Hiraku Sasaki

IFN-γ production downstream of NKT cell activation in mice infected with influenza virus enhances the cytolytic activities of both NK cells and viral antigen-specific CD8+ T cells.

Natural killer T (NKT) cell activation is responsible for eliminating pathogens. However, the biological functions of NKT cells against influenza virus are not fully understood. We therefore investigated the effects of NKT cells in viral infection using CD1d knockout (KO) mice. When CD1d KO or wild-type (WT) mice were infected with a sub-lethal dosage of the influenza virus, the survival rate of CD1d KO mice was significantly lower than for WT mice in association with delayed viral clearance in the lungs. Consistently, IFN-γ production in bronchoalveolar lavage fluid of CD1d KO mice was largely reduced compared to WT mice during infection. Moreover, the cytotoxic activities of NK cells and viral antigen-specific CD8(+) T cells were impaired in CD1d KO mice. It was concluded that activated NKT cell-induced IFN-γ release enhances both NK-cell activity and antigen-specific CD8(+) T cells to eliminate the influenza virus, thus leading to an enhanced survival.

Protection of chickens from fowl cholera by vaccination with recombinant adhesive protein of Pasteurella multocida

The recombinant adhesive protein (rCp39) of Pasteurella multocida strain P-1059 (serovar A:3) was prepared and purified with a hybrid condition of affinity chromatography. The rCp39 was highly protective for chickens from fowl cholera by challenge-exposure with parental strain P-1059 or heterologous strain X-73 (serovar A:1) compared to various kind of vaccines. Sixteen groups of ten chickens each were subcutaneously inoculated twice with 100, 200 or 400 microg proteins of rCp39, native Cp39, native outer membrane protein H (OmpH) or recombinant OmpH, or 100 microg proteins of crude capsular extract (CCE) of strains P-1059 or X-73 at 2 weeks interval. Five chickens of each group were challenge-exposed with each strain 2 weeks after the second inoculation. As the results, 60-100% protections were demonstrated in the chickens against both strains. Fishers exact test indicated no significant differences (P<0.05) in vaccine types and dosages. ELISA and Western blot analysis indicated that the chicken anti-rCp39 sera reacted to whole-cell lysate of parental or heterologous strains. In conclusion, rCp39 is a cross-protective recombinant adhesive antigen of P. multocida capsular serogroup A strains. Moreover, a hybrid condition of affinity chromatography was successfully demonstrated and protected the immunogenicity of recombinant protein.

A survey of ammonia‐assimilating micro‐organisms in cattle manure composting

Aims:  To evaluate the ammonia‐assimilating abilities of micro‐organisms isolated from cattle manure composting processes and to determine the distribution of cultivable species of ammonia‐assimilating micro‐organisms in microbial communities during the composting processes.

Molecular typing of Pasteurella pneumotropica isolated from rodents by amplified 16S ribosomal DNA restriction analysis and pulsed-field gel electrophoresis.

A total of 52 isolates of Pasteurella pneumotropica obtained from rodents were examined for their genetic heterogeneity. On the basis of DNA restriction analysis, including amplified 16S ribosomal DNA restriction analysis (ARDRA) and pulsed‐field gel electrophoresis (PFGE), differences were identified among the isolates. ARDRA typing with HaeIII revealed 4 different banding patterns of the P. pneumotropica isolates. Eighty‐two percent of the 23 isolates identified as a‐1 were derived from mice, whereas all the isolates identified as a‐3 were derived from rats. Most of the isolates, which showed hemolytic activity on blood agar, obtained from mice and rats, were identified as a‐2 and a‐4, respectively. By restriction analysis of genomic DNA, ApaI and NotI digestion differentiated 9 variants and an undiscriminating group. However, no close relation with regard to the phenotypic characteristics was observed among the variants. The isolates identified as a‐2 and a‐4 could not be distinguished by PFGE analysis. DNA restriction analysis revealed that the genetic diversity of the P. pneumotropica isolates was more complex than the phenotypic characteristics among the species, and that at least the P. pneumotropica isolates were clearly differentiated into 4 groups by ARDRA typing with HaeIII.

Prevalence and analysis of Pseudomonas aeruginosa in chinchillas

BackgroundChinchillas (Chinchilla laniger) are popular as pets and are often used as laboratory animals for various studies. Pseudomonas aeruginosa is a major infectious agent that causes otitis media, pneumonia, septicaemia enteritis, and sudden death in chinchillas. This bacterium is also a leading cause of nosocomial infections in humans. To prevent propagation of P. aeruginosa infection among humans and animals, detailed characteristics of the isolates, including antibiotic susceptibility and genetic features, are needed. In this study, we surveyed P. aeruginosa distribution in chinchillas bred as pets or laboratory animals. We also characterized the isolates from these chinchillas by testing for antibiotic susceptibility and by gene analysis.ResultsP. aeruginosa was isolated from 41.8% of the 67 chinchillas included in the study. Slide agglutination and pulsed-field gel electrophoresis discriminated 5 serotypes and 7 unique patterns, respectively. For the antibiotic susceptibility test, 40.9% of isolates were susceptible to gentamicin, 77.3% to ciprofloxacin, 77.3% to imipenem, and 72.7% to ceftazidime. DNA analyses confirmed that none of the isolates contained the gene encoding extended-spectrum β-lactamases; however, 2 of the total 23 isolates were found to have a gene similar to the pilL gene that has been identified in the pathogenicity island of a clinical isolate of P. aeruginosa.ConclusionsP. aeruginosa is widely spread in chinchillas, including strains with reduced susceptibility to the antibiotics and highly virulent strains. The periodic monitoring should be performed to help prevent the propagation of this pathogen and reduce the risk of infection from chinchillas to humans.

Identification and Characterization of Hemolysin-Like Proteins Similar to RTX Toxin in Pasteurella pneumotropica

Pasteurella pneumotropica is an opportunistic pathogen that causes lethal pneumonia in immunodeficient rodents. The virulence factors of this bacterium remain unknown. In this study, we identified the genes encoding two RTX toxins, designated as pnxI and pnxII, from the genomic DNA of P. pneumotropica ATCC 35149 and characterized with respect to hemolysis. The pnxI operon was organized according to the manner in which the genes encoded the structural RTX toxin (pnxIA), the type I secretion systems (pnxIB and pnxID), and the unknown orf. The pnxII gene was involved only with the pnxIIA that coded for a structural RTX toxin. Both the structural RTX toxins of deduced PnxIA and PnxIIA were involved in seven of the RTX repeat and repeat-like sequences. By quantitative PCR analysis of the structural RTX toxin-encoding genes in P. pneumotropica ATCC 35149, the gene expression of pnxIA was found to have increased from the early log phase, while that of pnxIIA increased from the late log to the early stationary phase. As expressed in Escherichia coli, both the recombinant proteins of PnxIA and PnxIIA showed weak hemolytic activity in both sheep and murine erythrocytes. On the basis of the results of the Southern blotting analysis, the pnxIA gene was detected in 82% of the isolates, while the pnxIIA gene was detected in 39%. These results indicate that the products of both pnxIA and pnxIIA were putative associations of virulence factors in the rodent pathogen P. pneumotropica.

Mice with Asthma Are More Resistant to Influenza Virus Infection and NK Cells Activated by the Induction of Asthma Have Potentially Protective Effects

PurposeThis study was conducted in order to investigate whether the virulence of the influenza virus infection is affected by asthma in mice.MethodsMice with asthma or control mice were infected with influenza virus. The survival rate, body weight, virus titer, cytokine profile, and cell infiltration in bronchoalveolar lavage fluid (BALF) were measured. The NK cell cytotoxicity was determined by a co-culture system with YAC-1 cells, and the effects of NK cells were observed by depletion of NK cells using anti-asialoGM1 serum. The virus-specific CD8+ T cell killing assay was also performed.ResultsWhen asthmatic or control mice were infected with non- and sub-lethal doses of influenza virus, the asthmatic mice were more resistant to the virus than control mice with regard to the survival rate, the remission of body weight loss, and the virus burden. Anti-viral cytokines and the NK cell number were increased in the BALF of asthmatic mice before the infection. The NK cell cytotoxicity in the asthmatic mice was significantly enhanced compared to that in control mice, and the depletion of NK cells in asthmatic mice was abrogated both the improved survival rate and the recovery of the body weight loss. The antigen-specific CD8+ T cell killing activity in asthmatic mice was also significantly increased following the infection compared to that in control mice.ConclusionNK cell activated by the induction of asthma and the subsequently activated antigen-specific CD8+ T cells could promptly eliminate the viral-infected cells, thus leading to improvements in the morbidity and mortality of influenza virus infection.

Molecular and virulence characteristics of an outer membrane-associated RTX exoprotein in Pasteurella pneumotropica

BackgroundPasteurella pneumotropica is a ubiquitous bacterium that is frequently isolated from laboratory rodents and causes various clinical symptoms in immunodeficient animals. Currently two RTX toxins, PnxIA and PnxIIA, which are similar to hemolysin-like high-molecular-weight exoproteins are known in this species. In this study, we identified and analyzed a further RTX toxin named PnxIIIA and the corresponding type I secretion system.ResultsThe RTX exoprotein, PnxIIIA, contains only a few copies of the RTX repeat-like sequence and 3 large repeat sequences that are partially similar to the outer membrane protein found in several prokaryotes. Recombinant PnxIIIA protein (rPnxIIIA) was cytotoxic toward J774A.1 mouse macrophage cells, whereas cytotoxicity was attenuated by the addition of anti-CD11a monoclonal antibody. rPnxIIIA could bind to extracellular matrices (ECMs) and cause hemagglutination of sheep erythrocytes. Binding was dependent on the 3 large repeat sequences in PnxIIIA. Protein interaction analyses indicated that PnxIIIA is mainly localized in the outer membrane of P. pneumotropica ATCC 35149 in a self-assembled oligomeric form. PnxIIIA is less cytotoxic to J774A.1 cells than PnxIA and PnxIIA.ConclusionsThe results implicate that PnxIIIA is located on the cell surface and participates in adhesion to ECMs and enhanced hemagglutination in the rodent pathogen P. pneumotropica.

Distribution of Ammonia Assimilating Bacteria In the Composting Process

Ammonia assimilating bacteria were isolated from composting processes and their abilities to assimilate ammonia were evaluated. In the prefermentation stage of the composting, 104.7 and 103.7 (CFU/ g) of ammonia assimilating bacteria were detected on the medium which contained ammonia as a sole nitrogen, at 37°C and 55°C, respectively. They were 104.6 and 103.2 in the end of the primary fermentation, and 105.1 and 103.2 in the end of the secondary fermentation, respectively. When isolates were purely cultivated in sterilized compost extract medium, many of them consumed ammonia not by nitrification but by assimilation. They still assimilated ammonia even in nonsterilized compost extract medium, i.e. mixed culture with the live microbial flora of the compost. However, isolates which showed high ammonia assimilating ability in the pure culture did not always show high abilities in nonsterilized compost extract media. Isolates which showed high ammonia assimilating ability in the nonsterilized medium were identified by analysis of 16S ribosomal DNA. Dominant species of ammonia assimilating microorganisms varied as composting proceeded.

The protective effects of lactoferrin against murine norovirus infection through inhibition of both viral attachment and replication.

The purpose of this study was to evaluate the effects of bovine lactoferrin against norovirus infection using mouse norovirus (MNV) and Raw264.7 cell in vitro. When Raw264.7 cells were infected with MNV in the presence or absence of lactoferrin, the cytotoxic damage to the infected Raw264.7 cells significantly and dose-dependently decreased and completely inhibited in the presence of 15 or 20 μg/well of lactoferrin as compared with the absence of lactoferrin. Correspondingly, the MNV titers in the culture medium and intracellularly were significantly decreased in infected Raw264.7 cells treated with lactoferrin compared to control infected Raw264.7 cells. The mechanisms responsible for the protective effects of lactoferrin against MNV infection were attributed to both its inhibition of the initial MNV attachment to cells and the subsequent interference with MNV replication. Moreover, it was revealed that lactoferrin could rapidly induce the expression of anti-viral cytokine mRNA, such as IFN-α and IFN-β which involved in inhibition of MNV replication in infected Raw264.7 cells, in the early phase of infection. It was concluded that lactoferrin exerts protective effects against MNV infection through inhibition of both viral attachment and replication. The present results provide evidence that lactoferrin may be useful as a preventive and/or therapeutic anti-norovirus agent.