JPH0222734B2 - - Google Patents
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- Publication number
- JPH0222734B2 JPH0222734B2 JP57011763A JP1176382A JPH0222734B2 JP H0222734 B2 JPH0222734 B2 JP H0222734B2 JP 57011763 A JP57011763 A JP 57011763A JP 1176382 A JP1176382 A JP 1176382A JP H0222734 B2 JPH0222734 B2 JP H0222734B2
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- 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
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- 239000007787 solid Substances 0.000 description 2
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- 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
- 241000271566 Aves Species 0.000 description 1
- 241001260012 Bursa Species 0.000 description 1
- 241000224483 Coccidia Species 0.000 description 1
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- 208000004232 Enteritis Diseases 0.000 description 1
- 241000059485 Escherichia coli O78 Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
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- 102000004142 Trypsin Human genes 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
- 229940024546 aluminum hydroxide gel Drugs 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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- 229910052708 sodium Inorganic materials 0.000 description 1
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- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
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Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (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、肝包膜炎、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、大腸菌O1、
O2およびO78の死菌体を活性成分とする抗大腸菌
症剤やその他の薬剤を鶏に投与することも知られ
ているが、従来は該抗大腸菌症剤やその他の薬剤
は胸筋等の筋肉や皮下等への注射または経口投与
によつて専ら投与されていた。しかしながら、か
かる従来の投与方法による場合には、大腸菌症の
予防効果が充分ではなかつた。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、大腸菌の死菌体を有効成分と
する抗大腸菌症剤を液状にし、これを鶏の総排泄
腔に投与することにより大腸菌症が極めて効果的
に予防できることを見い出した。
本発明では抗大腸菌症剤用の大腸菌として、動
物の臓器または糞便から分離したものを用い、例
としては大腸菌O1、O2およびO78が挙げられる。
本発明で用いる大腸菌はガンマー線照射、紫外
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
ることも妨たげない。また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、鶏のコクシジウム原虫、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Mycoplasma
gallisepticum、Clostridium perfringens、
Salmonella pullorum、Bifidobacterium、
Lactobacillusおよび大腸菌O1、O2、O78以外で
鶏の大腸菌症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。
次いで上記のようにして不活化した大腸菌また
はそれと上記したアジユバンド等の他の成分との
混合物を液体状態にして鶏の総排泄腔に投与す
る。鶏では、人間等とは異なり、排便、排尿、産
卵および精子の排泄が直腸のすぐ下に位置する１
つの排泄腔を経て行われるため、人間でいえば肛
門に相当する部分を、通常、総排泄腔と称してお
り、本発明における総排泄腔もかかる鶏における
総排泄腔をいう。投与に適当な液体状態として
は、不活化した大腸菌の死菌体を必要に応じてア
ジユバンド等のその他の成分と共に水、生理食塩
水、リン酸バツフアー（PBS）、組織倍養液等の
液体に分散または懸濁させたものを挙げることが
できる。そしてこの液状の抗大腸菌症剤を鶏の総
排泄腔に投与すると、抗大腸菌症剤が該総排泄腔
の接近して鶏の背側に位置する鶏のフアブリシウ
ス嚢内に浸透または吸収されて鶏の大腸菌症を極
めて効果的に予防することができる。投与の具体
的な方法としては、該液状の抗大腸菌症剤を鶏の
総排泄腔に滴下、注入、スプレーまたは塗布する
か、あるいは鶏の総排泄腔を該液状の抗大腸菌症
剤に浸す方法が挙げられる。その際には、鶏１羽
当たり大腸菌の死菌体の数で1.0×105個以上にな
るように、そして抗大腸菌症剤の液量では１羽当
たり0.002〜0.2mlになるようにして鶏の総排泄腔
に投与するのがよい。
大腸菌症の予防という点からは、本発明におけ
るような抗大腸菌症剤は、成長前の雛に投与され
ることが多いが、雛の総排泄腔は極めて小さく、
抗大腸菌症剤を座薬等の固形状にして雛の小さな
総排泄腔に挿入することは極めて困難である。し
たがつて、抗大腸菌症剤を固形状にして雛の総排
泄腔に挿入投与することは、多数の雛に対して抗
大腸菌症剤を順次投与していかなければならない
養鶏家等にとつて実質上不可能に近い。これに対
して、上記したように、本発明では液状の抗大腸
菌症剤を鶏（特に雛）の総排泄腔に単に滴下、注
入、スプレー、塗布等の方法または鶏（雛）の総
排泄腔を液状抗大腸菌症剤に浸すことによつてそ
の総排泄腔に投与することができるので、投与が
極めて簡単であり、未経験者でも容易に行うこと
ができる。
本発明に係る抗大腸菌症剤を本発明の接種方法
で行なえ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)〜（19）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O1を１羽当り1.2×109
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日目に斃死率および病変値について観察した。
結果は表１に示す。なお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O1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
ホルマリンを充分に除去し、遠心分離
（10000r.p.m20分間）を行ない、死菌体を集め、
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)に同様量鶏の
肛門を浸す。
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塗付す
る。
本発明(4)：大腸菌O1の2.5×1011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
えて死菌体浮遊液を得る〔試料(2)〕。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(5)：本発明(4)で用いた試料(2)に同様量鶏の
肛門を浸す。
本発明(6)：本発明(4)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)：大腸菌O1の1.0×1012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、放射線照射（３メガラツド）を行な
う〔試料(3)〕。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(8)：本発明(7)で用いた試料(3)に同様量、鶏
の肛門を浸す。
本発明(9)：本発明(7)で用いた試料(3)をPBSで４
倍に希釈し、鶏の肛門に約0.1ml塗付する。
本発明(10)：本発明(1)で用いた試料(1)をPBSで100
倍に希釈し、接種方法は本発明(1)と同様とし
た。
本発明(11)：本発明(1)で用いた試料(1)をPBSで100
倍に希釈し、接種方法は本発明(2)と同様とし
た。
本発明(12)：本発明(1)で用いた試料(1)をPBSで400
倍に希釈し、接種方法は本発明(3)と同様とし
た。
本発明(13)：本発明(4)で用いた試料(2)をPBSで
100倍に希釈し、接種方法は本発明(1)と同様と
した。
本発明(14)：本発明(4)で用いた試料(2)をPBSで
100倍に希釈し、接種方法は本発明(2)と同様と
した。
本発明(15)：本発明(4)で用いた試料(2)をPBSで
400倍に希釈し、接種方法は本発明(3)と同様と
した。
本発明(16)：本発明(7)で用いた試料(3)をPBSで
100倍に希釈し、接種方法は本発明(1)と同様と
した。
本発明(17)：本発明(7)で用いた試料(3)をPBSで
100倍に希釈し、接種方法は本発明(2)と同様と
した。
本発明(18)：本発明(7)で用いた試料(3)をPBSで
400倍に希釈し、接種方法は本発明(3)と同様と
した。
本発明(19)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、接種方法は本発明(1)と同様
とした。
本発明(20)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、接種方法は本発明(2)と同様
とした。
本発明(21)：本発明(1)で用いた試料(1)をPBSで
40000倍に希釈し、接種方法は本発明(3)と同様
とした。
本発明(22)：本発明(4)で用いた試料(2)をPBSで
10000倍に希釈し、接種方法は本発明(1)と同様
とした。
本発明(23)：本発明(4)で用いた試料(2)をPBSで
10000倍に希釈し、接種方法は本発明(2)と同様
とした。
本発明(24)：本発明(4)で用いた試料(2)をPBSで
40000倍に希釈し、接種方法は本発明(3)と同様
とした。
本発明(25)：本発明(7)で用いた試料(3)をPBSで
10000倍に希釈し、接種方法は本発明(1)と同様
とした。
本発明(26)：本発明(7)で用いた試料(3)をPBSで
10000倍に希釈し、接種方法は本発明(2)と同様
とした。
本発明(27)：本発明(7)で用いた試料(3)をPBSで
40000倍に希釈し、接種方法は本発明(3)と同様
とした。
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(4)で用いた試料(2)を鶏の胸筋に同
様量注射する。
対照(4)：本発明(7)で用いた試料(3)を鶏の胸筋に同
様量注射する。
対照(5)：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(6)：本発明(4)で用いた試料(2)を鶏に同様量経
口投与する。
対照(7)：本発明(7)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(1)で用いた試料(1)をPBSで100倍
に希釈し、接種方法は対照(2)と同様とした。
対照(9)：本発明(4)で用いた試料(2)をPBSで100倍
に希釈し、接種方法は対照(2)と同様とした。
対照(10)：本発明(7)で用いた試料(3)をPBSで100倍
に希釈し、接種方法は対照(2)と同様とした。
対照(11)：本発明(1)で用いた試料(1)をPBSで100倍
に希釈し、接種方法は対照(5)と同様とした。
対照(12)：本発明(4)で用いた試料(2)をPBSで100倍
に希釈し、接種方法は対照(5)と同様とした。
対照(13)：本発明(7)で用いた試料(3)をPBSで100
倍に希釈し、接種方法は対照(5)と同様とした。
対照(14)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、接種方法は対照(2)と同様と
した。
対照(15)：本発明(4)で用いた試料(2)をPBSで
10000倍に希釈し、接種方法は対照(2)と同様と
した。
対照(16)：本発明(7)で用いた試料(3)をPBSで
10000倍に希釈し、接種方法は対照(2)と同様と
した。
対照(17)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、接種方法は対照(5)と同様と
した。
対照(18)：本発明(4)で用いた試料(2)をPBSで
10000倍に希釈し、接種方法は対照(5)と同様と
した。
対照(19)：本発明(7)で用いた試料(3)をPBSで
10000倍に希釈し、接種方法は対照(5)と同様と
した。
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, hepatic capsulitis, arthritis, and diarrhea, and in severe cases, death can occur. Furthermore, this colibacillosis occurs collectively or sporadically, and is particularly common in broilers, causing great damage. For the prevention of such colibacillosis, E. coli O 1 ,
It is also known that anti-coliosis agents and other drugs containing killed O 2 and O 78 cells as active ingredients are administered to chickens; It was administered exclusively by intramuscular or subcutaneous injection or oral administration. However, when using such conventional administration methods, the preventive effect against coliosis was not sufficient. In addition, in the case of treatment, there are many problems with drug selection, administration methods, labor, etc.
It is hoped that something with excellent preventive effects will emerge. Therefore, the present inventors conducted research to solve the above-mentioned problem, and found that an anti-coliosis agent containing killed E. coli as an active ingredient was made into a liquid form, and this was administered into the cloaca of chickens. It has been found that coliosis can be extremely effectively prevented. In the present invention, Escherichia coli isolated from animal organs or feces is used as the anti-coliosis agent, and examples thereof include Escherichia coli O 1 , O 2 and O 78 . Escherichia coli used in the present invention is preferably inactivated by gamma irradiation, ultraviolet irradiation, or formalin treatment, but it can also be inactivated by treatment with phenol, acetone, alcohol, freeze-thaw, heating, ultrasound, or pressure. . Furthermore, aluminum hydroxide gel or an adjuband (immunostimulant) such as Freund's complete adjuband may be added to this product. It is also possible to mix this with immunogenic antigens for other diseases or non-pathogenic antigens and inoculate the mixture into the cloaca. The above-mentioned immunogenic antigens include chicken coccidia protozoa, vaccines against Newcatus disease, and the causative bacteria of infectious coryza.
Haemophilus paragallinarum, Mycoplasma, the causative agent of chicken mycoplasmosis
gallisepticum, Clostridium perfringens,
Salmonella pullorum, Bifidobacterium,
Other than Lactobacillus and E. coli O 1 , O 2 , O 78 , other O-antigen-bearing E. coli isolated from colibacillosis of chickens or non-pathogenic E. coli isolated from healthy chickens may be mentioned. Next, the E. coli inactivated as described above or a mixture of it and other ingredients such as the above-described adjuvant is made into a liquid and administered to the cloaca of the chicken. In chickens, unlike humans, defecation, urination, egg laying, and sperm excretion are located just below the rectum.
Since the cloaca is carried out through two cloacae, the part corresponding to the anus in humans is usually called the cloaca, and the cloaca in the present invention also refers to the cloaca in chickens. In a liquid state suitable for administration, inactivated dead E. coli cells can be mixed with other ingredients such as adjuvant as necessary in a liquid such as water, physiological saline, phosphate buffer (PBS), or tissue culture solution. Dispersion or suspension can be mentioned. When this liquid anti-coliosis agent is administered into the chicken's cloaca, it penetrates or is absorbed into the chicken's bursa Fabricius, which is located close to the cloaca and on the dorsal side of the chicken. Coliosis can be prevented very effectively. Specific methods of administration include dropping, injecting, spraying or applying the liquid anti-coliosis agent into the cloaca of chickens, or immersing the cloaca of chickens in the liquid anti-coliosis agent. can be mentioned. At that time, the number of dead E. coli cells per chicken should be at least 1.0 It is best to administer it to the cloaca of the body. From the perspective of preventing coliosis, the anti-coliosis agent as in the present invention is often administered to pre-grown chicks, but the cloaca of chicks is extremely small;
It is extremely difficult to form an anti-coliosis agent into a solid form such as a suppository and insert it into the small cloaca of chicks. Therefore, administering the anti-coliosis agent in solid form by inserting it into the cloaca of the chicks is useful for poultry farmers who need to sequentially administer the anti-coliosis agent to a large number of chicks. Virtually impossible. In contrast, as described above, in the present invention, a liquid anti-coliosis agent is simply dropped, injected, sprayed, applied, etc. into the cloaca of chickens (particularly chicks) or Since the drug can be administered into the cloaca by soaking it in a liquid anti-coliosis agent, administration is extremely simple and can be easily performed even by an inexperienced person. If the anti-coliosis agent of the present invention is administered by the inoculation method of the present invention, coliosis can be almost completely prevented without causing any side effects. Furthermore, although drug-resistant E. coli often appears in conventional drug administration methods, drug-resistant E. coli will not appear if the prevention method of the present invention is used. Next, examples will be given to demonstrate 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 (19) were inoculated with the sample by each method. These chickens were then challenged with E. coli O 1 at 1.2 x 10 9 per bird at 21 days of age.
Infect by intravenous inoculation. Control (1) is one in which only infection was performed without inoculating the sample. Mortality rate and lesion values were observed on day 7 after infection.
The results are shown in Table 1. The types of samples and inoculation methods are shown below. Present invention (1): Suspend 1.0×10 12 of E. coli O 1 in PBS, add formalin to 0.5% to make a killed bacteria suspension, and then thoroughly remove formalin using PBS. Remove and centrifuge (10000r.p.m for 20 minutes) to collect dead bacteria.
Suspend in PBS to make 10 ml [Sample (1)]. Apply this sample (1) to the anus of the chicken, 1 drop (approximately 0.025ml) per chicken.
Drip. Present invention (2): Dip the anus of a chicken in the same amount as sample (1) used in present invention (1). Present invention (3): 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 (4): 2.5×10 11 pieces of Escherichia coli O 1 were suspended in 2.5 ml of PBS and placed in a shear dish (Falcon model 1001 shear dish) using an ultraviolet germicidal lamp (Toshiba germicidal lamp).
GL15) Place the cells under 21.5cm, stir 4 times every 30 minutes, and irradiate them for 16 hours. Add PBS to 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 (5): Dip the anus of a chicken in the same amount as sample (2) used in present invention (4). Present invention (6): Sample (2) used in present invention (4) was dissolved in PBS for 4 hours.
Dilute it twice and apply about 0.1ml per chicken to the anus. Present invention (7): 1.0 × 10 12 pieces of E. coli O 1 in 10 ml of PBS
Float the specimen so that it becomes 200 ml, put it in a test tube, and irradiate it with radiation (3 megarads) using 60 Co as a radiation source [Sample (3)]. Add one drop (approximately 0.025 ml) of this sample to each chicken's anus. Present invention (8): Dip the anus of a chicken in the same amount as the sample (3) used in present invention (7). Present invention (9): The sample (3) used in the present invention (7) was dissolved in PBS for 4 hours.
Dilute it twice and apply about 0.1ml to the chicken's anus. Present invention (10): Sample (1) used in present invention (1) was diluted with PBS at 100%
The inoculation method was the same as that of the present invention (1). Present invention (11): Sample (1) used in present invention (1) was diluted with PBS at 100%
The inoculation method was the same as that of the present invention (2). Present invention (12): Sample (1) used in present invention (1) was diluted with PBS for 400 min.
The inoculation method was the same as that of the present invention (3). Present invention (13): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 100 times and the inoculation method was the same as in the present invention (1). Present invention (14): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 100 times and the inoculation method was the same as in the present invention (2). Present invention (15): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 400 times and the inoculation method was the same as in the present invention (3). Present invention (16): Sample (3) used in present invention (7) was dissolved in PBS.
It was diluted 100 times and the inoculation method was the same as in the present invention (1). Present invention (17): Sample (3) used in present invention (7) was dissolved in PBS.
It was diluted 100 times and the inoculation method was the same as in the present invention (2). Present invention (18): The sample (3) used in the present invention (7) was dissolved in PBS.
It was diluted 400 times and the inoculation method was the same as in the present invention (3). Present invention (19): The sample (1) used in the present invention (1) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (1). Present invention (20): The sample (1) used in the present invention (1) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (2). Present invention (21): Sample (1) used in present invention (1) was dissolved in PBS.
It was diluted 40,000 times and the inoculation method was the same as in the present invention (3). Present invention (22): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (1). Present invention (23): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (2). Present invention (24): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 40,000 times and the inoculation method was the same as in the present invention (3). Present invention (25): Sample (3) used in present invention (7) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (1). Present invention (26): The sample (3) used in the present invention (7) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (2). Present invention (27): Sample (3) used in present invention (7) was dissolved in PBS.
It was diluted 40,000 times and the inoculation method was the same as in the present invention (3). Control (1): Only infection is performed without inoculating the sample. Control (2): Inject the same amount of sample (1) used in the present invention (1) into the breast muscle of chickens. Control (3): Inject the same amount of sample (2) used in the present invention (4) into the breast muscle of chickens. Control (4): Inject the same amount of sample (3) used in the present invention (7) into the breast muscle of chickens. Control (5): Sample (1) used in the present invention (1) is orally administered in the same amount to chickens. Control (6): Sample (2) used in the present invention (4) is orally administered in the same amount to chickens. Control (7): Sample (3) used in the present invention (7) is orally administered in the same amount to chickens. Control (8): Sample (1) used in the present invention (1) was diluted 100 times with PBS, and the inoculation method was the same as control (2). Control (9): Sample (2) used in the present invention (4) was diluted 100 times with PBS, and the inoculation method was the same as control (2). Control (10): Sample (3) used in the present invention (7) was diluted 100 times with PBS, and the inoculation method was the same as control (2). Control (11): Sample (1) used in the present invention (1) was diluted 100 times with PBS, and the inoculation method was the same as control (5). Control (12): Sample (2) used in the present invention (4) was diluted 100 times with PBS, and the inoculation method was the same as control (5). Control (13): Sample (3) used in the present invention (7) was diluted with PBS at 100%
The inoculation method was the same as control (5). Control (14): Sample (1) used in the present invention (1) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as control (2). Control (15): Sample (2) used in the present invention (4) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as control (2). Control (16): Sample (3) used in the present invention (7) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as control (2). Control (17): Sample (1) used in the present invention (1) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as the control (5). Control (18): Sample (2) used in the present invention (4) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as the control (5). Control (19): Sample (3) used in the present invention (7) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as the control (5).
【表】【table】
【表】
実施例 ２
孵化後72時間以内の鶏（ブロイラー専用種ハバ
ード）10羽に下記に示す試料を接種し、また対照
(2)〜（19）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O2
を１羽当り５×108個で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つた
ものである。感染後７日目に斃死率および病変値
について観察した。結果は表２に示す。なお、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
ルマリンを充分に除去し、遠心分離（10000r.
p.m.20分間）を行ない、死菌体を集め、PBS
に浮遊させ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)：大腸菌O2の2.5×1011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
えて死菌体浮遊液を得る〔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(5)：本発明(4)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(6)：本発明(4)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量で
スプレーする。
本発明(7)：大腸菌O2の1.0×1012個をPBSで10ml
になるように浮遊させ、試験管に入れ、60Coを
線源として放射線照射（３メガラツト）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（0.025ml）滴下する。
本発明(8)：本発明(7)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(9)：本発明(7)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量で
スプレーする。
本発明(10)：本発明(1)で用いた試料(1)をPBSで100
倍に希釈し、接種方法は本発明(1)と同様とし
た。
本発明(11)：本発明(1)で用いた試料(1)をPBSで100
倍に希釈し、接種方法は本発明(2)と同様とし
た。
本発明(12)：本発明(1)で用いた試料(1)をPBSで400
倍に希釈し、接種方法は本発明(3)と同様とし
た。
本発明(13)：本発明(4)で用いた試料(2)をPBSで
100倍に希釈し、接種方法は本発明(1)と同様と
した。
本発明(14)：本発明(4)で用いた試料(2)をPBSで
100倍に希釈し、接種方法は本発明(2)と同様と
した。
本発明(15)：本発明(4)で用いた試料(2)をPBSで
400倍に希釈し、接種方法は本発明(3)と同様と
した。
本発明(16)：本発明(7)で用いた試料(3)をPBSで
100倍に希釈し、接種方法は本発明(1)と同様と
した。
本発明(17)：本発明(7)で用いた試料(3)をPBSで
100倍に希釈し、接種方法は本発明(2)と同様と
した。
本発明(18)：本発明(7)で用いた試料(3)をPBSで
400倍に希釈し、接種方法は本発明(3)と同様と
した。
本発明(19)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、接種方法は本発明(1)と同様
とした。
本発明(20)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、接種方法は本発明(2)と同様
とした。
本発明(21)：本発明(1)で用いた試料(1)をPBSで
40000倍に希釈し、接種方法は本発明(3)と同様
とした。
本発明(22)：本発明(4)で用いた試料(2)をPBSで
10000倍に希釈し、接種方法は本発明(1)と同様
とした。
本発明(23)：本発明(4)で用いた試料(2)をPBSで
10000倍に希釈し、接種方法は本発明(2)と同様
とした。
本発明(24)：本発明(4)で用いた試料(2)をPBSで
40000倍に希釈し、接種方法は本発明(3)と同様
とした。
本発明(25)：本発明(7)で用いた試料(3)をPBSで
10000倍に希釈し、接種方法は本発明(1)と同様
とした。
本発明(26)：本発明(7)で用いた試料(3)をPBSで
10000倍に希釈し、接種方法は本発明(2)と同様
とした。
本発明(27)：本発明(7)で用いた試料(3)をPBSで
40000倍に希釈し、接種方法は本発明(3)と同様
とした。
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(4)で用いた試料(2)を鶏の胸筋に同
様量注射する。
対照(4)：本発明(7)で用いた試料(3)を鶏の胸筋に同
様量注射する。
対照(5)：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(6)：本発明(4)で用いた試料(2)を鶏に同様量経
口投与する。
対照(7)：本発明(7)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(1)で用いた試料(1)をPBSで100倍
に希釈し、接種方法は対照(2)と同様とした。
対照(9)：本発明(4)で用いた試料(2)をPBSで100倍
に希釈し、接種方法は対照(2)と同様とした。
対照(10)：本発明(7)で用いた試料(3)をPBSで100倍
に希釈し、接種方法は対照(2)と同様とした。
対照(11)：本発明(1)で用いた試料(1)をPBSで100倍
に希釈し、接種方法は対照(5)と同様とした。
対照(12)：本発明(4)で用いた試料(2)をPBSで100倍
に希釈し、接種方法は対照(5)と同様とした。
対照(13)：本発明(7)で用いた試料(3)をPBSで100
倍に希釈し、接種方法は対照(5)と同様とした。
対照(14)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、接種方法は対照(2)と同様と
した。
対照(15)：本発明(4)で用いた試料(2)をPBSで
10000倍に希釈し、接種方法は対照(2)と同様と
した。
対照(15)：本発明(7)で用いた試料(3)をPBSで
10000位に希釈し、接種方法は対照(2)と同様と
した。
対照(17)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、接種方法は対照(5)と同様と
した。
対照(18)：本発明(4)で用いた試料(2)をPBSで
10000倍に希釈し、接種方法は対照(5)と同様と
した。
対照(19)：本発明(7)で用いた試料(3)をPBSで
10000倍に希釈し、接種方法は対照(5)と同様と
した。[Table] Example 2 Ten chickens (broiler breed Hubbard) within 72 hours after hatching were inoculated with the samples shown below, and a control
Samples (2) to (19) were inoculated using the respective methods.
These chickens were then challenged with E. coli O2 at 21 days of age.
Infect by intravenously inoculating 5 x 10 8 cells per bird. Control (1) is one in which only infection was performed without inoculating the sample. Mortality rate and lesion values were observed on day 7 after infection. The results are shown in Table 2. The types of samples and inoculation methods are shown below. Present invention (1): Suspend 1.0×10 12 E. coli O 2 cells in PBS, add formalin to 0.5% to make a dead bacteria suspension, and then thoroughly remove formalin using PBS. and centrifugation (10000r.
pm for 20 minutes), collect the dead bacteria, and add PBS.
Suspend the sample to 10 ml [Sample (1)]. This sample (1)
Add 1 drop (approximately 0.025 ml) per chicken to the anus of the 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): 2.5×10 11 pieces of E. coli O2 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) Place the cells under 21.5cm, stir 4 times every 30 minutes, and irradiate them for 16 hours. Add PBS to the inactivated and dried cells to make a dead cell suspension. Obtain [Sample (2)]. this sample
Add 1 drop (approximately 0.025ml) of (2) to each chicken's anus. Present invention (5): Inject the same amount of sample (2) used in present invention (4) into the cloaca of chickens. Present invention (6): Sample (2) used in present invention (4) was dissolved in PBS for 4 hours.
Dilute it twice and spray it into the anus of chickens at an amount of about 0.1ml per chicken. Invention (7): 1.0×10 12 pieces of E. coli O2 in 10ml of PBS
It was suspended in a test tube, and irradiated with radiation (3 Megarats) using 60 Co as a radiation source [Sample (3)]. Add 1 drop (0.025 ml) of this sample (3) to the anus of each chicken. Present invention (8): Inject the same amount of sample (3) used in present invention (7) into the cloaca of chickens. Present invention (9): The sample (3) used in the present invention (7) was dissolved in PBS for 4 hours.
Dilute it twice and spray it into the anus of chickens at an amount of about 0.1ml per chicken. Present invention (10): Sample (1) used in present invention (1) was diluted with PBS at 100%
The inoculation method was the same as that of the present invention (1). Present invention (11): Sample (1) used in present invention (1) was diluted with PBS at 100%
The inoculation method was the same as that of the present invention (2). Present invention (12): Sample (1) used in present invention (1) was diluted with PBS for 400 min.
The inoculation method was the same as that of the present invention (3). Present invention (13): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 100 times and the inoculation method was the same as in the present invention (1). Present invention (14): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 100 times and the inoculation method was the same as in the present invention (2). Present invention (15): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 400 times and the inoculation method was the same as in the present invention (3). Present invention (16): Sample (3) used in present invention (7) was dissolved in PBS.
It was diluted 100 times and the inoculation method was the same as in the present invention (1). Present invention (17): Sample (3) used in present invention (7) was dissolved in PBS.
It was diluted 100 times and the inoculation method was the same as in the present invention (2). Present invention (18): The sample (3) used in the present invention (7) was dissolved in PBS.
It was diluted 400 times and the inoculation method was the same as in the present invention (3). Present invention (19): The sample (1) used in the present invention (1) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (1). Present invention (20): The sample (1) used in the present invention (1) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (2). Present invention (21): Sample (1) used in present invention (1) was dissolved in PBS.
It was diluted 40,000 times and the inoculation method was the same as in the present invention (3). Present invention (22): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (1). Present invention (23): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (2). Present invention (24): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 40,000 times and the inoculation method was the same as in the present invention (3). Present invention (25): Sample (3) used in present invention (7) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (1). Present invention (26): The sample (3) used in the present invention (7) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (2). Present invention (27): Sample (3) used in present invention (7) was dissolved in PBS.
It was diluted 40,000 times and the inoculation method was the same as in the present invention (3). Control (1): Only infection is performed without inoculating the sample. Control (2): Inject the same amount of sample (1) used in the present invention (1) into the breast muscle of chickens. Control (3): Inject the same amount of sample (2) used in the present invention (4) into the breast muscle of chickens. Control (4): Inject the same amount of sample (3) used in the present invention (7) into the breast muscle of chickens. Control (5): Sample (1) used in the present invention (1) is orally administered in the same amount to chickens. Control (6): Sample (2) used in the present invention (4) is orally administered in the same amount to chickens. Control (7): Sample (3) used in the present invention (7) is orally administered in the same amount to chickens. Control (8): Sample (1) used in the present invention (1) was diluted 100 times with PBS, and the inoculation method was the same as control (2). Control (9): Sample (2) used in the present invention (4) was diluted 100 times with PBS, and the inoculation method was the same as control (2). Control (10): Sample (3) used in the present invention (7) was diluted 100 times with PBS, and the inoculation method was the same as control (2). Control (11): Sample (1) used in the present invention (1) was diluted 100 times with PBS, and the inoculation method was the same as control (5). Control (12): Sample (2) used in the present invention (4) was diluted 100 times with PBS, and the inoculation method was the same as control (5). Control (13): Sample (3) used in the present invention (7) was diluted with PBS at 100%
The inoculation method was the same as control (5). Control (14): Sample (1) used in the present invention (1) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as control (2). Control (15): Sample (2) used in the present invention (4) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as control (2). Control (15): Sample (3) used in the present invention (7) was mixed with PBS.
It was diluted to 10,000, and the inoculation method was the same as control (2). Control (17): Sample (1) used in the present invention (1) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as the control (5). Control (18): Sample (2) used in the present invention (4) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as the control (5). Control (19): Sample (3) used in the present invention (7) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as the control (5).
【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羽に下記に示す試料を接種し、また対照
(2)〜（19）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個で静脈内接種した感染させ
る。対照(1)は試料を接種せずに感染のみを行つた
ものである。感染後７日目に斃死率および病変値
について観察した。結果は表３に示す。なお、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O78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
ルマリンを充分に除去し、遠心分離（10000r.
p.m.20分間）を行ない、死菌体を集めてPBS
に浮遊させ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)：大腸菌O78の2.5×1011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
えて死菌体浮遊液を得る〔試料(2)〕。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(5)：本発明(4)で用い試料(2)を鶏の総排泄腔
に同様量注入する。
本発明(6)：本発明(4)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(7)：大腸菌O78の1.0×1012個をPBSで10ml
になるように浮遊させ、試験管に入れ、60Coを
線源として放射線照射（３メガラツド）を行な
う〔試料(3)〕。この試料を鶏の肛門に１羽当り
１滴（0.025ml）滴下する。
本発明(8)：本発明(7)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(9)：本発明(7)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(10)：本発明(1)で用いた試料(1)をPBSで100
倍に希釈し、接種方法は本発明(1)と同様とし
た。
本発明(11)：本発明(1)で用いた試料(1)をPBSで100
倍に希釈し、接種方法は本発明(2)と同様とし
た。。
本発明(12)：本発明(1)で用いた試料(1)をPBSで400
倍に希釈し、接種方法は本発明(3)と同様とし
た。
本発明(13)：本発明(4)で用いた試料(2)をPBSで
100倍に希釈し、接種方法は本発明(1)と同様と
した。
本発明(14)：本発明(4)で用いた試料(2)をPBSで
100倍に希釈し、接種方法は本発明(2)と同様と
した。
本発明(15)：本発明(4)で用いた試料(2)をPBSで
400倍に希釈し、接種方法は本発明(3)と同様と
した。
本発明(16)：本発明(7)で用いた試料(3)をPBSで
100倍に希釈し、接種方法は本発明(1)と同様と
した。
本発明(17)：本発明(7)で用いた試料(3)をPBSで
100倍に希釈し、接種方法は本発明(2)と同様と
した。
本発明(18)：本発明(7)で用いた試料(3)をPBSで
400倍に希釈し、接種方法は本発明(3)と同様と
した。
本発明(19)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、接種方法は本発明(1)と同様
とした。
本発明(20)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、接種方法は本発明(2)と同様
とした。
本発明(21)：本発明(1)で用いた試料(1)をPBSで
40000倍に希釈し、接種方法は本発明(3)と同様
とした。
本発明(22)：本発明(4)で用いた試料(2)をPBSで
10000倍に希釈し、接種方法は本発明(1)と同様
とした。
本発明(23)：本発明(4)で用いた試料(2)をPBSで
10000倍に希釈し、接種方法は本発明(2)と同様
とした。
本発明(24)：本発明(4)で用いた試料(2)をPBSで
40000倍に希釈し、接種方法は本発明(3)と同様
とした。
本発明(25)：本発明(7)で用いた試料(3)をPBSで
10000倍に希釈し、接種方法は本発明(1)と同様
とした。
本発明(26)：本発明(7)で用いた試料(3)をPBSで
10000倍に希釈し、接種方法は本発明(2)と同様
とした。
本発明(27)：本発明(7)で用いた試料(3)をPBSで
40000倍に希釈し、接種方法は本発明(3)と同様
とした。
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(4)で用いた試料(2)を鶏の胸筋に同
様量注射する。
対照(4)：本発明(7)で用いた試料(3)を鶏の胸筋に同
様量注射する。
対照(5)：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(6)：本発明(4)で用いた試料(2)を鶏に同様量経
口投与する。
対照(7)：本発明(7)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(1)で用いた試料(1)をPBSで100倍
に希釈し、接種方法は対照(2)と同様とした。
対照(9)：本発明(4)で用いた試料(2)をPBSで100倍
に希釈し、接種方法は対照(2)と同様とした。
対照(10)：本発明(7)で用いた試料(3)をPBSで100倍
に希釈し、接種方法は対照(2)と同様とした。
対照(11)：本発明(1)で用いた試料(1)をPBSで100倍
に希釈し、接種方法は対照(5)と同様とした。
対照(12)：本発明(4)で用いた試料(2)をPBSで100倍
に希釈し、接種方法は対照(5)と同様とした。
対照(13)：本発明(7)で用いた試料(3)をPBSで100
倍に希釈し、接種方法は対照(5)と同様とした。
対照(14)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、接種方法は対照(2)と同様と
した。
対照(15)：本発明(4)で用いた試料(2)をPBSで
10000倍に希釈し、接種方法は対照(2)と同様と
した。
対照(16)：本発明(7)で用いた試料(3)をPBSで
10000倍に希釈し、接種方法は対照(2)と同様と
した。
対照(17)：本発明(1)で用いた試料(1)をPBSで
10000倍に希釈し、接種方法は対照(5)と同様と
した。
対照(18)：本発明(4)で用いた試料(2)をPBSで
10000倍に希釈し、接種方法は対照(5)と同様と
した。
対照(19)：本発明(7)で用いた試料(3)をPBSで
10000倍に希釈し、接種方法は対照(5)と同様と
した。[Table] Example 3 Ten chickens (broiler breed Hubbard) within 72 hours after hatching were inoculated with the samples shown below, and a control
Samples (2) to (19) were inoculated using the respective methods.
These chickens are then infected at 21 days of age with E. coli O 78 inoculated intravenously at 1.4 x 10 10 cells per bird. Control (1) is one in which only infection was performed without inoculating the sample. Mortality rate and lesion values were observed on day 7 after infection. The results are shown in Table 3. The types of samples and inoculation methods are shown below. Present invention (1): Suspend 1.0×10 12 of E. coli O 78 in PBS, add formalin to 0.5% to make a killed bacteria suspension, and then thoroughly remove formalin using PBS. and centrifugation (10000r.
pm for 20 minutes), collect the dead bacteria and add to PBS.
Suspend the sample to 10 ml [Sample (1)]. This sample (1)
Add 1 drop (approximately 0.025 ml) per chicken to the anus of the 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. Invention (4): 2.5 × 10 11 pieces of E. coli O 78 were suspended in 2.5 ml of PBS, placed in a shear dish (model 1001 made by Falcon), and placed under an ultraviolet germicidal lamp (Toshiba germicidal lamp).
GL15) Place the cells under 21.5cm, stir 4 times every 30 minutes, and irradiate them for 16 hours. Add PBS to 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 (5): Inject the same amount of sample (2) used in present invention (4) into the cloaca of chickens. Present invention (6): Sample (2) used in present invention (4) was dissolved in PBS for 4 hours.
Dilute it twice and spray about 0.1ml per chicken into the anus. Present invention (7): 1.0×10 12 pieces of E. coli O 78 in 10ml of PBS
Float it so that it looks like this, put it in a test tube, and irradiate it with radiation (3 megarads) using 60 Co as a radiation source [Sample (3)]. Add 1 drop (0.025 ml) of this sample to the anus of each chicken. Present invention (8): Inject the same amount of sample (3) used in present invention (7) into the cloaca of chickens. Present invention (9): The sample (3) used in the present invention (7) was dissolved in PBS for 4 hours.
Dilute it twice and spray about 0.1ml per chicken into the anus. Present invention (10): Sample (1) used in present invention (1) was diluted with PBS at 100%
The inoculation method was the same as that of the present invention (1). Present invention (11): Sample (1) used in present invention (1) was diluted with PBS at 100%
The inoculation method was the same as that of the present invention (2). . Present invention (12): Sample (1) used in present invention (1) was diluted with PBS for 400 min.
The inoculation method was the same as that of the present invention (3). Present invention (13): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 100 times and the inoculation method was the same as in the present invention (1). Present invention (14): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 100 times and the inoculation method was the same as in the present invention (2). Present invention (15): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 400 times and the inoculation method was the same as in the present invention (3). Present invention (16): Sample (3) used in present invention (7) was dissolved in PBS.
It was diluted 100 times and the inoculation method was the same as in the present invention (1). Present invention (17): Sample (3) used in present invention (7) was dissolved in PBS.
It was diluted 100 times and the inoculation method was the same as in the present invention (2). Present invention (18): The sample (3) used in the present invention (7) was dissolved in PBS.
It was diluted 400 times and the inoculation method was the same as in the present invention (3). Present invention (19): The sample (1) used in the present invention (1) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (1). Present invention (20): The sample (1) used in the present invention (1) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (2). Present invention (21): Sample (1) used in present invention (1) was dissolved in PBS.
It was diluted 40,000 times and the inoculation method was the same as in the present invention (3). Present invention (22): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (1). Present invention (23): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (2). Present invention (24): Sample (2) used in present invention (4) was dissolved in PBS.
It was diluted 40,000 times and the inoculation method was the same as in the present invention (3). Present invention (25): Sample (3) used in present invention (7) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (1). Present invention (26): The sample (3) used in the present invention (7) was dissolved in PBS.
It was diluted 10,000 times and the inoculation method was the same as in the present invention (2). Present invention (27): Sample (3) used in present invention (7) was dissolved in PBS.
It was diluted 40,000 times and the inoculation method was the same as in the present invention (3). Control (1): Only infection is performed without inoculating the sample. Control (2): Inject the same amount of sample (1) used in the present invention (1) into the breast muscle of chickens. Control (3): Inject the same amount of sample (2) used in the present invention (4) into the breast muscle of chickens. Control (4): Inject the same amount of sample (3) used in the present invention (7) into the breast muscle of chickens. Control (5): Sample (1) used in the present invention (1) is orally administered in the same amount to chickens. Control (6): Sample (2) used in the present invention (4) is orally administered in the same amount to chickens. Control (7): Sample (3) used in the present invention (7) is orally administered in the same amount to chickens. Control (8): Sample (1) used in the present invention (1) was diluted 100 times with PBS, and the inoculation method was the same as control (2). Control (9): Sample (2) used in the present invention (4) was diluted 100 times with PBS, and the inoculation method was the same as control (2). Control (10): Sample (3) used in the present invention (7) was diluted 100 times with PBS, and the inoculation method was the same as control (2). Control (11): Sample (1) used in the present invention (1) was diluted 100 times with PBS, and the inoculation method was the same as control (5). Control (12): Sample (2) used in the present invention (4) was diluted 100 times with PBS, and the inoculation method was the same as control (5). Control (13): Sample (3) used in the present invention (7) was diluted with PBS at 100%
The inoculation method was the same as control (5). Control (14): Sample (1) used in the present invention (1) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as control (2). Control (15): Sample (2) used in the present invention (4) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as control (2). Control (16): Sample (3) used in the present invention (7) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as control (2). Control (17): Sample (1) used in the present invention (1) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as the control (5). Control (18): Sample (2) used in the present invention (4) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as the control (5). Control (19): Sample (3) used in the present invention (7) was mixed with PBS.
It was diluted 10,000 times and the inoculation method was the same as the control (5).
【表】【table】
【表】
実施例 ４
孵化後０〜72時間の鶏（ブロイラー専用種ハバ
ード）10羽に下記に示す試料を接種し、また対照
(4)〜（13）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42日令においてそれぞれの大
腸菌を用いて静脈内感染させた。対照(1)〜(3)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４に示す。なお試料の種類、接種方法および感
染大腸菌については下記に示す。
本発明(1)：大腸菌O2の1.0×1012個および大腸菌
O78の1.0×1012個を生理食塩水で10mlになるよ
うに混合し懸濁せしめ、試験管に入れ、60Coを
線源として放射線照射（３メガラツド）を行な
つた〔試料(1)〕。この試料を鶏の肛門に１羽当
り２滴（約0.05ml）滴下する。感染は大腸菌O2
を１羽当り５×108個とする。
本発明(2)：試料(1)を本発明(1)と同様に滴下する。
感染は大腸菌O78を１羽当り1.4×1010個とす
る。
本発明(3)：大腸菌O1の1.0×1012個と、大腸菌O2
の1.0×1012個および大腸菌O78の1.0×1012個を
生理食塩水で10mlになるようにまぜて懸濁せし
め、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
体浮遊液を得る〔試料(2)〕。この試料(2)を鶏の
肛門に１羽当り３滴（約0.075ml）滴下する。
感染は大腸菌O1を１羽当り1.2×109個とする。
本発明(4)：試料(3)を本発明(3)と同様に滴下する。
感染は大腸菌O2を１羽当り5.0×108個とする。
本発明(5)：試料(3)を本発明(3)と同様に滴下する。
感染は大腸菌O78を１羽当り1.4×1010個とす
る。
対照(1)：試料を接種せず、大腸菌O1を１羽当り
1.2×109個感染させる。
対照(2)：試料を接種せず、大腸菌O2を１羽当り
5.0×108個感染させる。
対照(3)：試料を接種せず、大腸菌O78を１羽当り
1.4×1010個感染させる。
対照(4)：本発明(1)で用いた試料(1)を鶏の胸筋に同
様量注射する。感染は大腸菌O2を１羽当り5.0
×108個とする。
対照(5)：本発明(1)で用いた試料(1)を鶏の胸筋に同
様量注射する。感染は大腸菌O78を１羽当り1.4
×1010個とする。
対照(6)：本発明(1)で用いた試料(1)を鶏に同様量経
口投与する。感染は大腸菌O2を１羽当り5.0×
108個とする。
対照(7)：本発明(1)で用いた試料(1)を鶏に同様量経
口投与する。感染は大腸菌O78を１羽当り1.0×
1010個とする。
対照(8)：本発明(3)で用いた試料(2)を鶏の胸筋に同
様量注射する。感染は大腸菌O1を１羽当り1.2
×109個とする。
対照(9)：本発明(3)で用いた試料(2)を鶏の胸筋に同
様量注射する。感染は大腸菌O2を１羽当り5.0
×108個とする。
対照(10)：本発明(3)で用いた試料(2)を鶏の胸筋に同
様量注射する。感染は大腸菌O78を１羽当り1.4
×1010個とする。
対照(11)：本発明(3)で用いた試料(2)を鶏に同様量経
口投与する。感染は大腸菌O1を１羽当り1.2×
109個とする。
対照(12)：本発明(3)で用いた試料(2)を鶏に同様量経
口投与する。感染は大腸菌O2を１羽当り5.0×
108個とする。
対照(13)：本発明(3)で用いた試料(2)を鶏に同様量
経口投与する。感染は大腸菌O78を１羽当り1.4
×1010個とする。[Table] Example 4 Ten chickens (broiler breed Hubbard) aged 0 to 72 hours after hatching were inoculated with the samples shown below, and a control
(4) to (13), samples were inoculated using the respective methods.
These chickens were then infected intravenously with the respective E. coli at 42 days of age. Controls (1) to (3) are those in which only infection was performed without inoculating the sample.
Mortality rate was observed on the 7th day after infection. The results are shown in Table 4. The types of samples, inoculation methods, and infected E. coli are shown below. Present invention (1): 1.0×10 12 pieces of E. coli O2 and E. coli
12 1.0×10 O 78 were mixed and suspended in physiological saline to a volume of 10 ml, placed in a test tube, and irradiated (3 megarads) using 60 Co as a radiation source [Sample (1) ]. Add 2 drops (approximately 0.05 ml) of this sample to each chicken's anus. The infection is E. coli O2
is 5×10 8 pieces per bird. Invention (2): Sample (1) is dropped in the same manner as in Invention (1).
Infection is with E. coli O 78 at 1.4 x 10 10 cells per bird. Present invention (3): 1.0×10 12 of E. coli O 1 and E. coli O 2
12 1.0× 10 cells of E. coli O 78 and 12 1.0× 10 cells of Escherichia coli O 78 were mixed and suspended in physiological saline to a total volume of 10 ml, placed in a shear dish (model 1001 made by Falcon), and placed under an ultraviolet germicidal lamp (Toshiba germicidal lamp). GL15)
Place it under 21.5 cm and stir 4 times every 30 minutes.
Further, the cells were irradiated for 16 hours in a stationary state, and PBS was added to the inactivated and dried cells to a volume of 2.5 ml to obtain a suspension of dead cells [Sample (2)]. Add 3 drops (approximately 0.075 ml) of this sample (2) to the anus of each chicken.
Infection is carried out at 1.2 x 10 9 Escherichia coli O 1 per bird. Invention (4): Sample (3) is dropped in the same manner as in Invention (3).
For infection, 5.0×10 8 Escherichia coli O 2 per bird. Invention (5): Sample (3) is dropped in the same manner as in Invention (3).
Infection is with E. coli O 78 at 1.4 x 10 10 cells per bird. Control (1): No sample inoculated, E. coli O 1 per bird
Infect 9 1.2×10. Control (2): No sample inoculated, E. coli O 2 per bird
Infect 8 5.0×10. Control (3): No sample inoculated, E. coli O 78 per bird
1.4×10 Infect 10 pieces. Control (4): Inject the same amount of sample (1) used in the present invention (1) into the breast muscle of chickens. Infection is E. coli O2 at 5.0 per bird.
×10 8 pieces. Control (5): Inject the same amount of sample (1) used in the present invention (1) into the breast muscle of chickens. Infection is E. coli O 78 at 1.4 per bird.
×10 10 pieces. Control (6): Sample (1) used in the present invention (1) is orally administered in the same amount to chickens. Infection is E. coli O2 at 5.0x per bird.
10 8 pieces. Control (7): Sample (1) used in the present invention (1) is orally administered in the same amount to chickens. Infection is E. coli O 78 at 1.0x per bird.
10 10 pieces. Control (8): Inject the same amount of sample (2) used in the present invention (3) into the pectoral muscle of chickens. Infection is E. coli O1 at 1.2 per bird.
×10 9 pieces. Control (9): Inject the same amount of sample (2) used in the present invention (3) into the breast muscle of chickens. Infection is E. coli O2 at 5.0 per bird.
×10 8 pieces. Control (10): Inject the same amount of sample (2) used in the present invention (3) into the breast muscle of chickens. Infection is E. coli O 78 at 1.4 per bird.
×10 10 pieces. Control (11): Sample (2) used in the present invention (3) is orally administered to chickens in the same amount. Infection is E. coli O 1 at 1.2x per bird.
10 9 pieces. Control (12): Sample (2) used in the present invention (3) is orally administered to chickens in the same amount. Infection is E. coli O2 at 5.0x per bird.
10 8 pieces. Control (13): Sample (2) used in the present invention (3) is orally administered in the same amount to chickens. Infection is E. coli O 78 at 1.4 per bird.
×10 10 pieces.
【表】
実施例 ５
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）滴下した。
試料(1)：Bifidobacterium、Lactobacillusおよび
非病原性大腸菌のそれぞれ１×1011個をPBS10
mlに混合懸濁せしめ、さらに実施例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(2)：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0.5％になるように
ホルマリンを加えて不活化し、PBSで３回洗
浄し、ホルマリンを除去し、PBSを加えて10
mlとし、これに実施例２の本発明(4)で用いた試
料(2)2.5mlを加えて懸濁混合せしめたもの。
試料(3)：Haemophilus paragallinarumおよび
Mycoplasma 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、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を得る。
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(4)：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(7)で用いた
試料(3)10mlを加えて混合せしめたもの。
試料(5)：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PBS10mlに浮遊させ
たものに実施例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
Bifidobacterium、Lactobacillus、非病原性大腸
菌（NS−244）、Clostridium perfringens、
Salmonella pullorum、Haemophilus
paragallinarum、Mycoplasma gallisepticum、
ニユーカツスル病生ワクチン、アイメリア・テネ
ラのスポロゾイトおよびアイメリア・ネカトリツ
クスのスポロゾイトの調製法は下記のとおりであ
る。試料(1)に用いたBifidobacterium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
行い、Bifidobacteriumの湿菌体を得る。
試料(1)に用いたLactobacillusは試料(1)に用い
たBifidobacteriumの調製法と同様に行なつた。
非病原性大腸菌は健康な鶏の腸管より分離し、
ハート・インフイユージヨンブロス（Difco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た非
病原性大腸菌の湿菌体を得る。
試料(2)に用いたClostridium perfringensは試
料(1)に用いたBifidobacteriumの調製法と同様に
行なつた。
試料(2)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
ユージヨンブイヨン（Difco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(3)に用いたHaemophilus paragallinarum
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(3)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の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、肝を細挫し、２倍量の蒸留水を
加えて１夜氷室に静置し、その後100℃で30分加
熱し過後NaClを0.5％、鶏血液を５％および10
％NaClを１％の量で加え、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1000U／ml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加えて寒天培地とした。分離し
たMycoplasma gallisepticumは５mlのPPLO増
殖培地（栄研）を用いて37℃で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℃において５日間培養し、次に
20000Gで30分間遠心分離して集収した。
試料(4)に用いたニユーカツスル病生ワクチン液
は化学及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(5)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％の次亜塩素酸ナトリウ
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
よそ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(5)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。[Table] Example 5 One sample per chicken was placed in the anus of 10 chickens (broiler breed Hubbard) within 72 hours after hatching.
A drop (approximately 0.025 ml) was added. Sample (1): 1 x 10 each of Bifidobacterium, Lactobacillus and non-pathogenic E. coli in PBS10
ml, and further added 10 ml of sample (1) used in Example 1 and suspended and mixed. Sample (2): Clostridium perfringens and
1 x 10 pieces each of Salmonella pullorum
Suspend in PBS, add formalin to inactivate to 0.5%, wash 3 times with PBS, remove formalin, add PBS and inactivate.
ml, and 2.5 ml of sample (2) used in the present invention (4) of Example 2 was added thereto and mixed for suspension. Sample (3): Haemophilus paragallinarum and
1x each of Mycoplasma 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.
After irradiation for 16 hours, add PBS to the inactivated and dried cells to a volume of 2.5 ml to obtain a suspension of dead cells.
Sample (2) used in this invention (4) of Example 3
Add 2.5 ml of and suspend and mix. Sample (4): New Katsuru disease live vaccine liquid 30ml
(1000 doses) and 10 ml of the sample (3) used in the present invention (7) of Example 1 was added and mixed. Sample (5): Eimeria tenella sporozoites
Sample (2) used in the present invention (4) of Example 3 was added to a suspension of 4,000,000 sporozoites and 4,000,000 sporozoites of Eimeria brunetsutei in 10 ml of PBS.
Add 2.5ml and suspend and mix. Used for samples (1), (2), (3), (4) and (5)
Bifidobacterium, Lactobacillus, non-pathogenic E. coli (NS-244), Clostridium perfringens,
Salmonella pullorum, Haemophilus
paragallinarum, Mycoplasma gallisepticum,
The method for preparing the live Eucatus disease vaccine, Eimeria tenella sporozoites, and Eimeria necatrix sporozoites is as follows. Bifidobacterium used in sample (1) was isolated from the intestinal tract of a chicken, and after culturing at 37°C for 20 hours using brain heart infusion broth (manufactured by Difco) containing sodium ascorbic acid and L-cysteine hydrochloride. , perform centrifugation at 10,000 rpm for 20 minutes to obtain wet Bifidobacterium cells. Lactobacillus used in sample (1) was prepared in the same manner as Bifidobacterium used in sample (1). Non-pathogenic E. coli was isolated from the intestinal tract of healthy chickens.
After culturing at 37°C for 18 hours using heart infusion broth (manufactured by Difco),
Wet cells of non-pathogenic E. coli are obtained by centrifugation at 10,000 rpm for 20 minutes. Clostridium perfringens used in sample (2) was prepared in the same manner as Bifidobacterium used in sample (1). Salmonella pullorum used for sample (2) was isolated from the feces of chickens suffering from chick dysentery, and incubated at 37°C using Heart Infusion Broth (manufactured by Difco).
After culturing for 18 hours, centrifugation is performed at 10,000 rpm for 20 minutes to obtain wet cells of Salmonella pullorum. Haemophilus paragallinarum used for sample (3)
was isolated from the nasal cavity of a chicken infected with infectious coryza, streak cultured on a thioyokolate agar medium (Climedeia, manufactured by Nissin Chemical Co., Ltd.), and the proliferated colonies were scraped off using a Conlage rod. Mycoplasma gallisepticum used in sample (3) 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 spread on an agar plate medium, and 0.1 ml was transferred to 2 ml of 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 made by finely crushing the thigh, breast, heart, and liver of one chicken, adding twice the amount of distilled water, leaving it in an icebox overnight, and then heating it at 100℃ for 30 minutes. NaCl 0.5%, chicken blood 5% and 10
Add %NaCl in an amount of 1%, 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 1000 U/ml was used. In addition, the agar plate medium used here was prepared by heating the solution (1) used when creating the liquid medium to approximately 60°C.
Add 1/10 amount of 15% agar solution heated and dissolved, heat briefly to dissolve completely, cool to 50℃, add 20% non-working horse serum, 5% thallium acetate 1% and penicillin 1000 U/ml. In addition, it was used as an agar medium. The isolated Mycoplasma gallisepticum was cultured at 37°C for 3 days using 5 ml of PPLO growth medium (Eiken), and then planted in 100 ml of PPLO medium (Eiken) at 37°C.
The cells were cultured for 3 days at
It was collected by centrifugation at 20,000G for 30 minutes. The live Newatus disease vaccine solution used in sample (4) was prepared by dissolving 1000 doses of the desiccation preventive solution in 30 ml of a solution for dissolving the live Newatus disease vaccine manufactured by Chemo-Serum Therapy Research Institute. The method for preparing Eimeria tenella sporozoites used for sample (5) 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 a 5% sodium hypochlorite solution are added to the oocysts, left for 15 minutes, and then centrifuged (2000 rpm, 3 minutes). The precipitate obtained is washed with water and then ground in a homogenizer. This ground material consists mostly of sporocysts. A 5% chicken bile and 0.25% trypsin PBS (-) solution is added to the sporocysts and digested in a 40°C bath for approximately 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 (5) are prepared according to the above-mentioned method for preparing sporozoites of Eimeria tenella. However, instead of Eimeria tenera, use Eimeria Brunetstei.
Claims (1)
たは組合わせからなる死菌体を有効成分として含
有する抗大腸菌症剤を鶏に投与して鶏の大腸菌症
を予防する方法において、該抗大腸菌症剤を液状
で鶏の総排泄腔に投与することを特徴とする鶏の
大腸菌症の予防法。 ２ 大腸菌の死菌体が大腸菌をガンマー線照射、
紫外線照射またはホルマリン処理で不活化するこ
とにより得られたものである特許請求の範囲第１
項記載の大腸菌症の予防法。 ３ 抗大腸菌症剤の投与菌量が、鶏１羽当たり
1.0×105個以上である特許請求の範囲第１項記載
の大腸菌症の予防法。 ４ 抗大腸菌症剤の鶏の総排泄腔への投与が、該
液状の抗大腸菌症剤を鶏の総排泄腔に滴下、注
入、スプレーまたは塗布するか、あるいは鶏の総
排泄腔を該液状の抗大腸菌症剤に浸すことにより
なされる特許請求の範囲第１項記載の大腸菌症の
予防法。[Scope of Claims] 1. Preventing colibacillosis in chickens by administering to chickens an anti-coliosis agent containing as an active ingredient killed cells of Escherichia coli O 1 , O 2 and O 78 , each singly or in combination. 1. A method for preventing colibacillosis in chickens, which comprises administering the anti-coliosis agent in liquid form to the cloaca of chickens. 2 The dead cells of E. coli are irradiated with gamma rays,
Claim 1, which is obtained by inactivation by ultraviolet irradiation or formalin treatment.
Methods for preventing colibacillosis as described in section. 3 The amount of bacteria administered with the anti-coliosis agent per chicken
The method for preventing coliosis according to claim 1, wherein the number of coliform bacteria is 1.0×10 5 or more. 4. Administration of the anti-coliosis agent to the cloaca of chickens is carried out by dropping, injecting, spraying or applying the liquid anti-coliosis agent into the cloaca of chickens, or by administering the liquid anti-coliosis agent to the cloaca of chickens. The method for preventing coliosis according to claim 1, which is carried out by soaking in an anti-coliosis agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57011763A JPS58131920A (en) | 1982-01-29 | 1982-01-29 | Prevention of poultry coli infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57011763A JPS58131920A (en) | 1982-01-29 | 1982-01-29 | Prevention of poultry coli infection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58131920A JPS58131920A (en) | 1983-08-06 |
| JPH0222734B2 true JPH0222734B2 (en) | 1990-05-21 |
Family
ID=11787011
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP57011763A Granted JPS58131920A (en) | 1982-01-29 | 1982-01-29 | Prevention of poultry coli infection |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58131920A (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH085802B2 (en) * | 1986-08-13 | 1996-01-24 | 日清製粉株式会社 | Poultry colibacillosis vaccine |
| ES2042718T3 (en) * | 1987-10-26 | 1993-12-16 | Akzo Nv | PROCEDURE FOR THE PREPARATION OF A VACCINE TO PROTECT POULTRY AGAINST SEPTICEMIA PRODUCED BY E. COLI. |
| JP2006131622A (en) * | 2004-10-05 | 2006-05-25 | Genichiro Soma | Medicinal agent |
| WO2017024059A1 (en) * | 2015-08-04 | 2017-02-09 | Vaxiion Therapeutics, Llc | Ionizing irradiation sterilization of bacterial minicell-based biopharmaceuticals and methods of use |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS52120116A (en) * | 1976-04-02 | 1977-10-08 | Kitasato Inst | Domestic animal colitis germ inactevated vaccine |
-
1982
- 1982-01-29 JP JP57011763A patent/JPS58131920A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS52120116A (en) * | 1976-04-02 | 1977-10-08 | Kitasato Inst | Domestic animal colitis germ inactevated vaccine |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58131920A (en) | 1983-08-06 |
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