## 8 **Murine EHEC Models**
Based on these experiments, the authors concluded that the toxins produced by EHEC and/or bacterial endotoxin, induce production of TNF\(\alpha\) to have an additive effect EHEC pathogenesis [107]. Priming of cells with pro-inflammatory mediators, especially TNF\(\alpha\), is well known to enhance subsequent cellular responses to pathogenic challenge, and this occurs both _in vitro_ and _in vivo_[120]. Similarly, TNF\(\alpha\) induced by bacterial endotoxin enhances subsequent Stx cytotoxicity in rat astrocytes and releases chemokines that may attract PMNs to contribute to neurological injury and loss of endothelial barrier function [121,122]. Though pre-treatment with antibiotics or the use of germ-free animals increases the likelihood of gut EHEC colonization and infection, it does not accurately depict the circumstance when normal gut flora is present as is the case with patients. To address this, alternative models were developed. C3H/HeJ mice have a TLR4 mutation, rendering them moderately immunocompromised and thus more susceptible to infection. These mice were fasted for 24 h prior to infection, and challenged with 1 \(\times\) 10\({}^{9}\) CFU/mL EHEC expressing Stx2 by oral gavage [108]. They developed gastrointestinal symptoms including loose, watery stools, neurological symptoms including ataxia and convulsions, and systemic symptoms including lethargy, anorexia and shivering with \(\sim\)40% mortality. Histologic examination of colon tissue demonstrated inflammatory infiltrates and necrotic foci. In the kidneys, they observed proliferation of glomerular mesangial cells, diffuse interstitial inflammation and necrosis of tubular cells. In addition, the authors noted fragmented red blood cells in the sick mice. Blood samples were not analyzed for markers of kidney function or for platelet numbers, so a full description of HUS pathology was not available [108]. Newly weaned BALB/c mice showed increased susceptibility to EHEC infection and Stx-mediated renal damage without pretreatment to alter commensal flora [109]. Between 22% and 27% of infected young animals developed high urea concentrations in the serum and all of these mice died. Mortality was associated with increased circulating neutrophils, followed by a significant decrease in all circulating leukocytes shortly before death. Colon pathology included leukocyte infiltration, and the kidney showed focal cortical necrosis with tubular epithelial swelling, although thrombotic microangiopathy was not evaluated. To initiate EHEC colonization and infection, murine models require much higher bacteria doses compared to humans ( murine \(\sim\)10\({}^{9}\) CFU _versus_ 10-100 organisms for humans). The observation that a subset of children infected with EHEC had an unbalanced diet prior to infection inspired [PERSON] _et al._ to hypothesize that protein calorie malnourished (PCM) mice would contract disease from lower doses of EHEC than conventional mice. Indeed, the minimal infectious dose of _E. coli_ in the PCM mice was lower at 2 \(\times\) 10\({}^{6}\) CFU. [110]. The infected PCM mice developed systemic and neurologic symptoms, no gastrointestinal symptoms were observed. BUN levels, but not creatinine, increased and acute kidney injury was minimal with mild renal tubular degeneration and normal glomeruli. Significant platelet changes were not observed [110]. The advantage of the PCM murine model is the lower inoculum dose with neurologic complications, but the animals do not appear to develop HUS.