In vitro investigation of the effects of Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 exopolysaccharides on tight junction damage caused by influenza virus infection.

It is a problem that influenza virus infection increases susceptibility to secondary bacterial infection in lungs leading to lethal pneumonia. We previously reported that exopolysaccharides (EPS) derived from Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1) could prevent against influenza virus infection followed by secondary bacterial infection in vitro. Therefore, the present study assessed whether EPS derived OLL1073R-1 protects the alveolar epithelial barrier disfunction caused by influenza virus infection. After A549 cells treated with EPS or without EPS were infected influenza virus A/Puerto Rico/8/34 (IFV) for 12 h, the levels of tight junction genes expression and inflammatory genes expression were measured by reverse transcription polymerase chain reaction. As results, EPS treatment could protect against low-titer IFV infection, but not high-titer IFV infection, followed by suppression of the increased expression of inflammatory cytokine gene levels and recovery of the decrease in the expression level of ZO-1 gene that was caused by low-titer IFV infection, leading to an improvement trend in the barrier function. Our findings showed that EPS derived from OLL1073R-1 could inhibit low-titer IFV infection leading to maintenance of the epithelial barrier function through the suppression of inflammatory cytokine genes expression.

Microbiological assessment of Suncus murinus bred and managed as laboratory animals.

Suncus murinus is gaining prominence as a laboratory animal; however, there is no generally accepted method for microbiological monitoring. This study aimed to apply non-serological microbiological monitoring of laboratory mice for S. murinus and identify the subdominant species obtained by culture methods for microbial assessment. Culture and PCR were used to test S. murinus for the laboratory mice test panels including 10 bacterial species and orthohantaviruses, all of which were negative. The species that grew sub-dominantly in rectal feces were identified as Aeromonas hydrophila, which is pathogenic to mammals. These results indicate that microbiological monitoring should be used to detect pathogens directly from S. murinus, not from sentinel animals, due to the host-specific microbial environment.

Effects of NKT Cells on Metabolic Disorders Caused by High-Fat Diet Using CD1d-Knockout Mice.

Purpose: The purpose of this study was to investigate whether NKT cells play an important role in preventing or exacerbating diseases caused by high-fat diet (HFD) using CD1d-knockout (KO) mice which lack NKT cells. Methods: Five-week-old male Balb/c (wild-type; WT) or CD1dKO mice were fed with control-diet (CTD) or HFD for 16 weeks. Results: The present study revealed four main findings. First, CD1dKO mice were susceptible to obesity caused by HFD in comparison to WT mice. Second, clinical conditions of fatty liver caused by HFD were comparable between CD1dKO mice and WT mice. Third, HFD-fed WT mice showed high levels of serum biochemical markers, involved in lipid metabolisms, in comparison to WT mice fed a CTD. Notably, the serum concentrations of ALT, T-CHO, TG and HDL-C in CD1dKO mice fed a HFD were almost comparable to those of CD1dKO mice fed a CTD. Fourth, the expression of peroxisome proliferator-activated receptor (PPAR) γ, low-density lipoprotein receptor (LDLR), CD36 of epididymal adipose tissue enhanced and proprotein convertase subtilisin/kexin type (PCSK) 9 in serum decreased. Conclusion: NKT cells were responsible for protection against HFD-induced obesity. However, CD1dKO mice were resistant to serum biochemical marker abnormalities after HFD feeding. One possible explanation is that the epididymal adipose tissue of CD1dKO mice could take up greater amounts of excess lipids in serum in comparison to WT mice.

Fecal microbiota transplantation from Suncus murinus, an obesity-resistant animal, to C57BL/6NCrSIc mice, and the antibiotic effects in the approach.

Introduction: Important studies on the relationship of the intestinal microbial flora with obesity have uncovered profound changes in the composition of the gut microbiota in obese individuals. Animal studies successfully altered body phenotypes by fecal microbiota transplantation (FMT). Methods: In this study, we analyzed the gut microbiome of Suncus murinus (S. murinus), a naturally obesity-resistant animal, and the changes of the gut flora of C57BL/6NCrSIc mice that received gut bacteria transplantation from S. murinus by 16S rRNA gene analysis method. And analyzed and discussed the possible impact of the use of antibiotics before transplantation on the outcome of transplantation. Results: Our results showed no significant changes in body weight in the FMT group compared to the control (AB) group, but large fluctuations due to antibiotics. There was no change in blood lipid levels between groups before and after FMT. The gut microbiota of S. murinus were enriched in Firmicutes and Proteobacteria, while Bacteroidetes were not detected, and fewer OTUs were detected in the intestine gut in comparison to other mouse groups. Statistically significant differences in alpha diversity were observed between the FMT group and other groups. Furthermore, a beta diversity analysis indicated an apparent structural separation between the FMT group and other groups. Conclusion: It was suggested that the gut flora of S. murinus was not well established in the gut trace of mice through FMT, and the administration of antibiotics before transplantation was an important factor affecting the overall composition of the gut flora. Although FMT of S. murinus failed to completely colonize the intestinal tract of the mice, it still had a certain effect on the establishment of the intestinal flora of the mice. The unpredictable effects of pre-transplantation antibiotics on the results of transplantation cannot be ignored.

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.

Seroprevalence of anti-mumps virus IgG antibody titer and voluntary mumps vaccination coverage in young adults in Japan.

In Japan, monovalent vaccine against mumps virus (MuV) infection was shifted to a voluntary basis vaccination due to the incidences of aseptic meningitis in the past. According to an analysis of a total of 409 participants aged 18-20 years, overall vaccination coverage rate was 48%. The mean anti-MuV IgG antibody titer of participants with medical history and more than two times vaccination was significantly higher than that in those without a medical history and unvaccinated and single vaccination, respectively. Seropositivity against MuV infection was >50% regardless of the number of vaccinations. Although these results suggest that seropositivity may persist due to asymptomatic infection, it is necessary to implement either a high vaccine coverage or routine vaccination for prevention of periodic mumps epidemics.

Persistence of Anti-Rubella Immunoglobulin G Antibody Titers in Young Adults Involved in a Short-Term Periodic Immunization in Japan.

In Japan, several rubella outbreaks in adults have erupted due to insufficient immunity against rubella virus (RUBV). Although selective immunization is being promoted along with routine rubella vaccination as its eradication strategy, serosurveillance against RUBV needs to be implemented in the generations corresponding to the vaccination transition period. In this study, a survey of anti-rubella immunoglobulin G (IgG) antibody titers was conducted among young adults involved in the transitional periods of the routine rubella vaccination program. Specifically, serosurveillance was performed in 370 healthy young adults aged 18-20 years, wherein their serum samples were analyzed using an enzyme immunoassay to determine rubella-specific IgG antibody titers. Although multiple regression analysis revealed significant differences only in medical history, more than 90% of participants exhibited seropositivity, excluding those who received a single-dose vaccine alone. Based on elapsed periods after the last vaccination, rubella-specific IgG antibody titers in less than a 6-year period were higher than those in more than a 10-year period. Although almost all study participants in the transitional period had seropositivity, the results may indicate that this persistence is related to past rubella outbreaks.

Penicillin-Binding Proteins and Associated Protein Mutations Confer Oxacillin/Cefoxitin Tolerance in Borderline Oxacillin-Resistant Staphylococcus aureus.

Among clinical isolates of Staphylococcus aureus, borderline oxacillin-resistant S. aureus (BORSA), which is mildly resistant to oxacillin (OXA) without harboring the mecA or mecC gene, is considered a risk factor for further resistance against multiple antibiotics. In this study, BORSA isolates and their derivatives were characterized through antibiotic susceptibility testing and mutation analysis of the genes encoding penicillin-binding proteins (PBPs) and their related proteins, including the promoter region. Eight BORSA isolates were confirmed to harbor the blaZ gene, and hyperproduction of blaZ-encoded penicillinase was predicted based on the minimum inhibitory concentrations (MICs). Of these, four derivative strains that were spontaneously selected based on viability on media containing high concentrations of OXA showed higher MICs than the parent isolates. The minimum bactericidal concentrations, MIC ratios, and TDtest results identified many strains with cefoxitin tolerance. Sequencing of pbp1, pbp2, pbp3, pbp4, gdpP, and yjbH, and the promoter of pbp4 revealed mutations in BORSA isolates and derivatives, despite their absence in parent isolates, suggesting that mutations in PBPs confer OXA/cefoxitin tolerance in BORSA strains.

NKT cells are responsible for the clearance of murine norovirus through the virus-specific secretory IgA pathway.

Norovirus infection cause epidemic nonbacterial gastroenteritis in patients. The immune mechanisms responsible for the clearance of virus are not completely understood. We examined whether NKT cells are effective against norovirus infection using CD1d KO mice. The body weights of 4-weeks-old CD1d KO mice that were infected with murine norovirus-S7 (MNV-S7) were significantly lower than those of non-infected CD1d KO mice. On the other hand, the body weights of infected WT mice were comparable to those of non-infected WT mice. Correspondingly, CD1d KO mice had an almost 1000-fold higher MNV-S7 burden in the intestine after infection in comparison to WT mice. The mechanism responsible for the insufficient MNV-S7 clearance in CD1d KO mice was attributed to reduced IFN-γ production early during MNV-S7 infection. In addition, the markedly impaired IL-4 production in CD1d KO mice resulted in an impaired MNV-S7-specific secretory IgA production after MNV-S7 infection which is associated with mucosal immunity. Thus, the present results provide evidence that NKT cells play an essential role in MNV-S7 clearance.

A Survey of Vaccine-Induced Measles IgG Antibody Titer to Verify Temporal Changes in Response to Measles Vaccination in Young Adults.

In Japan, sporadic measles cases increased rapidly in 2019 compared to the past six years. To clarify the persistence of immunity against measles in young adults, this study explored the persistence of immunoglobulin G (IgG) antibody titers against the measles virus in 17- to 24-year-old young participants who reside in the Chiba prefecture of Japan. Measles-specific IgG antibody titers, determined by enzyme immunoassay in serum samples collected from 506 participants, were assessed through statistical analyses. Multivariable regression analysis revealed that the distribution of measles IgG antibody titers was significantly correlated with a medical history of measles (P < 0.05), while there was no significant correlation between the number of vaccinations related to measles IgG titers. Furthermore, measles IgG titers tended to decrease, as revealed by the temporal change in IgG titers, during the elapsed period after the last vaccination (P = 0.08). These results indicate that periodic vaccination against measles is required to prevent sporadic measles infection in young and older adults.

Specific autoantigens identified by sera obtained from mice that are immunized with testicular germ cells alone.

There are various autoimmunogenic antigens (AIs) in testicular germ cells (TGCs) recognized as foreign by the body's immune system. However, there is little information of TGC-specific AIs being available. The aim of this study is to identify TGC-specific AIs. We have previously established that immunization using viable syngeneic TGC can also induce murine experimental autoimmune orchitis (EAO) without using any adjuvant. This study is to identify TGC-specific AIs by TGC liquid chromatography-tandem mass spectrometry analysis, followed by two-dimensional gel electrophoresis that reacted with serum IgG from EAO mice. In this study, we identified 11 TGC-specific AIs that reacted with serum from EAO mice. Real-time RT-PCR analysis showed that the mRNA expressions of seven TGC-specific AIs were significantly higher in only mature testis compared to other organs. Moreover, the recombinant proteins of identified 10 (except unnamed protein) TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction. These results indicated Atp6v1a, Hsc70t, Fbp1 and Dazap1 were candidates for TGC-specific AIs. Identification of these AIs will facilitate new approaches for understanding infertility and cancer pathogenesis and may provide a basis for the development of novel therapies.

Influenza virus infection causes neutrophil dysfunction through reduced G-CSF production and an increased risk of secondary bacteria infection in the lung.

The immunological mechanisms of secondary bacterial infection followed by influenza virus infection were examined. When mice were intranasally infected with influenza virus A and then infected with P. aeruginosa at 4 days after viral infection, bacterial clearance in the lung significantly decreased compared to that of non-viral infected mice. Neutrophils from viral infected mice showed impaired digestion and/or killing of phagocytized bacteria due to reduced myeloperoxidase (MPO) activity. G-CSF production in the lungs of viral infected mice was lower than that of non-viral infected mice after secondary bacterial infection. When viral infected mice were injected with G-CSF before secondary bacterial infection, the MPO activity of viral infected mice restored to the same level as that of non-infected mice. Bacteria clearance in viral infected mice was also recovered by G-CSF administration. Thus, neutrophil dysfunction caused by influenza virus is attributed to insufficient G-CSF production, which induces a secondary bacterial infection.

Identification of a virulence determinant that is conserved in the Jawetz and Heyl biotypes of [Pasteurella] pneumotropica.

[Pasteurella] pneumotropica is a ubiquitous bacterium frequently isolated from laboratory rodents. Although this bacterium causes various diseases in immunosuppressed animals, little is known about major virulence factors and their roles in pathogenicity. To identify virulence factors, we sequenced the genome of [P.] pneumotropica biotype Heyl strain ATCC 12555, and compared the resulting non-contiguous draft genome sequence with the genome of biotype Jawetz strain ATCC 35149. Among a large number of genes encoding virulence-associated factors in both strains, four genes encoding for YadA-like proteins, which are known virulence factors that function in host cell adherence and invasion in many pathogens. In this study, we assessed YadA distribution and biological activity as an example of one of virulence-associated factor shared, with biotype Jawetz and Heyl. More than half of mouse isolates were found to have at least one of these genes; whereas, the majority of rat isolates did not. Autoagglutination activity, and ability to bind to mouse collagen type IV and mouse fibroblast cells, was significantly higher in YadA-positive than YadA-negative strains. To conclude, we identified a large number of candidate genes predicted to influence [P.] pneumotropica pathogenesis.

The protective effects of intranasal administration of IL-12 given before influenza virus infection and the negative effects of IL-12 treatment given after viral infection.

To investigate whether the administration of IL-12 is effective against influenza virus infection, mice were intranasally administered IL-12 for three consecutive days and then infected with a non-lethal dose of the influenza virus. The IL-12-treated mice were more resistant to the virus than control mice with respect to the remission of body weight loss, virus burden, pro-inflammatory cytokine production, and inflammatory cell infiltration in the lungs. The number of NK cells and the level of NK cell cytotoxicity significantly increased in the lungs of the mice treated with IL-12 before infection compared to that observed in control mice, leading to promptly eliminate the viral-infected cells. Unexpectedly, all of mice that received IL-12 treatment after being infected with a non-lethal dose of the virus died as a result of their high virus burden and pro-inflammatory cytokine production in the lungs. One possibility of the mechanisms was considered to be activation of myeloid-derived suppressor cell (MDSC), which has immune suppressive function, in the lungs. Thus, IL-12 treatment has opposite effects depending on whether it is administered before or after infection. These results demonstrate the potential risks of immune modulating therapies such as administration of exogenous cytokine or neutralization of cytokine. J. Med. Virol. 88:1487-1496, 2016. © 2016 Wiley Periodicals, Inc.

Draft Genome Sequence of the Rodent Opportunistic Pathogen Pasteurella pneumotropica ATCC 35149T.

Pasteurella pneumotropica is an opportunistic pathogen in rodents that is commonly isolated from upper respiratory tracts in laboratory rodents. Here, we report the draft genome sequence of the P. pneumotropica type strain ATCC 35149, which was first isolated and characterized as biotype Jawetz.

Intranasal immunization with a non-adjuvanted adhesive protein descended from Pasteurella pneumotropica and its preventive efficacy against opportunistic infection in mice.

Intranasal vaccination is one of the most effective means of protecting against invading and colonizing pathogens because the vaccine elicits a mucosal immune response. The exploitation of vaccine adjuvants and delivery systems for intranasal vaccines is an important way to evoke antigen immunogenicity and elicit a better immune response at the mucosal sites. In the present study, we assessed the potential of intranasal immunization using a non-adjuvanted bacterial adhesive protein toward the host organs. We evaluated intranasal immunization with modified recombinant PnxIIIA (MP3) from Pasteurella pneumotropica and its preventive efficacy against opportunistic infection caused by P. pneumotropica, without using any adjuvants or delivery systems. The 100-kDa MP3 was confirmed to retain its immunogenicity and binding activity to collagen type I similar to the parent PnxIIIA. When MP3 was fused to green-fluorescent protein and inoculated into C57BL/6J mice intranasally, fluorescence intensity in the intranasal airway could be observed until 3 h after inoculation. Mice were intranasally immunized with MP3 at a maximum of 4 doses, with 7-day intervals. The antibody titer of serum IgG and IgA specific for MP3, as well as that of bronchoalveolar lavage fluid IgA, showed more than 9 (log2) after 3 or 4 rounds of immunization. Experimentally infecting immunized mice with P. pneumotropica resulted in the inability to isolate the bacterium from the nasal cavity, trachea, conjunctiva, or cecum with more than 3 doses in the immunized mice. Although the detection in each organ seldom changed with less than 2 rounds of immunization, unlike that observed in the non-immunized mice, the detection remarkably decreased with 3 or more rounds of immunization. These results suggest that intranasal immunization with a non-adjuvanted adhesive protein could have preventive effects against opportunistic infection by P. pneumotropica.

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-ß 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.

Mice with asthma are more resistant to influenza virus infection and NK cells activated by the induction of asthma have potentially protective effects.

PURPOSE: This study was conducted in order to investigate whether the virulence of the influenza virus infection is affected by asthma in mice. METHODS: Mice 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. RESULTS: When 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. CONCLUSION: NK 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.

Pathogenicity of Pasteurella pneumotropica in immunodeficient NOD/ShiJic-scid/Jcl and immunocompetent Crlj:CD1 (ICR) mice.

Pasteurella pneumotropica is an opportunistic pathogen in rodents. Natural infection in immunodeficient animals suggests that immunodeficiency is a major factor in P. pneumotropica pathogenesis. To understand this process, we performed clinical, pathological and bacteriological studies of immunodeficient NOD/ShiJic-scid/Jcl and immunocompetent Crlj:CD1 (ICR) mice experimentally infected with P. pneumotropica ATCC 35149. From 14 days postinoculation, some of P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice developed clinical signs of weight loss. Three of 10 P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice developed clinical signs of depression, ruffled coat, and weight loss and died at 27, 34, and 59 days postinoculation. At 35 days postinoculation, almost all P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice had lung abscesses. The bacteria were isolated from the upper and lower respiratory tracts, including the lungs, and blood. In contrast, P. pneumotropica-infected ICR mice exhibited no clinical signs or lesions. The bacteria were isolated from the upper, but not the lower respiratory tracts. We developed an animal model for understanding host interactions with P. pneumotropica.

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

BACKGROUND: Pasteurella 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. RESULTS: The 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. CONCLUSIONS: The 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.