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The transmission of simian immunodeficiency and Ebola viruses to humans in recent years has heightened awareness of the public health significance of zoonotic diseases of primate origin, particularly from chimpanzees. In this study, we analyzed 71 fecal samples collected from 2 different wild chimpanzee ( Pan troglodytes ) populations with different histories in relation to their proximity to humans. Campylobacter spp. were detected by culture in 19/56 (34%) group 1 (human habituated for research and tourism purposes at Mahale Mountains National Park) and 0/15 (0%) group 2 (not human habituated but propagated from an introduced population released from captivity over 30 years ago at Rubondo Island National Park) chimpanzees, respectively. Using 16S rRNA gene sequencing, all isolates were virtually identical (at most a single base difference), and the chimpanzee isolates were most closely related to Campylobacter helveticus and Campylobacter upsaliensis (94.7% and 95.9% similarity, respectively). Whole-cell protein profiling, amplified fragment length polymorphism analysis of genomic DNA, hsp60 sequence analysis, and determination of the mol% G+C content revealed two subgroups among the chimpanzee isolates. DNA-DNA hybridization experiments confirmed that both subgroups represented distinct genomic species. In the absence of differential biochemical characteristics and morphology and identical 16S rRNA gene sequences, we propose to classify all isolates into a single novel nomenspecies, Campylobacter troglodytis , with strain MIT 05-9149 as the type strain; strain MIT 05-9157 is suggested as the reference strain for the second C. troglodytis genomovar. Further studies are required to determine whether the organism is pathogenic to chimpanzees and whether this novel Campylobacter colonizes humans and causes enteric disease.

The incidence of gastric cancer is higher in men than women. Epidemiological studies suggest that female hormones reduce gastric cancer risk. We examined the effect of ovarian-dependent female hormones on Helicobacter pylori-induced gastric cancer in hypergastrinemic INS-GAS mice. Male and female sexually intact or ovariectomized (OVX) mice were inoculated with H.pylori SS1 or vehicle-only at 10 weeks of age, and tissues were evaluated at 16 or 28 weeks post-infection (WPI). A subset of OVX females were supplemented with 17beta-estradiol (E2), beginning at 16 WPI. Stomachs were evaluated by histopathology, Ki-67 proliferation index, H.pylori quantitative culture and quantitative polymerase chain reaction for messenger RNA expression of inducible nitric oxide synthase (iNOS) and inflammatory cytokines. Infected OVX females developed significantly more severe gastritis (P < 0.05) than infected intact females at both time points. E2 treatment in infected OVX females attenuated the severity of gastritis. Gastrointestinal intraepithelial neoplasia (GIN) developed in 42% of infected males and 10% of infected OVX females by 28 WPI, whereas infected intact females and E2-treated OVX females did not develop GIN. Infected OVX females showed significantly increased iNOS expression and epithelial cell proliferation when compared with intact, infected females. Likewise, interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta (IL-1beta) expression in infected OVX females were significantly increased at 28 WPI when compared with intact counterparts. E2 treatment in infected OVX females significantly decreased IL-1beta expression, increased IL-10 expression and reduced epithelial cell proliferation. These results demonstrate a protective effect of E2 in H.pylori-induced gastric cancer in a mouse model.

Bacterial cytolethal distending toxins (CDTs) containing DNase I-like activity can induce limited host DNA damage that leads to activation of the DNA-damage repair responses in cultured cell lines. However, in vivo experimental evidence linking CDTs to carcinogenesis is lacking. In this study, infection of A/JCr mice with an isogenic mutant of Helicobacter hepaticus lacking CDT activity (CDT mutant) induced chronic hepatitis comparable to wild-type H. hepaticus (Hh) infection at both 4 and 10 months post inoculation (MPI); however, the CDT mutant-infected mice did not develop hepatic dysplasic nodules at 10 MPI, whereas those infected with Hh did. There was no significant difference in hepatic colonization levels between the CDT mutant and Hh at both time points (P > 0.05). At 4 MPI, mice infected with Hh had significantly enhanced hepatic transcription of proinflammatory TNF-alpha, IFN-gamma and Cox-2, growth mediators IL-6 and TGF-alpha, anti-apoptotic Bcl-2 and Bcl-X(L), and increased hepatocyte proliferation (P < 0.05) compared with the control or the CDT mutant-infected mice. In addition, Hh infected male mice had upregulated hepatic mRNA levels of RelA (p65), p50, GADD45beta and c-IAP1, components of the NF-kappaB pathway compared with the CDT mutant-infected mice. At 10 MPI, Hh infection was associated with significant upregulation of IL-6 mRNA. Activation of the inflammatory NF-kappaB pathway and upregulation of proinflammatory cytokines plus IL-6 in the Hh but not in the CDT mutant-infected mice suggest that Hh CDT plays a key role in promoting the dysplastic changes in Hh-infected mouse livers.

The discovery of Helicobacter hepaticus and its role in hepatitis, hepatocellular carcinoma, typhlocolitis, and lower-bowel carcinoma in murine colonies was followed by the isolation and characterization of other Helicobacter spp. involved in enterohepatic disease. Colonization of mouse colonies with members of the family Helicobacteriaceae has become an increasing concern for the research community. From 2001 to 2005, shipments of selected gift mice from other institutions and mice received from specified commercial vendors were screened for Helicobacter spp. by culture of cecal tissue. The identities of the isolates were confirmed by genus-specific PCR, followed by species-specific PCR and restriction fragment length polymorphism analysis. Sequencing of the 16S rRNA gene was performed if the species identity was not apparent. The survey included 79 mice from 34 sources: 2 commercial sources and 16 research sources from the United States and 1 commercial source and 15 research sources from Canada, Europe, or Asia. Helicobacter spp. were cultured from the ceca of 62 of 79 mice. No Helicobacter spp. were found in mice from advertised Helicobacter -free production areas from two U.S. vendors. Multiple Helicobacter spp. were found in mice from one vendor's acknowledged Helicobacter -infected production area. The European commercial vendor had mice infected with novel Helicobacter sp. strain MIT 96-1001. Of the U.S. academic institutions, 6 of 16 (37%) had mice infected with Helicobacter hepaticus ; but monoinfection with H. bilis , H. mastomyrinus , H. rodentium , and MIT 96-1001 was also encountered, as were mice infected simultaneously with two Helicobacter spp. Non-U.S. academic institutions had mice that were either monoinfected with H. hepaticus , monoinfected with seven other Helicobacter spp., or infected with a combination of Helicobacter spp. This survey indicates that 30 of 34 (88%) commercial and academic institutions in Canada, Europe, Asia, Australia, and the United States have mouse colonies infected with Helicobacter spp. Mice from 20 of the 34 institutions (59%) were most commonly colonized with H. hepaticus alone or in combination with other Helicobacter spp. These results indicate that a broad range of Helicobacter spp. infect mouse research colonies. The potential impact of these organisms on in vivo experiments continues to be an important issue for mice being used for biomedical research.

Numbers of nonmigratory Canada geese have increased substantially in the past decade, and they have become a nuisance in some urban areas. Because of their close contact with humans in parks and areas adjacent to surface waterways, contact with their feces poses a zoonotic risk. A total of 97 geese from 10 separate geographic locales in the greater Boston area had their feces sampled for detection of Helicobacter spp. Identification of Helicobacter spp. based on 16S rRNA genus-specific helicobacter primers was noted in 39 of 97 (40.2%) DNA fecal extracts. Twenty-seven (27.8%) of these geese had helicobacters isolated from their feces. A urease-positive novel species, Helicobacter anseris , based on phenotypic, biochemical, and 16S rRNA analyses, was isolated from 20 geese from seven different flocks. A second, novel, urease-negative Helicobacter sp., H. brantae , was identified in seven geese. Four geese had both novel Helicobacter spp. cultured from their feces. Whether these two novel helicobacters pose a zoonotic risk, similar to other enteric helicobacters (e.g., H. canadensis , previously isolated from diarrheic and bacteremic humans and from geese in Europe), will require further studies.

Helicobacter spp. have been implicated in a variety of gastrointestinal tract diseases, including peptic ulcer disease, gastric cancer, and inflammatory bowel disease (IBD), in humans and animals. Although most models of IBD are experimentally induced, spontaneous or natural models of IBD are rare. Herein, we describe a long-term study of chronic, progressive lesions that develop in the distal portion of the large bowel of unmanipulated Syrian hamsters naturally infected with Helicobacter spp. Twenty-four Syrian hamsters of three age groups (group A, 1 month [n = 4], group B, 7–12 months [n = 12], group C, 18–24 months [n = 12]), underwent complete postmortem examination. Results of microbial isolation and polymerase chain reaction and restriction fragment length polymorphism analyses confirmed the presence of Helicobacter spp. infection in the distal portion of the large bowel of all animals. Additionally, confounding pathogens, such as Clostridium difficile, Lawsonia intracellularis, and Giardia spp. that can cause proliferative enteritis, were absent in the hamsters of this study. Histopathologic scores for inflammation ( P < 0.01), hyperplasia ( P < 0.01), and dysplasia ( P < 0.05) were significantly higher in the ileocecocolic (ICC) junction of animals in group C, relative to group A. Dysplastic lesions of various grades were detected in 5 of 11 hamsters in group C. Interestingly, the segment of the bowel that is usually colonized by Helicobacter spp. in hamsters had the most severe lesions. One hamster of group C developed a malignant fibrous histiocytoma, whereas another hamster developed a round cell sarcoma originating from the ICC junction. Thus, lesions in the distal portion of the large bowel of aging hamsters naturally colonized with Helicobacter spp. warrants developing the hamster as an animal model of IBD and potentially IBD-related cancer.

Helicobacter cetorum sp. nov. has been cultured from the stomach of Atlantic white-sided dolphins ( Lagenorhynchus acutus ) and the feces of Pacific white-sided ( L. obliquidens ) and Atlantic bottlenose ( Tursiops truncatus ) dolphins and a beluga whale ( Delphinapterus leucas ). H. cetorum has high homology to Helicobacter pylori as shown by 16S rRNA sequencing, and H. cetorum infection has been associated with gastritis and clinical signs in cetaceans. Because the prevalence of H. cetorum in wild populations is unknown, minimally invasive techniques for detecting H. cetorum were compared for 20 wild bottlenose dolphins sampled as part of a long-term health study. Fecal samples were tested for helicobacter by culture, Southern blotting, and PCR using genus-specific and H. cetorum- specific primers. An enzyme-linked immunosorbent assay (ELISA) was developed to measure H. cetorum immunoglobulin G (IgG). H. cetorum was cultured from 4 of 20 fecal samples, 7 samples were positive using Helicobacter sp. PCR, and 8 samples were positive for H. cetorum using species-specific primers. Two additional fecal samples were positive by Helicobacter sp. Southern blotting, suggesting infection with another helicobacter. All 20 sera contained high levels of IgG antibodies to H. cetorum that were significantly lowered by preabsorption of the sera with whole-cell suspensions of H. cetorum ( P < 0.02). Until the specificity of the serum ELISA can be determined by testing sera from dolphins confirmed to be uninfected, PCR and Southern blot screenings of feces are the most sensitive techniques for detection of H. cetorum , and results indicate there is at least a 50% prevalence of H. cetorum infection in these dolphins.

Since the recent discovery of Helicobacter cetorum in cetaceans and its role in the development of gastritis, speculation has existed as to whether pinnipeds have Helicobacter spp. associated gastritis and peptic ulcer disease. The gastric mucosa of 4 stranded harp seals Phoca groenlandica from the Massachusetts coastline were assessed for Helicobacter spp. by culture and PCR. We cultured 2 novel Helicobacter spp. from the pyloric antrum of 1 of the 4 harp seals studied, and identified these by PCR in 2 of the 4 seals. Both gram-negative bacterial isolates were catalase- and oxidase-positive. However, a fusiform helicobacter with flexispira morphology was urease-positive, and a spiral-shaped helicobacter was urease-negative. Slender, spiral and fusiform-shaped bacteria were detected in the gastric mucosa by the Warthin-Starry stain. Histopathologic analysis revealed mild diffuse lymphoplasmacytic gastritis within the superficial mucosa of the pyloric antrum of both infected seals. The 2 bacterial isolates were classified by 16S rRNA analysis; they clustered with other enteric helicobacters and represent 2 novel Helicobacter spp. The urease-negative bacterial isolate clustered with H. canis and the urease-positive isolate clustered with an isolate from a sea lion and isolates from sea otters. This cluster of pinniped isolates has 97 % similarity to a number of Helicobacter species, but appears to be most closely related to other helicobacters with flexispira morphology. These findings suggest that the novel Helicobacter spp. may play a role in the etiopathogenesis of gastrointestinal diseases in pinnipeds. To our knowledge, this represents the first isolation and characterization of a novel Helicobacter spp. from pinnipeds.

On the basis of biochemical, phenotypic, and 16S rRNA analyses, Helicobacter cinaedi was isolated from the colon, liver, and mesenteric lymph nodes of a 2-year-old rhesus monkey with chronic diarrhea. Histologically, the liver had mild to moderate biliary hyperplasia and hypertrophy with periportal inflammation and fibrosis. Colonic and cecal lesions consisted of diffuse chronic inflammation and glandular hyperplasia extending the length of the crypts. This is the first observation of H. cinaedi associated with active hepatitis and colitis in a nonhuman primate.

Helicobacter bilis and H. hepaticus, both urease-positive intestinal helicobacters of mice, have been shown experimentally to induce proliferative typhlocolitis in scid mice. We recently isolated a urease-negative Helicobacter sp. (H. sp.) that also induced proliferative typhlocolitis in pilot studies in scid mice. To determine the pathogenic potential of H. sp. in immunocompromised and immunocompetent mice, 5-week old male A/J or Tac:Icr:Ha(ICR)-scidfRF mice were inoculated by intraperitoneal (IP) injection with - 3 × 107 colony-forming units (CFU) of H. sp. Mice were necropsied at various time points postinoculation (PI). Sham-inoculated mice had no clinical, gross, or histopathological lesions. In contrast, scid mice inoculated IP with H. sp. had severe hemorrhagic diarrhea and decreased weight gain at 2, 7, and 18 weeks postinoculation (PI), with severe proliferative typhlocolitis, phlebothrombosis, and hepatitis. A/J mice had no clinical signs, but had mild to moderate proliferative typhlocolitis and moderate to marked cholangiohepatitis at 7 and 24 weeks PI. A/J mice infected with H. sp. developed robust immune responses of a predominant Th1 type. This report demonstrates that infection with a urease-negative helicobacter can cause inflammatory bowel disease (IBD) and hepatitis in scid and immunocompetent A/J mice. These results provide a new model of IBD and cholangiohepatitis associated with a specific urease-negative, novel H. species.

A novel helicobacter with the proposed name Helicobacter aurati (type strain MIT 97-5075c) has been isolated from the inflamed stomachs and ceca of adult Syrian hamsters. The new species is fusiform with multiple bipolar sheathed flagella and periplasmic fibers; it contains urease and gamma-glutamyl transpeptidase. By 16S rRNA sequencing and repetitive element PCR-based DNA fingerprinting, it was found that H. aurati represents a distinct taxon and clusters with Helicobacter muridarum , Helicobacter hepaticus , and Helicobacter sp. MIT 94-022. H. aurati was recovered from hamsters housed in various research and vendor facilities. Further studies are necessary to define its association with disease and other microbiota in hamsters, as well as its impact on research projects involving hamsters. H. aurati (GenBank accession number AF297868 ) can be used in animal experiments to define the factors that are important for gastric helicobacter pathogenesis.

A natural infection with Helicobacter pylori (H. pylori) in domestic cats (Felis cattus) less than 2 years of age has been well described in a closed colony of animals. Six cats from this colony that were serially evaluated by culture, polymerase chain reaction, and light and electron microscopy for a period of 3 years demonstrated persistent gastric colonization with a single cag(-) vac(+) strain of H. pylori. In these cats, as well as five other 5- to 6-year-old cats that were examined, a long-term infection resulted in chronic diffuse lymphofollicular atrophic gastritis with areas of mucosal dysplasia in the antrum and predominantly midsuperficial gastritis in the body and cardia. Topographically, the distribution of lesions was similar in both young and older cats and closely resembled that found in humans, with the most severe changes occurring in the gastric antrum. Few granulocytes and no significant elevation in mast cells were seen in older H. pylori-infected cats compared with uninfected controls; however, marked increases in interepithelial globule leukocytes and numerous active mucosal lymphoid follicles were present in infected animals. Indices of gastritis were significantly greater in older infected cats when compared with uninfected controls and younger cats (P0.05). The antral cell proliferation index of infected older cats was significantly (P = 0.021) greater than that of uninfected controls. Apoptotic indices of the gastric antrum and body of infected cats were significantly (P = 0.01) increased versus controls. Chronic infection with H. pylori in cats shares many features of long-term H. pylori infection in humans, including the development of preneoplastic processes. This similarity provides useful, comparative insights into host-pathogen interactions.

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.

Helicobacter cinaedi colonizes a wide host range, including rodents, and may be an emerging zoonotic agent. Colonization parameters, pathology, serology, and inflammatory responses to wild-type H. cinaedi (WT Hc ) were evaluated in B6.129P2- IL-10 tm1Cgn (IL-10 −/− ) mice for 36 weeks postinfection (WPI) and in C57BL/6 (B6) mice for 12 WPI. Because cytolethal distending toxin (CDT) may be a virulence factor, IL-10 −/− mice were also infected with the cdtB Hc and cdtB -N Hc isogenic mutants and evaluated for 12 WPI. Consistent with other murine enterohepatic helicobacters, WT Hc did not cause typhlocolitis in B6 mice, but mild to severe lesions developed at the cecocolic junction in IL-10 −/− mice, despite similar colonization levels of WT Hc in the cecum and colon of both B6 and IL-10 −/− mice. WT Hc and cdtB mutants also colonized IL-10 −/− mice to a similar extent, but infection with either cdtB mutant resulted in attenuated typhlocolitis and hyperplasia compared to infection with WT Hc ( P < 0.03), and only WT Hc infection caused dysplasia and intramucosal carcinoma. WT Hc and cdtB Hc mutant infection of IL-10 −/− mice elevated mRNA expression of tumor necrosis factor alpha, inducible nitric oxide synthase, and gamma interferon in the cecum, as well as elevated Th1-associated serum immunoglobulin G2a b compared to infection of B6 mice ( P < 0.05). Although no hepatitis was noted, liver samples were PCR positive at various time points for WT Hc or the cdtB Hc mutant in approximately 33% of IL-10 −/− mice and in 10 to 20% of WT Hc -infected B6 mice. These results indicate that WT Hc can be used to model inflammatory bowel disease in IL-10 −/− mice and that CDT contributes to the virulence of H. cinaedi .

Cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4) expressed at high levels by CD4+CD25+CD45RBlowregulatory T cells (Treg) is essential to their homeostatic and immunoregulatory functions. However, its relevance to anti-inflammatory roles of Treg in the context of colitogenic innate immune response during pathogenic bacterial infections has not been examined. We showed earlier in Rag2-deficient 129/SvEv mice that Treg cells are capable of suppressing colitis and colon cancer triggered byHelicobacter hepaticus, a widespread murine enterohepatic pathogen. Using this model, we now examined the effects of antibody blockade of CTLA-4 on Treg function during innate immune inflammatory response. Consistent with our previous findings, we found that a single adoptive transfer of Treg cells prior to infection prevented colitis development despite persistentH. hepaticusinfection in recipient mice. However, when infected mice were injected with anti-CTLA-4 antibody along with Treg cell transfer, they developed a severe acute colitis with poor body condition that was not observed in Rag2−/−mice without Treg cell transfer. Despite high numbers of Foxp3+Treg cells, evident by immunohistochemical analyses in situ, the CTLA-4 antibody-treated mice had severely inflamed colonic mucosa and increased rather than decreased expression levels of cytokines gamma interferon and interleukin-2. These findings indicate that antibody blockade of CTLA-4 clearly abrogates Treg cell ability to suppress innate immune-driven colitis and suggest that Treg cell CTLA-4 cognate interactions may be necessary to maintain homeostasis among cells of innate immunity.

Chronic idiopathic colitis is a common clinical entity in young captive rhesus monkeys. Eight isolates, cultured from five monkeys in colony 1 with endemic diarrhea and three from colony 2 without diarrhea, were grown under microaerobic conditions on selective agar and were classified by full 16S rRNA sequence, biochemical, and phenotypic analysis as a novel helicobacter, “ Helicobacter macacae ” (proposed name). All eight strains of H. macacae had 99.5% identical 16S rRNA sequences.

Helicobacter hepaticus, which induces chronic hepatitis and typhlocolitis in susceptible mouse strains, produces a cytolethal distending toxin (CDT) consisting of CdtA, CdtB, and CdtC. AcdtB-deficientH. hepaticusisogenic mutant (HhcdtBm7) was generated and characterized for colonization parameters in four intestinal regions (jejunum, ileum, cecum, and colon) of outbred Swiss Webster (SW) mice. Inactivation of thecdtBgene abolished the ability of HhcdtBm7 to colonize female mice at both 8 and 16 weeks postinfection (wpi), whereas HhcdtBm7 colonized all of four intestinal regions of three of five males at 8 wpi and then was eliminated by 16 wpi. Wild-type (WT)H. hepaticuswas detected in the corresponding intestinal regions of both male and female mice at 8 and 16 wpi; however, colonization levels of WTH. hepaticusin the cecum and colon of male mice were approximately 1,000-fold higher than in females (P< 0.0079) at 16 wpi. Infection with WTH. hepaticus, but not HhcdtBm7, at 8 wpi was associated with significantly increased mRNA level of ileal and cecal gamma interferon (IFN-γ) in females (P< 0.016 and 0.031 between WTH. hepaticus-infected and sham-dosed females, respectively). In contrast, the mRNA levels of IFN-γ were significantly higher in the colon (P< 0.0079) and trended to be higher in the cecum (P< 0.15) in the HhcdtBm7-colonized male mice versus the sham-dosed controls at 8 wpi. In addition, mRNA levels of ileal IFN-γ were significantly higher in the control females than males at 8 wpi (P< 0.016). There were significantly higher Th1-associated immunoglobulin G2a (IgG2a), Th2-associated IgG1 and mucosal IgA (P< 0.002, 0.002, 0.002, respectively) responses in the mice infected with WTH. hepaticuswhen compared to HhcdtBm7 at 16 wpi. Colonic interleukin-10 (IL-10) expressions at 16 wpi were significantly lower in both female and male mice colonized by WTH. hepaticusor in males transiently colonized through 8 wpi by HhcdtBm7 versus control mice (P< 0.0159). These lines of evidence indicate that (i)H. hepaticusCDT plays a crucial role in the persistent colonization ofH. hepaticusin SW mice; (ii) SW female mice are more resistant toH. hepaticuscolonization than male mice; (iii) there was persistent colonization of WTH. hepaticusin cecum, colon, and jejunum but only transient colonization ofH. hepaticusin the ileum of female mice; (iv)H. hepaticuscolonization was associated with down-regulation of colonic IL-10 production.

Background. A number of novel Helicobacter species have been isolated from both animals and humans. Many of these helicobacters colonize the lower gastrointestinal tract and hepatobiliary tract and are associated with diseases. Methods. A spiral-shaped bacterium, with bipolar single-sheathed flagella, was isolated from the liver and cecum of mastomys (the African rodent, Mastomys natalenis), from the feces and ceca of normal mice, and also from the cecum of a mouse with proctitis. 16S ribosomal RNA gene sequence analysis, restriction fragment length polymorphism (RFLP) and fluorophore-enhanced repetitive element polymerase chain reaction (FERP or rep-PCR) analysis were used to classify the organism. Results. The bacterium grew at 37 and 42°C under microaerobic conditions, rapidly hydrolyzed urea, and was catalase and oxidase positive. It did not reduce nitrate to nitrite, and was resistant to cephalothin and nalidixic acid. Like many other enterohepatic Helicobacter species, this organism expressed cytolethal distending toxin and causes cell distention. Conclusions. The organism was classified as a novel Helicobacter species for which we propose the name ‘Helicobacter mastomyrinus’. Although ‘H. mastomyrinus’, like Helicobacter hepaticus and Helicobacter bilis, colonizes the liver of rodents, the pathogenic potential of this novel helicobacter is unknown.

Although Helicobacter bilis infects mice worldwide, it is not known whether H. bilis causes enterohepatic disease in outbred Swiss Webster (SW) mice. Intestinal and liver specimens from four groups of 39 SW mice, five of which were treated with creatine in the drinking water, were obtained for culture for the presence of H. bilis and were analyzed as to whether infection status was associated with H. bilis seroconversion and/or hepatitis. Helicobacter bilis was isolated from the colon of all 27 mice of groups I-III, but only from the liver of one 12- to 13-month-old female mouse. Ten of 27 livers were H. bilis-positive based on polymerase chain reaction (PCR) analysis; 8 of 10 (80%) of the positive results were for older mice. Results of PCR analysis for H. bilis were negative, and H. bilis was not isolated from 12 control mice (group IV). Irrespective of treatment group or controls, severity of histologic lobular and periportal chronic inflammatory lesions in the liver of H. bilis-infected outbred mice ranged from minimally to moderately severe. Helicobacter bilis infection was associated with increased portal inflammation in group III mice, compared with age-matched, helicobacter-free, group IV mice (P0.03). A comparison of potential sex effects in group III mice indicated that H. bilis-infected female mice developed more severe portal inflammation than did H. bilis-infected male mice (P0.01). On the basis of results of an ELISA, 8 of 11, 6- to 8-month-old H. bilis-infected mice of group III seroconverted to H. bilis outer membrane antigen. Helicobacter bilis infection is associated with hepatitis in SW mice and can confound experimental results.

Helicobacter mustelae , the gastric pathogen of ferrets, produces an array of surface ring structures which have not been described for any other member of the genus Helicobacter , including H. pylori . The unique ring structures are composed of a protein named Hsr. To investigate whether the Hsr rings are important for colonization of the ferret stomach, ferrets specific pathogen free for H. mustelae were inoculated with an Hsr-deficient mutant strain or the wild-type H. mustelae strain. Quantitative cultures from antral biopsy specimens obtained at 3, 6, and 9 weeks postinoculation demonstrated no significant difference in the levels of bacteria in the ferrets that received the Hsr-negative strain and the ferrets infected with the parent strain. However, when the ferrets were biopsied at 12 and 15 weeks and necropsied at 18 weeks after infection, the levels of bacteria of the Hsr-negative strain in the stomach antrum were significantly reduced. This decline contrasted the robust antral colonization by the wild-type strain. The Hsr-negative strain did not efficiently colonize the gastric body of the study ferrets. Histological examination at 18 weeks postinoculation revealed minimal gastric inflammation in the animals that received the mutant H. mustelae strain, a finding consistent with its waning infection status, whereas lesions characteristic of helicobacter infection were present in ferrets infected with the wild-type strain. Scant colonization by the Hsr-negative H. mustelae strain at the end of the 18-week study, despite initial successful colonization, indicates an inability of the mutant to persist, perhaps due to a specific host response.

A novel helicobacter with the proposed name Helicobacter cetorum , sp. nov. (type strain MIT 99-5656; GenBank accession number AF 292378 ), was cultured from the main stomach of two wild, stranded Atlantic white-sided dolphins ( Lagenorhynchus acutus ) and from the feces of three captive cetaceans (a Pacific white-sided dolphin [ Lagenorhynchus obliquidens ]; an Atlantic bottlenose dolphin [ Tursiops truncatus ]; and a beluga whale [ Delphinapterus leucas ]). The infected captive cetaceans were either subclinical, or clinical signs included intermittent regurgitation, inappetance, weight loss, and lethargy. Ulcers were observed in the esophagus and forestomach during endoscopic examination in two of the three captive animals. In the third animal, esophageal linear erosions were visualized endoscopically, and histopathological evaluation of the main stomach revealed multifocal lymphoplasmacytic gastritis with silver-stained spiral-shaped bacteria. Helicobacter cetorum is a fusiform gram-negative bacterium with a single bipolar flagellum. The isolates grow under microaerobic conditions at 37 and 42°C but not at 25°C. H. cetorum is urease, catalase, and oxidase positive, and it is sensitive to cephalothin. The isolates from the wild, stranded dolphins were sensitive to nalidixic acid, whereas the isolates from the collection animals were resistant. By 16S rRNA sequencing it was determined that H. cetorum represented a distinct taxon that clusters most closely with H. pylori. Further studies are necessary to determine the role of H. cetorum in the development of gastric ulcers and gastritis of cetaceans. This is the first description and formal naming of a novel Helicobacter species from a marine mammal.

Woodchucks ( Marmota monax ) have a high incidence of hepatocellular carcinoma (HCC) associated with chronic infection with woodchuck hepatitis virus (WHV) and serve as a model of hepatitis B virus-associated HCC in humans. Helicobacter hepaticus , an enterohepatic helicobacter in mice, is known to cause hepatocellular adenomas and carcinomas in susceptible mouse strains. In long-term chemical bioassays conducted with B6C3F 1 mice, H . hepaticus has been regarded as a confounding factor because of its tumor-promoting activity. In order to determine if woodchucks harbor a Helicobacter sp. that might play a role in potentiating hepatic inflammation or neoplasia, a study was undertaken to determine whether woodchucks' livers were infected with a Helicobacter sp. Frozen liver samples from 20 (17 WHV-infected and 3 noninfected) woodchucks, 10 with WHV-associated hepatic tumors and 10 without tumors, were cultured by microaerobic techniques and analyzed by using genus- and species-specific helicobacter PCR primers. A 1,200-bp Helicobacter sp.-specific sequence was amplified from 14 liver samples. Southern hybridization confirmed the specific identity of the PCR products. Nine of the 10 livers with tumors had positive Helicobacter sp. identified by PCR, whereas 5 of the 10 livers without tumors were positive. By use of 16S rRNA species-specific primers for H . marmotae , two additional liver samples from the nontumor group had positive PCR amplicons confirmed by Southern hybridization. A urease-, catalase-, and oxidase-positive bacterium was isolated from one liver sample from a liver tumor-positive woodchuck. By 16S rRNA analysis and biochemical and phenotypic characteristics, the organism was classified as a novel Helicobacter sp. Subsequently, four additional bacterial strains isolated from feces of cats and characterized by biochemical, phenotypic, and 16S rRNA analysis were determined to be identical to the woodchuck isolate. We propose the name Helicobacter marmotae sp. nov. for these organisms. Further studies are required to ascertain if this novel Helicobacter sp. plays a tumor promotion role in hepadnavirus-associated tumors in woodchucks or causes enterohepatic disease in cats.

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.

The gastric fluid and feces of three belugas from the Mystic Aquarium were assessed for the presence of Helicobacter spp. Gastric fluid and feces from the two clinically healthy belugas were negative for helicobacter, and endoscopy performed on these animals revealed no lesions. However, a helicobacter isolate and PCR product similar to helicobacter strains previously recovered from dolphins were identified, respectively, from the feces and gastric fluid of a beluga manifesting intermittent inappetence and lethargy. Esophageal and forestomach ulcers were noted on endoscopy. This is the first report of novel Helicobacter spp. being identified from whales.

Chronic, idiopathic diffuse colitis is a well recognised clinical and pathological entity in captive rhesus monkeys. Six rhesus monkeys were diagnosed with clinically debilitating, chronic diarrhoea. Histologically, colonic tissues were characterised as chronic, moderate to severe colitis and typhlitis, with diffuse mononuclear inflammation of lamina propria, reactive lymphoid hyperplasia and multifocal micro-abscesses. Colonic tissues were cultured for Salmonella spp. and Shigella spp.; all results were negative. Samples were negative for Clostridium difficile A and B toxins, and special stains of colonic tissue for acid-fast bacteria were also negative. The six diarrhoeic monkeys tested gave negative results for serum IgG antibodies to herpes B virus, STLV, SRV and SIV. Colonic tissue from the six diarrhoeic and two clinically normal monkeys with histologically confirmed colitis from the same colony were also subjected to micro-aerobic culture. Micro-aerobic cultures from all eight monkeys incubated at 37 degrees C and 42 degrees C revealed pinpoint or spreading colonies on antibiotic-containing media. Bacteria were identified as gram-negative, oxidase positive and urease negative. Of the nine strains characterised biochemically, two separate biotypes (corresponding to different species by 16S rRNA analysis) were identified. One biotype (type 1), from non-diarrhoeic monkeys and the second biotype (type 2) from diarrhoeic animals with subclinical chronic colonic inflammation, differed by catalase activity, ability to reduce nitrate to nitrite and sensitivity to cephalothin. Complete 16S rRNA analysis of five of the nine strains characterised biochemically indicated that the organisms isolated were two novel Helicobacter spp. By electron microscopy, these novel helicobacters had spiral morphology with bipolar sheathed flagella. This is the first report describing the isolation of novel Helicobacter spp. from inflamed colons of rhesus monkeys. Studies are needed to determine whether these novel Helicobacter spp. play a causal role in the initiation and progression of chronic colitis in macaques. Further microbiological and histological analysis of this chronic idiopathic colitis syndrome in macaques may prove useful in understanding the aetiology and pathogenesis of inflammatory bowel disease in man.

Gastric ulcerations in dolphins have been reported for decades. Some of these lesions were associated with parasitic infections. However, cases of nonparasitic gastric ulcers with no clearly defined etiology also have been reported in wild and captive dolphins. Considerable speculation exists as to whether dolphins have Helicobacter -associated gastritis and peptic ulcer disease. The stomachs of seven stranded Atlantic white-sided dolphins, Lagenorhynchus acutus , and 1 common dolphin, Delphinus delphis , were assessed for the presence of Helicobacter species. Novel Helicobacter species were identified by culture in the gastric mucosa of two of the eight dolphins studied and by PCR in seven of the eight dolphins. The gram-negative organisms were urease, catalase, and oxidase positive. Spiral to fusiform bacteria were detected in gastric mucosa by Warthin Starry staining. Histopathology revealed mild to moderate diffuse lymphoplasmacytic gastritis within the superficial mucosa of the main stomach. The pyloric stomach was less inflamed, and bacteria did not extend deep into the glands. The lesions parallel those observed in Helicobacter pylori -infected humans. Bacteria from two dolphins classified by 16S rRNA analysis clustered with gastric helicobacters and represent a novel Helicobacter sp. most closely related to H. pylori . These findings suggest that a novel Helicobacter sp. may play a role in the etiopathogenesis of gastritis and gastric ulcers in dolphins. To our knowledge this represents the first isolation and characterization of a novel Helicobacter sp. from a marine mammal and emphasizes the wide host distribution and pathogenic potential of this increasingly important genus.

Fumarate reductase (FRD) is the key enzyme in fumarate respiration induced by anaerobic growth of bacteria. In Helicobacter pylori, this enzyme appears to be constitutively expressed under microaerobic conditions and is not essential for its survival in vitro. In this study, the role of FRD in the colonization of H. pylori was investigated using a mouse model. The frdA gene coding for subunit A of FRD, and two control genes, copA and copP associated with the export of copper out of H. pylori, were inactivated by insertion of the chloramphenicol acetyltransferase cassette into these individual genes. The isogenic mutants of H. pylori strain AH244 were obtained by natural transformation. Seventy-five ICR mice (15 mice/group) were orogastrically dosed with either the wild type H. pylori strain AH244, its isogenic mutants, or Brucella broth (negative control). Five mice from each group were killed at 2, 4 and 8 weeks post-inoculation (WPI), respectively. H. pylori colonization was not detected in mouse gastric mucosa infected with the frdA mutant at any time point in the study by both quantitative culture and PCR. In contrast, the mice inoculated with either wild type AH244, copA or copPH. pylori mutants became readily infected. These data indicate that FRD plays a crucial role in H. pylori survival in the gastric mucosa of mice. Given that FRD, present in all H. pylori strains, is immunogenic in H. pylori -infected patients and H. pylori growth in vitro can be inhibited by three anthelmintics (morantel, oxantel and thiabendazole), this enzyme could potentially be used both as a novel drug target as well as in the development of vaccines for H. pylori prevention and eradication.