Cloning and Expression of a Helicobacter bilis Immunoreactive Protein

In an effort to identify immunoreactive Helicobacter bilis antigens with potential for serodiagnosis, sera from mice experimentally infected with H. bilis were used to screen an H. bilis genomic DNA expression library. Among 17 immunoreactive clones, several contained sequences that encoded a predicted 167-kDa protein (P167). Five overlapping P167 peptides (P167A to P167E) of approximately 40 kDa each were generated and tested. Immune sera reacted with fragments P167C and P167D at dilutions of 1:1,600 and 1:6,400, respectively, and reacted with an H. bilis membrane extract at a dilution of 1:800 in an enzyme-linked immunosorbent assay. Sera from mice experimentally infected with H. hepaticus did not react with P167C and P167D. Sera from mice naturally infected with H. bilis but not sera from mice naturally infected with H. hepaticus reacted with P167C and P167D. Hyperimmune sera against P167C peptide reacted with recombinant P167C and with a 120-kDa band in H. bilis lysates but did not react with a protein of the same size on immunoblots prepared from H. hepaticus, H. muridarum, or unrelated Borrelia burgdorferi and Campylobacter jejuni whole-cell lysates. Nevertheless, the P167A, P167B, P167C, and P167D primers, but not the P167E primers, amplified DNA from H. hepaticus, and all five primer sets amplified DNA from H. muridarum. These results suggest that P167 is an immunodominant, H. bilis-specific antigen that may have potential for use in serodiagnosis.     

Differential detection of five mouse-infecting helicobacter species by multiplex PCR

Several species of helicobacter have been isolated from laboratory mice, including H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius, which appear to be the most common. The most widely used published method for molecular detection of these agents is PCR amplification of a conserved region of 16S rRNA, but differential speciation requires restriction enzyme digestion of the amplicons. This study was undertaken to determine PCR conditions that would simultaneously and specifically identify each of the five common species without restriction enzyme analyses. First, we designed novel and specific PCR primers for H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius, using sequences from the heterologous regions of 16S rRNA. Because of comigration of amplified products, we next identified P17, an H. bilis-specific protein; P25, an H. hepaticus-specific protein; and P30, an H. muridarum-specific protein by screening genomic DNA expression libraries of each species. Primers were designed from these three genes, plus newly designed, species-specific 16S rRNA primers for H. rodentium and H. typhlonius that could be utilized for a five-plex PCR. The sizes of the amplicons from H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius were 435, 705, 807, 191, and 122 bp, respectively, allowing simultaneous detection and effective discrimination among species.     

Cloning and Expression of an Immunogenic Membrane-Associated Protein of Helicobacter hepaticus for Use in an Enzyme-Linked Immunosorbent Assay

Helicobacter hepaticus is a bacterial pathogen that causes chronic active hepatitis and inflammatory bowel disease in mice. The purpose of this study was to develop a recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) to detect H. hepaticus-infected mice. A genomic library of H. hepaticus was constructed and was screened with sera from H. hepaticus-infected mice. A 459-bp open reading frame that coded for an 18-kDa immunoreactive protein, MAP18, was identified. The gene had high identity with genes coding for outer membrane proteins of other bacteria, and the predicted amino acid sequence of MAP18 had a putative membrane-trafficking signal sequence and a putative signal peptidase II cleavage site. The recombinant protein was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein, GST-MAP18, and purified by affinity chromatography. The 44-kDa fusion protein was detected on Western blots probed with sera from H. hepaticus-infected mice but was not detected on blots probed with sera from mice infected with Helicobacter muridarum or Helicobacter bilis or with sera from mice free of Helicobacter infection. The GST-MAP18 fusion protein was used as an antigen in an ELISA to detect anti-H. hepaticus antibodies in sera from infected mice. This ELISA was compared to an H. hepaticus-specific ELISA that uses a detergent extract of H. hepaticus as the antigen. Sera from mice naturally and experimentally infected with H. hepaticus, H. bilis, or H. muridarum and sera from mice free of Helicobacter infection were evaluated. Both ELISAs performed with a high specificity (98%); however, the detergent extract-based ELISA performed with a higher sensitivity (89%) than the recombinant protein-based ELISA (sensitivity, 66%). These data indicate that H. hepaticus carries a gene that encodes an immunogenic 18-kDa membrane-associated protein; however, antibodies to this protein are not detected in all infected mice.     

Development and Evaluation of a PCR-Based Assay for Detection of Haemobartonella felis in Cats and Differentiation of H. felis from Related Bacteria by Restriction Fragment Length Polymorphism Analysis

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats.     

Immune Responses to Bile-Tolerant Helicobacter Species in Patients with Chronic Liver Diseases, a Randomized Population Group, and Healthy Blood Donors

Bile-tolerant Helicobacter species such as Helicobacter pullorum, Helicobacter bilis, and Helicobacter hepaticus are associated with hepatic disorders in animals and may be involved in the pathogenesis of chronic liver diseases (CLD) in humans. Antibody responses to cell surface proteins of H. pullorum, H. bilis, and H. hepaticus in serum samples from patients with CLD, a randomized population group, and healthy blood donors were evaluated by using enzyme linked immunosorbent assay (ELISA). The results were compared with the antibody responses to Helicobacter pylori. For analysis of a possible cross-reactivity between bile-tolerant Helicobacter species and H. pylori, sera from a subpopulation of each group were absorbed with a whole-cell extract of H. pylori and retested by ELISA. Results before absorption showed that the mean value of the ELISA units for H. pullorum was significantly higher in patients with CLD than in healthy blood donors (P = 0.01). Antibody reactivity to cell surface protein of H. hepaticus was also significantly higher in the CLD patients than in the healthy blood donors and the population group (P = 0.005 and P = 0.002, respectively). Following the absorption, antibody responses to H. pullorum decreased significantly in all three groups (P = 0.0001 for CLD patients, P = 0.0005 for the population group, and P < 0.0001 for the blood donors), indicating that cross-reactivity between H. pylori and other Helicobacter spp. occurs. The antibody responses to H. hepaticus and H. bilis in CLD patients remained high following absorption experiments compared to ELISA results before absorption. The significance of this finding requires further investigations.     

Recombinant Helicobacter bilis Protein P167 for Mouse Serodiagnosis in a Multiplex Microbead Assay

Infection of mice with Helicobacter bilis is widespread in research and commercial mouse colonies. Therefore, sensitive, specific, and high-throughput assays are needed for rapid and accurate testing of mice in large numbers. This report describes a novel multiplex assay, based on fluorescent microbeads, for serodetection of H. bilis infection. The assay requires only a few microliters of serum to perform and is amenable to a high-throughput format. Individual microbead sets were conjugated to purified, H. bilis-specific, recombinant proteins P167C and P167D and bacterial membrane extracts from H. bilis and Helicobacter hepaticus. For detecting H. bilis infection in the microbead multiplex assay, P167C and P167D provided significantly higher sensitivities (94 and 100%, respectively) and specificities (100 and 95%, respectively) than membrane extract (78% sensitivity and 65% specificity). Microbead multiplex assay results were validated by enzyme-linked immunosorbent assay. Purified recombinant proteins showed low batch-to-batch variation; this feature allows for ease of quality control, assay robustness, and affordability. Thus, recombinant antigens are highly suitable in the multiplex microbead assay format for serodetection of H. bilis infection.     

PCR for Detection and Identification of Abiotrophia spp.

Members of the genus Abiotrophia, formerly known as nutritionally variant streptococci, are important pathogens causing septicemia and endocarditis. Cultivation and biochemical differentiation of Abiotrophia spp. are often difficult. Based on 16S rRNA sequences, two PCR assays for detection and identification of Abiotrophia spp. were developed. The first PCR assay was positive for all Abiotrophia spp. Subsequently performed restriction fragment length polymorphism analysis allowed the verification of the PCR amplicons and the differentiation of the three species. The second PCR assay was positive only for A. elegans, the most fastidious species of Abiotrophia. Both PCR assays were shown to be specific and sensitive and should facilitate the identification of Abiotrophia spp.     

Novel Primer-Probe Sets for Detection and Identification of Mycobacteria by PCR-Microarray Assay

A PCR-microarray assay was developed in which PCR primers and hybridization probes were designed for the 16S rRNA genes of 16 clinically relevant mycobacteria and for IS6110 of Mycobacterium tuberculosis. The assay, based on a multiplex PCR followed by hybridization with oligonucleotide probes, was tested against 16 Mycobacterium species and 70 clinical samples.     

Molecular method for Bartonella species identification in clinical and environmental samples

A new, efficient molecular method for detection of Bartonella, based on the 16S-23S rRNA intergenic spacer and 16S rRNA amplification by multiplex PCR combined with reverse line blotting, was designed. This assay could simultaneously detect 20 different known species and other Bartonella species not described previously.     

Discrimination of Burkholderia gladioli from Other Burkholderia Species Detectable in Cystic Fibrosis Patients by PCR

A procedure for molecular identification of Burkholderia gladioli is described. Specific 16S and 23S rRNA gene signature sequences were defined as primers for PCR. The method allows rapid and specific discrimination of B. gladioli from related species (B. cepacia, B. multivorans, B. vietnamiensis, B. mallei, B. pseudomallei, Ralstonia pickettii, and R. eutropha) and should contribute to the clarification of its role as a human pathogen, e.g., in cystic fibrosis.     

Direct polymerase chain reaction test for detection of Helicobacter pylori in humans and animals.

We designed a polymerase chain reaction (PCR) for amplifying the Helicobacter pylori gene encoding 16S rRNA. Primers for the specific detection of H. pylori were designed for areas of the 16S rRNA gene in which there is the least sequence homology between H. pylori and its closest relatives. The specificity of detection was confirmed by ensuring that the primers did not amplify DNA extracts from the campylobacters H. cinaedi, H. mustelae, and Wolinella succinogenes, which are the closest relatives of H. pylori, as determined by 16S rRNA sequencing. Serial dilution experiments revealed the detection of as little as 0.1 pg of DNA by PCR and 0.01 pg by nested PCR. H. pylori DNA was detected successfully in clinical paraffin-embedded and fresh gastric biopsy specimens from patients positive for the bacterium and also in fecal suspensions seeded with the organism. The DNA from the nonculturable coccoid form of H. pylori was also identified by the primers. Universal primers designed for highly conserved areas on the 16S rRNA gene enabled large amplification products to be produced for direct sequencing analysis. Gastric bacteria resembling H. pylori have been isolated from animals. DNA of these animal gastric bacteria amplified with H. pylori-specific primers yielded PCR products identical to those from human isolates of H. pylori, as confirmed by the use of a 20-base radiolabelled probe complementary to an internal sequence flanked by the H. pylori-specific primers. The results of PCR amplification and partial 16S rRNA gene sequence analysis strongly support the contention that the gastric organisms previously recovered from a pig, a baboon, and rhesus monkeys are H. pylori.     

Systemic Macrophage Depletion Inhibits Helicobacter bilis-Induced Proinflammatory Cytokine-Mediated Typhlocolitis and Impairs Bacterial Colonization Dynamics in a BALB/c Rag2?/? Mouse Model of Inflammatory Bowel Disease

Helicobacter bilis, an enterohepatic helicobacter, is associated with chronic hepatitis in aged immunocompetent inbred mice and inflammatory bowel disease (IBD) in immunodeficient mice. To evaluate the role of macrophages in H. bilis-induced IBD, Rag2^−/− BALB/c or wild-type (WT) BALB/c mice were either sham dosed or infected with H. bilis Missouri strain under specific-pathogen-free conditions, followed by an intravenous injection of a 0.2-ml suspension of liposomes coated with either phosphate-buffered saline (control) or clodronate (a macrophage depleting drug) at 15 weeks postinfection (wpi). At 16 wpi, the ceca of H. bilis-infected Rag2^−/− mice treated with control liposomes had significantly higher histopathological lesional scores (for cumulative typhlitis index, inflammation, edema, epithelial defects, and hyperplasia) and higher counts of F4/80⁺ macrophages and MPO⁺ neutrophils compared to H. bilis-infected Rag2^−/− mice treated with clodronate liposomes. In addition, cecal quantitative PCR analyses revealed a significant suppression in the expression of macrophage-related cytokine genes, namely, Tnfa, Il-1β, Il-10, Cxcl1, and iNos, in the clodronate-treated H. bilis-infected Rag2^−/− mice compared to the H. bilis-infected Rag2^−/− control mice. Finally, cecal quantitative PCR analyses also revealed a significant reduction in bacterial colonization in the clodronate-treated Rag2^−/− mice. Taken together, our results suggest that macrophages are critical inflammatory cellular mediators for promoting H. bilis-induced typhlocolitis in mice.     

6种常见呼吸道感染细菌16s rRNA基因的克隆与鉴定

目的：克隆并鉴定6种常见呼吸道感染细菌16srRNA基因，为制备基因芯片探针做准备。方法：设计、合成6种细菌16srRNA基因扩增引物；PCR扩增16srRNA基因目的片段；克隆16srRNA基因片段；最后对重组质粒进行鉴定。结果：（1）6种细菌16srRNA基因片段的PCR扩增结果：大肠埃希菌、金黄色葡萄球菌、肺炎链球菌、肺炎克雷伯杆菌、流感嗜血杆菌在1300bp处出现特异的目的片段；铜绿假单胞菌在1100bp出现特异的目的片段，与预计的片段大小吻合。（2）PCR产物分别与pMD18-T载体连接并转化JM109受体菌之后在Amp^r培养基表面生长，其中蓝色菌落为阴性克隆，白色菌落是阳性克隆。（3）各克隆质粒PCR鉴定及双酶切鉴定，结果均可见特异目的带。（4）克隆质粒的序列分析示：6种细菌克隆质粒部分16srRNA基因序列与GenBank中发表序列同源性为99．8％以上，说明细菌16srRNA基因已克隆成功。结论：成功克隆了大肠埃希菌、金黄色葡萄球菌、肺炎链球菌、肺炎克雷伯杆菌、流感嗜血杆菌及铜绿假单胞菌16srRNA基因片段，为制备基因芯片探针奠定了基础。     

Development of a real-time PCR assay for the specific detection and identification of Streptococcus pseudopneumoniae using the recA gene

We sequenced the evolutionarily conserved genes 16S rRNA, atpD, tuf, and recA from Streptococcus pseudopneumoniae, Streptococcus pneumoniae, Streptococcus mitis, and Streptococcus oralis. Phylogenetic analysis revealed that recA provided good resolution between these species, including discrimination of the novel species S. pseudopneumoniae. By contrast, the more conserved 16S rRNA, tuf and atpD are not sufficiently discriminatory. Therefore, recA sequences were used to develop a real-time PCR assay with a locked nucleic acid-mediated TaqMan probe for the specific detection and identification of S. pseudopneumoniae. The PCR assay showed excellent specificity and a detection limit of <10 genome copies for the detection and identification of S. pseudopneumoniae strains, which makes it a promising tool for molecular identification and epidemiological studies. In conclusion, this article describes for the first time a PCR assay for the specific identification of S. pseudopneumoniae.     

Chronic Hepatitis,Hepatic Dysplasia,Fibrosis, and Biliary Hyperplasia in Hamsters Naturally Infected with a Novel Helicobacter Classified in the H. bilis Cluster

We recently described helicobacter-associated progressive, proliferative, and dysplastic typhlocolitis in aging (18- to 24-month-old) Syrian hamsters. Other pathogens associated with typhlocolitis in hamsters, Clostridium difficile, Lawsonia intracellularis, and Giardia spp., were not indentified. The presence of Helicobacter genus-specific DNA was noted by PCR in cecal and paraffin-embedded liver samples from aged hamsters by the use of Helicobacter-specific PCR primers. By 16S rRNA analysis, the Helicobacter sp. isolated from the liver tissue was identical to the cecal isolates from hamsters. The six hamster 16S rRNA sequences form a genotypic cluster most closely related to Helicobacter sp. Flexispira taxon 8, part of the Helicobacter bilis/H. cinaedi group. Livers from aged helicobacter-infected hamsters showed various stages of predominantly portocentric and, to a lesser extent, perivenular fibrosis. Within nodules, there was cellular atypia consistent with nodular dysplasia. The livers also exhibited a range of chronic active portal/interface and lobular inflammation, with significant portal hepatitis being present. The inflammation was composed of a mixture of lymphocytes, neutrophils, and macrophages, indicative of its chronic-active nature in these aged hamsters infected with Helicobacter spp. The isolation of novel Helicobacter spp., their identification by PCR from the diseased livers of aged hamsters, and their taxonomic classification as belonging to the Helicobacter bilis cluster strengthen the argument that H. bilis and closely related Helicobacter spp. play an etiological role in hepatobiliary disease in both animals and humans.We recently described progressive, proliferative, and dysplastic typhlocolitis in aging (18- to 24-month-old) Syrian hamsters (31). The lesions in these hamsters were more severe in the older animals aged 7 to 24 months than in the younger, 1- to 6-month-old hamsters. The presence of Helicobacter spp. in the large bowel of all 24 hamsters included in the study was confirmed by culture and Helicobacter-specific PCR (31). Other pathogens associated with typhlocolitis in hamsters, Clostridium difficile, Lawsonia intracellularis, and Giardia spp., were not indentified in this study (31). Enterohepatic Helicobacter spp. are associated with the development of inflammatory bowel disease in mice and, as determined more recently, humans (2, 18, 27, 51). These helicobacters can also induce hepatitis and hepatocellular carcinoma in susceptible strains of mice (1, 14, 19). We undertook a study to examine in detail the histological profile of the livers of hamsters aged 18 to 24 months and to determine whether Helicobacter sp. DNA was present in their livers (31). In addition, we purchased a small number of 6-month-old hamsters from the same vendor which originally supplied the aged hamsters and examined them for the presence of Helicobacter spp. in their intestines and liver and whether inflammation was associated with the presence of Helicobacter spp. in these target tissues. We document that, with the exception of fibrosis, which does not develop in mice with any frequency, all of the hepatobioliary lesions noted in unmanipulated colony-maintained hamsters described in our report are consistent with the hepatic lesions noted in male A/JCr, ABF1 mice or B6C3F1 mice infected with Helicobacter hepaticus (14, 19, 20, 39). In general, the localization of hepatic fibrosis in these aged hamsters which had Helicobacter spp. in their livers correlated with the portocentric inflammation typically observed in helicobacter-infected animal models. We also classify the novel helicobacter isolates from the ceca and liver of hamsters and additional isolates from mice and gerbils as belonging to a novel genotype in the H. bilis cluster.     

Development of a PCR-Restriction Fragment Length Polymorphism Assay Using the Nucleotide Sequence of the Helicobacter hepaticus Urease Structural Genes ureAB

Infection with Helicobacter hepaticus causes chronic active hepatitis in certain strains of mice and is associated with hepatocellular carcinoma in A/JCr mice. Like the gastric helicobacters, H. pylori and H. mustelae, H. hepaticus possesses a high level of urease activity. However, the H. hepaticus urease structural gene sequences have not been previously determined, and the role of the urease enzyme in colonization and in pathogenesis is not known. PCR was used to amplify a portion of the urease structural genes from H. hepaticus genomic DNA. Amplified DNA fragments were cloned, and the nucleotide sequence was determined. The deduced amino acid sequence of the partial H. hepaticus ureA gene product was found to exhibit 60% identity and 75% similarity to the predicted H. pylori UreA. The deduced amino acid sequence of a partial H. hepaticus ureB gene product exhibited 75% identity and 87% similarity to the predicted H. pylori UreB. Diversity among H. hepaticus isolates was evaluated by means of a restriction fragment length polymorphism (RFLP) assay. The 1.6-kb fragments within the ureAB open reading frames, amplified from 11 independent isolates, were digested with the restriction endonuclease HhaI. Three distinct RFLP patterns were observed. Identical RFLP profiles were noted in sequential isolates of one strain of H. hepaticus during an 18 month in vivo colonization study, suggesting that the urease genes of H. hepaticus are stable. The urease genes among H. hepaticus strains were also well conserved, showing 98.8 to 99% nucleotide sequence identity among three isolates analyzed. These findings indicate that H. hepaticus has urease structural genes which are homologous to those of the gastric Helicobacter species and that these gene sequences can be used in a PCR and RFLP assay for diagnosis of this important murine pathogen.     

Morphological and genetic diversity of symbiotic cyanobacteria from cycads

The morphological and genetic diversity of cyanobacteria associated with cycads was examined using PCR amplification techniques and 16S rRNA gene sequence analysis. Eighteen symbiotic cyanobacteria were isolated from different cycad species. One of the symbiotic isolates was a species of Calothrix, a genus not previously reported to form symbioses with Cycadaceae family, and the remainder were Nostoc spp. Axenic cyanobacterial strains were compared by DNA amplification using PCR with either short arbitrary primers or primers specific for the repetitive sequences. Based on fingerprint patterns and phenograms, it was revealed that cyanobacterial symbionts exhibit important genetic diversity among host plants, both within and between cycad populations. A phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that most of the symbiotic cyanobacterial isolates fell into well‐separated clades. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Rapid Identification of Candida albicans and Other Human Pathogenic Yeasts by Using Short Oligonucleotides in a PCR

A PCR system that can quickly and accurately identify 14 species of human pathogenic yeasts was developed. The procedure distinguished between nine species of a closely related clade, Lodderomyces elongisporus, Candida parapsilosis, a new Candida sp., C. sojae, C. tropicalis, C. maltosa, C. viswanathii, C. albicans, and C. dubliniensis and between another five more divergent species, Pichia guilliermondii, C. glabrata, C. zeylanoides, C. haemulonii, and C. haemulonii type II. A rapid DNA extraction procedure that yields purified DNA in about 1 h is also described. The system uses uniform conditions with four primers for each reaction, two 40- to 50-mer universal primers that serve as a positive control and two 23- to 30-mer species-specific primers. Species-specific primers were derived from a 600-nucleotide variable region (D1/D2) at the 5′ end of the large-subunit (26S) ribosomal DNA gene and were generally designed to use mismatches at the 3′ end. Universal primers were developed from conserved nucleotide sequences in the small-subunit (18S) rRNA gene. In this system, a control 1,200- to 1,300-base DNA fragment was produced in all reactions and a smaller 114- to 336-base DNA fragment was produced if the chromosomal DNA from the target species was present. The PCR procedure is rapid and easy to interpret and may be used with mixed cultures.     

Identification of helicobacter species in human liver samples from patients with primary hepatocellular carcinoma

AIMS: Several studies have shown the presence of helicobacter species in the human biliary tract and in the intestinal tract of animals. Experimental infection by Helicobacter hepaticus in mice causes chronic hepatitis and hepatocellular carcinoma (HCC). This study investigated whether helicobacter species could be detected in the liver of patients with HCC. METHODS: Liver samples from 20 patients with primary liver carcinoma diagnosed by histopathology and 16 controls without primary liver carcinoma were studied. Histology with standard and immunohistochemical stains, culture, and polymerase chain reaction (PCR) amplification using helicobacter genus specific 16S rRNA primers were used to detect the presence of bacteria. Amplified products were identified by Southern hybridisation and sequencing. A search for other genes specific for Helicobacter pylori was also performed. RESULTS: Helicobacter species 16S rDNA was found in eight of 20 samples of primary liver carcinoma, whereas none of the controls harboured this rDNA. Six helicobacter specific PCR amplicons were sequenced and were found to have 98.5-99.0% similarity to the 16S rDNA of H pylori. Of the eight positive samples, seven were positive in PCR using 26 kDa protein primers and six showed morphological and immunohistochemical evidence of H pylori. The cagA and glmM genes were detected in only two samples. The vacA and rps4 genes were not detected. CONCLUSIONS: Helicobacter can be present in the liver of patients with primary liver carcinoma and is probably linked to the carcinogenic process in the liver.     

Pyogranulomatous Pneumonia in Goats Caused by an Undescribed Porphyromonas Species, “Porphyromonas katsikii”

A yet-undescribed bacterial species, tentatively named “Porphyromonas katsikii,” was isolated from individuals of a small goat herd with pyogranulomatous pneumonia during an outbreak of acute respiratory disease. The isolated bacteria grew in the form of black-pigmented colonies after 14 days of incubation under anaerobic conditions at 37°C on a tryptic soy blood agar medium. The bacteria were identified as a yet-undescribed Porphyromonas species by determination of the nucleotide sequence of the rrs 16S rRNA gene, and this species was tentatively named Porphyromonas katsikii. PCR amplification with specific primers for this yet-undescribed species revealed the presence of P. katsikii in the lung tissue of all affected animals, while no PCR signals were evidenced from the lungs of healthy goats or from goats with pasteurellosis caused by Mannheimia haemolytica. These data indicate P. katsikii as the causative agent of acute respiratory distress. P. katsikii is phylogenetically related to Porphyromonas somerae and Porphyromonas levii, which cause pathologies in humans and animals, respectively. P. katsikii was not detected by PCR from samples of the gingival pockets or of the faces of healthy goats.