## 8 **Murine EHEC Models**  
Relative to humans, mice are fairly resistant to EHEC infection, so murine models include stressors intended to increase susceptibility to intestinal bacterial colonization and infection. These approaches include pre-treatment with antibiotics, germ-free environments, and protein calorie malnutrition diets [117]. Antibiotics added to the animals' drinking water reduces or alters the normal intestinal facultative bacteria, increasing the likelihood of EHEC intestinal colonization by decreasing bacterial competition. An early study used streptomycin to study the colonization abilities of several _E. coli_ isolates [104] and [PERSON] _et al._ adopted streptomycin pre-treatment to develop a murine model of EHEC infection [105]. Streptomycin-treated male CD-1 mice were infected with either wild-type _E. coli_ O157:H7, a mutant strain that did not contain the plasmid encoding fimbiae, or both. Animals demonstrated colonization of the bacteria in all three conditions, though disease symptoms were not observed. However, mice inoculated with the mutant strain that had been previously recovered from the feces of one of the co-infection animals demonstrated symptoms of infection including loose stools, anorexia and lethargy between days 4-10 post-inoculation, with lethality following within a few days. Colonization without infection was the outcome unless the bacteria strain was host-adapted. The colons of these mice did not show gross or histological lesions, indicating that the animals did not die of hemorrhagic colitis. Rather, death appeared to be due to acute necrosis of proximal convoluted tubules, a lesion which is characteristic of toxin exposure in mice. However, the mice did not show evidence of glomerular damage, thrombotic microangiopathy, or thrombocytopenia [105]. It had been demonstrated previously that addition of mitomycin C to _in vitro_ EHEC cultures resulted in induction of phage expression and increased toxin production [118,119]. Young male ICR mice were pre-treated with streptomycin and mitomycin, then infected with EHEC strain E32511/HSC (Stx2c variant) pre-selected to be resistant to both antibiotics. The animals developed weakness and weight loss, but not diarrhea [106]. Microhemorrhages were observed in the brain and spinal cord, as were slight degeneration of tubules in the kidney. Blood urea nitrogen was significantly higher in experimental mice than in controls, suggesting renal injury, but serum creatinine levels did not change and light microscopy kidney pathology examinations were unremarkable. Acute encephalopathy with loss of blood brain barrier function, brain endothelial cell edema, and demyelination were predominant histologic features. Toxin antigen was detected in affected neurons, but it was not clear whether the neurologic symptoms were from primary neuronal lesions or secondary to hypoxia from thrombotic microangiopathy. The rationale behind antibiotic pretreatment of mice can be extended to include germ-free mouse models of EHEC infection. [PERSON] _et al._ showed that infection with a high dose (2 \(\times\) 10\({}^{9}\) CFU) of _E. coli_ O157:H7 was lethal within 7 days of inoculation for germ-free IQI mice. The mice became anorexic and urine retention was associated with renal tubular necrosis [107]. Interestingly, thrombocytopenia and leukocytosis were observed in infected animals. In order to elucidate a role for pro-inflammatory TNF\(\alpha\) in EHEC pathogenesis, the authors included a group of mice pretreated with the cytokine. The addition of TNF\(\alpha\) nearly doubled the lethality from 60% to 100% with the addition of neurologic symptoms. Furthermore, cytokine-treated mice demonstrated histopathologic changes in the kidney including proliferation of glomerular mesangial cells and microthrombosis, and increased cytokine responses in serum, kidney and brains.