鸭疫里默氏杆菌RIA_0940基因缺失株的构建及主要生物学特性测定

doi:10.16431/j.cnki.1671-7236.2022.12.006

Abstract

Abstract: 【Objective】 In this study, Riemerella anatipestifer RIA_0940 gene deletion strain was constructed, and its main biological characteristics were studied to explore potential functions of RIA_0940 gene.【Method】 Taking the isolated Riemerella anatipestifer RA-GD strain as the parent strain, the left and right homologous sequences and erythromycin resistance gene fragments were amplified, and the left homologous arm-ermR resistance gene box--right homologous arm fusion fragment was constructed.The RIA_0940 gene of RA-GD was deleted by natural transformation, and the gene deleted strains RA-GDΔRIA_ 0940 were screened and identified by PCR.Growth curves, LD₅₀ to ducklings, blood and tissues bacterial load and the ability of cell adhesion and invasion were tested and analyzed of wild-type strain RA-GD and deletion mutant RA-GDΔRIA_0940, respectively.【Result】 The results showed that the RA-GD RIA_0940 gene deletion strain was successfully constructed.The results of biological characteristics showed that the growth ability of strain RA-GDΔRIA_0940 in vitro was significantly or extremely significantly inhibited compared with its wild-type(P<0.05;P<0.01), the LD₅₀ of RA-GDΔRIA_0940 was 114 times that of wild-type strain.Compared with wild-type strain, the blood and tissues bacterial load of RA-GDΔRIA_0940 infected ducks were significantly or extremely significantly decreased (P<0.05;P<0.01);The adherence and invasion capacities of RA-GDΔRIA_0940 to Vero cells were extremely significantly decreased(P<0.01).【Conclusion】 In summary, RA-GDΔRIA_0940 deletion mutant was constructed successfully.The growth trend of the deletion strain was slow under the condition of in vitro culture.The pathogenicity to ducklings, the amount of bacteria in infected duck blood fluid and tissues, and the ability of adhesion and invasion to Vero cells were significantly or extremely significantly lower than those of the wild-type strain.The results laid a foundation for the further study of the molecular pathogenesis of Riemerella anatipestifer and the development of genetic engineering vaccine.

Key words: Riemerella anatipestifer; RIA_0940 gene; gene deletion; biological characteristics; virulence factor

CLC Number:

S852.61

LI Linlin, DONG Jiawen, ZHANG Junqin, HUANG Yunzhen, SUN Minhua. Construction and Main Biological Characteristics Determination of RIA_0940 Gene Deleted Strain of Riemerella anatipestifer[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4564-4572.

References

[1] SARVER C F, MORISHITA T Y, NERSESSIAN B.The effect of route of inoculation and challenge dosage on Riemerella anatipestifer infection in Pekin ducks (Anas platyrhynchos)[J].Avian Disease, 2005, 49(1):104-107.
[2] 陶志云, 朱春红, 施祖灏, 等.鸭疫里默氏菌感染对鸭肠道菌群结构的影响[J].中国畜牧兽医, 2021, 48(6):2129-2139. TAO Z Y, ZHU C H, SHI Z H, et al.Effect of Riemerella anatipestifer infection on the microflora of ducks[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(6):2129-2139.(in Chinese)
[3] 袁小远, 王友令, 王晓丽, 等.2015-2016年山东、河北地区鸭疫里默氏杆菌流行病学调查[J].中国家禽, 2017, 39(4):70-72. YUAN X Y, WANG Y L, WANG X L, et al.Epidemiological investigation on Riemerella anatipestifer in Shandong and Hebei province in 2015-2016[J].China Poultry, 2017, 39(4):70-72.(in Chinese)
[4] 万春和, 程龙飞, 陈红梅, 等.鸭疫里默氏菌铁依赖抑制子基因的原核表达[J].福建农业学报, 2017, 32(2):107-110. WAN C H, CHENG L F, CHEN H M, et al.Prokaryotic expression of RaDtxR gene of Riemerella anatipestifer[J].Fujian Journal of Agricultural Sciences, 2017, 32(2):107-110.(in Chinese)
[5] 吴征卓, 陈国权, 姚碧琼, 等.贵州省三穗县鸭疫里默氏杆菌的分离鉴定、毒力基因检测及耐药性分析[J].中国畜牧兽医, 2021, 48(11):4274-4283. WU Z Z, CHEN G Q, YAO B Q, et al.Isolation, identification, virus gene detection and antibiotic resistance analysis of Riemerella anatipestifer in Sansui county of Guizhou province[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(11):4274-4283.(in Chinese)
[6] HU Q, HAN X, ZHOU X, et al.OmpA is a virulence factor of Riemerella anatipestifer[J].Veterinary Microbiology, 2011, 150(3-4):278-283.
[7] WANG X, DING C, WANG S, et al.The AS87_04050 gene is involved in bacterial lipopoly-saccharide biosynthesis and pathogenicity of Riemerella anatipestifer[J].PLoS One, 2014, 9(10):e109962.
[8] YI H, YUAN B, LIU J, et al.Identification of a wza-like gene involved in capsule biosyn-thesis, pathogenicity and biofilm formation in Riemerella anatipestifer[J].Microbial Pathogenesis, 2017, 107:442-450.
[9] MIAO S, XING L, QI J, et al.Roles of the TonB1 and TonB2 proteins in haemin iron acquisition and virulence in Riemerella anatipestifer[J].Microbiology, 2015, 161(8):1592-1599.
[10] LU F, MIAO S, TU J, et al.The role of Ton B-dependent receptor TbdR1 in Riemerella anatipestifer in iron acquisition and virulence[J].Veterinary Microbiology, 2013, 167(3-4):713-718.
[11] LIU M, HUANG M, SHUI Y, et al.Roles of B739_1343 in iron acquisition and pathogenesis in Riemerella anatipestifer CH-1 and evaluation of the RA-CH-1ΔB739_1343 mutant as an attenuated vaccine[J].PLoS One, 2018, 13(5):e0197310.
[12] 梅世慧, 陈国权, 阎朝华, 等.鸭疫里默氏杆菌CAMP基因的克隆、原核表达及生物信息学分析[J].中国畜牧兽医, 2021, 48(12):4348-4360. MEI S H, CHEN G Q.YAN C H, et al.Cloning, prokaryotic expression and bioinformatics analysis of CAMP gene of Riemerella anatipestifer[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(12):4348-4360.(in Chinese)
[13] HU D, GUO Y, GUO J, et al.Deletion of the Riemerella anatipestifer type Ⅸ secretion system gene sprA results in differential expression of outer membrane proteins and virulence[J].Avian Pathology, 2019, 48(3):191-203.
[14] MALHI K K, WANG X, CHEN Z, et al.Riemerella anatipestifer gene AS87_08785 encodes a functional component, GldK, of the type Ⅸ secretion system[J].Veterinary Microbiology, 2019, 231:93-99.
[15] LI S, CHEN Q, GONG X, et al.RanB, a putative ABC-type multidrug efflux transporter contributes to aminoglycosides resistance and organic solvents tolerance in Riemerella anatipestifer[J]. Veterinary Microbiology, 2020, 243:108641.
[16] PARROW N L, FLEMING R E, MINNICK M F.Sequestration and scavenging of iron in infection[J].Infection and Immunity, 2013, 81(10):3503-3514.
[17] MIETHKE M, MARAHIEL M A.Siderophore-based iron acquisition and pathogen control[J].Microbiology and Molecular Biology Reviews, 2007, 71(3):413-451.
[18] LITWIN C M, CALDERWOOD S B.Role of iron in regulation of virulence genes[J].Clinical Microbiology Reviews, 1993, 6(2):137-149.
[19] HUANG M, LIU M, LIU J, et al.Functional characterization of Fur in iron metabolism, oxidative stress resistance and virulence of Riemerella anatipestifer[J].Veterinary Research, 2021, 52(1):48.
[20] LIAO H, CHENG X, ZHU D, et al.TonB energy transduction systems of Riemerella anatipestifer are required for iron and hemin utilization[J].PLoS One, 2015, 10(5):e0127506.
[21] WANG M, ZHANG P, ZHU D, et al.Identification of the ferric iron utilization gene B739_1208 and its role in the virulence of R.anatipestifer CH-1[J].Veterinary Microbiology, 2017, 201:162-169.
[22] TU J, LU F, MIAO S, et al.The siderophore-interacting protein is involved in iron acquisition and virulence of Riemerella anatipestifer strain CH3[J].Veterinary Microbiology, 2015, 168(2-4):395-402.
[23] TRINDADE I B, SILVA J M, FONSECA B M, et al.Structure and reactivity of a siderophore-interacting protein from the marine bacterium Shewanella reveals unanticipated functional versatility[J]. Journal of Biological Chemistry, 2019, 294(1):157-167.
[24] LIU M, ZHANG L, HUANG L, et al.Use of natural transformation to establish an easy knockout method in Riemerella anatipestifer[J]. Applied and Environmental Microbiology, 2017, 83(9):e00127-17.
[25] LIU M, TIAN X, WANG M, et al.Development of a markerless gene deletion strategy using rpsL as a counterselectable marker and characterization of the function of RA0C_1534 in Riemerella anatipestifer ATCC11845 using this strategy[J].PLoS One, 2019, 14(6):e0218241.
[26] ZHANG L, HUANG L, HUANG M, et al.Effect of nutritional determinants and TonB on the natural transformation of Riemerella anatipestifer[J].Frontiers in Microbiology, 2021, 12:644868.

Construction and Main Biological Characteristics Determination of RIA_0940 Gene Deleted Strain of Riemerella anatipestifer

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	SONG Yue, SU Shengjie, ZHANG Fan, BAI Fan, DAI Lingli, WANG Na, WANG Dawei, YANG Qianwen, ZHAO Shihua, ZHANG Yuemei. Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2479-2486.
[2]	CHEN Jianshi, ZHANG Ning, PU Fangxiong, MIAO Jianjia, ZENG Nan, LI Bingxue. Prediction of Bacillus anthracis Carrying Prophages and Analysis of Drug Resistance and Virulence [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2573-2584.
[3]	WANG Leping, PAN Yan, TAN Yizhou, LI Lei, HAN Kaiou, CAO Yajie, LI Jun, ZHOU Yuqing, LIAO Yuying, MA Dongxin, GONG Yu, PENG Hao, WANG Xiaoye. Isolation,Identification and Genome Analysis of Escherichia coli Phage vB_EcoP_GN07 [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1522-1531.
[4]	TIAN Changqing, ZHANG Kunzhong, QI Yumei, SHI Wenjing, DONG Zhijie, ZHANG Haohao, HE Zengwen, ZHI Ji, ZHAO Xuehui, CHONG Qian, XUE Huiwen, GOU Huitian. Isolation,Identification and Biological Characteristics Analysis of a Strain of Listeria monocytogenes from Environment [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1543-1555.
[5]	ZHU Xihui, LI Yanlan, WANG Zhiwei, PANG Zheyu, LI Yubao, LIU Cheng, SI Zhenshu, LU Jianbiao, CAO Shengliang, XUE Xijuan. Whole Genome Sequencing of a Resistant Riemerella anatipestifer Strain and Analysis of Antibiotic Resistance and Genetic Evolution [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1140-1149.
[6]	CHENG Longfei, LIAO Weilian, CHEN Hongmei, JIANG Nansong, FU Qiuling, WAN Chunhe, LIU Rongchang, SHI Shaohua, HUANG Yu, FU Guanghua. Screening and Biological Characteristics of Broad-spectrum Riemerella anatipestifer Phage [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1169-1176.
[7]	CHEN Dong, ZHANG Yanhao, ZHANG Yang, LI Sen, ZHOU Zutao, LI Zili. Isolation,Identification and Drug Sensitivity of 3 Strains of Riemerella anatipestifer [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1250-1258.
[8]	GAN Lu, ZHENG Huizhen, NUO Mingdalai, LIU Yan, JIN Min, HE Wenwen, WEN Licui, LI Yongchang, GAILIKE·Bayinchahan. Propagation of Trypanosoma evansi Yili Strain and Cloning, Expression and Bioinformatics Analysis of Its PFR Gene [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 469-478.
[9]	LIU Bin, LI Meng, XI Qianyun, SUN Jiajie, LUO Junyi, ZHANG Yongliang, CHEN Ting. Research Progress on Biological Characteristics of Hermetia illucens L. and Its Application in Poultry Production [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 586-597.
[10]	YIN Yangyan, PAN Yan, LI Xiaoning, LI Jun, LIAO Yuying, LI Changting, WEI Tianchao, PENG Hao. Study on Biological Characteristics and Antibacterial Activity in vitro of a Lactobacillus plantarum Strain [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 666-673.
[11]	WEI Chunyan, GUO Jia, ZHU Dexin, ZHANG Wei, ZHU Jiale, DEN Xingmei, JIA Sifeng, LIU Liangbo, ZHANG Hui. Construction of Deletion Strain of Brucella BPE159 Gene and Effect of BPE159 Protein on Expression of Cellular Autophagy Factors [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 26-36.
[12]	XU Jinge, LI Ting, CHEN Rujia, YU Bo, ZHANG Yanan, YANG Li, ZHAO Bin, PU Ling. Preparation and Immune Efficacy of Riemerella anatipestifer Bivalent Inactivated Vaccine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 290-299.
[13]	GAO Yefan, SONG Hanan, WANG Yunan, WU Yue, NIU Ruili, ZONG Xianchun, GUAN Weijun. Study on the Isolation, Culture and Differentiation Potential of Limbal Stem Cells from Beijing You Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3372-3381.
[14]	MA Guangqiang, LIU Lijuan, CHEN Guoquan, WU Liangtao, YANG Jun, LIU Junze, LI Xiaomeng, PAN Chengwen, ZHOU Bijun. Preparation of Propolis Inactivated Vaccine of Riemerella anatipestifer Epidemic Strain in Guizhou [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3163-3170.
[15]	ZHOU Yuqing, MA Dongxin, WU Jie, WANG Xiaoye. Isolation,Identification and Characterization Analysis of Broad-spectrum Escherichia coli O157∶H7 CVCC4050 Phage vB_EcoM_GXBP08 [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2735-2745.