CN107802658A - 灭活乳酸菌在防治家禽病毒性疾病药物中的应用 - Google Patents
灭活乳酸菌在防治家禽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家禽病毒性疾病药物中的新用途，属于生物医药领域，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家禽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家禽病毒性疾病药物中的新用途。
背景技术
近些年家禽病毒性疾病频发，给家禽养殖业造成重大损失，严重威胁着家禽养殖业的健康发展。常见的禽病毒性疾病包括：禽流感、新城疫、法氏囊、传染性支气管炎、腺病毒病、鸭肝炎、鸭细小病毒病、鸭黄病毒病、小鹅瘟、鹅副黏病毒病等。目前应对家禽病毒病常采用接种疫苗的方法，由于病毒不断变异，疫苗防控也往往失效。另外，一些新的病毒病突然爆发，尚无疫苗可控，如近年新发生的禽4型腺病毒病。目前抗病毒西药(如利巴韦林、金刚烷胺、吗啉双胍等)在兽医临床严格禁用。抗病毒中药成为了主要防治措施，如双黄连、清开灵、银翘散等，中药抗病毒虽疗效确切，但有时也很难达到理想效果。此外，一些免疫增强剂也常用于病毒病的辅助治疗，如黄芪多糖、转移因子、干扰素、卵黄抗体等。由于一些感染禽的病毒具有毒力强、容易变异和产生耐药性的特点，防治难度较大。因此寻找新型的防治禽病毒性疾病的药物非常重要。本发明发现将灭活的乳酸菌进行静脉给药可有效地防治家禽的病毒性疾病，目前国内外尚无相关报道。另外，灭活乳酸菌混悬液用于禽病毒病的防治，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家禽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，该药物静脉给药可以有效防治家禽的各种病毒性疾病。
灭活乳酸菌在制备防治家禽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。
用于本发明制备防治家禽病毒性疾病的灭活乳酸菌混悬液的乳酸菌种类包括：(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.lactis subsp.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。
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。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。每单位剂量(mL)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。所述防治家禽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，将灭活屎肠球菌对家禽翅下静脉给药，可以有效地防治鸡新城疫。说明灭活乳酸菌静脉给药可以用于家禽病毒性疾病的防治。
本发明的灭活乳酸菌混悬液经静脉给药可以用于家禽各种病毒性疾病的防治，包括：禽流感、新城疫、法氏囊、传染性支气管炎、腺病毒病、鸭肝炎、鸭细小病毒病、鸭黄病毒病、小鹅瘟、鹅副黏病毒病等。
灭活乳酸菌混悬液防治家禽病毒性疾病的机制目前尚不完全清楚，推测可能的机制是：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；也可能是灭活乳酸菌静脉给药后经家禽体内代谢产生了抗病毒物质；或者灭活乳酸菌静脉给药后可激活家禽的免疫系统，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可测定每mL屎肠球菌混悬液中含有屎肠球菌菌体数量约为108个。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未发生明显变化。
实施例2
将实施例1制备的混悬液稀释后制备10×浓度的灭活屎肠球菌混悬液，研究灭活屎肠球菌静脉给药对鸡新城疫的防治作用。选用10日龄白羽肉鸡随机分为新城疫对照组、灭活屎肠球菌高剂量组和灭活屎肠球菌低剂量组，每组30只。高剂量和低剂量灭活屎肠球菌组的给药剂量分别是翅下静脉注射10×的灭活屎肠球菌混悬液0.05mL/只和0.1mL/只。新城疫对照组翅下静脉注射生理盐水。对鸡翅下静脉注射给药24小时后攻毒，每组鸡肌肉注射含有新城疫病毒的尿囊液0.2mL。连续观察5天，记录死亡数。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，高剂量的灭活屎肠球菌静脉给药可以显著降低鸡的死亡数，说明灭活乳酸菌混悬液静脉给药可以用于家禽病毒性疾病的防治。
表1灭活屎肠球菌静脉给药对鸡新城疫的防治效果
注：**表示与新城疫对照组比较差异极显著P＜0.01，*表示与新城疫对照组比较差异显著P＜0.05。
Claims (10)
1.灭活乳酸菌在制备防治家禽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，所述的优选的，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：CICC6240)、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：Enterococcus faecium，保藏编号：CICC 6049)中的一种或几种。
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，所述防治家禽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1-6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1-8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，所述家禽病毒性疾病包括：禽流感、新城疫、法氏囊、传染性支气管炎、腺病毒病、鸭肝炎、鸭细小病毒病、鸭黄病毒病、小鹅瘟、鹅副黏病毒病。
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