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鶏コクシジウムEimeria tenellaにおける増殖，伝播および新規早熟株の作成と特徴付けに関する研究
https://az.repo.nii.ac.jp/records/5346
https://az.repo.nii.ac.jp/records/5346c6e83537-212e-404c-b78b-5512c08bf597
| 名前 / ファイル | ライセンス | アクション |
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diss_dv_kou0156 (4.6 MB)
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diss_dv_kou0156_jab&rev.pdf (173.3 kB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||||||
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| 公開日 | 2019-04-23 | |||||||||
| タイトル | ||||||||||
| タイトル | 鶏コクシジウムEimeria tenellaにおける増殖，伝播および新規早熟株の作成と特徴付けに関する研究 | |||||||||
| タイトル | ||||||||||
| タイトル | Studies on propagation and transmission of Eimeria tenella, and characterization of an attenuated precocious strain | |||||||||
| 言語 | en | |||||||||
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| 言語 | eng | |||||||||
| 資源タイプ | ||||||||||
| 資源タイプ | doctoral thesis | |||||||||
| アクセス権 | ||||||||||
| アクセス権 | open access | |||||||||
| アクセス権URI | http://purl.org/coar/access_right/c_abf2 | |||||||||
| 著者 |
Jarujareet, Wipaporn
× Jarujareet, Wipaporn
× Jarujareet, Wipaporn
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| 抄録 | ||||||||||
| 内容記述タイプ | Abstract | |||||||||
| 内容記述 | 鶏コクシジウム病は世界の養鶏産業において甚大な損害を与えている家畜原虫病の一つである。主な病原体はEimeria tenella、E. necatrix、E. maxima、E. brunettiおよびE. acervulinaの5種で、これらの中でE. tenellaの病原性が最も強い。コクシジウム原虫の“増殖”は生き残り戦略の一つとして重要で、鶏体内でより多くを増殖させた後、飼育環境に効率的に分散して、より多くが別の鶏に取り込まれることで種を保存・維持する。この原虫の“増殖”および“伝播”に関わる要因を明らかにすることは、本病の予防対策の検討と確立において重要である。本研究ではE. tenellaについて、次の4つの課題に取り組んだ。1) E. tenella 株の純化を目的としたオーシスト1個からの原虫増殖および本原虫の分子生物学的な同定のための核酸抽出法の確立、2) リボフラビンの飼料添加によるE. tenella感染鶏の排出オーシスト数および抗コクシジウム剤の薬効への影響、3) 鶏コクシジウムの伝播におけるゴキブリの役割、4）E. tenella早熟株の作成と病原性の減弱の確認およびワクチン株マーカーとしての蛍光蛋白遺伝子の移入、である。本論文はこれらの課題に関する研究について述べるものであり、4章から構成される。 第1章では、鶏コクシジウム病の病原体の分離と純化および分子生物学的識別について述べる。すなわち、E. tenella 感染鶏の糞便から1個のオーシストを分離し鶏に投与して継代を繰り返すことで純化された株を作出し、Multiplex PCR法で同定した。分子生物学的操作においては、オーシストおよびスポロシストの壁を破砕して核酸を抽出する必要があるが、E. tenellaのオーシスト壁は強固で破壊が容易でなく、核酸抽出の手段は確立されていない。本実験で、オーシスト壁の破壊に最も有効な方法として0.5㎜のガラスビーズを用いた約1分間の振盪が適当であることがわかった。 第2章では、リボフラビンの飼料添加によるE. tenella感染鶏の排出オーシスト数および抗コクシジウム剤の薬効に及ぼす影響について述べる。すなわち、実験鶏をリフラビン添加群、アンプロリウム添加群、未添加群、リボフラビンとアンプロリウム添加群などに分け、鶏の致死率、糞の性状および糞便１グラム当たりのオーシスト数（OPG）について群間の比較を行った。結果、リボフラビン添加群において、排出オーシスト数の有意な増加が認められた。さらに、リボフラビン添加が抗コクシジウム剤の効果を妨げないことが分かった。 第3章では、鶏コクシジウムの伝播における昆虫類の役割について述べる。当実験では、ワモンゴキブリ（Periplaneta americana）へのE. tenella の実験感染を行った。すなわち、E. tenellaオーシストをゴキブリに摂取させた後、消化管内および糞便内の原虫の存在をPCR法で確認し、また、オーシストを糞便検査で検出した。さらに回収したオーシストの鶏への感染性を調べた。結果、ゴキブリに投与後0日から4日までオーシストが腸に停留することが分かった。また、投与後3日のゴキブリ糞便内のオーシストを鶏に経口投与すると感染が成立することが実証された。以上のことから、ゴキブリが鶏コクシジウムを機械的に伝播する可能性が示唆された。 第4章では、E. tenellaの早熟弱毒株の作成について述べる。すなわち、感染鶏の糞便内に最も早く排泄されたオーシストを回収し、別の鶏に経口投与した。この、早熟オーシストを選択的に継代に用いる作業を繰り返し、継代を34代行った。結果、プレパテントピリオド（prepatent period)が180時間から132時間に減少した早熟株が得られた。この早熟株の病原性を調べると、感染鶏のオーシスト排泄期間およびOPG値は有意に減少していた。また、組織学的検索を行ったところ、早熟株では感染後96時間に成熟した第2世代のシゾントが認められ、感染後120時間にはガメートとオーシストの形成が認められた。これに対して、病原性の強い野生株では感染後96時間に未熟の第2世代シゾントしか観察されず、感染後120時間には未熟～成熟シゾントのみがみられた。これらの成績は、シゾント形成の段階において，早熟株は野生株よりも早く発育したことを示す。他方、この早熟株感染鶏の一部の糞便には潜血が認められたことから、当株の弱毒性を安定させるためにはさらに選択的継代を続ける必要あると考えられた。また、プラスミドベクターを用いて早熟株へ蛍光蛋白遺伝子を移入したところ、胞子未形成オーシストでは蛍光蛋白の発現が確認されたが、胞子形成オーシストでは確認されなかった。 以上のように、本研究は、鶏コクシジウムのオーシストからの簡易的核酸抽出法の一例を示し、また、リボフラビンの原虫増殖に対する影響およびゴキブリが原虫伝播にかかわる役割に関する基礎的な知見を提供した。その他に、新規早熟弱毒株の作成とその特徴付けに成功し、早熟株へ蛍光蛋白遺伝子移入を試みた。これらの成果は、原虫ワクチン開発の発展とコクシジウム病の制御に貢献するものである。 |
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| Abstract | ||||||||||
| 内容記述タイプ | Other | |||||||||
| 内容記述 | Chicken coccidiosis, which has a major impact on poultry production worldwide, is an intestinal protozoan disease. The five major pathogenic species are Eimeria tenella, E. necatrix, E. maxima, E. brunetti and E. acervulina. This thesis addresses the following questions on E. tenella in 4 studies. 1) Identification and purification of Eimeria species in the infected chicken has important implications for diagnosis and disease management as well as creation of new vaccines. Therefore, the first objective of this study is to establish a pure line of E. tenella by using single-oocyst isolation technique followed by confirmation with PCR. Since, the application of PCR technique for detecting Eimeria DNA has been made difficult by the remarkable toughness of oocyst and sporocyst wall, a simpler method to disrupt the oocyst of E. tenella to obtain the DNA for molecular identification is required. 2) Vitamins are known to be a necessary nutrient for the complete development of the parasite within the host. The aim of study II is to see whether addition of vitamin supplement in the basal diet will influence the oocyst production and on anticoccidial drug efficacy in chicken coccidiosis. 3) Sporulated oocyst, the infective stage of E. tenella is extremely resistant in environment. Infection occurs from ingestion of sporulated oocysts from fecal-contaminated food, water and fomites. The role of invertebrate in transmission of E.tenella has not been fully studied. Therefore, the objective of study III is to elucidate the role of cockroaches in the transmission of E. tenella oocysts through experimental infection. 4) Previous study stated that low-virulence attenuated precocious lines of E. tenella oocysts could be obtained through serial passage of precocious lines in chicken, which can then be used as a vaccine. The low-virulence attenuated precocious line vaccine must have reduced pathogenicity so that they do not show any clinical disease. The aim of study IV is to monitor the changes in biological characteristic of an attenuated precocious strain of E. tenella. In addition, transfection of the precocious line was tried out to investigate whether the transfected precocious line could express fluorescent protein throughout the life cycle for their potential use as a vaccine marker. Study I: Purification and molecular identification of Eimeria tenella A strain of E. tenella (Taiwan strain) was isolated from field sample by using single oocyst passage in chicken. The releasing of sporocysts was optimized by breaking the oocyst wall with glass beads of 0.5 mm diameter and vortexing time of 1 min. Identification of this E. tenella strain was performed with multiplex PCR. To ensure the purity of the strain used. Study II: Eimeria tenella oocyst excretion and riboflavin supplement in the diet of infected chicken To investigate the effect of riboflavin supplement in E. tenella-infected chickens, mortality, fecal consistency, and oocysts per gram of feces were monitored for groups of E. tenella-infected chickens administered a basal diet supplement with either riboflavin, the anti-coccidial drug amprolium, or with both compounds. It was demonstrated that addition of 0.8 g/kg of riboflavin to the basal diet can increase oocyst number in E. tenella-infected chicken, but has no effect on the efficacy of amprolium. Study III: Role of cockroach as transport host of chicken coccidiosis To evaluate the role of the American cockroach, Periplaneta americana as a transport host for E. tenella, cockroaches were orally fed with sporulated oocysts of E. tenella. Their feces and digestive tract were then examined for oocysts by sugar centrifugal flotation technique and PCR. Infectivity of oocysts from the digestive tract of infected cockroach or their feces was also evaluated by orally inoculating into chicken. E. tenella oocysts were found in the digestive tract and feces of cockroaches up to day four after ingestion of oocysts. Furthermore, oocysts that were recovered from the digestive tract and feces of cockroaches remained infective for four and three days after ingestion of oocysts, respectively. Study IV: Characterization of attenuated precocious line of Eimeria tenella The changes of biological characteristics of an attenuated precocious strain of E. tenella were studied. After 34 passages, the prepatent period of the precocious strain was reduced from 180 to 132 hours. The peak of oocysts output of this precocious strain was 3 days earlier than that of the parental strain. The pathogenicity of the precocious strain by means of lesion score, maximum of oocysts per gram of feces and oocyst output was significantly reduced when compared to the parent isolate. However, traces of bloody feces could still be seen in chickens infected with the precocious strain, which is almost the same as the parental isolate. The changes of biological characteristics of an attenuated precocious strain of E. tenella were studied. After 34 passages, the prepatent period of the precocious strain was reduced from 180 to 132 hours. The peak of oocysts output of this precocious strain was 3 days earlier than that of the parental strain. The pathogenicity of the precocious strain by means of lesion score, maximum of oocysts per gram of feces and oocyst output was significantly reduced when compared to the parent isolate. However, traces of bloody feces could still be seen in chickens infected with the precocious strain, which is almost the same as the parental isolate. Second-generation schizogony proceeds at a much faster rate in the precocious line, resulting in a predominance of mature second-generation of schizonts by 96 h and oocysts by 120 h after inoculation. The attempt of introducing fluorescent protein into the precocious line of E. tenella by cationic lipid-mediated transfection method was successful as the fluorescence was detected in unsporulated oocysts. However, as the oocyst continue to sporulate, the fluorescent protein could not be detected in the fully sporulated oocyst. Conclusions I. We have confirmed by PCR that the coccidian strain used was E. tenella and glass bead of diameter 0.5 mm is the most optimum for disrupting oocyt wall to obtain the nucleic acid for PCR. II. Addition of riboflavin at a dose of 0.8 g/kg to the basal diet could increase oocyst output in E. tenella-infected chickens, but has no effect on the efficacy of the amprolium anticoccidial drug. III. Presence of oocysts in feces of chicken that had been inoculated with either digestive tract or feces of P. americana demonstrated the capacity of cockroach to spread and transmit E. tenella to chicken. IV. After 34 passages of selection for the precocious strain, the prepatent periods was reduced from 180 to 132 hours. The maximum of OPG, oocyst output and lesion score were reduced but there were still traces of bloody feces like those seen in the parent isolate. Deletion of any stage of the life cycle was not confirmed but accelerated growth in second-generation shizogony was found in the precocious line. The attempt of introducing fluorescent protein into the precocious line of E. tenella was successful as the fluorescence was detected in unsporulated oocysts. However, no fluorescent protein was detected in the fully sporulated oocyst. |
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| 学位名 | ||||||||||
| 学位名 | 博士(獣医学) | |||||||||
| 学位授与機関 | ||||||||||
| 学位授与機関識別子Scheme | kakenhi | |||||||||
| 学位授与機関識別子 | 32701 | |||||||||
| 学位授与機関名 | 麻布大学 | |||||||||
| 学位授与年月日 | ||||||||||
| 学位授与年月日 | 2019-03-15 | |||||||||
| 学位授与番号 | ||||||||||
| 学位授与番号 | 甲第156号 | |||||||||
| Rights | ||||||||||
| 値 | 本論文の一部は以下のとおり公表されている。(Part of this dissertation has been published as follows.) Jarujareet,W.,Shigenoki,Y.,Taira,K.&Ooi,H.K(2018):Eimeria tenella oocyst excretion and riboflavin supplement in infected chicken. The Journal of Veterinary Medical Science, 80(9)1392-1394 DOI: 10.1292/jvms.18-0219 |
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| 出版タイプ | VoR | |||||||||
| 出版タイプResource | http://purl.org/coar/version/c_970fb48d4fbd8a85 | |||||||||
