Ivan Tatarov

Virulence of the Shiga Toxin Type 2-Expressing Escherichia coli O104:H4 German Outbreak Isolate in Two Animal Models

ABSTRACT In May 2011, a large food-borne outbreak was traced to an unusual O104:H4 enteroaggregative Escherichia coli (EAEC) strain that produced Shiga toxin (Stx) type 2 (Stx2). We developed a mouse model to study the pathogenesis and treatment for this strain and examined the virulence of the isolate for Dutch belted rabbits. O104:H4 strain C227-11 was gavaged into C57BL/6 mice at 109 to 1011 CFU/animal. The infected animals were then given water with ampicillin (Amp; 5 g/liter) ad libitum. The C227-11-infected, Amp-treated C57BL/6 mice exhibited both morbidity and mortality. Kidneys from mice infected with C227-11 showed acute tubular necrosis, a finding seen in mice infected with typical Stx-producing E. coli. We provided anti-Stx2 antibody after infection and found that all of the antibody-treated mice gained more weight than untreated mice and, in another study, that all of the antibody-treated animals lived, whereas 3/8 phosphate-buffered saline-treated mice died. We further compared the pathogenesis of C227-11 with that of an Stx-negative (Stx−) O104:H4 isolate, C734-09, and an Stx2− phage-cured derivative of C227-11. Whereas C227-11-infected animals lost weight or gained less weight over the course of infection and died, mice infected with either of the Stx− isolates did not lose weight and only one mouse died. When the Stx-positive (Stx+) and Stx2− O104:H4 strains were compared in rabbits, greater morbidity and mortality were observed in rabbits infected with the Stx2+ isolates than the Stx2− isolates. In conclusion, we describe two animal models for EAEC pathogenesis, and these studies show that Stx2 is responsible for most of the virulence observed in C227-11-infected mice and rabbits.

Escherichia coli O157:H7 Infection in Dutch Belted and New Zealand White Rabbits

Wun-Ju Shieh graduated from Taipei Medical University in 1979. He completed an internal medicine residency and infectious disease subspecialty training in 1986. He received a Master of Public Health from Harvard University in 1987, followed by a Ph.D. in Microbiology & Immunology from Vanderbilt University in 1992. Afterwards, he completed a combined anatomical and clinical pathology residency training at Vanderbilt University Medical Center and an infectious disease pathology fellowship at CDC. He has been working as a medical officer and pathologist at CDC since 1995. He has participated many outbreak investigations, and has published more than 120 papers in peer-review journals. The role of pathology in diagnosis of emerging viral zoonosesChi-Chao Chan earned her M.D. from Johns Hopkins University and ophthalmology residency from Stanford University School of Medicine. She has completed two post-doctoral fellowships: ophthalmic pathology at Wilmer Institute, Johns Hopkins and clinical ocular immunology at National Eye Institute, National Institutes of Health. She is the Chief of Immunopathology Section, Laboratory of Immunology and Head of Histopathology Core, National Eye Institute, the federal government medical research institute in the US. She has published 582 papers in peer-reviewed journals, 51 book chapters, and one textbook. She also serves as an editorial board member for 16 medical journals. Diagnosis of primary vitreoretinal lymphoma, a subtype of primary CNS lymphomaEnterohemorrhagic Escherichia coli (EHEC) produce one or more types of Shiga toxins and are foodborne causes of bloody diarrhea. The prototype EHEC strain, Escherichia coli O157:H7, is responsible for both sporadic cases and serious outbreaks worldwide. Infection with E. coli that produce Shiga toxins may lead to diarrhea, hemorrhagic colitis, or (less frequently) hemolytic uremic syndrome, which can cause acute kidney failure. The exact mechanism by which EHEC evokes intestinal and renal disease has not yet been determined. The development of a readily reproducible animal oral-infection model with which to evaluate the full pathogenic potential of E. coli O157:H7 and assess the efficacy of therapeutics and vaccines remains a research priority. Dutch belted (DB) rabbits are reported to be susceptible to both natural and experimental EHEC-induced disease, and New Zealand white (NZW) rabbits are a model for the intestinal manifestations of EHEC infection. In the current study, we compared the pathology caused by E. coli O157:H7 infection in DB and NZW rabbits. Both breeds of rabbits developed clinical signs of disease and intestinal lesions after experimental infection. In addition, one of the infected DB rabbits developed renal lesions. Our findings provide evidence that both breeds are susceptible to E. coli O157:H7 infection and that both may be useful models for investigating EHEC infections of humans.Lymphocytes play key roles in the chronic inflammation critical for T2D pathogenesis. We have shown T2D patients have an elevated ratio of pro- to anti-inflammatory T cells, and B cells that produce a pro-inflammatory cytokine profile. Thus lymphocytes promote T2D-associated inflammation. Numerous studies implicate the pro-inflammatory CD4+ T cell balance in T2D pathogenesis, but mechanisms that underlie elevated CD4+ T cell inflammation are poorly understood. We explored the possibility that the T2D-associated changes we identified in B cell function regulate T cell inflammation. We show that B cells control the T2D-associated increase in Th17-mediated inflammation in T2D patients and in obese/insulin resistant mice. Surprisingly, the disease-associated ability of B cells to regulate T cell function is contact-dependent, despite evidence that B cell cytokines hyper-secreted in T2D patients activate T cells. In contrast, elevated activation of Th1 cytokines is B cell-independent. We conclude that both T cell-intrinsic and T cell-extrinsic (B cell-dependent) changes regulate T cell inflammation in T2D. These data indicate that B cell depletion may partially curb T2D-associated T cell inflammation, and thus disease pathogenesis; however, combinatorial treatments aimed at multiple inflammatory axes may be required for favorable clinical outcomes.

Enhanced Virulence of the Escherichia coli O157:H7 Spinach-Associated Outbreak Strain in Two Animal Models Is Associated with Higher Levels of Stx2 Production after Induction with Ciprofloxacin

ABSTRACT Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and the hemolytic-uremic syndrome (HUS). STEC strains may produce Stx1a and/or Stx2a or variants of either toxin. A 2006 spinach-associated outbreak of STEC O157:H7 resulted in higher hospitalization and HUS rates than previous STEC outbreaks. The spinach isolate, strain K3995, contains both stx 2a and stx 2c. We hypothesized that the enhanced virulence of K3995 reflects the combination of stx 2 alleles (carried on lysogenic phages) and/or the amount of Stx2 made by that strain. We compared the virulence of K3995 to those of other O157:H7 isolates and an isogenic Stx2 mutant in rabbits and mice. We also measured the relative levels of Stx2 produced from those strains with or without induction of the stx-carrying phage. Some rabbits infected with K3995 exhibited intestinal pathology and succumbed to infection, while none of those infected with O157:H7 strain 2812 (Stx1a+ Stx2a+) died or showed pathological signs. Rabbits infected with the isogenic Stx2a mutant K3995 stx 2a::cat were not colonized as well as those infected with K3995 and exhibited no signs of disease. In the streptomycin-treated mouse model, more animals infected with K3995 died than did those infected with O157:H7 strain 86-24 (Stx2a+). Additionally, K3995 produced higher levels of total Stx2 and toxin phage DNA in cultures after phage induction than did 86-24. Our results demonstrate the greater virulence of K3995 compared to other O157:H7 strains in rabbits and mice. We conclude that this enhanced virulence is linked to higher levels of Stx2 expression as a consequence of increased phage induction.

A rabbit model of non-typhoidal Salmonella bacteremia

Bacteremia is an important cause of morbidity and mortality in humans. In this study, we focused on the development of an animal model of bacteremia induced by non-typhoidal Salmonella. New Zealand White rabbits were inoculated with a human isolate of non-typhoidal Salmonella strain CVD J73 via the intra-peritoneal route. Blood samples were collected at specific time points and at euthanasia from infected rabbits. Additionally, tissue samples from the heart, lungs, spleen, gastrointestinal tract, liver and kidneys were obtained at euthanasia. All experimentally infected rabbits displayed clinical signs of disease (fever, dehydration, weight loss and lethargy). Tissues collected at necropsy from the animals exhibited histopathological changes indicative of bacteremia. Non-typhoidal Salmonella bacteria were detected in the blood and tissue samples of infected rabbits by microbiological culture and real-time PCR assays. The development of this animal model of bacteremia could prove to be a useful tool for studying how non-typhoidal Salmonella infections disseminate and spread in humans.

N-glycolylneuraminic acid knockout reduces erythrocyte sequestration and thromboxane elaboration in an ex vivo pig-to-human xenoperfusion model

Wild‐type pigs express several carbohydrate moieties on their cell surfaces that differ from those expressed by humans. This difference in profile leads to pig tissue cell recognition of human blood cells causing sequestration, in addition to antibody‐mediated xenograft injury. One such carbohydrate is N‐glycolylneuraminic acid (Neu5Gc), a sialic acid molecule synthesized in pigs but not in humans. Here, we evaluate livers with and without Neu5Gc in an ex vivo liver xeno perfusion model.

Progress in Xenogeneic Lung Transplantation Using Multi-Transgenic Donor Pigs and Targeted Supportive Drug Treatments

Purpose Significant progress in genetic engineering has enabled the generation of knock-out pigs that additionally express multiple human “transgenes” chosen to address various known xenogeneic rejection pathways. We evaluated which transgene combinations are associated with prevention of lung xenograft injury in a rigorous life-supporting pig-to-baboon lung xenograft model. Methods GTKO.hCD46 porcine donor organs with up to 4 additional genetic modifications, including hCD55, hTBM, hEPCR, hTFPI, hCD47, hCD39, hHO-1, HLA-E, A20 and humanized vWF were used in 48 left single lung transplants into baboons. A rationally designed “platform drug regimen” consisted of steroids, sC1Inh, thromboxane synthase inhibitor, histamine receptor blockers, and anti-GPIb Fab. Desmopressin (DDAVP) was given to the donor animals prior to lung procurement, to deplete pig endothelial VWF. Immunosuppression consisted of aCD20, ATG, MMF, aCD40, coupled with anti-IL6R moAb and/or Alpha-1 Antitrypsin. Xenograft function was assessed intermittently by transplant blood flow measurements and radiographs. Results Xenogeneic lungs with 6 genetic modifications (6-GE) transplanted into baboons survived for up to 8d (hCD55.hEPCR.hTBM.hCD39) and 9d (hEPCR.hTBM.hCD47.HO-1), vs <12hrs for most 3- or 4-GE lungs. Several other donor transgene-combinations resulted in 47d survival. Lungs of GTKO.hCD46.hCD55.hEPCR.hCD47.hTFPI and a few 3- or 4-GE phenotypes (eg including HLA-E and hvWF) consistently exhibited life-supporting (LS) lung function for 24-30h, with relatively normal macro- and microscopic lung appearance until 2-3 d. Lung xenograft failure was usually associated with rebounding anti-pig antibody titer and loss of lung vascular barrier function leading to alveolar flooding and consolidation, consistent with “delayed xenograft rejection”. Accumulated evidence suggests that, in addition to anti-non-Gal antibody, recipient NK cells (HLA-E) and donor macrophage activation (thromboxane) each play important roles to drive the residual inflammation. Conclusion Combining multi-transgenic donor organs with mechanism-directed drug treatments significantly prolongs life-supportive lung xenograft function and recipient survival. Aggregation of existing targeted genetic modifications, along with antibody depletion and mechanism-based drug additions appear likely to successfully control known pathogenic pathways, and further advance lung xenotransplantation towards clinical application. United Therapeutics SRA. NIH U19A1090959.