【作者】 王建；

【导师】 杨小军；

【作者基本信息】 西北农林科技大学 ， 农业推广硕士（专业学位）， 2019， 硕士

【摘要】 家禽细菌性疾病多与革兰氏阴性菌有关,如沙门氏菌和大肠杆菌。抗生素类生长促进剂（AGPs）的使用对于降低细菌性疾病的风险和促进动物的生长具有重要作用,但长期使用AGPs会导致细菌产生耐药性、畜禽产品药物残留等危害。随着2020年AGPs全面退出饲料促生长类添加剂使用相关政策的明确,寻找新型安全的AGPs替代品成为当务之急。抗菌肽因其多靶点快速抑菌、抗菌谱广、不易使细菌产生耐药性等特性成为AGPs替代品的候选者之一。本试验首先在体外研究抗菌肽Api-PR19的杀菌效果和抗逆性,而后通过在肉鸡饲粮中添加抗菌肽Api-PR19,研究其对肉鸡的生长性能和肠道健康的影响,以期为抗菌肽Api-PR19在肉鸡无抗养殖中的应用提供技术支撑。试验一抗菌肽Api-PR19的体外杀菌活性和抗逆性研究本试验采用大肠杆菌ATCC25922、鼠伤寒沙门氏菌ATCC14028、巴氏杆菌ATCC19427、幽门螺旋杆菌ATCC43504和金黄色葡萄球菌ATCC25923,利用微量稀释法和微生物比浊法,离体检测抗菌肽Api-PR19的最小抑菌浓度（MIC）和杀菌效果。以大肠杆菌为指示菌,利用不同温度、pH、蛋白酶处理抗菌肽Api-PR19后,检测其杀菌活性。结果显示:1)抗菌肽Api-PR19对大肠杆菌ATCC25922、鼠伤寒沙门氏菌ATCC14028、巴氏杆菌ATCC19427、幽门螺旋杆菌ATCC43504和金黄色葡萄球菌ATCC25923的MIC分别是0.2、0.4、0.1、0.8和1.6μg/mL;2)抗菌肽Api-PR19在第4 h时,杀菌效能达到最高。1×MIC与10×MIC浓度的抗菌肽Api-PR19相比,杀菌效能并没有显著增加;3)不同温度处理抗菌肽Api-PR19的结果显示,随着温度的升高,抗菌肽Api-PR19的杀菌活性逐渐降低,在80℃时降低到原来杀菌活性的70%左右;不同pH处理抗菌肽Api-PR19的结果显示,随着pH值的降低,抗菌肽Api-PR19的杀菌活性逐渐降低,当pH=2时降低到原来杀菌活性的72%左右;不同蛋白酶处理抗菌肽Api-PR19的结果显示,胃蛋白酶（Pepsin,3000-3500 NFU）、胰蛋白酶（Trypsin,250 NFU）和α-糜蛋白酶（α-chymotrypsin,1200 U）处理抗菌肽Api-PR19后,其杀菌活性分别降低到原来的52%、63%和82%左右。本试验结果表明,抗菌肽Api-PR19在体外对大肠杆菌、鼠伤寒沙门氏菌、巴氏杆菌、幽门螺旋杆菌的生长有较好的抑制效果,对金黄色葡萄球菌的生长有一定抑制作用。抗菌肽Api-PR19具有耐高温、耐强酸的抗逆性特点,对α-糜蛋白酶和胰蛋白酶具有较好的抗性,但对胃蛋白酶较为敏感。试验二饲粮添加抗菌肽Api-PR19对肉鸡生长性能的影响本试验选用1日龄艾拔益加（AA）肉鸡630只,随机分为5个处理,每处理7个重复。5个处理分别为:1)对照组（CON）饲喂不含抗生素的基础饲粮;2)抗生素组（AGP）饲喂基础饲粮和4.96 mg/kg恩拉霉素;3)低剂量组（AP1）饲喂基础饲粮和100 mg/kg抗菌肽Api-PR19;4)中剂量组（AP2）饲喂基础饲粮和200 mg/kg抗菌肽Api-PR19;5)高剂量组（AP3）饲喂基础饲粮和300 mg/kg抗菌肽Api-PR19。结果显示:1)饲粮添加抗菌肽Api-PR19显著降低了肉鸡饲养前期（1-21 d）和饲养全期（1-42d）的料重比（P<0.05）,对饲养后期的料重比有改善的趋势（0.05<P<0.10）,且AP2组的料重比最低;2),饲粮添加抗菌肽Api-PR19对肉鸡胸腺指数、脾脏指数和42 d法氏囊指数无显著性影响,但AP1和AP2组显著降低了肉鸡第21 d法氏囊指数（P<0.05）;3)饲粮添加抗菌肽Api-PR19对肉鸡各阶段流感亚型H₅抗体水平和第14、21、28 d肉鸡的亚型H₉抗体水平无显著改善作用（P>0.05）,但显著提高了第7 d肉鸡亚型H₉抗体水平;4)由预测肉鸡饲粮中抗菌肽Api-PR19的最适添加水平回归方程得知,饲养前期抗菌肽Api-PR19的最适添加剂量为267.0 mg/kg（R²=0.424）,饲养后期的最适添加量为222.9 mg/kg（R²=0.325）。本试验结果表明,饲粮添加抗菌肽Api-PR19能够提高肉鸡生产性能,并为肉鸡早期免疫防线构建与完善起一定促进作用。试验三饲粮添加抗菌肽Api-PR19对肉鸡肠道健康的影响本试验分别于第21和42 d采集肉鸡十二指肠、空肠和回肠肠道黏膜样品和盲肠食糜样品。用4%的多聚甲醛固定第21和42 d冲洗后的十二指肠、空肠和回肠,固定好的肠段制成切片,经过苏木精-伊红（HE）染色,检测肠道形态;黏膜样品通过RT-qPCR技术测定肠道葡萄糖转运载体和氨基酸转运载体的mRNA表达水平;通过免疫组化检测分泌型免疫球蛋白（sIgA）在各肠段的表达情况;通过微生物的绝对定量,检测盲肠细菌总数和特定微生物数量。结果显示:1)抗菌肽Api-PR19对回肠肠道形态有显著改善作用;对饲养后期肉鸡（22-42 d）的十二指肠肠道形态有显著改善作用;2)抗菌肽Api-PR19显著上调了第21 d肉鸡十二指肠黏膜葡萄糖转运载体（GLUT2）和氨基酸转运载体（rBAT、y⁺LAT2、CAT1）基因相对表达量（P>0.05）,而对第42 d各肠段营养物质转运载体基因相对表达量无显著影响;3)抗菌肽Api-PR19对第21d和42 d肉鸡空肠及第21 d肉鸡十二指肠sIgA的分泌有改善作用（P>0.05）;4)抗菌肽Api-PR19显著减少了后期肉鸡盲肠有害菌大肠杆菌和空肠弯曲杆菌的数量（P>0.05）,并一定程度上维持了盲肠微生物的丰度。本试验结果表明:抗菌肽Api-PR19可改善肉鸡肠绒毛组织形态,增加小肠对营养物质的吸收,增强小肠免疫,并且减少盲肠有害微生物的数量,维持肠道微生态平衡,综合改善了肠道健康。综上所述,抗菌肽Api-PR19在体外有良好的杀菌效果和消化道抗逆性。抗菌肽Api-PR19在肉鸡饲粮中的添加改善了肉鸡肠绒毛组织结构,增加了小肠营养物质转运载体的表达,减少盲肠有害菌的数量,维持肠道微生态平衡,提高了肉鸡的生产性能。更多还原

【Abstract】 In modern poultry production,numerous bacterial diseases are associated with gram-negative bacteria,and representative of them have Salmonella and E.coli.Antibiotic growth promoters（AGPs）plays an important role in reducing the risk of bacterial diseases and promoting the growth of animals,but long-term use of AGPs will cause bacterial resistance,veterinary drug residue and other hazards.With the clarity of the policy that AGPs was withdrawn from the feed by 2020,it’s a priority to search for the new safe substitutes of AGPs.Antimicrobial peptides（AMP）are one of the candidates for AGPs substitutes because of the rapid bacteriostasis with multiple targets,broad-spectrum antibacterial activity and difficult to form drug resistance.In this study Apidaecin-derived peptide（Api-PR19）build by genetic engineering was selected to be an alternative to AGPs.Firstly,the antibacterial effect and stress resistance of Api-PR19 was detected in vivo;Secondly,we supplemented Api-PR19 into diet and detected the effects of Api-PR19 for growth performance and intestinal health of broilers,expected to provide a theoretical basis for the application of Api-PR19 in broiler breeding industry.Exp.1 Study on antimicrobial activity and stress resistance of Api-PR19 in vitroThe minimum inhibitory concentration（MIC）and antibacterial effect of Api-PR19 were tested by Escherichia coli ATCC25922,Salmonella typhi ATCC14028,Pasteurella ATCC19427,Helicobacter pylori ATCC43504 and Staphylicoccus aureus ATCC25923.We used Escherichia coli as the indicator,and explored the stress resistance of Api-PR19processed by different temperature,pH and protease.The results showed that:1)the MIC value of Api-PR19 against Escherichia coli ATCC25922,Salmonella typhi ATCC14028,Pasteurella ATCC19427,Helicobacter pylori ATCC43504 and Staphylicoccus aureus ATCC25923 were represented respectively 0.2、0.4、0.1、0.8 and 1.6μg/mL;2)Api-PR19reached the maximum bactericidal efficacy at 4 hour period,and compared with the 1 MIC,the 10 MIC showed no significant increase in bactericidal efficacy;3)With the increase of temperature,the bactericidal activity of Api-PR19 showed a tendency to reduce gradually,and Api-PR19 treated at 80℃decreased to about 70%of the primary antimicrobial activity;With the decrease of pH,the germicidal activity of Api-RP19 decreased gradually,and when pH=2,it reduced to about 72%of the pimary antimicrobial activity.Pepsin（3000-3500NFU）,Trypsin（250 NFU）andα-chymotrypsin（1200 U）reduced the antibacterial activity of Api-PR19 by about 52%,63%and 82%,respectively.The results showed in vitro Api-PR19had better antibacterial effect on Escherichia coli,salmonella typhi,Pasteurella and Helicobacter pylori,and had certain antibacterial effect on Staphylicoccus aureus.Api-PR19has the strongest bactericidal effect at 4-hour period.With the increase of concentration of Api-PR19,the bactericidal effect did not increase obviously.Api-PR19 are resistant to high temperature and strong acid,and has good resistance toα-chymotrypsin and trypsin,but poor resistance to pepsin.Exp.2 The effect of Api-PR19 on growth performance of broiler chickensA total of 630 one-day-old male chicks（AA）with similar body weight from a commercial hatchery were randomly divided into 5 groups consisting of 7 replicates with 18birds per replicate.Birds of each group received one of the following dietary treatments:1)control group（CON）fed basal diet;2)AGPs group（AGPs）fed basal diet supplemented with4.96 mg enramycin per kg diet;3)Low-level group（AP1）fed basal diet contaminated with100 mg Api-PR19 per kg diet;4)Middle-level group（AP2）fed basal diet contaminated with200 mg Api-PR19 per kg diet;5)High-level group（AP3）fed basal diet contaminated with300 mg Api-PR19 per kg diet.The results showed that:1)Adding Api-PR19 to the diet significantly reduced feed conversion rate（FCR）in the pre-feeding period（1-21 days）and the whole period of feeding（P<0.05）,and has a tendency to improve the late feeding stage（P<0.10）,and AP2 group had the lowest FCR;2)Adding Api-PR19 to feed had no significant effect on broiler index,spleen index and bursal index of 42 days broilers,but the bursal index of 21 days broilers in AP1 and AP2 groups was significantly lower（P<0.05）;3)Dietary supplementation with Api-PR19 had no significant effect on H₅ antibody levels in influenza subtypes at all stages of broilers,and subtype H₉ antibody levels at 14,21 and 28days did not significantly improve（P>0.05）,but Api-PR19 significantly increased the H₉antibody level at 7 day broiler subfamily;4)It was found that the optimal addition amount of Api-PR19 in the broiler diet was more accurate（related The coefficient is the highest,R²=0.496）,and the recommended value is 200.481 mg/kg.This experiment showed that the addition of Api-PR19 to the diet can improve feed conversion efficiency and improve broiler performance.It could be Api-PR19 improves the intestinal health of broilers;The results of bursal index and antibody titer reveal that Api-PR19 may play a regulatory role in the construction and improvement of early immune defense lines in broilers.Exp.3 The effect of Api-PR19 on intestine health of broiler chickensIn this experiment,duodenal,jejunal and ileal mucosal samples of broilers were collected at 21 and 42 days,as well as cecal chyme of 21 and 42 days,and fixed with paraformaldehyde at 21 and 42 days of age.The fixed intestines were sectioned and stained with hematoxylin-eosin（HE）to detect intestinal morphology.Mucosal samples were assayed for mRNA expression levels of gut glucose transporters and amino acid transporters by RT-qPCR.The expression of sIgA in each intestinal segment was detected by immunohistochemistry.The total number of cecal bacteria and the number of specific microorganisms are detected by absolute quantification of microorganisms.The results showed that:1)Api-PR19 had a significant improvement on the morphology of the ileum;it improved the shape of the duodenum in the broiler at the later feeding period;2)the Api-PR19 significantly up-regulated the relative expression of glucagon passive transporter（GLUT2）and amino acid transporter（rBAT,y+LAT2,CAT1）genes at the 21 day of age（P>0.05）,but had no significant effect on the expression of nutrient transporter genes at the later feeding period;3)The Api-PR19 improved the secretion of sIgA in jejunum and the pre-feeding broiler duodenal sIgA（P>0.05）;4)Api-PR19 significantly reduced the number of cecal Escherichia coli and campylobacter jejuni at the later feeding period（P>0.05）,and maintained the abundance of cecal microbes to some extent.The results of this experiment showed that Api-PR19 can improve the intestinal villus tissue morphology,increase the absorption of nutrients in the small intestine,enhance intestinal immunity,reduce the number of harmful microorganisms in the cecum,maintain intestinal micro-ecological balance,and comprehensively improve intestinal health,and thereby improve feed conversion efficiency and thus,enhanced broiler performance.In all,Api-PR19 has the superior of antimicrobial activity and stress resistance in vivo.Api-PR19 can improve the intestinal villus tissue morphology,increase the absorption of nutrients in the small intestine,reduce the number of harmful microorganisms in the cecum,maintain intestinal micro-ecological balance,and comprehensively improve intestinal health,and thereby improve feed conversion efficiency and broiler performance.更多还原