猪源抗菌肽对禽源沙门氏菌和大肠杆菌的抑菌作用

刘小龙; 赖隆永; 谭礼宁; 刘友霖; 徐磊; 吴昌标; 杨守深; 杨慧; 余勋信; 程龙飞; 黄瑜

doi:10.19303/j.issn.1008-0384.2019.05.010

猪源抗菌肽对禽源沙门氏菌和大肠杆菌的抑菌作用

doi: 10.19303/j.issn.1008-0384.2019.05.010

刘小龙^1,,
赖隆永¹,
谭礼宁¹,
刘友霖¹,
徐磊^{2, 3, ,},
吴昌标⁴,
杨守深⁵,
杨慧²,
余勋信²,
程龙飞⁶,
黄瑜⁶

1.
派生特(福州)生物科技有限公司, 福建福州 350500
2.
福建农业职业技术学院, 福建福州 350303
3.
南京天邦生物科技有限公司, 江苏南京 211102
4.
福建生物工程职业技术学院, 福建福州 350007
5.
龙岩学院生命科学学院, 福建龙岩 364012
6.
福建省农业科学院畜牧兽医研究所, 福建福州 350013

基金项目:

福建省科技计划项目 2019N0025

福建省科技计划项目 2017N0009

福建省中青年教师教育科研项目 JZ180531

福建省禽病防治重点实验室开放基金课题 KF201902

2019年度江苏省博士后科研资助计划苏人社发（2019）104号

福州市科技计划项目 2019-G-34

福建省动物保健与食品安全应用技术协同创新中心项目闽教科（2017）49号

福建省科技计划项目 2016S0002

福建省畜禽疫病防控技术重大研发平台项目 2014N2003

详细信息

作者简介:
刘小龙(1989-)，男，硕士，主要从事兽医传染病防治研究(E-mail: 798956657@qq.com)

通讯作者:
徐磊(1984-), 男, 博士, 教授, 主要从事畜禽传染病防治研究(E-mail:446326718@qq.com)

中图分类号: S852.6
计量
- 文章访问数: 1178
- HTML全文浏览量: 192
- PDF下载量: 67
- 被引次数: 0
出版历程
- 收稿日期: 2019-02-14
- 修回日期: 2019-04-15
- 刊出日期: 2019-05-28

Effect of Porcine Antibacterial Peptide against Avian Pathogenic Bacteria

1.
Present(Fuzhou) Biotech Co., Ltd., Fuzhou, Fujian 350500, China
2.
Fujian Vocational College of Agriculture, Fuzhou, Fujian 350303, China
3.
Nanjing Tianbang Bio-industry Company Limited, Nanjing, Jiangsu 211102, China
4.
Fujian Vocational College of Agriculture, Fuzhou, Fujian 350007, China
5.
College of Life Sciences, Longyan University, Longyan, Fujian 364012, China
6.
Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013, China

摘要

摘要: 目的明确猪源抗菌肽对禽源沙门氏菌和大肠杆菌的抑菌作用，为新型抗菌药物的研发和猪源抗菌肽的生产应用提供科学依据。方法以猪源抗菌肽为材料，采用液体培养基抑制测定法，结合体内外试验，分别测定猪源抗菌肽对禽源沙门氏菌和禽源大肠杆菌的抗菌活力值，研究其体内外抑菌作用。结果对不同浓度的抗菌肽抗菌活力值的测定结果表明，与0.5、0.2和0.1 mg·mL^-1的猪源抗菌肽相比较，1.0 mg·mL^-1的抗菌肽对含有10⁶ CFU·mL^-1的菌液抑菌效果最好；与阿莫西林、红霉素、四环素等15种抗生素相比较，猪源抗菌肽对禽源沙门氏菌FJS13株和禽源大肠杆菌FJE61株的抑菌圈最大，其抗菌效果最好；抗菌肽在雏鸡体内的抑菌试验表明，攻毒鸡皮下注射猪源抗菌肽有利于提高雏鸡的存活率，在雏鸡体内也具有一定的抑菌作用。结论猪源抗菌肽可用于抑制禽源沙门氏菌和大肠杆菌。
- 猪源抗菌肽 /
- 抗菌活力值 /
- 抑制作用 /
- 沙门氏菌 /
- 大肠杆菌 /
- 雏鸡
Abstract: Objective Activity of porcine antibacterial peptides (PAP) against avian Salmonella and Escherichia coli was determined for drug development. Method Inhibitive activity of PAP on avian pathogens was studied in vitro on an liquid medium and by an in vivo test. Results PAP at a concentration of 1.0 mg·mL^-1 showed a higher bacteriostatic effect on the solution contaning 10⁶ CFU·mL^-1 of the pathogens than at 0.5, 0.2 or 0.1 mg·mL^-1. Compared with 15 antibiotics, such as amoxicillin, erythromycin, tetracycline, etc., PAP induced the largest inhibition zone on the growth of avian Salmonella FJS13 and E. coli FJE61. In a challenging test on chicken, the PAP administration improved the survival rate of the subcutaneous injected birds. Conclusion PAP could be used as a bioagent to combat the infection on chickens by avian Salmonella and E. coli.
- porcine antibacterial peptide /
- antibacterial activity /
- inhibition /
- avian Salmonella /
- avian Escherichia coli /
- chicken

HTML全文

表 1 试验分组及给药

Table 1. Experimental groups and drug administration

组别 Group	数量 Quantity/只	攻毒Attack			24 h后治疗24 h Post treatment
组别 Group	数量 Quantity/只	A	B	C	D	E
沙门氏菌治疗组Salmonella treatment group	15	-	+	-	+	-
大肠杆菌治疗组E. coli treatment group	15	-	-	+	+	-
沙门氏菌模型组Salmonella model group	15	-	+	-	-	+
大肠杆菌模型组E. coli model group	15	-	-	+	-	+
对照组一Control group 1	15	+	-	-	-	+
对照组二Control group 2	15	+	-	-	+	-
注：A为生理盐水，B为沙门氏菌，C为大肠杆菌，D为猪源抗菌肽，E为生理盐水。“+”表示注射；“-”表示不注射。 Note:A.Saline，B.salmonella，C.E.coli，D.Porcine antibacterial peptide，E.Saline. “+” means injection; “-” means not injection.

下载: 导出CSV

表 2 不同质量浓度抗菌肽对禽源沙门氏菌的抗菌活力值U

Table 2. Antibacterial activity (U) of PAP in different concentrations on Salmonella

质量浓度 Concentration /(mg·mL^-1)	1 h	2 h	3 h	4 h	6 h	8 h	12 h	16 h	20 h	24 h
1.0	0.226±0.003a	0.292±0.005a	0.261±0.004a	0.271±0.006a	0.356±0.004a	0.466±0.004a	0.557±0.003a	0.685±0.005a	0.706±0.004a	0.696±0.005a
0.5	0.147±0.003b	0.276±0.004a	0.227±0.005b	0.274±0.004a	0.363±0.003a	0.473±0.005a	0.558±0.005a	0.675±0.004a	0.256±0.004c	0.169±0.004d
0.2	0.045±0.003c	0.155±0.005c	0.066±0.002d	0.034±0.005c	0.163±0.004b	0.176±0.005b	0.173±0.003b	0.155±0.005b	0.236±0.005c	0.233±0.004c
0.1	0.101±0.003b	0.207±0.005b	0.153±0.004c	0.108±0.003b	0.153±0.005b	0.097±0.005c	0.068±0.004c	0.059±0.004c	0.341±0.004b	0.323±0.006b
注：采用Duncan′s multiple range test方法分析，同列数据后不同字母表示差异显著(P＜0.05)。表 3同。 Note：Duncan′s multiple range test method was used to analy, different letters of the same column indicates a significant difference (P＜0.05).The same as Table 3.

下载: 导出CSV

表 3 不同质量浓度抗菌肽对禽源大肠杆菌的抗菌活力值(U)

Table 3. Antibacterial activity (U) of PAP in different concentrations on E. coli

质量浓度 Concentration /(mg·mL^-1)	1 h	2 h	3 h	4 h	6 h	8 h	12 h	16 h	20 h	24 h
1.0	0.243±0.004a	0.333±0.004a	0.312±0.004a	0.389±0.0052a	0.445±0.004a	0.502±0.004a	0.604±0.004a	0.709±0.004a	0.697±0.003a	0.689±0.005a
0.5	0.129±0.005b	0.313±0.004a	0.304±0.004a	0.382±0.0053a	0.413±0.004a	0.499±0.004a	0.595±0.005a	0.699±0.005a	0.335±0.006b	0.241±0.005b
0.2	0.075±0.004c	0.173±0.005b	0.172±0.004b	0.161±0.0041b	0.158±0.005b	0.165±0.004b	0.191±0.004b	0.122±0.005b	0.112±0.005c	0.132±0.005c
0.1	0.130±0.003b	0.106±0.005c	0.121±0.005c	0.139±0.0042b	0.141±0.004b	0.072±0.005c	0.072±0.005c	0.061±0.004c	0.352±0.004b	0.294±0.005b

下载: 导出CSV

表 4 抗菌肽对禽源沙门氏菌和大肠杆菌的抑菌试验结果

Table 4. Antibacterial test results of PAP on Salmonella and E. coli

药敏纸片/菌种 Drug sensitive paper/strain	沙门氏菌Salmonella		大肠杆菌Escherichia Coli
药敏纸片/菌种 Drug sensitive paper/strain	抑菌圈直径Inhibition zone diameter/mm	敏感性Sensitivity determination	抑菌圈直径Inhibition zone diameter/mm	敏感性Sensitivity determination
阿莫西林Amoxicillin	10	R	15	M
丁胺卡那霉素Amikacin	12	R	10	R
氟哌酸Norfloxacin	23	S	6	R
红霉素Eryphilin	8	R	6	R
卡那霉素Kanamycin	13	R	6	R
利福平Rifampin	9	R	20	S
链霉素Streptomycin	8	R	8	R
强力霉素Doxycycline	16	M	12	M
庆大霉素Gentamicin	12	M	12	M
壮观霉素Spectamycin	10	M	8	R
新霉素Neomycin	13	M	6	R
四环素Tetracycline	8	R	8	R
林可霉素Lincomycin	6	R	8	R
克林霉素Clindamycin	6	R	8	R
青霉素Penicillin	12	R	8	R
抗菌肽Antibacterial peptide	30	-	28	-
注：S为敏感，M为中介，R为耐受。 Note：S is sensitive, M is intermediary, and R is resistant.

下载: 导出CSV

表 5 不同菌液用量处理试验雏鸡的死亡情况

Table 5. Deaths of experimental chickens in groups with different bacterial conlentrations

菌株 Strain	菌液用量 Bacterial concentration/(CFU·mL^-1)	剂量 Dose /(mL·只^-1)	死亡率 Mortality rate/%
沙门氏菌 Salmonella	1.0×10⁴	0.5	0.0
	1.0×10⁵	0.5	40.0
	1.0×10⁶	0.5	53.3
	1.0×10⁷	0.5	66.7
	1.0×10⁸	0.5	86.7
	1.0×10⁹	0.5	100.0
大肠杆菌 E.coli	1.0×10⁴	0.5	0.0
	1.0×10⁵	0.5	33.3
	1.0×10⁶	0.5	46.7
	1.0×10⁷	0.5	60.0
	1.0×10⁸	0.5	80.0
	1.0×10⁹	0.5	100.0

下载: 导出CSV

表 6 各组试验雏鸡的存活情况

Table 6. Survival of chickens in each group

试验分组 Test group	数量 Quantity/只	存活数 Survival number/只	存活率 Survival rate/%	死亡率 Rate of death/%
沙门氏菌治疗组Salmonella treatment group	15	12	80.00	20.00
大肠杆菌治疗组E.coli treatment group	15	13	86.67	13.33
沙门氏菌模型组Salmonella model group	15	8	53.33	46.67
大肠杆菌模型组E. coli model group	15	7	46.67	53.33
对照组一Control group 1	15	15	100	0.0
对照组二Control group 2	15	15	100	0.0

下载: 导出CSV

参考文献(16)

[1]	刘诚, 黎满香, 卢帅, 等.抗菌肽研究进展[J].动物医学进展, 2011, 32(3):94-99. doi: 10.3969/j.issn.1007-5038.2011.03.021 LIU C, LI M X, LU S, et al. Progress in antibacterial peptides[J]. Progress in Animal Medicine, 2011, 32(3):94-99.(in Chinese) doi: 10.3969/j.issn.1007-5038.2011.03.021
[2]	魏中琴, 高雅婷, 马奔科, 等.抗菌肽的研究进展[J].黑龙江医药, 2015, 10(1):1-5. doi: 10.3969/j.issn.1008-0104.2015.01.001 WEI Z Q, GAO Y T, MA B K, et al. Research progress of antimicrobial peptides[J]. Heilongjiang Medicine, 2015, 10(1):1-5.(in Chinese) doi: 10.3969/j.issn.1008-0104.2015.01.001
[3]	韩新燕, 汪以真, 许梓荣.哺乳动物抗菌肽的研究进展[J].中国兽医学报, 2002, 15(2):205-208. http://d.old.wanfangdata.com.cn/Periodical/zgsyxb200202035 HAN X Y, WANG Y Z, XU Z R. Research progress of mammalian antimicrobial peptides[J]. Chinese Journal of Veterinary Medicine. 2002, 15(2):205-208.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zgsyxb200202035
[4]	ANDREAS R KOCZULLA. Robert Bals.Antimicrobial Peptides:current status and therapeutic potential[J]. Drugs, 2003, 63(4):389-460. http://cn.bing.com/academic/profile?id=cf38f64247c6e55a806d35e180b65cac&encoded=0&v=paper_preview&mkt=zh-cn
[5]	马卫明, 佘锐萍, 彭芳珍, 等.猪小肠抗菌肽的提取及部分生物学活性研究[J].科学技术与工程, 2004, 16(3):202-205. doi: 10.3969/j.issn.1671-1815.2004.03.010 MA W M, QI R P, PENG F Z, et al. Extraction and Biological Activity of Porcine Small Intestinal Antibacterial Peptides[J]. Science Technology and Engineering, 2004, 16(3):202-205.(in Chinese) doi: 10.3969/j.issn.1671-1815.2004.03.010
[6]	范志勇, 周定刚, 吴明夏, 等.猪小肠抗菌肽的抑菌作用研究[J].家畜生态学报, 2006, 27(6):99-101. doi: 10.3969/j.issn.1673-1182.2006.06.023 FAN Z Y, ZHOU D G, WU M X, et al. Antibacterial activity of porcine intestinal antimicrobial peptides[J]. Chinese Journal of Animal Ecology, 2006, 27(6):99-101.(in Chinese) doi: 10.3969/j.issn.1673-1182.2006.06.023
[7]	高明燕, 刘爱玲, 刘建军, 等.抗菌肽RP-07的抑菌作用及其稳定性研究[J].黑龙江畜牧兽医, 2015(7):189-191. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=hljxmsy201504062 GAO M Y, LIU A L, LIU J J, et al. Antibacterial activity and stability of antimicrobial peptide RP-07[J]. Heilongjiang Animal Husbandry and Veterinary Medicine, 2015(7):189-191.(in Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=hljxmsy201504062
[8]	POWERS J P, HANCOCK R E. The relationship between peptide structure and antibacterial activity[J]. Peptides, 2003, 24(11):1681-1691. http://med.wanfangdata.com.cn/Paper/Detail/PeriodicalPaper_PM15019199
[9]	单安山, 田昊天, 邵长轩, 等.抗菌肽抗细菌机理研究进展[J].东北农业大学学报, 2018, 8(3):84-94. doi: 10.3969/j.issn.1005-9369.2018.03.010 SHAN A S, TIAN H T, SHAO C X, et al. Advances in antibacterial mechanism of antibacterial peptides[J]. Journal of Northeast Agricultural University, 2018, 8(3):84-94.(in Chinese) doi: 10.3969/j.issn.1005-9369.2018.03.010
[10]	MOHAN K V. Antiviral activity of selected antimicrobial peptides against vaccinia virus[J]. Antiviral Research, 2010, 86(3):306-311. doi: 10.1016/j.antiviral.2010.03.012
[11]	BRADSHAW JEREMY. Cationic antimicrobial peptides:issues for potential clinical use[J]. Bio Drugs, clinicalimmunotherapeutics, biopharmaceuticals and gene therapy, 2003, 17(4):233-240. doi: 10.2165-00063030-200317040-00002/
[12]	徐磊, 刘友霖, 曾辉荣, 等.猪小肠复合抗菌肽的制备方法及用途: 中国, 201310343030.8[P].2015-08-11. XU L, LIU Y L, ZENG H R, et al. Preparation method and use of pig small intestine compound antimicrobial peptide: China, 201310343030.8[P].2015-08-11.(in Chinese)
[13]	Clinical and Laboratory Standards Institute(CLSI). Performance standards for antimicrobial susceptibility testing twentieth informational supplement[M]. USA, 2010.
[14]	董改琳.家禽大肠杆菌致病机理与防治措施[J].畜牧兽医科学, 2018, 5(4):64. doi: 10.3969/j.issn.2096-3637.2018.04.052 DONG G L. Pathogenic Mechanism and Control Measures of Escherichia Coli in Poultry[J]. Journal of Animal Husbandry and Veterinary Medicine, 2018, 5(4):64.(in Chinese) doi: 10.3969/j.issn.2096-3637.2018.04.052
[15]	朱健, 刘超群, 刘瑞奇, 等.抗菌肽免疫调节功能的研究进展[J].黑龙江畜牧兽医.2018, 24(1):77-79. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=hljxmsy201801019 ZHU J, LIU C Q, LIU R Q, et al. Research progress on immunomodulatory function of antimicrobial peptides[J]. Heilongjiang Animal Husbandry and Veterinary. 2018, 24(1):77-79.(in Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=hljxmsy201801019
[16]	卜艳玲.肠杆菌肽体外抑菌作用及在断奶仔猪生产中应用研究[D].沈阳: 沈阳农业大学, 2018. http://cdmd.cnki.com.cn/Article/CDMD-10157-1018993044.htm BU Y L. In vitro antibacterial effect of enterobacterin and its application in weaned pig production[D]. Shenyang: Shenyang Agricultural University, 2018.(in Chinese) http://cdmd.cnki.com.cn/Article/CDMD-10157-1018993044.htm