Publications by authors named "Yoshio Kiku"

23 Publications

  • Page 1 of 1

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

Effect of Lipopolysaccharide and Muramyl Dipeptide on Apoptosis of Bovine Mammary Gland Lymphocytes.

Animals (Basel) 2020 Jun 5;10(6). Epub 2020 Jun 5.

Department of Immunology, Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic.

The aim of this study was to evaluate whether apoptosis of lymphocytes is modulated by stimulation by lipopolysaccharide (LPS) of or muramyl dipeptide (MDP). Cell populations were obtained by lavaging of the mammary glands 24, 48, 72, and 168 hours following intramammary induced inflammation. The portion of apoptotic lymphocytes peaked at 48 hours after treatment with LPS or MDP. The analysis of CD44 expression of the same cell populations showed a higher percentage of CD44-positive lymphocytes 24- and 48-hours following induction of inflammation by LPS or MDP. The results demonstrate that during both experimental infection of bovine mammary glands with LPS or MDP, apoptosis of lymphocytes was induced in the initial phase of the inflammatory response and CD44 was also overexpressed at the beginning of inflammation. These data suggest a connection of lymphocyte apoptosis with the expression of CD44 receptors.
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http://dx.doi.org/10.3390/ani10060990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341217PMC
June 2020

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

Combined hepatocellular-cholangiocarcinoma in a cow.

J Vet Med Sci 2020 Jan 11;82(1):84-88. Epub 2019 Dec 11.

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

We examined a 10-year-old cow in which about half of the liver was displaced by malignant tissue consisting of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). Cytokeratin (CK) 18 and 7 were expressed in the latter. Metastasis was present in the hepatic, pancreaticoduodenal and mediastinal lymph nodes, where malignant cells had hepatocellular features, but more pleomorphic and atypical than in the primary lesion. Areas composed solely of CC cells or less-differentiated HCC cells were observed. In contrast, well-differentiated HCC cells were almost always admixed with the other two types, and may have had the ability to transform into CC cells and to dedifferentiate into less-differentiated cells. This report suggests that CK18 is an excellent marker for biliary differentiation in cattle.
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http://dx.doi.org/10.1292/jvms.19-0304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983657PMC
January 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

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

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

Flow cytometric analysis of peripheral blood mononuclear cells induced by experimental endotoxemia in horse.

J Vet Med Sci 2003 Aug;65(8):857-63

School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, Japan.

Cellular activation and functional cell surface markers were evaluated during experimentally-induced endotoxemia in healthy horses. Eight healthy adult horses were infused a low dose of endotoxin (lipopolysaccharide from Escherichia coli O26: B6, 30 ng/kg of body weight, IV) and five control horses were given an equivalent volume of sterile saline solution. Venous blood samples were collected for flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and to measure plasma endotoxin concentrations. Clinical signs of endotoxemia were recorded at 10, 20, 30, 40, 50 min, 1, 2, 3, 4, 8, 16, 24 and 48 hr after endotoxin or saline solution administration. Clinical findings characteristic of endotoxemia (tachycardia, tachypnea, increased rectal temperature, and leukopenia) occurred transiently in all horses administered endotoxin; however, plasma endotoxin concentrations were detectable in only 50% (4/8) of the endotoxin-infused horses. The percentage of CD4(+), CD5(+), and CD8(+) cells decreased while the percentage of CD14(+), IgM(+), and MHC class II(+) cells increased significantly after endotoxin infusion. Alterations in the immunophenotype of PBMCs from horses with experimentally-induced endotoxemia were associated with changes in vital signs, indicating that endotoxin altered the immuno balance.
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http://dx.doi.org/10.1292/jvms.65.857DOI Listing
August 2003

Effects of chlorpromazine, pentoxifylline and dexamethasone on mRNA expression of lipopolysaccharide-induced inflammatory cytokines in bovine peripheral blood mononuclear cells.

J Vet Med Sci 2002 Aug;64(8):723-6

Department of Veterinary Pathology, large animal Clincal Center, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, Japan.

The effects of chlorpromazine (CPZ), pentoxifylline (PTX) and dexamethasone (DEX) on mRNA expression of lipopolysaccharide (LPS)-induced proinflammatory cytokines were examined in bovine peripheral blood mononuclear cells (PBMCs) in vitro. The expression of inflammatory cytokine mRNAs was analyzed by RT-PCR and Southern blot hybridization in bovine PBMCs. CPZ and DEX decreased the expression of cytokine mRNA (such as interleukin-1 beta and tumor necrosis factor-alpha) after stimulation with LPS in a dose-dependent manner. However, pretreatment with PTX had no inhibitory effect on the mRNA expression of proinflammatory cytokines. These results indicated that pretreatment with CPZ and DEX might be effective to reduce the production of LPS-induced inflammatory cytokines in bovine PBMCs in vitro.
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http://dx.doi.org/10.1292/jvms.64.723DOI Listing
August 2002