Publications by authors named "Tomohito Hayashi"

38 Publications

Intramammary infection caused by Staphylococcus aureus increases IgA antibodies to iron-regulated surface determinant-A, -B, and -H in bovine milk.

Vet Immunol Immunopathol 2021 May 31;235:110235. Epub 2021 Mar 31.

Dairy Hygiene Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, 4 Hitsujigaoka, Toyohira, Sapporo, Hokkaido, 062-0045, Japan. Electronic address:

The aim of this study was to identify virulence factors that have high immunogenicity. An in vivo-expressed Staphylococcus aureus antigen was identified by probing bacteriophage expression libraries of S. aureus with antibodies in bovine mastitis milk. Eighteen clones were isolated, and their proteins were identified as 5 characterised proteins (IsdA, Protein A, IsdB, autolysin, and imidazole glycerol phosphate dehydratase) and 13 hypothetical proteins. We focused on IsdA, IsdB, and IsdH as virulence factors that have a high immunogenicity and are capable of inducing a specific humoral immune response in S. aureus-infected quarters. The optical density (OD) values of IsdA and IsdB IgA and IgG antibodies in milk affected by naturally occurring mastitis caused by S. aureus increased significantly compared to those in healthy milk. In the experimental infection study, the OD values of IsdA- and B-specific IgA and IgG antibodies were significantly increased from 2 to 4 weeks after S. aureus infection compared to day 0 (P < 0.05). On the other hand, we demonstrated that milk from natural and experimental intramammary infections caused by S. aureus are associated with significantly higher IgA levels against IsdH (P < 0.05), but no significant change in IgG levels. Our findings facilitated our understanding of the pathogenicity of S. aureus in bovine mastitis, as well as the mechanisms by which specific humoral immune responses to S. aureus infection are induced. In addition, the results obtained could provide insight into how bovine mastitis can be controlled, for example, through vaccination.
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http://dx.doi.org/10.1016/j.vetimm.2021.110235DOI Listing
May 2021

Rapid Detection From Clinical Mastitis Milk by Colloidal Gold Nanoparticle-Based Immunochromatographic Strips.

Front Vet Sci 2019 22;6:504. Epub 2020 Jan 22.

Dairy Hygiene Unit, Division of Pathology and Pathophysiology, Hokkaido Research Station, National Institute of Animal Health, National Agriculture and Food Research Organization, Sapporo, Japan.

Rapid diagnostic technologies for bovine mastitis caused by () are urgently needed. In the current study, we generated an anti-ribosomal protein-L7/L12 antibody to detect and an anti-ribosomal protein-L7/L12 antibody-coated immune-chromatographic strip (ICS) test. Moreover, we determined the ability of the ICS test to detect from milk samples collected from cows with clinical mastitis. The developed ICS reacted to in a bacteria load-dependent manner with a detection limit of ~10 CFU/mL. In the evaluation of possible cross-reactivity of the ICS test, six strains of coagulase-negative Staphylococci showed slightly positive reactions, although at a lower level; however, other bacteria were completely negative. Next, we investigated the sensitivity and specificity of the ICS test compared with the bacteriological culture method using milk samples from clinical bovine mastitis. The results of the experiments demonstrated that the ICS test had high sensitivity [100%, 95% confidence interval (CI): 91.3-100%] and specificity (91.9%, CI: 90.5-91.9%) compared with culture tests. In addition, the kappa statistic demonstrated that ICS tests showed substantial agreement (k = 0.77, CI: 0.66-0.87) with culture tests. Positive correlations were observed for the statistical analysis between ( gene) copy numbers and ICS test scores in mastitic milk infected by . Therefore, we assume that this new detection method using ICS may be useful as a highly sensitive -screening method for the diagnosis of bovine mastitis. Our findings support the ongoing effort to develop an ICS method for bovine -induced mastitis, which can contribute to the rapid diagnosis of this disease.
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http://dx.doi.org/10.3389/fvets.2019.00504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988510PMC
January 2020

Immunosuppression in Cows following Intramammary Infusion of Mycoplasma bovis.

Infect Immun 2020 02 20;88(3). Epub 2020 Feb 20.

Animal Health Laboratory, Graduate School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, Japan

is a destructive pathogen that causes large economic losses in rearing cattle for beef and dairy worldwide. causes suppression of and evades the host immune response; however, the mechanisms of host immune function involved in mastitis have not been elucidated. The purpose of this study was to elucidate the characteristics of the bovine immune response to mycoplasmal mastitis. We evaluated the responsiveness of the bovine mammary gland following infusion of Somatic cell counts and bacterial counts in milk from the infected quarter were increased. However, the proliferation of peripheral blood mononuclear cells (blood MNCs) and mononuclear cells isolated from -stimulated mammary lymph nodes (lymph node MNCs) did not differ from that in the unstimulated cells. Transcriptome analysis revealed that the mRNA levels of innate immune system-related genes in blood MNCs, complement factor D (CFD), ficolin 1 (FCN1), and tumor necrosis factor superfamily member 13 (TNFSF13) decreased following intramammary infusion of The mRNA levels of immune exhaustion-related genes, programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), lymphocyte activation gene 3 (LAG3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) of milk mononuclear cells (milk MNCs) in the infected quarter were increased compared with those before infusion. Increase in immune exhaustion-related gene expression and decrease in innate immune response-related genes of MNCs in quarters from cows were newly characterized by -induced mastitis. These results suggested that -induced mastitis affected the immune function of bovine MNCs, which is associated with prolonged duration of infection with .
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http://dx.doi.org/10.1128/IAI.00521-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035927PMC
February 2020

Staphylococcus aureus-specific IgA antibody in milk suppresses the multiplication of S. aureus in infected bovine udder.

BMC Vet Res 2019 Aug 9;15(1):286. Epub 2019 Aug 9.

Dairy Hygiene Unit, Division of Pathology and Pathophysiology, Hokkaido Research Station, National Institute of Animal Health, National Agriculture and Food Research Organization, 4 Hitsujigaoka, Toyohira, Sapporo, Hokkaido, 062-0045, Japan.

Background: Bovine mastitis caused by Staphylococcus aureus (S. aureus) is extremely difficult to control and new methods for its prevention and management are required. Nasal vaccines may prevent initial bovine mastitis infection caused by S. aureus. However, limited information is available regarding induction of mucosal immune response through nasal immunization with antigen and its suppression of S. aureus multiplication during bovine mastitis. This study sought to investigate whether induction of immunoglobulin A (IgA) in milk by nasal immunization could suppress multiplication of S. aureus in the bovine udder.

Results: Nasal immunization with formalin-killed S. aureus conjugated with a cationic cholesteryl-group-bearing pullulan-nanogel was performed. Anti-S. aureus-specific IgA antibodies were significantly more abundant in the milk of immunized cows than in non-immunized animals (P < 0.05). S. aureus counts in the quarter were negative in both non-immunized and nasal-immunized cows 1 week after mock infusion. In S. aureus-infused quarters, S. aureus multiplication was significantly suppressed in immunized compared with non-immunized cows (P < 0.05). Furthermore, a significant negative correlation was found between S. aureus-specific IgA antibodies and S. aureus counts in infused quarters of both non-immunized and nasal-immunized cows (r = - 0.811, P < 0.01).

Conclusion: In conclusion, the present study demonstrates that S. aureus-specific IgA antibodies in milk successfully suppressed the multiplication of S. aureus in infected bovine udders. Although the exact mechanism explaining such suppressive effect remains to be elucidated, nasal vaccines that can induce humoral immunity may help prevent initial infection with S. aureus and the onset of bovine mastitis.
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http://dx.doi.org/10.1186/s12917-019-2025-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688226PMC
August 2019

The bacterial load in milk is associated with clinical severity in cases of bovine coliform mastitis.

J Vet Med Sci 2019 Jan 26;81(1):107-112. Epub 2018 Nov 26.

Dairy Hygiene Unit, Division of Pathology and Pathophysiology, Hokkaido Research Station, National Institute of Animal Health, National Agriculture and Food Research Organization, 4 Hitsujigaoka, Toyohira, Sapporo, Hokkaido 062-0045, Japan.

We evaluated the relationship between the severity of coliform mastitis and bacterial load in 106 quarter milk samples. We found no significant relationship between somatic cell count and coliform bacterial load in milk in bovine clinical coliform mastitis. Results of the Cochran-Armitage test for trend in milk bacterial load proportions indicated a significant decreasing low group (P<0.001), increasing medium group (P<0.002) and increasing high group (P<0.02) with increasing clinical grade. The present study indicates that the coliform bacterial load in milk is significantly associated with clinical severity states in cases of bovine coliform mastitis, and can be a useful indicator for optimal management of this disease.
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http://dx.doi.org/10.1292/jvms.18-0581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361639PMC
January 2019

Cow's milk neutralizes the cytotoxicity of acrolein, a putative carcinogen in cigarette smoke.

J Vet Med Sci 2018 Aug 25;80(8):1301-1304. Epub 2018 Jun 25.

Dairy Hygiene Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, 4 Hitsujigaoka, Toyohira, Sapporo, Hokkaido 062-0045, Japan.

Cigarette smoke is a strong and independent risk factor for esophageal cancer, while the consumption of cow's milk has been proposed as a protective factor. The mechanistic role of milk in preventing cancer, however, has not been clarified. We focused our study on acrolein, an abundant unsaturated aldehyde present in cigarette smoke. Acrolein is a highly toxic compound and a putative carcinogen. Using a cell culture system, we found that (1) acrolein caused necrosis in Ramos Burkitt's lymphoma cells, (2) the necrosis was inhibited by preincubation of acrolein with milk, and (3) acrolein formed adducts with milk proteins. These results indicated the protective effects of cow's milk against acrolein-induced cytotoxicity via protein-acrolein adduct formation.
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http://dx.doi.org/10.1292/jvms.17-0603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115267PMC
August 2018

Phenotypic and functional analysis of bovine peripheral blood dendritic cells before parturition by a novel purification method.

Anim Sci J 2018 Jul 30;89(7):1011-1019. Epub 2018 Apr 30.

Cellular Biology Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan.

Dendritic cells (DCs) are specialized antigen presenting cells specializing in antigen uptake and processing, and play an important role in the innate and adaptive immune response. A subset of bovine peripheral blood DCs was identified as CD172a /CD11c /MHC (major histocompatibility complex) class II cells. Although DCs are identified at 0.1%-0.7% of peripheral blood mononuclear cells (PBMC), the phenotype and function of DCs remain poorly understood with regard to maintaining tolerance during the pregnancy. All cattle used in this study were 1 month before parturition. We have established a novel method for the purification of DCs from PBMC using magnetic-activated cell sorting, and purified the CD172a /CD11c DCs, with high expression of MHC class II and CD40, at 84.8% purity. There were individual differences in the expressions of CD205 and co-stimulatory molecules CD80 and CD86 on DCs. There were positive correlations between expression of cytokine and co-stimulatory molecules in DCs, and the DCs maintained their immune tolerance, evidenced by their low expressions of the co-stimulatory molecules and cytokine production. These results suggest that before parturition a half of DCs may be immature and tend to maintain tolerance based on the low cytokine production, and the other DCs with high co-stimulatory molecules may already have the ability of modulating the T-cell linage.
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http://dx.doi.org/10.1111/asj.13014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055732PMC
July 2018

Identification of a novel mechanism of action of bovine IgG antibodies specific for Staphylococcus aureus.

Vet Res 2018 02 26;49(1):22. Epub 2018 Feb 26.

International Education and Research Center for Food and Agricultural Immunology, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, 980-0845, Japan.

Staphylococcus aureus is a major pathogen that causes subclinical mastitis associated with huge economic losses to the dairy industry. A few vaccines for bovine mastitis are available, and they are expected to induce the production of S. aureus-specific antibodies that prevent bacterial adherence to host cells or promote opsonization by phagocytes. However, the efficacy of such vaccines are still under debate; therefore, further research focusing on improving the current vaccines by seeking additional mechanisms of action is required to reduce economic losses due to mastitis in the dairy industry. Here, we generated S. aureus-specific bovine IgG antibodies (anti-S. aureus) that directly inhibited bacterial growth in vitro. Inhibition depended on specificity for anti-S. aureus, not the interaction between Protein A and the fragment crystallizable region of the IgG antibodies or bacterial agglutination. An in vitro culture study using S. aureus strain JE2 and its deletion mutant JE2ΔSrtA, which lacks the gene encoding sortase A, revealed that the effect of anti-S. aureus was sortase-A-independent. Sortase A is involved in the synthesis of cell-wall-associated proteins. Thus, other surface molecules, such as membrane proteins, cell surface polysaccharides, or both, may trigger the inhibition of bacterial growth by anti-S. aureus. Together, our findings contribute insights into developing new strategies to further improve the available mastitis vaccine by designing a novel antigen on the surface of S. aureus to induce inhibitory signals that prevent bacterial growth.
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http://dx.doi.org/10.1186/s13567-018-0517-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828400PMC
February 2018

Detection of bovine mastitis pathogens by loop-mediated isothermal amplification and an electrochemical DNA chip.

J Vet Med Sci 2017 Dec 1;79(12):1973-1977. Epub 2017 Nov 1.

National Institute of Animal Health, National Agriculture and Food Research Organization, Sapporo, Hokkaido 062-0045, Japan.

Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program.
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http://dx.doi.org/10.1292/jvms.17-0263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5745174PMC
December 2017

Exfoliation rate of mammary epithelial cells in milk on bovine mastitis caused by Staphylococcus aureus is associated with bacterial load.

Anim Sci J 2018 Jan 11;89(1):259-266. Epub 2017 Sep 11.

Dairy Hygiene Unit, Division of Pathology and Pathophysiology, Hokkaido Research Station, National Institute of Animal Health, NARO, Sapporo, Hokkaido, Japan.

The exfoliation rate of mammary epithelial cells (MECs) in milk is affected by physiological, breeding and environmental factors. Little is known about the relationship between the MEC exfoliation into milk and mammary-infected Staphylococcus aureus (S. aureus) load on bovine mastitis caused by S. aureus. The aim of this study was to investigate the relationship between S. aureus load and the proportion of MEC exfoliation in milk using five substantial bovine mastitis models. In 64 randomly extracted milk samples from udders at 3-21 days after S. aureus infusion, there were various samples with different numbers of S. aureus counts and somatic cell counts. No significant correlations were found between the S. aureus counts and somatic cell count (r = 0.338). In contrast, a significant correlation was noted between S. aureus counts and the proportion of cytokeratin-positive cells in the milk from the infused udders (r = 0.734, P < 0.01). In conclusion, the increasing MEC exfoliation rate in milk from mastitis udders caused by S. aureus may contribute to reduced milk yield.
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http://dx.doi.org/10.1111/asj.12886DOI Listing
January 2018

Effect of intramammary infusion of recombinant bovine GM-CSF and IL-8 on CMT score, somatic cell count, and milk mononuclear cell populations in Holstein cows with Staphylococcus aureus subclinical mastitis.

Vet Res Commun 2017 Sep 9;41(3):175-182. Epub 2017 Mar 9.

Hokkaido Research Station, National Institute of Animal Health, NARO, 4 Hitsujigaoka, Toyohira, Sapporo, Hokkaido, 062-0045, Japan.

The effect of intramammary infusion of recombinant bovine granulocyte-macrophage colony-stimulating factor (rbGM-CSF) and interleukin-8 (rbIL-8) on mononuclear cell populations in quarters, somatic cell count (SCC) and the California Mastitis Test (CMT) score were investigated. From the selected cows with naturally occurring Staphylococcus aureus subclinical mastitis, one quarter of each cow were selected for the infusions of rbGM-CSF (400 μg/5 mL/quarter, n = 9), rbIL-8 (1 mg/5 mL/quarter, n = 9), and phosphate-buffered saline (5 mL/quarter, n = 7). The CMT score of both cytokines post infusion temporarily increased between days 0 and 1 and significantly decreased between days 7 and 14 compared to the preinfusion level. The SCC on day 14 after infusions of rbGM-CSF tended to be lower than that of the control group. The percentage of CD14+ cells increased on days 1 and 2 post infusion of rbGM-CSF. The percentage of CD4+ and CD8+ cells also increased on days 2 and 3, suggesting that the infusion of rbGM-CSF enhanced cellular immunity in the mammary gland. In contrast, the percentage of CD14+ cells decreased on days 0.25 and 1 post infusion of rbIL-8. No significant changes in the percentages of CD4+ and CD8+ cells in milk after infusion of rbIL-8 were evident during the experimental period, which suggested that rbIL-8 had little effect on the function of T cells in the mammary gland. These results indicated that rbGM-CSF and rbIL-8 decreased the CMT score by a different mechanism and may have a potential as therapeutic agents for subclinical mastitis.
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http://dx.doi.org/10.1007/s11259-017-9684-yDOI Listing
September 2017

The cell wall component lipoteichoic acid of Staphylococcus aureus induces chemokine gene expression in bovine mammary epithelial cells.

J Vet Med Sci 2016 Oct 20;78(9):1505-1510. Epub 2016 May 20.

Hokkaido Research Station, National Institute of Animal Health, NARO, Sapporo, Hokkaido 062-0045, Japan.

Staphylococcus aureus (SA) is a major cause of bovine mastitis, but its pathogenic mechanism remains poorly understood. To evaluate the role of lipoteichoic acid (LTA) in the immune or inflammatory response of SA mastitis, we investigated the gene expression profile in bovine mammary epithelial cells stimulated with LTA alone or with formalin-killed SA (FKSA) using cap analysis of gene expression. Seven common differentially expressed genes related to immune or inflammatory mediators were up-regulated under both LTA and FKSA stimulations. Three of these genes encode chemokines (IL-8, CXCL6 and CCL2) functioning as chemoattractant molecules for neutrophils and macrophages. These results suggest that the initial inflammatory response of SA infection in mammary gland may be related with LTA induced chemokine genes.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059380PMC
http://dx.doi.org/10.1292/jvms.15-0706DOI Listing
October 2016

Extracellular cyclophilin A possesses chemotaxic activity in cattle.

Vet Res 2015 Jul 11;46:80. Epub 2015 Jul 11.

Laboratory of Mucosal Immunology, Graduate School of Agricultural Science, Tohoku University, Miyagi, 981-8555, Japan.

Cyclophilin A (CyPA) was originally discovered in bovine thymocytes as a cytosolic binding protein of the immunosuppressive drug cyclosporine A. Recent studies have revealed that in mice and humans, CyPA is secreted from cells in injured or infected tissues and plays a role in recruiting inflammatory cells in those tissues. Here we found that in cattle abundant level of extracellular CyPA was observed in tissues with inflammation. To aid in investigating the role of extracellular CyPA in cattle, we generated recombinant bovine CyPA (rbCyPA) and tested its biological activity as an inflammatory mediator. When bovine peripheral blood cells were treated with rbCyPA in vitro, we observed that rbCyPA reacts with the membranous surface of granulocytes, monocytes and lymphocytes. Chemotaxis analysis showed that the granulocytes migrate toward rbCyPA and the migration is inhibited by pre-treatment with an anti-bovine CyPA antibody. These results indicate that, as for mice and humans, extracellular CyPA possesses chemotactic activity to recruit inflammatory cells (e.g., granulocytes) in cattle, and could thus be a potential therapeutic target for the treatment of inflammation.
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http://dx.doi.org/10.1186/s13567-015-0212-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498507PMC
July 2015

Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens.

J Vet Med Sci 2015 Aug 3;77(8):1007-9. Epub 2015 May 3.

Toshiba Corporation, 1, Komukai-Toshiba-cho, Saiwai-ku, Kawasaki, Kanagawa 212-8582, Japan.

A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.
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http://dx.doi.org/10.1292/jvms.14-0159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4565803PMC
August 2015

Antimicrobial susceptibilities of Mycoplasma isolated from bovine mastitis in Japan.

Anim Sci J 2014 Jan 21;85(1):96-9. Epub 2013 Nov 21.

Department of Animal Health 1, School of Veterinary Medicine, Azabu University, Sagamihara, Japan.

Mycoplasma spp. are highly contagious pathogens and intramammary Mycoplasma infection is a serious issue for the dairy industry. As there is no effective vaccine for Mycoplasma infection, control depends on good husbandry and chemo-antibiotic therapy. In this study, antimicrobial susceptibility of Mycoplasma strains recently isolated from cases of bovine mastitis in Japan was evaluated by minimum inhibitory concentration (MIC). All Mycoplasma bovis strains were sensitive to pirlimycin, danofloxacin and enrofloxacin, but not kanamycin, oxytetracycline, tilmicosin or tylosin. M. californicum and M. bovigenitalium strains were sensitive to pirlimycin, danofloxacin, enrofloxacin, oxytetracycline, tilmicosin and tylosin, but not to kanamycin. This is the first report to describe the MIC of major antimicrobial agents for Mycoplasma species isolated from bovine mastitis in Japan.
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http://dx.doi.org/10.1111/asj.12144DOI Listing
January 2014

Reliability in somatic cell count measurement of clinical mastitis milk using DeLaval cell counter.

Anim Sci J 2013 Dec 15;84(12):805-7. Epub 2013 Oct 15.

School of Veterinary Medicine, Azabu University, Sagamihara, Japan.

Somatic cell counts (SCC) measurements are typically performed using quantitative methods, such as the Breed method (Breed) and the Fossomatic method (FSCC). The DeLaval cell counter (DCC) developed recently is a quantitative somatic cell counter with a low initial cost and superior portability. However, since the DCC was specifically developed for measuring SCC of ≤ 4 × 10(6) cells/mL milk from bulk tanks or individual cows, its reliability for estimating SCC that exceed this concentration has not yet been clarified. This study therefore examined whether it is possible to accurately measure SCC by diluting milk samples with initial SCC of 4 × 10(6) cells/mL, as seen in clinical mastitis milk. We collected milk samples from 99 quarters of 99 Holstein cows with clinical mastitis. These milk samples were diluted 10-fold with saline and thoroughly mixed before performing SCC measurement with the DCC. The correlation coefficients of SCC measured by the FSCC, Breed and DCC methods indicated strong correlations between each pair of methods. The findings showed that DCC can be used to identify bovine clinical mastitis milk and is useful as a quantitative SCC measurement device on farm sites.
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http://dx.doi.org/10.1111/asj.12136DOI Listing
December 2013

Up-regulated expression of CXCL12 in human spleens with extramedullary haematopoiesis.

Pathology 2013 06;45(4):408-16

Department of Comprehensive Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

Aims: To determine the expression of CXCL12 in human spleens with extramedullary haematopoiesis (EMH) for clarifying the association of splenic haematopoietic stem cells (HSCs) with CXCL12, which has been demonstrated to be a marker of bone marrow niches.

Methods: We examined the expression of mRNA for CXCL12 by quantitative reverse transcription polymerase chain reaction (RT-PCR) and localised the CXCL12 protein by immunohistochemical staining in EMH negative and positive spleen samples from autopsy cases.

Results: Expression of CXCL12 was significantly higher in samples from EMH positive cases than those from EMH negative cases. CXCL12 was localised to the endothelial cells of the sinuses of the red pulp in EMH positive spleens while vascular endothelial cells of the white pulp expressed CXCL12 throughout the spleen. c-kit positive/CD34 negative cells were identified in contact with CXCL12 positive endothelial cells of sinuses in EMH positive cases, although the number was few. In contrast, erythroblastic islands were frequently observed in EMH positive cases and dominantly localised to the intrasinusoidal spaces in association with CD68 positive macrophages.

Conclusions: Our results suggest that endothelial cells of splenic sinuses expressing CXCL12 may contribute to attracting circulating haematopoietic precursor cells and constitute bone marrow niche-like regions of EMH in humans. Differentiating haematopoietic cells may move into intrasinusoidal spaces to form EMH foci such as erythroblastic accumulation.
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http://dx.doi.org/10.1097/PAT.0b013e3283613dbfDOI Listing
June 2013

Molecular-based identification of yeasts isolated from bovine clinical mastitis in Japan.

J Vet Med Sci 2013 26;75(3):387-90. Epub 2012 Oct 26.

National Institute of Animal Health, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-0856, Japan.

This study analyzed molecular-based identification of yeasts that associated with bovine clinical mastitis in Japan. Over 3,200 quarter milk samples from Holstein dairy cows collected in 2011 on Hokkaido and Honshu islands were examined. Yeast isolates were characterized by polymerase chain reaction amplification and sequencing of the D1/D2 region of the 26S rDNA. Molecular characterization confirmed that Candida spp. and Pichia spp. were most frequently isolated species. Our molecular analysis of mastitic milk samples demonstrated the prevalence of Pichia kudriavzevii(22/58) and Candida tropicalis(14/58). In addition, we demonstrated that molecular analysis of the D1/D2 region of the 26S rDNA is a rapid and reliable method for identifying clinically significant yeasts in dairy hygiene, including potentially new or emerging pathogenic species.
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http://dx.doi.org/10.1292/jvms.12-0362DOI Listing
September 2013

Effect of intramammary infusion of rbGM-CSF on SCC and expression of polymorphonuclear neutrophil adhesion molecules in subclinical mastitis cows.

Vet Res Commun 2012 Mar 5;36(1):21-7. Epub 2011 Nov 5.

National Institute of Animal Health, National Agriculture and Food Research Organization, 3-1-5 Kan-nondai, Tsukuba, Ibaraki 305-0856, Japan.

The effect of rbGM-CSF intramammary infusion on the subclinical mastitis was evaluated by the somatic cell count (SCC) and expression of adhesion molecules (CD62L and CD11b) on the surface of neutrophils (PMN) in blood and milk. Fifteen cows diagnosed to have subclinical mastitis were used in this study. Seven cows showed a decrease in the SCC (decreased group), whereas 8 cows showed an increase in the SCC (increased group) 7 days after infusion of rbGM-CSF compared to pre infusion level. The percentage of CD62+ cells tended to be lower and CD11b+cells tended to be higher at 6 h on blood PMN in the decreased group of cows. Increased group of cows showed opposite tendencies. The mean fluorescent intensity of these adhesion molecules expressed on PMN in blood and milk was similar in both groups. These results suggested some association between expression of adhesion molecules and changes in SCC by rbGM-CSF. Responsiveness of PMN adhesion molecules to rbGM-CSF might determine the changes in SCC of the subclinical mastitic cows after infusion of rbGM-CSF.
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http://dx.doi.org/10.1007/s11259-011-9506-6DOI Listing
March 2012

Increased concentration of high-mobility group box 1 protein in milk is related to the severity of bovine mastitis.

Vet Res Commun 2011 Jan 1;35(1):47-54. Epub 2010 Dec 1.

Research Institute for Biological Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan.

High-mobility group box 1 (HMGB1) protein is the major component of the nonhistone nuclear protein group and is involved in nucleosome stabilization and transcription regulation. HMGB1 has recently been focused on as a proinflammatory cytokine associated with various inflammatory diseases and as a target of anti-inflammatory therapy. Mastitis, a serious inflammatory disease of dairy cows, is caused by infection of the mammary gland and has detrimental effects on the quantity and quality of milk. By detecting the presence of HMGB1 in milk, we investigated the correlation between HMGB1 concentration and the severity of bovine mastitis, which was determined using the California Mastitis Test and somatic cell count (SCC). We detected a substantial amount of HMGB1 in mastitic milk but not in the milk from normal cows. We used the Spearman rank correlation coefficient to assess the relationship between HMGB1 concentration and SCC and found a significant correlation (n = 12, r = 0.975). Thus, we confirmed the positive correlation between HMGB1 concentration and SCC in milk, i.e., the severity of mastitis, which suggested that HMGB1 in milk is a new indicator of bovine mastitis.
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http://dx.doi.org/10.1007/s11259-010-9454-6DOI Listing
January 2011

Decrease in bovine CD14 positive cells in colostrum is associated with the incidence of mastitis after calving.

Vet Res Commun 2010 Feb;34(2):197-203

National Institute of Animal Health, 3-1-5 Kan-nondai, Tsukuba Ibaraki, 305-0856, Japan.

During the postpartum period there is a high incidence of mastitis in dairy cows. The reason for this increased risk of mastitis still remains unclear. Since leukocytes in colostrum have an important role in preventing the onset of mastitis, we investigated the leukocyte populations, which express CD4, CD8, CD14, CD21 or WC1, in colostrum as well as in blood obtained from 14 Holstein cows. Eight cows developed mastitis within a week after calving and the other 6 remained healthy. The percentage of CD14+ cells in colostrum was significantly lower in mastitic cows than in healthy cows. There were no significant differences in other marker positive cells either in the colostrum or in the blood. The CD14+ cells in colostrum play an important role of defense against invading microorganisms in the mammary glands. Our results suggested that the lower percentage of CD14+ cells in colostrum might predict the incidence of mastitis in the following period.
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http://dx.doi.org/10.1007/s11259-009-9339-8DOI Listing
February 2010

Peripheral blood leukocyte subpopulation of dairy cows with digital dermatitis and effect of hoof trimming with antibiotic treatment.

J Vet Med Sci 2009 Apr;71(4):391-5

School of Veterinary Medicine, Rakuno Gakuen University, Japan.

In the present study, 30 cows were used to evaluate the changes in the peripheral blood leukocyte subpopulation of dairy cows with digital dermatitis (DD) following hoof trimming and antibiotic treatment. The cows were divided into two groups; 18 cows (DD group) had DD on both hind feet, and 12 cows (control group) had four feet with no clinical abnormalities. The DD group was further divided into two groups based on the treatment; the antibiotic group (8 cows) was treated with only 2% lincomycin liquid spray once daily for 3 days, and the trimmed group (10 cows) received trimming of hooves as well as treatment with 2% lincomycin liquid spray. The plasma cortisol concentration was significantly higher in both DD groups before treatment than in the control group, and it decreased significantly after hoof trimming in the trimmed group. The number of CD3(+), CD4(+), WC1(+) and CD21(+) cells in both DD groups before treatment was significantly lower than that of the control group. The number of CD3(+), CD4(+), WC1(+) and CD21(+) cells in the trimmed group increased after treatment. These results indicated that cows with DD suffer from stress and reduced number of T and B cells. Treatment of DD with both hoof trimming and 2% lincomycin liquid spray was effective for reducing the stress and bringing the immune cell number back to the normal range.
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http://dx.doi.org/10.1292/jvms.71.391DOI Listing
April 2009

Seasonal influence on blood pressure in elderly normotensive subjects.

Hypertens Res 2008 Mar;31(3):569-74

Department of Preventive Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

The aim of this study was to examine whether or not fluctuations in blood pressure (BP) differ by season. Subjects were 45 elderly individuals (20 men and 25 women; mean age, 66.5+/-4.9 [SD] years). Each subject's BP was recorded with an ambulatory BP monitoring device for 24 h during each of the four seasons. Subjects also wore a portable weather meter to obtain ambient temperature, relative humidity, and barometric pressure simultaneously with BP. The relationships between meteorologic values and BP were investigated at various parts of the day. Seasonal differences in BP fluctuation around wake-up-time were analyzed by means of the Tukey's test. The difference between the pre-wake-up-time systolic BP and the wake-up-time systolic BP was significantly greater in winter than in summer (8.7 mmHg greater, p<0.001). The difference between pre-wake-up-time and wake-up-time systolic BP was significantly greater in autumn than in spring (9.4 mmHg greater, p<0.001) or summer (13.1 mmHg greater, p<0.001). The difference between pre-wake-up-time heart rate and wake-up-time heart rate did not differ statistically between seasons. In conclusion, the present study showed that the difference between pre-wake-up-time systolic BP and wake-up-time systolic BP was greatest in the colder seasons, i.e., autumn and winter. There appears to be a large fluctuation in wake-up-time in the colder seasons. Low ambient temperature likely induces this large fluctuation.
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http://dx.doi.org/10.1291/hypres.31.569DOI Listing
March 2008

Changes in peripheral blood mononuclear cells after calving in lactating cows with serious decrease of body weight before and after calving.

J Vet Med Sci 2008 Feb;70(2):153-8

School of Veterinary Medicine, Kitasato University, Towada, Aomori, Japan.

The aim of this study was to clarify the hormonal and immunological changes in periparturient dairy cows with excessive weight loss following calving. We analyzed leukocyte populations and the peripheral blood mononuclear cell (PBMC) mRNA of IFN-gamma, IL-4, growth hormone (GH)-receptor (R) and insulin -R and measured the GH and insulin concentrations. Ten dairy cows fed in one herd were used in this study and were divided into two groups. Five cows that experienced loss of body weight following calving of over 10% comprised the Weight Loss Group, and the remaining five cows, which experienced loss of body weight of 5% or less following calving, served as the Control Group. The Weight Loss Group had high serum cholesterol and low blood urea nitrogen levels compared with the Control Group throughout the period of observation. In regard to the leukocyte populations, there were significantly fewer CD4(+)CD45R-T cells in the Weight Loss Group 1 to 2 months after calving compared with the Control Group. The percentage of IFN-gamma/IL-4 among PBMCs in the Weight Loss Group tended to be lower than in the Control Group throughout the observation period. In the Weight Loss Group, the levels of GH-R during month 2 were lower than in the Control Group. We concluded that excessive loss of body weight in dairy cows following calving is associated with depressed cellular immune function.
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http://dx.doi.org/10.1292/jvms.70.153DOI Listing
February 2008

Effect of hoof trimming before the dry period on productive performance in perinatal dairy cows.

J Vet Med Sci 2008 Jan;70(1):95-8

School of Veterinary Medicine, Kitasato University, Aomori, Japan.

To investigate the effect of hoof trimming before the dry period, the hooves of 10 cows (trimmed group) were trimmed at 79.6 +/- 8.6 days before parturition and the hooves of 52 cows were left untrimmed (control group). Blood biochemistry and hormone concentrations were investigated for 6 cows from each group. The daily milk yield after parturition in the trimmed group tended to be higher than that of the control group. Between 0 and 30 days after parturition, the levels of non-esterified fatty acids were significantly lower stet, and the plasma glucose and glucose disposal rates were significantly higher in the trimmed group. The plasma cortisol concentration was stable before and after parturition in the trimmed group. Hoof trimming before the dry period appears to reduce stress and maintain the nutritional conditions of perinatal dairy cows.
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http://dx.doi.org/10.1292/jvms.70.95DOI Listing
January 2008

Influence of mental stress on cardiovascular function as evaluated by changes in energy expenditure.

Hypertens Res 2007 Nov;30(11):1019-27

Department of Preventive Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

We assessed the influence of mental stress on cardiovascular parameters, including systolic blood pressure (SBP), heart rate (HR) and sympathetic activity (LF/HF), with the use of a quantitative indicator of energy expenditure (EE). Forty-four male university students underwent a mental arithmetic test. Their EE was 1.3-fold that at rest in response to the test. Change in EE (DeltaEE) in response to the test was compared between subjects with high blood pressure (BP) and those without and between subjects with high salt intake and those without. Changes in SBP (DeltaSBP), changes in HR (DeltaHR) and changes in LF/HF (DeltaLF/HF) in relation to DeltaEE were represented by linear regression. Regression analysis showed that the coefficients of DeltaEE were positively related to the dependent variables DeltaSBP, DeltaHR and DeltaLF/HF. The slopes of the regression curves for the high-BP group (24-h SBP >or=127 mmHg) and the high-salt-intake group (>or=11 g/day) were steeper than those for the normal BP group and the low-salt-intake group (p=0.11 and p=0.01, respectively). Thus, we were able to determine the influence of mental stress on cardiovascular function. The influence of mental stress on cardiovascular function likely differs according to a subject's environmental conditions. Our study implied that high salt intake increases the sensitivity of cardiovascular functions to mental stress.
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http://dx.doi.org/10.1291/hypres.30.1019DOI Listing
November 2007

Cloning, expression, and characterization of the superantigen streptococcal pyrogenic exotoxin G from Streptococcus dysgalactiae.

Infect Immun 2007 Apr 5;75(4):1721-9. Epub 2007 Feb 5.

Department of Infectious Diseases, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan.

We identified seven novel variants of streptococcal pyrogenic exotoxin G (SPEGG), a superantigen, in Streptococcus dysgalactiae subsp. dysgalactiae or equisimilis isolates from clinical cases of infection in humans and animals. Phylogenetic analysis of the SPEGG variants indicated two clades in the dendrogram: one composed of variants derived from the bacteria isolated from the humans and the other composed of variants from the bacteria isolated from the animals. Bovine peripheral blood mononuclear cells (PBMCs) were stimulated effectively by recombinant SPEGGs (rSPEGGs) expressed in Escherichia coli, while human PBMCs were not stimulated well by any of the rSPEGGs tested. SPEGGs selectively stimulated bovine T cells bearing Vbeta1,10 and Vbeta4. Bovine serum showed reactivity to the rSPEGG proteins. These results indicated that SPEGGs have properties as superantigens, and it is likely that SPEGGs play a pathogenic role in animals.
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http://dx.doi.org/10.1128/IAI.01183-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1865666PMC
April 2007

Exclusion of influence of physical activity on ambulatory blood pressure.

Clin Exp Hypertens 2007 Jan;29(1):23-30

The Department of Public Health, Yokohama City University School of Medicine, Yokohama, Japan.

Objective: The basal blood pressure (BP) is more intimately related to hypertension severity than casual BP. BP values obtained by ambulatory blood pressure monitoring (ABPM) tend to be influenced by patients' physical activity because values are not always obtained with the patient at rest. The purpose of this study was to estimate the influence of physical activity on BP and determine daytime BP adjusted for activity as measured by ABPM.

Methods: This study targeted three clinically different groups: healthy medical students (HS, n = 40), patients with hypertension (HT, n = 20), and patients with diabetes mellitus (DM, n = 7). The subjects' BP, heart rate (HR), and physical activity level were measured by a noninvasive portable multi-biomedical recorder. To identify the influence of physical activity on BP in the three study groups, a least squares regression analysis of the relation between BP and ACT (an index of activity with acceleration) was performed for each group.

Results: ACT had a positive influence on systolic BP (SBP) in the HS, HT, and DM groups (R2 = 0.319, 0.576, 0.697, respectively). SBP adjusted for ACT (walking level) by means of the regression model with dummy variable was 0-24 mmHg lower than the value of SBP measured by ABPM, and daytime SBP (walking level) was overestimated by approximately 10 mmHg in comparison to the value of SBP at rest (ACT = 0).

Conclusion: Physical activity had a positive effect on SBP. The results showed that physical activity (walking-level) had a positive effect on SBP of about 10 mmHg.
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http://dx.doi.org/10.1080/10641960601096752DOI Listing
January 2007

Comparison of the insulin reaction of peripheral blood T cells between healthy Holstein dairy cows and JB during the periparturient period.

J Vet Med Sci 2006 Nov;68(11):1211-4

School of Veterinary Medicine and Animal Sciences, Kitasato University, Aamori, Japan.

To compare the changes in the insulin reaction of Holstein dairy cows and Japanese Black cows (JB) during the periparturient period, the insulin resistance test in vivo and lymphocytes proliferation with insulin in vitro were performed. Ten healthy Holstein dairy cows (Holstein group) and 10 healthy JB cows (JB group) used in this study were observed on days 60, 40, and 20 before calving and days 7 and 20 after calving. In insulin resistance reaction in vivo and in vitro, a low insulin-stimulated glucose disposal rate and lymphocyte proliferation with insulin were observed in the Holstein group compared with the JB group during the experimental period. An analysis of the lymphocytes cultured with insulin showed that the percentage of CD4+CD45R- T cells in the Holstein group was significantly lower than that of the JB group before day 20. These findings indicate that T cells reaction to insulin in healthy periparturient Holstein cows is lower than that in Japanese Black.
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http://dx.doi.org/10.1292/jvms.68.1211DOI Listing
November 2006

Comparison of two different nutritive conditions against the changes in peripheral blood mononuclear cells of periparturient dairy cows.

J Vet Med Sci 2006 Nov;68(11):1161-6

School of Veterinary Medicine and Animal Sciences, Kitasato University, Aamori, Japan.

To clarify the relationship between cellular immune status and nutritive condition in periparturient dairy cows, feeding content, blood profiles, and immune condition were observed in cows from two dairy herds with different types of feed content. Immunological analyses such as leukocyte population and peripheral blood mononuclear cell (PBMC) mRNA of IFN-gamma, TNF-alpha, IL-4, and IL-10, quantified by real-time RT-PCR were performed. With regard to feed content during dry periods, there were six cows in the herd with insufficient non-structural carbohydrate (NFC) intake (group I) and six cows in the herd with sufficient NFC intake (group II). Significantly lower levels of blood glucose were observed in group I between weeks -12 and 16 compared with group II. Serum cholesterol level was significantly lower in group I between weeks 2 and 10 than in group II. The numbers of CD3+ and CD4+ T cells in group I were significantly lower than those in group II in weeks 6 and 14. The numbers of CD21+ B cells were significantly lower in group I than in group II in weeks -16, -12, 2, and 10. On the other hand, the CD4+/CD8+ ratio in group II was significantly higher than group I between weeks 2 and 14. The IFNgamma/IL-4 mRNA rate in group I was significantly lower than group II in week 6. We concluded that cellular immune depression occurrs after calving in dairy cows with low nutritional status in the periparturient period.
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http://dx.doi.org/10.1292/jvms.68.1161DOI Listing
November 2006