200843789 九、發明說明 【發明所屬之技術領域】 本發明關於土壤絲菌屬之細菌於製造用於對抗魚之非 土壤絲菌型細菌感染之疫苗上的用途。 【先前技術】 過去數十年間可見列全世界食用之魚量大爲增加。而 食入之冷水性魚類（諸如鮭魚、多寶魚（turbot)、比目魚及 鳕魚）和熱帶魚類（諸如亞洲海鱸魚、吳郭魚（tilapia)、虱 目魚、紅鮒魚、琥珀鰺、斑魚及海鱺）均是如此增加。 因此，可以見到魚類養殖場之數量及規模在增加中， 以滿足市場增加之需求。 自例如畜牧業可知：大量動物密集生活在一起時很容 易受所有種類之疾病入侵，甚至出現那些在開始有大規模 商業農場前很少人知道或見到或甚至是從不知道的疾病。 魚類養殖場亦有此相同情況。被發現可使魚致病之細菌係 屬於，如弧菌屬（Vibrio)、美人魚發光桿菌 (Photobacterium damselae)、屈撓桿菌屬 (Tenacibaculum)、黃桿菌屬（Flavobacterium)、黏液菌屬 (Flexibacter)、纖維菌屬（Cytophaga)、鏈球菌屬 (Streptococcus)、乳球菌屬（Lactococcus)或愛德華氏菌屬 (Edwardsiella) 〇 知名之具商業重要性的魚病原菌之實例爲如泰國專利 申請案TH92 840中所描述之最近發現引起大肚症候群（Big 200843789200843789 IX. Description of the Invention [Technical Field] The present invention relates to the use of a bacterium of the genus Trichoderma for the manufacture of a vaccine for combating non-fertilizing bacterial infections of fish. [Prior Art] The amount of fish consumed worldwide has increased significantly over the past few decades. Ingested cold-water fish (such as salmon, turbot, flounder and squid) and tropical fish (such as Asian sea bass, tilapia, milkfish, red snapper, amber, Both spotted fish and sea bream are so increased. Therefore, it can be seen that the number and scale of fish farms are increasing to meet the increasing demand of the market. For example, from the livestock industry, a large number of animals are vulnerable to the invasion of all kinds of diseases when they live together intensively, and even those diseases that few people know or see or even never know before starting a large-scale commercial farm. This is also the case in fish farms. Bacteria found to cause disease in fish, such as Vibrio, Photobacterium damselae, Tenacibaculum, Flavobacterium, Flexibacter, An example of a commercially important fish pathogen of the genus Cytophaga, Streptococcus, Lactococcus or Edwardsiella is as described in the Thai patent application TH92 840. Description of the recent findings of the cause of the big belly syndrome (Big 200843789
Belly syndrome)的細菌（此新穎細菌之一種實例（BB E3F1) 已存放於 Collection National ede Cultures deBelly syndrome) (an example of this novel bacterium (BB E3F1) has been deposited in Collection National ede Cultures de
Microorganisms(CNCM)5Institut Pasteur，25 Rue du Docteur Reoux，F-75724 Paris Cedex 15,法國，存放編號 CNCM 1-3257)、鰻弧菌（Vibrio anguillarum)、美人魚發光 桿菌殺魚亞種(Photobacterium damselae subspecies piscicida)、海屈撓桿菌（Tenacibaculummaritimum)、柱狀 g 黃桿菌（Flavobacterium columnare)、海膝鏈球菌 (Streptococcus iniae)、艱難鏈球菌(Streptococcus difficile)、無乳鏈球菌（Streptococcus agalactiae)、停乳 鏈球菌（Streptococcus dysgalactiae)、格氏乳球菌 (Lactococcus garviae)、遲鈍愛德華氏菌（Edwardsiella 1汪1：(1&)及鲶魚愛德華氏菌（£(1>¥&1(15丨611&丨(^&1111^)。 這些細菌對至少一種下列品種之魚具致病性：紅鮒 魚、琥珀鰺、嘉鱲魚、亞洲海鱸魚、斑魚、紅鯛、吳郭 φ 魚、鯰魚及鯉魚。 疫苗可用來拮抗某些這些病原，然而，並無拮抗其他 病原之疫苗。在這類情況中，以抗生素治療爲拮抗感染僅 有之治療法。目前爲止，大部分抗生素之用途爲治療性。 預防性治療包括，如在種魚產卵前爲其腹膜內注射抗生素 並在卵硬化期間在水中倂入抗生素。 然而，從生態學觀點，因爲有對不同抗生素之抗性的 報告之事實，使用抗生素並非較佳方法。 顯然地，對於新穎、更有效之拮抗魚致病性細菌的疫 -5- 200843789 苗確實有需要。再者，能擁有保護魚而拮抗數種細菌菌種 之多價疫苗甚至更佳。 本發明的目的之一係提供這類疫苗。 【發明內容】 令人驚訝地，現在發現：土壤絲菌屬之細菌可在魚中 提供顯著之拮抗非土壤絲菌型魚致病性革蘭氏陰性細菌的 Φ 保護水準。 此在魚中拮抗非土壤絲菌型魚致病性細菌之保護作用 甚至可在無非土壤絲菌型魚致病性細菌之存在下藉由包含 土壤絲菌之疫苗誘導出。 胃 再者，當將土壤絲菌以菌苗（b a c t e r i η)及/或活減毒形 式投給時，其可提供此保護作用。 此保護作用之一種吸引人之處在於當經由口服投給細 菌或藉由浸沒法接種疫苗時亦可取得此保護作用。 φ 更令人驚訝地，即使這類浸沒疫苗中包含爲去活化形 式之土壤絲菌時亦可取得顯著之保護水準。一般而言，與 利用活減毒細菌進行浸沒疫苗接種相較下，以去活化之細 菌進行浸沒疫苗接種的效果明顯較差。 此技藝（如 NL 73 06964、DT 2713-680、ΒΕ-86 1 -782) 中已曾描述土壤絲菌在哺乳動物中作爲佐劑（即，免疫系 統之一般，但非特異性刺激劑）之效果。然而，首先，此 點僅有在哺乳動物中之描述。據吾人所知，未曾有在魚中 之此現象的描述，最可能的原因哺乳動物和魚之免疫系統 -6- 200843789 非常不同。 再者，即使在哺乳動物中，爲了保護動物拮抗特殊抗 原（如：細菌），這類由土壤絲菌成分所誘導之一般但非特 異性刺激僅可與該特殊抗原一起使用。於該觀點中，土壤 絲菌成分僅爲傳統佐劑之另一形式。 僅供作爲例不’當將土壤絲菌成分與拮抗細菌疾病之 疫苗一起投服時，預期土壤絲菌成分可改良對疫苗之免疫 • 反應。當然，不能期待土壤絲菌成分可在無疫苗存在時提 供拮抗細菌疾病之保護作用，即取代疫苗。 土壤絲菌屬細菌可提供顯著之拮抗非土壤絲菌型魚致 病性細菌的保護水準這種出人意表之發現背後的作用機制 * 目前仍未知。然而，可假定菌體表面存有或附有一種所有 土壤絲所共有之成分，此成分爲在魚中拮抗其他細菌之交 叉特異性免疫力的強力刺激劑。魚中之交叉特異性免疫力 在拮抗革蘭氏陰性細菌（諸如弧菌、美人魚發光桿菌、屈 φ 撓桿菌、黃桿菌、黏液菌或愛德華氏菌）上最顯著。此觀 點中，交叉特異性意指：由土壤絲菌誘導並提供拮抗非土 壤絲菌種之保護作用。 因此，本發明關於土壤絲菌屬之細菌於製造用於對抗 魚中非土壤絲菌型革蘭氏陰性細菌感染之疫苗上的用途。 供製造這類疫苗時，細菌之狀態（存活或去活化）並不 真的重要。重要的是該在魚中拮抗非土壤絲菌型魚致病性 細菌之交叉特異性免疫力的刺激劑仍然存在的事實。此可 經由使用全細菌製品確定。如上述，製品中之細菌爲存 200843789 活、死亡或甚至是切成片段（如：將其通過法式細胞破碎 器壓碎）並不重要。 活減毒細菌非常合適，因爲其確定攜帶刺激該拮抗非 土壤絲菌型魚致病性細菌之交叉特異性免疫力的因子。活 減毒細菌優於菌苗之處在於其可容易投給而不需佐劑。再 者，其自行複製至某種程度直至免疫系統令其停止，因 此，投給之菌體數可較少。因此，於一較佳形式中，本發 B 明關於根據本發明之土壤絲菌屬之細菌的用途，其中該土 壤絲菌爲活減毒土壤絲菌。 另一方面，當這些細菌爲菌苗形式時，刺激該拮抗非 土壤絲菌型魚致病性細菌之交叉特異性免疫力的因子亦存 β 於細菌上。菌苗優於活減毒細菌之處在於其非常安全。 因此，於一等佳之形式中，本發明關於根據本發明之 土壤絲菌屬之細菌的用途，其中該土壤絲菌爲菌苗之形 式。 # 本技藝已知數種土壤絲菌屬之菌種，如：星形土壤絲 菌（N.asteroides)、殺鮭土 壤絲菌（N.salmonicida)、克索特 土 壤絲菌（N.crassostreae)、巴西土 壤絲菌（N.brasiliensis )、羅多克倫土壤絲菌（N.rhodocrans)、暗色土壤絲菌 (N.opaca)、紅色土壤絲菌（N.rubra)及獅土壤絲菌 (N.seriolae)(正式名稱爲卡帕其土壤絲菌（N.kampachi))。 於一較佳之形式中，供根據本發明使用之土壤絲菌種 爲鯽土壤絲菌。 較佳地，製造之疫苗係用於對抗魚之非土壤絲菌感 -8- 200843789 染，其中引起該非土壤絲菌型細菌感染之細菌爲引起大肚 症候群（Big Belly syndrome)之細菌或弧菌屬、美人魚發 光桿菌、屈撓桿菌屬、黃桿菌屬、黏液菌屬或愛德華氏菌 屬之魚致病性細菌。 更佳地，製造之疫苗係用於對抗魚之非土壤絲菌感 染，其中引起該非土壤絲菌型細菌感染之細菌爲引起大肚 症候群之細菌、鰻弧菌、美人魚發光桿菌殺魚亞種、海屈 φ 撓桿菌、柱狀黃桿菌、海黏液菌、遲鈍愛德華氏菌或鯰魚 愛德華氏菌。 最佳地，製造之疫苗係用於對抗魚之非土壤絲菌感 染，其中引起該非土壤絲菌型細菌感染之細菌爲引起大肚 ' 症候群之細菌或柱狀黃桿菌。 根據本發明製造之疫苗可根據本技藝之技術人士所熟 知的技術從細菌培養開始製備。關於魚疫苗及其製造方法 之評論文章有，如：S 〇 m m e r s e t，I ·，K r 〇 s s 0 y，B ·，B i e r i n g 5 E.and Frost?P.in Expert Review of Vaccines 4 : 89-101 (2005)，Buchmann，K.，Lindenstr0m，T. and Bresciani，in J.Acta P arasitologica 46 · 71-81(2001)Vinitnantharat? S . 5Gravningen，K . and Greger,E. in Advances in veterinary medicine 41 : 53 9-550( 1 999)及 Anderson，D.P· in Developments in Biological Standardization 90 ·· 2 5 7-265( 1 997)所著者。 活減毒細菌爲較其野生型對應菌種不具致病性，但仍 可誘出有效之免疫反應之細菌。 -9- 200843789 減毒菌株可順著本技藝長久所知之習知途徑，諸如化 學致哭變、UV-放射線照射等或定點突變（site-directed mutagenesis)取得。 此處定義之菌苗爲一種去活化形式。用於去活化之方 法顯示出與菌苗之活性並無相關性。用於去活化之習知方 法（諸如熱處理，以福馬林、二乙烯亞胺、硫柳汞，等處 理，這些均爲本技藝所熟知之處理）均同等適用。利用例 如法式細胞破碎器，藉由物理壓力將細菌去活化可提供同 樣適合用於製造根據本發明之疫苗的起始物質。 基本上，根據本發明製造之疫苗包含供根據本發明使 用之有效量的細菌及藥學上可接受之載體。此文所使用之 “有效”一詞係定義爲足夠在靶的魚中誘出免疫反應的 量。 所投給之土壤絲菌菌體的量將取決於投服途徑 '是否 存有佐劑及投給時機。 此外，本技藝之技術熟習人士可在上述參考資料及下 列資訊中（尤其是實例中）找到足夠之指導。 一般而言’以菌苗爲基礎之根據本發明製造的疫田可 藉由注射投給1〇3至101G隻細菌（宜爲1〇6至1〇9’更宜爲 介於108和109之間）之劑量。超過iO10細菌之劑量雖然 在免疫學上適合使用，但在商業理由上較無吸引力。在根 據本發明製造及供口服之疫苗中的細菌量方面’下列實例 將提供充足之指導。 然而，在土壤絲菌之情況中’由於菌體之分支特性’ -10- 200843789 因此很難藉由計算總菌體數來正確測定在一體積內之土壤 絲菌菌體數。因此，從實例中亦顯示出抗原濃度係以 ODU/耄升表示。ODU/毫升係依下述測定：抗原濃度 (ODU/毫升）=(((〇D66〇).l +(〇D660).2)/2)-0.2118)/0.〇〇18x DFxlO6，其中（〇D66Q).l+(〇D66()).2 爲二次 〇D66q 測量之 〇D66〇値且其中DF爲稀釋因子。 以活減毒細菌爲基礎之根據本發明製造的疫苗可以較 φ 低劑量投給，因爲該細菌在投給後仍將持續複製一段時 間。 適合用於根據本發明使用之疫苗中的藥學上可接受之 載體的實例有無菌水、生理食鹽水、水性緩衝劑（諸如 ^ PBS)等。另外，根據本發明之疫苗可含有其他添加劑，諸 如佐劑、安定劑、抗氧化劑及其他如下述之添加劑。 供根據本發明使用之疫苗（尤其是含有菌苗者）亦可含 有稱爲佐劑之免疫刺激物質，以較佳之形式呈現。一般而 φ 言，佐劑包含以非特異性方式增強宿主之免疫反應的物 質。本技藝已知多種不同之佐劑。常用於魚及貝類養殖場 之佐劑的實例爲胞壁醯二肽、脂多醣、數種葡聚醣及聚醣 和卡波姆（Carb οροί)®。適合用於魚及貝類疫苗之佐劑的大 規模檢視列於 Jan Raa(Reviews in Fisheries Science 4(3): 229-288(1 996))之評論文章中。 疫苗亦可包含“載劑”。載劑爲細菌黏附至其上，而 不需與其共價結合之化合物。這類載劑爲，如生物微膠 囊、微-藻酸鹽、脂質體及大分子物（macrosols)(此均爲本 -11 - 200843789 技藝所已知）。 這類載劑之一種特殊形式（其中該抗原部分包埋在載 劑中）係稱爲ISCOM(歐洲專利案EP 109.942、EP 1 80.564、EP 242.3 80) ° 另外，疫苗可包含一或多種合適之界面活性化合物或 乳化劑，如司邦（Span)或吐溫（Tween)。 適合用於油包水乳劑之油性佐劑爲，如礦物油或可代 謝之油。礦物油爲，如Bayol®、Marcol®及Drakeol®。 非礦物油佐劑之實例爲，如Montanide-IS.A-763 -A。 可代謝之油有，如蔬菜油（諸如花生油及大豆油），動物油 (諸如魚油角鯊烷及角鯊烯）和生育醇及其衍生物。 合適之佐劑爲，如w/o乳劑、ο/w乳劑及w/o/w複乳 劑。 以水爲基礎之奈米粒佐劑的實例爲，如Montanide-IMS-22 1 2。 通常，疫苗與安定劑（如防止有降解傾向之蛋白質降 解）混合，以增強疫苗之耐儲時間或改良凍乾之效率。可 用之安定劑爲，如 SPGA((Bovarnik et al; J.Bacteriology 5 9 : 5 09( 1 95 0))、碳水化合物（如山梨醇、甘露醇、海藻 糖、澱粉、蔗糖、葡萄聚糖或葡萄糖）、蛋白質（諸如白蛋 白或酪蛋白或其降解產物）及緩衝劑（諸如鹼金屬磷酸 鹽）。 另外，可將疫苗懸浮在生理學上可接受之稀釋劑中。 不言而喻，輔助之其他方式、加入載劑成分或稀釋劑、乳 -12- 200843789 化或穩定蛋白質亦包含在本發明中。 可應用之本投服方法有多種，均爲技藝所已知。根據 本發明使用之土壤絲菌疫苗宜經由注射、浸沒、浸泡或經 由口服投給。 僅供作爲例示，若現在用來製造對抗魚之非土壤絲菌 感染之疫苗的土壤絲菌爲，如活減毒細菌，則因投服容 易，該疫苗可，如經由浸沒或浸泡疫苗接種法投服。這類 Φ 疫苗通常係藉由浸沒接種法來施用。 另一方面，若現在用來製造對抗魚之非土壤絲菌感染 之疫苗的土壤絲菌爲菌苗形式，則口服及，如經腹膜內途 徑施用爲具吸引力之投服方法。尤其是，在經腹膜內途徑 β 施用之情況中宜存有佐劑。 一般而言，若可經由加入佐劑來改良疫苗，則投給方 法宜爲經腹膜內途徑投給。從免疫學觀點來看，以菌苗經 腹膜內途徑接種疫苗爲非常有效之接種疫苗之途徑，尤其 • 是因爲可倂入佐劑。 利用去活化細菌之浸沒接種疫苗法即使無佐劑存在時 仍顯示出可提供令人驚訝之拮抗非土壤絲菌型魚致病性革 蘭氏陰性細菌之免疫水準。 此投給途徑由於容易投服疫苗因而較佳。 投服之擬定計劃可根據標準之接種疫苗操作令其最優 化。本技藝之技術熟習人士可知如何處理或者可在上述文 章中找到指導。 欲接受疫苗接種之魚的年齡並無嚴格限制，但很清楚 -13- 200843789 地，個人會希望儘可能早地（即在暴露於病原之前）進行拮 抗非土壤絲菌型魚致病性細菌之疫苗接種。 一般而言，以上述之土壤絲菌爲基礎的疫苗係在其正 常應投服之時刻投服，以防止被非土壤絲菌型革蘭氏陰性 細菌感染。實際上，此表示魚係在儘可能早之時期進行疫 苗接種。 特別是，當魚仍小時（如介於2及5克之間），浸沒疫 φ 苗接種法爲接種疫苗之選擇。若需要或想要時，5克以上 之魚亦可藉由注射來接種疫苗。 在經口投服方面，疫苗宜與適合口服之載體（即，纖 維素、食物或可代謝之物質，諸如α-纖維素或不同之植 * 物或動物來源的油）混合。將疫苗投至高濃度之生物餌 料’再將生物餌料銀魚亦爲一種吸引人之方法。供口服遞 送根據本發明之疫苗的特佳食物載體爲可將疫苗包囊在其 中之生物餌料。 • 較佳地，供根據本發明使用之土壤絲菌爲鯽土壤絲菌 種。 除了 土壤絲菌外亦加上至少一種非土壤絲菌型魚致病 性微生物或病毒、這類微生物或病毒之抗原或編碼這類抗 原之遺傳物質來製造疫苗是有利的。 合倂使用土壌絲菌及至少一種非土壤絲菌型魚致病性 細菌來製造疫苗可具有建立較持久之拮抗該非土壤絲菌型 魚致病性細菌的特殊保護作用之利處。 合倂使用土壤絲菌及至少一種魚致病性病毒來製造疫 -14 - 200843789 苗可具有同時取得拮抗細菌感染及拮抗該魚致病性病毒之 感染的保護作用之利處。 因此，本較佳體系之較佳形式係關於土壤絲菌及至少 一種非土壤絲菌型魚致病性微生物或魚致病性病毒、這類 微生物或病毒之抗原或編碼這類抗原之遺傳物質於製造疫 苗之用途。 市售之重要的熱帶及/或地中海魚的魚病原之實例有 Φ 鰻弧菌、美人魚發光桿菌殺魚亞種、海屈撓桿菌、黃桿菌 種、黏液菌種、格氏乳球菌、遲鈍愛德華氏菌、鯰魚愛德 華氏菌、海豚鏈球菌、艱難鏈球菌、無乳鏈球菌、停乳鏈 ~ 球菌、病毒性出血性敗血病病毒、病毒性壞死病毒、虹彩 ‘病毒（iridovirus)及錦鯉疱瘆病毒。 因此，本較佳體系之更佳形式中，其他微生物或病毒 係選自下列之魚病原：鰻弧菌、美人魚發光桿菌殺魚亞 種、海屈撓桿菌、黃桿菌種、黏液菌種、格氏乳球菌、遲 φ 鈍愛德華氏菌、鯰魚愛德華氏菌、海豚鏈球菌、艱難鏈球 菌、無乳鏈球菌、停乳鏈球菌、病毒性出血性敗血病病 毒、病毒性壞死病毒、鯉春季病毒血症病毒或錦鯉疱疹病 毒。 下列實例提供大量如何使用土壤絲菌屬之細菌來製造 用於對抗魚之非土壤絲菌感染的疫苗的指導。 【實施方式】 實例1 : -15- 200843789 由測試菌種引起之大肚感染（亦稱爲BB感染，見上述 參考資料）爲亞洲海鱸幼魚養殖場中主要的感染之一。絕 大多數之BB-病爆發係發生在培養過程中之稍早階段。 BB-培養 以0 · 5 %福馬林將生長在巧克力瓊脂上且收成在胰蛋 白腺/酵母菌肉湯中之BB-菌培養去活化，測得之〇D66〇爲 0.5。未計算總菌體數或進行其他定量方法主要係由於所 討論之菌體具有強多形性性質。 鯽土壤絲菌之培養 使用以福馬林去活化之全菌體發酵槽製造之鯽土壤絲 菌（9.1xl08ODU/毫升）作爲免疫刺激抗原。 所使用之鯽土壤絲菌抗原係衍生自鯽土壤絲菌之野生 株。依下述測定鯽土壤絲菌抗原之抗原濃度： 抗原濃度（ODU/毫升）= (((Οϋ66〇)·1 +(OD66()).2)/2) -0.2118)/0.0018xDFxl06，其中（OD66〇).l+( OD66〇).2 爲不 同批之同一獅土壤絲菌且DF爲稀釋因子。 此批之抗原強度爲9.1xl08ODU/毫升。 鯛土壤絲菌亦稱爲免疫刺激劑（IS)。 下文中，BB-培養及土壤絲菌-培養稱爲“抗原”。 使用之動物 使用在實驗開始時平均體重爲0.5克之亞洲海鱸 -16- 200843789 (Lates calcarifer)。將魚安置於26 土 2°C之全濃度海水中， 最大密度爲20公斤/米3魚槽體積。 自檢疫槽收集隨手取得之二組110隻魚並依序分配在 各別組中。將魚浸沒並置於分派之半槽內。魚並未個別標 不 ° 浸沒處置 • 在接種疫苗前令魚飢餓24小時。捕捉每組1 1 0隻魚 並轉移至各別處置組中。給予之處置、分派之槽及處置之 名稱列於表1中。B B -抗原及土壤絲菌二種抗原溶液分別 稀釋約1 0及1 0 0倍。 將魚浸沒在迨些最終懸浮夜中1 5分鐘，此時，監測 pH値、02及溫度。浸沒後將魚轉移至如表1中指示之其 指定槽中。 • 表1 :所使用之處置及分派之槽_ 處理 體積 ，•抗原丨· 體積 海水 魚數 槽 BB-疫苗(BB) 140毫升 1，360毫升 110 5F05 免疫刺激劑(IS) 50毫升 4,950毫升 110 5F03 對照組 - 5,000毫升 110 5F04 挑戰培養之製備方法 將B B -株分種在巧克力瓊脂上並在2 6 °C培育一整夜。 接著，將長成之菌收集在0.22微米無菌過濾之海水中。 將所產生之懸浮液調整至〇〇66〇爲1.5 73並以此懸浮液作 -17- 200843789 爲最高挑戰懸浮液。另外，在無菌海水中自此起始懸浮液 再製備4種1 0倍稀釋液並用來注射魚。 挑戰 浸沒處理後3週，用網自每一條件之處置組（BB; 土 壌絲菌或對照組）捕捉1 0至1 2隻魚，在A q u i - S中麻醉並 經腹膜內途徑注射〇· 1毫升製備好之稀釋液。注射後立即 Φ 將魚轉移至其指定之槽部分，再使其自麻醉中回復。 每一挑戰稀釋液之所有三注射組（即，包含1 0隻浸沒 於BB之魚的組、包含1 0隻浸沒於土壤絲菌之魚的組及 包含1 0 -1 2隻對照組魚之組）均置於同一槽內，所有注射 * 相同之挑戰懸浮液者均置於同一槽中，其中此三組係藉由 置於槽中之垂直分隔板保持分隔。 共使用5種挑戰濃度，範圍係從未稀釋之懸浮液（稱 爲“純液”）至稀釋10,000倍之純液。 死亡率之觀察 每日觀察魚共3週且每日至少收集一次死亡或明顯瀕 臨死亡之魚。檢查出現典型之大肚病病徵之魚的代表數並 檢查內臟器官之吉姆薩（Giemsa)染色（其在受BB感染之魚 中通常爲成塊混染）以確定引起發病之器官。在21天觀察 期結束時收集所有剩餘之魚並評估出現典型之B B病病徵 的情況。若出現病徵時，這些魚在評估結果中便歸類爲 “ BB陽性”。 -18- 200843789 結果 結果之解釋 根據下式計算相關之存活百分比（RPS)來評估二組處 置組（BB及土壤絲菌）與對照組相較下之死亡率： RPS = 接種疫苗組中之％死亡率 ~~if照,組之％死亡率~ }χ100 挑戰後之死亡率 在每一挑戰劑量下於不同處置組及對照組中每曰觀察 到之死亡率呈現於第1圖中（5個連續圖）。 ' 該5個連續圖明確顯示出挑戰劑量與第一次死亡出現 之間的明確相關性。另外，使用較高劑量可造成1 00%死 亡率，然而，在較低劑量組中，至少在一些組中有些魚通 過挑戰存活下來。 # 從這些圖中之第三個觀察結果爲：在大部分情況中， BB-浸沒組顯示出最高峰之死亡率且在BB組中此高峰通 常較其他組早出現。 所有死亡之魚清楚顯示出典型之大肚病病徵且在所有 魚樣本中明顯出現BB-菌。 效力之評估 根據所得之死亡率圖形，計算每一條件之RP S値。 結果表示於第2圖中。 -19- 200843789 結果顯示出在二組使用最低之挑戰濃度組中取得保護 作用’其中累積之對照組死亡率範圍係在5 8.3 - 8 3.3 %。 (在3個最高劑量中之累積的對照組死亡率造成對照組 100%死亡率）。再者，BB處置組顯示出負RPS値，此表 示透過浸沒法以BB-抗原處置時對於接下去之挑戰具負面 效果。 與獅土壤絲菌抗原一起浸泡可明確誘出拮抗BB挑戰 φ 之保護作用。此點非常値得注意，因爲免疫刺激係在挑戰 前三週進行且刺激係透過浸沒，以去活化之鯽土壤絲菌進 行，而挑戰透過注射進行時會造成對照組高死亡率。 • 實例2 : 由柱狀黃桿菌引起之“魚類爛鰓病”爲引起大部分培 養之淡水魚種死亡的主要疾病。吳郭魚爲極易受影響之品 種，本實例使用其作爲標靶品種。 φ 使用以福馬林去活化之全菌體發酵槽製造之獅土壤絲 菌（9x1 08ODU/毫升）作爲非特異性免疫刺激劑（亦稱爲免疫 刺激劑）。 挑戰菌株 自美國密西西比州運河鯰分離出野生柱狀黃桿菌分離 物。 自印尼吳郭魚分離出野生柱狀黃桿菌分離物。 -20- 200843789 表2 :免疫刺激劑濃度、使用之魚數、施用時間及免Microorganisms (CNCM) 5 Institut Pasteur, 25 Rue du Docteur Reoux, F-75724 Paris Cedex 15, France, storage number CNCM 1-3257), Vibrio anguillarum, Photobacterium damselae subspecies piscicida ), Tenacibaculummaritimum, Flavobacterium columnare, Streptococcus iniae, Streptococcus difficile, Streptococcus agalactiae, Streptococcus dysgalactiae (Streptococcus dysgalactiae), Lactococcus garviae, Edward Edwards (Edwardsiella 1 (1&) and E. sinensis (£(1>¥&1(15丨611&丨(( ^&1111^) These bacteria are pathogenic to at least one of the following species: red snapper, amber, Jiayu, Asian sea bass, spotted fish, red dragonfly, Wu Guo φ fish, squid and squid Vaccines can be used to antagonize certain of these pathogens, however, there are no vaccines that antagonize other pathogens. In such cases, antibiotic treatment is used to antagonize infections only. Therapeutic methods. Until now, most antibiotics have been used therapeutically. Prophylactic treatment includes, for example, intraperitoneal injection of antibiotics before the fish are laid and injecting antibiotics into the water during egg hardening. However, from an ecological point of view Because of the fact that there are reports of resistance to different antibiotics, the use of antibiotics is not a preferred method. Obviously, there is a need for a new and more effective vaccine against the pathogenic bacteria of fish -5-200843789. It is even more preferable to have a multivalent vaccine that protects fish against several bacterial species. One of the objects of the present invention is to provide such a vaccine. [Summary of the Invention] Surprisingly, it has now been found that bacteria of the genus Geobacteria can be Provides significant antagonism in the fish against the Φ protection level of pathogenic Gram-negative bacteria in non-fertilizing strains of fish. This antagonizes the protection of pathogenic bacteria in non-fertilizing strains of fish in fish even in soil without soil In the presence of bacterial pathogenic bacteria, it is induced by a vaccine containing soil bacterium. In the stomach, when the soil bacterium is bacterily (bacteri η) and/or live When administered to a toxic form, which may provide this protection. At an attractive protective effect of this is that when administered via the oral bacteria or by immersion vaccination can achieve this protection. φ Even more surprisingly, even if such immersion vaccines contain soil strains of deactivated form, significant levels of protection can be achieved. In general, immersion vaccination with deactivated bacteria is significantly less effective than immersion vaccination with live attenuated bacteria. This technique (eg, NL 73 06964, DT 2713-680, ΒΕ-86 1 -782) has been described as an adjuvant in mammals (ie, a general but non-specific stimulator of the immune system). effect. However, first of all, this point is only described in mammals. As far as we know, there has never been a description of this phenomenon in fish, the most likely cause is the very different mammalian and fish immune system -6- 200843789. Furthermore, even in mammals, in order to protect animals against specific antigens (e.g., bacteria), such general but non-specific stimuli induced by soil muscle components can only be used with the particular antigen. In this view, the soil mycelium component is only another form of traditional adjuvant. For example only when the soil flora component is administered with a vaccine against bacterial disease, the soil flora component is expected to improve the immune response to the vaccine. Of course, it is not expected that the soil flora component will provide protection against bacterial disease in the absence of a vaccine, i.e., replace the vaccine. The mechanism behind the unexpected discovery that soil genus bacteria can provide significant protection against the physiologic levels of non-fertilizing bacteria-derived bacteria is still unknown. However, it can be assumed that the surface of the cells is present or attached with a component common to all soil filaments, which is a potent stimulator that antagonizes the cross-specific immunity of other bacteria in fish. Cross-specific immunity in fish is most pronounced on antagonizing Gram-negative bacteria such as Vibrio, mermaid, bacterium, bacterium, bacterium, or bacterium. In this view, cross-specificity means that it is induced by soil bacterium and provides protection against non-soil species. Accordingly, the present invention relates to the use of a bacterium of the genus Trichoderma for the manufacture of a vaccine for combating a non-fertilizing gram-negative bacterial infection in fish. The state of the bacteria (survival or deactivation) is not really important when making such vaccines. Importantly, this stimulator that antagonizes the cross-specific immunity of non-fertilizing strains of pathogenic bacteria in fish still exists. This can be determined by using a whole bacterial preparation. As mentioned above, it is not important that the bacteria in the product survive, die or even cut into pieces (eg, crush them through a French cell disruptor). Live attenuated bacteria are very suitable because they are determined to carry a factor that stimulates the cross-specific immunity that antagonizes the pathogenic bacteria of non-fertilizing fish. Live attenuated bacteria are superior to vaccines in that they can be easily administered without adjuvant. Furthermore, it replicates to some extent until the immune system stops it, so the number of cells administered can be reduced. Accordingly, in a preferred form, the invention relates to the use of a bacterium of the genus Geotrichum according to the invention, wherein the soil bacterium is a live attenuated soil bacterium. On the other hand, when these bacteria are in the form of a bacterin, the factor which stimulates the cross-specific immunity against the pathogenic bacteria of the non-soil bacterium is also present on the bacteria. The vaccine is superior to live attenuated bacteria in that it is very safe. Accordingly, in a preferred form, the invention relates to the use of a bacterium of the genus Geotrichum according to the invention, wherein the soil bacterium is in the form of a bacterium. # This technique is known for several species of soil genus, such as N. asteroides, N. salmonicida, and N. crassostreae. , N. brasiliensis, N. rhodocrans, N. opaca, N. ruba, and lion soil bacteria (N) .seriolae) (formally known as N. kampachi). In a preferred form, the soil strain for use in accordance with the present invention is Agrobacterium tumefaciens. Preferably, the vaccine is prepared for combating non-soil bacterium feelings of fish - 8 - 1987 789 789, wherein the bacteria causing the infection of the non-soil bacterium are bacteria or Vibrio causing Big Belly syndrome A faecal pathogenic bacterium of the genus, the mermaid luminescent bacterium, the genus Curvus, the genus Flavobacterium, the genus Mucus or the genus Edwards. More preferably, the vaccine is used for combating non-soil bacterium infection of fish, wherein the bacteria causing the infection of the non-soil bacterium are the bacteria causing the big belly syndrome, Vibrio anguillarum, mermaid bacterium, subsp. Helicobacter pylori, Flavobacterium columnum, sea slime, retarded Edwards or E. sinensis. Most preferably, the vaccine is produced for combating non-soil bacterium infection of fish, wherein the bacterium causing the infection of the non-fertilizing bacterium is a bacterium causing the 'big belly' syndrome or a bacterium of the bacterium. Vaccines made in accordance with the present invention can be prepared starting from bacterial culture according to techniques well known to those skilled in the art. Comments on fish vaccines and their methods of manufacture are: S 〇mmerset, I ·, K r 〇ss 0 y, B ·, Biering 5 E. and Frost? P.in Expert Review of Vaccines 4 : 89- 101 (2005), Buchmann, K., Lindenstr0m, T. and Bresciani, in J. Acta P arasitologica 46 · 71-81 (2001) Vinitnantharat? S. 5 Gravningen, K. and Greger, E. in Advances in veterinary medicine 41 : 53 9-550 (1 999) and Anderson, DP·in Developments in Biological Standardization 90 ·· 2 5 7-265 (1 997). Live attenuated bacteria are bacteria that are less pathogenic than their wild-type counterparts, but can still induce an effective immune response. -9- 200843789 Attenuated strains can be obtained by conventional means known in the art, such as chemical crying, UV-radiation, or site-directed mutagenesis. The bacterin defined herein is in a deactivated form. The method used for deactivation showed no correlation with the activity of the vaccine. Conventional methods for deactivation (such as heat treatment, treatment with formalin, diethyleneimine, thimerosal, etc., which are well known in the art) are equally applicable. Deactivation of the bacteria by physical pressure, such as a French cell disruptor, can provide a starting material that is equally suitable for use in the manufacture of a vaccine according to the invention. Essentially, a vaccine made in accordance with the present invention comprises an effective amount of a bacterium and a pharmaceutically acceptable carrier for use in accordance with the present invention. The term "effective" as used herein is defined as an amount sufficient to elicit an immune response in a target fish. The amount of soil mycelia that is administered will depend on the route of administration 'whether there is an adjuvant and the timing of the administration. In addition, those skilled in the art can find sufficient guidance in the above references and in the following information (especially in the examples). In general, a vaccine produced according to the present invention based on the vaccine can be administered by injection of from 1 to 3 to 101 G bacteria (preferably from 1 to 6 to 1 9), preferably between 108 and 109. Dosage). Dose over iO10 bacteria, although immunologically suitable for use, is less attractive for commercial reasons. The following examples will provide sufficient guidance in terms of the amount of bacteria in a vaccine manufactured and supplied orally according to the present invention. However, in the case of soil bacterium, 'because of the branching characteristics of the bacterium' -10- 200843789, it is therefore difficult to accurately determine the number of soil bacterium in a volume by calculating the total number of cells. Therefore, it is also shown from the examples that the antigen concentration is expressed in ODU/耄. ODU/ml is determined by the following antigen concentration (ODU/ml) = (((〇D66〇).l +(〇D660).2)/2)-0.2118)/0.〇〇18x DFxlO6, where 〇D66Q).l+(〇D66()).2 is the 〇D66〇値 measured by the secondary 〇D66q and where DF is the dilution factor. A vaccine made in accordance with the present invention based on live attenuated bacteria can be administered at a lower dose than φ because the bacteria will continue to replicate for a period of time after administration. Examples of pharmaceutically acceptable carriers suitable for use in a vaccine for use in accordance with the present invention are sterile water, physiological saline, aqueous buffers such as PBS, and the like. Further, the vaccine according to the present invention may contain other additives such as an adjuvant, a stabilizer, an antioxidant, and other additives as described below. Vaccines for use in accordance with the present invention (especially those containing bacterins) may also contain an immunostimulating substance called an adjuvant, which is preferably presented. Generally, φ, an adjuvant contains a substance that enhances the host's immune response in a non-specific manner. A variety of different adjuvants are known in the art. Examples of adjuvants commonly used in fish and shellfish farms are cell wall dipeptides, lipopolysaccharides, several glucans and glycans, and Carb οροί®. A large-scale review of adjuvants suitable for use in fish and shellfish vaccines is listed in a review article by Jan Raa (Reviews in Fisheries Science 4(3): 229-288 (1 996)). Vaccines may also contain "carriers." The carrier is a compound to which the bacteria adhere, without the need to covalently bind thereto. Such carriers are, for example, biological microcapsules, microalginates, liposomes, and macrosols (as known in the art of this -11-200843789). A particular form of such a carrier, in which the antigen is partially embedded in the carrier, is referred to as ISCOM (European Patent Publication EP 109.942, EP 1 80.564, EP 242.3 80). Additionally, the vaccine may comprise one or more suitable An interfacially active compound or emulsifier such as Span or Tween. Oily adjuvants suitable for use in water-in-oil emulsions are, for example, mineral oils or oils which are identifiable. Mineral oils such as Bayol®, Marcol® and Drakeol®. An example of a non-mineral oil adjuvant is, for example, Montanide-IS.A-763-A. Metabolizable oils such as vegetable oils (such as peanut oil and soybean oil), animal oils (such as fish oil squalane and squalene) and tocopherols and their derivatives. Suitable adjuvants are, for example, w/o emulsions, ο/w emulsions and w/o/w double emulsions. An example of a water-based nanoparticle adjuvant is, for example, Montanide-IMS-22 1 2 . Typically, the vaccine is mixed with a stabilizer (e.g., to prevent degradation of the protein with a tendency to degrade) to enhance the shelf life of the vaccine or to improve the efficiency of lyophilization. The stabilizers that can be used are, for example, SPGA ((Bovarnik et al; J. Bacteriology 5 9 : 5 09 (1 95 0)), carbohydrates (such as sorbitol, mannitol, trehalose, starch, sucrose, dextran or Glucose), protein (such as albumin or casein or its degradation products) and buffer (such as alkali metal phosphate). In addition, the vaccine can be suspended in a physiologically acceptable diluent. Other means, addition of carrier ingredients or diluents, milk-12-200843789 or stabilized proteins are also encompassed by the present invention. There are a variety of methods of administration that are applicable, and are known in the art. The soil bacterium vaccine should be administered by injection, immersion, soaking or by oral administration. For example only, if the soil bacterium that is currently used to make a vaccine against non-soil bacterium infection of fish is, for example, live attenuated bacteria, Because of the ease of administration, the vaccine can be administered, for example, via immersion or immersion vaccination. Such Φ vaccines are usually administered by immersion inoculation. On the other hand, if used now to fight against fish The soil strain of the soil filaria infection vaccine is in the form of bacterin, and oral administration and, if administered by the intraperitoneal route, are attractive methods of administration. In particular, it should be present in the case of intraperitoneal route β administration. In general, if the vaccine can be modified by the addition of an adjuvant, the administration method is preferably administered intraperitoneally. From the immunological point of view, it is very effective to vaccinate the vaccine via the intraperitoneal route. The vaccination route, in particular, is because the adjuvant can be invaded. The immersion vaccination method using deactivated bacteria shows that it can provide surprisingly antagonistic non-soil bacterium-type pathogenic Gram even in the absence of adjuvant. The immunization level of the negative bacteria. This route of administration is preferred because of the ease of administration of the vaccine. The proposed plan for the administration can be optimized according to standard vaccination procedures. Those skilled in the art will know how to deal with or can The article finds guidance. The age of the fish to be vaccinated is not strictly limited, but it is clear that -13-43,739,789, individuals will want to be as early as possible (ie Vaccination against pathogenic bacteria other than soil strains before exposure to the pathogen. In general, the above-mentioned soil-based bacteria-based vaccine is administered at a time when it is normally expected to be taken to prevent it from being Non-soil-type gram-negative bacterial infections. In fact, this means that the fish line is vaccinated as early as possible. In particular, when the fish is still small (eg between 2 and 5 grams), immersion φ The seedling inoculation method is the choice of vaccination. If necessary or desired, more than 5 grams of fish can also be vaccinated by injection. In oral administration, the vaccine should be compatible with the carrier suitable for oral administration (ie, cellulose, Food or metabolizable substances, such as alpha-cellulose or oils of different plant or animal origin. Mixing the vaccine to a high concentration of biological bait 'and then using the biological bait silverfish is also an attractive method. A particularly preferred food carrier for oral delivery of a vaccine according to the invention is a biological bait in which the vaccine can be encapsulated. • Preferably, the soil bacterium for use in accordance with the present invention is a soil filth. In addition to soil filariasis, it is advantageous to add at least one non-fertilizing bacterium type pathogenic microorganism or virus, an antigen of such a microorganism or virus, or a genetic material encoding such an antigen to produce a vaccine. The use of soil mites and at least one non-fertilizing bacterium for pathogenic bacteria to produce vaccines may have the advantage of establishing a more durable antagonism against the non-fertilizing bacterium-derived bacteria. Combining the use of soil bacterium and at least one fish-borne virus to produce the disease -14 - 200843789 The seedling can have the protective effect of simultaneously obtaining an antagonistic bacterial infection and antagonizing the infection of the fish-borne virus. Thus, a preferred form of the preferred system relates to soil filature and at least one non-fertilizing bacterium pathogenic microorganism or porcine pathogenic virus, antigens of such microorganisms or viruses, or genetic material encoding such antigens For the purpose of manufacturing vaccines. Examples of fish pathogens of tropical and/or Mediterranean fish that are commercially available are Φ Vibrio cholerae, mermaid bacterium, subtilis, sea bacterium, yellow bacillus, mucus strain, Lactococcus lactis, retarded Edward Salmon, Salmonella elegans, Streptococcus faecalis, Streptococcus mutans, Streptococcus agalactiae, lactating chain ~ cocci, viral hemorrhagic septicemia virus, viral necrosis virus, iridovirus and nectar Prion. Thus, in a preferred form of the preferred system, the other microorganism or virus is selected from the group consisting of the following fish pathogens: Vibrio anguillarum, mermaid photobacterium, subspecies, sea flexor, yellow bacillus, mucus species, Lactococcus, Lactobacillus blunt, E. faecalis, Streptococcus faecalis, Streptococcus mutans, Streptococcus agalactiae, Streptococcus dysgalactiae, Viral hemorrhagic septicemia virus, Viral necrosis virus, 鲤 spring Viremia virus or koi herpes virus. The following examples provide guidance on how to use bacteria of the genus Geotrichum to produce vaccines against non-soil bacterium infections of fish. [Examples] Example 1: -15- 200843789 The big belly infection caused by the test species (also known as BB infection, see above) is one of the major infections in the juvenile fish farms of Asia. The vast majority of BB-disease outbreaks occur at an early stage in the culture process. BB-culture The BB-bacteria grown on the chocolate agar and harvested in the trypsin/yeast broth were deactivated with 0. 5 % of the formalin, and the D66 was determined to be 0.5. The total number of cells not counted or other quantitative methods are mainly due to the strong polymorphic nature of the cells in question. Culture of soil filariasis A soil bacterium (9.1 x 10 08 ODU/ml) manufactured by a full-cell fermentation tank activated by formalin was used as an immunostimulating antigen. The soil sputum antigen system used was derived from a wild strain of Agrobacterium tumefaciens. Determine the antigen concentration of the soil sputum antigen according to the following: antigen concentration (ODU/ml) = (((Οϋ66〇)·1 +(OD66()).2)/2) -0.2118)/0.0018xDFxl06, where OD66〇).l+( OD66〇).2 is a different batch of the same soil soil bacterium and DF is the dilution factor. The antigen intensity of this batch was 9.1 x 108 ODU/ml. Agrobacterium tumefaciens is also known as an immunostimulant (IS). Hereinafter, BB-culture and soil filament-culture are referred to as "antigens". Animals used Asian sea otter -16-200843789 (Lates calcarifer) with an average body weight of 0.5 g at the beginning of the experiment was used. The fish was placed in a total concentration of 26 ° 2 ° C seawater, the maximum density of 20 kg / m 3 fish tank volume. Two sets of 110 fish obtained from the quarantine tank were collected and distributed in the respective groups. Immerse the fish and place it in the assigned half tank. The fish are not individually labeled. ° Immersion disposal • Let the fish starve for 24 hours before vaccination. Capture 110 fish per group and transfer to separate treatment groups. The names of disposals, distribution slots and disposals given are listed in Table 1. The B B -antigen and soil bacterium two antigen solutions were diluted by about 10 and 100 times, respectively. The fish were immersed in these final suspension nights for 15 minutes, at which time pH 値, 02 and temperature were monitored. After immersion, the fish were transferred to their designated tanks as indicated in Table 1. • Table 1: Disposal and Dispensing Tanks used _ treatment volume, • antigen 丨 · volume of marine fish number trough BB-vaccine (BB) 140 ml 1,360 ml 110 5F05 immunostimulant (IS) 50 ml 4,950 ml 110 5F03 Control group - 5,000 ml 110 5F04 Preparation method of challenge culture The BB-strain was planted on chocolate agar and incubated overnight at 26 °C. Next, the grown bacteria were collected in 0.22 micron sterile filtered seawater. The resulting suspension was adjusted to 7366〇 to 1.573 and this suspension was used as the highest challenge suspension for -17-200843789. In addition, four 10-fold dilutions were prepared from the starting suspension in sterile seawater and used to inject fish. Three weeks after the challenge immersion treatment, 10 to 12 fish were captured from the treatment group (BB; Mycelium or control group) of each condition, anesthetized in A qui - S and injected intraperitoneally. 1 ml of the prepared dilution. Immediately after injection Φ Transfer the fish to its designated trough section and allow it to recover from anesthesia. All three injection groups of each challenge dilution (ie, a group containing 10 fish immersed in BB, a group containing 10 fish immersed in soil filaria, and containing 10 - 12 control fish) Groups) are all placed in the same tank, and all challenge suspensions with the same * are placed in the same tank, wherein the three groups are kept separated by vertical partitions placed in the tank. A total of five challenging concentrations were used, ranging from undiluted suspensions (referred to as "pure") to 10,000 times diluted pure. Observation of Mortality Fish were observed daily for 3 weeks and at least one death or apparently dying fish was collected daily. Examine the representative number of fish with typical symptoms of the big belly and examine the Giemsa staining of the internal organs (which is usually a block mixed in BB infected fish) to determine the organ causing the disease. All remaining fish were collected at the end of the 21-day observation period and assessed for signs of typical B B disease. In the event of a symptom, these fish are classified as “BB positive” in the assessment results. -18- 200843789 Interpretation of results The percentage of survival (RPS) calculated by the following formula was used to evaluate the mortality of the two treatment groups (BB and soil filaria) compared with the control group: RPS = % of the vaccinated group Mortality ~~if photos, % mortality in the group ~ }χ100 The mortality rate after the challenge was observed in each of the different treatment groups and the control group at each challenge dose in the first picture (5 Continuous graph). The five consecutive graphs clearly show a clear correlation between the challenge dose and the first death. In addition, the use of higher doses can cause a 100% mortality rate, however, in the lower dose group, at least some of the groups survive by challenge. # The third observation from these figures is that, in most cases, the BB-immersion group showed the highest peak mortality and this peak in the BB group usually appeared earlier than the other groups. All dead fish clearly showed typical symptoms of a big belly disease and BB-bacteria were evident in all fish samples. Evaluation of effectiveness Based on the resulting mortality pattern, the RP S値 for each condition is calculated. The results are shown in Figure 2. -19- 200843789 The results showed that protection was achieved in the two groups using the lowest challenge concentration group. The cumulative control group mortality range was 5 8.3 - 83.3%. (The cumulative control mortality in the 3 highest doses resulted in 100% mortality in the control group). Furthermore, the BB treatment group showed a negative RPS 値, which indicates that the BB-antigen treatment by the immersion method has a negative effect on the next challenge. Soaking with the lion soil antigen antigen can clearly induce the protective effect of antagonizing the BB challenge φ. This is very important because the immune stimuli are performed in the first three weeks of the challenge and the stimuli are immersed in the deactivated soil sputum, and the challenge is high mortality in the control group. • Example 2: “Fish rot disease” caused by Flavobacterium columna is the main cause of death of most cultivated freshwater species. Wu Guoyu is a highly susceptible species, and this example uses it as a target species. φ A soil-fertilizing bacterium (9x1 08 ODU/ml) manufactured by a formalin-activated whole cell fermentation tank was used as a non-specific immunostimulating agent (also known as an immunostimulant). Challenge Strains The isolates of Flavobacterium sinensis were isolated from the canal of Mississippi, USA. The isolate of Flavobacterium sinensis was isolated from Indonesian koi fish. -20- 200843789 Table 2: Immunostimulant concentration, number of fish used, application time and exemption
疫刺 激後之分佈 免疫刺激劑濃度 (0DU/毫升） 施用方式 槽 免疫-1 9x106 第一次挑戰前ό天 4F07A 免疫-2 9xl06 第一次挑戰前1天 4F07B 對照組 4F02B 這些實驗係在實驗開始時平均體重約5克之吳郭魚 B (Oreochromis 種）中進行。 ，實驗期間之水質條件 •鹽度： 免疫刺激後4 - 6 p p t * /挑戰後0 p p tDistribution of immunostimulant concentration after epidemic stimulation (0DU/ml) Administration method trough immunization-1 9x106 First challenge before the day 4F07A Immunization-2 9xl06 1st day before the first challenge 4F07B Control group 4F02B These experiments were started at the beginning of the experiment When the average body weight is about 5 grams, it is carried out in Wu Guoyu B (Oreochromis species). Water quality conditions during the experiment • Salinity: 4 - 6 p p t * after immunostimulation / 0 p p t after challenge
•溫度： 26°C +/-3°C •槽： 免疫刺激後5 00升/挑戰後50升 *第一次挑戰前一天將所有槽換成淡水且維持在淡水 中直到結束所有挑戰。 實驗設計 動物之處置組分配 共選擇隨手取得之240隻魚。 免疫刺激計劃 在免疫刺激前令魚飢餓至少24小時，以確保腸道完 全清空，而藉此減少由浸沒程序引起之壓力。實驗包括3 組（2組免疫刺激劑組及1組對照組），各由80隻魚組成。 -21 - 200843789 經由1 5分鐘之浸沒暴露將2組各80隻魚進行免疫刺激。 對照組之80隻魚則進行假暴露。將所有來自相同免疫組 之魚浸沒在免疫刺激劑浴中1 5分鐘。使用前將去活化之 獅土壤絲菌培養稀釋1 : 1 00。免疫刺激劑浴之總體積爲5 升。將對照組魚浸在5升淡水中一段相同之時間。根據表 2進行免疫刺激。記錄1 5分鐘暴露期間之水質（pH、溫度 及DO)。浸沒後立即將魚轉移至其被指派之槽，再使其恢 挑戰接種物之製備方法 柱狀黃桿菌挑戰株 利用1小瓶（1毫升）<-50°C之貯藏培養將柱狀黃桿菌 美國株再活化。收集0.5毫升之瓶內物，將其在蒸餾水中 稀釋10倍。將1%接種在100毫升噬纖維菌肉湯中。將肉 湯在26 °c培育22小時。當取得0.43 3之OD6 6〇時，使用 該培養來接種較大體積之噬纖維菌肉湯。接種量爲1 %之 培養體積。將培養在26°C培育約1 7小時，利用攪拌棒通 氣。當取得0.405之OD66〇時，使用該培養進行挑戰。藉 由標準塗抹法將每份1 00微升之1 0倍稀釋的細菌懸浮液 塗抹在噬纖維菌瓊脂上，再在26°C培育24-48小時以測 定在挑戰培養中之株落形成單位的數目。 將一部分<-50 °C之貯藏培養在噬纖維菌瓊脂上劃線， 以將柱狀黃桿菌亞洲株再活化。將培養盤在26 t培育約 24小時。將生長物收集在DIH20中直到達到約0.18-0.2 0 -22- 200843789 之〇D66G。接著，將收集之生長物在蒸餾水中稀釋10倍 並接種入含有微量元素之修改的噬纖維菌肉湯中。接種量 爲1%之培養體積。將肉湯在26 °C培育16-20小時，利用 攪拌棒通氣。取得約0.3 50-0.3 95之OD66G。藉由標準塗 抹法將每份100微升之10倍稀釋的細菌懸浮液塗抹在噬 纖維菌瓊脂上，再在26 °C培育24-48小時以測定在挑戰 培養中之株落形成單位的數目。 挑戰 在第6天及免疫刺激後第1天與二種挑戰株一起浸沒 來進行挑戰。在第一次挑戰方面，自各處置組收集40隻 | 魚並分成2組各20隻魚。將這些魚浸沒挑戰1 5分鐘（美 國株）或3 0分鐘（亞洲株）。有2組處置組及1組對照組， 因此，共使用3x20隻魚以各挑戰株進行浸沒挑戰。美國 株及亞洲株之挑戰懸浮液分別爲5升及2升。在第8及3 φ 天和第3及2週（分別是免疫-1及免疫-2)之重複挑戰中， 僅使用亞洲株，並依前述關於此菌株之相同程序進行。將 魚自處置室轉移至挑戰容器中進行挑戰。挑戰後，將魚轉 移入挑戰槽內。表3中指出不同挑戰組之分佈的檢視。 -23- 200843789 表3 :挑戰後不同挑戰組之分佈 挑戰 菌株 組別 魚/挑戰 之數目 挑戰 第1次 使用 之槽 挑戰 第2次 使用 之槽 挑戰 第3次 使用 之槽 亞洲株 免疫-1 20 第6天 7F14 第8天 7F14 第3週 7F11 免疫-2 20 第1天 7F15 第3天 7F15 第2週 7F12 對照組 20 NA 7F24 NA 7F24 NA 7F13 美國株 免疫-1 20 第6天 7F11 ND 免疫-2 20 第1天 7F12 對照組 20 NA 7F19 NA :未施用，ND :未做 挑戰後之觀察 挑戰後觀察魚2 -1 0天並記錄出現之死亡率。將死魚 移出槽並檢查之。注意每日之死魚數。 外部檢查包含在特殊記錄紙上指出是否出現鰓及/或 皮膚病害。自代表性之魚數目中採取鰓/皮膚之病害，進 行細菌學分析並平皿接種在噬纖維菌瓊脂上。將培養盤在 26°C培育24-48小時並評估是否出現典型之柱狀黃桿菌生 長（平坦、假根、群集、黏附及黃色）。 效力 以相關存活百分比表示免疫刺激之效力。使用下列 式。 RPS=(1- 疫苗接種組之％死亡率 對照組之％死亡率 結果 -24- 200843789 安全性 在免疫刺激之期間或免疫刺激後未立即觀察到死亡。 在3週之觀察期間內未觀察到死亡。 挑戰濃度 用於製備挑戰浴所使用之挑戰培養的濃度及第3週、 第3週重複及第4週之挑戰的有效挑戰濃度列於表4中。 φ 所有挑戰懸浮液在使用時均爲純液。 表4挑戰培養之CFU測定及使用之挑戰浴 挑戰時間點 刺激後之時間 挑戰培養 (CFU/毫升） 挑戰浴 (CFU/毫升） 1 第6及第1天 l.lxlO9 4.3χ108 8χ107 2·2χ108 2 第8及第3天 ΝΑ 5·4χ108 ΝΑ 1·5χ108 3 第3週及第4週1 ΝΑ 4.9x108 ΝΑ 3·7χ107 NA :未施用，D :天，Wk :週 *免疫-1及免疫-2組之各別時間點 挑戰後之死亡率 挑戰後未立即（浸沒後2小時內）觀察到死亡。然而’ 所使用之挑戰模型誘使死亡非常快速地開始。在所有挑戰 濃度中，挑戰後4-6小時可觀察到第一次死亡。魚將出現 典型之浮腫、出血的鰓且所有魚將顯現不同程度之鰓壞 死。自約6小時後可見到典型之皮膚及鰭之病害。死亡總 是在挑戰當天出現，在第2天達到高峰。之後’僅見到很 少或無魚死亡。所有死亡之魚顯示出典型之疾病病徵且幾 -25- 200843789 乎所有再分離物均爲陽性（除非，在平皿接種鰓時已生長 過度）。因爲所有魚均出現臨床病徵，所有死亡均被視爲 係由挑戰有機體造成。以不同之挑戰株（美國株及亞洲株） 在不同時間點由柱狀黃桿菌挑戰造成之累積死亡率呈現於 第3至6圖中。 效力 _ 不論所使用之挑戰株爲何，在整個試驗期之所有挑戰 時點中，二組免疫組均顯示出較對照組顯著降低之死亡率 (2-尾費雪精確檢定，p<〇. 05)。當以美國挑戰株挑戰魚 ~ 時，第6天及免疫刺激後之第1天可取得l〇〇%RPS値（對 '照組中爲60%死亡率）。以亞洲株挑戰時，不同免疫組的 RP S値列於第7圖中。當以亞洲挑戰株挑戰時，免疫-1之 RPS値較高（刺激後第6天，RPS = 80)，免疫-2之RPS値 較低（剌激後第1天，RPS = 25)，對照組之死亡率爲 • 100%。然而，二組均與對照組顯著不同（Ρ<〇·〇5)。當在第 3及第8天進行挑戰時，二組之RPS値均良好（對照組之 RPS = 60，死亡率爲85%)。免疫-2在第1天之RPS値較低 表示非特異性免疫刺激早早開始。當在免疫刺激後之第2 和3週進行挑戰時，二組之RPS値均良好（免疫_1(第3週） 及免疫-2(第2週）之RPS値分別爲RPS = 73.3及 RPS = 60.0) 〇 結論 -26- 200843789 總之，以柱狀黃桿菌挑戰之吳郭魚，利用根據本發明 製造之去活化的鯽土壤絲菌疫苗（其係藉由浸沒投服）可在 進行刺激後3週取得卓越之RPS値。 【圖式簡單說明】 第1圖：在免疫刺激後利用不同挑戰劑量進行BB挑 戰後之3週內，每一疫苗條件之每日死亡率。BB vax :經 接種大肚疫苗，Immuno-S :經獅土壤絲菌免疫刺激。 第2圖：與對照組相較時，各BB挑戰濃度下之每一 處理條件的RPS値。Vacc ··經接種大肚疫苗，IS :經獅 土壤絲菌免疫刺激。 第3圖：第1次挑戰（第6天及免疫刺激後第1天）時 由柱狀黃桿菌亞洲挑戰株誘出之％累積死亡率（n = 20)。 第4圖：第2次挑戰（第8天及免疫刺激後第3天）時 由柱狀黃桿菌亞洲挑戰株誘出之％累積死亡率（n = 20)。 第5圖··第3次挑戰（第3週及免疫刺激後第2週）時 由柱狀黃桿菌亞洲挑戰株誘出之％累積死亡率（n = 20)。 第6圖：第1次挑戰（第6天及免疫刺激後第1天）時 由柱狀黃桿菌美國挑戰株誘出之％累積死亡率（n = 20)。 第7圖：利用柱狀黃桿菌亞洲挑戰株在不同挑戰時I占 以免疫刺激劑取得之RPS値（n = 20)。 -27-• Temperature: 26°C +/-3°C • Slot: 500 liters after immune stimulation/50 liters after challenge * All slots were replaced with fresh water and maintained in fresh water the day before the first challenge until all challenges were met. Experimental Design Distribution of Animal Disposal Group A total of 240 fish were obtained. Immune Stimulation Program Let the fish starve for at least 24 hours before the immune stimulus to ensure that the intestines are completely emptied, thereby reducing the stress caused by the immersion procedure. The experiment consisted of 3 groups (2 groups of immunostimulating agents and 1 group of control groups), each consisting of 80 fish. -21 - 200843789 Two groups of 80 fish were immunostimulated via 15 minutes of immersion exposure. 80 fish in the control group were given false exposure. All fish from the same immunization group were immersed in the immunostimulant bath for 15 minutes. Dilute 1 : 00 of cultured lion soil strain before deactivation. The total volume of the immunostimulant bath is 5 liters. The control fish was immersed in 5 liters of fresh water for the same period of time. Immunostimulation was performed according to Table 2. Water quality (pH, temperature and DO) during the 15 minute exposure period was recorded. Immediately after immersion, the fish are transferred to the tank to which they are assigned, and then the challenge is used to prepare the inoculum. The Flavobacterium citrate challenge strain is cultured in 1 vial (1 ml) <-50 °C. The US strain is reactivated. Collect 0.5 ml of the contents and dilute them 10 times in distilled water. 1% was inoculated in 100 ml of fibrin broth. The broth was incubated at 26 °C for 22 hours. This culture was used to inoculate a larger volume of the fibrin broth when the OD6 6 0.4 of 0.43 3 was obtained. The inoculum size is 1% of the culture volume. The culture was incubated at 26 ° C for about 17 hours and ventilated using a stir bar. When the OD66 0.4 of 0.405 was obtained, the culture was used for the challenge. Each 100 μl of the 10-fold diluted bacterial suspension was applied to the fibroblast agar by standard smearing and incubated at 26 ° C for 24-48 hours to determine the colony forming units in challenge culture. Number of. A portion of the <-50 °C storage culture was streaked on the fibrin agar to reactivate the Agrobacterium tumefaciens strain. The plates were incubated at 26 t for approximately 24 hours. The growth was collected in DIH20 until about 0.18-0.2 0 -22-200843789 〇 D66G was reached. Next, the collected growth was diluted 10-fold in distilled water and inoculated into a modified fibrin broth containing trace elements. The inoculum size was 1% of the culture volume. The broth was incubated at 26 °C for 16-20 hours and ventilated using a stir bar. Obtain an OD66G of approximately 0.350-0.395. Each 100 μl of 10-fold diluted bacterial suspension was applied to the fibroblast agar by standard smearing and incubated at 26 ° C for 24-48 hours to determine the number of colony forming units in challenge culture. . Challenge Challenge was carried out on day 6 and on the first day after the immune challenge with the two challenge strains. In terms of the first challenge, 40 fish were collected from each disposal group and divided into 2 groups of 20 fish each. Immerse these fish for 15 minutes (US strain) or 30 minutes (Asia strain). There were two treatment groups and one control group. Therefore, a total of 3x20 fish were used to challenge the challenge with each challenge strain. The challenge suspensions for US strains and Asian strains are 5 liters and 2 liters, respectively. In the repeated challenge of the 8th and 3rd φ days and the 3rd and 2nd weeks (Immune-1 and Immune-2, respectively), only the Asian strain was used, and the same procedure as described above for this strain was carried out. Transfer the fish from the treatment room to the challenge container for challenge. After the challenge, move the fish into the challenge slot. A review of the distribution of the different challenge groups is indicated in Table 3. -23- 200843789 Table 3: Distribution of different challenge groups after challenge Challenges of strain group fish/challenge challenge First use tank challenge Second use tank challenge Third use tank Asian strain immunity-1 20 Day 6 7F14 Day 8 7F14 Week 3 7F11 Immunization-2 20 Day 1 7F15 Day 3 7F15 Week 2 7F12 Control Group 20 NA 7F24 NA 7F24 NA 7F13 US strain Immunization-1 20 Day 6 7F11 ND Immunization - 2 20 Day 1 7F12 Control group 20 NA 7F19 NA : Not administered, ND: Observation without challenge After observation, the fish were observed for 2 - 10 days and the mortality was recorded. Remove the dead fish from the tank and inspect it. Note the number of dead fish per day. External inspections are included on special recording paper to indicate the presence of imperfections and/or skin conditions. A disease of sputum/skin was taken from the representative number of fish, bacteriologically analyzed and plated on a fibrin agar. The plates were incubated at 26 °C for 24-48 hours and evaluated for the appearance of typical L. columnar growth (flat, pseudo root, cluster, adhesion and yellow). Efficacy The efficacy of immunostimulation is expressed as a percentage of relative survival. Use the following formula. RPS=(1-% mortality in the vaccination group. % mortality in the control group -24-200843789 Safety was not observed immediately during the period of immunostimulation or after immunostimulation. No observation was observed during the 3-week observation period. Death. Challenge Concentrations The concentration of challenge cultures used to prepare the challenge bath and the effective challenge concentrations for the 3rd, 3rd, and 4th week challenges are listed in Table 4. φ All challenge suspensions are in use Table 4 Challenge CFU for challenge culture and challenge of use Bath challenge time point after stimulation time challenge culture (CFU/ml) Challenge bath (CFU/ml) 1 Day 6 and Day l.lxlO9 4.3χ108 8χ107 2·2χ108 2 Days 8 and 3ΝΑ5·4χ108 ΝΑ1·5χ108 3 Week 3 and Week 4 1 ΝΑ 4.9x108 ΝΑ 3·7χ107 NA: Not applied, D: Day, Wk: Week*Immunity-1 And the immunization-2 group at each time point after the challenge of mortality after the challenge was not immediately (within 2 hours after immersion) observed death. However, the challenge model used induced the death to start very quickly. In all challenge concentrations The first time can be observed 4-6 hours after the challenge The fish will show typical edema, hemorrhage, and all fish will show varying degrees of necrosis. Typical skin and fin disease can be seen after about 6 hours. Death always appears on the day of the challenge, on the second day. After reaching the peak, 'only few or no fish died. All dead fish showed typical disease symptoms and several -25-200843789 all re-separators were positive (unless they were overgrown when the plate was inoculated) Because all fish have clinical signs, all deaths are thought to be caused by challenging organisms. The cumulative mortality caused by different challenge strains (US strains and Asian strains) at different time points by the challenge of Flavobacterium columnum is presented. Figures 3 to 6. Efficacy _ Regardless of the challenge strain used, the two groups of immunization groups showed a significantly lower mortality rate than the control group at all challenge points throughout the trial period (2-tail Fisher Accurate Verification) , p < 〇. 05). When challenged fish with the American challenge strain, on the 6th day and the first day after the immune stimulation, 10% RPS 可 (60% mortality in the 'group) was obtained. In Asia At the challenge of the strain, the RP S of the different immunization groups is listed in Figure 7. When challenged with the Asian challenge strain, the RPS of immunization-1 is higher (6 days after stimulation, RPS = 80), immunization-2 RPS値 was lower (on day 1 after stimulation, RPS = 25), and the mortality of the control group was • 100%. However, both groups were significantly different from the control group (Ρ<〇·〇5). On the 8th day of challenge, both groups had good RPS sputum (RPS = 60 in the control group and 85% mortality). The lower RPS 免疫 of immunization-2 on day 1 indicates that non-specific immune stimuli begin early. When challenged at 2 and 3 weeks after immunostimulation, both groups had good RPS ( (immunization_1 (week 3) and immunization-2 (week 2) RPS RP were RPS = 73.3 and RPS, respectively. = 60.0) 〇Conclusion-26- 200843789 In summary, Wu Guoyu, challenged with Flavobacterium columnum, can be stimulated after using the deactivated A. sinensis vaccine (which is submerged) by the present invention. 3 weeks to achieve excellence in RPS値. [Simplified Schematic] Figure 1: Daily mortality for each vaccine condition within 3 weeks after the BB challenge with different challenge doses after immune stimulation. BB vax: Inoculated with a big belly vaccine, Immuno-S: immunostimulated by S. serrata. Figure 2: RPS 每一 for each treatment condition at each BB challenge concentration compared to the control group. Vacc · · Inoculated with a big belly vaccine, IS: immune stimulation by lion soil. Figure 3: % cumulative mortality (n = 20) induced by the Flavobacterium columnar Asian challenge strain at the first challenge (day 6 and day 1 after immunostimulation). Figure 4: % cumulative mortality (n = 20) induced by the Flavobacterium columnar Asian challenge strain at the 2nd challenge (Day 8 and day 3 after immunostimulation). Figure 5··3rd challenge (3rd week and 2nd week after immunostimulation) % cumulative mortality (n = 20) induced by the Flavobacterium columnar Asia challenge strain. Figure 6: % cumulative mortality (n = 20) induced by the Flavobacterium columnarum American challenge strain on the first challenge (day 6 and day 1 after immunostimulation). Figure 7: RPS値 (n = 20) obtained with an immunostimulant at different challenges using the A. faecalis strain. -27-