JPH0315607B2 - - Google Patents
Info
- Publication number
- JPH0315607B2 JPH0315607B2 JP1815182A JP1815182A JPH0315607B2 JP H0315607 B2 JPH0315607 B2 JP H0315607B2 JP 1815182 A JP1815182 A JP 1815182A JP 1815182 A JP1815182 A JP 1815182A JP H0315607 B2 JPH0315607 B2 JP H0315607B2
- Authority
- JP
- Japan
- Prior art keywords
- present
- sample
- chickens
- same amount
- control
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 241000287828 Gallus gallus Species 0.000 claims description 172
- 235000013330 chicken meat Nutrition 0.000 claims description 167
- 241000588724 Escherichia coli Species 0.000 claims description 54
- 238000000034 method Methods 0.000 claims description 21
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 20
- 210000003555 cloaca Anatomy 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 238000007598 dipping method Methods 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 238000005507 spraying Methods 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims 1
- 238000010422 painting Methods 0.000 claims 1
- 239000008363 phosphate buffer Substances 0.000 description 67
- 210000000436 anus Anatomy 0.000 description 46
- 210000000481 breast Anatomy 0.000 description 39
- 210000003205 muscle Anatomy 0.000 description 38
- 239000000243 solution Substances 0.000 description 20
- 208000015181 infectious disease Diseases 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 13
- 239000002609 medium Substances 0.000 description 13
- 210000002976 pectoralis muscle Anatomy 0.000 description 11
- 210000003046 sporozoite Anatomy 0.000 description 9
- 239000007921 spray Substances 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 8
- 229920001817 Agar Polymers 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 239000002244 precipitate Substances 0.000 description 7
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- 241000186000 Bifidobacterium Species 0.000 description 6
- 241000223924 Eimeria Species 0.000 description 6
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- 229960005486 vaccine Drugs 0.000 description 5
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- 241000186660 Lactobacillus Species 0.000 description 4
- 241000607683 Salmonella enterica subsp. enterica serovar Pullorum Species 0.000 description 4
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
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- 238000001035 drying Methods 0.000 description 2
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- 239000008103 glucose Substances 0.000 description 2
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- 229940049954 penicillin Drugs 0.000 description 2
- 208000008494 pericarditis Diseases 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 206010039083 rhinitis Diseases 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
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- 230000001954 sterilising effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- HQOJMTATBXYHNR-UHFFFAOYSA-M thallium(I) acetate Chemical compound [Tl+].CC([O-])=O HQOJMTATBXYHNR-UHFFFAOYSA-M 0.000 description 2
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- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 108010082340 Arginine deiminase Proteins 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 241001455504 Escherichia coli O2 Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
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- 108091022930 Glutamate decarboxylase Proteins 0.000 description 1
- 102000008214 Glutamate decarboxylase Human genes 0.000 description 1
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- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 108010048581 Lysine decarboxylase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 102000052812 Ornithine decarboxylases Human genes 0.000 description 1
- 108700005126 Ornithine decarboxylases Proteins 0.000 description 1
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- 206010071301 Perihepatitis Diseases 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
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- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 description 1
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- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
本発明は鶏の大腸菌症を免疫的に予防し得る方
法に関する。
鶏の大腸菌症は特定の血清型を有する大腸菌の
感染により発症する。この病気の種類としては、
大腸菌性敗血症をはじめ、関節炎、出血性腸炎等
が挙げられる。このものに感染した鶏は気嚢炎、
心膜炎、関節炎および下痢等の症状を示して重症
の場合は死に至る。またこの大腸菌症は集団的ま
たは散発的に発生するものであり特にブロイラー
での発生が多く、被害の大きいものである。
かかる大腸菌症の予防のためには薬剤の投与が
実施されているが、薬剤耐性菌が多く出現してい
るために効果はあまり期待できない。また、治療
の場合も薬剤の選択、投与方法、労力等に問題が
多く、優れた予防効果を有するものの出現が望ま
れている。
そこで本発明者らは前記の問題を解決すべく研
究を行なつた結果、大腸菌を鶏の総排泄腔より接
種することにより大腸菌症を予防し得ることを見
い出した。
本発明の大腸菌は動物の臓器または糞便から分
離されたものであり、例としては大腸菌O1、O2
および大腸菌（E.coli）NS−244（微工研菌寄第
5757号）が挙げられる。
大腸菌NS−244は牛の糞便を大腸菌の選択培地
であるDHL寒天〔栄研化学(株)製〕上に画線培養
し出現したコロニーより得られた一株である。こ
のものの菌学的性質を述べる。
１ 形態学的所見
1.0〜4.0×0.4〜0.7μ
グラム陰性棹菌
運動性あり
芽胞を形成しない
２ 培地上の所見
肉エキス培地で容易に発育する
肉エキス１％、ペプトン１％および塩化ナト
リウム0.3％を含有する寒天培地での生育良好
コロニーは低い凸状を示し、無色でＳ型、か
なり不透明、周辺はまるい
３ 生物学的性状
生育PH3.0〜10.0（至適PH7.0）
生育温度20〜40℃（至適温度37℃）
好気性および通性嫌気性
４ 非抗酸性
カタラーゼ 陽性
オキシターゼ 陰性
KCN培地における発育は認められない
β−ガラクトシターデ 陽性
ブドウ糖からのガス産生あり
インドールの生成あり
TSI培地での硫化水素生成せず
ウレアーゼ生成せず
ゼラチン液化能なし
フエニルアラニン・デアミナーゼ 陰性
リジン・デカルボキシラーゼ 陽性
オルニチン・デカルボキシラーゼ 陽性
アルギニン・ジヒドロラーゼ 陰性
グルタミン酸デカルボキシラーゼ 陽性
MR反応 陽性
VP反応 陰性
単一炭素源として酢酸を利用するがクエン酸を
利用できない
４ 炭水化物からの酸産生能
ブドウ糖 陽性
アラビノース 陽性
セロビオース 陰性
乳糖 陽性
麦芽糖 陽性
ラフイノース 陰性
ラムノース 陽性
白糖 陰性
トレハロース 陽性
キシロース 陽性
アドニツト 陰性
ズルシツト 陽性
エスクリン 陰性
マンニツト 陽性
ソルビツト 陽性
グリセリン 陽性
サリシン 陰性
イノシツト 陰性
以上の菌学的性状から「Bergey's Manual of
Determinative Bacteriology」第８版の分類の
基準にしたがつて既知の菌株との異同を検討した
結果、本発明において使用する菌株（NS−244）
は大腸菌（Escherichia coli）と同定された。
この大腸菌NS−244の鶏に対する病原性を調べ
た結果、全く病原性がないことを確認した。すな
わち大腸菌NS−244の生菌を３週令の鶏10羽に１
羽当り1.1×1011個で静脈内投与した。７日後、
鶏の状態を観察したが死亡例は０であり、大腸菌
症の特徴的な病変である心膜炎や肝包膜炎の出現
は全く認められなかつた。
本発明で用いる大腸菌NS−244は生菌の状態で
用いることができるが、不活化したものを用いた
方が扱い易い。本発明で用いる大腸菌O1、O2は
不活化して用いる。大腸菌NS−244と大腸菌O1、
O2は紫外線照射、ガンマー線照射またはホルマ
リン処理で不活化するのがよいが、フエノール、
アセトン、アルコール、凍結融解、加熱、超音波
あるいは圧力などで処理して不活化することもで
きる。さらに、このものに水酸化アルミニウムゲ
ル、フロインドの完全アジユバンドのようなアジ
ユバンド（免疫賦活剤）を加えることも妨げな
い。さらにこのものに他の疾病の免疫的抗原や非
病原性の抗原を混合して総排泄腔に接種すること
もできる。前記の免疫的抗原とは、鶏コクシジウ
ム原虫、ニユーカツスル病などのワクチン、伝染
性コリーザの原因菌であるHaemophilus
paragallinarum、鶏マイコプラズマ病の原因菌
であるMycoplisma gallisepticum、Clostridium
perfringens、Salmonella pullorum、
Bifidobacterium、Lactobacillusおよび大腸菌
O1、O2、O78以外で大腸菌症に罹患した鶏から分
離される他のＯ抗原を有する大腸菌または健康な
鶏から分離される非病原性大腸菌などが挙げられ
る。
次いで前記大腸菌を鶏に接種する。接種に適当
な状態としては抗大腸菌症剤を水、生理食塩水、
PBS（リン酸バツフアー）および組織培養液など
の溶液に懸濁せしめたもの、または前記の不活化
処理そしてまたアジユバンド処理を行なつたもの
が例として挙げられる。このものを鶏の総排泄腔
に接種する。接種の具体的な方法としては鶏の肛
門、総排泄腔あるいはフアブリシウス嚢内に抗大
腸菌剤を上記の溶液に懸濁せしめ１羽当り0.002
ml〜0.2mlを滴下、注入、塗布、スプレーまたは
肛門部を浸すことによつて投与する方法が挙げら
れる。抗大腸菌症剤の接種菌量は鶏１羽当り1.0
×105個以上を用いる。
本発明に係る抗大腸菌症剤を本発明の接種方法
で使用すれば副作用を起こすことなく大腸菌症を
ほとんど完全に予防し得る。さらに、従来の薬剤
投与法では、薬剤耐性大腸菌の出現が多かつた
が、本発明の予防方法を用いれば、薬剤耐性大腸
菌の出現はない。
次に本発明の効果を示すための実施例を挙げ
る。
実施例 １
０日令の鶏（ブロイラー専用種ハバード）10羽
の肛門に試料を接種し、また対照(2)〜(9)は試料を
それぞれの方法で接種した。次いでこれらの鶏に
21日令において大腸菌O78を１羽当り1.4×1010個
静脈内接種して感染させる。対照(1)は試料を接種
せずに感染のみを行なつたものである。感染後７
日目に斃死率について観察した。結果は表１に示
す。試料の種類および接種方法については下記に
示す。
本発明(1)：大腸菌NS−244の４×1011個をPBS10
mlに懸濁せしめた懸濁液〔試料(1)〕を鶏の肛門
に１羽当り１滴（約0.025ml）滴下する。
本発明(2)：本発明(1)で用いた試料(1)をPBSで４
倍に希釈し、鶏の肛門に１羽当り約0.1ml塗付
する。
本発明(3)：本発明(1)で用いた試料(1)をPBSで100
倍に希釈し、鶏の肛門に１羽当り１滴（約
0.025ml）滴下する。
本発明(4)：本発明(1)で用いた試料(1)をPBSで400
倍に希釈し、鶏の肛門に１羽当り約0.1ml塗付
する。
本発明(5)：大腸菌NS−244の４×1011個をPBSに
懸濁せしめ、さらにホルマリンを0.5％になる
ように加えて死菌懸濁液とした後、PBSを用
いてホルマリンを十分に除去し、遠心分離
（10000rpm、20分間）を行ない、死菌体を集め
そしてPBSに浮遊させて10mlとする〔試料
(2)〕。この試料(2)を鶏の肛門に１羽当り１滴
（約0.025ml）滴下する。
本発明(6)：本発明(5)で用いた試料(2)をPBSで４
倍に希釈し、鶏の肛門に１羽当り約0.1ml塗付
する。
本発明(7)：本発明(5)で用いた試料(2)をPBSで100
倍に希釈し、鶏の肛門に１羽当り１滴（約
0.025ml）滴下する。
本発明(8)：本発明(5)で用いた試料(2)をPBSで400
倍に希釈し、鶏の肛門に１羽当り約0.1ml塗付
する。
本発明(9)：大腸菌NS−244の1.2×1012個を
PBS2.5mlに浮遊させ、シヤーレ（Falcon製
1001型シヤーレ）に入れ、紫外線殺菌灯（東芝
殺菌ランプ GL15）21.5cm下に置き、30分毎
に撹拌を４回行ない、さらに静置状態で16時間
照射し、不活化乾燥した菌体に2.5mlになるよ
うにPBSを加えて死菌体浮遊液〔試料(3)〕を
得る。この試料を鶏の肛門に１羽当り１滴（約
0.025ml）滴下する。
本発明(10)：本発明(9)で用いた試料(3)をPBSで４
倍に希釈し、鶏の肛門に１羽当り約0.1ml塗付
する。
本発明（11）：本発明(9)で用いた試料(3)をPBSで
100倍に希釈し、鶏の肛門に１羽当り１滴（約
0.025ml）滴下する。
本発明（12）：本発明(9)で用いた試料(3)をPBSで
400倍に希釈し、鶏の肛門に１羽当り約0.1ml塗
付する。
本発明（13）：大腸菌NS−244の1.2×1012個を
PBSで10mlになるように浮遊させ、試験管に
入れ、 60Coを線源としそして放射線照射（３
メガラツド）を行なつた〔試料(4)〕。この試料
(4)を鶏の肛門に１羽当り１滴（約0.025ml）滴
下する。
本発明（14）：本発明（13）で用いた試料(4)を
PBSで４倍に希釈し、鶏の肛門に１羽当り約
0.1ml塗付する。
本発明（15）：本発明（13）で用いた試料(4)を
PBSで100倍に希釈し、鶏の肛門に１羽当り１
滴（約0.025ml）滴下する。
本発明（16）：本発明（13）で用いた試料(4)を
PBSで400倍に希釈し、鶏の肛門に１羽当り約
0.1ml塗付する。
対照(1)：試料を接種せず、感染のみを行なう。
対照(2)：本発明(1)で用いた試料(1)を鶏の胸筋に同
様量注射する。
対照(3)：本発明(1)で用いた試料(1)を鶏に同様量経
口投与する。
対照(4)：本発明(5)で用いた試料(2)を鶏の胸筋に同
様量注射する。
対照(5)：本発明(5)で用いた試料(2)を鶏に同様量経
口投与する。
対照(6)：本発明(9)で用いた試料(3)を鶏の胸筋に同
様量注射する。
対照(7)：本発明(9)で用いた試料(3)を鶏に同様量経
口投与する。
対照(8)：本発明（13）で用いた試料(4)を鶏の胸筋
に同様量注射する。
対照(9)：本発明（13）で用いた試料(4)を鶏に同様
量経口投与する。
The present invention relates to a method for immunologically preventing colibacillosis in chickens. Colibacillosis in chickens is caused by infection with Escherichia coli having a specific serotype. The type of this disease is
These include Escherichia coli sepsis, arthritis, and hemorrhagic enteritis. Chickens infected with this substance develop air sacculitis.
Symptoms include pericarditis, arthritis, and diarrhea, which can lead to death in severe cases. In addition, this coliosis occurs collectively or sporadically, and is particularly common in broiler chickens, causing great damage. Drugs are administered to prevent such colibacillosis, but because of the emergence of many drug-resistant bacteria, it is not expected to be very effective. Furthermore, in the case of treatment, there are many problems in the selection of drugs, administration methods, labor, etc., and it is hoped that a drug with excellent preventive effects will emerge. The present inventors conducted research to solve the above-mentioned problem and found that coliosis can be prevented by inoculating E. coli through the cloaca of chickens. The E. coli of the present invention is isolated from animal organs or feces, and examples include E. coli O 1 , O 2
and Escherichia coli (E.coli) NS-244
5757). Escherichia coli NS-244 is a strain obtained from colonies that appear after streak culture of cow feces on DHL agar (manufactured by Eiken Chemical Co., Ltd.), which is a selective medium for Escherichia coli. The mycological properties of this substance will be described. 1 Morphological findings 1.0-4.0 x 0.4-0.7 μ Gram-negative rods Motile Does not form spores 2 Findings on culture medium Easily grows in meat extract medium Meat extract 1%, peptone 1% and sodium chloride 0.3% Good growth on agar medium containing Colonies are low convex, colorless, S-shaped, quite opaque, and round around 3. Biological properties Growth pH 3.0 - 10.0 (optimal pH 7.0) Growth temperature 20 - 40℃ (optimal temperature 37℃) Aerobic and facultative anaerobic 4 Non-acid-fast Catalase positive Oxidase negative No growth observed in KCN medium β-galactositade positive Gas production from glucose Indole formation No hydrogen sulfide generation No urease generation No ability to liquefy gelatin Phenylalanine deaminase negative Lysine decarboxylase positive Ornithine decarboxylase positive Arginine dihydrolase negative Glutamate decarboxylase positive MR reaction positive VP reaction negative Acetic acid as a single carbon source Ability to produce acid from carbohydrates Glucose positive Arabinose positive Cellobiose negative Lactose positive Maltose positive Raffinose negative Rhamnose positive White sugar negative Trehalose positive Inosite Negative Based on the above mycological properties, "Bergey's Manual of
As a result of examining the differences from known bacterial strains according to the classification criteria of "Determinative Bacteriology" 8th edition, the strain used in the present invention (NS-244) was identified.
was identified as Escherichia coli. As a result of examining the pathogenicity of E. coli NS-244 to chickens, it was confirmed that it was not pathogenic at all. In other words, live bacteria of E. coli NS-244 should be added to 1 per 10 chickens aged 3 weeks.
It was administered intravenously at 1.1×10 11 per bird. 7 days later,
The condition of the chickens was observed, and there were no deaths, and no signs of pericarditis or hepatic capsulitis, which are characteristic lesions of coliform bacteria, were observed. Escherichia coli NS-244 used in the present invention can be used in a live state, but it is easier to handle if it is inactivated. E. coli O 1 and O 2 used in the present invention are inactivated. E. coli NS-244 and E. coli O 1 ,
O 2 is best inactivated by ultraviolet irradiation, gamma ray irradiation, or formalin treatment, but phenol,
It can also be inactivated by treatment with acetone, alcohol, freeze-thaw, heat, ultrasound, or pressure. Furthermore, aluminum hydroxide gel or an adjuband (immunostimulant) such as Freund's complete adjuband may be added to this product. Furthermore, this can be mixed with immunogenic antigens of other diseases or non-pathogenic antigens and inoculated into the cloaca. The above-mentioned immunogenic antigens include chicken coccidia protozoa, vaccines such as Newcatus disease, and Haemophilus, which is the causative agent of infectious coryza.
paragallinarum, Mycoplisma gallisepticum, which is the causative agent of chicken mycoplasmosis, Clostridium
perfringens, Salmonella pullorum,
Bifidobacterium, Lactobacillus and E. coli
Other than O 1 , O 2 , and O 78 , examples include E. coli having other O antigens isolated from chickens suffering from colibacillosis, or nonpathogenic E. coli isolated from healthy chickens. Next, the E. coli is inoculated into chickens. Appropriate conditions for inoculation are anticoliosis agents in water, physiological saline,
Examples include those suspended in solutions such as PBS (phosphate buffer) and tissue culture medium, or those subjected to the above-mentioned inactivation treatment and also adjuvant treatment. This is inoculated into the cloaca of chickens. The specific method of inoculation is to suspend an anti-coliform agent in the above solution into the anus, cloaca or bursa of Fabricius of the chicken, and inject 0.002 ml per chicken.
Examples include methods of administering ml to 0.2 ml by dropping, injecting, applying, spraying, or dipping the anal area. The amount of inoculum of anti-coliosis agent is 1.0 per chicken.
×10 Use 5 or more. If the anti-coliosis agent of the present invention is used in the inoculation method of the present invention, coliosis can be almost completely prevented without causing side effects. Further, with conventional drug administration methods, drug-resistant E. coli often appears, but if the prevention method of the present invention is used, drug-resistant E. coli will not appear. Next, examples will be given to show the effects of the present invention. Example 1 A sample was inoculated into the anus of 10 0-day-old chickens (broiler breed Hubbard), and controls (2) to (9) were inoculated with the sample by each method. Then to these chickens
At 21 days of age, each bird is infected by intravenously inoculating 1.4×10 10 E. coli O 78 cells. Control (1) is one in which only infection was performed without inoculating the sample. 7 days after infection
Mortality rates were observed on the following day. The results are shown in Table 1. The types of samples and inoculation methods are shown below. The present invention (1): 4 × 10 11 pieces of E. coli NS-244 were added to PBS10
ml suspension [sample (1)] is placed into the anus of each chicken (approximately 0.025 ml). Present invention (2): Sample (1) used in present invention (1) was dissolved in PBS for 4 hours.
Dilute it twice and apply about 0.1ml per chicken to the anus. Present invention (3): Sample (1) used in present invention (1) was diluted with PBS at 100%
Dilute to 2 times, add 1 drop per chicken to the anus (approx.
0.025ml) dropwise. Present invention (4): Sample (1) used in present invention (1) was diluted with PBS for 400 min.
Dilute it twice and apply about 0.1ml per chicken to the anus. Present invention (5): Suspend 4 x 10 11 of E. coli NS-244 in PBS, add formalin to 0.5% to make a killed bacteria suspension, and then remove formalin sufficiently using PBS. Centrifuge (10,000 rpm, 20 minutes), collect dead bacteria, and suspend in PBS to make 10 ml [sample
(2)〕. Add 1 drop (approximately 0.025 ml) of this sample (2) to the anus of each chicken. Present invention (6): Sample (2) used in present invention (5) was dissolved in PBS for 4 hours.
Dilute it twice and apply about 0.1ml per chicken to the anus. Present invention (7): Sample (2) used in present invention (5) was diluted with PBS at 100%
Dilute to 2 times, add 1 drop per chicken to the anus (approx.
0.025ml) dropwise. Present invention (8): Sample (2) used in present invention (5) was diluted with PBS for 400 min.
Dilute it twice and apply about 0.1ml per chicken to the anus. Present invention (9): 1.2×10 12 pieces of E. coli NS-244
Suspend in 2.5 ml of PBS, and
1001 model), placed under an ultraviolet sterilizing lamp (Toshiba sterilizing lamp GL15) 21.5 cm, stirred 4 times every 30 minutes, and irradiated for 16 hours in a stationary state to inactivate and dry the bacterial cells. Add PBS to make a total of 1 ml to obtain a dead cell suspension [sample (3)]. Apply this sample to the anus of each chicken (approximately 1 drop per bird).
0.025ml) dropwise. Present invention (10): Sample (3) used in present invention (9) was dissolved in PBS for 4 hours.
Dilute it twice and apply about 0.1ml per chicken to the anus. Present invention (11): The sample (3) used in the present invention (9) was dissolved in PBS.
Dilute 100 times and apply 1 drop per chicken (approx.
0.025ml) dropwise. Present invention (12): The sample (3) used in the present invention (9) was dissolved in PBS.
Dilute 400 times and apply approximately 0.1ml per chicken to the anus. Invention (13): 1.2×10 12 pieces of E. coli NS-244
Suspend the volume in PBS to 10 ml, put it in a test tube, use 60 Co as a radiation source, and irradiate it (3
[Sample (4)] this sample
Add 1 drop (approximately 0.025ml) of (4) to each chicken's anus. Present invention (14): Sample (4) used in present invention (13)
Dilute it 4 times with PBS and apply it to the anus of each chicken.
Apply 0.1ml. Present invention (15): Sample (4) used in the present invention (13)
Dilute 100 times with PBS and apply 1 ml per chicken anus.
Add a drop (approximately 0.025ml). Present invention (16): Sample (4) used in present invention (13)
Dilute it 400 times with PBS and apply it to the anus of the chicken.
Apply 0.1ml. Control (1): Only infection is performed without inoculating the sample. Control (2): The same amount of sample (1) used in the present invention (1) is injected into the pectoral muscle of chickens. Control (3): Sample (1) used in the present invention (1) is orally administered to chickens in the same amount. Control (4): Inject the same amount of sample (2) used in the present invention (5) into the breast muscle of chickens. Control (5): Sample (2) used in the present invention (5) is orally administered in the same amount to chickens. Control (6): Inject the same amount of sample (3) used in the present invention (9) into the breast muscle of chickens. Control (7): Sample (3) used in the present invention (9) is orally administered to chickens in the same amount. Control (8): Inject the same amount of sample (4) used in the present invention (13) into the breast muscle of chickens. Control (9): Sample (4) used in the present invention (13) is orally administered in the same amount to chickens.
【表】【table】
【表】
実施例 ２
０日令の鶏（ブロイラー専用種ハバード）10羽
の肛門に試料を接種し、また対照２〜19及び対照
21〜38は試料をそれぞれの方法で接種した。対照
１、20は試料は接種しない。次いでこれらの鶏に
21日令において、本発明１〜27の鶏と対照１〜19
の鶏は大腸菌O2を１羽当り5.0×108個静脈内接種
し、本発明28〜54の鶏と対照20〜38の鶏は大腸菌
O78を１羽当り1.4×1010個静脈内接種して感染さ
せる。感染後７日目に斃死率について観察した。
結果は表２に示す。試料の種類および接種方法に
ついては下記に示す。
本発明(1)：大腸菌NS−244の1.2×1012個および
大腸菌O2の1.0×1012個をPBSに懸濁せしめ、
さらにホルマリンを0.5％になるように加え、
死菌懸濁液とした後、PBSを用いてホルマリ
ンを十分に除去し遠心分離（10000rpm、20分
間）を行ない、死菌体を集めそしてPBSに浮
遊させて10mlとする〔試料(1)〕。この試料(1)を
鶏の肛門に１羽当り１滴（約0.025ml）滴下す
る。
本発明(2)：本発明(1)で用いた試料(1)を鶏の総排泄
腔に同様量注入する。
本発明(3)：本発明(1)で用いた試料(1)をPBSで４
倍に希釈し、鶏の肛門に１羽当り約0.1mlスプ
レーする。
本発明(4)：本発明(1)で用いた試料(1)をPBSで100
倍に希釈し、鶏の肛門に１羽当り１滴（約
0.025ml）滴下する。
本発明(5)：本発明(1)で用いた試料(1)をPBSで100
倍に希釈し、鶏の総排泄腔に同様量注入する。
本発明(6)：本発明(1)で用いた試料(1)をPBSで400
倍に希釈し、鶏の肛門に１羽当り約0.1mlスプ
レーする。
本発明(7)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、鶏の肛門に１羽当り１滴
（約0.025ml）滴下する。
本発明(8)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、鶏の総排泄腔に同様量注入
する。
本発明(9)：本発明(1)で用いた試料(1)をPBSで
40000倍に希釈し、鶏の肛門に１羽当り約0.1ml
スプレーする。
本発明(10)：大腸菌NS−244の1.2×1012個および
大腸菌O2の1.0×1012個をPBS2.5mlに混合して
浮遊させ、シヤーレ（Falon製1001型シヤー
レ）に入れ、紫外線殺菌灯（東芝殺菌ランプ
GL 15）21.5cm下に置き、30分毎に撹拌を４回
行ない、さらに静置状態で16時間照射し、不活
化乾燥した菌体に2.5mlになるようにPBSを加
え死菌体浮遊液〔試料(2)〕を得る。この試料を
鶏の肛門に１羽当り１滴（約0.025ml）滴下す
る。
本発明（11）：本発明(10)で用いた試料(2)を鶏の総
排泄腔に同様量注入する。
本発明（12）：本発明(10)で用いた試料(2)をPBSで
４倍に希釈し、鶏の肛門に１羽当り約0.1mlス
プレーする。
本発明（13）：本発明(10)で用いた試料(2)をPBSで
100倍に希釈し、鶏の肛門に１羽当り１滴（約
0.025ml）滴下する。
本発明（14）：本発明(10)で用いた試料(2)をPBSで
100倍に希釈し、鶏の総排泄腔に同様量注入す
る。
本発明（15）：本発明(10)で用いた試料(2)をPBSで
400倍に希釈し、鶏の肛門に１羽当り約0.1mlス
プレーする。
本発明（16）：本発明(10)で用いた試料(2)をPBSで
10000倍に希釈し、鶏の肛門に１羽当り１滴
（約0.025ml）滴下する。
本発明（17）：本発明(10)で用いた試料(2)をPBSで
10000倍に希釈し、鶏の総排泄腔に同様量注入
する。
本発明（18）：本発明(10)で用いた試料(2)をPBSで
40000倍に希釈し、鶏の肛門に１羽当り約0.1ml
スプレーする。
本発明（19）：大腸菌NS−244の4.0×1011個およ
び大腸菌O2の1.0×1012個をPBSで10mlになる
ように混合浮遊させ、試験管に入れ、 60Coを
線源とし放射線照射（３メガラツド）を行なつ
た〔試料(3)〕。この試料(3)を鶏の肛門に１羽当
り１滴（約0.025ml）滴下する。
本発明（20）：本発明（19）で用いた試料(3)を鶏
の総排泄腔に同様量注入する。
本発明（21）：本発明（19）で用いた試料(3)を
PBSで４倍に希釈し、鶏の肛門に１羽当り約
0.1mlスプレーする。
本発明（22）：本発明（19）で用いた試料(3)を
PBSで100倍に希釈し、鶏の肛門に１羽当り１
滴（約0.025ml）滴下する。
本発明（23）：本発明（19）で用いた試料(3)を
PBSで100倍に希釈し、鶏の総排泄腔に同様量
注入する。
本発明（24）：本発明（19）で用いた試料(3)を
PBSで400倍に希釈し、鶏の肛門に１羽当り約
0.1mlスプレーする。
本発明（25）：本発明（19）で用いた試料(3)を
PBSで10000倍に希釈し、鶏の肛門に１羽当り
１滴（約0.025ml）滴下する。
本発明（26）：本発明（19）で用いた試料(3)を
PBSで10000倍に希釈し、鶏の総排泄腔に同様
量注入する。
本発明（27）：本発明（19）で用いた試料(3)を
PBSで40000倍に希釈し、鶏の肛門に１羽当り
約0.1mlスプレーする。
本発明（28）：本発明(1)と全く同様に行なう。
本発明（29）：本発明(2)と全く同様に行なう。
本発明（30）：本発明(3)と全く同様に行なう。
本発明（31）：本発明(4)と全く同様に行なう。
本発明（32）：本発明(5)と全く同様に行なう。
本発明（33）：本発明(6)と全く同様に行なう。
本発明（34）：本発明(7)と全く同様に行なう。
本発明（35）：本発明(8)と全く同様に行なう。
本発明（36）：本発明(9)と全く同様に行なう。
本発明（37）：本発明(10)と全く同様に行なう。
本発明（38）：本発明（11）と全く同様に行なう。
本発明（39）：本発明（12）と全く同様に行なう。
本発明（40）：本発明（13）と全く同様に行なう。
本発明（41）：本発明（14）と全く同様に行なう。
本発明（42）：本発明（15）と全く同様に行なう。
本発明（43）：本発明（16）と全く同様に行なう。
本発明（44）：本発明（17）と全く同様に行なう。
本発明（45）：本発明（18）と全く同様に行なう。
本発明（46）：本発明（19）と全く同様に行なう。
本発明（47）：本発明（20）と全く同様に行なう。
本発明（48）：本発明（21）と全く同様に行なう。
本発明（49）：本発明（22）と全く同様に行なう。
本発明（50）：本発明（23）と全く同様に行なう。
本発明（51）：本発明（24）と全く同様に行なう。
本発明（52）：本発明（25）と全く同様に行なう。
本発明（53）：本発明（26）と全く同様に行なう。
本発明（54）：本発明（27）と全く同様に行なう。
対照(1)：試料を接種せず、感染のみを行なう。
対照(2)：本発明(1)で用いた試料を鶏の胸筋に同様
量注射する。
対照(3)：本発明(1)で用いた試料を鶏に同様量経口
投与する。
対照(4)：本発明(4)で用いた試料を鶏の胸筋に同様
量注射する。
対照(5)：本発明(4)で用いた試料を鶏に同様量経口
投与する。
対照(6)：本発明(7)で用いた試料を鶏の胸筋に同様
量注射する。
対照(7)：本発明(7)で用いた試料を鶏に同様量経口
投与する。
対照(8)：本発明(10)で用いた試料を鶏の胸筋に同様
量注射する。
対照(9)：本発明(10)で用いた試料を鶏に同様量経口
投与する。
対照(10)：本発明（13）で用いた試料を鶏の胸筋に
同様量注射する。
対照（11）：本発明（13）で用いた試料を鶏に同
様量経口投与する。
対照（12）：本発明（16）で用いた試料を鶏の胸
筋に同様量注射する。
対照（13）：本発明（16）で用いた試料を鶏に同
様量経口投与する。
対照（14）：本発明（19）で用いた試料を鶏の胸
筋に同様量注射する。
対照（15）：本発明（19）で用いた試料を鶏に同
様量経口投与する。
対照（16）：本発明（22）で用いた試料を鶏の胸
筋に同様量注射する。
対照（17）：本発明（22）で用いた試料を鶏に同
様量経口投与する。
対照（18）：本発明（25）で用いた試料を鶏の胸
筋に同様量注射する。
対照（19）：本発明（25）で用いた試料を鶏に同
様量経口投与する。
対照（20）：試料を接種せず、感染のみを行なう。
対照（21）：本発明(1)で用いた試料を鶏の胸筋に
同様量注射する。
対照（22）：本発明(1)で用いた試料を鶏に同様量
経口投与する。
対照（23）：本発明(4)で用いた試料を鶏の胸筋に
同様量注射する。
対照（24）：本発明(4)で用いた試料を鶏に同様量
経口投与する。
対照（25）：本発明(7)で用いた試料を鶏の胸筋に
同様量注射する。
対照（26）：本発明(7)で用いた試料を鶏に同様量
経口投与する。
対照（27）：本発明(10)で用いた試料を鶏の胸筋に
同様量注射する。
対照（28）：本発明(10)で用いた試料を鶏に同様量
経口投与する。
対照（29）：本発明（13）で用いた試料を鶏の胸
筋に同様量注射する。
対照（30）：本発明（13）で用いた試料を鶏に同
様量経口投与する。
対照（31）：本発明（16）で用いた試料を鶏の胸
筋に同様量注射する。
対照（32）：本発明（16）で用いた試料を鶏に同
様量経口投与する。
対照（33）：本発明（19）で用いた試料を鶏の胸
筋に同様量注射する。
対照（34）：本発明（19）で用いた試料を鶏に同
様量経口投与する。
対照（35）：本発明（22）で用いた試料を鶏の胸
筋に同様量注射する。
対照（36）：本発明（22）で用いた試料を鶏に同
様量経口投与する。
対照（37）：本発明（25）で用いた試料を鶏の胸
筋に同様量注射する。
対照（38）：本発明（25）で用いた試料を鶏に同
様量経口投与する。[Table] Example 2 Samples were inoculated into the anus of 10 0-day-old chickens (broiler breed Hubbard), and controls 2 to 19 and controls
Nos. 21 to 38 were inoculated with samples using their respective methods. Controls 1 and 20 are not inoculated with the sample. Then these chickens
At 21 days old, chickens of the invention 1 to 27 and controls 1 to 19
The chickens were intravenously inoculated with 5.0×10 8 E. coli O2 per bird, and the chickens of the present invention 28-54 and the control chickens 20-38 were inoculated with E. coli O2.
Each bird is infected by intravenously inoculating 1.4 x 10 10 O 78 . Mortality rate was observed on the 7th day after infection.
The results are shown in Table 2. The types of samples and inoculation methods are shown below. Present invention (1): 1.2×10 12 E. coli NS-244 and 1.0×10 12 E. coli O 2 were suspended in PBS,
Furthermore, formalin was added to 0.5%,
After making a suspension of killed bacteria, remove formalin thoroughly using PBS, perform centrifugation (10,000 rpm, 20 minutes), collect dead bacteria, and suspend in PBS to make 10 ml [Sample (1)] . Add 1 drop (approximately 0.025 ml) of this sample (1) to the anus of each chicken. Present invention (2): Inject the same amount of the sample (1) used in the present invention (1) into the cloaca of chickens. Present invention (3): Sample (1) used in present invention (1) was dissolved in PBS for 4 hours.
Dilute it twice and spray about 0.1ml per chicken into the anus. Present invention (4): Sample (1) used in present invention (1) was diluted with PBS at 100%
Dilute to 2 times, add 1 drop per chicken to the anus (approx.
0.025ml) dropwise. Present invention (5): Sample (1) used in present invention (1) was diluted with PBS at 100%
Dilute it twice and inject the same amount into the cloaca of chickens. Present invention (6): Sample (1) used in present invention (1) was diluted with PBS for 400 min.
Dilute it twice and spray about 0.1ml per chicken into the anus. Present invention (7): The sample (1) used in the present invention (1) was dissolved in PBS.
Dilute it 10,000 times and apply one drop (approximately 0.025ml) per chicken into the anus. Present invention (8): The sample (1) used in the present invention (1) was dissolved in PBS.
Dilute it 10,000 times and inject the same amount into the cloaca of chickens. Present invention (9): The sample (1) used in the present invention (1) was dissolved in PBS.
Dilute 40,000 times and apply approximately 0.1ml per chicken anus.
Spray. Present invention (10): 1.2 × 10 12 pieces of E. coli NS-244 and 1.0 × 10 12 pieces of E. coli O 2 were mixed and suspended in 2.5 ml of PBS, placed in a shear dish (Falon model 1001 shear dish), and sterilized with ultraviolet light. Light (Toshiba Germicidal Lamp)
GL 15) Place the cell under 21.5 cm, stir 4 times every 30 minutes, and irradiate for 16 hours in a still state. Add PBS to 2.5 ml of the inactivated and dried cells to make a dead cell suspension. Obtain [sample (2)]. Add one drop (approximately 0.025 ml) of this sample to each chicken's anus. Present invention (11): Inject the same amount of sample (2) used in present invention (10) into the cloaca of chickens. Present invention (12): Dilute sample (2) used in present invention (10) 4 times with PBS and spray about 0.1 ml per chicken into the anus. Present invention (13): The sample (2) used in the present invention (10) was dissolved in PBS.
Dilute 100 times and apply 1 drop per chicken (approx.
0.025ml) dropwise. Present invention (14): Sample (2) used in the present invention (10) was dissolved in PBS.
Dilute it 100 times and inject the same amount into the cloaca of chickens. Present invention (15): Sample (2) used in the present invention (10) was dissolved in PBS.
Dilute it 400 times and spray about 0.1ml per chicken into the anus. Present invention (16): Sample (2) used in the present invention (10) was dissolved in PBS.
Dilute it 10,000 times and apply one drop (approximately 0.025ml) per chicken into the anus. Present invention (17): The sample (2) used in the present invention (10) was dissolved in PBS.
Dilute it 10,000 times and inject the same amount into the cloaca of chickens. Present invention (18): Sample (2) used in the present invention (10) was dissolved in PBS.
Dilute 40,000 times and apply approximately 0.1ml per chicken anus.
Spray. Present invention (19): 4.0 x 10 11 pieces of E. coli NS-244 and 1.0 x 10 12 pieces of E. coli O 2 were mixed and suspended in PBS to a volume of 10 ml, placed in a test tube, and exposed to radiation using 60 Co as a radiation source. Irradiation (3 megarads) was performed [sample (3)]. Add 1 drop (approximately 0.025 ml) of this sample (3) to the anus of each chicken. Present invention (20): Inject the same amount of the sample (3) used in the present invention (19) into the cloaca of chickens. Present invention (21): Sample (3) used in the present invention (19)
Dilute it 4 times with PBS and apply it to the anus of each chicken.
Spray 0.1ml. Present invention (22): Sample (3) used in the present invention (19)
Dilute 100 times with PBS and apply 1 ml per chicken anus.
Add a drop (approximately 0.025ml). Present invention (23): Sample (3) used in the present invention (19)
Dilute 100 times with PBS and inject the same amount into the cloaca of chickens. Present invention (24): Sample (3) used in the present invention (19)
Dilute it 400 times with PBS and apply it to the anus of the chicken.Approx.
Spray 0.1ml. Present invention (25): Sample (3) used in the present invention (19)
Dilute it 10,000 times with PBS and apply 1 drop (approximately 0.025 ml) per chicken to the anus. Present invention (26): Sample (3) used in the present invention (19)
Dilute it 10,000 times with PBS and inject the same amount into the cloaca of chickens. Present invention (27): Sample (3) used in the present invention (19)
Dilute it 40,000 times with PBS and spray about 0.1ml per chicken into the anus. Invention (28): Performed in exactly the same manner as Invention (1). Invention (29): Performed in exactly the same manner as Invention (2). Invention (30): Performed in exactly the same manner as Invention (3). Present invention (31): Performed in exactly the same manner as present invention (4). Invention (32): Performed in exactly the same manner as Invention (5). Invention (33): Performed in exactly the same manner as Invention (6). Present invention (34): Performed in exactly the same manner as present invention (7). Invention (35): Performed in exactly the same manner as Invention (8). Invention (36): Performed in exactly the same manner as Invention (9). Invention (37): Performed in exactly the same manner as Invention (10). Present invention (38): Performed in exactly the same manner as the present invention (11). Invention (39): Performed in exactly the same manner as Invention (12). Invention (40): Performed in exactly the same manner as Invention (13). Present invention (41): Performed in exactly the same manner as the present invention (14). Present invention (42): Performed in exactly the same manner as the present invention (15). Present invention (43): Performed in exactly the same manner as the present invention (16). Present invention (44): Performed in exactly the same manner as the present invention (17). Present invention (45): Performed in exactly the same manner as the present invention (18). Present invention (46): Performed in exactly the same manner as the present invention (19). Present invention (47): Performed in exactly the same manner as the present invention (20). Present invention (48): Performed in exactly the same manner as the present invention (21). Present invention (49): Performed in exactly the same manner as the present invention (22). Present invention (50): Performed in exactly the same manner as the present invention (23). Present invention (51): Performed in exactly the same manner as the present invention (24). Present invention (52): Performed in exactly the same manner as the present invention (25). Present invention (53): Performed in exactly the same manner as the present invention (26). Present invention (54): Performed in exactly the same manner as the present invention (27). Control (1): Only infection is performed without inoculating the sample. Control (2): Inject the same amount of the sample used in the present invention (1) into the pectoral muscle of chickens. Control (3): The same amount of the sample used in the present invention (1) is orally administered to chickens. Control (4): The same amount of the sample used in the present invention (4) is injected into the pectoral muscle of chickens. Control (5): The same amount of the sample used in the present invention (4) is orally administered to chickens. Control (6): Inject the same amount of the sample used in the present invention (7) into the breast muscle of chickens. Control (7): The same amount of the sample used in the present invention (7) is orally administered to chickens. Control (8): Inject the same amount of the sample used in the present invention (10) into the breast muscle of chickens. Control (9): The same amount of the sample used in the present invention (10) is orally administered to chickens. Control (10): Inject the same amount of the sample used in the present invention (13) into the breast muscle of chickens. Control (11): The same amount of the sample used in the present invention (13) is orally administered to chickens. Control (12): Inject the same amount of the sample used in the present invention (16) into the breast muscle of chickens. Control (13): The same amount of the sample used in the present invention (16) is orally administered to chickens. Control (14): Inject the same amount of the sample used in the present invention (19) into the breast muscle of chickens. Control (15): The same amount of the sample used in the present invention (19) is orally administered to chickens. Control (16): Inject the same amount of the sample used in the present invention (22) into the breast muscle of chickens. Control (17): The same amount of the sample used in the present invention (22) is orally administered to chickens. Control (18): Inject the same amount of the sample used in the present invention (25) into the breast muscle of chickens. Control (19): The same amount of the sample used in the present invention (25) is orally administered to chickens. Control (20): No sample is inoculated, only infection is performed. Control (21): Inject the same amount of the sample used in the present invention (1) into the pectoral muscle of chickens. Control (22): The same amount of the sample used in the present invention (1) is orally administered to chickens. Control (23): Inject the same amount of the sample used in the present invention (4) into the breast muscle of chickens. Control (24): The same amount of the sample used in the present invention (4) is orally administered to chickens. Control (25): Inject the same amount of the sample used in the present invention (7) into the breast muscle of chickens. Control (26): The same amount of the sample used in the present invention (7) is orally administered to chickens. Control (27): Inject the same amount of the sample used in the present invention (10) into the breast muscle of chickens. Control (28): The same amount of the sample used in the present invention (10) is orally administered to chickens. Control (29): Inject the same amount of the sample used in the present invention (13) into the breast muscle of chickens. Control (30): The same amount of the sample used in the present invention (13) is orally administered to chickens. Control (31): Inject the same amount of the sample used in the present invention (16) into the breast muscle of chickens. Control (32): The same amount of the sample used in the present invention (16) is orally administered to chickens. Control (33): Inject the same amount of the sample used in the present invention (19) into the breast muscle of chickens. Control (34): The same amount of the sample used in the present invention (19) is orally administered to chickens. Control (35): Inject the same amount of the sample used in the present invention (22) into the pectoral muscle of chickens. Control (36): The same amount of the sample used in the present invention (22) is orally administered to chickens. Control (37): Inject the same amount of the sample used in the present invention (25) into the breast muscle of chickens. Control (38): The same amount of the sample used in the present invention (25) is orally administered to chickens.
【表】【table】
【表】【table】
【表】
実施例 ３
孵化後72時間以内の鶏（ブロイラー専用種ハバ
ード）10羽の肛門に試料を接種し、また対照(2)〜
(10)及び対照（12）〜（20）及び対照（22）〜
（30）は試料をそれぞれの方法で接種した。対照
(1)，（11），（21）は試料を接種しない。次いでこ
れらの鶏に21日令において、本発明(1)〜(9)と対照
(1)〜(10)の鶏は大腸菌O1を１羽当り1.2×109個静脈
内接種し、本発明(10)〜（18）と対照（11）〜
（20）の鶏は大腸菌O2を１羽当り5.0×108個静脈
内接種し、そして本発明（19）〜（27）と対照
（21）〜（30）の鶏は大腸菌O78を１羽当り1.4×
1010個静脈内接種して感染させる。感染後７日目
に斃死率について観察した。結果は表３に示す。
試料の種類、接種方法については下記に示す。
本発明(1)：大腸菌NS−244の3.6×1012個、大腸
菌O1の3.0×1012個および大腸菌O2の3.0×1012
個をPBSに混合懸濁せしめ、さらにホルマリ
ンを0.5％になるように加え死菌懸濁液とした
後、PBSを用いてホルマリンを十分に除去し、
遠心分離（10000rpm、20分間）を行ない、死
菌体を集めそしてPBSに浮遊させて10mlとす
る〔試料(1)〕。この試料(1)に鶏の肛門を１羽当
り約0.075mlとなるように浸す。
本発明(2)：本発明(1)で用いた試料(1)をPBSで100
倍に希釈したもので、鶏の肛門を同様量になる
ように浸す。
本発明(3)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈したもので、鶏の肛門を同様量
になるように浸す。
本発明(4)：大腸菌NS−244の3.0×1012個、大腸
菌O1の3.0×1012個および大腸菌O2の3.0×1012
個をPBS2.5mlに混合して浮遊させ、シヤーレ
（Falon製1001型シヤーレ）に入れ、紫外線殺
菌灯（東芝殺菌ランプGL15）21.5cm下に置き、
30分毎に撹拌を４回行ない、さらに静置状態で
16時間照射し、かくして不活化乾燥した菌体に
2.5mlになるようにPBSを加えて死菌体浮遊液
〔試料(2)〕を得る。この試料(2)に鶏の肛門を１
羽当り0.075mlになるように浸す。
本発明(5)：本発明４(4)用いた試料(2)をPBSで100
倍に希釈したもので、鶏の肛門を同様量になる
ように浸す。
本発明(6)：本発明(4)で用いた試料(2)をPBSで
10000倍に希釈したもので、鶏の肛門を同様量
になるように浸す。
本発明(7)：大腸菌NS−244の3.6×1012個、大腸
菌O1の3.0×1012個および大腸菌O2の3.0×1012
個をPBS10mlに浮遊させ、試験管に入れ、
60Coを線源とし放射線照射（３メガラツド）を
行なつた〔試料(3)〕。この試料(3)に鶏の肛門を
１羽当り0.075mlになるように浸す。
本発明(8)：本発明(7)で用いた試料(3)をPBSで100
倍に希釈したものに、鶏の肛門を同様量になる
ように浸す。
本発明(9)：本発明(7)で用いた試料(3)をPBSで
10000倍に希釈したものに、鶏の肛門を同様量
になるように浸す。
本発明(10)：本発明(1)と全く同様に行なう。
本発明（11）：本発明(2)と全く同様に行なう。
本発明（12）：本発明(3)と全く同様に行なう。
本発明（13）：本発明(4)と全く同様に行なう。
本発明（14）：本発明(5)と全く同様に行なう。
本発明（15）：本発明(6)と全く同様に行なう。
本発明（16）：本発明(7)と全く同様に行なう。
本発明（17）：本発明(8)と全く同様に行なう。
本発明（18）：本発明(9)と全く同様に行なう。
本発明（19）：本発明(1)と全く同様に行なう。
本発明（20）：本発明(2)と全く同様に行なう。
本発明（21）：本発明(3)と全く同様に行なう。
本発明（22）：本発明(4)と全く同様に行なう。
本発明（23）：本発明(5)と全く同様に行なう。
本発明（24）：本発明(6)と全く同様に行なう。
本発明（25）：本発明(7)と全く同様に行なう。
本発明（26）：本発明(8)と全く同様に行なう。
本発明（27）：本発明(9)と全く同様に行なう。
対照(1)：試料を接種せず、感染のみを行なう。
対照(2)：本発明(1)で用いた試料を鶏の胸筋に同様
量注射する。
対照(3)：本発明(2)で用いた試料を鶏の胸筋に同様
量注射する。
対照(4)：本発明(3)で用いた試料を鶏の胸筋に同様
量注射する。
対照(5)：本発明(4)で用いた試料を鶏の胸筋に同様
量注射する。
対照(6)：本発明(5)で用いた試料を鶏の胸筋に同様
量注射する。
対照(7)：本発明(6)で用いた試料を鶏の胸筋に同様
量注射する。
対照(8)：本発明(7)で用いた試料を鶏の胸筋に同様
量注射する。
対照(9)：本発明(8)で用いた試料を鶏の胸筋に同様
量注射する。
対照(10)：本発明(9)で用いた試料を鶏の胸筋に同様
量注射する。
対照（11）：試料を接種せず、感染のみを行なう。
対照（12）：本発明(10)で用いた試料を鶏の胸筋に
同様量注射する。
対照（13）：本発明（11）で用いた試料を鶏の胸
筋に同様量注射する。
対照（14）：本発明（12）で用いた試料を鶏の胸
筋に同様量注射する。
対照（15）：本発明（13）で用いた試料を鶏の胸
筋に同様量注射する。
対照（16）：本発明（14）で用いた試料を鶏の胸
筋に同様量注射する。
対照（17）：本発明（15）で用いた試料を鶏の胸
筋に同様量注射する。
対照（18）：本発明（16）で用いた試料を鶏の胸
筋に同様量注射する。
対照（19）：本発明（17）で用いた試料を鶏の胸
筋に同様量注射する。
対照（20）：本発明（18）で用いた試料を鶏の胸
筋に同様量注射する。
対照（21）：試料を接種せず、感染のみを行なう。
対照（22）：本発明（19）で用いた試料を鶏の胸
筋に同様量注射する。
対照（23）：本発明（20）で用いた試料を鶏の胸
筋に同様量注射する。
対照（24）：本発明（21）で用いた試料を鶏の胸
筋に同様量注射する。
対照（25）：本発明（22）で用いた試料を鶏の胸
筋に同様量注射する。
対照（26）：本発明（23）で用いた試料を鶏の胸
筋に同様量注射する。
対照（27）：本発明（24）で用いた試料を鶏の胸
筋に同様量注射する。
対照（28）：本発明（25）で用いた試料を鶏の胸
筋に同様量注射する。
対照（29）：本発明（26）で用いた試料を鶏の胸
筋に同様量注射する。
対照（30）：本発明（27）で用いた試料を鶏の胸
筋に同様量注射する。[Table] Example 3 Samples were inoculated into the anus of 10 chickens (broiler breed Hubbard) within 72 hours after hatching, and controls (2) to
(10) and control (12) to (20) and control (22) to
(30) inoculated the samples using their respective methods. contrast
(1), (11), and (21) do not inoculate the sample. Then, at 21 days old, these chickens were given the present invention (1) to (9) and a control.
The chickens of (1) to (10) were intravenously inoculated with 1.2 x 109 E. coli O1 per bird, and the present invention (10) to (18) and the control (11) to
The chickens in (20) were intravenously inoculated with 5.0 × 10 8 E. coli O2 per bird, and the chickens of the present invention (19) to (27) and controls (21) to (30) were inoculated with 1 E. coli O 78 per bird. 1.4× per wing
10 Infect by intravenously inoculating 10 cells. Mortality rate was observed on the 7th day after infection. The results are shown in Table 3.
The types of samples and inoculation methods are shown below. Present invention (1): 3.6×10 12 of E. coli NS-244, 3.0×10 12 of E. coli O 1 and 3.0×10 12 of E. coli O 2
After mixing and suspending the cells in PBS and adding formalin to 0.5% to make a killed bacteria suspension, formalin was sufficiently removed using PBS.
Perform centrifugation (10,000 rpm, 20 minutes), collect dead bacteria, and suspend in PBS to make 10 ml [sample (1)]. Dip the anus of a chicken into this sample (1) to a volume of approximately 0.075ml per bird. Present invention (2): Sample (1) used in present invention (1) was diluted with PBS at 100%
Dip the anus of the chicken in the same amount of diluted solution. Present invention (3): Sample (1) used in present invention (1) was dissolved in PBS.
Dilute the solution 10,000 times and soak the chicken's anus in the same amount. Present invention (4): 3.0×10 12 of E. coli NS-244, 3.0×10 12 of E. coli O 1 and 3.0×10 12 of E. coli O 2
Mix the cells with 2.5 ml of PBS, suspend them, put them in a shear dish (Falon model 1001 shear dish), place them under an ultraviolet germicidal lamp (Toshiba germicidal lamp GL15) 21.5 cm,
Stir 4 times every 30 minutes, then leave to stand still.
Irradiated for 16 hours, thus inactivating and drying the bacterial cells.
Add PBS to 2.5 ml to obtain a dead cell suspension [sample (2)]. Add one chicken anus to this sample (2).
Soak so that each wing is 0.075ml. Invention (5): Sample (2) used in Invention 4 (4) was added to PBS at 100%
Dip the anus of the chicken in the same amount of diluted solution. Present invention (6): Sample (2) used in present invention (4) was dissolved in PBS.
Dilute the solution 10,000 times and soak the chicken's anus in the same amount. Present invention (7): 3.6×10 12 of E. coli NS-244, 3.0×10 12 of E. coli O 1 and 3.0×10 12 of E. coli O 2
Suspend the cells in 10ml of PBS and put them in a test tube.
Irradiation was performed using 60 Co as a radiation source (3 megarads) [Sample (3)]. Dip the anus of a chicken into this sample (3) to a volume of 0.075ml per bird. Present invention (8): Sample (3) used in present invention (7) was diluted with PBS at 100%
Soak the anus of a chicken in the same amount of diluted solution. Present invention (9): Sample (3) used in present invention (7) was dissolved in PBS.
Soak the anus of a chicken in the same amount of diluted 10,000 times. Invention (10): Performed in exactly the same manner as Invention (1). Invention (11): Performed in exactly the same manner as Invention (2). Invention (12): Performed in exactly the same manner as Invention (3). Invention (13): Performed in exactly the same manner as Invention (4). Invention (14): Performed in exactly the same manner as Invention (5). Invention (15): Performed in exactly the same manner as Invention (6). Present invention (16): Performed in exactly the same manner as present invention (7). Invention (17): Performed in exactly the same manner as Invention (8). Invention (18): Performed in exactly the same manner as Invention (9). Invention (19): Performed in exactly the same manner as Invention (1). Invention (20): Performed in exactly the same manner as Invention (2). Invention (21): Performed in exactly the same manner as Invention (3). Invention (22): Performed in exactly the same manner as Invention (4). Invention (23): Performed in exactly the same manner as Invention (5). Invention (24): Performed in exactly the same manner as Invention (6). Invention (25): Performed in exactly the same manner as Invention (7). Invention (26): Performed in exactly the same manner as Invention (8). Present invention (27): Performed in exactly the same manner as the present invention (9). Control (1): Only infection is performed without inoculating the sample. Control (2): Inject the same amount of the sample used in the present invention (1) into the pectoral muscle of chickens. Control (3): Inject the same amount of the sample used in the present invention (2) into the pectoral muscle of chickens. Control (4): The same amount of the sample used in the present invention (3) is injected into the breast muscle of chickens. Control (5): Inject the same amount of the sample used in the present invention (4) into the breast muscle of chickens. Control (6): Inject the same amount of the sample used in the present invention (5) into the pectoral muscle of chickens. Control (7): Inject the same amount of the sample used in the present invention (6) into the pectoral muscle of chickens. Control (8): Inject the same amount of the sample used in the present invention (7) into the breast muscle of chickens. Control (9): Inject the same amount of the sample used in the present invention (8) into the pectoral muscle of chickens. Control (10): Inject the same amount of the sample used in the present invention (9) into the breast muscle of chickens. Control (11): No sample is inoculated, only infection is performed. Control (12): Inject the same amount of the sample used in the present invention (10) into the breast muscle of chickens. Control (13): Inject the same amount of the sample used in the present invention (11) into the breast muscle of chickens. Control (14): Inject the same amount of the sample used in the present invention (12) into the breast muscle of chickens. Control (15): Inject the same amount of the sample used in the present invention (13) into the breast muscle of chickens. Control (16): Inject the same amount of the sample used in the present invention (14) into the breast muscle of chickens. Control (17): Inject the same amount of the sample used in the present invention (15) into the breast muscle of chickens. Control (18): Inject the same amount of the sample used in the present invention (16) into the breast muscle of chickens. Control (19): Inject the same amount of the sample used in the present invention (17) into the breast muscle of chickens. Control (20): Inject the same amount of the sample used in the present invention (18) into the breast muscle of chickens. Control (21): Only infection is performed without inoculating the sample. Control (22): Inject the same amount of the sample used in the present invention (19) into the breast muscle of chickens. Control (23): Inject the same amount of the sample used in the present invention (20) into the breast muscle of chickens. Control (24): Inject the same amount of the sample used in the present invention (21) into the breast muscle of chickens. Control (25): Inject the same amount of the sample used in the present invention (22) into the breast muscle of chickens. Control (26): Inject the same amount of the sample used in the present invention (23) into the breast muscle of chickens. Control (27): Inject the same amount of the sample used in the present invention (24) into the breast muscle of chickens. Control (28): Inject the same amount of the sample used in the present invention (25) into the breast muscle of chickens. Control (29): Inject the same amount of the sample used in the present invention (26) into the pectoral muscle of chickens. Control (30): Inject the same amount of the sample used in the present invention (27) into the breast muscle of chickens.
【表】【table】
【表】
実施例 ４
孵化後72時間以内の鶏（ブロイラー専用種ハバ
ード）10羽の肛門に下記に示す試料を１羽当り１
滴（約0.025ml）の量で滴下した。
試料(1)：ニユーカツスル病生ワクチン液30ml
（1000ドーズ）に実施例１の本発明(1)で用いた
試料(1)10mlを懸濁せしめたもの。
試料(2)：アイメリア・テネラのスポロゾイト
4000000個とアイメリア・ブルネツテイのスポ
ロゾイト4000000個とを実施例１の本発明(1)で
用いた試料(1)10mlに懸濁せしめたもの。
試料(3)：Bifidobacterium、Lactobacillusおよび
非病原性大腸菌の各１×1011個をPBS10mlに混
合懸濁せしめ、さらに実施例３の本発明(1)で用
いた試料(1)10mlを加えて懸濁混合せしめたも
の。
試料(4)：Clostridium perfringensおよび
Salmonella pullorumのそれぞれ１×1011個を
PBSに懸濁せしめ、さらにホルマリンを0.5％
になるように加え、死菌懸濁液とした後、ホル
マリンを十分に除去し、遠心分離（10000rpm、
20分間）を行ない、死菌体を集めてPBSに浮
遊させたものに実施例３の本発明(1)で用いた試
料(1)を10ml懸濁混合せしめたもの。
試料(5)：Haemophilue paragallinarumおよび
Mrcoplasma gallisepticumのそれぞれ１×
1010個をPBS2.5mlに浮遊させ、シヤーレ
（Falcon製1001型シヤーレ）に入れ、紫外線殺
菌灯（東芝殺菌ランプGL15）21.5cm下に置き、
30分毎に撹拌を４回行ない、さらに静置状態で
16時間照射し、その後2.5mlになるようにPBS
を加えて死菌体浮遊液を得、このものに実施例
３の本発明(4)で用いた試料(2)を2.5ml加えて懸
濁混合せしめたもの。
試料(1)、(2)、(3)、(4)および(5)に用いたニユーカ
ツスル病生ワクチン、アイメリア・テネラのスポ
ロゾイト、アイメリア・ブルネツテイのスポロゾ
イト、Bifidobacterium、Lactobacillus、非病原
性大腸菌、Clostridium、perfringens、
Salmonella pullorum、Haemophilus
paragallinarum、Mycoplasma gallisepticumの
調製法は下記のとおりである。
試料(1)に用いたニユーカツスル病生ワクチン液
は化学及び血清療法研究所製のニユーカツスル病
生ワクチンの溶解用液30ml中に乾燥予防液を1000
ドーズ分になるように溶解して調製した。
試料(2)に用いたアイメリア・テネラのスポロゾ
イトの調製法は次のとおりである。アイメリア・
テネラの成熟オーシストを経口投与した鶏の糞、
盲腸壁および盲腸内容物を集め、３％重クロム酸
カリウム溶液に浮遊せしめそして28℃において２
日間培養した。得られた成熟オーシストを含む溶
液を遠心分離（3000rpm、10分間）しそして沈澱
を集める。次いでこの沈澱を水で洗浄し且つ飽和
食塩水を添加する。この溶液を遠心分離
（3000rpm、10分間）し、上澄をとり、この上澄
を水で希釈し、希釈液を更に遠心分離
（1000rpm、３分間）しそして沈澱を集める。こ
の沈澱はほとんどオーシストのみよりなる。前記
オーシストに水および５％の次亜鉛素酸ナトリウ
ム液を加え且つ15分間放置した後、遠心分離
（2000rpm、３分間）を行う。得られた沈澱を水
で洗浄し、次いでホモジナイザーで磨砕する。こ
の磨砕物はほとんどスポロシストよりなる。この
スポロシストに５％鶏胆汁および0.25％トリプシ
ンPBS（−）溶液を加えそして40℃の温浴中でお
よび1.5時間消化せしめる。得られた消化液を遠
心分離して沈澱を集め、そしてこの沈澱をPBS
（−）溶液で洗浄する。このものはほとんどスポ
ロゾイトよりなる。これにPBS（−）溶液を加え
た。
試料(2)で用いたアイメリア・ブルネツテイのス
ポロゾイトは上記のアイメリア・テネラのスポロ
ゾイト調製法に準じて行なう。ただしアイメリ
ア・テネラの代わりに、アイメリア・ブルネツテ
イを使用する。
試料(3)に用いたBifidobateriumは鶏の腸管か
ら分離し、アスコルビン酸ソーダとＬ−システイ
ン塩酸塩加ブレイン・ハートインフイユージヨン
ブロス（Difco Laboratory社製）を用いて37℃
で20時間培養した後、10000rpmにおいて20分間
遠心分離を行つてBifidobacteriumの湿菌体を得
る。
試料(3)に用いたLactobacillusは試料(3)に用い
たBifidobacteriumの調製法と同様に行なつた。
試料(3)に用いた非病原性大腸菌は健康な鶏の腸
管より分離し、ハート・インフユージヨンブロス
（Difco Laboratory社製）を用いて37℃で18時間
培養した後、10000rpmにおいて20分間遠心分離
を行なつて非病原性大腸菌の湿菌体を得る。
試料(4)に用いたClostreidium perfringensは試
料(3)に用いたBifidobacteriumの調製法と同様に
行なつた。
試料(4)に用いたSalmonella pullorumは雛白痢
に罹患した鶏の糞便より分離し、ハート・インフ
イユージヨンブイヨン（Difco Laboratory社製）
を用いて37℃で18時間培養した後、10000rpmに
おいて20分間遠心分離を行なつてSalmonella
pullorumの湿菌体を得る。
試料(5)に用いたHaemophilus
pararagllinarumは伝染性コリーザに罹患した鶏
の鼻腔より分離し、チヨコレート寒天培地〔日清
化学(株)製クリメデイア〕で画線培養し、増殖した
コロニーをコンラージ棒を用いてかき取つた。
試料(5)に用いたMycoplasma gallisepticum
は、鶏呼吸器性マイコプラズマ病に罹患した鶏の
気嚢より分離した。分離方法は「Bull.Nat.Inst.
Anim.Hlth.」No.53（1966年８月）第10頁に準じて
行なつた。すなわち、気嚢の内側を滅菌綿棒でぬ
ぐい、それを0.5％イーストエクストラクト
（Difco社製）加燐酸緩衝食塩液１mlに振り出し、
この培養材料の１白金耳を寒天平板培地に塗抹す
るとともに0.1mlを２mlを液体培地に移植した。
平板培地は乾燥を防ぐ目的で水でしめらした脱脂
綿を入れたデシケーター中に収め、液体培地はそ
のままそれぞれ37℃で培養した。平板培地は６日
後に50倍の拡大で集落の有無を観察した。液体培
地は10日間観察し、その間培地の黄変したものは
その都度平板培地に塗抹培養して集落の有無を検
査した。ここで使用した液体培地は、鶏１羽分の
もも肉、胸肉、心、肝を細坐し、２倍量の蒸溜水
を加えて１夜永室に静置し、その後100℃に30分
加熱し、過後NaClを0.5％、鶏血液を５％そし
て10％NaClを１％加え、100℃30分加熱し、PH
7.8に修正後過および過滅菌を行なつた液
(1)に、非働性馬血清20％、10％ブドウ糖１％、１
％フエノールレツド0.2％、５％酢酸タリウム１
％およびペニシリン1000μ／mlを加えたものを使
用した。また、ここで使用した寒天平板培地は、
液体培地作成時に使用した液１を約60℃に加熱
し、15％寒天液の加熱溶解したもの1/10量を加え
て短時間加熱して完全に溶解させ、50℃に冷却
し、非働性馬血清20％、５％酢酸タリウム１％お
よびペニシリン1000U／mlを加えて寒天培地とし
た。分離したMycoplasma gallisepticumは５ml
PPLO増殖培地（栄研）を用いて37℃において３
日間培養し、次に100mlのPPLO培地（栄研）に
植え次いで37℃で３日間培養し、次に3000mlの
PPLO増殖培地に植え、次いで37℃で５日間培養
し、次に20000Gで30分間遠心分離して集収した。[Table] Example 4 One sample per chicken was placed in the anus of 10 chickens (broiler breed Hubbard) within 72 hours after hatching.
It was added dropwise (approximately 0.025 ml). Sample (1): New Katsuru disease live vaccine solution 30ml
(1000 doses) in which 10 ml of the sample (1) used in the present invention (1) of Example 1 was suspended. Sample (2): Eimeria tenella sporozoites
4,000,000 sporozoites and 4,000,000 sporozoites of Eimeria brunetsutei were suspended in 10 ml of sample (1) used in the present invention (1) of Example 1. Sample (3): Mix and suspend 1 x 10 each of Bifidobacterium, Lactobacillus and non-pathogenic E. coli in 10 ml of PBS, and then add 10 ml of sample (1) used in the present invention (1) of Example 3 and suspend. A cloudy mixture. Sample (4): Clostridium perfringens and
1 x 10 pieces each of Salmonella pullorum
Suspend in PBS and add 0.5% formalin.
After making a dead bacteria suspension, remove formalin thoroughly and centrifuge (10,000 rpm,
20 minutes), the dead bacteria were collected and suspended in PBS, and 10 ml of the sample (1) used in the present invention (1) of Example 3 was suspended and mixed. Sample (5): Haemophilue paragallinarum and
1x each of Mrcoplasma gallisepticum
10 10 pieces were suspended in 2.5 ml of PBS, placed in a shear dish (Falcon model 1001 shear dish), and placed under an ultraviolet germicidal lamp (Toshiba germicidal lamp GL15) 21.5 cm.
Stir 4 times every 30 minutes, then leave to stand still.
Irradiate for 16 hours, then add PBS to 2.5 ml
was added to obtain a suspension of dead bacterial cells, and 2.5 ml of sample (2) used in the present invention (4) of Example 3 was added to this suspension and mixed. Live Eucatus disease vaccine used for samples (1), (2), (3), (4) and (5), Eimeria tenella sporozoites, Eimeria brunetsutei sporozoites, Bifidobacterium, Lactobacillus, non-pathogenic Escherichia coli, Clostridium ,perfringens,
Salmonella pullorum, Haemophilus
The preparation method for paragallinarum and Mycoplasma gallisepticum is as follows. The live Newatus disease vaccine solution used for sample (1) was prepared by adding 1,000 ml of the dry preventive solution to 30 ml of the Newatus disease live vaccine dissolution solution manufactured by the Chemical and Serum Therapy Research Institute.
It was prepared by dissolving it to the desired dose. The method for preparing Eimeria tenella sporozoites used for sample (2) is as follows. Eimeria・
Feces from chickens to which mature tenella oocysts were orally administered;
The cecal wall and cecal contents were collected, suspended in a 3% potassium dichromate solution and incubated at 28°C for 2 hours.
Cultured for 1 day. The resulting solution containing mature oocysts is centrifuged (3000 rpm, 10 minutes) and the precipitate is collected. The precipitate is then washed with water and saturated brine is added. The solution is centrifuged (3000 rpm, 10 minutes), the supernatant is removed, the supernatant is diluted with water, the diluted solution is further centrifuged (1000 rpm, 3 minutes), and the precipitate is collected. This precipitate consists almost exclusively of oocysts. Water and 5% sodium hypozinc chlorate solution are added to the oocysts, and the mixture is left to stand for 15 minutes, followed by centrifugation (2000 rpm, 3 minutes). The precipitate obtained is washed with water and then ground in a homogenizer. This ground material consists mostly of sporocysts. The sporocysts are added with 5% chicken bile and 0.25% trypsin in PBS (-) and digested in a 40°C bath for 1.5 hours. The resulting digestive fluid was centrifuged to collect the precipitate, and this precipitate was added to PBS.
(-) Wash with solution. This substance consists mostly of sporozoites. A PBS(-) solution was added to this. The sporozoites of Eimeria brunettii used in sample (2) are prepared according to the above-mentioned method for preparing sporozoites of Eimeria tenella. However, instead of Eimeria tenera, use Eimeria Brunetstei. The Bifidobaterium used for sample (3) was isolated from the intestinal tract of chickens and incubated at 37°C using brain heart infusion broth (manufactured by Difco Laboratory) containing sodium ascorbic acid and L-cysteine hydrochloride.
After culturing for 20 hours, centrifugation is performed at 10,000 rpm for 20 minutes to obtain wet Bifidobacterium cells. Lactobacillus used in sample (3) was prepared in the same manner as Bifidobacterium used in sample (3). The non-pathogenic E. coli used in sample (3) was isolated from the intestinal tract of a healthy chicken, cultured at 37°C for 18 hours using heart infusion broth (manufactured by Difco Laboratory), and then centrifuged at 10,000 rpm for 20 minutes. Separation is performed to obtain wet cells of non-pathogenic E. coli. Clostreidium perfringens used in sample (4) was prepared in the same manner as Bifidobacterium used in sample (3). Salmonella pullorum used for sample (4) was isolated from the feces of a chicken suffering from chick dysentery, and was added to Heart Infection Broth (manufactured by Difco Laboratory).
After culturing at 37°C for 18 hours using
Obtain wet bacterial cells of pullorum. Haemophilus used for sample (5)
pararagllinarum was isolated from the nasal cavity of a chicken infected with infectious coryza, streak cultured on a tyokolate agar medium (Climedeia, manufactured by Nissin Chemical Co., Ltd.), and the proliferated colonies were scraped off using a Conlage rod. Mycoplasma gallisepticum used for sample (5)
was isolated from the air sac of a chicken infected with avian respiratory mycoplasmosis. The separation method is “Bull.Nat.Inst.
Anim.Hlth.'' No. 53 (August 1966), page 10. That is, wipe the inside of the air sac with a sterile cotton swab, shake it out in 1 ml of 0.5% yeast extract (manufactured by Difco) phosphate buffered saline,
One platinum loopful of this culture material was smeared onto an agar plate medium, and 0.1 ml and 2 ml were transferred to a liquid medium.
The plate culture medium was placed in a desiccator containing absorbent cotton moistened with water to prevent it from drying out, and the liquid culture medium was cultured as it was at 37°C. The plate culture medium was observed for the presence or absence of colonies under 50x magnification after 6 days. The liquid culture medium was observed for 10 days, during which time any yellowed medium was cultured by smearing on a plate medium to examine the presence or absence of colonies. The liquid medium used here was prepared by placing the thigh, breast, heart, and liver of one chicken in a bowl, adding twice the amount of distilled water, and leaving it in a room overnight, then heating it to 100℃ for 30 minutes. After heating, add 0.5% NaCl, 5% chicken blood and 1% NaCl, heat at 100℃ for 30 minutes, and adjust the pH.
7.8 Liquid that has been subjected to filtration and oversterilization after modification
(1), non-working horse serum 20%, 10% glucose 1%, 1
% phenol red 0.2%, 5% thallium acetate 1
% and penicillin 1000μ/ml were used. In addition, the agar plate medium used here was
Heat Solution 1 used to create the liquid medium to about 60℃, add 1/10 amount of 15% agar solution heated and dissolved, heat for a short time to dissolve completely, cool to 50℃, and inactivate. 20% horse serum, 5% thallium acetate 1%, and 1000 U/ml penicillin were added to prepare an agar medium. 5ml of isolated Mycoplasma gallisepticum
3 at 37℃ using PPLO growth medium (Eiken)
Cultured for 1 day, then planted in 100 ml of PPLO medium (Eiken), cultured at 37℃ for 3 days, then 3000 ml of culture medium (Eiken).
They were plated in PPLO growth medium, then cultured at 37°C for 5 days, and then harvested by centrifugation at 20000G for 30 minutes.
Claims (1)
分とする抗大腸菌症剤を鶏の総排泄腔に接種する
ことを特徴とする鶏の大腸菌症の予防法。 ２ 大腸菌NS−244の生菌または死菌体と、大腸
菌O1あるいはO2あるいはO1とO2の死菌体の組み
合わせからなる抗大腸菌症剤を鶏の総排泄腔に接
種することを特徴とする鶏の大腸菌症の予防法。 ３ 大腸菌の不活化がガンマー線照射、紫外線照
射またはホルマリンでの処理でなされたものであ
る特許請求の範囲第１項または第２項記載の大腸
菌症の予防法。 ４ 抗大腸菌症剤の接種菌量が１羽当り1.0×105
個以上である特許請求の範囲第１項または第２項
記載の大腸菌症の予防法。 ５ 抗大腸菌症剤の接種方法が滴下、注入、スプ
レー、浸漬または塗付によりなされる特許請求の
範囲第１項または第２項記載の大腸菌症の予防
法。[Scope of Claims] 1. A method for preventing coliosis in chickens, which comprises inoculating the cloaca of chickens with an anti-coliosis agent containing live or killed Escherichia coli NS-244 as an active ingredient. 2. An anti-coliosis agent consisting of a combination of live or dead Escherichia coli NS-244 and dead Escherichia coli O 1 or O 2 or O 1 and O 2 is inoculated into the cloaca of chickens. A method for preventing coliosis in chickens. 3. The method for preventing coliosis according to claim 1 or 2, wherein E. coli is inactivated by gamma irradiation, ultraviolet irradiation, or treatment with formalin. 4 The amount of inoculum of anti-coliosis agent is 1.0×10 5 per bird.
The method for preventing coliosis according to claim 1 or 2, wherein the amount of coliform bacteria is 1 or more. 5. The method for preventing coliosis according to claim 1 or 2, wherein the method of inoculating the anti-coliosis agent is by dropping, injecting, spraying, dipping or painting.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1815182A JPS58135819A (en) | 1982-02-09 | 1982-02-09 | Prevention of colibacillosis of poultry |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1815182A JPS58135819A (en) | 1982-02-09 | 1982-02-09 | Prevention of colibacillosis of poultry |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58135819A JPS58135819A (en) | 1983-08-12 |
| JPH0315607B2 true JPH0315607B2 (en) | 1991-03-01 |
Family
ID=11963607
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1815182A Granted JPS58135819A (en) | 1982-02-09 | 1982-02-09 | Prevention of colibacillosis of poultry |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58135819A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69016129T2 (en) * | 1989-08-18 | 1995-05-24 | Akzo Nobel Nv | Vaccine against Escherichia coli. |
-
1982
- 1982-02-09 JP JP1815182A patent/JPS58135819A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58135819A (en) | 1983-08-12 |
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