Alexis García

The Rabbit as a New Reservoir Host of Enterohemorrhagic Escherichia coli

We investigated the prevalence of enterohemorrhagic Escherichia coli (EHEC) in rabbits acquired from two commercial vendors and a local petting zoo. Fecal samples from 34 Dutch Belted (DB) and 15 New Zealand White (NZW) rabbits were cultured; and isolates were biotyped, serotyped, tested by polymerase chain reaction (PCR), and genotyped by repetitive-element sequence–based PCR (Rep-PCR). Seven (25%) of 28 DB rabbits acquired from one commercial source were positive for EHEC, including O153:H- and O153:H7. One (9%) of 11 NZW rabbits from the same source was positive for eae-, stx1+ O153 strains. In contrast, six DB rabbits from another commercial source and four rabbits from a petting zoo were negative for EHEC. Rep-PCR demonstrated that the O153 EHEC and O145 enteropathogenic E. coli were two distinct clones. Our study indicates that rabbits are a new reservoir host of EHEC that may pose a zoonotic risk for humans.

Renal Injury Is a Consistent Finding in Dutch Belted Rabbits Experimentally Infected with Enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) produces Shiga toxin (Stx) and causes renal disease in humans. Dutch Belted (DB) rabbits naturally infected with EHEC O153 develop hemolytic-uremic syndrome-like disease. The aims of this study were to experimentally reproduce O153-induced renal disease in DB rabbits and investigate bacterial and host factors involved in pathogenesis. The pathogenicity of E. coli O157:H7 was also investigated in rabbits. The stx1AB region of O153 was sequenced. By use of liquid chromatography-tandem mass spectrometry, we identified homologs of the Stx receptor, globotriaosylceramide (Gb3), in rabbit kidney extracts. Infected rabbits developed clinical signs and intestinal and kidney lesions. Renal pathological changes consisted of intimal swelling, perivascular edema, erythrocyte fragmentation, capillary thickening, luminal constriction, leukocytic infiltration, mesangial deposits, and changes in Bowmans capsule and space. Sequence analysis of a approximately 7-kb region of the O153 chromosome indicated homology to the Stx1-producing bacteriophage H19B. Our findings indicate that DB rabbits are suitable for the study of the renal manifestations of EHEC infection in humans.

A Naturally Occurring Rabbit Model of Enterohemorrhagic Escherichia coli–Induced Disease

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis and hemolytic-uremic syndrome (HUS) in humans. The exact mechanism by which EHEC induces disease remains unclear because of the lack of a natural animal model for the disease. An outbreak of bloody diarrhea and sudden death was investigated in a group of Dutch belted rabbits. Two of these rabbits harbored enteropathogenic E. coli O145:H(-), and 1 rabbit was coinfected with EHEC O153:H(-). A partial Shiga toxin 1 gene (stx1) fragment from E. coli O153:H(-) was confirmed by Southern blot and sequence analysis. Toxin production was demonstrated by a HeLa cell cytotoxicity assay. Histopathologic findings in all affected rabbits included erosive and necrotizing enterocolitis with adherent bacterial rods, proliferative glomerulonephritis, tubular necrosis, and fibrin thrombi within small vessels and capillaries. Our findings provide evidence for a naturally occurring animal model of EHEC-induced systemic disease that closely resembles human HUS.

Biology and Diseases of Rabbits

Beginning in 1931, an inbred rabbit colony was developed at the Phipps Institute for the Study, Treatment and Prevention of Tuberculosis at the University of Pennsylvania. This colony was used to study natural resistance to infection with tuberculosis (Robertson et al., 1966). Other inbred colonies or well-defined breeding colonies were also developed at the University of Illinois College of Medicine Center for Genetics, the Laboratories of the International Health Division of The Rockefeller Foundation, the University of Utrecht in the Netherlands, and Jackson Laboratories. These colonies were moved or closed in the years to follow. Since 1973, the U.S. Department of Agriculture has reported the total number of certain species of animals used by registered research facilities (1997). In 1973, 447,570 rabbits were used in research. There has been an overall decrease in numbers of rabbits used. This decreasing trend started in the mid-1990s. In 2010, 210,172 rabbits were used in research. Despite the overall drop in the number used in research, the rabbit is still a valuable model and tool for many disciplines.

Genetic Susceptibility to Chronic Hepatitis Is Inherited Codominantly in Helicobacter hepaticus-Infected AB6F1 and B6AF1 Hybrid Male Mice, and Progression to Hepatocellular Carcinoma Is Linked to Hepatic Expression of Lipogenic Genes and Immune Function-Associated Networks

ABSTRACT Helicobacter hepaticus causes hepatitis in susceptible strains of mice. Previous studies indicated that A/JCr mice are susceptible and C57BL/6NCr mice are resistant to H. hepaticus-induced hepatitis. We used F1 hybrid mice derived from A/J and C57BL/6 matings to investigate their phenotype and determine their hepatic gene expression profile in response to H. hepaticus infection. F1 hybrid mice, as well as parental A/J and C57BL/6 mice, were divided equally into control and H. hepaticus-infected groups and euthanized at 18 months postinoculation. Hepatic lesions were evaluated histologically and the differential hepatic gene expression in F1 mice was determined by microarray-based global gene expression profiling analysis. H. hepaticus-infected parental strains including A/J and C57BL/6 mice, as well as F1 mice, developed significant hepatitis. Overall, hepatocellular carcinomas or dysplastic liver lesions were observed in 69% of H. hepaticus-infected F1 male mice and H. hepaticus was isolated from hepatic tissues of all F1 mice with liver tumors. Liver tumors, characterized by hepatic steatosis, developed in livers with high hepatitis scores. To identify gene expression specific to H. hepaticus-induced hepatitis and progression to hepatocellular carcinoma in F1 mice, a method using comparative group transcriptome analysis was utilized. The canonical pathway most significantly enriched was immunological disease. Fatty acid synthase and steaoryl-coenzyme A desaturase, the two rate-limiting enzymes in lipogenesis, were upregulated in neoplastic relative to dysplastic livers. This study suggests a synergistic interaction between hepatic steatosis and infectious hepatitis leading to hepatocellular carcinoma. The use of AB6F1 and B6AF1 mice, as well as genetically engineered mice, on a C57BL/6 background will allow studies investigating the role of chronic microbial hepatitis and steatohepatitis in the pathogenesis of liver cancer.

Hepatobiliary Inflammation, Neoplasia, and Argyrophilic Bacteria in a Ferret Colony

Hepatobiliary disease was diagnosed in eight of 34 genetically unrelated cohabitating pet ferrets (Mustela putorios furo) during a 7-year period. The eight ferrets ranged in age from 5 to 8 years and exhibited chronic cholangiohepatitis coupled with cellular proliferation ranging from hyperplasia to frank neoplasia. Spiral- shaped argyrophilic bacteria were demonstrated in livers of three ferrets, including two with carcinoma. Sequence analysis of a 400-base pair polymerase chain reaction product amplified from DNA derived from fecal bacteria from one ferret demonstrated 98% and 97% similarity to Helicobacter cholecystus and Helicobacter sp. strain 266-11, respectively. The clustering of severe hepatic disease in these cohabitating ferrets suggests a possible infectious etiology. The role of Helicobacter species and other bacteria in hepatitis and/or neoplasia in ferrets requires further study.

Helicobacter hepaticus–Induced Liver Tumor Promotion Is Associated with Increased Serum Bile Acid and a Persistent Microbial-Induced Immune Response

Chronic microbial infection influences cancer progression, but the mechanisms that link them remain unclear. Constitutive androstane receptor (CAR) is a nuclear receptor that regulates enzymes involved in endobiotic and xenobiotic metabolism. CAR activation is a mechanism of xenobiotic tumor promotion; however, the effects of chronic microbial infection on tumor promotion have not been studied in the context of CAR function. Here, we report that CAR limits the effects of chronic infection-associated progression of liver cancer. CAR knockout (KO) and wild-type (WT) male mice were treated with or without the tumor initiator diethylnitrosamine (DEN) at 5 weeks of age and then orally inoculated with Helicobacter hepaticus (Hh) or sterile media at 8 weeks of age. At approximately 50 weeks postinoculation, mice were euthanized for histopathologic, microbiological, molecular, and metabolomic analyses. Hh infection induced comparable hepatitis in WT and KO mice with or without DEN that correlated with significant upregulation of Tnfα and toll receptor Tlr2. Notably, DEN-treated Hh-infected KO mice exhibited increased numbers of liver lobes with dysplasia and neoplasia and increased multiplicity of neoplasia, relative to similarly treated WT mice. Enhanced tumor promotion was associated with decreased hepatic expression of P450 enzymes Cyp2b10 and Cyp3a11, increased expression of Camp, and increased serum concentrations of chenodeoxycholic acid. Together, our findings suggest that liver tumor promotion is enhanced by an impaired metabolic detoxification of endobiotics and a persistent microbial-induced immune response.

17β-Estradiol suppresses Helicobacter pylori-induced gastric pathology in male hypergastrinemic INS-GAS mice

Helicobacter pylori-associated gastric cancer is male predominant and animal studies suggest that sex hormones influence gastric carcinogenesis. We investigated the effects of 17β-estradiol (E2) or castration on H.pylori-induced gastritis in male INS-GAS/FVB/N (Tg(Ins1-GAS)1Sbr) mice. Comparisons were made to previously evaluated sham (n = 8) and H.pylori-infected (n = 8), intact male INS-GAS mice which had developed severe corpus gastritis accompanied by atrophy, hyperplasia, intestinal metaplasia and dysplasia of the epithelium within 16 weeks postinfection (all P < 0.01). Castration at 8 weeks of age had no sparing effect on lesions in uninfected (n = 5) or H.pylori-infected mice (n = 7) but all lesion subfeatures were attenuated by E2 in H.pylori-infected mice (n = 7) (P < 0.001). Notably, inflammation was not reduced but glandular atrophy, hyperplasia, intestinal metaplasia and dysplasia were also less severe in uninfected, E2-treated mice (n = 7) (P < 0.01). Attenuation of gastric lesions by E2 was associated with lower messenger RNA (mRNA) expression of interferon (IFN)-γ (P < 0.05) and interleukin (IL)-1β (P < 0.004), and higher IL-10 (P < 0.02) as well as decreased numbers of Foxp3(+) regulatory T cells when compared with infected intact males. Infected E2-treated mice also developed higher Th2-associated anti-H.pylori IgG1 responses (P < 0.05) and significantly lower Ki-67 indices of epithelial proliferation (P < 0.05). E2 elevated expression of mRNA for Foxp3 (P < 0.0001) and IL-10 (P < 0.01), and decreased IL-1β (P < 0.01) in uninfected, intact male mice compared with controls. Therefore, estrogen supplementation, but not castration, attenuated gastric lesions in H.pylori-infected male INS-GAS mice and to a lesser extent in uninfected mice, potentially by enhancing IL-10 function, which in turn decreased IFN-γ and IL-1β responses induced by H.pylori.

Intravenous Shiga toxin 2 promotes enteritis and renal injury characterized by polymorphonuclear leukocyte infiltration and thrombosis in Dutch Belted rabbits.

Enterohemorrhagic Escherichia coli (EHEC) infection causes hemolytic uremic syndrome, a leading cause of acute renal failure in children. Dutch Belted (DB) rabbits are susceptible to EHEC-induced disease. Using real-time quantitative RT-PCR we measured the renal mRNA expression of cytokines and fibrinolytic factors in DB rabbits challenged with intravenous Shiga toxin 2 (Stx2) (1200 ng/kg). Group 1 rabbits received an incremental dose during an 8-day period whereas Group 2 rabbits received a single dose. Group 1 rabbits developed mild disease. In contrast, Group 2 rabbits developed severe diarrhea, higher levels of circulating polymorphonuclear leukocytes, increased mean platelet volume, and increased fibrinogen levels. Group 2 rabbits developed polymorphonuclear leukocyte infiltration in the intestine and kidney as well as glomerular congestion, luminal constriction, and mesangial glomerulonephropathy. These renal lesions were associated with up-regulation of interleukin-8 (P<0.006), plasminogen activator inhibitor-1 (P<0.04), and tissue plasminogen activator (P<0.05). Circulating Stx2 promoted dose-dependent enteritis and renal injury characterized by inflammation and impaired fibrinolysis leading to thrombosis.

Helicobacter pylori infection does not promote hepatocellular cancer in a transgenic mouse model of hepatitis C virus pathogenesis

Helicobacter pylori (H. pylori) and hepatitis C virus (HCV) infect millions of people and can induce cancer. We investigated if H. pylori infection promoted HCV-associated liver cancer. Helicobacter-free C3B6F1 wild-type (WT) and C3B6F1-Tg(Alb1-HCVN)35Sml (HT) male and female mice were orally inoculated with H. pylori SS1 or sterile media. Mice were euthanized at ~12 mo postinoculation and samples were collected for analyses. There were no significant differences in hepatocellular tumor promotion between WT and HT mice; however, HT female mice developed significantly larger livers with more hepatic steatosis than WT female mice. H. pylori did not colonize the liver nor promote hepatocellular tumors in WT or HT mice. In the stomach, H. pylori induced more corpus lesions in WT and HT female mice than in WT and HT male mice, respectively. The increased corpus pathology in WT and HT female mice was associated with decreased gastric H. pylori colonization, increased gastric and hepatic interferon gamma expression, and increased serum Th1 immune responses against H. pylori. HT male mice appeared to be protected from H. pylori-induced corpus lesions. Furthermore, during gastric H. pylori infection, HT male mice were protected from gastric antral lesions and hepatic steatosis relative to WT male mice and these effects were associated with increased serum TNF-α. Our findings indicate that H. pylori is a gastric pathogen that does not promote hepatocellular cancer and suggest that the HCV transgene is associated with amelioration of specific liver and gastric lesions observed during concurrent H. pylori infection in mice.