1290047 九、發明說明： 【發明所屬之技術領域】 本發明提供一種結合有效量之去活化豬黴漿菌及去活化細菌 性抗原之疫苗及其製法，係可應用於豬隻畜養之技術領域相較於 單獨的豬黴漿菌疫苗或細菌性抗原疫苗，本發明之疫苗可更全面 預防豬隻感染肺炎，具有更優良的功效。 【先前技術】 豬黴漿菌性肺炎係屬高傳染率低死亡率的慢性病，本病臨床 上的發生率在25〜93%之間，屠體肺臟檢查陽性率為28〜80%，感染 豬的生長效率亦降低14〜16%，若飼養情形為高密度飼養，且處於 通風不良且多濕和氣溫變化大的氣候時，對於本病的發生和漫延 將極為嚴重，導致豬隻的飼料換肉率降低、生長遲緩、豬隻的發 炎性反應，以及產生免疫抑制作用，更因經常伴隨併發如胸膜肺 炎放射桿菌、巴斯德桿菌或豬鏈球菌等的二次感染，最後往往造 成嚴重的損失，成為養豬產業成本提高的重要病因之一。 常見的豬黴漿菌有三種：豬肺炎黴漿菌（挪a hyopneumoniae)、錄箅’钣 1菌（Mycoplasma fiyorhinis)反 Μ· ，其中緒肺炎黴衆菌是造成赭黴毁茵肺炎（swine enzootic pneumonia，SEP)的重要病原，豬鼻黴漿菌是造成多發 性漿膜炎及關節炎的病原，而#. //occw/are則未被證實會引起 疾病。豬黴漿菌肺炎過去均由豬肺炎黴漿菌所引起，最近卻慢慢 轉變為單純豬鼻黴漿菌或上述兩種黴漿菌混合感染所造成。台灣 在1996年以前從感染豬黴漿菌肺炎之豬肺臟所分離出的黴漿菌僅 有豬肺炎黴漿菌一種，豬鼻黴漿菌在國外的病例報告主要由感染 關節炎之豬隻的關節液中分離出，並不會引起豬黴漿菌肺炎，因 此在豬隻疾病中並不是一個很重要的病原，台灣過去未曾出離出 此病原，但是自1996年開始，台灣動物科學研究所黴漿菌研究室 1290047 在豬肺炎病例中，肺臟所分離出黴漿菌利用西方免疫點潰法與 ATCC標準菌株比較後，可藉由抗體結合反應確定為豬鼻黴漿菌。 動物科技研究所平常接受全省各地豬場委託進行黴漿菌分離 鑑定，發現在豬隻肺炎病例中分離出豬鼻黴漿菌的機率越來越 高。在2001年242個病例及2002年205個病例中，豬肺炎黴漿 菌感染率由2001年的46. 8%下降到2002年的15.8%，而豬鼻黴漿 菌感染率則由2001年的65. 5%上升到2002年的79.2%，混合豬肺 炎黴漿菌及豬鼻黴漿菌感染的豬隻感染率在2001年為14. 4%而在 2002年為15%。顯示在國内的豬肺炎病例中，由肺臟分離出豬鼻 黴漿菌的情況越來越多；由其感染率也可觀察到豬肺炎黴漿菌的 感染率降低的很快，豬鼻黴漿菌的感染率快速上升，混合感染率 上升的速度卻沒有顯著的變化，此種情況顯示豬鼻黴聚菌正逐漸 取代豬肺炎黴漿菌，成為豬黴漿菌肺炎的主要病原。過去認為豬 黴漿菌中除了豬肺炎黴漿菌之外，其他的黴漿菌並不會引起豬黴 聚菌肺炎，而目前的情況卻不再如此，在上面分離的病例中，2001 年有一個病例為單純的豬鼻黴漿菌感染，而2002年則有5個豬鼻 黴漿菌單純感染的病例，顯示豬鼻黴漿菌已經可以單獨誘發豬黴 漿菌肺炎。 由現場實務經驗得知，本病於感染過後，再度遭受感染的機 率相當低，顯示豬隻本身對此疾病病原的免疫性相當良好，故認 為使用疫苗接種方式防範此疾病，應是一種非常可行的防治措施。 另外，除了豬黴漿菌肺炎之外，豬肺炎也可經由其他細菌性 途徑而感染，例如：巴斯德桿菌或胸膜 肺炎放射桿菌等。巴斯德桿 菌引起的肺炎多為化膿性支氣管肺炎或纖維素性肺炎，其傳染的 途徑尚不是很清楚，在致病的角色上可以是主凶或幫凶，發病過 程不似胸膜肺炎放射桿菌的急性，通常以慢性較多，由於是化膿 1290047 性支氣管肺炎，在支氣管内常發現大量膿性粘稠分泌物。胸膜肺 炎放射桿菌是豬胸膜肺炎常見的重要病原體，傳染的途徑為接觸 傳染，依據豬隻免疫力、環境狀態和豬隻接觸病原體之含量等影 響，使豬隻呈現不同的臨床症狀。感染急性豬胸膜肺炎的病例， 其臨床症狀包括沒有食慾、體溫上升、呼吸困難和突然死亡等。 感染後24〜36小時可能致死，也可能於暴斃前未發現任何臨床 症狀。當豬急性臨床症狀未死亡者，其後可發展成為慢性豬胸膜 肺炎，呈現自發性咳漱現象。豬霍亂沙門氏桿菌 c/zo/eraesiz/s*)感染的臨床症狀有敗血症或大小腸炎，亦時有爆 發肺炎、腦膜腦炎或慢性消耗性疾病等症狀。 就目前台灣地區而言，對於感染肺炎豬隻的治療，一般是採 取於飼料中添加抗生素之類的藥物做為防治方法，但是長期在飼 料中添加抗生素，容易產生抗藥性菌種，導致用藥上的困擾，以 及造成肉品中抗生素殘留等問題，嚴重影響肉品衛生，此外就實 務經驗也顯示利用餵食藥物以達預防的效果，並不如預期的理想。 因此，開發一有效預防豬肺炎之疫苗係為目前刻不容緩的工 作。 【發明内容】 有鑒於習知技術之不足，本發明之一目的係提供一種結合豬 黴漿菌及細菌性抗原之疫苗，係包括一有效量之去活化豬黴漿菌 以及一有效量之去活化細菌性抗原。 本發明之另一目的係為製備前述結合豬黴漿菌及細菌性抗原 疫苗之方法，其步驟係包括··培養豬黴漿菌；將前述豬黴漿菌以 福馬林進行去活化處理；培養細菌性抗原；將前述細菌性抗原以 福馬林進行去活化處理；將前述去活化之細菌性抗原進行濃縮； 以及混合前述步驟製得的豬黴漿菌及細菌性抗原。 本發明之再一目的係關於一種可增進疫苗效力之培養基，係 1290047 包括Hank’s solution 500毫升、蒸餾水12, 000毫升、Bacto腦心浸出 培養液（Difco) 82克、Bacto PPLO broth 87克、酵母萃取6〇〇毫升、酚 紅45毫升、枯草菌素2·5克、盤尼西林或甲氧苯青黴素2· 5克及去活化的 豬血清4000至5000克，該培養基較佳係用於培養含豬黴漿菌之疫苗，例如， 但不限於本發明之疫苗。 本發明之又一目的提供一種用於預防豬隻感染肺炎之醫藥組 合物，係包括一有效量之本發明疫苗與一醫藥可接受之載體。 【實施方式】 本發明係提供一種可用於預防豬隻感染肺炎的疫苗，具體而 言，，本發明t疫苗係由一有效量之去活化豬黴黎菌及一有效量之 去活化細g性抗原而組成，其中前述豬黴漿㈣可選自豬鼻徽浆 菌、豬肺炎黴漿g或其組合；而前述細菌性抗原係可選自巴斯德 桿菌⑽价㈣/化卵/⑽地）、豬霍亂沙門氏桿g (似麵心沒 咖f心）、胸膜肺炎放射桿菌（如—7/似 p/ew印加/ac)、其任二種之組合或其三種之組合。 月ϋ述疫田之痛液混合體稽[•仓，丨炎4 & 1:4。十15。間。丨為細讀性抗原:豬黴聚菌介於 ,rm包含豬黴聚菌及細菌性抗原所組成的菌液之外， 尚可進-^包含醫藥可接受之载體、佐劑或稀釋劑。其中前述之 載體、佐剤或稀釋劑佔整體疫苗 為娜；前述之菌液佔；；广體積比例為〇%至5〇%，較佳為 較佳為灘_。 ，崎積曝画至耀， 1二，<者%/本發明之疫苗在提供給一對象後能獲得有 以⑽炎為例，彳目⑽體内或體外獲得預期的活性。 果包含症狀的杨、不適感的舒緩、病程縮減、加速痤癒、死亡 1290047 率減低、育成率及換肉率的增加等。提供給對象的精確劑量必須 依疾病的程度或症狀及對象之品種來決定，例如：對象的當時健 康狀況、年齡、體重及對藥物的忍受度。熟悉此領域之人士能依 據前述或其他因子來決定適當的劑量。 正如本技術領域者所習知，不同的微生物野外分離株常在其 基因序列上有微小的變異，然而當此變異不影響其蛋白質合成、 結構，或是主要作用功能區或時，該微生物之他種野外分離株即 使基因序列無法百分之百相同，其基本的生理功能並不會有所改 變。但是同源性的比較如果是針對全部的基因來進行比對，在實 行上是非常巨大的工程，不太可行，目前國際上常使用16S ribosomal RNA (16S rRNA)來進行細菌型別的分辨，因此，在相 似性的比較上可以16S rRNA來進行比對而得到其同源性。所以， 本發明之疫苗所使用的黴漿菌與細菌性抗原並不限定於本發明所 使用的野外分離株，其他野外分離菌株在下述條件内，亦可用來 作成本發明之疫苗。 前述豬鼻黴漿菌係包括菌株ATIT-7或與菌株ATIT-7同源性 (相似性）高達至少80%、至少85%、至少90%、至少95%或至少99% 之豬黴漿菌菌株，前述豬鼻黴漿菌菌株ATIT-7係於民國92年5 月8日寄存於食品工業發展研究所之菌種保存及研究中心，寄存 編號為 BCRC 910223。 前述豬肺炎徽漿:菌係包括菌株PR IT-5或與菌株PRIT-5同源 性（相似性）高達至少80%、至少85%、至少90%、至少95%或至少 99%之豬黴漿菌菌株，前述豬肺炎黴漿菌菌株PRIT-5係於民國85 年3月14日寄存於食品工業發展研究所之菌種保存及研究中心， 寄存編號為CCRC910045。 前述之巴斯德桿菌除本發明使用之野外分離株外，亦可使用 與該野外分離株同源性（相似性）高達至少80%、至少85%、至少 1290047 90%、至少95%或至少99%之菌株。 前述之豬霍亂沙門氏桿菌除本發明使用之野外分離株外，亦 可使用與該野外分離株同源性（相似性）高達至少80%、至少85%、 至少90%、至少95%或至少99%之菌株。 前述之胸膜肺炎放射桿菌除本發明使用之野外分離株外，亦 可使用與該野外分離株同源性（相似性）高達至少80%、至少85%、 至少90%、至少95%或至少99%之菌株。 前述序列之同源性百分比係指相似位置數除以總比對殘基 數之結果，比對的方式可視需要利用本技術領域者所習知之比對 程式或軟體來完成。 為了比對兩序列之間的同源性，需將兩序列依照最合適的位 置排列，例如：為了達到最合適的位置排列，可在其中一個序列 中插入缺口（ gap);或者是可忽略位於缺口位置的非相似性片段， 以期符合和另一序列的比對。兩個序列不需為同等長度，比對的 序列可以長於或短於參考序列，且比對序列相似的序列位置可位 於該參考序列的任何部位。 以下係透過實施例更進一步說明本發明之技術特徵與優點。 實施例··本發明疫苗之製備 一、疫苗菌株 1. PRIT-5菌株，為豬肺炎黴漿菌，其係申請人於民國79年4 月24日提出之另一專利申請案，已核准專利並於民國80 年4月21日公告，該菌株於民國85年3月14日寄存於食 品工業發展研究所，寄存編號為CCRC910045。1290047 IX. Description of the Invention: [Technical Field] The present invention provides a vaccine for deactivating porcine mold and deactivated bacterial antigen in combination with an effective amount thereof, and a method for preparing the same, which can be applied to the technical field of pig breeding Compared with the single porcine mold vaccine or the bacterial antigen vaccine, the vaccine of the invention can more completely prevent pigs from contracting pneumonia, and has better efficacy. [Prior Art] Porcine mycoplasma pneumonia is a chronic disease with high infection rate and low mortality. The clinical incidence rate of this disease is between 25~93%, and the positive rate of carcass lung examination is 28~80%. Infected pigs The growth efficiency is also reduced by 14 to 16%. If the breeding situation is high-density breeding and the climate is poorly ventilated and humid and the temperature changes greatly, the occurrence and spread of the disease will be extremely serious, resulting in the feed change of the pigs. Reduced meat rate, growth retardation, inflammatory response in pigs, and immunosuppressive effects, as well as secondary infections often accompanied by concurrent infections such as Pneumocystis pneumoniae, Pasteurella or Streptococcus suis, often resulting in serious Loss has become one of the important causes of the cost increase in the pig industry. There are three common porcine molds: Helicobacter pneumoniae (N. a hyopneumoniae), and co'箅1 My (Mycoplasma fiyorhinis) , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , sw sw sw sw sw sw sw An important pathogen of pneumonia, SEP), a pathogen causing multiple serositis and arthritis, and #.occw/are has not been confirmed to cause disease. Pig mycoplasma pneumonia has been caused by Mycoplasma pneumoniae in the past, but it has recently been transformed into a simple mixed infection of Porphyridae or the above two molds. Taiwan's mycoplasma isolated from the lungs of pigs infected with hyphae pneumonia before 1996 was only one of the porcine pneumoniae, and the case of pig sclerotia in foreign countries was mainly reported by pigs infected with arthritis. It is isolated from the joint fluid and does not cause porcine mycoplasma pneumonia. Therefore, it is not a very important pathogen in pig diseases. Taiwan has not left this pathogen in the past, but since 1996, Taiwan Animal Science Research has been carried out. Mycoplasma Research Laboratory 1290047 In the case of swine pneumonia, the mycoplasma isolated from the lungs was determined to be Rhizoctonia solani by antibody binding reaction after comparison with the ATCC standard strain by Western immunological point collapse. The Animal Science and Technology Research Institute has generally accepted the identification of mycoplasma isolates from pig farms throughout the province, and found that the chances of isolating the pig's nose mold in pig pneumonia cases are getting higher and higher. In 242 cases in 2001 and 205 cases in 2002, the infection rate of Mycoplasma pneumoniae decreased from 46.8% in 2001 to 15.8% in 2002, while the infection rate of Rhizoctonia solani was from 2001. 65. 5% rose to 79.2% in 2002. The infection rate of pigs infected with mixed porcine pneumoniae and porcine nasal bacterium was 14.4% in 2001 and 15% in 2002. It is shown that in the domestic cases of swine pneumonia, there are more and more cases of the isolation of Porcine Helicobacter pylori from the lungs; the infection rate of the porcine pneumoniae can also be observed to decrease rapidly. The infection rate of the bacterium was rapidly increased, but the rate of increase of the mixed infection rate did not change significantly. This situation indicates that the porcine porcine mold is gradually replacing the porcine pneumoniae, which is the main pathogen of porcine mycoplasma pneumonia. In the past, it was considered that except for the porcine pneumoniae, other mycoplasma bacteria did not cause hyphae pneumonia, but the current situation is no longer the case. In the case of the above separation, in 2001 One case was a simple infection of Rhizopus oryzae, while in 2002 there were 5 cases of Rhizopus oryzae infection alone, indicating that Rhizoctonia solani can induce porcine mycoplasma pneumonia alone. It is known from on-the-spot experience that the probability of re-infection after the infection is quite low, indicating that the pig itself is quite immune to the pathogen of the disease, so it is considered to be very feasible to use the vaccination method to prevent the disease. Control measures. In addition, in addition to porcine mycoplasma pneumonia, porcine pneumonia can also be infected via other bacterial routes, such as: Pasteurella or Pneumocystis pneumoniae. Most of the pneumonia caused by Pasteurella is suppurative bronchopneumonia or fibrinous pneumonia. The route of infection is not very clear. It may be the main accomplice or accomplice in the role of the disease. The pathogenesis is not as acute as that of P. pneumoniae. Usually, it is more chronic. Because it is suppurative 1290047 bronchopneumonia, a large amount of purulent and viscous secretions are often found in the bronchi. Pleuropneumonia is a common pathogen of porcine pleuropneumonia. The route of infection is contact infection. According to the immunity of pigs, the state of the environment and the content of pigs exposed to pathogens, pigs present different clinical symptoms. In clinical cases of acute porcine pleuropneumonia, there are no appetite, rising body temperature, difficulty breathing and sudden death. It may be fatal 24 to 36 hours after infection, and no clinical symptoms may be found before violent. When the acute clinical symptoms of pigs are not dead, they can develop into chronic porcine pleuropneumonia, which presents with spontaneous cough. The clinical symptoms of Salmonella choleraesuis c/zo/eraesiz/s*) are sepsis or large-scale enteritis, and there are also symptoms such as pneumonia, meningoencephalitis or chronic wasting disease. As far as Taiwan is concerned, the treatment of pigs infected with pneumonia is generally based on the use of antibiotics such as antibiotics in the feed as a method of prevention, but long-term addition of antibiotics to the feed is prone to develop resistant strains, leading to medication. The troubles, as well as the problem of antibiotic residues in meat, have seriously affected meat hygiene. In addition, practical experience has also shown that the use of feeding drugs for preventive effects is not as good as expected. Therefore, the development of an effective vaccine against swine pneumonia is currently an urgent task. SUMMARY OF THE INVENTION In view of the deficiencies of the prior art, one object of the present invention is to provide a vaccine combining the porcine mold and bacterial antigen, comprising an effective amount of deactivated porcine mold and an effective amount. Activate bacterial antigens. Another object of the present invention is to prepare the aforementioned method for combining a porcine mold and a bacterial antigen vaccine, the steps of which include: cultivating a porcine mold; the above-mentioned porcine mold is deactivated by using fumarin; a bacterial antigen; the aforementioned bacterial antigen is deactivated by formalin; the deactivated bacterial antigen is concentrated; and the porcine mold and bacterial antigen prepared in the above step are mixed. A further object of the invention relates to a medium for enhancing the efficacy of a vaccine comprising 1290047 comprising Hank's solution 500 ml, distilled water 12 000 ml, Bacto brain leaching medium (Difco) 82 g, Bacto PPLO broth 87 g, yeast extract 6 ml, phenol red 45 ml, subtilin 2.5 g, penicillin or methicillin 2.5 g and deactivated pig serum 4000 to 5000 g, the medium is preferably used for culture of pig mold A vaccine for bacteria, such as, but not limited to, a vaccine of the present invention. A further object of the present invention is to provide a pharmaceutical composition for preventing infection of pigs with pneumonia comprising an effective amount of the vaccine of the present invention and a pharmaceutically acceptable carrier. [Embodiment] The present invention provides a vaccine which can be used for preventing pigs from contracting pneumonia. Specifically, the t vaccine of the present invention is deactivated by an effective amount of deactivated Rhizoctonia solani and an effective amount of deactivated fine g And the composition of the antigen, wherein the aforementioned porcine mold (4) may be selected from the group consisting of Rhizoctonia solani, porcine pneumoniae g or a combination thereof; and the aforementioned bacterial antigen system may be selected from the group consisting of Pasteurella (10) valence (four) / oxidized egg / (10) ), pig cholera Shamen's rod g (like face no heart), pleural pneumoniae (such as -7 / like p / ew Inca / ac), a combination of any two or a combination of the three. The monthly misunderstanding of the painful liquid mixture of the epidemic field [• Cang, Yan Yan 4 & 1:4. Tenteen. between.丨 is a fine-grained antigen: in the case of porcine mold, rm contains bacteriological liquid composed of porcine mold and bacterial antigen, and can contain pharmaceutically acceptable carrier, adjuvant or diluent. . The carrier, the adjuvant or the diluent mentioned above is the whole vaccine; the aforementioned bacterial liquid accounts for; the broad volume ratio is 〇% to 5%, preferably □. , 崎 积 引 画 画 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , The symptoms include Yang, the relief of discomfort, the reduction of the course of the disease, the accelerated healing, the death rate of 1290047, the increase of the breeding rate and the rate of meat exchange. The exact dose to be administered to a subject must be determined by the degree or condition of the disease and the variety of the subject, for example, the subject's current state of health, age, weight, and tolerance to the drug. Those skilled in the art will be able to determine the appropriate dosage based on the foregoing or other factors. As is known in the art, different microbial field isolates often have minor variations in their gene sequences, but when the mutation does not affect its protein synthesis, structure, or major functional domain, His field isolates do not change their basic physiological functions even if the gene sequence is not 100% identical. However, if the comparison of homology is to compare all the genes, it is a very huge project in practice, and it is not feasible. At present, 16S ribosomal RNA (16S rRNA) is often used internationally for bacterial type discrimination. Therefore, in the comparison of similarity, 16S rRNA can be aligned to obtain homology. Therefore, the mycoplasma and bacterial antigens used in the vaccine of the present invention are not limited to the field isolates used in the present invention, and other field isolates can be used as vaccines for the invention under the following conditions. The aforementioned Rhizoctonia solani includes strain ATIT-7 or homologous (similarity) with strain ATIT-7 up to at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of porcine mycoplasma The strain, the aforementioned strain of the genus Rhizopus oryzae ATIT-7 was deposited on May 8, 1992 in the Center for Culture and Conservation of the Institute of Food Industry Development, and the registration number is BCRC 910223. The aforementioned swine pneumonia syrup: the strain includes the strain PR IT-5 or the homology (similarity) with the strain PRIT-5 up to at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of the porcine mold The bacterial strain, the above-mentioned strain PRIM-5 of the porcine pneumoniae strain was deposited in the Culture and Conservation Research Center of the Food Industry Development Research Institute on March 14, 1985. The registration number is CCRC910045. The aforementioned Pasteurella may also use at least 80%, at least 85%, at least 1290047 90%, at least 95% or at least homology (similarity) to the field isolate in addition to the field isolate used in the present invention. 99% of the strains. The aforementioned Salmonella choleraesuis can also use at least 80%, at least 85%, at least 90%, at least 95% or at least homology (similarity) to the field isolate in addition to the field isolate used in the present invention. 99% of the strains. The aforementioned P. pneumoniae may also use homology (similarity) up to at least 80%, at least 85%, at least 90%, at least 95% or at least 99 in addition to the field isolate used in the present invention. % of the strain. The percent homology of the foregoing sequences is the result of dividing the number of similar positions by the number of residues in the total alignment, and the manner of alignment can be accomplished by using a comparison program or software as is known in the art. In order to align the homology between the two sequences, the two sequences need to be arranged according to the most suitable position. For example, in order to achieve the most suitable positional arrangement, a gap may be inserted in one of the sequences; or it may be negligible. A non-similar segment of the gap position, in order to match the alignment with another sequence. The two sequences need not be of equal length, the aligned sequences may be longer or shorter than the reference sequence, and the sequence positions similar to the aligned sequences may be located anywhere in the reference sequence. The technical features and advantages of the present invention are further illustrated by the following examples. EXAMPLES·Preparation of vaccine of the present invention 1. Vaccine strain 1. PRIT-5 strain is a porcine pneumoniae, which is another patent application filed by the applicant on April 24, 79, and has been approved. It was announced on April 21, 1980 in the Republic of China. The strain was deposited with the Food Industry Development Research Institute on March 14, 1985. The deposit number is CCRC910045.
2. ATIT-7菌株，為從本省感染豬隻肺臟中所分離的豬鼻黴漿 菌菌株，經過培養測定後，發現其增殖非常快速，在培養 基中35至38°C培養16小時，其菌體數可達109 CCU/mL 以上，其他分離的豬鼻黴漿菌菌株則落於108至109CCU/mL 1290047 間。目前AT IT-7菌株已於民國92年5月8日寄存於食品 工業發展研究所，寄存編號為BCRC910223。 3.巴斯德桿菌、豬霍亂沙門氏桿菌及胸膜肺炎放射桿菌為野 外分離株。 二、培養基製備 1.豬黴漿菌培養基（F r i i s培養基）配方及製備如下：2. ATIT-7 strain, which is a strain of Rhizopus oryzae isolated from the lungs of infected pigs in the province, was found to have a very rapid proliferation after culture and was cultured in the medium at 35 to 38 ° C for 16 hours. The number of bodies can reach 109 CCU/mL or more, and other isolated strains of Rhizoctonia solani fall between 108 and 109 CCU/mL 1290047. At present, the AT IT-7 strain has been deposited with the Food Industry Development Research Institute on May 8, 1992. The registration number is BCRC910223. 3. Pasteurella, Salmonella choleraesuis and Pneumocystis pneumoniae are wild isolates. 2. Preparation of the medium 1. The formula and preparation of the porcine mold medium (F r i i s medium) are as follows:
Hank^s solution 500 毫升 蒸飽水 12,000 毫升 Bacto brain heart infusion 82 M. (Difco) Bacto PPLO broth 87 1 酵母萃取（Yeast extract) 600 毫升 酣紅（Phenol Red) 45 毫升 枯草菌素（Bacitracin) 2A 克_ 盤尼西林或甲氧苯青黴素 2A (Methicillin) 豬血淸 4,000 至 5,000 毫升 (豬血淸先在56°C進行1至2次30分鐘的不活化） 培養基配好後調整pH値到7.4至7.6，然後以0.2 μιη 的Millipore濃心過濾、。 2.細菌培養基配方及製備如下： 以Difco胰化絡蛋白大豆培養液（Trypticase Soy Broth，TSB) 30 g溶解於1公升蒸顧水中，再經過滅菌 處理即可使用。 三、疫苗製備 1290047 1·豬黴漿菌製備：菌株PRIT-5及ATIT-7各自培養於前述豬 黴漿菌培養基中，於37°C恆溫培養箱中震盪培養，當菌液 變黃後將其取出，利用光電比色計測定，菌株PRIT-5 0. D55〇 介於0· 08至0· 16間，菌株ATIT-7 0.D㈣介於〇. 14至0. 33 間，取出的菌液加入0.1至〇· 2%的福馬林在37°C中進行1 小時不活化處理，再放入2至8°C中靜置8至24小時。 2·其他細菌製備：自-201：冰箱中取出巴斯德桿菌（以下簡稱 PM)、豬霍亂沙門氏桿菌（以下簡稱sal)及胸膜肺炎放射 桿菌（以下簡稱AP)，分別在5毫升腦心浸出培養液（Brain heart infusion broth, BHI broth)(胸膜肺炎放射桿菌使 用含有菸鹼胺腺呤雙核酸（nicotinamide adenine dinucleotide，NAD)胰化絡蛋白大豆培養液（TrypucaseHank^s solution 500 ml distilled water 12,000 ml Bacto brain heart infusion 82 M. (Difco) Bacto PPLO broth 87 1 Yeast extract 600 ml of blush (Phenol Red) 45 ml of bacitracin 2A g _ Penicillin or methicillin 2A (Methicillin) pig blood 淸 4,000 to 5,000 ml (porcine blood sputum first at 56 ° C for 1 to 2 times 30 minutes of inactivation) After the medium is adjusted, adjust the pH 7.4 to 7.4 to 7.6, It was then filtered through a 0.2 μιη Millipore concentrate. 2. Bacterial medium formulation and preparation are as follows: 30 g of Difco Trypticase Soy Broth (TSB) is dissolved in 1 liter of steamed water and sterilized. 3. Vaccine preparation 1290047 1. Preparation of porcine mold: The strains PRIT-5 and ATIT-7 were cultured in the above-mentioned porcine mold culture medium, and cultured in a constant temperature incubator at 37 ° C. When the bacterial liquid turned yellow, The sample was taken out and determined by a photoelectric colorimeter. The strain PRIT-5 0. D55〇 was between 0·08 and 0·16, and the strain ATIT-7 0.D (four) was between 〇. 14 and 0.33. The solution was added with 0.1 to 2% of formalin for 1 hour at 37 ° C for inactivation, and then placed at 2 to 8 ° C for 8 to 24 hours. 2. Preparation of other bacteria: From -201: Take out the Pasteurella (hereinafter referred to as PM), Salmonella choleraesuis (hereinafter referred to as sal) and Pneumocystis pneumoniae (hereinafter referred to as AP) in the refrigerator, respectively, in the 5 ml brain Brain heart infusion broth (BHI broth) (Pycnopharemia pneumoniae uses a nicotinamide adenine dinucleotide (NAD) pancreatic fossil protein soy broth (Trypucase)
Soy Broth, TSB))中增菌18-24小時。在新的腦心浸出培 養液中加入1%(V/V)增菌菌液，以120rpm震盪培養4一8小 時，待細菌濃度達到lx 1〇9〜5x 109CFU/ml之間時，加入 0·2%-3%(V/V)的福馬林原液（38%)不活化菌體8—16小時， 將各菌液以離心方式濃縮8一15倍（3〇〇〇—5〇〇〇卬^離心 15 - 30分鐘）並懸浮於lx PBS巾，同時將不活化的菌體劃在 培養基上，不得長菌。 3·混合囷液一製備··取步驟！所製備之黴漿菌不活化菌液 500-800 €升，可為菌株ATIT—7或pRn—5單獨或兩者混合 使用’其使用或混合的方式及比例請參照中華民國專利第 〇九二一-六八八四號中請案之内容，以及加人步驟2所 製備的細讀濃縮菌液各5-15毫升混合均勻。 •疫苗製作··將步驟3所製備的菌液與佐劑混合，1中菌液 2所5=广為5°，(V/V)，#劑所佔比例為 25-_m) ’再以祕器充㈣拌均勻，製成疫 12 1290047 °c備用。 四、 疫苗使用方法 每頭仔緒進行肌肉注射2-3次，每次注射一劑量，第一次 注射在1-3週齡，第二次注射在3-5週齡，第三次注射在5-7 週齡，注射前疫苗必須混合均勻。 五、 疫苗安全性試驗 1 ·小鼠安全性試驗··製備甲（豬鼻黴漿菌+PM+AP+Sal)、乙（豬 肺炎黴漿菌+PM+AP+Sal)及丙（豬鼻黴漿菌+豬肺炎黴漿菌 +PM+AP+Sal)等三種疫苗，取42隻BALB/c小鼠，隨機取 6隻作為對照組，另外36隻隨機分為6組，每組6隻，將 A、B及C三種疫苗以皮下注射或腹腔注射方式各注射0.5 毫升，注射後觀察14天，在觀察期間均無任何不良反應 且健存。 六、 疫苗效力試驗 1.小鼠抗體力價測定：取BALB/c四週齡雌性小鼠80隻， 經過隨機分為8組，每組10隻，分別為： A:四合一組（豬肺炎黴漿菌（Mh) + PM + AP+Sal) B:三合一組（磷酸鹽緩衝溶液(PBS) + PM+AP+Sal) C :三合一 Friis 組(Friis 培養基+ PM+AP+Sal) D : Mh 組 E : AP 組 F : Sal 組 G : PM 組 Η :對照組(Ffiis培養基） 小鼠以皮下注射方式免疫兩次，在第二次免疫後一星 1290047 期，以麻醉方式進行眼窩採血，收集的血液在室溫下放置 1小時後，再放入4°C中冷藏過夜，隔天以3000 r.p.m·離 心30分鐘，離心後將上清液取出放入新的離心管中，利 用直接式ELISA法免疫定量分析之。 第一圖係顯示小鼠血清豬肺炎黴漿菌抗體測定結果， 其中A為四合一疫苗（Mh+AP+Sal + PM)，D為Mh單劑 疫苗，Η為對照組。第二圖係顯示小鼠血清胸膜肺炎放射 桿菌抗體測定，其中Α為四合一疫苗（Mh+AP+Sal + PM)，B 為三合一疫苗（PBS +AP+Sal + PM)，C 為含有 Friis 培養基的三合一疫苗，E為AP單劑疫苗，Η為對照組。 第三圖係顯示小鼠血清霍亂沙門氏桿菌抗體測定，其中A 為四合一疫苗（Mh+AP+Sal+PM)，B .為三合一疫苗（AP + Sal+PM)，C為含有Friis的三合一疫苗，F為Sal單劑 疫苗，Η為對照組。第四圖係顯示小鼠血清巴斯德桿菌抗 體測定，其中Α為四合一疫苗（Mh+AP+Sal + PM)，Β為 三合一疫苗（AP + Sal + PM)，C為添加Friis的三合一疫 苗，G為PM單劑疫苗，Η為對照組。於各圖結果中，單 價疫苗與多價疫苗所產生的血清抗體量均無太大差異，顯 示當合併多種抗原於一多價疫苗中時，抗原之間並不會互 相抑制其他抗原抗體的產生，亦即相對於單價疫苗而言， 本發明之多價疫苗可廣泛地預防多種病原的入侵，且其對 於每種病原的預防效能並不會被減弱。 另外，本發明亦發現當比較三合一組及三合一 Friis 組的多價疫苗，三合一 Friis組對於豬霍亂沙門氏桿菌抗 體（比較第三圖B和C攔）及巴斯德桿菌抗體（比較第四 圖B和C欄）有明顯的增加，然對於胸膜肺炎放射桿抗體 (比較第二圖B* C攔）則無顯著差異，推論原因可能為 14 1290047 黴漿菌專用的Friis培養基中含有大量血清，具有保護抗 原的效果，從而促使三合一 Friis組的多價疫苗比三合一 組的多價疫苗在抗體的誘發上具有較佳的效果。 2·仔豬免疫試驗：取三週齡仔豬12頭，分為四組每組3頭， 第一組以四合一疫苗（Mh+PM+AP+Sal)免疫，第二組以 三合一疫苗（磷酸鹽緩衝溶液（PBS) + PM + AP + Sal)免 疫，第三組以含有Friis培養基之三合一疫苗（Friis培養 基+ PM+AP+Sal)免疫，第四組為對照組以Friis培養基 免疫。以第一次免疫表示為第1天，在第15天再補強一 次，在第25天進行採血，收集後血清利用直接式elISA 法免疫定量分析，其結果如第五圖所示。 第五圖係顯示仔豬血清豬肺炎黴漿菌（Mh)、胸膜肺炎 放射桿菌（AP)、霍亂沙門氏桿菌（Sai)、巴斯德桿菌（pM) 之抗體測定，其中4 in 1為四合一疫苗（Mh+AP+Sal + PM)，3 in 1 為三合一疫苗（AP+Sai + pM),3 inF 為含 Friis 培養基的三合一疫苗，Fdis為接種Friis培養基的對照組。 結果發現，於各種抗體測試結果中，結果最好的仍屬四合 一疫苗及含Friis的三合一疫苗，結果與小鼠血清測試的 結果相符。 3.田間試驗：在豬場的使用上除了小豬使用外，亦可使用 於母豬’使小豬獲件移行抗體來增進對疾病的抵抗力。另 外目前疫苗的使用均為第1、3週各施行一次免疫，但是 由於豬鼻黴漿菌在6日齡小豬就可分離到，因此本疫苗將 以1、3、5週來施行會有較好的效果。為了解疫苗在田間 使用的免疫保護效果，於南部某種豬場（仔豬生產約4〇〇〇 頭/月）測試，在使用前仔豬的育成率為78%，使用後育成 15 1290047 率提高到87.6%，有顯著的提升。 常見豬隻感染肺炎的病原有許多種，單獨黴漿菌疫苗或單獨 細菌性抗原僅能就一種病原作預防，正因如此，單價疫苗的預防 效果一直不甚理想。本發明中將豬黴漿菌合併細菌性抗原，例 如：巴斯德桿菌、豬霍亂沙門氏桿菌、胸膜肺炎放射桿菌等，作 成的二合一、三合一、四合一、五合一之多價疫苗，以便廣泛而 有效地預防常見黴漿菌及細菌傳染的豬隻肺炎，本發明之多價疫 苗其誘發抗體的效果與單獨黴漿菌疫苗或單獨細菌性抗原疫苗 效果相同甚至更好，且能廣泛的預防多種病原。本發明亦發現當 以黴漿菌培養基（Friis培養基）與細菌性多價疫苗相混合，所製 成的疫苗也比單純的多價細菌性疫苗效果更好，推論可能原因為 黴漿菌培養基中的某些成分，具有保護抗原或刺激免疫反應的效 果，本發明之多價疫苗不但省時、省工、省錢，亦顯示多價疫苗 的實用性。 上述實施例係用來詳細敘述本發明之内容，惟不宜用以限制 本發明於所揭示之特定形式。本發明之範疇係以所附之申請專利 範圍所定義為基準，並且包括不脫離本發明之精神與範圍之所有 修飾與類似之變更。 1290047 【圖式之簡單說明】 第一圖係顯示小鼠血清豬肺炎黴漿菌抗體測定結果。 第二圖係顯示小鼠血清胸膜肺炎放射桿菌抗體測定結果。 第三圖係顯示小鼠企清霍亂沙門氏桿菌抗體測定結果。 第四圖係顯示小鼠血清巴斯德桿菌抗體測定結果。 第五圖係仔豬血清豬肺炎黴漿菌、胸膜肺炎放射桿菌、霍亂 沙門氏桿菌及巴斯德桿菌之抗體測定。Soy Broth, TSB)) is enriched for 18-24 hours. Add 1% (V/V) of the enriched bacteria solution to the new brain heart leaching culture solution, and incubate at 120 rpm for 4-8 hours. When the bacterial concentration reaches lx 1〇9~5x 109 CFU/ml, add 0. · 2%-3% (V/V) of the formalin solution (38%) does not activate the bacteria for 8-16 hours, and the bacteria liquid is concentrated by centrifugation 8-15 times (3〇〇〇-5〇〇〇)卬^ Centrifuge for 15 - 30 minutes) and suspend in lx PBS towel, while inactive cells are placed on the medium, not long bacteria. 3. Mixing sputum one preparation · take the steps! The prepared moldy fungus does not activate the bacterial liquid 500-800 liters, and can be used for the strain ATIT-7 or pRn-5 alone or in combination. 'The method and proportion of its use or mixing can refer to the Republic of China Patent No. 〇92 The content of the case in the case of No. 6-8, and the 5-15 ml of each of the fine-grained concentrated bacteria prepared in Step 2 were uniformly mixed. • Vaccine production · Mix the bacterial solution prepared in step 3 with an adjuvant. In the case of 1 bacterial solution 2, 5 = 5°, (V/V), and the proportion of #剂 is 25-_m) The secret device is filled (four) and mixed evenly, and the epidemic 12 1290047 °c is prepared for use. IV. Vaccine Usage Each injection is performed 2-3 times intramuscularly, one dose per injection, the first injection is 1-3 weeks old, the second injection is 3-5 weeks old, and the third injection is in the third injection. At 5-7 weeks of age, the vaccine must be mixed evenly before injection. V. Vaccine Safety Test 1 · Mouse Safety Test··Preparation of A (porcine nasal fungus + PM+AP+Sal), B (porcine pneumoniae +PM+AP+Sal) and C (pig nose) Three vaccines, including mycoplasma + porcine pneumoniae + PM + AP + Sal, were taken from 42 BALB/c mice, 6 were randomly selected as the control group, and 36 were randomly divided into 6 groups, 6 in each group. The vaccines of A, B and C were injected 0.5 ml each by subcutaneous injection or intraperitoneal injection, and observed for 14 days after injection. No adverse reactions occurred during the observation period and survived. 6. Vaccine efficacy test 1. Determination of mouse antibody valence: 80 BALB/c four-week female mice were randomly divided into 8 groups, 10 in each group, respectively: A: four-in-one group (pig pneumonia) Mold fungus (Mh) + PM + AP+Sal) B: Triad (phosphate buffer solution (PBS) + PM+AP+Sal) C: 3-in-1 Friis group (Friis medium + PM+AP+Sal) D : Mh group E : AP group F : Sal group G : PM group Η : control group (Ffiis medium) The mice were immunized twice by subcutaneous injection, and after the second immunization, one star 1290047, anesthesia Blood was collected from the orbit, and the collected blood was allowed to stand at room temperature for 1 hour, then refrigerated at 4 ° C overnight, centrifuged at 3000 rpm for 30 minutes every other day, and after centrifugation, the supernatant was taken out into a new centrifuge tube. Immunoquantitative analysis by direct ELISA. The first panel shows the results of mouse serum S. pneumoniae antibody assay, in which A is a four-in-one vaccine (Mh+AP+Sal + PM), D is a Mh single-dose vaccine, and sputum is a control group. The second panel shows the mouse serum Pleuropneumoniae antibody assay, in which Α is a four-in-one vaccine (Mh+AP+Sal + PM), B is a three-in-one vaccine (PBS +AP+Sal + PM), C is A three-in-one vaccine containing Friis medium, E is a single dose of AP vaccine, and sputum is a control group. The third panel shows the serum S. cholerae antibody assay in mice, where A is a four-in-one vaccine (Mh+AP+Sal+PM), B. is a three-in-one vaccine (AP + Sal+PM), and C is contained. Friis's three-in-one vaccine, F is a single dose of Sal, and sputum is the control group. The fourth panel shows the mouse serum Pasteurella antibody assay, in which Α is a four-in-one vaccine (Mh+AP+Sal + PM), Β is a three-in-one vaccine (AP + Sal + PM), and C is added Friis The three-in-one vaccine, G is a single dose of PM vaccine, and sputum is the control group. In the results of each graph, the amount of serum antibody produced by the monovalent vaccine and the multivalent vaccine did not differ much, indicating that when multiple antigens were combined in a multivalent vaccine, the antigens did not inhibit each other from producing other antigens. That is, the multivalent vaccine of the present invention can broadly prevent the invasion of various pathogens with respect to the monovalent vaccine, and its preventive efficacy against each pathogen is not attenuated. In addition, the present inventors have also found that when comparing multivalent vaccines in the triad and triad Friis groups, the three-in-one Friis group for Salmonella choleraesuis antibody (compare the third panel B and C) and Pasteurella There was a significant increase in antibodies (compare the fourth panel in columns B and C), but there was no significant difference in the pleural pneumonia radiograph antibody (compared to the second panel B* C). The reason for the inference might be 14 1290047 for fungi-specific Friis The medium contains a large amount of serum, which has the effect of protecting the antigen, thereby promoting the multivalent vaccine of the three-in-one Friis group to have a better effect in inducing the antibody than the three-in-one multivalent vaccine. 2. Piglet immunization test: 12 piglets of 3 weeks old were divided into 4 groups of 3 heads, the first group was immunized with 4 in 1 vaccine (Mh+PM+AP+Sal), and the second group was treated with 3 in 1 vaccine. (Phosphate buffer solution (PBS) + PM + AP + Sal) immunized, the third group was immunized with a three-in-one vaccine containing Friis medium (Friis medium + PM + AP + Sal), and the fourth group was a control group with Friis medium. Immunity. The first immunization was expressed as the first day, and the re-reinforcement was performed on the 15th day, and the blood was collected on the 25th day. The collected serum was immunologically quantitatively analyzed by the direct elISA method, and the results are shown in Fig. 5. The fifth picture shows the antibody assay of piglet serum M. pneumoniae (Mh), P. pleuropneumoniae (AP), Salmonella cholerae (Sai), and Pasteurella (pM), of which 4 in 1 is quadruple. One vaccine (Mh+AP+Sal + PM), 3 in 1 is a three-in-one vaccine (AP+Sai + pM), 3 inF is a three-in-one vaccine containing Friis medium, and Fdis is a control group inoculated with Friis medium. It was found that among the various antibody test results, the best results were still a four-in-one vaccine and a three-in-one vaccine containing Friis, and the results were consistent with the results of the mouse serum test. 3. Field trials: In addition to the use of piglets in pig farms, they can also be used in sows to enable piglets to acquire antibodies to enhance disease resistance. In addition, the current vaccine is administered once every 1st and 3rd week, but since the pig's nose fungus can be isolated in 6-day-old piglets, the vaccine will be administered in 1, 3, and 5 weeks. Better results. In order to understand the immune protection effect of the vaccine in the field, the pig breeding rate was 78% in some pig farms in the south (the piglet production was about 4 fish/month), and the breeding rate was increased to 87.6 after use. %, there is a significant improvement. Many common diseases of pigs infected with pneumonia, single mycobacteria vaccine or bacterial antigen alone can only prevent one pathogen, which is why the prevention effect of monovalent vaccine has not been satisfactory. In the present invention, the porcine mold is combined with a bacterial antigen, such as: Pasteurella, Salmonella choleraesuis, Pneumocystis pneumoniae, etc., made in two-in-one, three-in-one, four-in-one, five-in-one Multivalent vaccines for the broad and effective prevention of common mycobacteria and bacterial infection of swine pneumonia. The multivalent vaccine of the present invention has the same effect as or induces an antibody against a single mycobacterial vaccine or a bacterial antigen alone vaccine. And can prevent a wide range of pathogens. The present invention also finds that when the mycoplasma culture medium (Friis medium) is mixed with the bacterial multivalent vaccine, the prepared vaccine is also better than the simple multivalent bacterial vaccine, and the reason may be that the mycoplasma culture medium is in the medium. Some of the components have the effect of protecting the antigen or stimulating the immune response. The multivalent vaccine of the present invention not only saves time, labor, and money, but also shows the practicability of the multivalent vaccine. The above-described embodiments are intended to be illustrative of the present invention and are not intended to limit the invention in the particular form disclosed. The scope of the invention is defined by the scope of the appended claims, and all modifications and equivalents are 1290047 [Simple description of the schema] The first panel shows the results of the determination of mouse serum porcine pneumoniae antibody. The second panel shows the results of the mouse serum Pleuropneumoniae antibody assay. The third panel shows the results of the antibody assay for Salmonella choleraesuis in mice. The fourth panel shows the results of the mouse serum Pasteurella antibody assay. The fifth picture shows the determination of antibodies against piglet serum, M. pneumoniae, P. pleuropneumoniae, Salmonella cholerae and Pasteurella.
1717