PPARD and Interferon Gamma Promote Transformation of Gastric Progenitor Cells and Tumorigenesis in Mice.

BACKGROUND & AIMS: The peroxisome proliferator-activated receptor delta (PPARD) regulates cell metabolism, proliferation, and inflammation and has been associated with gastric and other cancers. Villin-positive epithelial cells are a small population of quiescent gastric progenitor cells. We expressed PPARD from a villin promoter to investigate the role of these cells and PPARD in development of gastric cancer. METHODS: We analyzed gastric tissues from mice that express the Ppard (PPARD1 and PPARD2 mice) from a villin promoter, and mice that did not carry this transgene (controls), by histology and immunohistochemistry. We performed cell lineage-tracing experiments and analyzed the microbiomes, chemokine and cytokine production, and immune cells and transcriptomes of stomachs of these mice. We also performed immunohistochemical analysis of PPARD levels in 2 sets of human gastric tissue microarrays. RESULTS: Thirty-eight percent of PPARD mice developed spontaneous, invasive gastric adenocarcinomas, with severe chronic inflammation. Levels of PPARD were increased in human gastric cancer tissues, compared with nontumor tissues, and associated with gastric cancer stage and grade. We found an inverse correlation between level of PPARD in tumor tissue and patient survival time. Gastric microbiomes from PPARD and control mice did not differ significantly. Lineage-tracing experiments identified villin-expressing gastric progenitor cells (VGPCs) as the origin of gastric tumors in PPARD mice. In these mice, PPARD up-regulated CCL20 and CXCL1, which increased infiltration of the gastric mucosa by immune cells. Immune cell production of inflammatory cytokines promoted chronic gastric inflammation and expansion and transformation of VGPCs, leading to tumorigenesis. We identified a positive-feedback loop between PPARD and interferon gamma signaling that sustained gastric inflammation to induce VGPC transformation and gastric carcinogenesis. CONCLUSIONS: We found PPARD overexpression in VPGCs to result in inflammation, dysplasia, and tumor formation. PPARD and VGPCs might be therapeutic targets for stomach cancer.

Protective role of commensal bacteria in Sjögren Syndrome.

CD25 knock-out (CD25KO) mice spontaneously develop Sjögren Syndrome (SS)-like inflammation. We investigated the role of commensal bacteria by comparing CD25KO mice housed in conventional or germ-free conditions. Germ-free CD25KO mice have greater corneal barrier dysfunction, lower goblet cell density, increased total lymphocytic infiltration score, increased expression of IFN-γ, IL-12 and higher a frequency of CD4+IFN-γ+ cells than conventional mice. CD4+ T cells isolated from female germ-free CD25KO mice adoptively transferred to naive immunodeficient RAG1KO recipients caused more severe Sjögren-like disease than CD4+ T cells transferred from conventional CD25KO mice. Fecal transplant in germ-free CD25KO mice reversed the spontaneous dry eye phenotype and decreased the generation of pathogenic CD4+IFN-γ+ cells. Our studies indicate that lack of commensal bacteria accelerates the onset and severity of dacryoadenitis and generates autoreactive CD4+T cells with greater pathogenicity in the CD25KO model, suggesting that the commensal bacteria or their metabolites products have immunoregulatory properties that protect exocrine glands in the CD25KO SS model.

Timing of gestational exposure to Zika virus is associated with postnatal growth restriction in a murine model.

BACKGROUND: Vertical transmission of Zika virus leads to infection of neuroprogenitor cells and destruction of brain parenchyma. Recent evidence suggests that the timing of infection as well as host factors may affect vertical transmission. As a result, congenital Zika virus infection may only become clinically apparent in the postnatal period. OBJECTIVE: We sought to develop an outbred mouse model of Zika virus vertical transmission to determine if the timing of gestational Zika virus exposure yields phenotypic differences at birth and through adolescence. We hypothesized that later gestational inoculations would only become apparent in adolescence. STUDY DESIGN: To better recapitulate human exposures, timed pregnant Swiss-Webster dams (n = 15) were subcutaneously inoculated with 1 × 104 plaque-forming units of first passage contemporary Zika virus HN16 strain or a mock injection on embryonic day 4, 8, or 12 with bioactive antiinterferon alpha receptor antibody administered in days preceding and proceeding inoculation. The antibody was given to prevent the robust type I interferon signaling cascade that make mice inherently resistant to Zika virus infection. At birth and adolescence (6 weeks of age) offspring were assessed for growth, brain weight, and biparietal head diameters, and Zika virus viral levels by reverse transcription-polymerase chain reaction or in situ hybridization. RESULTS: Pups of Zika virus-infected dams infected at embryonic days 4 and 8 but not 12 were growth restricted (P < .003). Brain weights were significantly smaller at birth (P = .01) for embryonic day 8 Zika virus-exposed offspring. At 6 weeks of age, biparietal diameters were smaller for all Zika virus-exposed males and females (P < .05), with embryonic day 8-exposed males smallest by biparietal diameter and growth-restriction measurements (weight >2 SD, P = .0007). All pups and adolescent mice were assessed for Zika virus infection by reverse transcription-polymerase chain reaction. Analysis of all underweight pups reveled 1 to be positive for neuronal Zika virus infection by in situ hybridization, while a second moribund animal was diffusely positive at 8 days of age by Zika virus infectivity throughout the brain, kidneys, and intestine. CONCLUSION: These findings demonstrate that postnatal effects of infection occurring at single time points continue to be detrimental to offspring in the postnatal period in a subset of littermates and subject to a window of gestational susceptibility coinciding with placentation. This model recapitulates frequently encountered clinical scenarios in nonendemic regions, including the majority of the United States, where travel-related exposure occurs in short and well-defined windows of gestation. Our low rate of infection and relatively rare evidence of congenital Zika syndrome parallels human population-based data.

Sjögren-Like Lacrimal Keratoconjunctivitis in Germ-Free Mice.

Commensal bacteria play an important role in the formation of the immune system but their role in the maintenance of immune homeostasis at the ocular surface and lacrimal gland remains poorly understood. This study investigated the eye and lacrimal gland phenotype in germ-free and conventional C57BL/6J mice. Our results showed that germ-free mice had significantly greater corneal barrier disruption, greater goblet cell loss, and greater total inflammatory cell and CD4⁺ T cell infiltration within the lacrimal gland compared to the conventionally housed group. A greater frequency of CD4⁺IFN-γ⁺ cells was observed in germ-free lacrimal glands. Females exhibited a more severe phenotype compared to males. Adoptive transfer of CD4⁺ T cells isolated from female germ-free mice into RAG1KO mice transferred Sjögren-like lacrimal keratoconjunctivitis. Fecal microbiota transplant from conventional mice reverted dry eye phenotype in germ-free mice and decreased CD4⁺IFN-γ⁺ cells to levels similar to conventional C57BL/6J mice. These findings indicate that germ-free mice have a spontaneous lacrimal keratoconjunctivitis similar to that observed in Sjögren syndrome patients and demonstrate that commensal bacteria function in maintaining immune homeostasis on the ocular surface. Thus, manipulation of intestinal commensal bacteria has the potential to become a novel therapeutic approach to treat Sjögren Syndrome.

Helicobacter Species Identified in Captive Sooty Mangabeys (Cercocebus atys) with Metastatic Gastric Adenocarcinoma.

BACKGROUND: Of all human cancers, gastric carcinoma is the one of the leading causes of death. Helicobacter pylori is considered a major etiologic agent of this disease. Spontaneously occurring gastric carcinoma is a rare diagnosis in nonhuman primates. A 2011 case report documented a high incidence of gastric adenocarcinoma in a closed colony of captive sooty mangabeys (Cercebus atys). However, H. pylori infection was not detected in these animals. MATERIALS AND METHODS: In this study, using archived formalin-fixed, paraffin-embedded stomach sections of these animals alternative methodologies were used to identify H. pylori and other non-H. pylori Helicobacter species. In addition, two additional cases of sooty mangabeys with metastatic gastric carcinoma are characterized. RESULTS: Using fluorescent in situ hybridization, we identified gastric H. suis in 75% of archived and new gastric carcinoma cases. In the two newly reported cases, H. suis and a novel Helicobacter species were detected via PCR and sequence analysis of the 16S rRNA gene. H. pylori was not identified in any of the gastric carcinoma cases via FISH and/or PCR and sequence analysis of Helicobacter spp. in DNA from of available tissues. CONCLUSIONS: This report is the first to characterize Helicobacter species infection in spontaneous gastric carcinoma with metastatic potential in nonhuman primates.

Helicobacter canis colonization in sheep: a Zoonotic link.

BACKGROUND: Helicobacter canis has been associated with hepatobiliary and gastrointestinal disease in dogs, cats, and humans. Infection has not been documented in other species. MATERIALS AND METHODS: Sheep feces subjected to microaerobic culture. Isolates were characterized by genus-specific PCR, restriction fragment length polymorphism, biochemical profiling, and 16S rRNA sequence analysis. RESULTS: Helicobacter canis was isolated from sheep feces and confirmed by the above methods. These isolates are distinct from other sheep-origin enterohepatic Helicobacter species previously isolated. CONCLUSIONS: This study identifies sheep as H. canis reservoirs potentially important in zoonotic or foodborne transmission.

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

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.

Enzootic enteropathogenic Escherichia coli infection in laboratory rabbits.

Enteropathogenic Escherichia coli (EPEC) is the most important cause of persistent diarrhea in children, particularly in developing countries. Animals serve as pathogenic E. coli reservoirs, and compelling evidence for cross-species EPEC transmission exists. In this report, enzootic EPEC infection associated with up to 10.5% diarrhea-associated morbidity in a large laboratory Dutch Belted rabbit colony was investigated. These rabbits were obtained from a commercial vendor and had acute diarrhea following shipment. Fecal culture of 20 rabbits yielded 48 E. coli isolates, 83% of which were eae positive. Repetitive sequence-based PCR (REP-PCR) and serologic analysis identified a single disease-associated EPEC O145:H2 strain. In sampled rabbits, EPEC-positive culture and the presence of diarrhea were significantly associated. This strain displayed a localized adherence-like HEp-2 cell adherence pattern, as seen in diarrheic human infant EPEC isolates. Treatment was instituted with the fluoroquinolone antibiotic enrofloxacin, to which all isolates were susceptible. Preshipment parenteral enrofloxacin administration reduced diarrhea-associated morbidity 22-fold and mortality 12-fold in subsequent deliveries. This report emphasizes the zoonotic potential of animal EPEC strains and the need for virulence determinant-based screening of E. coli isolates from diarrheic animals.

Diet-derived metabolites and mucus link the gut microbiome to fever after cytotoxic cancer treatment.

Not all patients with cancer and severe neutropenia develop fever, and the fecal microbiome may play a role. In a single-center study of patients undergoing hematopoietic cell transplant (n = 119), the fecal microbiome was characterized at onset of severe neutropenia. A total of 63 patients (53%) developed a subsequent fever, and their fecal microbiome displayed increased relative abundances of Akkermansia muciniphila, a species of mucin-degrading bacteria (P = 0.006, corrected for multiple comparisons). Two therapies that induce neutropenia, irradiation and melphalan, similarly expanded A. muciniphila and additionally thinned the colonic mucus layer in mice. Caloric restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Antibiotic treatment to eradicate A. muciniphila before caloric restriction preserved colonic mucus, whereas A. muciniphila reintroduction restored mucus thinning. Caloric restriction of unirradiated mice raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila in vitro with propionate reduced utilization of mucin as well as of fucose. Treating irradiated mice with an antibiotic targeting A. muciniphila or propionate preserved the mucus layer, suppressed translocation of flagellin, reduced inflammatory cytokines in the colon, and improved thermoregulation. These results suggest that diet, metabolites, and colonic mucus link the microbiome to neutropenic fever and may guide future microbiome-based preventive strategies.

A comprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies.

A collection of 130 new plant cell wall glycan-directed monoclonal antibodies (mAbs) was generated with the aim of facilitating in-depth analysis of cell wall glycans. An enzyme-linked immunosorbent assay-based screen against a diverse panel of 54 plant polysaccharides was used to characterize the binding patterns of these new mAbs, together with 50 other previously generated mAbs, against plant cell wall glycans. Hierarchical clustering analysis was used to group these mAbs based on the polysaccharide recognition patterns observed. The mAb groupings in the resulting cladogram were further verified by immunolocalization studies in Arabidopsis (Arabidopsis thaliana) stems. The mAbs could be resolved into 19 clades of antibodies that recognize distinct epitopes present on all major classes of plant cell wall glycans, including arabinogalactans (both protein- and polysaccharide-linked), pectins (homogalacturonan, rhamnogalacturonan I), xyloglucans, xylans, mannans, and glucans. In most cases, multiple subclades of antibodies were observed to bind to each glycan class, suggesting that the mAbs in these subgroups recognize distinct epitopes present on the cell wall glycans. The epitopes recognized by many of the mAbs in the toolkit, particularly those recognizing arabinose- and/or galactose-containing structures, are present on more than one glycan class, consistent with the known structural diversity and complexity of plant cell wall glycans. Thus, these cell wall glycan-directed mAbs should be viewed and utilized as epitope-specific, rather than polymer-specific, probes. The current world-wide toolkit of approximately 180 glycan-directed antibodies from various laboratories provides a large and diverse set of probes for studies of plant cell wall structure, function, dynamics, and biosynthesis.

Comparison of Four Beddings for Ammonia Control in Individually Ventilated Mouse Cages.

Ammonia control is an important characteristic of rodent bedding materials. Among natural bedding materials, corncob bedding provides excellent ammonia control but contains estrogenic compounds and is ingested by mice. By comparison, processed cellulose bedding products are biologically inert and harbor fewer bacteria but historically have shown low absorbency or poor ammonia control. New cellulose products have been developed to address these shortcomings. Over a 2-wk period, we evaluated intracage ammonia levels in mouse IVC using 4 bedding types: shaved aspen, corncob, virgin pelleted cellulose, and refined virgin diced cellulose. Ammonia levels were measured by using 3 methods: colored reagent tubes, colorimetric paper strips, and a photoionization detector. Corncob, pelleted cellulose, and diced cellulose showed better ammonia control than aspen as early as 4 d after cage changing and throughout the 2-wk measurement period. In addition, pelleted and diced cellulose products resulted in lower ammonia levels than corncob at the end of the 14-d cage-change interval. Our data indicate that pelleted or refined diced cellulose are viable alternatives to natural bedding products in IVC to limit the risk of exposure of mice to high ammonia levels.

Helicobacter suis and Helicobacter pylori infection in a colony of research macaques: characterization and clinical correlates.

Introduction. Helicobacter suis (Helicobacter heilmannii type 1) commonly infects nonhuman primates but its clinical importance is in question.Aim. To characterize H. suis infection in a colony of rhesus macaques (Macaca mulatta) used in cognitive neuroscience research.Hypothesis/Gap Statement. Inquiries into the nature of Helicobacter suis in nonhuman primates are required to further define the organism's virulence and the experimental animal's gastric microbiome.Methodology. Animals with and without clinical signs of vomiting and abdominal pain (n=5 and n=16, respectively) were evaluated by histology, culture, PCR amplification and sequencing, fluorescent in situ hybridization (FISH) and serology. Three of the five animals with clinical signs, an index case and two others, were evaluated before and after antimicrobial therapy.Results. The index animal had endoscopically visible ulcers and multifocal, moderate, chronic lymphoplasmacytic gastritis with intraglandular and luminal spiral bacteria. Antimicrobial therapy in the index animal achieved histologic improvement, elimination of endoscopically visible ulcers, and evident eradication but clinical signs persisted. In the other treated animals, gastritis scores were not consistently altered, gastric bacteria persisted, but vomiting and abdominal discomfort abated.Nineteen of 21 animals were PCR positive for H. suis and five animals were also PCR positive for H. pylori. Organisms were detected by FISH in 17 of 21 animals: 16S rRNA sequences of two of these were shown to be H. suis. Mild to moderate lymphoplasmacytic gastritis was seen in antrum, body and cardia, with antral gastritis more likely to be moderate than that of the body.Conclusion. No clear association between the bacterial numbers of Helicobacter spp. and the degree of inflammation was observed. H. suis is prevalent in this colony of Macaca mulatta but its clinical importance remains unclear. This study corroborates many of the findings in earlier studies of H. suis infection in macaques but also identifies at least one animal in which gastritis and endoscopically visible gastric ulcers were strongly associated with H. suis infection. In this study, serology was an inadequate biomarker for endoscopic evaluation in diagnosis of H. suis infection.

Cytotoxic Escherichia coli strains encoding colibactin and cytotoxic necrotizing factor (CNF) colonize laboratory macaques.

BACKGROUND: Many Escherichia coli strains are considered to be a component of the normal flora found in the human and animal intestinal tracts. While most E. coli strains are commensal, some strains encode virulence factors that enable the bacteria to cause intestinal and extra-intestinal clinically-relevant infections. Colibactin, encoded by a genomic island (pks island), and cytotoxic necrotizing factor (CNF), encoded by the cnf gene, are genotoxic and can modulate cellular differentiation, apoptosis and proliferation. Some commensal and pathogenic pks+ and cnf+ E. coli strains have been associated with inflammation and cancer in humans and animals. RESULTS: In the present study, E. coli strains encoding colibactin and CNF were identified in macaque samples. We performed bacterial cultures utilizing rectal swabs and extra-intestinal samples from clinically normal macaques. A total of 239 E. coli strains were isolated from 266 macaques. The strains were identified biochemically and selected isolates were serotyped as O88:H4, O25:H4, O7:H7, OM:H14, and OM:H16. Specific PCR for pks and cnf1 gene amplification, and phylogenetic group identification were performed on all E. coli strains. Among the 239 isolates, 41 (17.2%) were pks+/cnf1-, 19 (7.9%) were pks-/cnf1+, and 31 (13.0%) were pks+/cnf1+. One hundred forty-eight (61.9%) E. coli isolates were negative for both genes (pks-/cnf1-). In total, 72 (30.1%) were positive for pks genes, and 50 (20.9%) were positive for cnf1. No cnf2+ isolates were detected. Both pks+ and cnf1+ E. coli strains belonged mainly to phylogenetic group B2, including B21. Colibactin and CNF cytotoxic activities were observed using a HeLa cell cytotoxicity assay in representative isolates. Whole genome sequencing of 10 representative E. coli strains confirmed the presence of virulence factors and antibiotic resistance genes in rhesus macaque E. coli isolates. CONCLUSIONS: Our findings indicate that colibactin- and CNF-encoding E. coli colonize laboratory macaques and can potentially cause clinical and subclinical diseases that impact macaque models.

Adult-onset, chronic, cyclic thrombocytopenia in a Rhesus macaque (Macaca mulatta) after dengue virus vaccination and viral challenge.

An 8-year-old, male Rhesus macaque (Macaca mulatta), previously used for dengue virus (DENV) vaccine research with viral challenge, was presented with adult-onset, chronic, cyclic thrombocytopenia. Platelet number, morphology, and function were evaluated by automated hematology, peripheral blood smears, electron microscopy, flow cytometry, and impedance aggregometry. Bone marrow was evaluated by cytology. Both serum anti-dengue nonstructural protein 1 (NS1) antibodies and anti-platelet antibodies were detected by ELISA. Platelet characterization showed a lack of aggregation to all agonists (ADP, ASP, and collagen), increased activation with increased expression of surface marker (HLA-ABC), and an absence of surface receptor GPIX during clinical episodes of petechiae and ecchymoses, even in the presence of normal platelet counts. Bone marrow aspirates identified potential mild megakaryocytic hypoplasia. All platelet functions and morphologic attributes were within normal limits during clinically normal phases. Presence of anti-dengue NS1 serum antibodies confirmed a positive DENV titer 8 years postvaccination. Based on the history and clinical findings, a primary differential diagnosis for this chronic, cyclic platelet pathology was autoimmune platelet destruction with potential bone marrow involvement.

Differences in virulence between two serotypes of Ichthyophthirius multifiliis.

Naive channel catfish Ictalurus punctatus were infected by 2 isolates of the parasitic ciliate Ichthyophthirius multifiliis that differed in virulence. The isolates, NY1 and G5, Serotypes A and D, respectively, express different surface immobilization-antigens. The virulence of the 2 isolates was compared using tail-fin infections to quantitate parasite numbers and by analysis of the survival of infected fish. Although NY1 infected fish at a lower level than G5, all NY1-infected fish died, but 51% of G5-infected fish survived. The greater virulence of NY1 is apparently a consequence of its shorter life cycle, which results in overwhelming reinfection of fish before they can develop a protective immune response. This report represents the first experimental evidence for differences in virulence between serotypes of I. multifiliis.

Enterohepatic Helicobacter spp. in cats with non-haematopoietic intestinal carcinoma: a survey of 55 cases.

Several enterohepatic Helicobacter spp. (EHS) have been isolated from cats. Despite the reported association between EHS infection and intestinal neoplasia in other species, this association has not been explored in cats. In this study, 55 non-haematopoietic feline intestinal carcinoma cases were histopathologically evaluated. In contrast with prior reports, large intestinal (LI) carcinoma was observed with greater frequency (61 %) relative to small intestinal (SI) carcinoma (35 %). There was a significant association between intestinal location and animal gender. Of males examined, 83 % had LI carcinoma, while no such trend was observed in females. Previously described associations between Siamese breed and intestinal carcinoma could not be definitively confirmed, although the Siamese breed may be predisposed to SI carcinoma location. Of all carcinomas examined in this study, 62 % were classified as adenocarcinoma, although mucinous adenocarcinoma (28 %) and solid carcinoma (11 %) were also identified. Tumours were all moderately or poorly differentiated. When considered by intestinal location and histopathologic classification, LI adenocarcinoma was associated with significantly advanced mean age (13 years) when compared to SI adenocarcinoma and LI mucinous adenocarcinoma (mean, 9 years in both cases), which were also frequently encountered. To determine whether EHS might play a role in feline intestinal neoplasia, Helicobacter genus- and species-specific fluorescence in situ hybridization was performed. Of these carcinoma cases, 56 % were positive for Helicobacter spp. and one or more species-specific assay for Helicobacterbilis, Helicobactercanis or Helicobactermarmotae. The presence of EHS was significantly associated with both LI location (68 %) and mucinous adenocarcinoma (92 %). These findings suggest a role for intestinal bacteria in non-haematopoietic feline intestinal neoplasia.

Helicobacter bilis and Helicobacter trogontum: infectious causes of abortion in sheep.

The aim of our study was to determine the association of Helicobacter spp. that had flexispira morphology with ovine abortion, and to understand the importance of these organisms as a cause of ovine abortion in New Zealand. A retrospective diagnostic survey was carried out on laboratory submissions from ovine abortion outbreaks. A comparison was made of the proportion of laboratory submissions where Helicobacter spp. were detected from flocks that had no other agent identified (group A) with a group that had a known cause of abortion identified (group B). This latter group was considered to be a negative control, given the premise that Helicobacter spp. were not causing abortions and that Helicobacter spp. should be present at a lower rate in the group. Where no diagnosis had been made, aborted material was positive for Helicobacter spp. with flexispira morphology in 8 submissions (20%, 8/40) from 5 of the 31 survey farms (16%, 5/31). Helicobacter spp. were not detected in any of the 18 submissions from the 17 control farms (group B). Helicobacter spp. were confirmed by 16S ribosomal RNA sequencing of 3 of the Helicobacter spp. isolated by culture from the livers of aborted sheep fetuses, and 7 of the 8 where samples were positive in a Helicobacter PCR assay. The Helicobacter spp. were identified as Helicobacter trogontum (Flexispira taxon 5 genotype) and Helicobacter bilis (Flexispira taxon 8 genotype). The findings support Helicobacter spp. being a probable causative agent of ovine abortions in New Zealand.

Postnatal epigenetic regulation of intestinal stem cells requires DNA methylation and is guided by the microbiome.

BACKGROUND: DNA methylation is an epigenetic mechanism central to development and maintenance of complex mammalian tissues, but our understanding of its role in intestinal development is limited. RESULTS: We use whole genome bisulfite sequencing, and find that differentiation of mouse colonic intestinal stem cells to intestinal epithelium is not associated with major changes in DNA methylation. However, we detect extensive dynamic epigenetic changes in intestinal stem cells and their progeny during the suckling period, suggesting postnatal epigenetic development in this stem cell population. We find that postnatal DNA methylation increases at 3' CpG islands (CGIs) correlate with transcriptional activation of glycosylation genes responsible for intestinal maturation. To directly test whether 3' CGI methylation regulates transcription, we conditionally disrupted two major DNA methyltransferases, Dnmt1 or Dnmt3a, in fetal and adult intestine. Deficiency of Dnmt1 causes severe intestinal abnormalities in neonates and disrupts crypt homeostasis in adults, whereas Dnmt3a loss was compatible with intestinal development. These studies reveal that 3' CGI methylation is functionally involved in the regulation of transcriptional activation in vivo, and that Dnmt1 is a critical regulator of postnatal epigenetic changes in intestinal stem cells. Finally, we show that postnatal 3' CGI methylation and associated gene activation in intestinal epithelial cells are significantly altered by germ-free conditions. CONCLUSIONS: Our results demonstrate that the suckling period is critical for epigenetic development of intestinal stem cells, with potential important implications for lifelong gut health, and that the gut microbiome guides and/or facilitates these postnatal epigenetic processes.

Helicobacter hepaticus infection promotes hepatitis and preneoplastic foci in farnesoid X receptor (FXR) deficient mice.

Farnesoid X receptor (FXR) is a nuclear receptor that regulates bile acid metabolism and transport. Mice lacking expression of FXR (FXR KO) have a high incidence of foci of cellular alterations (FCA) and liver tumors. Here, we report that Helicobacter hepaticus infection is necessary for the development of increased hepatitis scores and FCA in previously Helicobacter-free FXR KO mice. FXR KO and wild-type (WT) mice were sham-treated or orally inoculated with H. hepaticus. At 12 months post-infection, mice were euthanized and liver pathology, gene expression, and the cecal microbiome were analyzed. H. hepaticus induced significant increases hepatitis scores and FCA numbers in FXR KO mice (P<0.01 and P<0.05, respectively). H. hepaticus altered the beta diversity of cecal microbiome in both WT and FXR KO mice compared to uninfected mice (P<0.05). Significant upregulation of ß-catenin, Rela, Slc10a1, Tlr2, Nos2, Vdr, and Cyp3a11 was observed in all FXR KO mice compared to controls (P<0.05). Importantly, H. hepaticus and FXR deficiency were necessary to significantly upregulate Cyp2b10 (P<0.01). FXR deficiency was also a potent modulator of the cecal microbiota, as observed by a strong decrease in alpha diversity. A significant decrease in Firmicutes, particularly members of the order Clostridiales, was observed in FXR KO mice (P<0.05 and FDR<5%, ANOVA). While FXR deficiency strongly affects expression of genes related to immunity and bile acid metabolism, as well as the composition of the microbiome; however, its deficiency was not able to produce significant histopathological changes in the absence of H. hepaticus infection.

Enteropathogenic Escherichia coli prevalence in laboratory rabbits.

Rabbit-origin enteropathogenic Escherichia coli (EPEC) causes substantial diarrhea-associated morbidity and has zoonotic potential. A culture-based survey was undertaken to ascertain its prevalence. EPEC was isolated from 6/141 (4.3%) commercially-acquired laboratory rabbits. Three of these did not have diarrhea or EPEC-typical intestinal lesions; they instead had background plasmacytic intestinal inflammation. Asymptomatically infected rabbits may function as EPEC reservoirs.