) and eltAB (1,274-bp PCR product). Molecular sizes (kilobase pairs) are denoted by arrowheads to the right.

FIG. 2.

Immunoblot of periplasmic protein extracts for E. coli heat-labile enterotoxin (LT). Lanes: 1, recipient strain WAM2317; 2, single-crossover mutant; 3, eltAB clone CB2091; 4, LT⁺ wild-type 2534-86; 5, LT⁻ (ΔeltAB) mutant MUN284; 6, complemented mutant MUN287; 7, LT⁻ (ΔeltAB) mutant MUN285; 8, purified LT_h (Sigma); 9, negative control DH5α. Protein extracts were standardized for total protein concentration. The blot was probed sequentially with rabbit CT antiserum and horseradish peroxidase-conjugated goat anti-rabbit IgG. Detection was done by enhanced chemiluminescence. Numbers to the right denote molecular masses (kilodaltons).

FIG. 3.

Immunoblot of culture supernatants for STb. Lanes: 1, negative control G58-1; 2, LT⁺ wild-type 2534-86; 3, recipient strain WAM2317; 4, LT⁻ (ΔeltAB) mutant MUN285; 5, complemented mutant MUN287; 6, negative control NADC 2290; 7, STb⁺ clone NADC 2329; 8, molecular mass marker; 9, vector control NADC 2787; 10, STb⁻ mutant clone NADC 4924. Samples were standardized for total protein concentration. The blot was probed with rabbit anti-STb serum (12) and horseradish peroxidase-conjugated goat anti-rabbit IgG. Detection was done by enhanced chemiluminescence. Numbers to the right denote molecular masses (kilodaltons).

FIG. 4.

Competitive ELISA to detect EAST1 expression. Bars: 1, blank; 2 to 4, EAST1 N-terminal synthetic peptide at 100 ng (bar 2), 50 ng (bar 3), and 10 ng (bar 4); 5, negative control DH5α; 6, negative control G58-1; wild-type 2534-86; 8, recipient strain WAM2317; 9, LT⁻ (ΔeltAB) mutant MUN285; 10, complemented mutant MUN287; 11, wild-type enteroaggregative E. coli strain 17-2; 12, EAST1⁺ clone BF-3. Optical densities (O.D.) at 490 nm are averages from three independent assays. Cytoplasmic protein samples of each strain were run in triplicate in each assay. Protein extracts were standardized for total protein concentration. EAST1 N-terminal synthetic peptide, prototype enteroaggregative E. coli strain 17-2, and astA clone BF-3 were used as EAST1⁺ controls.

FIG.5.

Effects of inoculation of gnotobiotic piglets with isogenic LT⁺ and LT⁻ ETEC strains. Results are presented as the least-squares (LS) means ± standard error. Superscripts A to C refer to statistical comparisons of LS means of different treatment groups (inoculation with different bacterial strains), and X to Y refer to statistical comparisons of total moribund ETEC, total nonmoribund ETEC, and G58-1 (control) groups, respectively. Total moribund ETEC and total nonmoribund ETEC include all data combined, respectively, for moribund and nonmoribund animals inoculated with ETEC strains. LS means with unlike superscripts (e.g., A versus B or X versus Y) are significantly different based on the general t values (P < 0.05). Blood and serum analyte concentrations were determined on the p.i. specimen obtained immediately prior to euthanasia; differences between the p.i. and preinoculation values were reported as the change in concentration. (A) Rate of change in body weight. (B) Change in HCT. (C) Change in serum bicarbonate (HCO3−) concentration. (D) Ileal colonization score. (E) Intestinal ischemia score. (F) Blood bacterial concentration.