Reiko Nagata

Neutralization of Interleukin-10 Significantly Enhances Gamma Interferon Expression in Peripheral Blood by Stimulation with Johnin Purified Protein Derivative and by Infection with Mycobacterium avium subsp. paratuberculosis in Experimentally Infected Cattle with Paratuberculosis

ABSTRACT Monoclonal antibody neutralization of interleukin-10 (IL-10) increased Johnin purified protein derivative-induced whole-blood gamma interferon (IFN-γ) secretion 23-fold and also increased IFN-γ secretion ninefold following in vitro Mycobacterium avium subsp. paratuberculosis infection of peripheral blood mononuclear cells. These results demonstrate the suppressive effect of IL-10 on immune responses to M. avium subsp. paratuberculosis infection in cattle.

Expression Cloning of Gamma Interferon-Inducing Antigens of Mycobacterium avium subsp. paratuberculosis

ABSTRACT Three recombinant proteins, Map10, Map39, and Map41, produced based on nucleotide sequences obtained from the screening of Mycobacteriumavium subsp. paratuberculosis genomic library expressed in Escherichiacoli significantly elicited gamma interferon production in peripheral blood mononuclear cells from infected cattle. Two of these proteins were members of the PPE protein family.

Limited surveillance of fumonisins in brown rice and wheat harvested in Japan.

Fumonisins are mycotoxins mainly produced by Fusarium verticillioides, which is a major contaminant of corn. However, there are sporadic reports of fumonisin contamination in wheat worldwide. The rice adherent fungus Gibberella fujikuroi is taxonomically closely related to F. verticillioides. Therefore, the potential risk of fumonisin contamination in rice and wheat is significant. Previously, a sensitive detection method utilizing liquid chromatography with tandem electrospray mass spectrometry (LC-ESI-MS-MS) was developed for the determination of fumonisins in brown rice. In the present study, the incidence of fumonisins in brown rice and wheat harvested in Japan was investigated using LC-ESI-MS-MS. Forty-eight rice samples and 47 wheat samples were screened and analyzed for the major B-type fumonisins: fumonisin B1 (FB1) and fumonisin B2 (FB2). About 1 kg of rice or wheat seed was divided into three subsamples, and 10 g from each subsample was used for the analysis. The limits of detection were 0.012 and 0.011 mg/kg for FBt and FB2, respectively, in rice samples and 0.010 and 0.008 mg/kg for FB1 and FB2, respectively, in wheat samples. The mean (standard deviation) recoveries of FB1 spiked at 0.50 mg/kg into toxin-free rice and wheat samples were 77.6 (4.2)% and 84.5 (3.1)%, respectively. One of the wheat samples was positive for FBt with a value greater than the limit of detection,but no fumonisin was found in any of the rice samples. This is the first report of fumonisins detected in Japanese wheat.

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

ABSTRACT Johnes 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 Johnes 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.

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

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 Johnes 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 Johnes disease.

Detection and Confirmation of Mycobacterium avium subsp. paratuberculosis in Direct Quantitative PCR Positive Fecal Samples by the Manual Fluorescent MGIT Culture System

ABSTRACT 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.

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

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 hosts 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.

Preparation of an In-House Reference Material Containing Fumonisins in Thai Rice and Matrix Extension of the Analytical Method for Japanese Rice

Mycotoxin contamination in rice is less reported, compared to that in wheat or maize, however, some Fusarium fungi occasionally infect rice in the paddy field. Fumonisins are mycotoxins mainly produced by Fusarium verticillioides, which often ruins maize. Rice adherent fungus Gibberella fujikuroi is taxonomically near to F. verticillioides, and there are sporadic reports of fumonisin contamination in rice from Asia, Europe and the United States. Therefore, there exists the potential risk of fumonisin contamination in rice as well as the need for the validated analytical method for fumonisins in rice. Although both natural and spiked reference materials are available for some Fusarium mycotoxins in matrices of wheat and maize, there are no reference materials for Fusarium mycotoxins in rice. In this study, we have developed a method for the preparation of a reference material containing fumonisins in Thai rice. A ShakeMaster grinding machine was used for the preparation of a mixed material of blank Thai rice and F. verticillioides-infected Thai rice. The homogeneity of the mixed material was confirmed by one-way analysis of variance, which led this material to serve as an in-house reference material. Using this reference material, several procedures to extract fumonisins from Thai rice were compared. Accordingly, we proved the applicability of an effective extraction procedure for the determination of fumonisins in Japanese rice.

Systemic mycobacteriosis caused by ‘Mycobacterium avium subspecies hominissuis’ in a 14-month-old Japanese black beef steer

A 14-month-old Japanese black beef steer presented with severe chronic diarrhea and emaciation and was euthanized. Postmortem examination showed thickened and corrugated intestinal mucosa and enlarged granulomatous mesenteric lymph nodes with caseating necrosis. Numerous epithelioid cells and multinucleated giant cells infiltrated in the lamina propria and the submucosal tissue of the intestines. These cells were also observed in the systemic organs. Many acid-fast bacilli were detected in the cytoplasm of these cells and were identified as ‘Mycobacterium avium subsp. hominissuis’ (Mah) on the basis of the results of molecular examinations and immunohistochemistry. These findings indicate that Mah can cause systemic mycobacteriosis, and this unique infection needs to be distinguished from Johne’s disease and tuberculosis in cattle.

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

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.