CN118216620A - 牛碱性磷酸酶bIAP作为饲料添加剂用于抵抗产气荚膜梭菌感染肉鸡所致感染症状的应用 - Google Patents
牛碱性磷酸酶bIAP作为饲料添加剂用于抵抗产气荚膜梭菌感染肉鸡所致感染症状的应用 Download PDFInfo
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Abstract
本发明涉及农业生物技术领域，具体涉及牛碱性磷酸酶bIAP作为饲料添加剂用于抵抗产气荚膜梭菌感染肉鸡所致感染症状的应用。向肉鸡饲料中添加牛来源的肠碱性磷酸酶（bIAP）可达到提高肉鸡对产气荚膜梭菌感染的抵抗能力，提升肉鸡健康水平，一方面可改善肉鸡因产气荚膜梭菌感染导致的生长减缓现象，提高肉鸡的生长性能；第二方面还可降低产气荚膜梭菌感染引起的炎症反应水平，缓解免疫器官肿大，降低血清炎症因子水平；最后，bIAP对产气荚膜梭菌引起的肠道菌群紊乱起到了恢复作用。
Description
技术领域
本发明涉及农业生物技术领域，具体涉及牛碱性磷酸酶bIAP作为饲料添加剂用于抵抗产气荚膜梭菌感染肉鸡所致感染症状的应用。
背景技术
产气荚膜梭菌是肉鸡肠道菌群中的一种条件性致病菌，也是一种食源性致病菌，可导致人和动物发病。目前，禁止使用抗生素生长促进剂，因此寻找绿色高效的抗生素替代物作为预防/治疗产气荚膜梭菌感染成为亟待解决的问题。
饲用酶制剂已被证明在帮助预防/治疗动物致病菌感染与改善动物体健康方面具有巨大潜力。肠道碱性磷酸酶IAP 是一种经糖基磷脂酰肌醇键锚定于肠上皮细胞顶膜上的糖蛋白。肠道病原菌分为革兰氏阴性菌和革兰氏阳性菌，目前IAP的抗感染能力研究多局限于革兰氏阴性致病菌，且研究动物为哺乳类动物，在禽类如肉鸡上的研究、以及针对革兰氏阳性病原菌的研究较为局限。饲喂bIAP能使小鼠抵御革兰氏阴性菌鼠伤寒沙门氏菌对肠道的感染和所致腹泻，这是因为革兰氏阴性菌的一大毒力因子是LPS，它除了在进入血液中引起动物机体发热，还会刺激肠道发生炎症。bIAP通过将LPS上的磷酸基团水解从而将其钝化，从而使内毒素失去和肠道上皮细胞受体结合的能力，减轻其对动物肠道上皮的直接致炎作用。此外，肠道炎症发生过程中所伴随肠道屏障通透性的提升，此时LPS可进入体内；内毒素是动物体内轻度炎症的主要诱因，而由于bIAP可跨越肠道屏障进入体液循环，因此它在体内对LPS的水解可进一步降低体内轻度炎症水平。因此，bIAP对LPS的降解和解除其毒性可解释为什么bIAP能够抵御鼠伤寒沙门氏菌这种革兰氏阴性菌的感染。
同鼠伤寒沙门氏菌不同，产气荚膜梭菌为革兰氏阳性菌，它并不产生内毒素，其致病机制为外毒素而非革兰氏阴性菌一样的内毒素LPS，因此依据现有技术的教导，理论上bIAP无法像抗伤寒沙门氏菌感染一样，在动物中抵御革兰氏阳性菌产气荚膜梭菌的感染。
发明内容
本发明的目的是提供牛来源的碱性磷酸酶bIAP作为饲料添加剂的应用。
根据本发明的技术方案，提供了牛来源的碱性磷酸酶bIAP作为饲料添加剂用于以下用途的应用：
抵抗产气荚膜梭菌感染肉鸡所致生产性能下降；
降低产气荚膜梭菌感染肉鸡所致炎症反应和肠道损伤；
降低产气荚膜梭菌感染肉鸡所致肠道菌群紊乱。
根据本发明的技术方案，所述牛来源的碱性磷酸酶bIAP的添加量为1000 U/kg~5000 U/kg。
根据本发明的技术方案，所使用的牛来源的肠碱性磷酸酶为利用重组里氏木霉菌株发酵制得，其序列如SEQ ID No：1所示。
针对以上问题，结合目前肉鸡养殖中所面临的肠道革兰氏阳性病原菌产气荚膜梭菌感染风险，本发明将里氏木霉重组表达的bIAP添加至肉鸡小麦日粮中，并进行产气荚膜梭菌攻毒感染，以探究bIAP的添加是否可用以预防肉鸡产气荚膜梭菌感染以及改善感染症状。结果表明，向肉鸡饲料中添加牛来源的肠碱性磷酸酶（bIAP）可达到提高肉鸡对产气荚膜梭菌感染的抵抗能力，提升肉鸡健康水平，一方面可改善肉鸡因产气荚膜梭菌感染导致的生长减缓现象，提高肉鸡的生长性能；第二方面还可降低产气荚膜梭菌感染引起的炎症反应水平，缓解免疫器官肿大，降低血清炎症因子水平；最后，bIAP对产气荚膜梭菌引起的肠道菌群紊乱起到了恢复作用。
附图说明
图1显示bIAP对21日龄肉鸡免疫器官肿大的缓解，其中，统计结果均为和对照NC组相比较，“*”为差异显著(P ＜ 0.05)；
图2显示bIAP 对21日龄肉鸡血清中炎症因子浓度的降低，统计结果均为和对照NC组相比较，其中，“ns”为差异不显著，“*”为差异显著(P ＜ 0.05)，“***”为差异极显著(P＜ 0.001)；
图3显示bIAP对肉鸡肠道损伤的保护；
图4-1和图4-2 显示在门和属水平bIAP对肉鸡肠道菌群紊乱的调控。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例使用牛来源的肠碱性磷酸酶，为利用重组里氏木霉菌株发酵制得，其序列如SEQ ID No：1所示。
SEQ ID No：1：
LIPAEEENPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYRTIGVSAAARYNQCNTTRGNEVTSVINRAKKAGKAVGVVTTTRVQHASPAGAYAHTVNRNWYSDADLPADAQKNGCQDIAAQLVYNMDIDVILGGGRMYMFPEGTPDPEYPDDASVNGVRKDKQNLVQEWQAKHQGAQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLAEMTEAALQVLSRNPRGFYLFVEGGRIDHGHHDGKAYMALTEAIMFDNAIAKANELTSELDTLILVTADHSHVFSFGGYTLRGTSIFGLAPGKALDSKSYTSILYGNGPGYALGGGSRPDVNGSTSEEPSYRQQAAVPLASETHGGEDVAVFARGPQAHLVHGVQEETFVAHIMAFAGCVEPYTDCNLPAPA。
以下实施例所使用基础饲料按质量百分比计的原料组成及占比如表1所示，基础日粮营养成分含量如表2所示。
表1：基础日粮配方
| 原料名称 | 含量（%） |
| 小麦 | 64.22 |
| 豆粕 | 28.8 |
| 大豆油 | 2.3 |
| 磷酸氢钙 | 1.7 |
| DL-蛋氨酸 | 0.17 |
| L-赖氨酸 | 0.18 |
| 苏氨酸 | 0.15 |
| 石粉 | 1.1 |
| 食盐 | 0.35 |
| 多维预混料 | 0.03 |
| 微量元素预混料 | 0.2 |
| 氯化胆碱（50%） | 0.3 |
| 二氧化钛 | 0.5 |
。
表 2基础日粮营养成分含量
| 营养物质 | 含量（%） |
| 代谢能 ME/(MJ/kg) | 2.80 |
| 钙(%) | 1.00 |
| 有效磷(%) | 0.44 |
| 食盐(%) | 0.30 |
| 蛋氨酸(%) | 0.50 |
| 赖氨酸(%) | 1.13 |
| 苏氨酸(%) | 0.89 |
。
本申请所提供的如上配方的养殖饲料中，bIAP添加量（按酶活性单位计）分别1000U/kg以及5000 U/kg。本申请中，除添加酶外的原料质量百分比之和为1.13100%。以下以具体实施例进行说明，实施例中所用原料均可通过市购获得。
实施例1 饲料中添加bIAP抵抗产气荚膜梭菌感染所致生产性能下降
肉鸡的生长性能是衡量其经济效益的重要指标，产气荚膜梭菌感染后，肉鸡因严重的肠道炎症导致腹泻、精神萎靡等症状，引起肉鸡采食量下降，平均日增重降低，料肉比升高的现象。可以通过测量平均日增重、平均日采食量反映bIAP对产气荚膜梭菌感染肉鸡的生长性能的改善作用。
试验采用随机分组法，选取288只体重相近的健康AA肉仔鸡，随机分为4个处理组，每组6个重复（每个重复12羽）。实验分为阴性对照组饲喂基础日粮、阳性对照组饲喂基础日粮并进行产气荚膜梭菌攻毒、添加1000 U/kg与5000 U/kg bIAP基础日粮的攻毒组四个处理组。以掺入基础饲粮方式喂饲21天。基础日粮参照NRC(1994)肉鸡营养需要进行配制，见表2。肉鸡养殖于实验鸡舍，自由采食和饮水，1-3日龄24 h光照，4-7日龄23 h光照，8-21日龄20 h光照，定时清扫卫生，定期对舍内消毒，避免交叉感染，免疫及饲养管理遵照实验鸡场规定执行。
按照表1中饲料配方，配置成4种饲料，如表3所示，分别命名为阴性对照组NC、阳性对照组PC、低剂量bIAP组、高剂量bIAP组。对照组饲喂表1中基础饲料，实验组按照表3分为低剂量组和高剂量组分别添加bIAP冻干粉1000 U/kg以及5000 U/kg。
表3实验分组
| 实验分组 | 处理 |
| NC | 基础日粮＋无菌培养基 |
| PC | 基础日粮＋产气荚膜梭菌 |
| 低剂量组 | 基础日粮＋1000 U/kg bIAP＋产气荚膜梭菌 |
| 高剂量组 | 基础日粮＋5000 U/kg bIAP＋产气荚膜梭菌 |
。
实验第0、21日，分别对各组肉鸡进行空腹称重并记录，准确记录饲养期内肉鸡的采食量，参照中华人民共和国农业行业标准（NY/T 823-2020-家禽生产性能名词术语和度量统计方法）测定肉鸡生产性能。计算公式为：日采食量（g/d/只）=全期耗料/饲养只日数；日均增重（g/d/只）=总增重/饲养只日数；耗料增重比=全程消耗饲料总量/总增重，不同添加bIAP对养殖肉鸡生长性能的比较结果见表4。
表4 添加bIAP对肉鸡生长性能的影响
| 处理 | 平均日采食量 (g/d) | 平均日增重(g/d) | 料肉比(F/G) |
| NC | 29.34±4.98bc | 15.5±0.51bc | 1.89±0.07a |
| PC | 26.78±5.37a | 11.2±0.87a | 2.39±0.05bc |
| 低剂量组 | 27.75±3.67ab | 12.53±0.64ab | 2.21±0.05ab |
| 高剂量组 | 28.97±2.36bc | 15.82±0.66bc | 1.87±0.06a |
由表4可知，在21日龄，与阳性对照组相比，bIAP各剂量组肉鸡平均日增重显著升高(P ＜ 0.05)；平均日采食量随剂量增加而增加；料重比随bIAP添加量增加而降低，其中高剂量组料重比最低；结果表明，在肉仔鸡日粮中添加bIAP可显著提高产气荚膜梭菌感染肉鸡采食量及日增重，并降低料重比。
实施例2 添加bIAP降低产气荚膜梭菌感染所致炎症反应和肠道损伤
由实施例1可知，bIAP添加可对抗产气荚膜梭菌感染后肉鸡日增重下降所致损失。此外，产气荚膜梭菌可引起坏死性肠炎，并释放多种毒素引发全身性炎症，进而刺激免疫器官肿大。按照实施例1进行肉鸡养殖实验，各实验组分别在第21日龄随机抽取12只鸡称重后，取肝脏、脾脏、胸腺和法氏囊，用滤纸吸干水分后称重，计算免疫器官指数：免疫器官指数=免疫器官重量/体重。
各组于第21日龄随机抽取12只鸡，电击处死后取血，每只试鸡约5 mL，静置后，经过高速离心机15 min处理后，放置于-20℃下保存备用。血清中免疫球蛋白的测定采用相应的酶联免疫吸附法试剂盒测定。将试剂盒在室温中平衡20 min，取出待测血清，按照试剂盒说明书规范操作。通过测定特定波长的吸光度值，参照标准曲线即可算出血清中促炎因子白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）以及细胞坏死因子-α（TNF-α）的含量，同时测定抗炎因子白细胞介素-10（IL-10）的含量。肝脏、胸腺、法氏囊和脾脏是家禽免疫系统重要的免疫器官，在感染期间，通过免疫器官指数可以初步衡量机体炎症水平。血清炎症因子的含量与动物机体免疫功能也有密切的关系。添加bIAP对肉鸡免疫功能的提升如图1和2，可见bIAP能够在产气荚膜梭菌攻毒实验中对肉鸡免疫器官的肿大起到缓解作用（图1），降低血清中炎症因子的浓度。
各组于21日龄采取十二指肠、空肠及回肠固定样品，包埋后制作切片观察。产气荚膜梭菌感染会引起肉鸡肠道绒毛损伤变短，丰度降低。添加bIAP对肉鸡肠道损伤的保护如图3所示，可见bIAP能很好地缓解产气荚膜梭菌攻毒感染后肉鸡的肠道损伤。
以上结果表明，在饲粮中添加bIAP能够缓解肉鸡机体的炎症反应，降低炎症水平，其中高添加剂量的作用效果更为显著。
实施例3 添加bIAP对肉鸡中产气荚膜梭菌感染所致肠道菌群紊乱起到调控作用
基于实施例1与实施例2结果，由于产气荚膜梭菌的感染会引起肉鸡肠道菌群的紊乱，因此在21日龄每个处理选取6只肉鸡，电击处死后解剖，采取盲肠微生物至无菌离心管中，于液氮中迅速冷冻并进行微生物测序分析，如图4-1和图4-2所示，bIAP的添加在门水平上降低了拟杆菌门（Bacteroidetes）的丰度，5000 U/kg的BIAP提高了厚壁菌门（Firmicutes）的丰度，同时降低了变形菌门（Proteobacteria）的丰度；在属水平上，5000U/kg的BIAP显著提升乳酸杆菌属（Lactobacillus）的丰度，并显著降低大肠杆菌属（Escherichia）的丰度。总体而言，bIAP改善了肠道菌群的紊乱，降低致病菌丰度，维持肠道菌群健康，bIAP对产气荚膜梭菌感染肉鸡肠道菌群紊乱的调节作用。
以上实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。
Claims (3)
1.牛来源的碱性磷酸酶bIAP作为饲料添加剂用于以下用途的应用：
抵抗产气荚膜梭菌感染肉鸡所致生产性能下降；
降低产气荚膜梭菌感染肉鸡所致炎症反应和肠道损伤；或
降低产气荚膜梭菌感染肉鸡所致肠道菌群紊乱。
2.根据权利要求1所述的应用，其特征在于，所述牛来源的碱性磷酸酶bIAP在饲料中的添加量为1000 U/kg~5000 U/kg。
3.根据权利要求1所述的应用，其特征在于，所述牛来源的肠碱性磷酸酶的氨基酸序列如SEQ ID No：1所示。
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| US20110206654A1 (en) * | 2008-08-29 | 2011-08-25 | The General Hospital Corporation | Methods of Modulating Gastrointestinal Tract Flora Levels with Alkaline Phosphatase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090010912A1 (en) * | 2004-02-04 | 2009-01-08 | Pharmaaware Sepsis B.V. | Use of Alkaline Phosphatase for the Detoxification of Lps Present at Mucosal Barriers |
| US20110206654A1 (en) * | 2008-08-29 | 2011-08-25 | The General Hospital Corporation | Methods of Modulating Gastrointestinal Tract Flora Levels with Alkaline Phosphatase |
| US20180326018A1 (en) * | 2017-03-21 | 2018-11-15 | Synthetic Biologics, Inc. | Alkaline phosphatase formulations |
| US20210189358A1 (en) * | 2018-06-18 | 2021-06-24 | Synthetic Biologics, Inc. | Methods of making alkaline phosphatase agents |
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