Differential virulence of clinical and bovine-biased enterohemorrhagic Escherichia coli O157:H7 genotypes in piglet and Dutch belted rabbit models

Abstract

Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) is an important cause of food and waterborne illness in the developed countries. Cattle are a reservoir host of EHEC O157 and a major source of human exposure through contaminated meat products. Shiga toxins (Stxs) are an important pathogenicity trait of EHEC O157. The insertion sites of the Stx-encoding bacteriophages differentiate EHEC O157 isolates into genogroups commonly isolated from cattle but rarely from sick humans (bovine-biased genotypes [BBG]) and those commonly isolated from both cattle and human patients (clinical genotypes [CG]). Since BBG and CG share the cardinal virulence factors of EHEC O157 and are carried by cattle at similar prevalences, the infrequent occurrence of BBG among human disease isolates suggests that they may be less virulent than CG. We compared the virulence potentials of human and bovine isolates of CG and BBG in newborn conventional pig and weaned Dutch Belted rabbit models. CG-challenged piglets experienced severe disease accompanied by early and high mortality compared to BBG-challenged piglets. Similarly, CG-challenged rabbits were likely to develop lesions in kidney and intestine compared with the BBG-challenged rabbits. The CG strains used in this study carried stx2 and produced significantly higher amounts of Stx, whereas the BBG strains carried the stx2c gene variant only. These results suggest that BBG are less virulent than CG and that this difference in virulence potential is associated with the Stx2 subtype(s) carried and/or the amount of Stx produced.

Fig 1

Clinical scores of piglets challenged with EHEC strains belonging to different genotypes. Five animals were challenged with each genotype (CG-1, CG-3, BBG-5, and BBG-6). Groups not sharing the specific letters shown in bars differed significantly (P = 0.023, Kruskal-Wallis one-way ANOVA with Dunn's multiple comparison).

Fig 2

Kaplan-Meier survival curves for piglet groups challenged with different EHEC O157 genogroups. CG strains differ significantly from BBG strains according to the log rank test (P = 0.002).

Fig 3

Tissue from a piglet challenged with the positive-control Sakai strain exhibiting mortality on day 6 p.i. (A) Section of the brain stained with Luxol-fast blue stain showing affected blood vessel (arrows, magnified in panels B and C) with vacuolation of adjacent tissues (arrowhead) caused by focal myelin degeneration. (B) Affected blood vessel in brain showing microhemorrhage (long arrow), pyknosis (short arrow), and hyperplasia (arrowhead). (C) Affected blood vessel in brain showing perivascular edema. Bar = 100 μm.

Fig 4

Serosal hyperemia and hemorrhage in the area of the distal cecum adjacent to the junction with the proximal colon in a Dutch Belted rabbit infected with EHEC O157 strain EDL933 on day 7 p.i.

Fig 5

(A) Sham-inoculated normal rabbit cecum. (B) EHEC O157 strain E5252 (CG-3)-infected rabbit cecum with submucosal edema and acute necrotizing heterophilic vasculitis (arrow). (C) Higher magnification of submucosal vascular lesion in B showing heterophilic vasculitis and perivasculitis with fibrinoid vascular degeneration/necrosis and intimal proliferation. (D) Normal rabbit glomerulus. (E) Glomerulus of a rabbit infected with EHEC O157 strain E5252 demonstrating mild capillary thickening (arrows) and few heterophils in distended capillaries. (F) Glomerulus of a rabbit infected with EHEC O157 strain E5252 showing global edematous swelling, luminal constriction, decreased numbers of erythrocytes (“bloodless glomerulus”), and fragmentation of erythrocytes (arrow). All tissue sections from rabbits on day 7 p.i. Bars = ∼150 μm (A and B) and ∼50 μm (C, D, E, and F).

Fig 6

Stx ELISA OD₄₅₀ scores by EHEC O157 genotypes following uninduced, nalidixic acid-induced, or carbadox-induced growth in broth enrichment culture. Five EHEC O157 strains were tested in each genotype (CG-1, CG-3, BBG-5, and BBG-6). All strains are nalidixic acid resistant. *, significantly different from all the other values (P ≤ 0.05), three-way (growth conditions, nalidixic susceptibility, and genogroup) GLM with a Tukey-Kramer multiple comparison test.