JPH054066B2 - - Google Patents
Info
- Publication number
- JPH054066B2 JPH054066B2 JP60145705A JP14570585A JPH054066B2 JP H054066 B2 JPH054066 B2 JP H054066B2 JP 60145705 A JP60145705 A JP 60145705A JP 14570585 A JP14570585 A JP 14570585A JP H054066 B2 JPH054066 B2 JP H054066B2
- Authority
- JP
- Japan
- Prior art keywords
- strain
- pasteurella multocida
- mutant strain
- pasteurella
- fermentable
- 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 - Lifetime
Links
- 241000606856 Pasteurella multocida Species 0.000 claims description 53
- 229940051027 pasteurella multocida Drugs 0.000 claims description 53
- 229930006000 Sucrose Natural products 0.000 claims description 14
- 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 claims description 12
- 241000282898 Sus scrofa Species 0.000 claims description 10
- 208000015181 infectious disease Diseases 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 7
- 229960001212 bacterial vaccine Drugs 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 101710194807 Protective antigen Proteins 0.000 claims description 3
- 230000034659 glycolysis Effects 0.000 claims description 3
- 230000002458 infectious effect Effects 0.000 claims description 3
- 239000003471 mutagenic agent Substances 0.000 claims description 3
- 206010034107 Pasteurella infections Diseases 0.000 claims description 2
- 201000005115 pasteurellosis Diseases 0.000 claims description 2
- 150000003445 sucroses Chemical class 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 description 12
- 230000002008 hemorrhagic effect Effects 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 8
- 229960005486 vaccine Drugs 0.000 description 8
- 229920001817 Agar Polymers 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 6
- 241000282887 Suidae Species 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 239000003053 toxin Substances 0.000 description 6
- 231100000765 toxin Toxicity 0.000 description 6
- 241000588779 Bordetella bronchiseptica Species 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000002956 necrotizing effect Effects 0.000 description 4
- 210000001944 turbinate Anatomy 0.000 description 4
- 206010003694 Atrophy Diseases 0.000 description 3
- 241000700198 Cavia Species 0.000 description 3
- 230000037444 atrophy Effects 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 231100000518 lethal Toxicity 0.000 description 3
- 230000001665 lethal effect Effects 0.000 description 3
- 230000001338 necrotic effect Effects 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 231100000219 mutagenic Toxicity 0.000 description 2
- 230000003505 mutagenic effect Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 108091008717 AR-A Proteins 0.000 description 1
- 201000008283 Atrophic Rhinitis Diseases 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 206010012559 Developmental delay Diseases 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000606860 Pasteurella Species 0.000 description 1
- 108010059749 Pasteurella multocida toxin Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010039088 Rhinitis atrophic Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
(産業上の利用分野)
本発明は、豚パスツレラ感染症を予防するパス
ツレラ・ムルトシダ変異株、生菌ワクチン及びそ
の菌株を取得法に関する。
(従来の技術)
豚の萎縮性鼻炎(以下、ARと略称する)は鼻
甲介の萎縮・変形をきたし、流行性肺炎の併発、
発育遅延などをもたらすために畜産経営上大きな
問題になつている。最近、AR重篤化にはボルデ
テラ・ブロンキセプチカ菌株に加えて、出血性壊
死毒素産生パスツレラ・ムルトシダ
(pasteurella multocida)菌株が関与することが
実証されている(de Jong、M.F.et.al.Intl.Pig
Vet.Soc.1980 Cogress、Denmark、Proceedings
P.221;PedersenK.B.& Barfod、K.Nord.Vet.
Met.1981、33、p.513−522.を参照)。近年、AR
予防のために、ボルデテラ・ブロンキセプチカ死
菌ワクチンが使用されているが、充分な効果を上
げるに至つていない。ARの予防に、パスツレ
ラ・ムルトシダ毒素ワクチンが有効であるという
報告も最近出されている(de Jong et.al.、Intl.
Pig Vet.Soc.1980 Cogress、Mexico、
Proceedings P.119)。
(問題点を解決するための手段)
本発明者等は、上述のような現状に鑑み、出血
性壊死毒素産生パスツレラ・ムルトシダの感染を
防御することによつて、ARを防御できる有効で
安全な生菌ワクチンを開発すべく種々研究を重ね
た。その結果、該ARを防御できる有効なパスツ
レラ・ムルトシダ生菌ワクチン用菌株を見出だし
たもので、その菌株は、白糖非発酵性及び温度感
受性を有する変異株である。而して、本発明は、
パスツレラ・ムルトシダ変異株は、生菌ワクチン
として有効且つ安全に利用できることを見出だし
た。
更に本発明は、該白糖非発酵性及び温度感受性
を有するパスツレラ・ムルトシダ変異株の取得法
を提供するもので、感染防御抗原を有するパスツ
レラ・ムルトシダ菌株を変異誘起剤で処理した
後、白糖加糖分解培地で培養し、安定な白糖非発
酵性変異株を選択し、次で、この白糖非発酵性変
異株に紫外線照射を行つた後培養し、安定な温度
感受性変異株を選択することを特徴とする白糖非
発酵性及び温度感受性を有する。
(実施例)
本発明の実施例につき以下詳述する。
本発明によれば、生菌ワクチンとしての使用に
適するパスツレラ・ムルトシダ変異株は、下記の
取得法によつて得られる。即ち、出血性壊死毒素
産生パスツレラ・ムルトシダ菌株を変異誘起剤例
えば、紫外線、ニトロソグアニジンなどの変異手
段により、糖(例えば、白糖、マンニトールな
ど)非発酵性、その他の遺伝標識(例えば、カタ
ラーゼ陰性など)の単独または複数の遺伝標識及
び温度感受性(30℃より高い温度では発育が抑制
される性質)を有する変異株を多数分離し、この
中から動物試験により、安全性と免疫原性の高い
変異株を選択することによつて、下記に詳述する
ように、生菌ワクチン用に適した白糖非発酵性及
び前記の温度感受性を有するパスツレラ・ムルト
シダ変異菌が得られる。その代表例であるパスツ
レラ・ムルトシダDZ10菌を微工研に寄託した。
更に詳述すれば、本発明の上記取得法で用いる
出発菌株は、血清型Dの出血性壊死毒素産生パス
ツレラ・ムルトシダの野外株が好ましいが、パス
ツレラ・ムルトシダの感染防御抗原を有していれ
ば、如何なるパスツレラ・ムルトシダ株であつて
も良い。
液状培地及び固型培地としてはパスツレラ・ム
ルトシダ菌株が発育できる公知のいずれの培地も
用いられる。
本発明によつて得られる代表例として上記した
変異株パスツレラ・ムルトシダDZ10株は野外分
離親株パスツレラ・ムルトシダDZ株の変異株で
ある。野外分離親株パスツレラ・ムルトシダDZ
株は養豚場で重篤なARに罹つた豚の鼻汁より分
離し、バージー・マニユアル(Bergey′s
Manual of Determinative Bacteriology、8th
Editon,p.370−372、1975)により同定された血
清型Dで出血性壊死毒素産生株のうちの1菌株で
ある。パスツレラ・ムルトシダDZ10株はその野
外分離親株DZ株と比較するとき、下記第1表に
示す生物学的特性を有する。
(Industrial Application Field) The present invention relates to a Pasteurella multocida mutant strain, a live bacteria vaccine, and a method for obtaining the strain for preventing swine Pasteurella infection. (Prior art) Pig atrophic rhinitis (hereinafter abbreviated as AR) causes atrophy and deformation of the nasal turbinates, resulting in complications such as epidemic pneumonia and
It has become a major problem in livestock management because it causes developmental delays. Recently, it has been demonstrated that in addition to Bordetella bronchiseptica strains, hemorrhagic necrotoxin-producing Pasteurella multocida strains are involved in the severity of AR (de Jong, MFet.al.Intl.Pig
Vet.Soc.1980 Cogress, Denmark, Proceedings
P.221; PedersenK.B. & Barfod, K.Nord.Vet.
(See Met. 1981, 33, p. 513-522.) In recent years, AR
A killed Bordetella bronchiseptica vaccine has been used for prevention, but it has not been sufficiently effective. There have also been recent reports that Pasteurella multocida toxin vaccine is effective in preventing AR (de Jong et.al., Intl.
Pig Vet.Soc.1980 Cogress, Mexico,
Proceedings P.119). (Means for Solving the Problems) In view of the above-mentioned current situation, the present inventors have developed an effective and safe method that can protect against AR by preventing infection with hemorrhagic necrotizing toxin-producing Pasteurella multocida. Various studies were conducted to develop a live bacterial vaccine. As a result, we found an effective Pasteurella multocida live bacteria vaccine strain that can protect against AR, and this strain is a mutant strain that is non-fermentable and temperature sensitive. Therefore, the present invention
It has been found that the Pasteurella multocida mutant strain can be used effectively and safely as a live bacterial vaccine. Furthermore, the present invention provides a method for obtaining a Pasteurella multocida mutant strain that is non-fermentable and temperature sensitive, in which a Pasteurella multocida strain having an infectious protective antigen is treated with a mutagenic agent, and then saccharide glycolysis is performed. The method is characterized in that a stable white sugar non-fermenting mutant strain is selected by culturing in a medium, and then the white sugar non-fermenting mutant strain is irradiated with ultraviolet light and then cultured to select a stable temperature-sensitive mutant strain. White sugar has non-fermentable properties and temperature sensitivity. (Example) Examples of the present invention will be described in detail below. According to the present invention, a Pasteurella multocida mutant strain suitable for use as a live bacterial vaccine is obtained by the following acquisition method. That is, a hemorrhagic necrotoxin-producing Pasteurella multocida strain is mutagenized with mutagenic agents such as ultraviolet light and nitrosoguanidine, so that sugars (e.g., sucrose, mannitol, etc.), non-fermentable, and other genetic markers (e.g., negative for catalase, etc.) ), we isolated a large number of mutant strains with single or multiple genetic markers and temperature sensitivity (the property that growth is suppressed at temperatures higher than 30°C), and from among these, we conducted animal tests to identify mutations with high safety and immunogenicity. By selecting the strain, a Pasteurella multocida mutant with white sugar non-fermentability and temperature sensitivity as described above is obtained, which is suitable for use in live vaccines, as detailed below. A representative example of this, Pasteurella multocida DZ10, was deposited at the Microtech Institute. More specifically, the starting strain used in the above acquisition method of the present invention is preferably a field strain of serotype D hemorrhagic necrotizing toxin-producing Pasteurella multocida, but it may be used as long as it has the infection-protective antigen of Pasteurella multocida. , any Pasteurella multocida strain. As the liquid medium and the solid medium, any known medium in which the Pasteurella multocida strain can grow can be used. The mutant strain Pasteurella multocida strain DZ10 described above as a representative example obtained by the present invention is a mutant strain of the field-isolated parent strain Pasteurella multocida strain DZ. Field isolated parent strain Pasteurella multocida DZ
The strain was isolated from the nasal secretions of pigs suffering from severe AR at a pig farm, and was collected from Bergey's Manual.
Manual of Determinative Bacteriology, 8th
This strain is one of the serotype D hemorrhagic necrotizing toxin-producing strains identified by Edison, p. 370-372, 1975). Pasteurella multocida strain DZ10 has the biological properties shown in Table 1 below when compared with its field-isolated parent strain DZ strain.
【表】【table】
【表】
以下、更に詳細な実施例により本発明を説明す
る。
実施例 1
先ず、白糖非発酵性の遺伝標識を有するパスツ
レラ・ムルトシダ変異株の分離について述べる。
AR症状を示す豚の鼻汁から分離した血清型D
で、出血性壊死毒素産生パスツレラ・ムルトシダ
菌株のうちの1株をDZ株と称する。パスツレ
ラ・ムルトシダDZ株をYPC液体培地(熊谷等
編:豚病学、第2判、465頁、近代出版、1982年)
で液体培養(37℃、18時間)した菌液を生理的食
塩液で遠心洗浄後、再び生理的食塩液に懸濁し
た。これに変異誘起剤ニトロソグアニジン水溶液
を100マイクログラム/mlになるように加え、菌
の生存率が約100分の1になるまで37℃で加温し
た。直ちに、この処理菌液の希釈液を寒天平板培
地に塗沫し、37℃、2日間培養した。成育した集
落を1個づつ拾い、マイクロタイター・プレート
(96穴)の穴に分注・固化させた白糠加糠分解寒
天培地中に移植した。37℃、3日間培養後、白糖
加糖分解寒天培地が黄変しなかつた白糖非発酵性
集落を多数拾つた。単集落分離を繰り返した後、
安定な白糖非発酵性変異株を選択した。
次に、この白糖非発酵性変異株の1株を液体培
養(37℃、18時間)した菌液を生理的食塩液で洗
浄後、生理的食塩液に懸濁した。この懸濁菌液の
5mlをペトリ皿(9cm直径)に入れ、菌の生存率
が100〜1000分の1になるまで紫外線照射を行つ
た。異変を安定化するために、この処理菌株を液
体培養(30℃)し、細胞分裂を数回行わせた。こ
の培養菌液の希釈液をYPC寒天平板培地に塗沫
し、30℃で培養した。発育した菌集落を2枚の
YPC寒天平板培地にレプリカし、1枚を40℃で、
もう1枚を30℃で培養(3日間)した。30℃で発
育し、40℃で殆ど発育が認められなかつた集落を
多数拾つた。単集落分離を繰り返した後、安定な
温度感受性変異株を選択した。そのうちの1株を
パスツレラ・ムルトシダDZ10株と名付けた(微
工研菌寄第8226号)。野外分離親株パスツレラ・
ムルトシダDZ株とこの本発明の変異株パスツレ
ラ・ムルトシダDZ10株の各培養温度における液
体培地で18時間培養した場合の発育を添付図面に
示した。仝図から明らかなように、本発明の変異
株は、図示のような温度感受性を具備しているこ
とが分る。
次にこの本発明のパスツレラ・ムルトシダ
DZ10株の出血性壊死毒素産生性を、その親株パ
スツレラ・ムルトシダDZ株と比較した。菌株を
30℃で液体培養し、その培養上清の無菌濾過液
(0.45nmミリポアフイルター)の0.1mlをモルモ
ツト(SPF、ハートレー、250〜300g体重、雌)
に皮内注射し、48時間後に判定した。パスツレ
ラ・ムルトシダDZ株の培養濾過液の注射では、
直径15〜25mmの出血性壊死円が形成され、モルモ
ツトは3日後に死亡した。これに対し、パスツレ
ラ・ムルトシダDZ10株の培養濾過液の注射では、
直径15〜25mmの出血性壊像は殆ど認められず、モ
ルモツトの死亡も観察されなかつた(2週間観
察)。
致死毒性試験:
実施例1によつて得られたパスツレラ・ムルト
シダDZ10株のマウスに対する致死毒性をその親
株パスツレラ・ムルトシダDZ株と比較した。
YPs寒天平板培地で培養(30℃)した菌集落を生
理的食塩液に懸濁した。この懸濁液の0.5mlをマ
ウス(SPF、ICR、雌、4週令)腹腔内に注射し
た。経時的に5日目まで生死を観察した結果を第
2表に示した。親株パスツレラ・ムルトシダDZ
株に比べ、得られたパスツレラ・ムルトシダ
DZ10株はマウスに対する致死毒性が著しく低下
していることが確認された。[Table] The present invention will be explained below with more detailed examples. Example 1 First, the isolation of a Pasteurella multocida mutant strain having a genetic marker for non-fermenting white sugar will be described.
Serotype D isolated from the nasal secretions of pigs exhibiting AR symptoms
One strain of Pasteurella multocida that produces hemorrhagic necrotic toxin is called the DZ strain. Pasteurella multocida DZ strain in YPC liquid medium (edited by Kumagai et al.: Swine Pathology, 2nd edition, 465 pages, Kindai Publishing, 1982)
The bacterial suspension cultured in liquid (37°C, 18 hours) was centrifugally washed with physiological saline, and then suspended in physiological saline again. An aqueous mutagenic nitrosoguanidine solution was added to this at a concentration of 100 micrograms/ml, and the mixture was heated at 37°C until the survival rate of the bacteria was approximately 1/100. Immediately, a diluted solution of this treated bacterial solution was spread on an agar plate medium and cultured at 37°C for 2 days. The grown colonies were picked up one by one, dispensed into the holes of a microtiter plate (96 holes), and transplanted into a solidified white rice bran-added agar medium. After culturing at 37°C for 3 days, a large number of white sugar non-fermenting colonies that did not turn yellow on the white sugar glycolysis agar medium were picked up. After repeating single colony separation,
A stable white sugar non-fermenting mutant strain was selected. Next, a liquid culture (37° C., 18 hours) of this sucrose non-fermenting mutant strain was washed with physiological saline, and then suspended in physiological saline. 5 ml of this bacterial suspension was placed in a Petri dish (9 cm diameter) and irradiated with ultraviolet rays until the survival rate of the bacteria was reduced to 1/100 to 1/1000. In order to stabilize the mutation, this treated strain was cultured in liquid (30°C) and subjected to cell division several times. A diluted solution of this culture was spread on a YPC agar plate medium and cultured at 30°C. The developed bacterial colony is shown in two pictures.
Replica onto YPC agar plate medium, place one plate at 40℃,
The other plate was cultured at 30°C (3 days). We found many colonies that grew at 30°C and showed almost no growth at 40°C. After repeated single colony isolations, stable temperature-sensitive mutants were selected. One of the strains was named Pasteurella multocida strain DZ10 (Feikoken Bacterium No. 8226). Field-isolated parent strain Pasteurella
The growth of Pasteurella multocida strain DZ and this mutant strain of the present invention, Pasteurella multocida strain DZ10, when cultured for 18 hours in a liquid medium at each culture temperature is shown in the attached drawing. As is clear from the figure, the mutant strain of the present invention has the temperature sensitivity shown in the figure. Next, Pasteurella multocida of the present invention
The hemorrhagic necrotic toxin production of strain DZ10 was compared with that of its parent strain Pasteurella multocida strain DZ. strain
Liquid culture was performed at 30°C, and 0.1 ml of the sterile filtrate (0.45 nm Millipore filter) of the culture supernatant was added to guinea pigs (SPF, Hartley, 250-300 g body weight, female).
was injected intradermally and assessed 48 hours later. In the injection of culture filtrate of Pasteurella multocida strain DZ,
A hemorrhagic necrotic circle with a diameter of 15-25 mm was formed, and the guinea pigs died after 3 days. On the other hand, injection of culture filtrate of Pasteurella multocida strain DZ10,
Almost no hemorrhagic necrosis with a diameter of 15 to 25 mm was observed, and no death of guinea pigs was observed (observation for 2 weeks). Lethal toxicity test: The lethal toxicity of Pasteurella multocida strain DZ10 obtained in Example 1 to mice was compared with its parent strain Pasteurella multocida strain DZ.
Bacterial colonies cultured on YPs agar plate medium (30°C) were suspended in physiological saline. 0.5 ml of this suspension was intraperitoneally injected into a mouse (SPF, ICR, female, 4 weeks old). Table 2 shows the results of observing the survival and death over time up to the 5th day. Parent stock Pasteurella multocida DZ
Pasteurella multocida obtained compared to the strain
It was confirmed that the DZ10 strain has significantly reduced lethal toxicity to mice.
【表】【table】
【表】
実施例 2
実施例1によつて得られたパスツレラ・ムルト
シダDZ10株の生菌ワクチンの感染防御能と安全
性をマウスで確認した。マウス(SPF、ICR、
雌、3週令)を対象群(10匹)と免疫群(10匹)
とに分けた。
パスツレラ・ムルトシダDZ10株をYPC寒天平
板培地で培養(30℃、18時間)後、生理的食塩液
に懸濁し、その0.05ml(マウス当たり3×106個
の菌数)を免疫群のマウスの鼻腔内に摂取した。
2週間後に、対照群及び免疫群の全てのマウスの
鼻腔内に、野外分離パスツレラ・ムルトシダDZ
株の培養菌液0.05ml(マウス当たり2×105個の
菌数)の摂取・攻撃した。マウスの生存匹数を15
日間観察した結果を第3表に示した。このように
本発明のパスツレラ・ムルトシダDZ10株は出血
性壊死毒素産生性の強毒なパスツレラ・ムルトシ
ダ菌株の感染に対して優れた防御能を有し、また
免疫群のマウスには異常な臨床症状が全く認めら
れなかつたことから安全性の高い菌株であること
が確認された。[Table] Example 2 The ability and safety of the live vaccine of Pasteurella multocida strain DZ10 obtained in Example 1 to prevent infection were confirmed in mice. Mouse (SPF, ICR,
Female, 3 weeks old) in control group (10 animals) and immunized group (10 animals)
It was divided into two parts. Pasteurella multocida strain DZ10 was cultured on YPC agar plate medium (30°C, 18 hours), suspended in physiological saline, and 0.05 ml (3 x 10 6 bacteria per mouse) was added to mice in the immunized group. Ingested intranasally.
After 2 weeks, field isolated Pasteurella multocida DZ was administered intranasally to all mice in the control and immunized groups.
The mice were ingested and challenged with 0.05 ml of a culture of the strain (2 x 10 5 bacteria per mouse). Number of surviving mice is 15
The results of daily observation are shown in Table 3. As described above, the Pasteurella multocida strain DZ10 of the present invention has an excellent protective ability against infection with a highly virulent Pasteurella multocida strain that produces hemorrhagic necrotizing toxin, and also shows abnormal clinical symptoms in mice in the immunized group. It was confirmed that the strain was highly safe as no was observed at all.
【表】
実施例 3
実施例2に示したと同様な条件下でパスツレ
ラ・ムルトシダDZ10株生菌ワクチンの感染防御
効果試験を行つた。但し、攻撃をパスツレラ・ム
ルトシダDZ株とボルデテラ・ブロンキセプチカ
SI株(夫々、マウス当たり2×105個の菌数)の
同時接種で行つた。結果を第4表に示した。この
ように、本発明のパスツレラ・ムルトシダDZ10
株はARの原因株である強毒なパスツレラ・ムル
トシダ菌株及びボルデテラ・ブロンキセプチカ菌
株の同時感染にも防御効果のあることが示され
た。[Table] Example 3 Under the same conditions as shown in Example 2, a test for the protection against infection of the Pasteurella multocida strain DZ10 live bacteria vaccine was conducted. However, the attack is Pasteurella multocida strain DZ and Bordetella bronchiseptica.
Simultaneous inoculation of the SI strain (2×10 5 bacteria per mouse, respectively) was performed. The results are shown in Table 4. Thus, Pasteurella multocida DZ10 of the present invention
The strain was also shown to have a protective effect against co-infection with the highly virulent Pasteurella multocida strain and Bordetella bronchiseptica strain, which are the causative strains of AR.
【表】
実施例 4
実施例1によつて得られたパスツレラ・ムルト
シダDZ10株生菌ワクチンの感染防御効果試験を
豚について行つた。子豚9頭を感染対照群3頭、
ワクチン接種感染群3頭、非接種非感染対照群3
頭の3群に分けた。7日令の子豚の鼻腔内にパス
ツレラ・ムルトシダDZ10株の培養菌液(豚1頭
当たり1.0×108個の菌数)を接種した。ワクチン
接種後2週目にボルデテラ・ブロンキセプチカSI
株(強毒)の培養菌液(豚1頭当たり1.2×108個
の菌数)で経鼻攻撃し、更に1週間後と2週間後
にパスツレラ・ムルトシダDZ株(強毒)の培養
菌液(豚1頭当たり4.0×107個の菌数)で経鼻攻
撃した。その後7週間目(豚12週令時)に全頭の
豚を剖検した。感染対照群3頭には、重度の鼻甲
介萎縮が認められた。これに対して、ワクチン接
種・感染群3頭には軽微な鼻甲介萎縮が認められ
ただけであつた。非接種・非感染対照群3頭の鼻
甲介は正常であつた。以上の成績により、パスツ
レラ・ムルトシダDZ10株の生菌ワクチンとして
の有効性が確認された。
(発明の効果)
このように本発明によるときは、感染防御抗原
を有するパスツレラ・ムルトシダ菌株より変異誘
起手段により白糖非発酵性と温度感受性を有する
パスツレラ・ムルトシダ変異株が得られ、該変異
株は、これを正ワクチンとして使用すると豚の
ARに対する優れた予防効果をもたらす。[Table] Example 4 An infection prevention effect test of the Pasteurella multocida DZ10 live bacterial vaccine obtained in Example 1 was conducted on pigs. 9 piglets, 3 infected control group,
3 animals in vaccinated infected group, 3 animals in non-vaccinated uninfected control group
The head was divided into three groups. A culture solution of Pasteurella multocida strain DZ10 (1.0 x 10 8 bacteria per pig) was inoculated into the nasal cavity of a 7-day-old piglet. Bordetella bronchiseptica SI 2 weeks after vaccination
A culture of Pasteurella multocida strain (highly virulent) (1.2 x 10 8 bacteria per pig) was administered through the nose, and one and two weeks later, a culture of Pasteurella multocida strain DZ (highly virulent) was challenged. (4.0 x 10 7 bacteria per pig) was intranasally challenged. After 7 weeks (when the pigs were 12 weeks old), all pigs were necropsied. Severe nasal turbinate atrophy was observed in three dogs in the infected control group. In contrast, only slight nasal turbinate atrophy was observed in the three dogs in the vaccinated/infected group. The nasal turbinates of the three animals in the non-inoculated and non-infected control group were normal. The above results confirmed the effectiveness of Pasteurella multocida strain DZ10 as a live bacterial vaccine. (Effects of the Invention) According to the present invention, a Pasteurella multocida mutant strain having white sugar non-fermentability and temperature sensitivity can be obtained by mutagenic means from a Pasteurella multocida strain having an infectious protective antigen, and the mutant strain is , when this is used as a positive vaccine, pigs
Provides excellent preventive effects against AR.
図面は。本発明菌株 温度感受性を示すグラフ
である。
The drawing is. It is a graph showing the temperature sensitivity of the present invention strain.
Claims (1)
レラ・ムルトシダ変異株。 2 白糖非発酵性及び温度感受性を有するパスツ
レラ・ムルトシダ変異株で、豚パスツレラ感染症
を予防する生菌ワクチン。 3 感染防御抗原を有するパスツレラ・ムルトシ
ダ菌株を変異誘起剤で処理した後、白糖加糖分解
培地で培養し、安定な白糖非発酵性変異株を選択
し、次で、この白糖非発酵性変異株に紫外線照射
を行つた後培養し、安定な温度感受性変異株を選
択することを特徴とする白糖非発酵性及び温度感
受性を有するパスツレラ・ムルトシダ変異株の取
得法。[Scope of Claims] 1. A Pasteurella multocida mutant strain that is non-fermentable and temperature sensitive. 2 A live bacterial vaccine for preventing swine Pasteurella infection using a Pasteurella multocida mutant strain that is non-fermentable and temperature sensitive. 3. After treating a Pasteurella multocida strain having an infectious protective antigen with a mutagenic agent, culturing it in a sucrose glycolysis medium to select a stable sucrose non-fermenting mutant strain, this sucrose non-fermenting mutant strain 1. A method for obtaining a Pasteurella multocida mutant strain that is non-fermentable and temperature sensitive, which comprises culturing after irradiation with ultraviolet rays and selecting a stable temperature-sensitive mutant strain.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP60145705A JPS626673A (en) | 1985-07-04 | 1985-07-04 | Preparation of strain for live vaccine of pastdurella multocida, live vaccine and strain thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP60145705A JPS626673A (en) | 1985-07-04 | 1985-07-04 | Preparation of strain for live vaccine of pastdurella multocida, live vaccine and strain thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS626673A JPS626673A (en) | 1987-01-13 |
JPH054066B2 true JPH054066B2 (en) | 1993-01-19 |
Family
ID=15391208
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP60145705A Granted JPS626673A (en) | 1985-07-04 | 1985-07-04 | Preparation of strain for live vaccine of pastdurella multocida, live vaccine and strain thereof |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS626673A (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6790954B2 (en) * | 2002-01-29 | 2004-09-14 | Immunogen, Inc. | Mutant Actinosynnema pretiosum strain with increased maytansinoid production |
-
1985
- 1985-07-04 JP JP60145705A patent/JPS626673A/en active Granted
Also Published As
Publication number | Publication date |
---|---|
JPS626673A (en) | 1987-01-13 |
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