[show abstract][hide abstract] ABSTRACT: Noroviruses cause most cases of acute viral gastroenteritis worldwide. The lack of a cell culture infection model for human norovirus necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict norovirus inactivation. Murine norovirus (MNV) may be used to construct a small animal model for studying the biology and pathogenesis of noroviruses because MNV is the only norovirus that replicates in cell culture and a small animal model. However, recent studies have shown that natural MNV infection is widespread in laboratory mouse colonies. We investigated MNV infection in both conventional and specific pathogen-free (SPF) genetically modified mice from Japan and the US, and commercial mice from several animal breeders in Japan, using serological and molecular techniques. MNV antibodies were detected in 67.3% of conventional mice and 39.1% of SPF mice from Japan and 62.5% of conventional mice from the US. MNV antibodies were also found in 20% of commercial SPF C57BL/6 mice from one of three breeders. Partial gene amplification of fecal isolates from infected animals showed that the isolates were homologous to reported MNV sequences. These results suggest that both conventional and SPF laboratory mice, including commercial mice, are widely infected with MNV, which might require considerable attention as an animal model of human disease.
[show abstract][hide abstract] ABSTRACT: Klebsiella pneumoniae usually shows intrinsic resistance to ampicillin and other beta-lactams. bla(SHV) is thought to be a key beta-lactamase gene responsible for this intrinsic resistance to ampicillin. Nevertheless, surveys of clinical strains reveal that some isolates of K. pneumoniae that carry bla(SHV) remain susceptible to ampicillin. To explore susceptibility to ampicillin in relation to bla(SHV) in K. pneumoniae, we analysed the existence and transcription of bla(SHV) and determined beta-lactamase activity as well as the susceptibilities to clinically relevant beta-lactams, including ampicillin in 160K. pneumoniae isolates from China. In total, 141 isolates (88.1%) were detected as bla(SHV)-positive, 20 (14.2%) of which were found to be broadly susceptible to all beta-lactams tested, including ampicillin. Among the 20 broadly susceptible isolates, sequencing of bla(SHV) revealed synonymous point mutations in 19 isolates and a premature stop codon in 1 isolate. Reverse transcription-polymerase chain reaction failed to detect bla(SHV) mRNA in five isolates (25%). The results demonstrate that differential expression of bla(SHV) in clinical isolates of K. pneumoniae can affect susceptibility to ampicillin.
International Journal of Antimicrobial Agents 04/2007; 29(3):344-7. · 4.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Adult T-cell leukemia (ATL) is an aggressive neoplasm caused by human T-cell leukemia virus type I (HTLV-I). The nuclear transcription factor, NF-kappaB, is induced by HTLV-I and is central to the ensuing neoplasia. To examine the effect of a novel NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on ATL in vivo, we developed an improved severe combined immunodeficiency (SCID) mouse model for ATL. Five-week-old SCID mice in which natural killer (NK) cell activity had been eliminated were inoculated intraperitoneally with the HTLV-I-infected cell lines, TL-Om1, MT-1, MT-2 and HUT-102. No engraftment of TL-Om1 cells and little tumorigenesis of MT-1 cells were detected 40 days after injection. In contrast, inoculation of mice with MT-2 and HUT-102 cells elicited high mortality, 100% frequency of gross tumor formation and tumor cell infiltration of various organs, all of which were reduced by coadministration of DHMEQ during the inoculation. Moreover, tumors from mice treated with DHMEQ had a high frequency of apoptosis. These results suggest that DHMEQ induces apoptosis in HTLV-I-transformed cells in vivo, resulting in inhibition of tumor formation and organ infiltration, thereby enhancing survival.
[show abstract][hide abstract] ABSTRACT: Reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the nucleocapsid (N) gene of mouse hepatitis virus (MHV) successfully detected 36 strains from the faeces of mice that had been housed in animal facilities in Japan from 2000 to 2003. Subsequent restriction fragment length polymorphism (RFLP) analysis, using the enzymes Acc I, Alu I, EcoR I and Mbo I, demonstrated that these strains could be divided into five distinct subgroups and that the same MHV strains were detected from closely related mouse facilities. Furthermore, strains from the same facility showed the same RFLP pattern, irrespective of the year of detection, but this pattern varied between different locations. This study shows that RFLP analyses are a rapid and useful method for differentiation of MHV strains.
[show abstract][hide abstract] ABSTRACT: Lactobacilli are the predominant microorganisms in the vaginal flora of human beings, and are known to play an important role in protecting them from genital infections. On the other hand, the composition of the vaginal flora differs among laboratory animal species, and lactobacilli are not the predominant vaginal microorganism in many laboratory animals. We speculated that the vaginal flora of chimpanzees would be more similar to those of human beings than to those of other animal species, because chimpanzees are phylogenetically close to human beings, and their reproductive physiology is similar to that of human beings. To clarify our speculation, we examined the development of the vaginal flora in chimpanzees (Pan troglodytes). Streptococci, lactobacilli, and members of the family Bacteroidaceae were the most predominant bacteria in the vagina of mature chimpanzees (9 to 22 years old). During development of the vaginal flora of chimpanzees, the total number of bacteria increased with age and reached a plateau just before sexual maturity (5 to 7 years of age; juvenile period). Lactobacilli were already one of the predominant bacteria before sexual maturity. In mature chimpanzees, the total number of bacteria (aerobes and anaerobes) in the vagina was highest during the swelling phase of the menstrual cycle. During the swelling phase in mature chimpanzees, streptococci, lactobacilli, and Bacteroidaceae were the most frequently isolated (100%) organisms, and the total number of organisms recovered from vaginal specimens from these three groups was the highest. In mature chimpanzees in which the number of bacteria was the highest, lactobacilli were the predominant bacteria. Taken together, these results suggest that these three bacterial groups (streptococci, lactobacilli, and Bacteroidaceae) are indigenous to the vagina of chimpanzees, and chimpanzees would be the most suitable laboratory animals for studying the role of lactobacilli in the vagina of human beings.
Comparative medicine 01/2005; 54(6):705-12. · 1.12 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human T cell leukemia virus type I (HTLV-I), the etiological agent of adult T cell leukemia, integrates into the host genome as a provirus. Multiple defective copies of the integrated provirus are often present in the host genome. For this reason it is difficult to clone the intact provirus from HTLV-I-infected cells using conventional techniques. Here, we used overlapping polymerase chain reaction (PCR) to construct a full-length provirus of HTLV-I directly from an HTLV-I-transformed cell line, MT-2, which contains multiple defective proviruses. First, four overlapping proviral HTLV-I fragments (1.4-3.9 kb each) were constructed from genomic MT-2 DNA using PCR. Next, the complete HTLV-I proviral DNA (9 kb) was generated from these fragments using asymmetric PCR and cloned into a plasmid vector. 293 T cells transfected with this plasmid produced virus-like particles, and we show that these particles are capable of infecting a human T cell line. We propose that this cloning technique constitutes a powerful tool for constructing infectious molecular clones from cells of patients infected with HTLV-I or other viruses.
[show abstract][hide abstract] ABSTRACT: Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell neoplasm. The health of ATL patients rapidly deteriorates resulting in death; however, the induction of death in a small animal model due to tumor has not yet been reported. SCID mice, 5 weeks old, younger than those previously used, which were inoculated with ATL cells, eliminated NK cell activity and showed rapid tumor formation resulting in death. Age is the crucial factor influencing tumor formation and death in the SCID mice model for cancer.
[show abstract][hide abstract] ABSTRACT: We examined quantitatively the vaginal flora of conventionally reared mice, rats, hamsters, rabbits and dogs, species that are widely used as laboratory animals. Vaginal specimens were examined according to the method of analyzing intestinal flora (Mitsuoka's procedure). The total number of bacteria (aerobes and anaerobes) and the prevalence of specific bacteria were determined. The total number of bacteria was highest during estrus and lowest during diestrus or anestrus in mice, rats, hamsters, and dogs. The most predominant bacteria during estrus were streptococci in mice; gram-negative rods (GNR), streptococci, and members of the family Bacteroidaceae in rats; GNR, Bacteroidaceae and gram-positive anaerobic cocci in hamsters, and Bacteroidaceae in dogs. The increase in the total number of bacteria during estrus was caused by an increase of predominant bacteria in the vagina. Aerobes were more predominant than anaerobes in mice, and number of aerobes was comparable to that of anaerobes in rats and dogs. On the other hand, in hamsters, anaerobes were more predominant than aerobes and the total number of bacteria was highest among the laboratory animals (mice, rats, hamsters, rabbits, and dogs). However, in rabbits, bacteria were not isolated from about 90% of the vaginal specimens. Rabbits do not have cyclic reproductive stages and are usually in precoital status in the laboratory. In precoital rabbits, vaginal epithelium manifests few signs of secretion. Therefore, we suspect that the vaginal environment in precoital rabbits is comparable to that during diestrus or anestrus in mice, rats, hamsters, and dogs. These results suggest that the vaginal flora of laboratory animals is influenced by the estrous cycle, and probably by mucous secretion. Our data imply that vaginal flora differ among laboratory animals species, and researchers need to take into consideration the estrous cycle of laboratory animals when studying their vaginal flora.
Comparative medicine 09/2003; 53(4):404-12. · 1.12 Impact Factor
[show abstract][hide abstract] ABSTRACT: It has been suspected that embryos stored in liquid nitrogen tanks may become contaminated with murine pathogens, if some pathogens had been introduced to the tanks accidentally. To examine this, we stored tubes containing embryos with tubes containing mouse hepatitis virus (MHV) or Pasteurella pneumotropica in liquid nitrogen tanks and examined whether progeny mice derived from the embryos were contaminated with the pathogens or not. After storing for 6 months or 1 year the frozen embryos were thawed and implanted into the oviducts of pseudopregnant female mice, and the mice were bred in vinyl isolators. We could not detect serum antibodies to MHV and isolate Pasteurella pneumotropica in the progeny mice, suggesting that cross-contamination between tubes in a liquid nitrogen tank scarcely occurs.
[show abstract][hide abstract] ABSTRACT: We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) resulted in acute hepatic failure accompanying extremely elevated viral growth in the liver in interferon-gamma-deficient BALB/c (BALB-GKO), but not C57BL/6 (B6-GKO) mice. To examine the basis of the strain difference against MHV infection in interferon-gamma-deficient mice, viral replication in primary hepatocyte cultures from BALB/c and B6 mice with or without the IFN-gamma gene was compared in vitro. The MHV replication in BALB/c hepatocytes with or without the IFN-gamma gene was significantly higher than that in B6 hepatocytes with or without the IFN-gamma gene, suggesting that there is a strain difference in MHV replication in hepatocytes. Since a significant difference in MHV replication in hepatocytes was not observed between wild type and IFN-gamma-deficient mice of the same genetic background, the phenomenon is thought to be independent of IFN-gamma. However, pretreatment of hepatocytes with recombinant mouse interferon-gamma inhibited MHV replication in a dose-dependent fashion. The results are discussed with respect to the pathology of MHV infection in mice with or without the IFN-gamma gene.
[show abstract][hide abstract] ABSTRACT: A total of 713 strains of fecal Escherichia coli (E. coli) isolated from laboratory animals in the colonies of 4 research laboratories and 4 commercial breeders in Japan in 1994 were examined in regard to resistance to 8 antibacterial agents. The incidence of resistance to sulfadimethoxine (Su), streptomycin (Sm), ampicillin, cephaloridine, tetracycline, chloramphenicol, kanamycin, and gentamicin was 99.9%, 32.5%, 6.7%, 0.7%, 7.0%, 2.6%, 6.6% and 0.7%, respectively. These results indicated that Su and Sm resistance are penetrating into normal E. coli strains isolated from laboratory animals.
Journal of Veterinary Medical Science 01/2003; 64(12):1133-5. · 0.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Familial amyloidotic polyneuropathy (FAP) is a hereditary disease characterized by the systemic accumulation of amyloid fibrils. A mutant transthyretin (TTR) gene is mainly responsible for the disease. However, the variable age of onset and low penetrance might be due to environmental factors, one of which is the intestinal flora. Three types of intestinal flora were introduced into a transgenic (Tg) mouse FAP model, 6.0-hMet30. The CV1 and CV2 group transgenic mice were transferred with the intestinal flora from two different mouse facilities housed under conventional conditions, and the SPF group transgenic mice were kept under specific pathogen free conditions in our facility. All the mice were maintained under controlled temperature, humidity and bacterial conditions. Over a period of 28 months, amyloid was not deposited in the SPF and CV1 groups. In contrast, amyloid was deposited in the esophagus and small intestine of two of the three CV2 mice at 18 months. Many neutrophils infiltrated the lesions. The numbers of tissue neutrophils were higher in the CV2 group than in the SPF and CV1 groups at 18 months. The CV2 flora included fewer gram-positive anaerobic cocci as well as higher proportions of yeasts, staphylococci and enterobacteriaceae compared with the SPF and CV1 flora. These findings suggest that the intestinal flora plays an important role in amyloid deposition.
[show abstract][hide abstract] ABSTRACT: We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) persists in interferon-gamma (IFN-gamma)-deficient C57BL/6 (B6-GKO) mice and results in subacute fatal peritonitis, which bears a resemblance to feline infectious peritonitis. To examine the role of other host factors in MHV infection in mice, IFN-gamma-deficient mice with a BALB/c background (BALB-GKO) were infected intraperitoneally with MHV and compared with B6-GKO mice. In contrast to B6-GKO mice, BALB-GKO mice died within 1 week due to acute hepatic failure. The viral titer of the liver in BALB-GKO mice was significantly higher than that in B6-GKO mice. All hepatocytes in BALB-GKO mice were necrotic at 5 days post-infection, which was clearly distinct from large but limited lesion in the liver from infected B6-GKO mice. The serum alanine aminotransferase activity of infected BALB-GKO mice were higher than that of B6-GKO mice and was paralleled with the severity of the pathological changes and viral titers in infected mice. Administration of exogenous IFN-gamma to BALB-GKO partially inhibited the acute death. These results indicate that BALB-GKO and B6-GKO mice clearly show different diseases following MHV infection, although wild type counterparts of both mice apparently showed the same clinical course after MHV infection.
Virus Research 03/2002; 83(1-2):169-77. · 2.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) persists in interferon-γ (IFN-γ)-deficient C57BL/6 (B6-GKO) mice and results in subacute fatal peritonitis, which bears a resemblance to feline infectious peritonitis. To examine the role of other host factors in MHV infection in mice, IFN-γ-deficient mice with a BALB/c background (BALB-GKO) were infected intraperitoneally with MHV and compared with B6-GKO mice. In contrast to B6-GKO mice, BALB-GKO mice died within 1 week due to acute hepatic failure. The viral titer of the liver in BALB-GKO mice was significantly higher than that in B6-GKO mice. All hepatocytes in BALB-GKO mice were necrotic at 5 days post-infection, which was clearly distinct from large but limited lesion in the liver from infected B6-GKO mice. The serum alanine aminotransferase activity of infected BALB-GKO mice were higher than that of B6-GKO mice and was paralleled with the severity of the pathological changes and viral titers in infected mice. Administration of exogenous IFN-γ to BALB-GKO partially inhibited the acute death. These results indicate that BALB-GKO and B6-GKO mice clearly show different diseases following MHV infection, although wild type counterparts of both mice apparently showed the same clinical course after MHV infection.
Virus Research - VIRUS RES. 01/2002; 83(1):169-177.
[show abstract][hide abstract] ABSTRACT: The nucleotide sequences of the coding region of the nucleocapsid (N) gene of 12 mouse hepatitis virus (MHV) strains recently found in animal facilities in Japan were analyzed. Nucleotide sequencing was performed directly on polymerase chain reaction (PCR) products amplified by reverse transcription (RT) and polymerase chain reaction (RT-PCR) analysis from fecal samples or isolated viruses. Phylogenetic analysis of these MHV strains along with those reported previously indicated that sequence analysis of the N gene was a useful tool for differentiation of MHV strains,although most MHV strains in Japanese facilities were phylogenetically close. Results suggested that interchange of mice infected with MHV among facilities provided opportunities of introduction of MHV into otherwise MHV-free facilities and that the source of MHV infection could be traced by use of nucleotide analysis of the N gene.
Comparative medicine 09/2001; 51(4):319-25. · 1.12 Impact Factor
[show abstract][hide abstract] ABSTRACT: To understand the virus-cell interactions that occur during murine coronavirus infection, six murine cell lines (A3-1M, B16, CMT-93, DBT, IC-21 and J774A.1) were inoculated with eight murine coronaviruses, including prototype strains of both polytropic and enterotropic biotypes, and new isolates. All virus strains produced a cytopathic effect (CPE) with cell-to-cell fusion in B16, DBT, IC-21 and J774A.1 cells. The CPE was induced most rapidly in IC-21 cells and was visible microscopically in all cell lines tested. In contrast, the coronaviruses produced little CPE in A3-1M and CMT-93 cells. Although most virus-infected cells, except KQ3E-infected A3-1M, CMT-93 and J774A.1 cells, produced progeny viruses in the supernatants when assayed by plaque formation on DBT cells, the kinetics of viral replication were dependent on both the cell line and virus strain; replication of prototype strains was higher than that of new isolates. There was no significant difference in replication of enterotropic and polytropic strains. B16 cells supported the highest level of viral replication. To determine the sensitivity of the cell lines to murine coronaviruses, the 50% tissue culture infectious dose of the coronaviruses was determined with B16, DBT, IC-21 and J774A.1 cells, and compared to that with DBT cells. The results indicate that IC-21 cells were the most sensitive to murine coronaviruses. These data suggest that B16 and IC-21 cells are suitable for large-scale preparation and isolation of murine coronaviruses, respectively.