1株宽噬菌谱鸡白痢沙门氏菌噬菌体的分离及生物学特性分析

doi:10.16431/j.cnki.1671-7236.2018.09.030

摘要/Abstract

摘要：

本研究采用双层琼脂平板法从现代化肉鸡养殖场采集的粪便、污水和垫料样品中分离宽噬菌谱鸡白痢沙门氏菌烈性噬菌体，并通过透射电子显微镜观察、噬菌谱分析、热稳定性、pH稳定性、最佳感染复数及一步生长曲线等对分离纯化的噬菌体进行生物学特性分析。结果显示，试验成功分离纯化出一株鸡白痢沙门氏菌噬菌体，命名STP98-a。该噬菌体噬菌斑直径4~5 mm，圆形透明，无晕圈，其头部偏椭圆形，长径约61 nm，横径约67 nm，尾长约112 nm，直径约10 nm，属于长尾噬菌体科；能裂解不同血清型鸡白痢沙门氏菌和肠炎沙门氏菌肠亚种，噬菌谱宽；在30~60℃保持高活性；在pH 3.0~12.0稳定；最佳感染复数为0.001；一步生长曲线结果显示，其感染宿主菌潜伏期为5 min，裂解期为135 min，裂解量为93。综上所述，STP98-a为一株宽噬菌谱鸡白痢沙门氏菌烈性噬菌体，可用于鸡白痢沙门氏菌噬菌体制剂的研发。

关键词: 鸡白痢沙门氏菌噬菌体; 宽噬菌谱; 分离; 生物学特性

Abstract:

The study was aimed to separate a Salmonella Pullorum lytic phage with wide host range using the double-layer agar culture method.The feces,sewage and padding were collected from the modern broiler farms.And the biological parameters of the lytic phage were assayed,including the transmission electron microscopy observation,determination of host range,thermal stability,pH stability,optimal multiplicity of infection (MOI) and one-step growth curve drawing.The results showed that a lytic phage against Salmonella Pullorum was successfully isolated and purified which was named as STP98-a.Its plaques were round and transparent and the sizes were about 4 to 5 mm in diameter.It had an elliptical head with approximately 61 nm in diameter,67 nm in transverse diameter and had a long tail with 112 nm in length,10 nm in diameter,indicating it belonged to Siphoviridae.Phage STP98-a could lyse Salmonella Pullorum and Salmonella enterica subsp.enterica with a wide host range.The phage was stable with a wide range of temperature (30 to 60℃) and pH (3.0 to 12.0).The optimal MOI was 0.001.The one-step growth curve showed that the latent time and burst time of STP98-a were 5 and 135 min,respectively,and the burst size was 93.In conclusion,STP98-a was a Salmonella Pullorum lytic phage with wide host range that could serve as a promising phage preparations against Salmonella Pullorum.

Key words: Salmonella Pullorum lytic phage; wide host range; isolation; biological properties

中图分类号:

S855.1

汪祥燕, 辛国芹, 董佩佩, 刘元香, 徐海燕. 1株宽噬菌谱鸡白痢沙门氏菌噬菌体的分离及生物学特性分析[J]. 《中国畜牧兽医》, 2018, 45(9): 2575-2581.

WANG Xiangyan, XIN Guoqin, DONG Peipei, LIU Yuanxiang, XU Haiyan. Isolation and Biological Properties Analysis of a Salmonella Pullorum Lytic Phage with Wide Host Range[J]. , 2018, 45(9): 2575-2581.

参考文献

[1] AN H,ZHANG D H,CHEN X L,et al.Isolation and identification of Salmonella Pullorum[J].Agricultural Science & Technology,2012,13(3):661-663.
[2] 徐三兰.浅议鸡白痢沙门氏菌病的诊治[J].中国畜禽种业,2016,6:155-156.XU S L.Discussion on the diagnosis and treatment of Salmonella Pullorum disease[J].The Chinese Livestock and Poultry Breeding,2016,6:155-156.(in Chinese)
[3] HUR J, JAWALE C,LEE J H.Antimicrobial resistance of Salmonella isolated from food animals:A review[J].Food Research International,2012,45(2):819-830.
[4] 梁惠.鸡白痢沙门氏菌的分离鉴定及耐药性分析[J].山东畜牧兽医,2016,37(12):15-16.LIANG H.The study on identification and drug resistance of Salmonella Pullorum[J].Shandong Journal of Animal Science and Veterinary Medicine,2016,37(12):15-16.(in Chinese)
[5] SMITH H W, HUGGINS M B.Effectiveness of phages in treating experimental Escherichia coli diarrhea in calves,piglets and lambs[J].Journal of General Microbiology,1983,129(8):2659-2675.
[6] HENDRIX R W.Bacteriophage genomics[J].Current Opinion in Microbiology,2003,6(5):506-511.
[7] 刘童,黄海燕,徐嘉良.噬菌体在食品安全领域的研究进展[J].食品科技,2015,40(8):296-300.LIU T,HUANG H Y,XU J L.Research progress of phage application in the field of food safety[J].Food Science and Technology,2015,40(8):296-300.(in Chinese)
[8] 朱育玮,李玉保,王守荣,等.噬菌体及其治疗细菌感染的研究进展[J].中国畜牧兽医,2015,42(3):769-773.ZHU Y W,LI Y B,WANG S R,et al.Research progress on bacteriophage and the therapy in bacterial infection[J].China Animal Husbandry & Veterinary Medicine,2015,42(3):769-773.(in Chinese)
[9] HAWKINS C,HARPER D,BURCH D,et al.Topical treatment of Pseudomonas aeruginosa otitis of dogs with a bacteriophage mixture:A before/after clinical trial[J].Veterinary Microbiology,2010,146(3-4):309-313.
[10] WATANABE R,MATSUMOTO T,SANO G,et al.Efficacy of bacteriophage therapy against gut-derived sepsis caused by Pseudomonas aeruginosa in mice[J].Antimicrobial Agents and Chemotherapy,2007,51(2):446-452.
[11] 安瑞.噬菌体治疗的前世、今生与未来——对话微生物学界噬菌体专家[J].科学通报,2017,62(23):2577-2580.AN R.The past,present and future of phage therapy:Dialogues with bacteriophage experts in microbio-logy[J].Chinese Science Bulletin,2017,62(23):2577-2580.(in Chinese)
[12] 萨姆布鲁克,拉赛尔著.黄培堂译.分子克隆实验指南[M].北京:科学出版社,2002.SAMBROOK J,RUSSELL D W(EDITOR).HUANG P T(TRANSLATER).Guide of Molecular Clone[M].Beijing:Science Press,2002.(in Chinese)
[13] SHARMA S,CHATTERJEE S,DATTA S,et al.Bacteriophages and its applications:An overview[J].Folia Microbiologica,2017,62(1):17-55.
[14] 席利萌,李陇平,徐坤,等.致仔猪痢疾沙门氏菌特异性噬菌体的分离与纯化[J].西北农林科技大学学报(自然科学版),2012,40(5):1-4.XI L M,LI L P,XU K, et al.Isolation and purification of bacteriophage against Salmonella associated piglets dysentery[J].Journal of Northwest A & F University (Natural Science Edtion),2012,40(5):1-4.(in Chinese)
[15] 邹尚书,许家园,翁亚彪,等.1株肠炎沙门氏菌噬菌体的污水分离及生物学特性研究[J].中国畜牧兽医,2014, 41(3):119-123.ZOU S S,XU J Y,WENG Y B,et al.Isolation and biological characteristics of a phage against Salmonella enteritidis from swage water[J].China Animal Husbandry & Veterinary Medicine,2014,41(3):119-123.(in Chinese)
[16] 李涵依,车晨,耿亚,等.鸡白痢沙门氏菌O12特异性噬菌体的分离及生物学特性研究[J].西南民族大学学报(自然科学版),2014,40(2):186-191.LI H Y,CHE C,GENG Y,et al.Study on isolation and biological characterization of bacteriophage of Salmonella O12 for Pullorum[J].Journal of Southwest University(Nationalities Natural Science Edition),2014,40(2):186-191.(in Chinese)
[17] 江艳华,李风铃,王联珠,等.一株沙门氏菌裂解性噬菌体的分离鉴定及生物学特性[J].微生物学通报,2015, 42(3):534-542.JIANG Y H,LI F L,WANG L Z,et al.Isolation,identification and biological properties of a lytic phage against Salmonella[J].Microbiology China,2015,42(3):534-542.(in Chinese)
[18] 李萌,韩峰,林洪,等.一株沙门氏菌烈性噬菌体的分离纯化与生理特性研究[J].水产科学,2013,9:531-535.LI M,HAN F,LIN H,et al.Isolation,identification and physiological characterization of lytic phage against bacterium Salmonella sp.[J].Fisheries Science,2013,9:531-535.(in Chinese)
[19] 包红朵,张辉,王冉.宽噬菌谱肠炎沙门氏菌噬菌体的生物学特性[J].江苏农业学报,2011,27(5):1117-1121.BAO H D,ZHANG H,WANG R.Biological properties of a Salmonella enteritidis lytic phage with wide host range[J].Jiangsu Journal of Agricultural Sciences,2011,27(5):1117-1121.(in Chinese)
[20] 包红朵,张鹏禹,周艳,等.沙门氏菌裂解性噬菌体的分离鉴定及其生物学特性[J].食品科学,2015,5:131-136.BAO H D,ZHANG P Y,ZHOU Y,et al.Isolation and biological characteristics of lytic Salmonella bacteriophage[J].Food Science,2015,5:131-136.(in Chinese)
[21] 邢颖,朱芷葳,张利环,等.鸡白痢沙门菌噬菌体的分离及电镜分析[J].中国家禽,2015,37(8):55-57.XING Y,ZHU Z W,ZHANG L H,et al.Separation and electron microscope analysis of Salmonella Pullorum phage[J].China Poultry,2015,37(8):55-57.(in Chinese)
[22] 赵影,刘秀侠,李金敏,等.一株鼠伤寒沙门氏菌噬菌体的分离鉴定及对小鼠的防制效果研究[J].中国畜牧兽医,2018,45(1):234-241.ZHAO Y,LIU X X,LI J M, et al.Isolation and identification of a phage against Salmonella Typhimurium and its control effect on mice[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):234-241.(in Chinese)
[23] 冯烨,刘军,孙洋,等.噬菌体最新分类与命名[J].中国兽医学报,2013,33(12):1954-1958.FENG Y,LIU J,SUN Y,et al.An introduction to current classification and nomenclature of bacterial viruses[J].Chinese Journal of Veterinary Science,2013,33(12):1954-1958.(in Chinese)