JP3775481B2 - Method for mass culture of atypical Aeromonas salmonicida (Aeromonasalmonicida) - Google Patents

Description

この表から分かるようにワクチンの実投与の３倍量でも何ら異常は認められず、本ワクチンが安全である事を確認した。なお、比較試験として無菌リン酸緩衝液生理食塩水（ＰＢＳ）０．６ｍｌを腹腔内投与した（比較試験（０１７））。
【００２０】
＜実験例２ ワクチン投与による凝集抗体価の確認＞
（０１８） 本発明の不活化ワクチン（１０ｍｇ／ｍｌ）および免疫補助剤（アジュバント）（１０ｍｇ／ｍｌ）の等量混合液０．２ｍｌを供試鯉の腹腔内に投与し（実験例２−１）、常法通り２週間飼育した後、尾静脈よりヘパリンで処理した注射筒で採血した。なお、比較試験として無菌ＰＢＳ０．２ｍｌを同様腹腔内に投与した（比較試験（０１８））。
（０１９） （０１８）で記載した同様の方法で、実投与量の２倍量の不活化ワクチン（実験例２−２）および３倍量の不活化ワクチン（実験例２−３）免疫補助剤と混合して投与し、投与量により凝集抗体価について検討した。次いで常法通り２週間飼育したのち、尾静脈よりヘパリン処理した注射筒により採血した。
（０２０） （０１８）および（０１９）で得られたヘパリン化血液を常法通り血清を調整した。
（０２１） 得られた血清の抗体化はマイクロタイター法で行なった。即ち、ＰＢＳで段階的に希釈した血清２５μｌを各ウェルに流し込み、次いで不活化ワクチン懸濁液（１０ｍｇ／ｍｌ）２５μｌを各ウェルに添加した。充分に混合した後、一昼夜室温で放置して凝集反応を行なった。尚、対照実験として、正常な鯉にワクチンの代わりにＰＢＳを腹腔内に投与し、２週間飼育後、採血して血清を調整した。生じた凝集は目視で確認し、凝集が生じなくなったウェルを１〜１２のスコアーで評価した。なお、凝集価はＬｏｇ_２で表示した。
（０２２） この結果を表２に示す。 表中、（＋）は凝集陽性を（−）は陰性を示す。この際、Ｂｌａｎｋとは、不活化ワクチン（菌体 １０ｍｇ／ｍｌ）２５μｌとＰＢＳ ２５μｌのみをウエルに添加したものである。

（０２３） 不活化ワクチン０．２ｍｌ（実投与量）の投与量で、飼育２週間後の凝集抗体価はスコア１０（１：１０２４）であり、この抗体力価は不活化ワクチンの投与量による差は認められなかった。不活化ワクチン０．２ｍｌ（実投与量）の投与量で充分に抗体が産生されている事を確認できた。
【００２１】
＜実験例３ チャレンジ試験による有効性の確認＞
チャレンジ試験に供試した被検鯉は３０日間１７℃、一日一回の投餌で予備飼育を行い、体重測定、外観観察を行い異常がない事を確認した。
（０２４） 実験群としてワクチン非投与・非感染群１１尾（陰性対照 比較試験１）、ワクチン非投与・感染群１１尾（陽性対照 実施例１）およびワクチン投与・感染群１１尾（陽性対照 実施例２）の３群に群別した。
各被検鯉は１７℃に保温した５０Ｌ水槽で通気しながら飼育した。
（０２５） 感染スケジュールは、（０１８）で調整した不活化ワクチンを腹腔に投与して２週間後にチャレンジ試験に供した。
（０２６） 感染は−８５℃で保存したＡ．ｓａｌｍｏｎｉｃｉｄａ懸濁液を急速溶解させ、直接ハートインヒュージョン寒天培地に塗抹し、２４℃ ５日間培養した菌体を用いた。

（０２７） 培養菌体を無菌ＰＢＳで掻きとり、同無菌緩衝液で洗浄した。洗浄菌体を滅菌したＴｒｉｓ−ＨＣｌ緩衝液（ｐＨ８．３ ０．０１Ｍ）３．０Ｌに均一になるよう均一に懸濁し、これを浸漬感染液とした。
（０２８） 浸漬感染液のＡ．ｓａｌｍｏｎｉｃｉｄａ 生菌濃度は６．７ｘ１０^６ＣＦＵ／ｍｌとした。
（０２９） １７℃に保持した感染液３．０Ｌに被検鯉１１尾を１時間浸漬し、感染させた。 感染後、１７℃に保った５０Ｌの水槽に移し、飼育した。なお、給餌は一日一回とした。
（０３０） Ａ．ｓａｌｍｏｎｉｃｉｄａ 感染後の被検鯉の死亡の推移を図１に示す。ワクチン非投与・感染群は感染後、８日目で斃死が始まり、２８日で全数斃死した。しかし、ワクチン投与・非感染群では２８日間の飼育で２尾のみが斃死したに過ぎない。
（０３１） 表３は感染後２８日目の死亡推移の結果を纏めたものである。
ワクチン投与・非感染群の感染後飼育２８日目のＲＰＳ値（相対生存率）は８１．８％であり、その有意性をＦｉｓｈｅｒの直接確立計算値より求めた。その結果、表３に示すように、直接確立計算値は０．０２％となり、統計学的にもその有意性を確認した。

【００２２】
＜実験例４ 斃死鯉の潰瘍性炎症の確認＞
（０３２） Ａ．ｓａｌｍｏｎｉｃｉｄａの感染により斃死した鯉に発症した潰瘍性炎症部位の平均数をワクチン非投与・感染群およびワクチン投与・感染群別に纏めたのが表３である。
（０３３） 表４に示すように、ワクチン非投与・感染群の斃死鯉に発症した潰瘍性炎症患部数は１尾当たり平均４．８の炎症患部が検出されるのに反し、ワクチン投与・感染群の斃死鯉での炎症患部数は１尾当たり１箇所の炎症が検出されたに過ぎない。統計学的にも有意差が認められ、ワクチンにより潰瘍性炎症の発症を抑制していることが確認された。
【００２３】
＜実験例５ 斃死鯉より感染菌の回収＞
（０３４） 斃死鯉の体表潰瘍患部および腎臓、肝・膵臓より感染菌の回収を行なった。

（０３５） 体表の潰瘍部患部からは無菌的に切除し、また腎臓、肝・膵臓は無菌的に摘出し、定法とおりホモゲナイズした後、無菌ＰＢＳで段階的希釈を行いハートインヒュージョン寒天培地あるいは馬脱繊維血（日本バイオテスト研究所製）を含むトリプトソーヤ寒天培地（日本製薬製）で検出した。培養は２４℃、４〜５日間行った。
（０３６） この結果を表５に示す。

（０３７） 本表に示すように、ワクチン非投与・感染群の斃死鯉で発症した全例の体表部潰瘍性炎症患部から感染菌であるＡ．ｓａｌｍｏｎｉｃｉｄａは回収されたが、腎臓、肝・膵臓からの感染菌の回収は１１尾中３尾に過ぎなかった。
ワクチン投与・感染群で斃死した被検鯉の体表患部（尾鰭）からはＡ．ｓａｌｍｏｎｉｃｉｄａは検出できなかった。
【００２４】
＜実験例６ Ａ．ｓａｌｍｏｎｉｃｉｄａの大量培養の検討＞
（０３８）ブドウ糖０．１％、食塩０．０５％、リン酸水素２カリウム０．２５％を基礎培地として種々の有機体窒素について検討した。その結果、表６に示すように、Ａ．ｓａｌｍｏｎｉｃｉｄａの生育にはハートインヒュージョンおよびブレインハートインヒュージョンが至適有機窒素体であると認めた。そのうち、酵母エキス、ポリペプトンおよび肉エキスではＡ．ｓａｌｍｏｎｉｃｉｄａの増殖は認められなかった。
（０３９） ハートインヒュージョン２．５％を基本培地として、種々の糖類１．０％の影響について検討したところ、ブドウ糖、ショ糖、マンニット、乳糖では殆ど菌の増殖効果は認められず、わずかにグリセリンに菌の増殖促進が認められたに過ぎない。
（０４０） 次に通気量の影響について検討した。５０ｍｌの三角フラスコにハートインヒュージョン２，５％５ｍｌ、１５ｍｌ、３０ｍｌおよび５０ｍｌとそれぞれに攪拌子を入れて高圧滅菌後、Ａ．ｓａｌｍｏｎｉｃｉｄａ ０．１ｍｌを接種し、２４℃５日間低速攪拌しながら培養を行なった。その結果、培地容量（培地通気量）に関係なく、培地１ｍｌ当りの菌体の収量はほぼ一定であった。しかし、接種菌量を５０倍にしたところ、通気量の高い培地（培地容量の少ない培地）で著しい菌の増殖が認められた。
（０４１） （０３８），（０３９），（０４０）の結果から、ハートインヒュージョンと通気量および通気量と接種菌量間に交互作用の有無をも考慮して、表７に示す第２水準のＬ_１６（２^１５）の割り付けに基づいた実験計画法により５０ｍｌの三角フラスコレベルで培養条件の検討を行なった。
【表６】

その結果、通気と接種菌量間に交互作用が認められ、接種菌量を多くして通気攪拌培養する事が好ましく、従って、Ａ．ｓａｌｍｏｎｉｃｉｄａを大量培養する至適培養条件はハートインヒュージョンブイヨンで３日間静置培養し、次いで２日間攪拌培養に切り替えることにより多量の菌体が得られることを認めた。この際の菌体の収量の９５％の信頼限界は２００ｍｇ±１１ｍｇ／Ｌ（乾燥重量）である。
【表７】

[0001]
BACKGROUND OF THE INVENTION
The present invention is intended to enhance the ability of bioprotective activity by mixing administration of an inactivated vaccine and a non-oil-based adjuvant, thereby protecting against bacterial infection. atypical Aeromonassalmonicida (Aeromonas salmonicida) bacterial cells non-oil adjuvant the bacteria were inactivated by formalin treatment is, for example, by mixing an aluminum salt granules adjuvant, and intraperitoneally administered to the subject fish example Nishikikoi, biodefense It enhances the protective activity against infection by increasing its function. In addition, A. Salmonicida is not limited to a specific strain, but is an atypical A. cerevisiae that is a causative bacterium of "New hole disease". Salmonica refers to all genera.
[0002]
[Prior art]
As for the fish disease vaccine, publication number 11-332558 is a preventive vaccine against enterococcal infections of yellowtail, publication number 8-40932 is a prevention vaccine against Staphylococcus infections, A prophylactic vaccine for vitiligo virus, and an open-air iridovirus infection vaccine for fish are disclosed in Japanese Patent Application Laid-Open No. 9-176043.
[0003]
On the other hand, a well-killed disease with ulcerative inflammation on the surface of the body has been prevalent in farmed nishikikoi, causing serious damage. This disease is similar to the “hole disease” that was prevalent in goldfish and Nishiki Koi in the 1970s, but the ulcerative inflammation in the trunk is the same as the symptoms of “hole disease”. It is characterized by ulcerative inflammation in the base, lip, and vagina, and is highly lethal, which is different from the conventional “perforated disease”. ".
[0004]
This disease also occurs in small fish such as this year-old fish, is highly lethal, has not been effective with the administration of antibacterial agents for fisheries and thermal treatment that were effective in conventional treatment, and the size of the affected ulcers Regardless, it leads to death. In the event of a “new hole disease,” the pond must be disinfected, the bactericidal basin must be completely isolated, and measures must be taken to prevent infection of other basins.
Therefore, investigation of the cause of the onset and establishment of preventive treatment measures have become urgent issues.
[0005]
[Means for Solving the Problems]
Recently, Wakabayashi et al. Isolated a strain from the ulcer inflammation site of spontaneously affected Nishiki Koi, and the strain was atypical A. cerevisiae by phenotype and 16S rRNA gene sequence. It was identified as salmonicida, and its strong pathogenic “new puncture disease” was recognized by subcutaneous injection, and this strain was assumed to be the causative bacterium. This bacterium is highly transmissible, and it is essential to administer a vaccine to prevent the spread of “new hole disease”.
[0006]
However, A. Salmonicida is said to migrate to VBNC (Vable but non-culturable but not cultivatable) even if it is a typical form showing good growth. Moreover, it was impossible to obtain a large amount of cells by culturing a large amount of atypical Aeromonas salmonicida, which is difficult to culture.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The inventor has intensively studied and succeeded in obtaining a large amount of bacterial cells by carrying out a sift-up of the amount of aeration with a heart infusion medium during the culture. Furthermore, the immune adjuvant (Adjuvant) used for raising the antibody titer by intraperitoneal administration generally uses an oil-based base material, so that an ulcer develops at the site of inoculation, which may impair the commercial value of Nishiki Koi. was there. The inventor has further studied and succeeded in a method in which ulcer does not develop at the inoculation site by using non-oil-based Adjuvant aluminum salt without inflammation.
[0008]
Next, atypical A.I. The production of the salmonicida vaccine will be described in detail. The affected area, kidney, spleen, and liver of the Raden disease of “New hole disease” are removed, and sterile physiological saline is added to the removed organ and pulverized while cooling with a homogenizer. Decant the grinding liquid and collect the supernatant. The obtained supernatant is smeared on 3% horse defibrinated blood agar and cultured at 25 ° C. for 5 days.
[0009]
Depending on the biochemical properties and serological classification of the obtained strain, atypical A. Confirm that it is salmonicida. Furthermore, the pathogenicity of the strain is examined by subcutaneous inoculation.
[0010]
The causative bacteria identified by pathogenicity and serological confirmation are suspended in 1.0 ml of heart infusion and Bren heart infusion liquid medium to 10 ² to 10 ⁸ CFU, preferably 10 ⁶ CFU, and at −85 ° C. save. In culturing the strain, the strain stored at -85 ° C is rapidly dissolved at 20 ° C to 50 ° C, preferably 40 ° C. After completely dissolving, immediately cool on ice.
[0011]
0.1 ml of this lysed suspension is inoculated into a test tube containing 5 ml of heart infusion or brain heart infusion liquid medium, and cultured at 18 ° C. to 25 ° C., preferably 24 ° C. for 2 to 7 days, preferably 5 days, This is referred to as seed culture.
Next, the seed culture is transplanted in advance into a 100 ml Erlenmeyer flask medium containing 50 ml of the above medium and cultured at 18 ° C. to 25 ° C., preferably 24 ° C. for 2 to 7 days, preferably 5 days. This is referred to as pre-seed culture.
[0012]
Next, 950 ml of Heart-infusion and Bren-Heart-infusion liquid medium as a main culture and inoculated into a 1000 ml Erlenmeyer flask containing a stirrer and left at 18 ° C. to 25 ° C., preferably 24 ° C. for 2 to 7 days, preferably 3 days The culture is followed by stirring and culture at 18 ° C. to 25 ° C., preferably 24 ° C., for 2 to 5 days, preferably 3 days.
[0013]
The obtained culture suspension is aseptically collected by centrifugation (2500 × G to 5000 × G, preferably 3000 × G, 10 to 30 minutes, preferably 20 minutes (3500 rpm, 20 minutes for KOKUSAN H-103N).
[0014]
Next, the bacteria are washed several times with sterile phosphate buffered saline, preferably 2 to 3 times. The washed cells are suspended in sterile water and adjusted to a concentration of 50 mg to 500 mg (wet weight), preferably 100 mg (wet weight), and then formalin is gradually added. It is adjusted to 3 to 0.7%, preferably 0.5%. In this formalin treatment, the reaction is carried out with stirring at 18 ° C. to 25 ° C., preferably 24 ° C. for 18 to 30 hours, preferably 24 hours.
[0015]
The formalin-treated suspension is washed with aseptic purified water or aseptic distilled water 3 to 10 times, preferably 5 to 7 times, and no formalin remains. Confirm that there is no viable Salmonicida.
[0016]
After confirmation, adjust to a suspension of 1 mg / ml (sterile water) and aseptically fill vials.
[0017]
【Example】
<Example 1 Production of vaccine>
(001) Atypical A. cerevisiae isolated from the affected part of the body surface of the carp affected by “new puncture disease”. Salmonicida was added to a heart infusion bouillon solution (Nissui Pharmaceutical Co., Ltd.) with 1% bacterial powder agar (Nissui Pharmaceutical Co., Ltd.) washed with purified water several times, and autoclaved at 121 ° C for 20 minutes. Pour and solidify. In this Petri dish medium, A. Salmonida is smeared and cultured for 24 hours 4 days.
(002) Pour 10 ml of sterilized heart infusion broth into the petri dish obtained in (001) and scrape bacteria aseptically. Carefully transfer 0.5 ml of the resulting bacterial suspension (OD ₆₀₀ 0.5) to a 1 ml sterile bayer and store at -85 ° C.
(003) A. stored at −85 ° C. Salmonida was rapidly dissolved in water at about 40 ° C. and then stored in ice.
(004) Six test tubes containing 5 ml of heart infusion liquid medium were autoclaved at 121 ° C. for 20 minutes, allowed to cool, then inoculated with the dissolution preservation solution obtained in (003), and allowed to stand at 24 ° C. for 5 days. Cultured. This was the seed culture before
(005) The pre-seed culture obtained in (004) is transplanted to six 100 ml Erlenmeyer flasks containing 50 ml of heart infusion liquid medium. Subsequently, it was statically cultured at 24 ° C. for 7 days and used as a seed culture.
(006) A 950 ml heart infusion liquid medium and a 1000 ml Erlenmeyer flask with a stirring bar inserted were sterilized under the same conditions as described above, and the preculture obtained in (005) was inoculated. Next, the culture was allowed to stand at 24 ° C. for 3 days, and this was placed on a multi stirrer (manufactured by Slow stirrer SW-800N-1NISSIN), and stirred at 24 ° C. for 3 days according to the scale 6. Observed turbidity, OD600 1.10).
(007) 6 L of the culture solution obtained in (006) was collected with a centrifuge (KOKUSAN H-103N) at 3500 rpm and 20. The obtained cells were washed with aseptic purified water three times (wet body 50 g).
(008) The microbial cells (wet body 50 g) obtained in (007) were further washed once with 300 ml of sterile phosphate buffered saline (PBS) by the method described in (007).
(009) PBS-washed cells (wet 50 g) were suspended in 400 ml of sterile PBS.
(010) The suspension obtained in (009) was carefully added with stirring so that the concentration of formalin was 0.5%, and reacted at 24 ° C. for 24 hours.
(011) The formalin-treated cells obtained in (010) were repeatedly washed 4-5 times with sterile PBS.
(012) The washed cells (820 mg as dry cells) obtained in (011) were suspended in 10 ml of sterile PBS. To this suspension A. It was confirmed in accordance with the hygiene test method that no living Salmonida and formalin remained. This suspension was appropriately diluted to adjust the concentration to 10 mg / ml.
(013) The Bayer bottle obtained in (012) and sterilized with the suspension was aseptically filled with 1.0 ml and stored refrigerated. This was designated as an inactivated vaccine.
[0018]
<Example 2 Preparation of immune adjuvant (adjuband) (014) 16.7 g of aluminum sulfate (Al ₂ (SO 4) ₃ .18H ₂ O) is dissolved in 25 ml of distilled water in a 500 ml container. Then 1N caustic soda (NaOH) is gradually added and mixed. The resulting precipitate is washed several times with distilled water. Next, the precipitate was passed through a dusting machine (manufactured by Model S-1 National) to form fine particles.
(015) The fine particle suspension obtained in (014) was a suspension that did not precipitate after standing for at least 10 minutes.
(016) This dry weight was weighed and adjusted to exactly 10 mg / ml in sterile PBS and filled into sterile Bayer bottles. Store this in the refrigerator.
[0019]
<Experimental Example 1 Safety Test>
(017) A test solution prepared by mixing equal amounts of the inactivated vaccine obtained in (013) and the immune adjuvant obtained in (016) into a test cage of this year-old fish having an average weight of 40 g is 0 .2 ml (Experimental Example 1 (017) 0.2) as a basic quantity, and twice the quantity (Experimental Example 1 (017) 0.4) and three times the quantity (Experimental Example 1 (017) 0.6) The animals were administered intraperitoneally to 5 groups of tails and reared at 17 ° C. for 1 month with aeration in a 50 L water tank. The safety was examined by observing the number of survivors and abnormalities of the trunk, measuring body weight, and finding autopsy. The results are shown in Table 1.

As can be seen from this table, no abnormality was observed even at 3 times the actual dose of the vaccine, confirming that the vaccine was safe. As a comparative test, 0.6 ml of sterile phosphate buffered saline (PBS) was intraperitoneally administered (Comparative test (017)).
[0020]
<Experimental example 2 Confirmation of aggregated antibody titer by vaccine administration>
[0181] 0.2 ml of an equal volume mixture of the inactivated vaccine of the present invention (10 mg / ml) and an immune adjuvant (adjuvant) (10 mg / ml) was administered into the abdominal cavity of a test tube (Experimental Example 2-1) ), Reared for 2 weeks as usual, and then blood was collected from the tail vein with a syringe treated with heparin. As a comparative test, 0.2 ml of sterile PBS was administered intraperitoneally in the same manner (Comparative test (018)).
(019) In the same manner as described in (018), an inactivated vaccine having twice the actual dose (Experimental Example 2-2) and an inactivated vaccine having three times the actual dose (Experimental Example 2-3) Were mixed and administered, and the aggregated antibody titer was examined according to the dose. Subsequently, after raising for 2 weeks as usual, blood was collected from a tail vein through a syringe treated with heparin.
(020) Serum was prepared from heparinized blood obtained in (018) and (019) as usual.
[021] The obtained serum was antibodyized by the microtiter method. That is, 25 μl of serum serially diluted with PBS was poured into each well, and then 25 μl of inactivated vaccine suspension (10 mg / ml) was added to each well. After thorough mixing, the mixture was allowed to stand at room temperature for a whole day and night to conduct an agglutination reaction. As a control experiment, PBS was administered intraperitoneally to a normal rabbit instead of the vaccine, and after 2 weeks of breeding, blood was collected to prepare serum. The resulting agglomeration was visually confirmed, and wells in which agglutination did not occur were evaluated with a score of 1-12. The aggregation value was expressed as Log ₂ .
[022] The results are shown in Table 2. In the table, (+) indicates aggregation positive and (-) indicates negative. At this time, the Blank is obtained by adding only 25 μl of inactivated vaccine (bacteria 10 mg / ml) and 25 μl of PBS to the well.

(023) Aggregated antibody titer after 2 weeks of breeding at a dose of 0.2 ml of inactivated vaccine (actual dose) is score 10 (1: 1024), and this antibody titer depends on the dose of inactivated vaccine There was no difference. It was confirmed that the antibody was sufficiently produced at a dose of 0.2 ml of inactivated vaccine (actual dose).
[0021]
<Experimental example 3 Confirmation of effectiveness by challenge test>
The test rods used for the challenge test were preliminarily raised by feeding once a day at 17 ° C. for 30 days, and weighed and observed the appearance to confirm that there was no abnormality.
[0243] 11 vaccine non-administered / non-infected groups (negative control comparative test 1), 11 vaccine non-administered / infected groups (positive control Example 1) and 11 vaccine-administered / infected groups (positive control) They were grouped into 3 groups of Example 2).
Each test cage was bred with aeration in a 50 L water tank kept at 17 ° C.
(025) The infection schedule was such that the inactivated vaccine prepared in (018) was administered intraperitoneally and subjected to a challenge test 2 weeks later.
(026) Infection was stored at -85 ° C. Salmonica suspension was rapidly dissolved, smeared directly on a heart infusion agar medium, and cultured at 24 ° C. for 5 days.

[027] The cultured cells were scraped with sterile PBS and washed with the same sterile buffer. The washed cells were uniformly suspended in 3.0 L of sterilized Tris-HCl buffer (pH 8.3 0.01 M) so as to be a uniform immersion infection solution.
(028) A. of immersion infection liquid The concentration of viable salmonicida was 6.7 × 10 ⁶ CFU / ml.
[029] Eleven test rods were immersed in 3.0 L of an infectious solution maintained at 17 ° C for 1 hour to be infected. After infection, it was transferred to a 50 L water tank maintained at 17 ° C. and reared. Feeding was performed once a day.
(030) A.1. The transition of the death of the test sputum after salmonicida infection is shown in FIG. In the non-vaccine-administered / infected group, drowning started on the 8th day after infection, and all drowned on the 28th. However, in the vaccine-administered / non-infected group, only 2 dogs were moribund after 28 days of breeding.
(031) Table 3 summarizes the results of the death transition on the 28th day after infection.
The RPS value (relative survival rate) on the 28th day after breeding of the vaccine-administered / non-infected group was 81.8%, and its significance was determined from Fisher's directly established calculated value. As a result, as shown in Table 3, the directly established calculated value was 0.02%, and the significance was confirmed statistically.

[0022]
<Experimental example 4 Confirmation of ulcerative inflammation of moribund>
(032) A.1. Table 3 summarizes the average number of ulcerative inflammatory sites that developed in the moribund pupae due to salmonicida infection, by vaccine non-administered / infected group and vaccine administered / infected group.
(033) As shown in Table 4, the number of ulcerative inflammatory lesions that developed in moribund in the non-vaccine-infected / infected group was detected on average, but 4.8 inflammatory lesions per fish, whereas vaccine-administered / infected The number of inflammatory lesions in the moribund group of the group was only one inflammation detected per fish. A statistically significant difference was observed, and it was confirmed that the vaccine suppressed the onset of ulcerative inflammation.
[0023]
<Experimental Example 5 Recovery of Infecting Bacteria from Dying Death>
(034) Infectious bacteria were collected from the ulcer affected area of the moribund and the kidney, liver, and pancreas.

(035) Aseptically excise from the affected part of the ulcer on the body surface, and aseptically remove the kidney, liver and pancreas, homogenize as usual, and then serially dilute with sterile PBS to form heart infusion agar medium or Detection was performed with tryptosa agar medium (manufactured by Nippon Pharmaceutical Co., Ltd.) containing equine defibrinated blood (manufactured by Nippon Biotest Laboratories). The culture was performed at 24 ° C. for 4 to 5 days.
(036) The results are shown in Table 5.

(037) As shown in this table, A. cerevisiae which is an infectious bacterium from ulcerative inflammatory lesions of body surface in all cases that developed with moribund mortality in the vaccine non-administered group. Salmonica was recovered, but only 3 out of 11 infections were recovered from the kidney, liver and pancreas.
From the body surface affected area (caudal fin) of the subject moribund in the vaccinated / infected group A. Salmonicida could not be detected.
[0024]
<Experimental Example 6 A. Study of large-scale culture of salmonicida>
[038] Various organic nitrogens were examined using glucose 0.1%, sodium chloride 0.05%, and dipotassium hydrogen phosphate 0.25% as a basal medium. As a result, as shown in Table 6, A.I. It was recognized that heart infusion and brain heart infusion were the optimal organic nitrogen bodies for the growth of salmonicida. Among them, yeast extract, polypeptone and meat extract are A. No growth of salmonicida was observed.
(039) Using Heart Infusion 2.5% as the basic medium, the effects of 1.0% of various saccharides were examined. Glucose, sucrose, mannitol, and lactose showed almost no bacterial growth effect. Only glycerin was found to promote bacterial growth.
(040) Next, the influence of the air flow rate was examined. Into a 50 ml Erlenmeyer flask, Heart Infusion 2,5% 5 ml, 15 ml, 30 ml and 50 ml were placed in a stir bar and sterilized under high pressure. 0.1 ml of salmonicida was inoculated and cultured at 24 ° C. for 5 days with low speed stirring. As a result, the yield of bacterial cells per 1 ml of the medium was almost constant regardless of the medium volume (medium aeration volume). However, when the amount of inoculated bacteria was increased 50 times, significant bacterial growth was observed in a medium with a high aeration rate (a medium with a small medium volume).
(041) From the results of (038), (039), and (040) , the second level shown in Table 7 is considered in consideration of the presence or absence of interaction between the heart infusion, the aeration amount, and the aeration amount and the inoculum amount. The culture conditions were examined at the 50 ml Erlenmeyer flask level by the experimental design method based on the allocation of L ₁₆ (2 ¹⁵ ).
[Table 6]

As a result, an interaction is observed between aeration and the amount of inoculated bacteria, and it is preferable to increase the amount of inoculated bacteria and culture with aeration and agitation. The optimum culture conditions for culturing salmonicida in large quantities were confirmed to be a large amount of bacterial cells obtained by static culture for 3 days in a heart infusion broth and then switching to agitation culture for 2 days. The 95% confidence limit of the yield of the bacterial cells at this time is 200 mg ± 11 mg / L (dry weight).
[Table 7]

Claims (3)

Atypical Aeromonas salmonicida ( Aeromonas salmonicida ) isolated from a naturally occurring “new hole disease” inflamed area was first heated to 18 ° C using a heart-infusion medium supplemented with glycerin, yeast extract and nonionic surfactant. mass culture method of atypical Aeromonas salmonicida (Aeromonas salmonicida), characterized in that standing 2-7 days of incubation at to 25 ° C., stirred for 2-5 days in culture and then at 18 ° C. to 25 ° C.. The atypical Aeromonas salmonicida according to claim 1, wherein the concentration of the heart infusion medium is 2.5%, and the addition amounts of glycerin, yeast extract and nonionic surfactant are 10.0%, 0.5% and 0.05%, respectively. ( Aeromonas salmonicida ) mass culture method. The method for mass culture of atypical Aeromonas salmonicida ( Aeromonas salmonicida ) according to claim 1 or 2, wherein the culture is stationary at 24 ° C for 3 days and stirred at 24 ° C for 2 days.