WEKO3
アイテム
ブリの類結節症に対する細胞性免疫機構に関する研究
https://fra.repo.nii.ac.jp/records/2003230
https://fra.repo.nii.ac.jp/records/2003230f093b1cc-009b-47ee-ad38-d2cf8a4acfdd
| Item type | 紀要論文 / Departmental Bulletin Paper(1) | |||||
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| 公開日 | 2024-04-25 | |||||
| タイトル | ||||||
| タイトル | ブリの類結節症に対する細胞性免疫機構に関する研究 | |||||
| 言語 | ja | |||||
| タイトル | ||||||
| タイトル | Studies on Cellular Immune Response of Yellowtail Seriola quinqueradiata against Pseudotuberculosis | |||||
| 言語 | en | |||||
| 言語 | ||||||
| 言語 | jpn | |||||
| キーワード | ||||||
| 言語 | en | |||||
| 主題Scheme | Other | |||||
| 主題 | Yellowtail; Seriola quinqueradiata; Pasteurella piscicida; pseudotuberculosis in yellowtail; cellular immune response | |||||
| 資源タイプ | ||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_6501 | |||||
| 資源タイプ | departmental bulletin paper | |||||
| アクセス権 | ||||||
| アクセス権 | metadata only access | |||||
| アクセス権URI | http://purl.org/coar/access_right/c_14cb | |||||
| 著者 |
浜口, 昌巳
× 浜口, 昌巳 |
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| 抄録 | ||||||
| 内容記述タイプ | Abstract | |||||
| 内容記述 | Pseudotuberculosis, caused by the bacterium Pasteurella piscicida, occurs every year in cultured yellowtail, Seriola quinqueradiata, in Japan resulting in great economic losses to marine fish farms. Various drugs have been used to control this disease, but resistant strains of the bacterium have recently appeared. A vaccine using formalin-killed bacterin (FKB) has been developed. However, the efficacy of this vaccine is low and thus its use for yellowtail culture is impractical. The reason for this is that the bacterium is capable of intracellular growth in the phagocytic cells of yellowtail. This suggests that the cellular immune response, consisting of an interaction between lymphocytes and phagocytic cells, may play an important role in controlling this disease. Thus, a killed bacterin vaccine like FKB would not be expected to be effective against such intracellular pathogens because it does not activate the cellular immune response. The aim of the present study was to investigate the cellular immune response of yellowtail in order to develop a practical vaccine against pseudotuberculosis in yellowtail. Yellowtail lymphocytes were identified by fluorescent antibody techniques, and their reactivity to various mitogenic substances, their distribution in yellowtail and their function in controlling the disease were examined. Two types of lymphocytes were found. One type possessed surface membrane immunoglobulin (smlg) and the cell surface antigens of thymic lymphocytes (CATL). These cells were capable of cap formation at 4°C with an optimum at 18-25°C. Antigen-bound smIg disappeared within 3 to 4.5 hrs at 25°C, and reappeared after 6 to 24 hrs in RPMI 1640 medium without anti-yellowtail immunoglobulin M rabbit IgG. However, it did not reappear in an excess of anti-yellowtail immunoglobulin M rabbit IgG. Cap formation was inhibited by iodoacetamide, sodium azide, dinitrophenol and potassium cyanide. Cap formation did not occur with monovalent antibody. The smlg cells had a mitogenic reactivity against pokeweed mitogen (PWM) and bacterial lipopolysaccharide (LPS). The data show that the smlg cells of yellowtail are similar to mammalian B lymphocytes. The other type of lymphocyte possessed CATL, but no smlg, and showed mitogenic reactivity against concanavarin A (Con A)and phytohemagglutinin (PHA). These lymphocytes did not cross react with mouse la antigen. The first type of lymphocyte occurred mainly in the spleen and liver, whereas the second type occurred in the thymus and kidney. In the blood, the first type constituted 60.5± 8.3% of the lymphocytes, the second type 36.6±4.2%. In the kidney, antibody producing cells were mainly observed. Both types of lymphocytes were present in the thymus. However, the different functions of these lymphocytes developed upon maturation after leaving the thymus. The immune response against the bacterium was controlled by these lymphocytes. The first type of lymphocyte was capable of antigen recognition, immunological memory and antibody-production. In vivo tests showed that the second type was very important in reducing mortalities from the disease. Yellowtail phagocyte cells were prepared and observed, and their killing of the intracellular bacteria was examined. To prepare neutrophils and macrophages that were phagocytic, the changes in number and composition of peritoneal exudate cells (PEC) were followed after injection of yellowtail with P. piscicida FKB or proteose-peptone (PP). The increase of PEC after injection of FKB was greater than after injection of PP. Most of the cells in the peritoneal cavity of healthy fish were lymphocytes. After injection of either of the two substances, neutrophils, eosinophils and basophils appeared first, followed by macrophages. The percentage of lymphocytes gradually decreased. Macrophages appeared earlier in fish injected with FKB than in fish injected with PP. The peak number of neutrophils in PEC was observed 6 hrs after injection of FKB and 12 hrs after injection of PP. The peak number of macrophages in PEC was observed 48 hrs after injection of FKB and 192 hrs after injection of PP. Accordingly, neutrophil and macrophage preparations were made at these times. Phagocytosis of the bacterium by neutrophils required opsonization of the serum, but phagocytosis by macrophages did not. However, specific antibody significantly increased the phagocytic activity and phagocytic index of both cells. After addition of complement, in both cells neither phagocytic activity nor phagocytic index changed. In the presence of specific antibody and complement, phagocytic activity and phagocytic index were significantly higher than in the control, and higher than in the previously mentioned tests. These results suggest that in yellowtail the activation of the classical pathway of complement by antigenantibody reaction is important in opsonization of the bacteria. Moreover, phagocytosis of macrophages was induced by the supernatant of a lymphocyte culture. This indicates that an interaction between macrophages and lymphocytes occurs in yellowtail. After phagocytosis, the bacterial pathogen grows gradually inside the phagocytic cells and the disease develops. The fate of the bacterium after phagocytosis and the interactions between the phagocytic cells and the bacterium were studied. In addition, methods for assessing the amount of killing of intracellular bacteria by yellowtail phagocytic cells were investigated. The following method was found applicable. A mixture of phagocytic cells from yellowtail kidney, a suspension of viable bacteria and normal yellowtail serum was prepared and incubated at 25℃ for 30 min. Then, 100 μg/ml streptomycin and 1,500 units/ml penicillin G were added to the mixture, which was incubated again at 25°C for 15 min. The phagocytic cells and the remaining extracellular bacteria were separated by centrifugation at 5,900 × g for 5 min on Percoll at 1.070 specific gravity. Phagocytic cells were washed three times by modified L-15, and resuspended in modified L-15 at a density of 1 × 107 cells/ml. After incubation at 25℃ for 60 min, the phagocytic cells were destroyed by exposure to 15% sodium chloride solution containing 0.2% non-ionic detergent, BL-25, at 0°C for 15 min. The number of viable bacteria in the destroyed phagocytic cells was counted by the pour plate method using BHI agar containing 1.0% sodium chloride. Avirulent and low virulent strains of the bacterium were killed by neutrophils and macrophages. However, high virulent strains of the bacterium were killed only by macrophages obtained from fish surviving the disease. In addition, killing of intracellular bacteria by macrophages was induced by the supernatant of a culture of lymphocytes obtained from fish surviving the disease and from fish immunized by the live bacterium. The data indicate that the function of macrophages was controlled by lymphocytes products. Moreover, the lymphocytes obtained from fish surviving the disease and from fish immunized by the live bacterium produced substances effective in controlling the disease, and the cellular immune response, that is, the interaction between lymphocytes and macrophages, was stimulated by the live bacterium. The efficacy of vaccines and substances in activating the cellular immune response was examined in vivo. Because the virulent live bacterium has a high risk for practical use as a vaccine, attenuated live bacterin (ALB) was tested instead. ALB, as well as FKB and heat-killed bacterins (HKB) were prepared from the bacterium and their efficacy in immersion vaccination against pseudotuberculosis in yellowtail was compared. The ALB preparation stimulated the highest phagocytic activity in the fish and was the most effective in protecting the fish. The results suggest that the efficacy of ALB vaccination is based on the activation of phagocytic cells. In general, ribosomal vaccines have a high capability of stimulating the cellular immune response. Therefore, the efficacy of ribosomal and other antigens prepared from the bacterium was compared. Yellowtail were immunized twice by intraperitoneal injection using these antigens to determine the effects on exposure to the virulent bacterium. The antigen preparations used were ribosomal antigen P and S (RBP and RBS), outer membrane fraction (OMF), lipopolysaccharide (LPS), precipitated antigens (PCA), and extracellular products (ECP), with FKB as a control. Three weeks after the last immunization, RBP resulted in the highest level of phagocytic activity among the immunized groups, although it resulted in the lowest level of specific antibody against the bacterium LPS and in the lowest level of serum opsonization. The RBP preparation was the most effective of the antigen preparations. Levamisole has a immunostimulant effects on tumor immunity, based on cellular immune response, in animals. Yellowtail injected levamisole before exposing the bacterium was enhanced resistance to the disease. The date indicate that the resistance to the disease in yellowtail was based on cellular immune response. In summary, vaccines prepared from attenuated live bacterin and from RBP of the bacterium were more effective than various killed bacterins of the bacterium because they have a high capability of activating mature lymphocytes. The results suggest that yellowtail has a cellular immune system consisting of lymphocytes and macrophages like mammals and that the control of pseudotuberculosis in yellowtail may be possible based on the activation of the cellular immune response with ALB and RBP prepared from P. piscicida. | |||||
| 言語 | en | |||||
| bibliographic_information |
ja : 南西海区水産研究所研究報告 en : Bulletin of Nansei National Fisheries Research Instituite 巻 24, p. 27-151, ページ数 125, 発行日 1991-03 |
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| 出版者 | ||||||
| 出版者 | 南西海区水産研究所 | |||||
| 言語 | ja | |||||
| 出版者 | ||||||
| 出版者 | Nansei National Fisheries Research Instituite | |||||
| 言語 | en | |||||
| item_10002_source_id_9 | ||||||
| 収録物識別子タイプ | PISSN | |||||
| 収録物識別子 | 0388-841X | |||||
| item_10002_source_id_11 | ||||||
| 収録物識別子タイプ | NCID | |||||
| 収録物識別子 | AN00181988 | |||||
| 情報源 | ||||||
| 識別子タイプ | Local | |||||
| 関連識別子 | nnf_k_24_27 | |||||
| 関連サイト | ||||||
| 識別子タイプ | URI | |||||
| 関連識別子 | https://agriknowledge.affrc.go.jp/RN/2010501355 | |||||
| 言語 | ja | |||||
| 関連名称 | 日本農学文献記事索引(agriknowledge) | |||||
