Helicobacter apodemus sp. nov., a new Helicobacter species identified from the gastrointestinal tract of striped field mice in Korea.

A novel Helicobacter species was identified from the gastrointestinal tract of the Korean striped field mouse (Apodemus agrarius). Biochemical testing, ultrastructure characterization, and 16S rRNA gene sequence analysis suggested that this bacterium represents a distinct taxon. The bacterium was positive for urease activity, susceptible to cephalothin and nalidixic acid, and weakly positive for oxidase and catalase activity. Electron microscopy revealed that the bacterium has spirally curved rod morphology with singular bipolar nonsheathed flagella. Genotypically, the isolated bacterial strains (YMRC 000215, YMRC 000216, and YMRC 000419) were most closely related to a reference strain of Helicobacter mesocricetorum (97.25%, 97.32%, and 97.03% 16S rRNA sequence similarities, respectively). The 16S rRNA sequences of these strains were deposited into GenBank under accession numbers AF284754, AY009129, and AY009130, respectively. We propose the name Helicobacter apodemus for this novel species.

[New Helicobacters other than H. pylori].

Since discovery of Helicobacter pylori, more than 30 species non-H. pylori Helicobacter spp. (NHPH) have been reported. Those NHPH were now classified into gastric Helicobacter spp. and enterohepatic Helicobacter spp.(EHS). Gastric NHPH show tight spiral and long shape in the gastric mucosa, and we can distinguish from H. pylori by light microscope. Some gastric NHPH may be zoonosis and cause gastritis in human. H. hepaticus and H. cinaedi belongs in EHS were detected in human diseases. H. hepaticus may be associated with hepatobiliary diseases in humans. Surprisingly, it was reported that H. cinaedi infection was associated with atrial arrhythmias and atherosclerosis. Many NHPH will be recognized as human pathogen in the future.

Electron microscopic, genetic and protein expression analyses of Helicobacter acinonychis strains from a Bengal tiger.

Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy) and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms) as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5-6 µm) and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers) from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections.

Bacteremia caused by a novel helicobacter species in a 28-year-old man with X-linked agammaglobulinemia.

We report on a case of bacteremia caused by a previously unknown urease-negative Helicobacter strain, IMMIB HP-28/08, isolated from blood cultures of a 28-year-old man with X-linked agammaglobulinemia. The identification of the isolate was based on 16S rRNA gene sequencing. In the phylogenetic tree, the isolate fell into a cluster which included Helicobacter canadensis, Helicobacter equorum, and Helicobacter pullorum. This is the first report of bacteremia caused by this fastidious organism. Further investigations are necessary to determine the potential role of this species as a pathogen of bloodstream infections.

Detection of atypical cultivable canine gastric Helicobacter strain and its biochemical and morphological characters in naturally infected dogs.

Helicobacter-like organisms are frequently found in canine stomachs, but the relationship between such organisms and gastric pathology has not been established. However, some such organisms have zoonotic importance. The aims of this study were to evaluate the morphological and biochemical characteristics of cultivable canine gastric Helicobacter-like organisms (GHLOs) in pets and stray dogs and their prevalence in these two groups of dogs. Specimens were taken by gastroscopy from 30 clinically healthy stray dogs and 30 pet dogs. Cultures were positive from biopsies of 11/30 of stray and 6/30 of pet dogs. The isolated Helicobacters were observed by light microscopy and studied by biochemical, physiological and PCR analysis. Some of the isolated GHLO's displayed atypical shapes that were similar to Helicobacter pylori or Helicobacter acinonychis in stray dogs' cytological examinations. They had 2-3 helices and were smaller than other canine GHLOs. One of these atypical Helicobacter strains was cultured. It was not possible to distinguish such strains by routine PCR and biochemical evaluations. Electron microscopy showed a smaller Helicobacter (2 microm in length) with 2 or 3 helixes. This study demonstrates that not all canine gastric Helicobacters are 5-15 microm in length, as has been previously proposed, and portrays the need for further investigation of canine GHLOs.

Helicobacter canis bacteraemia in a 7-month-old child.

On the basis of biochemical, phenotypic and 16S rRNA analyses, Helicobacter canis was isolated and identified from an otherwise healthy 7-month-old girl with intermittent fever. Blood cultures signalled bacterial growth after 5 days that was characterized as small gram-negative spiral rods. Subculturing on Colombia plates with 5% sheep blood, chocolate agar and brucella agar, aerobically and anaerobically as well as in a microaerophilic atmosphere, showed scanty growth after an additional 4 days. Secondarily seeded with fluid from the original bottle, the paediatric blood bottles repeatedly signalled growth after one night's incubation, whereas the conventially treated bottles did not support growth after 7 days' incubation. From the secondary seeded paediatric bottles a pure culture was isolated on chocolate agar plates, and identified as H. canis. This case indicates that blood culture systems should be compared and improved for their capacity to detect Helicobacter and related pathogenic bacteria species. Further studies are also needed to determine the importance of H. canis as a primary pathogen, and the role of cats in the possible zoonotic spread of H. canis to humans.

Helicobacter callitrichis sp. nov., a novel Helicobacter species isolated from the feces of the common marmoset (Callithrix jacchus).

A slowly growing microaerophilic Helicobacter species was isolated from the feces of the common marmoset (Callithrix jacchus). This bacterium possessed a pair of nonsheathed bipolar flagella, was positive for oxidase, catalase and alkaline phosphatase activities, but was negative for gamma-glutamyltranspeptidase and urease activity and for nitrate reduction. The bacterium was susceptible to nalidixic acid and resistant to cephalotine and did not hydrolyze hippurate. On the basis of phenotypic characteristics, 16S rRNA gene sequence analysis and whole-cell protein profiles, the isolate represents a new species of the genus Helicobacter, for which the name Helicobacter callitrichis sp. nov. is proposed; the type strain of the new species is R-204(T) (GenBank accession number AY192526).

Extension of the species Helicobacter bilis to include the reference strains of Helicobacter sp. flexispira taxa 2, 3 and 8 and Finnish canine and feline flexispira strains.

The evolution and taxonomy of enterohepatic Helicobacter species with flexispira morphology were studied by a polyphasic approach including phenotypic characterization, analysis of 16S rRNA and ureB gene sequences and dot-blot DNA-DNA hybridization of whole genomic DNA. In addition, available phylogenetic data on the HSP60 gene were used in the analysis. The study included 14 Finnish canine and feline flexispira strains, the reference strains of Helicobacter sp. flexispira taxa 2, 3 and 8 and Helicobacter bilis ATCC 51630(T). Phenotypically, all canine and feline strains were similar to H. bilis. Analysis of 16S rRNA gene sequences of these strains revealed a similarity of 97-99.5 %. Similarity of ureB nucleotide and amino acid sequences within the studied flexispira group was 97-100 % and 99-100 %, respectively, revealing close relatedness. ureB sequences of Helicobacter hepaticus had only 64-66 % similarity to the flexispira group. The similarity to Helicobacter trogontum was 81.5-82.1 %. High levels of DNA-DNA hybridization between the strains were found in dot-blot tests. Polyphasic analysis of the phenotypic and genotypic characteristics of the Finnish flexispira strains and the reference strains of taxa 2, 3 and 8 showed that they differed from other Helicobacter species and are members of the previously described species H. bilis. In addition, canine strain F56 differed in all phylogenetic analyses from the H. bilis group and probably represents a novel Helicobacter species.

A novel enterohepatic Helicobacter species 'Helicobacter mastomyrinus' isolated from the liver and intestine of rodents.

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 degrees 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.

Comparison of Helicobacter spp. in Cheetahs (Acinonyx jubatus) with and without gastritis.

Chronic gastritis causes significant morbidity and mortality in captive cheetahs but is rare in wild cheetahs despite colonization by abundant spiral bacteria. This research aimed to identify the Helicobacter species that were associated with gastritis in captive cheetahs but are apparently commensal in wild cheetahs. Helicobacter species were characterized by PCR amplification and sequencing of the 16S rRNA, urease, and cagA genes and by transmission electron microscopy of frozen or formalin-fixed paraffin-embedded gastric samples from 33 cheetahs infected with Helicobacter organisms (10 wild without gastritis and 23 captive with gastritis). Samples were screened for mixed infections by denaturant gel gradient electrophoresis of the 16S rRNA gene and by transmission electron microscopy. There was no association between Helicobacter infection and the presence or severity of gastritis. Eight cheetahs had 16S rRNA sequences that were most similar (98 to 99%) to H. pylori. Twenty-five cheetahs had sequences that were most similar (97 to 99%) to "H. heilmannii" or H. felis. No cheetahs had mixed infections. The ultrastructural morphology of all bacteria was most consistent with "H. heilmannii," even when 16S rRNA sequences were H. pylori-like. The urease gene from H. pylori-like bacteria could not be amplified with primers for either "H. heilmannii" or H. pylori urease, suggesting that this bacteria is neither H. pylori nor "H. heilmannii." The cagA gene was not identified in any case. These findings question a direct role for Helicobacter infection in the pathogenesis of gastritis and support the premise that host factors account for the differences in disease between captive and wild cheetah populations.

Observation of Helicobacter-like organisms in gastric mucosa of grey foxes (Urocyon cinereoargenteus) and bobcats (Lynx rufus).

Archival specimens of gastric mucosa of 10 raccoons (Procyon lotor), 9 porcupines (Erethizon dorsatum), 6 grey foxes (Urocyon cinereoargenteus), 6 bobcats (Lynx rufus), 4 skunks (Mephitis mephitis), and 3 black bears (Ursus americanus) were microscopically examined for evidence of Helicobacter-like organisms. Such organisms were seen in the specimens from the grey foxes and bobcats only. Histochemical stains (modified Steiner and carbol fuchsin methods) revealed long spiral organisms within lumina of gastric glands; however, neither gross nor microscopic lesions were observed. By electron microscopy (EM), the organisms were found to be free in the glandular lumina and were seen occasionally in the cytoplasm of gastric epithelial cells. Morphologically, 2 different phenotypes of spiral organisms were identified by EM. The organisms associated with bobcats appeared to be more tightly coiled than those seen in grey foxes. The presence of Helicobacter-like organisms in the gastric mucosa of grey foxes has not previously been described.

Natural and experimental Helicobacter infections.

Current information about Helicobacter infections in humans and various domestic, wild, and research animal species that have been used or have the potential to be used as animal models of human disease is presented. The Helicobacter genus now includes at least 26 formally named species, with additional novel species in the process of being characterized. The natural history, host range with zoonosis potential, pathology, and diagnostic techniques are presented, along with examples of how Helicobacter infection has interfered with unrelated in vivo research. Current recommendations for deriving and managing helicobacter-free animal colonies for research are provided.

Isolation and characterization of novel Helicobacter spp. from the gastric mucosa of harp seals Phoca groenlandica.

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.

Helicobacter muricola sp. nov., a novel Helicobacter species isolated from the ceca and feces of Korean wild mouse (Mus musculus molossinus).

A slowly growing microaerophilic Helicobacter strain was isolated from the ceca and fecal pellets of Korean wild mice (Mus musculus molossinus). This bacterial strain possessed a pair of nonsheathed bipolar flagella, was positive for urease, catalase and oxidase, and reduced nitrate to nitrite. It proved susceptible to nalidixic acid and resistant to cephalodine, and did not hydrolyze hippurate. On the basis of phenotypic characteristics and 16S rRNA gene sequence analysis, the isolate represents a new species of the genus Helicobacter, for which the name Helicobacter muricola sp. nov. is proposed; the type strain of the new species is w-06T.

Captive rhesus monkeys (Macaca mulatta) are commonly infected with Helicobacter cinaedi.

Helicobacter cinaedi may cause proctocolitis or bacteremia in homosexual men infected with human immunodeficiency virus or occasionally in other immunocompromised hosts. There are scattered reports of H. cinaedi isolated from a variety of animal hosts, but to date only hamsters have been found to be a common natural reservoir. Microaerophillic cultures of feces from 5 of 16 asymptomatic rhesus monkeys (Macaca mulatta) (31%) were positive for a curved gram-negative rod. A polyphasic taxonomic approach was used to identify the organism as H. cinaedi. These results show that H. cinaedi frequently colonizes asymptomatic captive rhesus monkeys, which may serve as another potential reservoir for human infection.

Fatal gastrointestinal infection with 'Flexispira rappini'-like organisms in a cat.

A 4-month-old male British Blue cat with catarrhal to haemorrhagic enteritis showed massive colonization of the stomach, small intestine and caecum with spiral-shaped bacilli. In the stomach, organisms were located in foveolae and gland lumina and within unaltered and degenerate epithelial cells. Inflammatory infiltration was moderate and T cell dominated. In the intestine, bacilli were found in the gut lumen, berween villi, in crypt lumina and within epithelial cells. Degeneration of crypt epithelial cells as well as crypt dilation and moderate to massive macrophage-dominated infiltration of the mucosa and submucosa were observed. Immunohistochemically, bacilli were positive with an antibody against Helicobacter. Ultrastructurally, the organisms strongly resembled 'Flexispira rappini', a spiral-shaped Helicobacter species known as a normal intestinal colonizer in dogs and mice.

Cholangiohepatitis and inflammatory bowel disease induced by a novel urease-negative Helicobacter species in A/J and Tac:ICR:HascidfRF mice.

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 approximately 3 x 10(7) 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 cholangio-hepatitis associated with a specific urease-negative, novel H. species.

Helicobacter winghamensis sp. nov., a novel Helicobacter sp. isolated from patients with gastroenteritis.

From 1997 to 1999 seven isolates of Campylobacter-like organisms from five patients that were exhibiting symptoms of gastroenteritis, including fever, stomach malaise, and diarrhea, were investigated. The organisms were isolated from stool samples and found to exhibit a diverse colony morphology; hence multiple isolates were submitted from one of the patients. All isolates were found to be identical. The organisms were catalase, urease, alkaline phosphatase, and nitrate negative but oxidase and indoxyl acetate positive. They grew at 37 degrees C but not at 42 degrees C, and three of the isolates from two different patients were sensitive to nalidixic acid and cephalothin. Full 16S rRNA sequence analysis not only grouped these organisms within the Helicobacter genus but also differentiated them from previously identified Helicobacter species. The closest relative by phylogenetic analysis was Helicobacter sp. flexispira taxon 1. Electron microscopy showed that these isolates had one or two bipolar flagella; however, the periplasmic fibers, a characteristic of the known Helicobacter sp. flexispira taxa, were not observed. The present isolates also lacked a flagellar sheath, a trait shared with four other Helicobacter spp., H. canadensis, H. mesocricetorum, H. pullorum, and H. rodentium. On the basis of the unique phenotypic properties of these isolates and 16S rRNA sequence analysis, we propose the classification of a new Helicobacter species, Helicobacter winghamensis sp. nov.

Novel Helicobacter species isolated from rhesus monkeys with chronic idiopathic colitis.

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.