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マレック病の免疫方法に関する研究 : とくに七面鳥ヘルペスウイルスに由来するワクチンの開発について
https://az.repo.nii.ac.jp/records/3176
https://az.repo.nii.ac.jp/records/3176df23fd75-4d1e-47bd-bc6e-63871c6db0bf
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diss_dv_otsu0102 (9.9 MB)
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diss_dv_otsu0102_jab&rev (543.3 kB)
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diss_dv_otsu0102_jab.pdf (305.8 kB)
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diss_dv_otsu0102_eab.pdf (477.4 kB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||||||
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| 公開日 | 2013-01-22 | |||||||||
| タイトル | ||||||||||
| タイトル | マレック病の免疫方法に関する研究 : とくに七面鳥ヘルペスウイルスに由来するワクチンの開発について | |||||||||
| タイトル | ||||||||||
| タイトル | Studies on immunization against Marek's disease : development of herpes virus of turkey vaccine | |||||||||
| 言語 | en | |||||||||
| 言語 | ||||||||||
| 言語 | jpn | |||||||||
| 資源タイプ | ||||||||||
| 資源タイプ | thesis | |||||||||
| 著者 |
藤川, 英雄
× 藤川, 英雄
× Fujikawa, Hideo
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| 抄録 | ||||||||||
| 内容記述タイプ | Abstract | |||||||||
| 内容記述 | マレック病(以下MDと略す)はヘルペス型ウイルスによって起る鶏の伝染性疾患で、最初に報告したのはハンガリーのMarekである。1907年に白血病の研究を行なったマレックは神経病変を示すものは他の白血病とは異なることを病理学的に証明した。その後MDは欧米において若齢ひなに1950年後半から1960年前半にかけ集団的に発生し注目されたが、現在では世界各国に流行発生している。我国においてもMDは1963～1964年頃より発生しはじめ、今日では全国的に蔓延し、そのためひなの育成率低下の大きな原因となった。ことにひなは30～120日齢時に発病し死亡するため養鶏業界に甚大な被害を与えた。 MDの病原体は1967年Churchill and Biggsにより分離され、細胞随伴性の強いヘルペスウイルス(以下MDHVと略す)であることが明らかにされた。MDの予防に関する研究は、病原体が分離され、各種の性状が検討されはじめると同時に始められ、ワクチンの研究はもとより、遺伝的抵抗性を持つ鶏の作出および閉鎖環境下での飼育が検討された。後二者ではそれぞれよい成績が得られたが、実際養鶏場での実施には経済的負担が著しく大きいため、ワクチンの開発を望む声が高かった。ワクチンについてはヨーロッパでは強毒を弱毒化したMDHVあるいは自然界より分離した弱毒MDHVを用いるもの、またアメリカではMDHVと抗原的にきわめて類似しており、かつ鶏に対して病原性を示さない七面鳥ヘルペスウイルス(以下HVTと略す)をMDワクチンとして利用しようと云う試みがなされた。いずれのワクチンも野外ではMDの予防に効果のあることが報告された。いつぽう我国においてはまだMDワクチンの開発はされていない。そこで著者はMDワクチンの開発を目的に本研究に着手した。 まず、本研究を進めるにあたり攻撃用の強毒ウイルスが必要であり、またMDの予防方法を確立するためにはMDHVそのものの性状をも検討する必要が認められたので、野外からMDHVの分離を試みた。その結果強毒MDHVは6株分離され、またこの分離株の2・3の性状を明らかにした。つづいて分離MDHVを用い九州地方におけるMDの疫学をゲル内沈降反応を用い検討を行なった。その成績からMDHVは1964年頃より九州地方にはすでに広く浸潤していたことがわかった。 次にHVTのMDに対する発病防止効果に着目し、日本で飼育されている七面鳥からHVTの分離を試みた。その結果7群中2群より多数のウイルスが分離され、その代表株(YT-7株と命名)の性状を詳細に検討したところ、HVTと同定された。HVTを用いてMDワクチンの研究を進め、ワクチンが実用化されるため必要な諸条件を検討し、凍結および乾燥ワクチンの試作に成功した。また試作ワクチンを用いて野外における本ワクチンの効果とその影響が詳細に検討された。さらにワクチン接種時の省力化を目的としMDおよびニューカッスル病(以下NDと略す)混合生ワクチンを試作して実験室内および野外応用試験を行ない、実用可能な成績が得られた。 以下著者が検討し得られた成績の概要について述べる。 1)野外からのMDHVの分離およびその性状 1970年8月佐賀県の某養鶏場において臨床的にMDと診断された120日齢の鶏10羽より腎臓の直接培養でMDHVの分離を試みたところ9例中6例よりウイルスが分離できた。分離ウイルスの代表株(SD-3株と命名)について2・3の性状が検討された。分離ウイルスは鶏、アヒルおよび七面鳥由来の初代培養細胞に病原性を示し、屈光性の強い円形細胞の集族を特徴とするCPEを出現させたが細胞随伴性の強い性質を示した。また感染細胞は癒合して多核巨細胞となり、核内にはA型の封入体が認められた。しかし兎、モルモット、マウス、豚、牛腎、牛睾丸およびマウス胎児の初代細胞、猿、豚およびハムスター腎、ハムスター肺、入羊膜の継代細胞ではウイルスは増殖せず、CPEも認められなかった。分離ウイルスを発育鶏卵の漿尿膜上に接種するとポックを形成し増殖したがIUDR添加により増殖は阻害された。電顕により形態学的な追求を行なったところヘルペス型のウイルス粒子が認められた。次に分離ウイルスの赤血球凝集性が鶏、モルモット、兎、牛および馬血球を用いて検討されたが、MDHVの凝集する血球は認められなかった。分離ウイルスを1日齢SPFひなの腹腔内に接種したところ、70日齢時までに33例中2例に症状を、10例に肉眼病変を、また全例に組織学的に変化が認められた。病変の部位は肝、脾、腎、肺および腺胃に著明で、神経には特に多発する傾向は認められなかった。また接種および不接種同居鶏全例からウイルスが回収され、ゲル内沈降抗体は50%のものが陽性を示した。 2)MDの疫学的検討 1964年に九州各県から収集された544例(A群)および1968～1971年に収集された871例(B群)計1415例の血清についてゲル内沈降反応により抗体調査が行なわれた。その結果A群の血清では検査全県に陽性例が認められ、陽性率は低い県で15.4%、また高い県で44.8%であった。B群の血清では0～84.3%の陽性率が得られた。このことからMDHVはすでに1964年頃より九州地方に広く浸潤していたことが明らかになった。次に抗体陽性率は日齢により差があり、1～30日齢では1%であったが90～120日齢時になると72.4%を示し、日齢の進むとともに高くなった。鶏の系統別にみるとハンドレスでは10%の陽性率であったがデカルブでは68.8%と高く、系統により陽性率に差がみられた。次いで熊本県内で得られた1～3日齢のひな7群について移行抗体の保有状況を検討したところ、1群を除き55～83%の陽性率が得られた。これらMDHVの移行抗体は12日齢まで認められたが、15日齢以上では陰性となった。またウイルス感染後の能動抗体の出現は早いもので30日齢、遅いものでは100日齢から認められた。その他七面鳥、アヒル、鳩、牛、犬および人の血清にはMDHVに対する抗体は認めなかった。次にふ化後日齢毎にウイルス分離試験を行なったところ、汚染環境下では9日齢のひなから、消毒の行なわれた環境下では28～33日齢時からウイルスが検出された。 3)野外からのHVTの分離およびその性状 健康な七面鳥の血液、腎および毛のう部からウイルスの分離を試みたところ、熊本県収集の37例は全例陰性であったが、山口県収集の43例中39例の血液から分離できた。この分離ウイルスの代表株(YT-7株)について2・3の性状が検討された。 分離ウイルスは鶏胎児線維芽細胞(以下CEFと略す)およびアヒル胎児線維芽細胞(以下DEFと略す)に屈光性の強いCPEを現わした。感染細胞は多核巨細胞となり、核内にA型の封入体を形成した。またウイルスは細胞随伴性が強く、発育鶏卵で増殖し、漿尿膜上にはポックを形成した。しかしIUDR添加により増殖は阻害された。分離ウイルスを電顕により形態学的に追求したところヘルペス型のウイルス粒子が認められた。次に鶏、モルモツト、兎、牛および馬の血球を用いて分離ウイルスのHA性を検討したが凝集した血球はなかった。その他分離ウイルスは実験用小動物(乳のみマウス、成熟マウス、乳のみモルモツトおよび兎)に対して病原性を示さず、また哺乳動物由来の初代(兎、モルモツト、マウス、豚、牛の各腎、マウス胎児および牛睾丸)および継代(猿、豚、ハムスター腎、ハムスター肺および人羊膜)細胞では増殖しなかった。次に分離ウイルスを1日齢SPFひな20羽の腹腔内に接種し70日間臨床症状を観察したが、臨床症状を示したものは1例もなく正常に発育した。また70日齢時の剖検および組織学的所見においても全例変化は認められず、抗体は全例陽性であった。 4)HVT凍結生ワクチンの野外応用試験 HVT(FC-126株)を用いてMD凍結生ワクチンを作成し産卵鶏および肉用鶏に対する野外応用試験が行なわれた。 産卵鶏に対する接種試験は熊本県内の5養鶏場をえらび接種群10,174羽、対照群4,972羽を対象とし、ワクチンは1,000PFU/羽を腹腔内あるいは皮下に接種し、180日間観察した。この間死亡とう汰されたもの全例についてその原因が病理組織学的に検討された。その結果ワクチン接種後臨床的に異常を示すひなは認められなかった。供試鶏の死亡とう汰の割合は接種群3.3～10.1%、平均7.5%であったが対照群では6.1～23.2%、平均14,4%で、接種群では対照群に比較し死亡とう汰率が48.1%減少した。これらの死亡とう汰率を推計学的(χ^2 -test)に検討すると両群間に有意差(P<0.001)が認められた。死亡とう汰原因の主なものは虚弱、事故、カンニバリズム、発育不良、MD、白血病および慢性呼吸器病などであった。虚弱、事故および卵巣異常を除いたその他の死亡とう汰の原因は接種群では対照群よりいずれも少なかった。MD発生数はワクチン接種群で1.1%および対照群では7.1%で両群間には推計学的に有意差(P<0.001)が認められ、接種群のMD発生数は対照群に比較し84%減少した。ワクチンウイルスは180日間91.4～100%回収され、また低率ではあるが同居感染が認められた。ワクチン接種鶏でも野外MDHVの感染を阻止できず重感染が成立していた。MDHV抗原によるゲル内沈降抗体陽性率、伝染性気管支炎ウイルス中和抗体価、マイコプラズマガリセプチカム凝集抗体陽性率およびND-HI抗体価には接種群および対照群との間に差を認めず、また体重の増加率および産卵率においても差は認められなかった。 肉用鶏に対する接種試験は4養鶏場をえらび接種群103,509羽、対照群36,021羽を対象とし、ワクチンは1000PFU/羽を1羽量として1,1/2および1/4羽量を皮下に接種し49～70日間観察した。さらにまた出荷食鶏処理場で腫瘍病変の出現状況が観察された。その結果、死亡とう汰率では接種および対照群で差を認めなかったが、腫瘍発生数は対照群に比較し接種群で88.7%減少し、推計学的に両群間に有意差(P<0.001)を認めた。次に各鶏群について、各回50～150羽宛体重を測定し、その平均値とばらつきを求めた。その結果接種群の平均体重は対照群に比較し10～100g重く、また体重のばらつきも少なかった。ワクチンウイルスは17日齢時から回収され、またゲル内沈降抗体も33日齢から陽性となった。飼料要求率は接種群で0.06～0.11、平均0.09少なく、経費計算をすると1羽当り14.38円の増収があった。 以上の成績からHVTを用いた凍結生ワクチンはMD発病予防および肉用鶏の飼料要求率を減少させるため、きわめて有効なワクチンであると思われる。 5)HVT凍結乾燥ワクチンに関する検討 HVT分離株(YT-7株)はDEFおよびCEEで継代を重ねると細胞フリーウイルスの量が次第に増加し、DEF12代-CEF40代継代株では6.5×10^6 PFU/mlの細胞フリーウイルスが得られるようになった。また細胞フリーウイルスの性状を検討したところ原株と同じ性状が得られた。この継代ウイルスを用いて凍結乾燥ワクチンが作成れ、1日齢SPFひなに200～9,000PFU/羽接種した。その結果接種群は全例臨床的に異常を示すものはなく、ゲル内沈降抗体は15週齢時80%の陽性率であり、ワクチンウイルスは100%のものから回収された。また3週齢時に攻撃試験を行なったところ対照群は80%にMD病変が認められたが、ワクチン接種鶏ではMD病変を示すものはなかった。次に同居感染試験を行なったところ実験室内試験では同居感染は成立しなかった。つづいて、10^1.0 ～10^5.0 PFU/羽のウイルスを1日齢の移行抗体陰性および陽性ひなに接種しウイルスの回収試験を行なった。その結果抗体陰性ひなでは10^1.0 PFU/羽接種群においてもワクチンウイルスは回収された。しかし大量(10^2.0 ～10^5.0 PFU/羽)接種群に比較し、その回収率は低かった。また移行抗体保有ひな群は10^3.0 PFUのワクチンウイルスを接種した場合、ウイルスの回収時期がおくれた。 このワクチンについて5養鶏場のひな13,965羽(接種群10,384羽および対照群3,581羽)を用いて野外応用試験が行なわれた。観察は150日間行ない、この間死亡とう汰したもの全例についてその原因を病理組織学的に検討した。 ワクチンは皮下に0.2ml(1,600PFU/羽)宛接種された。その結果ワクチン接種により異常を示したひなは認められなかった。死亡とう汰数は接種群468例(4.5%)、対照群271例(7.6%)で減少率は41.8%であった。MD発生数は、接種群117例(1.13%)、対照群133例(3.71%)で、MDの減少率は69.7%を示し、両群間には推計学的に有意差(P<0,001)を認めた。ワクチンウイルスは150日間、79～90%のものから回収され、また5群中1群に同居感染した例があった。ゲル内沈降抗体は121～150日齢時に72%の陽性率を示した。またワクチン接種鶏は野外MDHVの感染を阻止せず重感染が成立していた。 これらのことからこの乾燥ワクチンは凍結ワクチンと同様に十分野外に応用できるものと思われる。 6)MD(HVT).ND混合生ウイルスワクチンに関する試験 HVTおよびニユーカツスル病ウイルス(以下NDVと略す)B_1 株二種混合生ワクチンを作成し安全性および免疫原性が検討された。供試ひなはSPFおよび市販の1日齢ひなで、1羽量(HVT1200～2200PFU/羽、NDV10^6.1 ～10^6.5 EID50/羽)の混合ワクチンを皮下、筋肉および腹腔内に接種した。その結果混合ワクチンおよびMD単味ワクチン接種鶏との間にMDワクチンウイルス回収率、MD中和価および攻撃試験による発病防禦に差はみられず、また混合ワクチンおよびND単味ワクチン接種鶏の間にもND-HI価および攻撃試験による耐過率に差がなかった。接種部位は腹腔内接種が皮下および筋肉内に比べてよかった。混合ワクチン接種後3日毎にMDワクチンウイルスの回収試験を行なったところ6日目では86～100%回収され、MD単味ワクチン接種群と比較し差を認めなかった。また混合ワクチンの接種は、その後に再接種されたND単味生ワクチンの免疫効果に影響を与えなかった。NDワクチンウイルスの同居感染は市販およびSPFひなともに点鼻したND単味ワクチン群より皮下接種した混合ワクチン群で起りにくかった。市販およびSPFひなを用いて安全試験を行なったところ、市販ひなでは全例接種反応は陰性であったが、SPFひなではND単味ワクチンと同程度の軽い一過性の呼吸器症状(5～10分間に1～2回クシヤミ)が認められた。しかし反応を示したひなは発育には影響をうけなかった。次に混合ワクチンを用いて2養鶏場のひな5,092羽(混合接種群3,014羽、MD単味接種群2,078羽)に野外応用試験を行ない、観察は120日間実施した。その結果混合ワクチン接種後接種反応が認められたものは1例もなく、120日齢時の育成率は96.1および98.8%と良好であった。またこれらの接種ひなからはMDワクチンウイルスが100%回収され、ND-HI抗体の上昇も認められた。次に試験管内でHVTおよびNDVB_1 株の干渉の有無を検討したところ相互に干渉は起さなかった。 以上の成績から、この混合ワクチンは実用可能なものと考える。 |
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| Abstract | ||||||||||
| 内容記述タイプ | Other | |||||||||
| 内容記述 | Marek's disease (MD) is an avian infectious disease caused by an avian herpes virus. The first description of the disease was shown in 1907 by a Hungarian scientist, Marek who found the evidence, in the pathological studies on avian leukosis, that there was a lymphomatosis with the neural enlargement which was distinguished from other leukosis. From 1950s to 1960s, MD appeared in Europe and the United States of America, but the disease spreads in all over the world at present time. In Japan, MD was recognized in 1963 to 1964, and nowadays the disease is prevalent in all over the country. MD is one of the most principal diseases causing decrease in weight gain and feed conversion or increase in the rate of dead and culled birds. The chickens exposed to MD virus infection die at 30 to 120 days of age, which has been causing a great deal of economic loss in all areas of intensive poultry production. The discovery of a viral agent closely associated with MD was done by Churchill and Biggs in 1967, and the virus was found to be a highly cell associated herpes virus (MDHV). As soon as MDHV was isolated, studies on the control of MD were started almost simultaneously with the investigation on the properties of MDHV, and three methods of control of MD were available. They were the filtered air positive pressure system of management, selection for genetically controlled resistance and vaccination. Some success in reducing the incidence of MD was obtained by the former two methods, but it was too expensive to put them in practical use in poultry farms, therefore, the development of MD vaccine was strongly desired. In Europe, two types of MD vaccines were developed. One was prepared from an attenuated MDHV and another was from a naturally avirulent MDHV isolated from a normal flock. On the other hand, in the United States of America, an another type of MD vaccine was produced, which was prepared from a herpes virus of turkey (HVT) being no pathogenic for chickens and related to MDHV in its antigenicity. All of these vaccines were found to be effective in the field. In Japan, as MD vaccine had not been developed, the author began to study for the purpose of producing the vaccine. At first time, isolation of a virulent MDHV needed as a challenge virus was attempted, and from the birds closely associated with MD, 6 viral agants were isolated. These isolates were identified to be MDHV by the examination of their properties. The epzootiological surveys on MD in Kyushu district were taken by the agar gel precipitation test, from which it was evident that MD had already appeared in the district in 1964. On the other hand, the blood of the turkeys bred in Japan were examined for isolation of HVT, as which is effective in reducing the incidence of MD, and viral agents were isolated from the 2 flocks of the 7 ones examined. Some properties of these isolates were examined, and as there was no difference in their properties, an isolate was selected as a representative strain among them (YT-7). Further investigations on the properties of strain YT-7 were done in detail, and this virus proved to be a HVT. The requirements for developing MD vaccine were studied with the YT-7 strain of HVT. After the fundamental studies, 2 types of HVT vaccines were prepared, namely one was cell-associated HVT and another was cell-free HVT. The safety and immunogenicity for chickens of these vaccines were tested in detail in several poultry farms. Additionally for the purpose of decrease in vaccination times, a Newcastle disease (ND)-MD combined vaccine was developed. From the studies in the laboratory and field, the combined vaccine was considered to be effective for controlling these disease. It is the intention of this paper to show the summary of the studies on MD vaccines. 1) Isolation of MDHV and it's properties In August, 1970, Some birds of flock consisted of 120-day-old chickens in a poultry form in Saga prefecture showed the clinical signs associated with MD, and from the 6 cases of 9 ones examined, viral agents were isolated. A few properties of strain SD-3, which was selected from these isolates as a representative virus, were examined, and the isolate was identifed to be a MDHV. The primary cells in culture of chicken, duck and turkey were susceptible to MDHV infection, and these infected cells showed refractile and rounded CPE. The infectivity of strain SD-3 was highly cell-associated. The infected cells showed syncytial formation, and Cowdry type A intranuclear inclusion bodies were recognized among them. While a thorough attempt to propagate strain SD-3 in a wide variety of mammalian primary cells of rabbit, quinea pig, mouse, swine and bovine kidneys, bovine testicle and mouse embryo and cell lines of green monkey kidney (GMC), swine kidneys (PK-15, PS), hamster lung(HmLu), hamster kidney (BHK-21), and human amnion (FL) was unsuccessful. Strain SD-3 propagated in chorio allantoic membrane (CAM) and formed pocks on CAM, and the pock formation was inhibited by the treatment of IUDR. In additon, the cells infected with strain SD-3 was examined by electron microscopy, and herpes virus particles were seen in the infected cells. The hemoagglutination (HA) test was done with the erythrocytes from chicken, guinea pig, rabbit, cattle and horse, but strain SD-3 agglutinated none of these erythocytes. When one-day-old SPF chickens were inoculated intraperitoneally with strain SD-3, the clinical symptoms, growth lesions and microscopic lesions of MD were recognized in 2, 10 and all cases respectively among the 33 birds tested at 70 days after the inoculation. The lesions of MD appeared chiefly in the organs of liver, spleen, kidney, lung and gizzad, but very few cases of the neural lesions were observed. MDHV was isolated from all of the chickens in the inoculated and contact exposed groups, but the gel precipitating antibody was detected in only 50% cases among the chickens. 2) The epizootiological survey on Marek's disease Both of the sera taken from the 544 birds in 1964 in Kyushu district (A group) and the 871 ones in 1968 to 1971 in the same district (B group) were investigated for the gel precipitating antibody to MDHV. In A group, the antibody was detected in the sera from the chickens of all of the prefectures surveyed, and the positive rate were 15.4 to 44.8%. Meanwhile, in B group, the positive rate were 0 to 84.3%. This showed that MD had already spread widely in Kyushu district in 1964. The appearance of the gel precipitating antibody to MDHV was depended on the age of birds. The positive rate of the antibody in 1 to 30-day-old chickens was 1%, but it increased to 72.4% at 90 to 120 days of age. There was a variation in the appearance of the gel precipitating antibody among the genetically different chickens. Incidentally, the positive rate in Handless and Dekalb were 10 and 68.8% respectively. Seven groups consisted of 1 to 3-day-old chickens in Kumamoto prefecture were examined for the maternal antibody to MDHV by the agar gel precipitation test. The antibody was detected in the sera from the 6 groups except one group, and the positive rate were 55 to 83%. The maternal antibody was detectable until 12 days of age, but at 15 or more days of age, it become undetectable. Antibody responce by MDHV infection occured at 30 to 100 days of age. No evidence of the gel precipitating antibody to MDHV was detected in the sera from turkey, duck, pigeon, cattle, dog or human being. Virus isolation tests from the chickens being hatched and bred under the hygienic and no disinfectant circumstances were done. MDHV was isolated at 28 to 33 days of age under the hygienic condition, and at 9 days of age under the dirty condition. 3) Isolation of HVT and it's Properties An attempt to isolate HVT from the materials of blood, kidney and feather follicles from the normal turkeys reared in Kyushu and Yamaguchi districts was done. Virus was isolated from the blood from 39 of 43 turkeys raised in Yamaguchi prefecture, but no virus was detected in the materials from Kumamoto prefecture. A virus strain was selected from these isolates as a representative strain (YT-7), and strain YT-7 was proved to be a HVT from it's properties. Strain YT-7 produced refractile CPE in chicken embryo fibroblast (CEF) and duck embryo fibroblast (DEF), and syncytial formation and Cowdry type A intranuclear inclusion bodies were seen in the infected cells. The infectivity of strain YT-7 was highly cell associated. Strain YT-7 propagated in chicken embryonated eggs and made pocks on the CAM. These propagation of strain YT-7 were inhibited by adding IUDR in the medium. The cells infected with strain YT-7 were examined by electron microscopy, and in which a number of herpes virus particles were seen. HA activity of strain YT-7 was tested with the erythrocytes prepared from chicken, guinea pig, rabbit, cattle and horse, but none of them used was agglutinated by the stain. A thorough attempt to propagate strain YT-7 in a wide variety of small animals (suckling and adult mice, suckling guinea pig and rabbit), mammalian primary cells (rabbit, guinea pig, mouse, swine and bovine kidneys, mouse embryo fibroblast and bovine testicle) and mammalian cell lines (GMC, PK-15, PS, BHK-21, HmLu and FL) was unsuccessful. Twenty one-day-old SPF chickens were inoculated intraperitoneally with strain YT-7, and these chickens were observed for the clinical signs for 70 days postinoculation. During the observation, none of the 20 chickens showed any clinical signs, and neither gross lesions nor microscopic ones at necropsy were observed, but antibody response to HVT was detected in the sera from all the chickens. 4) A field experiment of Marek's disease vaccine (freeze-type) A preparation of Marek's disease vaccine (freeze-type) was made with strain FC 126 of HVT. The vaccine was tested for safety and immunogenicity for layers and broilers in the field. The field experiment of the vaccine for layers was done in 5 poultry farms in Kumamoto prefecture. Each of 10,174 chickens was inoculated intraperitoeally or subcutaneously with 1,000 PFU of the vaccine (inoculated group), and 4,972 chickens were applied to the control group. These chickens were observed for clinical symptoms for 180 days postinoculation. No birds showed any clinical signs due to the inoculation. All of the birds which died or were culled during the observation were examined histopathologically for the cause of them. The rate of death and culling in the vaccinated and control groups were 3.3 to 10.1% (7.5% on the average) and 6.1 to 23.2% (14.4% on the average) respectively. The rate of diseased birds in the vaccinated group compared with that in the control group was decreased by 48.1% (P<0.001). The causes of death and culling were weakness, accident, cannibalism, bad growth, MD, avian leucosis, chronic respiratory disease and so forth. The number of these causes except weakness, accident and ovarian disease in the vaccinated group was fewer than that in the control group. The rate of MD occurence in the vaccinated and control groups were 1.1 and 7.1% respectively, and the rate in the former compared with that in the latter was decreased by 84% (P<0.001). The vaccinal virus was reisolated from 91.4 to 100% of the vaccinated birds and from the control ones though it was just a few rate. MDHV was isolated even from the vaccinated birds, and that showed the vaccine could not prevent MDHV infection in the field. Between the vaccinated and control groups, there were no significant differences in a variety of MD-gel precipitating, IB-neutralizing, Mycoplasma gallisepticum-agglutinating and Newcastle disease-hemagglutinating antibody titers and the rate of weight gain and egg laying. In addition, the field test of the vaccine for broilers was done in the 4 poultry farms in Kumamoto prefecture. A total number of 103,509 chickens were inoculated subcutaneously with a variety of 1 (1,000 PFU), 1/2 and 1/4 doses of the vaccine (vaccinated group) and a total number of 36,021 chickens were applied to the control group. Observation was done for 49 to 70 days postinoculation, and at the time of slaughter in the processing plants, they were examined for MD lesions. There was no significant difference in the rate of death and culling in both of the groups, but the rate of tumor lesions was lower in the vaccinated group than that in the control one. The decreased rate in the former compared with the latter was 88.7% (P<0.001). Fifty to 150 birds were sampled at random at a proper interval from the vaccinated and control groups. The weight of the birds in the vaccinated group was heavier by 10 to 100 gs than that in the control group, and the variance in weight in the former was smaller than that in the latter. The vaccinal virus was reisolated from the vaccinated birds at 17 or more days of age, and gel precipitating antibody responce to HVT was detected at 33 or more days of age. The ratio of feed demand in vaccinated group was lower by an average of 0.09 (0.06 to 0.11) than that in the control group, and economic income in the vaccinated group was larger by 14.38 yen/chicken than that in the control group. These results indicate that this vaccine is highly usefull for the prevention of MD and decrease in feed demand. 5) Studies on a lyophilized HVT vaccine Strain YT-7 of HVT was passed serially in DEF and CEF, and with undergoing the passage, cell free virus increased. The titer of cell free virus at the passage level of DEF 12-CEF40 was 6.5 x 10^6 PFU/ml, and cell free HVT had the same properties with cell associated HVT. A preparation of cell free HVT was lyophilized. One-day-old SPF chickens were inoculated with 200 to 9000 PFU of the vaccine. None of them showed any clinical symptoms due to the vaccination. Gel precipitating antibody to HVT was detected in 80% of the sera from the vaccinated birds at 15 weeks postinoculation, and the vaccinal virus was reisolated from all of the birds. The vaccinated chickens were challenged with a pathogenic MDHV at 3 weeks of age. None of them had the lesions of MD, but the lesions were shown in 80% of the control group. Contact transmission of HVT from the donor to the pen mate was not evident. In addition, the effect of the maternal antibody to MDHV on immunization with the vaccine was tested with one-day-old chickens. The vaccinal virus was reisolated from the maternal antibody negative chickens even when they were inoculated with only 10^1.0 PFU of the vaccine, but the recovery rate was lower than that when they were inoculated with 1o^2.0 to 10^5.0 PFU of the vaccine. On the other hand, when the maternal antibody possitive chickens were inoculated with 10^3.0 PFU of the vaccine, it needed more days to reisolate the vaccinal virus from them. The vaccine was tested for safety and immunogenicity with a total number of 13,965 chickens in 5 poultry farms in Kumamoto prefecture. A total number of 10,384 chickens were inoculated subcutaneouly with 1600 PFU/0.2 ml of the vaccine, and the remainder 3,581 chickens were applied to the control group. Observation was done for 150 days postinoculation, and the birds which died or being culled during the observation were examined histopathologically for the causal agents. None of the vaccinated chicken showed any clinical symptoms due to the inoculation. The rate of death and culling in the inoculated and control groups were 4.5 and 7.6% respectively. MD occured in 117 cases (1.13%) of the vaccinated birds, and in 133 ones (3.71%) of the control birds. The rate in the former compared with the latter was decreased by 69.7% (P<0.001). The vaccinal virus was reisolated from 79 to 90% of the vaccinated birds for 150 days postinoculation, and contact transmission of HVT from the donors to the pen mates was recognized in only one group among the 5 groups. Gel precipitating antibody responce to HVT at 121 to 150 days of age was detected in 72% of the sera from the vaccinated birds. MDHV infection occured in the vaccinated groups, and HVT and MDHV persisted in the same host for long time. These results indicate that the lyophilized HVT vaccine has the same level of safety and efficacy for the prevention of MD in the field. 6) Studies on MD (HVT)-ND combined live virus vaccine A MD-ND combined vaccine was prepared from HVT and the B_1 strain of Newcastle disease virus. The vaccine was examined for safety and imunogenicity with conventional and SPF one-day-old chickens. They were inoculated with the combined vaccine involving 1200 to 2200 PFU of HVT and 10^6.1 to 10^6.5 EID_50 of NDV per chicken by the routes of hypoderm, muscle and abdominal cavity. Between the combined vaccine inoculated group and MD vaccine inoculated group, there were no differences in the rate of HVT recovery, neutralizing antibody responce to HVT and protective efficacy against the challenge with a pathogenic MDHV. Similarly, between the combined vaccine inoculated group and ND vaccine inoculated group, there were no differences in ND-HI antibody responce and protective efficacy against the challenge with a pathogenic NDV. For the route of vaccination, intraperitoneal inoculation was better than other methos. HVT was reisolated at 6 days of age in 86 to 100% of the vaccinated chickens, and the recovery rate was almost equal to that of the chickens inoculated with MD vaccine. The combined vaccine inoculation gave no inhivition to the secondary antibody responce to NDV. Contact transmission of ND vaccinal virus from the donors inoculated intranarially with ND vaccine to the pen mates was more detectable than that from the donors inoculated subcutaneously with the combined vaccine to the pen mates. Safety for conventional and SPF chickens of the combined vaccine was tested. None of the conventional chickens inoculated with the vaccine showed any clinical symptoms, but some SPF chickens inoculated with the vaccine showed slight temporary respiratory symptoms (1 to 2 sneezes per 5 to 10 minutes), though such signs were recognized when SPF chickens were vaccinated with ND vaccine, which gave no significant influences on the growth of the chickens. In addition to these experimental studies, a field experiment of the combined vaccine was done with a total number of 5,092 chickens in 2 poultry farms in Kumamoto prefecture. A total number of 3,014 chickens were inoculated subcutaneously with the combined vaccine, and a total number of 2,078 ones were applied to the MD vaccine inoculated group. None of the chickens inoculated with these vaccines showed any clinical symptoms due to the vaccination. Observation was done for 120 days postinoculation. The rate of death and culling at 120 days of age were 1.2 to 3.9% in the combined vaccine inoculated groups and, almost same level of the rate was recognized in the MD vaccine inoculated group. HVT was reisolated from all of the vaccinated birds, and ND-HI antibody responce was detected in all of them. In vitro assay on interference between HVT and NDV was done, but no evidence of the interference was detected. From these results, it can be said that the combined vaccine is suficientry and practically usable in the field. |
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| 学位名 | ||||||||||
| 学位名 | 獣医学博士 | |||||||||
| 学位授与機関 | ||||||||||
| 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 | 1977-03-14 | |||||||||
| 学位授与番号 | ||||||||||
| 学位授与番号 | 乙第102号 | |||||||||
| 著者版フラグ | ||||||||||
| 出版タイプ | AM | |||||||||
| 出版タイプResource | http://purl.org/coar/version/c_ab4af688f83e57aa | |||||||||
