Publications by authors named "Yasuyuki Mori"

45 Publications

Complete Genome Sequence of Mycobacterium avium subsp. Strain 42-13-1, Isolated in Japan.

Microbiol Resour Announc 2021 Apr 15;10(15). Epub 2021 Apr 15.

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

Here, we report the complete genome sequence of subsp. strain 42-13-1, isolated from cattle presenting with chronic diarrhea caused by Johne's disease in Japan, which was assembled via long- and short-read hybrid assembly.
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http://dx.doi.org/10.1128/MRA.00084-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050961PMC
April 2021

The enhancement of Th1 immune response by anti-PD-L1 antibody in cattle infected with Mycobacterium avium subsp. paratuberculosis.

J Vet Med Sci 2021 Feb 7;83(2):162-166. Epub 2020 Dec 7.

Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan.

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteritis of ruminants. Previous studies have shown that programmed death-ligand 1 (PD-L1) is associated with the disease progression, and PD-L1 blockade activates MAP-specific Th1 responses in vitro. Here, we performed anti-PD-L1 antibody administration using 2 MAP-infected cattle at the late subclinical stage of infection. After administration, bacterial shedding was reduced or maintained at a low level. Additionally, MAP-specific Th1 cytokine production was upregulated, and CD69 expression was increased in T cells. Collectively, the treatment has a potential as a novel control method against Johne's disease.
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http://dx.doi.org/10.1292/jvms.20-0590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7972883PMC
February 2021

A Novel Real-Time PCR-Based Screening Test with Pooled Fecal Samples for Bovine Johne's Disease.

J Clin Microbiol 2020 11 18;58(12). Epub 2020 Nov 18.

Division of Bacterial and Parasitic Disease, National Institute of Animal Health, National Agriculture and Food Research Organization, Tsukuba, Japan.

Johne's disease (JD) is an economically important infectious disease in livestock farming caused by subsp. As an alternative to serological tests, which are used mainly for the screening of whole herds, we developed a novel ResoLight-based real-time PCR (RL-PCR) assay with pooled fecal samples for the detection of fecal shedders in cattle herds. The RL-PCR assay included an internal amplification control (IC) which was amplified using the same primer pair as the target molecule subsp. IS and differentiated based on melting temperatures. Individual fecal suspensions were pooled and concentrated by centrifugation to avoid a loss of sensitivity by the dilution effect. Combined with a DNA extraction kit (Johne-PureSpin; FASMAC), no inhibition of PCR amplification was observed with up to 15 fecal samples in a pool. The detection limit of RL-PCR at a pool size of 10 was 10 subsp. organisms per gram of feces, which was comparable to that of individual testing. A total of 2,654 animals in 12 infected herds were screened by individual antibody-enzyme-linked immunosorbent assay (ELISA) and the RL-PCR assay using pooled feces. Fifty animals were diagnosed with JD through the screening by RL-PCR, compared with only 5 by ELISA (which were also positive in RL-PCR). In 7 JD-free herds, the results of 4 out of 327 pools (1.2%) were invalid due to the lack of IC amplification, and then animals were confirmed negative individually. Our results suggest that implementation of herd screening by pooled RL-PCR would advance the monitoring and control of JD in cattle herds.
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http://dx.doi.org/10.1128/JCM.01761-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685889PMC
November 2020

Alternating oligo(,-phenylenes) ruthenium catalyzed diol-diene benzannulation: orthogonality to cross-coupling enables nanographene and PAH construction.

Chem Sci 2018 Oct 30;9(40):7866-7873. Epub 2018 Aug 30.

University of Texas at Austin , Department of Chemistry , Austin , TX 78712 , USA . Email: ; Email:

Ruthenium(0) catalyzed diol-diene benzannulation is applied to the conversion of oligo(-phenylene vinylenes) , and to alternating oligo(,-phenylenes) , . Orthogonality with respect to conventional palladium catalyzed biaryl cross-coupling permits construction of -bromo-terminated alternating oligo(,-phenylenes) , , which can be engaged in Suzuki cross-coupling and Scholl oxidation. In this way, structurally homogeneous nanographenes are prepared. Nanographene , which incorporates 14 fused benzene rings, was characterized by single crystal X-ray diffraction. In a similar fashion, -bromo-terminated oligo(-phenylene ethane diol) , which contains a 1,3,5-trisubstituted benzene core, is converted to the soluble, structurally homogeneous hexa--hexabenzocoronene . A benzothiophene-terminated pentamer was prepared and subjected to Scholl oxidation to furnish the helical bis(benzothiophene)-fused picene derivative . The steady-state absorption and emission properties of nanographenes , ,,,, and were characterized. These studies illustrate how orthogonality of ruthenium(0) catalyzed diol-diene benzannulation with respect to classical biaryl cross-coupling streamlines oligophenylene and nanographene construction.
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http://dx.doi.org/10.1039/c8sc03236jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194800PMC
October 2018

Prostaglandin E Induction Suppresses the Th1 Immune Responses in Cattle with Johne's Disease.

Infect Immun 2018 05 23;86(5). Epub 2018 Apr 23.

Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.

Johne's disease, caused by subsp. , is a bovine chronic infection that is endemic in Japan and many other countries. The expression of immunoinhibitory molecules is upregulated in cattle with Johne's disease, but the mechanism of immunosuppression is poorly understood. Prostaglandin E (PGE) is immunosuppressive in humans, but few veterinary data are available. In this study, functional and kinetic analyses of PGE were performed to investigate the immunosuppressive effect of PGE during Johne's disease. PGE treatment decreased T-cell proliferation and Th1 cytokine production and upregulated the expression of immunoinhibitory molecules such as interleukin-10 and programmed death ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) from healthy cattle. PGE was upregulated in sera and intestinal lesions of cattle with Johne's disease. stimulation with Johnin purified protein derivative (J-PPD) induced cyclooxygenase-2 (COX-2) transcription, PGE production, and upregulation of PD-L1 and immunoinhibitory receptors in PBMCs from cattle infected with subsp. Therefore, Johnin-specific Th1 responses could be limited by the PGE pathway in cattle. In contrast, downregulation of PGE with a COX-2 inhibitor promoted J-PPD-stimulated CD8 T-cell proliferation and Th1 cytokine production in PBMCs from the experimentally infected cattle. PD-L1 blockade induced J-PPD-stimulated CD8 T-cell proliferation and interferon gamma production Combined treatment with a COX-2 inhibitor and anti-PD-L1 antibodies enhanced J-PPD-stimulated CD8 T-cell proliferation , suggesting that the blockade of both pathways is a potential therapeutic strategy to control Johne's disease. The effects of COX-2 inhibition warrant further study as a novel treatment of Johne's disease.
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http://dx.doi.org/10.1128/IAI.00910-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913856PMC
May 2018

Copper-Catalyzed Stereodefined Construction of Acrylic Acid Derivatives from Terminal Alkynes via CO Insertion.

Org Lett 2017 02 8;19(4):854-857. Epub 2017 Feb 8.

Graduate School of Engineering, Nagasaki University , 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.

IPrCuCl catalyzes the CO insertion reaction undergone by a dialkylvinylborane intermediate derived from alkynyltrialkylborate by a 1,2-alkyl group migration to afford α-alkyl acrylic acids with excellent regio- and stereoselectivities.
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http://dx.doi.org/10.1021/acs.orglett.6b03860DOI Listing
February 2017

Comparison of fecal pooling methods and DNA extraction kits for the detection of Mycobacterium avium subspecies paratuberculosis.

Microbiologyopen 2016 Feb 15;5(1):134-42. Epub 2015 Dec 15.

Hygienics Division, National Livestock Breeding Center, Nishigo, Nishi-Shirakawa, Fukushima, 961-8511, Japan.

The aim of the study was to develop a sensitive method using quantitative real-time polymerase chain reaction (qPCR) with pooled fecal samples for the screening of Johne's disease (JD). Manufacturer-specified and our new pooling method in combination with five commercial kits for DNA extraction and purification were compared. Different volumes of pooled fecal suspensions were tested, and the results were compared for individual samples and three pool sizes (5, 10, and 50 samples); each of the fecal suspensions, which were prepared from healthy dairy and beef cattle was spiked with 0, 10, 100, or 1000 cultured Mycobacterium avium subspecies paratuberculosis (MAP) organisms or was mixed with fecal suspensions from experimentally infected cattle. The MAP DNA detection proportion with our pooling method in combination with Johne-Spin kit (Fasmac, Japan) was 100% for all models and all pool sizes, except for the low shedder model with a pool size of 50. There was no loss of sensitivity in pools of 10 subjects or less by using the new method. These results suggest that new method is a sensitive, practical, and cost-effective screening test for the detection of MAP-infected cattle and the monitoring of JD-free herds.
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http://dx.doi.org/10.1002/mbo3.318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767428PMC
February 2016

Bovine Immunoinhibitory Receptors Contribute to Suppression of Mycobacterium avium subsp. paratuberculosis-Specific T-Cell Responses.

Infect Immun 2016 01 19;84(1):77-89. Epub 2015 Oct 19.

Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan

Johne's disease (paratuberculosis) is a chronic enteritis in cattle that is caused by intracellular infection with Mycobacterium avium subsp. paratuberculosis. This infection is characterized by the functional exhaustion of T-cell responses to M. avium subsp. paratuberculosis antigens during late subclinical and clinical stages, presumably facilitating the persistence of this bacterium and the formation of clinical lesions. However, the mechanisms underlying T-cell exhaustion in Johne's disease are poorly understood. Thus, we performed expression and functional analyses of the immunoinhibitory molecules programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) and lymphocyte activation gene 3 (LAG-3)/major histocompatibility complex class II (MHC-II) in M. avium subsp. paratuberculosis-infected cattle during the late subclinical stage. Flow cytometric analyses revealed the upregulation of PD-1 and LAG-3 in T cells in infected animals, which suffered progressive suppression of interferon gamma (IFN-γ) responses to the M. avium subsp. paratuberculosis antigen. In addition, PD-L1 and MHC-II were expressed on macrophages from infected animals, consistent with PD-1 and LAG-3 pathways contributing to the suppression of IFN-γ responses during the subclinical stages of M. avium subsp. paratuberculosis infection. Furthermore, dual blockade of PD-L1 and LAG-3 enhanced M. avium subsp. paratuberculosis-specific IFN-γ responses in blood from infected animals, and in vitro LAG-3 blockade enhanced IFN-γ production from M. avium subsp. paratuberculosis-specific CD4(+) and CD8(+) T cells. Taken together, the present data indicate that M. avium subsp. paratuberculosis-specific T-cell exhaustion is in part mediated by PD-1/PD-L1 and LAG-3/MHC-II interactions and that LAG-3 is a molecular target for the control of M. avium subsp. paratuberculosis-specific T-cell responses.
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http://dx.doi.org/10.1128/IAI.01014-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694020PMC
January 2016

An outbreak of Mycobacterium genavense infection in a flock of captive diamond doves (Geopelia cuneata).

Avian Dis 2014 Sep;58(3):383-90

Two diamond doves (Geopelia cuneata) in a flock of 23 birds housed in an aviary in a zoo in central Japan were found dead as a result of mycobacteriosis. Fecal samples of the remaining doves were positive for mycobacterial infection, and thus they were euthanatized. Clinical signs and gross pathology, including weight loss and sudden death and slight enlargement of the liver and intestine, were observed in a small number of birds (3/23). Disseminated histiocytic infiltration of either aggregates or sheets of epithelioid cells containing acid-fast bacilli, in the absence of caseous necrosis, were observed in different organs of the infected doves, especially lungs (23/23), intestines (9/23), livers (7/23), and hearts (6/23). Mycobacterium sp. was isolated from the livers of three birds (3/23). DNA extracted from frozen liver and formalin-fixed, paraffin-embedded tissues (5/23) were used for amplification of the gene encoding mycobacterial 65-kDa heat shock protein (hsp65). The causative Mycobacterium species was identified by PCR-restriction fragment length polymorphism analysis. Mycobacterium genavense infection was confirmed in three of the diamond doves. Moreover, partial 16S rDNA gene sequencing revealed 100% identity across the three samples tested, and 99.77% nucleotide homology of the isolate sequence to M. genavense. The main route of M. genavense infection in the diamond doves was most likely airborne, suggesting a potential zoonotic risk of airborne transmission between humans and birds.
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http://dx.doi.org/10.1637/10775-011714-Reg.1DOI Listing
September 2014

Detection and confirmation of Mycobacterium avium subsp. paratuberculosis in direct quantitative PCR positive fecal samples by the manual fluorescent MGIT culture system.

J Vet Med Sci 2014 Jan 20;76(1):65-72. Epub 2013 Sep 20.

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

An efficient protocol for the manual fluorescent MGIT culture system combined with rapid confirmation of Mycobacterium avium subsp. paratuberculosis (MAP) growth in the broth culture was established and evaluated for the detection of viable MAP in direct quantitative PCR (QPCR) positive bovine feces. Manually detected fluorescence emissions from MGIT tubes were analyzed objectively using an open source software, ImageJ. For molecular confirmation of MAP growth, DNA samples harvested by simply boiling the broth, an inexpensive and time- and labor-saving DNA preparation method, yielded adequate results. The sheep strain of MAP required longer incubation time relative to the cattle strain, suggesting that the MGIT system may not support well the growth of ovine isolates as described previously. Of 61 direct QPCR positive bovine feces, the recovery rate of MAP in the MGIT system (62.3%) was significantly higher (P<0.05) than that using 7H10 agar-based slants (44.3%). The time to obtain a final result for fecal culture by the MGIT system was several weeks earlier compared to solid media. In MGIT culture positive samples, the time to detect fluorescence was correlated with the DNA quantity detected in fecal QPCR. As a positive result in the direct fecal QPCR test does not mean fecal excretion of viable MAP, bacterial isolation by fecal culture could be conducted to verify the QPCR result. For this purpose, the manual MGIT system is a sensitive and rapid culture method at least for bovine samples.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979941PMC
http://dx.doi.org/10.1292/jvms.13-0366DOI Listing
January 2014

Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease.

Vet Immunol Immunopathol 2013 Oct 5;155(4):253-8. Epub 2013 Jul 5.

Bacterial and Parasitic Disease Research Division, National Institute of Animal Health, NARO, 3-1-5, Kannondai, Tsukuba, Ibaraki 305-0856, Japan. Electronic address:

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded "echA12_2" in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2-7months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.
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http://dx.doi.org/10.1016/j.vetimm.2013.06.020DOI Listing
October 2013

Deoxynivalenol impairs the immune functions of neutrophils.

Mol Nutr Food Res 2013 Jun 21;57(6):1026-36. Epub 2013 Feb 21.

INRA-UMR1331, Toxalim, Research Centre in Food Toxicology, Toulouse, France.

Scope: Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., is toxic to many animal species, with pigs being the most sensitive species to the toxin. The aim of the present study was to determine the effects of DON on pig polymorphonuclear cells (PMNs), the first line of defense against infection.

Methods And Results: PMNs isolated from pig blood samples were stimulated with LPS to mimic infection. DON (0.5-10 μM) altered three main functions of pig PMNs: LPS-induced secretion of IL-8, chemotaxis, and phagocytosis capability. This alteration of PMN properties was due to apoptotis induced by DON exposure. Using Western blot and flow cytometry, we demonstrated that this process included the permeabilization of the mitochondrial outer membrane and the activation of caspase-3. The effect of DON was mediated by the phosphorylation of the p38 mitogen-activated protein kinase within the first 30 min of exposure.

Conclusion: This study provides evidence that low concentrations of DON can alter the immune functions of porcine PMNs and suggests the involvement of p38 mitogen-activated protein kinase in the signal transduction pathway. These immunosuppressive effects of DON may have implications for humans and/or animals when eating contaminated food/feed.
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http://dx.doi.org/10.1002/mnfr.201200755DOI Listing
June 2013

Detection of antibody responses against Mycobacterium avium subsp. paratuberculosis stress-associated proteins within 30 weeks after infection in cattle.

Vet Immunol Immunopathol 2012 Nov 11;150(1-2):101-11. Epub 2012 Sep 11.

Bacterial and Parasitic Disease Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.

In this study, humoral immune responses in cattle against Mycobacterium avium subsp. paratuberculosis (MAP) stress-associated recombinant proteins were assessed longitudinally by ELISA during the first 30 weeks after MAP infection. A total of 11 MAP genes previously identified by proteomic analysis were selected for cloning and expression. These included possible general stress-associated proteins of MAP and proteins expressed in vivo in MAP-infected sheep at an early stage of infection. An increase in the antibody levels against 5 recombinant antigen preparations (MAP1027c, MAP1339, MAP1588c, MAP1589c and MAP2411) was seen in MAP-infected calves (n=16) but not in control calves (n=3) over the time examined. Antibody responses were recorded as early as two weeks post-inoculation, and 87.5% of the inoculated cattle responded to at least one of the five immunogenic antigen preparations within the first 30 weeks of infection, suggesting that these proteins identified in the in vitro models of stress were also expressed in vivo in MAP-infected cattle at a relatively early stage after infection and therefore stimulate the host's immune system. It has been assumed that the sensitivity of antibody ELISA tests is dependent on the stage of infection and the age of the animals. However, we have provided some evidence that humoral immunity occurs at an early stage of paratuberculosis and can be detected using appropriate antigens such as MAP stress-associated proteins.
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http://dx.doi.org/10.1016/j.vetimm.2012.09.003DOI Listing
November 2012

Decreased expression of matrix metalloproteinase-9 and increased expression of tissue inhibitors of matrix metalloproteinase-1 in paratuberculosis-infected cattle in the ELISA-negative subclinical stage.

Anim Biotechnol 2011 Jan;22(1):44-9

Research Team for Paratuberculosis, National Institute of Animal Health, Tsukuba, Japan.

We investigated the gene expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitors of matrix metalloproteinases-1 (TIMP-1) in peripheral blood cells from infected cattle with Mycobacterium avium subsp. paratuberculosis (Map) in the ELISA-negative subclinical stage compared with uninfected control cattle. Significant decreased MMP-9 expression and increased TIMP-1 expression were found in peripheral blood cells from Map-infected cattle after stimulation with Map lysate and Map purified protein derivative (PPD) than in control cattle by real-time RT-PCR analysis. In contrast to the uninfected controls, the activity of MMP-9 was also decreased in peripheral blood cell culture supernatants from Map-infected cattle at 24 hr after Map lysate and MapPPD stimulation by gelatin zymography analysis. As a result, the MMP-9 may play an important role in the development of Mycobacterium avium subsp. paratuberculosis disease.
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http://dx.doi.org/10.1080/10495398.2010.536096DOI Listing
January 2011

Absorbed EVELISA: a diagnostic test with improved specificity for Johne's disease in cattle.

Foodborne Pathog Dis 2010 Nov 12;7(11):1291-6. Epub 2010 Aug 12.

Center for Wildlife Health, Department of Forestry, Wildlife, and Fisheries, The University of Tennessee, Knoxville, Tennessee 37996, USA.

The use of enzyme-linked immunosorbent assays (ELISAs) is recommended for Johne's disease (JD) control in dairy herds. In 2006, we developed a novel ELISA test for JD, named EVELISA (ELISA using ethanol extract of Mycobacterium avium subsp. paratuberculosis), which showed higher sensitivity than commercial ELISA tests. To further investigate the performance of EVELISA, we obtained 38 serum samples from cattle in a JD-free herd with suspected cases of serological false-positive reactions. When these samples were tested using the EVELISA and a commercial ELISA test, more than 70% of the samples were falsely identified as JD positive. Antibodies in the serum samples reacted strongly with antigens of various environmental mycobacteria, suggesting the presence of cross-reactive antibodies in the samples. The possible cross reactions in the EVELISA were inhibited markedly by the use of Mycobacterium phlei antigens for antibody absorption. When these samples were tested, 8 samples were classified as positive for JD by the EVELISA with the antibody absorption, whereas 27 samples were classified as positive for JD by the commercial ELISA. For an estimation of tentative sensitivity and specificity, the ELISA tests were performed on 38 serum samples from JD-negative herds with no suspected cases of serological false-positive reaction and 68 samples from cattle diagnosed as positive for M. avium subsp. paratuberculosis infection by fecal culture test. Sensitivity and specificity of the EVELISA with preabsorption of serum with M. phlei ("ethanol vortex absorbed-ELISA" or EVA-ELISA) were estimated to be 97.1% and 100%, respectively, whereas those of the commercial ELISA were 48.5% and 97.4%, respectively. Further, in 85 fecal culture-negative cattle in JD-positive herds, higher sensitivity of the EVA-ELISA than the commercial ELISA was demonstrated by a Bayesian analysis. This study indicates that the EVA-ELISA may form a basis for a sensitive diagnostic test with a higher level of specificity than that of the current commercial ELISA test.
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http://dx.doi.org/10.1089/fpd.2010.0541DOI Listing
November 2010

[Study on the effects of HTST pasteurization temperatures on Mycobacterium avium subsp. paratuberculosis in an industrial fluid milk-processing system].

Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku 2010 (128):81-4

Johne disease is ruminant chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). The domestic animals infected with this pathogen present severe weight loss due to chronic diarrhea and a reduction in lactation yield. These result in enormous economic loss since the affected animals are subsequently subject to artificial selections and disinfection of the environment are absolutely necessary. Furthermore, MAP has been suspected to have pathological relationship to Crohn's disease, human chronic granulomatous enteritis. The bacterium grows slower on solid culture and its colony becomes visible after two months of culture. In Japan, there has been almost no investigation on pasteurization temperature of commercial milk using MAP. It comes from the fact that the growth rate of MAP is very slow and that MAP is a related species to Mycobacterium tuberculosis, which pasteurization condition has been well defined. The studies on the pasteurization conditions of commercial milk have been mainly targeted to reduce the risk of infection to Coxiella and Mycobacterium tuberculosis. However, there has been a concern about the possibility that MAP is remained in pasteurized milk because MAPs form an aggregate and the bacterium at its center may not receive enough heat to get pasteurized. From these reasons, the present study aims to investigate validity of the current pasteurization conditions of commercial milk by implementing experimental pasteurization at various pasteurization temperatures using milk experimentally infected with MAP, and to clarify if MAP is eliminated at these temperatures in order to achieve smooth enforcement of the current ministry order. We conducted plant pasteurization experiment at four pasteurization conditions (high temperature, short time (HTST); 82, 77, 72 degrees C for 15 seconds and low temperature, long time (LTLT); 63 degrees C for 30 minutes) using two MAP strains, ATCC19698 and OKY-20. In conclusion, there appeared no colony of the two MAP strains formed from the milk pasteurized at the four pasteurization conditions examined.
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May 2011

A specific induction of interleukin-10 by the Map41 recombinant PPE antigen of Mycobacterium avium subsp. paratuberculosis.

Vet Immunol Immunopathol 2010 May 13;135(1-2):71-78. Epub 2009 Nov 13.

Research Team for Paratuberculosis, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.

Interleukin-10 (IL-10) is not only an essential immunoregulator in host immunity, but also it accounts for the intracellular survival of mycobacteria because of its inhibitory activity against anti-mycobacterial functions of macrophage. It has been also indicated that blood cells from calves infected with Mycobacterium avium subsp. paratuberculosis (Map) produce a large amount of IL-10 after stimulation with Map antigen, and it leads to suppression of Interferon-gamma (IFN-gamma) production in T-cells. This characteristic expression of IL-10 in Map-infected cattle seems to be playing important roles in the pathogenesis of Johne's disease caused by Map, and could be an important diagnostic indicator. The aim of this study was to investigate the diagnostic significance of IL-10 production from blood cells stimulated by a PPE (Proline-Proline-Glutamic acid) protein family of Map. The recombinant PPE protein, Map41, which has been reported as one of the IFN-gamma inducing antigens of Map, also strongly induced IL-10 from macrophages obtained from infected calves. The elicited IL-10 production in response to Map41 from experimentally infected calves was as early as 2 weeks after the inoculation of Map, and the IL-10 production was detected earlier than that of IFN-gamma. The blood cells from calves immunized with Map produced higher amounts of IL-10 against Map41 stimulation than those of calves immunized with various Mycobacterium species. Furthermore, this IL-10 induction also showed high specificity to Map in guinea pigs experimentally infected with various Mycobacterium species. These observations suggest that IL-10 assay is a useful diagnostic method in the early stage of Johne's disease.
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http://dx.doi.org/10.1016/j.vetimm.2009.11.002DOI Listing
May 2010

Immune response of gnotobiotic piglets against Mycoplasma hyopneumoniae.

J Vet Med Sci 2008 Oct;70(10):1065-70

Research Team for Advanced Biologicals, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.

In this study, several cytokine responses were investigated during Mycoplasma hyopneumoniae (Mhp) infection using a gnotobiotic infection model. We found that several inflammatory cytokines (IL-1beta, IL-8, IL-18, and TNF-alpha) and an anti-inflammatory cytokine IL-10 were induced from peripheral blood mononuclear cells (PBMC) of germ-free (GF) piglets stimulated with heat killed Mhp whole antigens, but no IFN-gamma and IL-4 were induced by Mhp. After the intranasal infection of Mhp, IL-1beta, IL-8, IL-18, and IFN-gamma were also detected in the broncho-alveolar lavage fluids (BALF). The antigen-specific IFN-gamma and IL-10 responses after infection of Mhp were gradually suppressed during Mhp infection as well as non-specific immune response to concanavalin A (ConA) and lipopolysacchalide (LPS) at early stage of infection. These results suggested that Mhp infection modulates the immune response of pigs by inducing several cytokines, and causes immuno-suppression of pigs in a gnotobiotic condition.
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http://dx.doi.org/10.1292/jvms.70.1065DOI Listing
October 2008

Susceptibility of germ-free pigs to challenge with protease mutants of Salmonella enterica serovar Typhimurium.

Immunobiology 2007 14;212(7):577-82. Epub 2007 Jun 14.

Department of Immunology and Gnotobiology, Institute of Microbiology of Academy of Sciences of the Czech Republic, 549 22 Novy Hradek, Czech Republic.

Salmonella protease mutants, clpP and especially htrA, are candidate live oral vaccines in humans. A functional and mature immune system is, however, required to cope with them in mice. Here, we test the cytokine response of highly susceptible germ-free pigs to infection with Salmonella Typhimurium clpP and htrA mutants. Cytokine levels (IL-4, IL-10, IL-18 and IFN-gamma) were measured by ELISA in plasma and washes from the terminal small bowel 24h after oral challenge. Unlike the infection with the wild type strain, no IFN-gamma response and low IL-18 intestinal levels were found in pigs infected with the protease mutants. Despite this and regardless of partially reduced ability of htrA and clpP mutants to invade and multiply in a 3D4 porcine macrophage-like cell line, both the mutants were as virulent as was the wild type LT2 strain and caused fatal septicaemia in germ-free pigs. IFN-gamma and IL-18 response therefore did not correlate with the virulence of Salmonella Typhimurium. Our results indicate that htrA and clpP attenuations should be used with caution in populations in which an increased number of immunocompromised individuals can be expected.
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http://dx.doi.org/10.1016/j.imbio.2007.05.001DOI Listing
November 2007

Corticotropin-releasing hormone and urocortin expression in peripheral blood cells from experimentally infected cattle with Mycobacterium avium subsp. paratuberculosis.

Microbes Infect 2007 Jul 17;9(9):1061-9. Epub 2007 May 17.

Research Team for Paratuberculosis, National Institute of Animal Health, 3-1-5 Kan-nondai, Tsukuba 305-0856, Japan.

Urocortin (UCN) is a new neuropeptide of the corticotrophin-releasing hormone (CRH) family which plays an important role in immune responses. Mycobacterium avium subspecies paratuberculosis (Map) is the etiological agent of paratuberculosis (Johne's disease). The role of UCN or CRH in the pathogenesis of Map-infection is unknown. In the present study, we first cloned the bovine UCN gene and demonstrated the profile of UCN or CRH expression in peripheral blood cells from Map-infected cattle and uninfected controls by real-time reverse transcription-polymerase chain reaction (RT-PCR) and ELISA analysis. These data are the first observations of the characteristic kinetics of these neuropeptides in Map-infection. UCN or CRH expression in non-stimulated blood samples from infected cattle was higher than that in similarly treated samples from uninfected controls; however, exposure to Map lysate and live Map resulted in down-regulated expression of UCN in infected cattle compared to their counterparts from uninfected controls. These results have provided a direction in understanding the pathogenesis of paratuberculosis and improving diagnostic methods for Map-infection.
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http://dx.doi.org/10.1016/j.micinf.2007.04.017DOI Listing
July 2007

Detection of Mycobacterium avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture.

Vet Microbiol 2007 Nov 13;125(1-2):36-48. Epub 2007 May 13.

Faculty of Veterinary Science, University of Sydney, Camden, NSW, Australia.

The aims of this study were to develop a new real-time quantitative PCR (QPCR) assay based on IS900 for detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP) DNA in faeces, and to use this to detect infected sheep. Both the C and S strains of MAP were detected by the QPCR assay, and no cross reactions were detected with 51 other species of mycobacteria including 10 which contained IS900-like sequences. One copy of IS900 fragment cloned into plasmid pCR2.1 and 1 fg of MAP genomic DNA were consistently detected, while in spiked faecal samples the detection limit was 10 viable MAP per gram of ovine faeces. A total of 506 individual ovine faecal samples and 27 pooled ovine faecal samples with known culture results were tested. The QPCR assay detected 68 of 69 BACTEC culture positive individual faeces and there was a strong relation between time to detection in culture and DNA quantity measured by QPCR (r= -0.70). In pooled faecal samples, QPCR also agreed with culture (kappa=0.59). MAP DNA was detected from some culture negative faecal samples from sheep exposed to MAP, suggesting that the QPCR has very high analytical sensitivity for MAP in faecal samples and detects non-viable MAP in ovine faeces. None of the faecal samples from 176 sheep that were not exposed to MAP were positive in QPCR. This is the first report of a direct faecal QPCR assay that has similar sensitivity to a gold standard radiometric culture assay.
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http://dx.doi.org/10.1016/j.vetmic.2007.05.002DOI Listing
November 2007

IL-18 expression in pigs following infection with Mycoplasma hyopneumoniae.

J Interferon Cytokine Res 2006 Sep;26(9):637-44

Department of Immunology, National Institute of Animal Health, Tsukuba, Ibaraki, 305-0856, Japan.

Little is known about the detail of the immune response during infection of pigs with Mycoplasma hyopneumoniae (Mhp). To further understand this important porcine pathogen, we examined the interleukin-18 (IL- 18) response in experimentally infected piglets. We found that large amounts of IL-18 were produced in the bronchoalveolar lavage fluids (BALF) of pigs experimentally infected with Mhp. However, the concentration of interferon-gamma (IFN-gamma) in the same BALF was negatively correlated with that of IL-18. The antibody response against Mhp was found to be associated with the IL-18 concentration in the BALF. Immunohistochemical staining revealed that both IL-18 and IL-18 receptor alpha chain (IL-18Ralpha) were present in macrophages and plasma cells in the lungs of Mhp-infected pigs. Lung mononuclear cells isolated from pneumonic lesions secreted IL-18 and prostaglandin E(2) (PGE(2)) in vitro, and PGE(2) production was enhanced by stimulation with IL-18. These results indicate that IL-18 produced in the pig lung contributes to the development of innate and acquired immune responses against Mhp as a proinflammatory cytokine rather than as an IFN-gamma-inducing factor and may be involved in immunomodulation in pigs.
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http://dx.doi.org/10.1089/jir.2006.26.637DOI Listing
September 2006

A highly sensitive and subspecies-specific surface antigen enzyme- linked immunosorbent assay for diagnosis of Johne's disease.

Clin Vaccine Immunol 2006 Aug;13(8):837-44

Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries, the University of Tennessee, Knoxville, Tennessee 37996-1071, USA.

Johne's disease (JD), or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Control of JD could be accomplished by diagnosis and good animal husbandry, but this is currently not feasible because commercially available diagnostic tests have low sensitivity levels and are incapable of diagnosing prepatent infections. In this study, a highly sensitive and subspecies-specific enzyme-linked immunosorbent assay was developed for the diagnosis of JD by using antigens extracted from the surface of M. avium subsp. paratuberculosis. Nine different chemicals and various intervals of agitation by vortex were evaluated for their ability to extract the surface antigens. Various quantities of surface antigens per well in a 96-well microtiter plate were also tested. The greatest differences in distinguishing between JD-positive and JD-negative serum samples by ethanol vortex enzyme-linked immunosorbent assay (EVELISA) were obtained with surface antigens dislodged from 50 microg/well of bacilli treated with 80% ethanol followed by a 30-second interval of agitation by vortex. The diagnostic specificity and sensitivity of the EVELISA were 97.4% and 100%, respectively. EVELISA plates that had been vacuum-sealed and then tested 7 weeks later (the longest interval tested) had diagnostic specificity and sensitivity rates of 96.9 and 100%, respectively. In a comparative study involving serum samples from 64 fecal culture-positive cattle, the EVELISA identified 96.6% of the low-level fecal shedders and 100% of the midlevel and high-level shedders, whereas the Biocor ELISA detected 13.7% of the low-level shedders, 25% of the mid-level shedders, and 96.2% of the high-level shedders. Thus, the EVELISA was substantially superior to the Biocor ELISA, especially in detecting low-level and midlevel shedders. The EVELISA may form the basis for a highly sensitive and subspecies-specific test for the diagnosis of JD.
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http://dx.doi.org/10.1128/CVI.00148-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1539126PMC
August 2006

A novel enzyme-linked immunosorbent assay for diagnosis of Mycobacterium avium subsp. paratuberculosis infections (Johne's Disease) in cattle.

Clin Vaccine Immunol 2006 May;13(5):535-40

Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries, The University of Tennessee, P.O. Box 1071, Knoxville, TN 37901-1071, USA.

Enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis, were developed using whole bacilli treated with formaldehyde (called WELISA) or surface antigens obtained by treatment of M. avium subsp. paratuberculosis bacilli with formaldehyde and then brief sonication (called SELISA). ELISA plates were coated with either whole bacilli or sonicated antigens and tested for reactivity against serum obtained from JD-positive and JD-negative cattle or from calves experimentally inoculated with M. avium subsp. paratuberculosis, Mycobacterium avium subsp. avium, or Mycobacterium bovis. Because the initial results obtained from the WELISA and SELISA were similar, most of the subsequent experiments reported herein were performed using the SELISA method. To optimize the SELISA test, various concentrations (3.7 to 37%) of formaldehyde and intervals of sonication (2 to 300 s) were tested. With an increase in formaldehyde concentration and a decreased interval of sonication, there was a concomitant decrease in nonspecific binding by the SELISA. SELISAs prepared by treating M. avium subsp. paratuberculosis with 37% formaldehyde and then a 2-s burst of sonication produced the greatest difference (7x) between M. avium subsp. paratuberculosis-negative and M. avium subsp. paratuberculosis-positive serum samples. The diagnostic sensitivity and specificity for JD by the SELISA were greater than 95%. The SELISA showed subspecies-specific detection of M. avium subsp. paratuberculosis infections in calves experimentally inoculated with M. avium subsp. paratuberculosis or other mycobacteria. Based on diagnostic sensitivity and specificity, the SELISA appears superior to the commercial ELISAs routinely used for the diagnosis of JD.
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http://dx.doi.org/10.1128/CVI.13.5.535-540.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1459654PMC
May 2006

Bovine IL-18 ELISA: detection of IL-18 in sera of pregnant cow and newborn calf, and in colostrum.

J Immunoassay Immunochem 2005 ;26(3):203-13

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

In this study, we examined the concentration of bovine IL-18 in the sera of pregnant cows, their fetuses and newborn calves, and in colostrum in order to examine the role of IL-18 in bovine pregnancy and the neonatal period. A sandwich-ELISA to quantify bovine IL-18 was established using anti-porcine IL-18 monoclonal antibodies, which cross-reacted with bovine IL-18, and used it to measure the concentration of bovine IL-18 in the sera of pregnant cows, their fetuses and newborn calves, and in colostrum. Significant levels of IL-18 were detected in the sera of pregnant cows, but not in the sera obtained from the corresponding fetuses, umbilical arteries and veins. After birth, IL-18 levels in the sera of 1-day and 1-week old calves were low, and significantly increased in the sera of 1-month and 4-month old calves. IL-18 was also detected in colostrum, with the concentration of IL-18 in the first colostrum produced after delivery being the highest, and then decreasing depending on the number of milkings. Furthermore, the serum IL-18 concentration of newborn calves was increased after the oral administration of colostrum. These results suggest that IL-18 during bovine pregnancy and in the newborn period may play important roles in the maintenance of pregnancy and in the maturation of neonatal immunity.
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http://dx.doi.org/10.1081/IAS-200062487DOI Listing
September 2005

Expression cloning of gamma interferon-inducing antigens of Mycobacterium avium subsp. paratuberculosis.

Infect Immun 2005 Jun;73(6):3778-82

Immune System Section, Department of Immunology, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki, Japan 305-0856.

Three recombinant proteins, Map10, Map39, and Map41, produced based on nucleotide sequences obtained from the screening of Mycobacterium avium subsp. paratuberculosis genomic library expressed in Escherichia coli significantly elicited gamma interferon production in peripheral blood mononuclear cells from infected cattle. Two of these proteins were members of the PPE protein family.
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http://dx.doi.org/10.1128/IAI.73.6.3778-3782.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1111817PMC
June 2005

Effect of bacterial virulence on IL-18 expression in the amnion infected with Escherichia coli.

Am J Reprod Immunol 2005 May;53(5):255-60

Department of Immunology and Gnotobiology, Institute of Microbiology, Academy of Sciences of the Czech Republic, 549 22 Nový Hrádek, Czech Republic.

Problem: The upregulation of inflammatory substances threatens pregnancy. Interleukin-18 (IL-18) is elevated in women who miscarried. The purpose of this study was to develop a pig model of chorioamnionitis to study the effect of bacterial virulence on IL-18 response in experimentally infected amnion.

Method Of Study: A total of 20,000 colony-forming units of Escherichia coli (an enteropathogenic O55 strain, EPEC or O86 non-pathogenic strain) were administered into the amniotic cavity of pig fetuses at 70% of gestation for 10 hr. Fetal amniotic fluid samples were analyzed for IL-18 levels by enzyme-linked immunosorbent assay. The expression of IL-18 was studied also by immunohistochemistry on cryostat sections through amniotic membranes and pathological changes were observed by electron microscopy.

Results: Both E. coli strains propagated in amniotic fluids and reached similar counts. Only EPEC, however, caused a significant increase of IL-18 amniotic fluid levels (P < 0.001) and cytokine expression in the amniotic epithelium.

Conclusions: The levels of IL-18 in infected amniotic fluids correlated with bacterial virulence and pathological changes in the amnion.
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http://dx.doi.org/10.1111/j.1600-0897.2005.00273.xDOI Listing
May 2005

Serological diagnosis of enzootic pneumonia of swine by a double-sandwich enzyme-linked immunosorbent assay using a monoclonal antibody and recombinant antigen (P46) of Mycoplasma hyopneumoniae.

Vet Microbiol 2005 Feb 23;105(3-4):251-9. Epub 2004 Dec 23.

Zen-noh Institute of Animal Health, 7 Ohja-machi, Sakura, Chiba 285-0043, Japan.

To facilitate the control of enzootic pneumonia (EP) of swine caused by Mycoplasma hyopneumoniae, the complement fixation (CF) test has been used for the detection of M. hyopneumoniae antibodies. However, the CF test is a cumbersome and time-consuming technique and cross-reactivity are major drawbacks associated with this method. To circumvent these drawbacks, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA), consisting of purified monoclonal antibody (Mab) against the 46 kDa surface antigen (P46) of M. hyopneumoniae and recombinant P46 protein expressed in Escherichia coli, for the detection of antibodies to M. hyopneumoniae in serum samples from pigs experimentally inoculated with M. hyopneumoniae and from naturally infected pigs, and compared the practical usefulness of ELISA using the CF test. In experimentally inoculated pigs, the CF and ELISA antibodies were detected at almost the same time, and a good correlation was demonstrated between the CF test and the ELISA. In a survey conducted on field samples, the seropositivity by ELISA in pigs of age 2-6 months was increased. At the time of slaughter, approximately 80% of the animals were seropositive for ELISA. However, a gradual decrease in the prevalence of ELISA positive samples was observed in sows with increasing parity. No correlation was seen between the results obtained with the two methods in the clinical samples. The CF test appears to have limited value for the diagnosis of EP in conventional herds because nonspecific reactions were frequently observed. Therefore, this ELISA is a useful alternative to the CF test currently used for the diagnosis of EP.
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http://dx.doi.org/10.1016/j.vetmic.2004.11.006DOI Listing
February 2005

Expression and one-step purification of bovine interleukin-21 (IL-21) in silkworms using a hybrid baculovirus expression system.

Biotechnol Lett 2004 Sep;26(18):1453-8

Department of Immunology, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.

A hybrid baculovirus, a hybrid of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of bovine interleukin-21 (IL-21) in silkworms. A recombinant hybrid baculovirus containing the full length of the cDNA of bovine interleukin-21 was constructed and used to infect silkworm larvae or silkmoth pupae. After the infection of the virus, bovine mature IL-21 was produced in the haemolymph or pupal cell lysates. A one-step purification of bovine mature IL-21 from haemolymph using a cation exchange column gave 0.5 mg. IL-21 from 30 ml haemolymph. The bovine IL-21 produced by silkworms strongly induced NK cell proliferation using a human NK cell-line, NK0, and enhanced the lymphokine activated killer (LAK) activity of bovine peripheral blood mononuclear cells.
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http://dx.doi.org/10.1023/B:BILE.0000045643.66758.9dDOI Listing
September 2004

Generation of multinucleated giant cells in vitro from bovine monocytes and macrophages.

J Vet Med Sci 2004 Sep;66(9):1065-9

National Institute of Animal Health, Tsukuba, Japan.

The generation of multinucleated giant cells (MGC) from cells of the bovine monocyte-macrophage lineage was investigated. Freshly isolated monocytes were incubated with the conditioned medium (CM) of peripheral blood mononuclear cell cultures treated with Concanavalin A for 1-4 days (CM1 to CM4). Only CM1 generated MGC despite similar concentrations of IFNgamma in all CMs. Nevertheless, MGC formation from monocytes was enhanced by adding either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), MGC formations from macrophages were observed only when macrophages were cultured with GM-CSF plus CM. These results indicate that several mechanisms to generate MGC from bovine monocytes-macrophage lineage cells exist, and that GM-CSF is a major mediator of MGC formation in cattle.
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http://dx.doi.org/10.1292/jvms.66.1065DOI Listing
September 2004