CN107802657A - 灭活乳酸菌在防治猪病毒性疾病药物中的应用 - Google Patents
灭活乳酸菌在防治猪病毒性疾病药物中的应用 Download PDFInfo
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Abstract
本发明涉及一种灭活乳酸菌在防治猪病毒性中的新用途，属于生物医药领域。这种药物的主要成分是灭活乳酸菌，可以通过将活的乳酸菌经过常规方法灭活制备。灭活乳酸菌静脉给药可以用于猪的各种病毒性疾病的预防、治疗或辅助治疗，具有广阔的应用前景。
Description
技术领域
本发明属于生物医药领域，尤其涉及灭活乳酸菌在防治猪病毒性疾病中的新用途。
背景技术
这些年猪的病毒性疾病给养猪业造成了巨大经济损失。猪的常见病毒性疾病包括：猪口蹄疫、伪狂犬病、繁殖与呼吸综合征、细小病毒病、猪瘟、猪圆环、日本脑炎、猪流感、猪肠病毒综合征、猪传染性胃肠炎、猪流行性腹泻等。猪的多种病毒性疾病可造成机体免疫抑制，引起细菌继发感染而出现死亡；一些病毒性疾病还会引起母猪繁殖障碍，出现流产、死胎、木乃伊胎等；有的病毒还可以垂直传播影响商品猪的生产性能；仔猪的一些病毒性腹泻发病率高、死亡率高，危害严重；猪口蹄疫还是人畜共患病，影响人的健康。
目前应对猪病毒性疾病主要依靠疫苗，但由于病毒变异等原因，疫苗防控也往往失效。除了使用疫苗预防猪的病毒性疾病外，在临床上还使用一些中药，如清瘟败毒散、双黄连注射液等进行猪病毒性疾病的防治，但往往效果不理想。此外，一些免疫调节剂也用于猪病毒性疾病防治，如如黄芪多糖、转移因子，但往往只能起到辅助治疗作用。另外，一些抗病毒药物如金刚烷胺、利巴韦林、吗啉双胍等在兽医临床上已禁用。所以，目前对于猪病毒性疾病的防治非常困难，多年来这些病毒性疾病困扰着养猪业的健康发展，寻找新型的防治猪病毒性疾病的药物非常重要。
本发明发现灭活乳酸菌静脉注射给药可有效地防治猪的病毒性疾病，目前国内外尚无相关报道。另外，灭活乳酸菌混悬液用于猪病毒性疾病的防治，无有害物质残留，不存在食品安全问题，未来具有广阔的应用前景。
发明内容
本发明提供了一种全新的可以用于猪病毒性疾病防治的灭活乳酸菌混悬液，这种药物的主要成分为灭活的乳酸菌，该药静脉给药可以有效地防治猪的各种病毒性疾病。
灭活乳酸菌在制备防治猪病毒性疾病药物中的用途，其特征在于，对所述的灭活乳酸菌进行革兰氏染色，在油镜下观察，灭活乳酸菌保持完整菌体形态，药物的给药方式为注射给药。所述的保持完整菌体形态是指与灭活前活菌菌体的轮廓和形态基本一致。所说的基本一致实质上是指乳酸菌灭活过程中菌体细胞壁可能会出现轻微变化，比如部分表面成分的流失，但这种变化微乎其微或很少发生。
所述乳酸菌种类包括：(1)乳杆菌属：德氏乳杆菌(L.delbrueckii)、保加利亚乳杆菌(L.bulgaricus)、瑞士乳杆菌(L.helviticus)、嗜酸乳杆菌(L.acido phlus)、格氏乳杆菌(L.gasseri)、唾液乳杆菌(L.salivarius)、植物乳杆菌(L.plantarum)、罗伊氏乳杆菌(L.reuteri)、短乳杆菌(L.brevis)、干酪乳杆菌(L.casei)、发酵乳杆菌(L.fementi)等；(2)片球菌属：如乳酸片球菌(P.acidi1actic)、戊糖片球菌(P.pentasiaceus)、小片球菌(P.parvulus)等；(3)明串球菌属：肠膜明串球菌(L.mesenteroides)及其乳脂亚种(L.cremoris)和葡聚糖亚种(Leuc.dextranicun)、乳酸明串球菌(L.lactis)、酒明串球菌(L.oenos)等；(4)肠球菌属：屎肠球菌(E.faecium)、粪肠球菌(E.faecalis)等；(5)乳球菌属：乳酸乳球菌乳酸亚种(L.lactis subsp.lactis)、乳酸乳球菌乳脂亚种(L.lactissubsp.cremoris)、乳酸乳球菌叶蝉亚种(L.lactis subsp.hordniae)等；(6)链球菌属：乳酸链球菌(S.lactis)、丁二酮乳酸链球菌(S.diacetilactis)、乳酪链球菌(S.creamoris)、嗜热乳链球菌(S.thermophilus)等；(7)双歧杆菌属：两歧双歧杆菌(B.bifidum)、长双歧杆菌(B.longum)、短双歧杆菌(B.breve)、婴儿双歧杆菌(B.infantis)、青春双歧杆菌(B.adolescentis)、动物双歧杆菌(B.animalis)等；(8)其他种属的乳酸菌。
优选的，乳酸菌选自乳酸乳球菌乳酸亚种(拉丁名称：Lactococcus lactissubsp.Lactis，保藏编号：CICC 6246)、植物乳杆菌植物亚种(拉丁名称：Lactobacillusplantarum subsp.Plantarum，保藏编号：CICC 6240)、长双歧杆菌(拉丁名称：Bifidobacterium longum，保藏编号：CICC 6196)、短乳杆菌(拉丁名称：Lactobacillusbrevis，保藏编号：CICC 6239)、屎肠球菌(拉丁名称：Enterococcus faecium，保藏编号：CICC 6049)。灭活乳酸菌为一种或两种以上灭活乳酸菌混合物。注射给药时，每ml剂量药物中包含灭活乳酸菌完整菌体数量为105—1012个。所述的注射给药为静脉注射给药。
利用高温高压灭活、紫外线灭活、化学试剂灭活或辐射灭活中的任一种灭活方法得到所述灭活乳酸菌。
所述防治猪病毒性疾病药物可以制成粉针剂或混悬注射剂。
本发明采用常规方法灭活了屎肠球菌，发现灭活的屎肠球菌仍然可以进行革兰氏染色，油镜下观察灭活的屎肠球菌与活的屎肠球菌保持一致的菌体轮廓和形态。然后对灭活屎肠球菌生理盐水混悬液进行离心，弃上清留沉淀，提取DNA，采用PCR技术仍可以扩增出16S rDNA基因片段，通过测序还可以进行乳酸菌种类鉴别。进一步研究发现，将灭活屎肠球菌对猪耳静脉给药，可以有效地治愈发病的口蹄疫，而且对未发病的猪具有预防口蹄疫发生的作用。研究还发现将灭活的植物乳杆菌植物亚种给猪耳静脉给药，可以预防猪蓝耳病发生。以上说明灭活乳酸菌静脉给药可以用于猪病毒性疾病的防治。
本发明的灭活乳酸菌混悬液经静脉给药可以用于猪各种病毒性疾病的防治，包括：猪口蹄疫、猪繁殖呼吸综合征、猪瘟、猪圆环病毒病、猪细小病毒病、猪伪狂犬、猪传染性胃肠炎、猪流行性腹泻、猪日本脑炎、猪水疱性病、猪流感、猪痘等。灭活乳酸菌静脉给药，可能与病毒发生相互作用，阻断病毒感染组织器官；也可能灭活乳酸菌静脉给药后经猪机体代谢产生了抗病毒物质；灭活乳酸菌静脉给药后可激活猪的免疫系统，间接发挥抗病毒作用等。
具体实施方式
实施例1
将屎肠球菌(购于中国工业微生物菌种保藏管理中心，拉丁名称：Enterococcusfaecium，保藏编号：CICC 6049)接种于MRS培养基，于37℃温箱培养24小时，然后3000转离心5分钟，去除上层培养液保留沉淀，加入无菌生理盐水清洗沉淀，离心5分钟，重复清洗3次后，加入无菌生理盐水，与沉淀混匀。取一定量的屎肠球菌生理盐水混悬液，于分光光度计690nm处测量其OD值，当用无菌生理盐水稀释的最终浓度的OD值为0.38时，将这样稀释浓度的屎肠球菌生理盐水混悬液作为1倍(1×)浓度，经过THOMA细菌计数板进行细菌计数测定每mL混悬液中含有约108个屎肠球菌菌体。本实验配制了1×浓度的屎肠球菌生理盐水混悬液。取少量的1×浓度的屎肠球菌生理盐水混悬液，进行革兰氏染色，在油镜下观察活菌的形态。此后将配制好的1×浓度的屎肠球菌生理盐水混悬液于121℃、压力0.12MPa，灭活15min，得到灭活屎肠球菌混悬液，取少量灭活屎肠球菌混悬液进行革兰氏染色，在油镜下观察灭活菌体的形态，发现灭活乳酸菌保持完整菌体形态，与其灭活前活菌菌体轮廓和形态一致，灭活前后完整细菌数量没有发生明显变化。
实施例2
将实施例1制备的混悬液稀释后获得10×浓度的灭活屎肠球菌混悬液，检测灭活屎肠球菌混悬液对猪口蹄疫的防治作用。某猪场共有17头母猪。一头母猪发生口蹄疫，四肢溃烂，紧急耳静脉推注10×浓度灭活屎肠球菌混悬液5mL，第二天该猪分娩，又静脉推注灭活屎肠球菌混悬液5mL；第4天该猪起卧、采食正常，口蹄疫被治愈。此后又对其中的7头未发病的母猪均在分娩前后的两天内各静脉推注了10×浓度的灭活乳酸菌生理盐水混悬液5ml，而其余9头母猪没有给药，作为对照。在后续的观察中发现，静脉注射药物的7头母猪均未发生口蹄疫，而未静注药物的9头母猪中，7头发生了口蹄疫，只有2头未发生口蹄疫。采用SPSS 11.5软件对数据进行卡方检验，结果见表1。由表1可知，给药组与未给药组相比较差异极显著。以上说明，灭活乳酸菌静脉给药对猪口蹄疫具有良好的防治作用。
表1灭活屎肠球菌静脉给药对母猪口蹄疫发病的影响
注：**表示与未给药组比较差异极显著P＜0.01，*表示与未给药组比较差异显著P＜0.05。
实施例3
将植物乳杆菌植物亚种(购于中国工业微生物菌种保藏管理中心，拉丁名称：Lactobacillus plantarum subsp.Plantarum，保藏编号：CICC 6240)，根据实施例1方法制备10×浓度的灭活植物乳杆菌植物亚种混悬液，检测灭活植物乳杆菌植物亚种静脉给药对猪蓝耳病的防治作用。购买了16头健康猪随机分为正常组、蓝耳病模型组、预防给药组和治疗给药组，每组4头。采用每头猪耳后肌肉注射蓝耳病病毒液2mL，制备猪蓝耳病模型。预防给药组的猪耳静脉注射10×浓度的灭活植物乳杆菌植物亚种混悬液1mL，第二天进行攻毒制备蓝耳病模型；治疗给药组的猪在攻毒制备蓝耳病模型后的当天耳静脉注射10×浓度的灭活植物乳杆菌植物亚种混悬液1mL，蓝耳病模型组在猪攻毒制备蓝耳病模型后的当天耳静脉注射1mL无菌生理盐水。正常组猪耳静脉注射1mL无菌生理盐水。此后观察了10天猪的采食量和外观变化，结果见表2。蓝耳病模型组的4头猪均发病，表现为炸毛消瘦，采食量明显下降；治疗给药组的4头猪均发病，也表现为炸毛消瘦，采食量下降；而预防给药组4头猪均无发病症状，即无炸毛消瘦现象，食量较正常组慢，但采食量没有下降。以上说明，灭活植物乳杆菌植物亚种混悬液可以有效地防治猪蓝耳病的发生。
表2灭活植物乳杆菌植物亚种静脉给药对蓝耳病发病的影响
Claims (10)
1.灭活乳酸菌在制备防治猪病毒性疾病药物中的用途，其特征在于，对所述的灭活乳酸菌进行革兰氏染色，在油镜下观察，灭活乳酸菌保持完整菌体形态，与其灭活前活菌菌体轮廓和形态一致，药物的给药方式为注射给药。
2.根据权利要求1的用途，其特征在于，所述的乳酸菌选自乳杆菌属、肠球菌属、乳球菌属、双歧杆菌属、明串球菌属、链球菌属。
3.根据权利要求1所述的用途，其特征在于，所述乳酸菌选自乳酸乳球菌乳酸亚种(拉丁名称：Lactococcus lactis subsp.Lactis，保藏编号：CICC 6246)、植物乳杆菌植物亚种(拉丁名称：Lactobacillus plantarum subsp.Plantarum，保藏编号：CICC 6240)、长双歧杆菌(拉丁名称：Bifidobacterium longum，保藏编号：CICC 6196)、短乳杆菌(拉丁名称：Lactobacillus brevis，保藏编号：CICC 6239)、屎肠球菌(拉丁名称：Enterococcusfaecium，保藏编号：CICC 6049)。
4.如权利要求1所述的用途，其特征在于，灭活乳酸菌为一种或两种以上灭活乳酸菌混合物。
5.根据权利要求1所述的用途，其特征在于，利用高温高压灭活、紫外线灭活、化学试剂灭活或辐射灭活中的任一种灭活方法得到所述灭活乳酸菌。
6.根据权利要求1所述的用途，其特征在于，所述防治猪病毒性疾病药物可以制成粉针剂或混悬注射剂。
7.根据权利要求6所述的用途，其特征在于，所述的注射给药为静脉注射给药。
8.根据权利要求7所述的用途，其特征在于，注射给药时，每ml剂量药物中包含灭活乳酸菌完整菌体数量为105—1012个。
9.根据权利要求8任一项所述的注射剂，其特征在于，所述的注射剂按照如下方法制备：将乳酸菌进行常规液体培养基培养12-36小时后，3000-5000转离心保留沉淀，再将沉淀充分清洗后，配制成所需浓度混悬液，于120-122℃、压力0.1-0.2MPa，灭活15-30min得到灭活乳酸菌注射剂。
10.根据权利要求1-9任一项所述的用途，其特征在于，所述猪病毒性疾病包括：猪口蹄疫、猪繁殖呼吸综合征、猪瘟、猪圆环病毒病、猪细小病毒病、猪伪狂犬、猪传染性胃肠炎、猪流行性腹泻、猪日本脑炎、猪水疱性病、猪流感、猪痘。
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