Genomic and pathogenic characterization of RTX toxin producing Rodentibacter sp. that is closely related to Rodentibacter haemolyticus.

Rodentibacter spp. are opportunistic pathogens that are often isolated from the upper respiratory tracts of laboratory rodents. In particular, R. pneumotropicus and R. heylii require considerable caution in rodent colonies, as they cause lethal pneumonia in rodents. A new species, R. haemolyticus, has recently been classified in the genus, and a very closely related strain, Rodentibacter sp. strain JRC, has been isolated in Japan. This study focused on strain JRC by performing genomic and pathogenic analyses. Draft genome sequencing of strain JRC identified several genes coding for putative virulent proteins, including hemolysin and adhesin. Furthermore, we found a new RTX (repeats-in-structural toxin) toxin gene in the genome, which was predicted to produce a critical virulence factor (RTXIA) similar to Enterobacteriaceae. The concentrated culture supernatant containing RTX toxin (RTXIA) showed cytotoxicity toward RAW264.7 cells. Pre-incubation with anti-CD11a attenuated the cytolysis, suggesting that the concentrated culture supernatant containing RTXIA is cell surface LFA-1 mediated cytolysin. Experimental infection of strain JRC intranasally with 5 female BALB/c-Rag2-/- mice showed 60% lethality and was not significantly different from those of R. pneumotropicus ATCC 35149T using the log-rank test. Combined with our finding that RTXIA has an almost identical amino acid sequence (98% identity) to that of R. haemolyticus 1625/19T, these results strongly suggest that RTXIA-producing strain JRC (and related R. haemolyticus) is pathogenic to immunodeficient rodents, and both agents should be excluded in laboratory rodent colonies.

Mouse resources at the RIKEN BioResource Research Center and the National BioResource Project core facility in Japan.

The RIKEN BioResource Research Center (BRC) was established in 2001 as a comprehensive biological resource center in Japan. The Experimental Animal Division, one of the BRC infrastructure divisions, has been designated as the core facility for mouse resources within the National BioResource Project (NBRP) by the Japanese government since FY2002. Our activities regarding the collection, preservation, quality control, and distribution of mouse resources have been supported by the research community, including evaluations and guidance on advancing social and research needs, as well as the operations and future direction of the BRC. Expenditure for collection, preservation, and quality-control operations of the BRC, as a national core facility, has been funded by the government, while distribution has been separately funded by users' reimbursement fees. We have collected over 9000 strains created mainly by Japanese scientists including Nobel laureates and researchers in cutting-edge fields and distributed mice to 7000 scientists with 1500 organizations in Japan and globally. Our users have published 1000 outstanding papers and a few dozen patents. The collected mouse resources are accessible via the RIKEN BRC website, with a revised version of the searchable online catalog. In addition, to enhance the visibility of useful strains, we have launched web corners designated as the "Mouse of the Month" and "Today's Tool and Model." Only high-demand strains are maintained in live colonies, while other strains are cryopreserved as embryos or sperm to achieve cost-effective management. Since 2007, the RIKEN BRC has built up a back-up facility in the RIKEN Harima branch to protect the deposited strains from disasters. Our mice have been distributed with high quality through the application of strict microbial and genetic quality control programs that cover a globally accepted pathogens list and mutated alleles generated by various methods. Added value features, such as information about users' publications, standardized phenotyping data, and genome sequences of the collected strains, are important to facilitate the use of our resources. We have added and disseminated such information in collaboration with the NBRP Information Center and the NBRP Genome Information Upgrading Program. The RIKEN BRC has participated in international mouse resource networks such as the International Mouse Strain Resource, International Mouse Phenotyping Consortium, and Asian Mouse Mutagenesis and Resource Association to facilitate the worldwide use of high-quality mouse resources, and as a consequence it contributes to reproducible life science studies and innovation around the globe.

A novel Filobacterium sp can cause chronic bronchitis in cats.

BACKGROUND: Cilia-associated respiratory bacillus (CARB; now known as Filobacterium rodentium gen. nov., sp. nov.) is a primary pathogen of rodents. A CARB-like organism was reported in post-mortem lung samples of cats using light and electron microscopy. Here we explore by molecular procedures if a Filobacterium sp. is a part of the normal feline lower respiratory microbiome and whether it could in some cats contribute to the development of chronic bronchial disease. METHODOLOGY: A Filobacterium sp. was identified in three Czech cats clinically diagnosed as having chronic neutrophilic bronchitis. Bronchoalveolar lavage fluid (BALF) specimens obtained from these cats were subjected to panbacterial 16S rDNA PCR followed by Sanger sequencing of the V5 to V8 region. After these cats were treated with specific antimicrobials, their clinical signs resolved promptly, without recurrence. Next, BALF specimens from 13 Australian and 11 Italian cats with lower respiratory disease and an additional 16 lung samples of Italian cats who died of various causes were examined using next generation sequencing (NGS). Subsequently, a Filobacterium-specific qPCR assay was developed and used to re-test BALF specimens from the 11 Italian cats and lung tissue homogenates from the additional 16 deceased cats. PRINCIPAL FINDINGS: An amplicon of 548 bp with 91.24% sequence agreement with Filobacterium rodentium was obtained from all three patients, suggesting the novel Filobacterium sp. was the cause of their lower respiratory disease. The novel Filobacterium sp., which we propose to call F. felis, was detected in 3/3 Czech cats with chronic neutrophilic bronchitis, 13/13 Australian cats and 6/11 Italian cats with chronic lower respiratory disease, and 14/16 necropsy lung specimens from Italian cats. NGS and qPCR results all showed identical sequences. The Filobacterium sp. was sometimes the preponderant bacterial species in BALF specimens from cats with lower airway disease. There was an association between the presence of large numbers (greater than 105 organisms/mL) of Filobacterium and the presence of neutrophilic and/or histiocytic inflammation, although only a subset of inflammatory BALF specimens had F. felis as the preponderant organism. CONCLUSION: The novel Filobacterium sp. comprises a finite part of the normal feline lower respiratory microbiome. Under certain circumstances it can increase in absolute and relative abundance and give rise to neutrophilic and/or histiocytic bronchitis, bronchiolitis and bronchopneumonia. These findings strongly suggest that F. felis could be an underdiagnosed cause of feline bronchial disease.

Filobacterium rodentium gen. nov., sp. nov., a member of Filobacteriaceae fam. nov. within the phylum Bacteroidetes; includes a microaerobic filamentous bacterium isolated from specimens from diseased rodent respiratory tracts.

Strain SMR-CT, which was originally isolated from rats as the SMR strain, had been named 'cilia-associated respiratory bacillus' ('CAR bacillus'). 'CAR bacillus' was a Gram-stain-negative, filamentous argentophilic bacterium without flagella. SMR-CT grew at 37 °C under microaerobic conditions, showed gliding activity, hydrolysed urea and induced chronic respiratory diseases in rodents. The dominant cellular fatty acids detected were iso-C15 : 0 and anteiso-C15 : 0. The DNA G+C content was 47.7 mol%. 16S rRNA gene sequence analysis revealed SMR-CT and other strains of 'CAR bacillus' isolated from rodents all belonged to the phylum Bacteroidetes. The nearest known type strain, with 86 % 16S rRNA gene sequence similarity, was Chitinophaga pinensis DSM 2588T in the family Chitinophagaceae. Strain SMR-CT and closely related strains of 'CAR bacillus' rodent-isolates formed a novel family-level clade in the phylum Bacteroidetes with high bootstrap support (98-100 %). Based on these results, we propose a novel family, Filobacteriaceae fam. nov., in the order Sphingobacteriales as well as a novel genus and species, Filobacterium rodentium gen. nov., sp. nov., for strain SMR-CT. The type strain is SMR-CT ( = JCM 19453T = DSM 100392T).

Polymorphisms influencing expression of dermonecrotic toxin in Bordetella bronchiseptica.

Bordetella bronchiseptica is a pathogenic bacterium causing respiratory infections in a broad range of mammals. Recently, we determined the whole genome sequence of B. bronchiseptica S798 strain isolated from a pig infected with atrophic rhinitis and found four single-nucleotide polymorphisms (SNPs) at positions -129, -72, +22, and +38 in the region upstream of dnt encoding dermonecrotic toxin (DNT), when compared with a rabbit isolate, RB50. DNT is known to be involved in turbinate atrophy observed in atrophic rhinitis. Immunoblotting, quantitative real-time PCR, and ß-galactosidase reporter assay revealed that these SNPs resulted in the increased promoter activity of dnt and conferred the increased ability to produce DNT on the bacteria. Similar or identical SNPs were also found in other pig isolates kept in our laboratory, all of which produce a larger amount of DNT than RB50. Our analysis revealed that substitution of at least two of the four bases, at positions -72 and +22, influenced the promoter activity for dnt. These results imply that these SNPs are involved in the pathogenicity of bordetellae specific to pig diseases.

A broadly reactive one-step SYBR Green I real-time RT-PCR assay for rapid detection of murine norovirus.

A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1-ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was completed within 1 h, and the reliable detection limit was 10 copies of MNV plasmid or 0.063 median tissue culture infective doses per milliliter of RAW264 cell culture-propagated viruses. The diagnostic performance of the assay was evaluated using 158 mouse fecal samples, 91 of which were confirmed to be positive. The melting curve analysis demonstrated the diversity of MNV in the samples. This is the first report of a broadly reactive one-step SYBR Green I real-time RT-PCR assay for detecting of MNVs. The rapid and sensitive performance of this assay makes it a powerful tool for diagnostic applications.

Detection of murine norovirus by reverse transcription loop-mediated isothermal amplification.

Murine norovirus (MNV) has considerable genetical and biological diversity and is recognized worldwide as the most common contaminant in laboratory mouse colonies. This study developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method with the potential to detect a broad range of MNV. RT-LAMP, using a set of five primers containing mixed bases, obtained results under isothermal conditions at 62°C for 90min. Sensitivity of RT-LAMP was 50-fold less than that of two-step TaqMan real-time reverse transcription-polymerase chain reaction (TaqMan RT-PCR). Diagnostic performance of RT-LAMP on RNA extracted from mouse fecal specimens was compared with TaqMan RT-PCR and nested RT-PCR. MNV was detected in 54 of 120 mouse fecal specimens by RT-LAMP, and RT-LAMP had an estimated sensitivity and specificity of 96.4% and 100% compared with TaqMan RT-PCR, and 94.7% and 100% compared with nested RT-PCR. RT-LAMP, which does not require expensive instruments, might be useful for the screening of mice actively or persistently infected with MNV.

Reverse transcription-loop-mediated isothermal amplification for the detection of rodent coronaviruses.

Mouse hepatitis virus (MHV) is one of the most prevalent viruses detected in laboratory mouse colonies. Enterotropic strains predominate in natural infections, and molecular techniques for the detection of MHV shedding in feces are powerful enough to diagnose active infections. A reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technique was developed for the detection of rodent coronaviruses within 90 min. The specificity of this technique was confirmed by its ability to detect all 17 different strains of MHV and 6 strains of rat coronaviruses as well as its failure to detect human, bovine, and porcine coronaviruses nonspecifically. The sensitivity of RT-LAMP was 3.2-fold higher than that of reverse transcription-polymerase chain reaction (RT-PCR) and 31.6-fold lower than that of nested RT-PCR. An evaluation of the diagnostic performance of RT-LAMP performed in duplicate using mouse fecal specimens showed that the sensitivity and specificity with respect to nested RT-PCR were 85.7% and 100%, respectively. RT-LAMP assays would be suitable for monitoring active MHV infection in mouse colonies.

Indirect ELISA and indirect immunofluorescent antibody assay for detecting the antibody against murine norovirus S7 in mice.

To evaluate murine norovirus (MNV) infection in laboratory mice, we attempted to develop an enzyme-linked immunosorbent assay (ELISA) system and an indirect immunofluorescent antibody (IFA) assay for detecting the anti-MNV-S7 antibody in mice. MNV-S7, which was isolated in Japan, was used in both assays. The antigen for ELISA was prepared by ultracentrifugation of culture supernatants of RAW 264 cells infected with MNV-S7. Positive sera were obtained from 6-week-old, female C57BL/6JJcl mice inoculated orally with MNV-S7. IFA against infected RAW 264 cells was able to discriminate positive sera from negative sera. Indirect ELISA was performed using 96-well ELISA plates coated with formalin-treated MNV-S7 antigen. In this ELISA system, mouse sera obtained 2 weeks after infection or later showed significantly high OD values and were judged positive. An equal level of anti-MNV-S7 antibody response was observed in BALB/cAJcl, C57BL/6JJcl, DBA/2JJcl, and Jcl:ICR mice; whereas, C3H/HeJJcl mice demonstrated slightly lower antibody production 4 weeks after infection. We also used this ELISA system to evaluate 77 murine serum samples obtained from 15 conventional mouse rooms in research facilities in Japan and found that approximately half of the serum samples contained antibody to MNV-S7. We found that some serum samples were negative for antibodies to mouse hepatitis virus and Mycoplasma pulmonis but positive for antibody to MNV-S7. The results suggest that the MNV infection is more prevalent than other infections such as mouse hepatitis virus and Mycoplasma pulmonis in conventional mouse colonies in Japan, as is the case in other areas of the world.

The mouse resources at the RIKEN BioResource center.

Mice are one of the most important model organisms for studying biological phenomena and diseases processes in life sciences. The biomedical research community has succeeded in launching large scale strategic knockout mouse projects around the world. RIKEN BRC, a comprehensive government funded biological resource center was established in 2001. RIKEN BRC has been acting as the core facility for the mouse resources of the National BioResource Project (NBRP) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan since 2002. RIKEN BRC is a founding member of the Federation of International Mouse Resources (FIMRe) together with the Jackson Laboratory, the European Mouse Mutant Archive, and other centers, and has participated in the International Mouse Strain Resource (IMSR) to distribute mouse strains worldwide. With the support of the scientific community, RIKEN BRC has collected over 3,800 strains including inbred, transgenic, knockout, wild-derived, and ENU-induced mutant strains. Excellent mouse models for human diseases and gene functions from academic organizations and private companies are distributed through RIKEN BRC. To meet research and social needs, our mice will be rederived to a specific pathogen-free state, strictly monitored for their health, and accurately tested for their genetic modifications and backgrounds. Users can easily access our mouse resources through the internet and obtain the mouse strains for a minimal fee. Cryopreservation of embryos and sperm is used for efficient preservation of the increasing number of mouse resources. RIKEN BRC collaborates with FIMRe members to support Japanese scientists in the use of valuable mouse resources from around the world.

Differences and similarities in the developmental status of embryo-derived stem cells and primordial germ cells revealed by global expression profiling.

Embryonic germ-line cells are unipotent cells that give rise to either sperm or oocytes. However, pluripotent stem cells can be derived from primordial germ cells (PGCs) or spermatogonia, suggesting that germ-line cells retain a capacity for pluripotency. Here, we made genome-wide comparisons of the gene expression profiles of freshly isolated PGCs, in vitro-formed PGCs (iPGCs), and other stem cell lines, including embryonic stem cells (ESCs), embryonic germ cells (EGCs) and germ-line stem (GS) cells. Comparing PGC with ESC, 382 genes/transcripts were significantly up-regulated in ESC, while 188 were elevated in PGC. This suggests that PGCs possess transcription program distinct from that of ESC, although both share expression of many pluripotency-associated genes. Our micro-array analysis showed that the analyzed samples could be classified into two groups: one consisting of all the ESCs and most of EGCs, and the other containing PGC samples, iPGC, one type of female EGC and GS cells. We then identified "signature" genes for the two groups, and used them to characterize GS cells, EGC, and iPGCs, and revealed developmental status of each cell type. The relationships between PGCs and stem cells derived from embryos or germ cells are discussed in light of these findings.

Detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by ELISA using purified recombinant nucleoprotein.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.

Genes encoded within 8q24 on the amplicon of a large extrachromosomal element are selectively repressed during the terminal differentiation of HL-60 cells.

Human acute myeloblastic leukemia HL-60 cells become resistant to differentiation during long-term cultivation. After 150 passages, double minute chromosomes (dmins) found in early-passaged cells are replaced by large extrachromosomal elements (LEEs). In a DNA library derived from a purified fraction of LEEs, 12.6% (23/183) of clones were assigned to 8q24 and 9.2% (17/183) were assigned to 14q11 in the human genome. Fluorescence in situ hybridization (FISH) revealed a small aberrant chromosome, which had not been found in early-passaged cells, in addition to the purified LEEs. We determined that each LEE consisted of six discontinuous segments in a region that extended for 4.4Mb over the 8q24 locus. Five genes, namely, Myc (a proto-oncogene), NSMCE2 (for a SUMO ligase), CCDC26 (for a retinoic acid-dependent modulator of myeloid differentiation), TRIB1 (for a regulator of MAPK kinase) and LOC389637 (for a protein of unknown function), were encoded by the amplicon. Breaks in the chromosomal DNA within the amplicon were found in the NSMCE2 and CCDC26 genes. The discontinuous structure of the amplicon unit of the LEEs was identical with that of dmins in HL-60 early-passaged cells. The difference between them seemed, predominantly, to be the number (10-15 copies per LEE versus 2 or 3 copies per dmin) of constituent units. Expression of the Myc, NSMCE2, CCDC26 and LOC389637 and TRIB1 genes was constitutive in all lines of HL-60 cells and that of the first four genes was repressed during the terminal differentiation of early-passaged HL-60 cells. We also detected abnormal transcripts of CCDC26. Our results suggest that these genes were selected during the development of amplicons. They might be amplified and, sometimes, truncated to contribute to the maintenance of HL-60 cells in an undifferentiated state.

Lymphocytic choriomeningitis infection undetected by dirty-bedding sentinel monitoring and revealed after embryo transfer of an inbred strain derived from wild mice.

Persistent LCMV infection in wild-derived MAI/Pas mice housed under conventional conditions remained undetected for a decade, despite periodic health monitoring using dirty-bedding sentinels. When MAI/Pas mice were rederived by embryo transfer, recipient mothers produced antiLCMV antibodies, which first revealed the presence of the virus in the colony. Before this information was obtained, MAI/Pas mice had been shipped to another facility, undergone cesarean rederivation there, and been introduced into the recipient barrier. The foster mothers of rederived pups were LCMV-negative according to enzyme-linked immunosorbent assay, but sera of both cesarean-rederived MAI/Pas mice and their foster mothers were positive for LCMV infection by immunofluorescent assay (IFA). LCMV was isolated from the MAI/Pas mice, and its genomic RNA was sequenced. Examination of animal technicians in contact with LCMV-infected mice and of other mouse samples by IFA or a reverse transcriptase-polymerase chain reaction test (or both) revealed that neither the workers nor other animals had been infected with LCMV. Experimental data showed that LCMV transmission from persistently infected mice to naïve ones occurred only after direct contact of animals housed in the same cage. This experience demonstrates the importance of careful viral monitoring in the transfer of laboratory rodents between institutions, the limitation of dirty-bedding sentinels for detection of LCMV infection, and the inadequacy of cesarean rederivation for elimination of enzootic LCMV infection. 111