副猪嗜血杆菌小鼠毒力和仔猪毒力的相关性分析

doi:10.11843/j.issn.0366-6964.2020.024

摘要/Abstract

摘要： 旨在对我国最为流行的血清4、5、12和13型副猪嗜血杆菌（Haemophilus parasuis， HPS）（共36株）进行BALB/c小鼠和仔猪毒力试验的比较研究。小鼠毒力试验结果表明，4种血清型菌株的LD₅₀分别介于9.80×10⁷~4.60×10⁹、2.10×10⁸~8.85×10⁹、4.81×10⁷~7.01×10⁹和1.75×10⁸~8.45×10⁸ CFU；整体毒力表现强弱依次是13、4、12、5型，但仅在5型与13型菌株之间毒力具有显著差异（P<0.05）。仔猪毒力试验结果表明，同一血清型中不同菌株的毒力具有明显差异，均存在强毒和弱毒菌株；整体毒力表现强弱依次是5、13、4、12型；但5型与4型（P=0.039）和12型（P=0.033）之间具有显著差异（P<0.05），与13型差异不显著（P=0.241）。综合对比分析HPS的小鼠和仔猪毒力试验结果，可以得出3个结论：1）4种国内流行性血清型菌株的整体毒力由强到弱依次是5、13、4和12型；2）但同一血清型中均存在强毒和弱毒菌株，HPS的血清型与其毒力之间不具有相关性；3） HPS虽能致死BALB/c小鼠，但其毒力结果与仔猪毒力试验结果并不一致，表明其作为替代模型具有一定的缺陷。

关键词: 副猪嗜血杆菌, 血清型, 毒力, 小鼠, 仔猪, 相关性

Abstract: In this study, we compared the virulence of the most prevalent Haemophilus parasuis (36 strains) serovars 4, 5, 12, and 13 in China in BALB/c mice and piglets models. The results of mice virulence tests showed that the LD₅₀values of the four serotype strains were 9.80×10⁷-4.60×10⁹CFU (serovar 4),2.10×10⁸-8.85×10⁹ CFU (serovar 5),4.81×10⁷-7.01×10⁹ CFU (serovar 12) and 1.75×10⁸-8.45×10⁸ CFU (serovar 13), respectively. Serovar 13 was the most highly virulent serovar in mice, followed by serovars 4, 12, 5. However, there was a significant difference in virulence only between serovars 5 and 13 (P<0.05).The results of piglet virulence tests showed that the virulence of different strains in the same serovar were remarkably different, including virulent strains and weak strains. Serovar 5 was the most highly virulent in piglet, followed by serovars 13, 4, 12,but there were significant differences between serovars 5 and 4 (P=0.039), serovars 5 and 12 (P=0.033), and there was no significant difference between serovars 5 and 13 (P=0.241). Comparing the results of HPS virulence tests on mice and piglets, three conclusions can be drawn:first, the overall virulence of the four domestic epidemic serotypes from strong to weak were serovars 5, 13, 4 and 12 in turn; Second, there were both strong and weak strains in the same serotype, so there was no correlation between the serovar and virulence of HPS. Third, although HPS can kill BALB/c mice, its virulence result was not consistent with the piglet virulence test, indicating that it has some defects as an alternative model.

Key words: Haemophilus parasuis, serotype, virulence, mice, piglet, correlation

中图分类号:

S852.612

李飞跃, 陈坤鹏, 丁文文, 薛云, 王臣, 司丽芳, 赵战勤. 副猪嗜血杆菌小鼠毒力和仔猪毒力的相关性分析[J]. 畜牧兽医学报, 2020, 51(6): 1408-1418.

LI Feiyue, CHEN Kunpeng, DING Wenwen, XUE Yun, WANG Chen, SI Lifang, ZHAO Zhanqin. Correlation between Virulence of Haemophilus parasuis in Mice and Piglet[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1408-1418.

参考文献 40

[1]	陈溥言. 兽医传染病学[M]. 6版. 北京:中国农业出版社, 2015:212-269.CHEN P Y. Veterinary infectious disease[M]. 6th ed. Beijing:China Agriculture Press, 2015:212-269. (in Chinese)
[2]	MA L N, WANG L Y, CHU Y F, et al. Characterization of Chinese Haemophilus parasuis isolates by traditional serotyping and molecular serotyping methods[J]. PLoS One, 2016, 11(12):e0168903.
[3]	KIELSTEIN P, RAPP-GABRIELSON V J. Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts[J]. J Clin Microbiol, 1992, 30(4):862-865.
[4]	TADJINE M, MITTAL K R, BOURDON S, et al. Development of a new serological test for serotyping Haemophilus parasuis isolates and determination of their prevalence in North America[J]. J Clin Microbiol, 2004, 42(2):839-840.
[5]	CAI X W, CHEN H C, BLACKALL P J, et al. Serological characterization of Haemophilus parasuis isolates from China[J]. Vet Microbiol, 2005, 111(3-4):231-236.
[6]	CASTILLA K S, DE GOBBI D D S, MORENO L Z, et al. Characterization of Haemophilus parasuis isolated from Brazilian swine through serotyping, AFLP and PFGE[J]. Res Vet Sci, 2012, 92(3):366-371.
[7]	LUPPI A, BONILAURI P, DOTTORI M, et al. Haemophilus parasuis serovars isolated from pathological samples in Northern Italy[J]. Transbound Emerg Dis, 2013, 60(2):140-142.
[8]	RúBIES X, KIELSTEIN P, COSTA L, et al. Prevalence of Haemophilus parasuis serovars isolated in Spain from 1993 to 1997[J]. Vet Microbiol, 1999, 66(3):245-248.
[9]	MORIKOSHI T, KOBAYASHI K, KAMINO T, et al. Characterization of Haemophilus parasuis isolated in Japan[J]. Jpn J Vet Sci, 1990, 52(3):667-669.
[10]	ANGEN Ø, SVENSMARK B, MITTAL K R. Serological characterization of Danish Haemophilus parasuis isolates[J]. Vet Microbiol, 2004, 103(3-4):255-258.
[11]	RAPP-GABRIELSON V J, KOCUS G J, CLARK J T, et al. Virulence of different serovars of Haemophilus parasuis for cesarean-derived, colostrum-deprived pigs[M]//DOACHIE W, LAINSON F A, HODGSON J C. Haemophilus, Actinobacillus, and Pasteurella. New York:Plenum Press, 1995:204.
[12]	METCALF D S, MACINNES J I. Differential expression of Haemophilus parasuis genes in response to iron restriction and cerebrospinal fluid[J]. Can J Vet Res, 2007, 71(3):181-188.
[13]	HOWELL K J, WEINERT L A, CHAUDHURI R R, et al. The use of genome wide association methods to investigate pathogenicity, population structure and serovar in Haemophilus parasuis[J]. BMC Genomics, 2014, 15(1):1179.
[14]	BROCKMEIER S L, REGISTER K B, KUEHN J S, et al. Virulence and draft genome sequence overview of multiple strains of the swine pathogen Haemophilus parasuis[J]. PLoS One, 2014, 9(8):e103787.
[15]	GALOFRÉ-MILÀ N, CORREA-FIZ F, LACOUTURE S, et al. A robust PCR for the differentiation of potential virulent strains of Haemophilus parasuis[J]. BMC Vet Res, 2017, 13(1):124.
[16]	LI G, NIU H, ZHANG Y H, et al. Haemophilus parasuis cytolethal distending toxin induces cell cycle arrest and p53-dependent apoptosis[J]. PLoS One, 2017, 12(5):e0177199.
[17]	NIELSEN R. Pathogenicity and immunity studies of Haemophilus parasuis serotypes[J]. Acta Vet Scand, 1993, 34(2):193-198.
[18]	AMANO H, SHIBATA M, KAJIO N, et al. Pathogenicity of Haemophilus parasuis serovars 4 and 5 in contact-exposed pigs[J]. J Vet Med Sci, 1996, 58(6):559-561.
[19]	VAHLE J L, HAYNES J S, ANDREWS J J. Experimental reproduction of Haemophilus parasuis infection in swine:clinical, bacteriologic, and morphologic findings[J]. J Vet Diagn Invest, 1995, 7(4):476-480.
[20]	OLIVEIRA S, GALINA L, BLANCO I, et al. Naturally-farrowed, artificially-reared pigs as an alternative model for experimental infection by Haemophilus parasuis[J]. Can J Vet Res, 2003, 67(2):146-150.
[21]	蔡旭旺. 副猪嗜血杆菌的分离鉴定及诊断方法与灭活疫苗的研究[D]. 武汉:华中农业大学, 2006.CAI X W. Isolation and characterization of Haemophilus parasuis and development of its diagnostic method and Inactivated bacterin[D]. Wuhan:Huazhong Agricultural University, 2006. (in Chinese)
[22]	ZHAO Z Q, LIU H S, XUE Y, et al. Analysis of efficacy obtained with a trivalent inactivated Haemophilus parasuis serovars 4, 5, and 12 vaccine and commercial vaccines against Glässer's disease in piglets[J]. Can J Vet Res, 2017, 81(1):22-27.
[23]	MOROZUMI T, HIRAMUNE T, KOBAYASHI K. Experimental infections of mice and guinea pigs with Haemophilus parasuis[J]. Natl Inst Anim Health Q (Tokyo), 1982, 22(1):23-31.
[24]	赵国振, 李晓华, 戴爱玲, 等. 闽西地区副猪嗜血杆菌的分离鉴定及毒力研究[J]. 黑龙江畜牧兽医, 2015(9):10-13, 17.ZHAO G Z, LI X H, DAI A L, et al. Isolation and identification of Haemophilus parasuis and the study of its virulence in the west of Fujian Province[J]. Heilongjiang Animal Science and Veterinary Medicine, 2015(9):10-13, 17. (in Chinese)
[25]	YU J, ZHANG Y Y, DU Y J, et al. The BALB/c mouse infection model for improving the Haemophilus parasuis serotyping scheme[J]. Res Vet Sci, 2016, 109:166-168.
[26]	高鹏程, 储岳峰, 赵萍, 等. 副猪嗜血杆菌对部分常用实验动物的致病性试验[J]. 畜牧兽医学报, 2009, 40(1):138-141.GAO P C, CHU Y F, ZHAO P, et al. Pathogenic experiment of Haemophilus parasuis to certain experimental animals[J]. Acta Veterinaria et Zootechnica Sinica, 2009, 40(1):138-141. (in Chinese)
[27]	贺云霞, 徐慧, 叶飞, 等. 副猪嗜血杆菌15个血清型标准分离株对小鼠毒力的研究[J]. 黑龙江畜牧兽医, 2011(17):90-91.HE Y X, XU H, YE F, et al. Study on the virulence of 15 standard isolates of Haemophilus parasuis in mice[J]. Heilongjiang Animal Science and Veterinary Medicine, 2011(17):90-91. (in Chinese)
[28]	王占伟, 刘茂军, 王丽, 等. 副猪嗜血杆菌对豚鼠和小鼠的致病力研究[J]. 中国兽医杂志, 2015, 51(5):80-82.WANG Z W, LIU M J, WANG L, et al. Pathogenicity test for Haemophilus parasuis using guinea pigs and mice[J]. Chinese Journal of Veterinary Medicine, 2015, 51(5):80-82. (in Chinese)
[29]	TIAN H B, FU F, LI X S, et al. Identification of the immunogenic outer membrane protein A antigen of Haemophilus parasuis by a proteomics approach and passive immunization with monoclonal antibodies in mice[J]. Clin Vaccine Immunol, 2011, 18(10):1695-1701.
[30]	HOWELL K J, PETERS S E, WANG J H, et al. Development of a multiplex PCR assay for rapid molecular serotyping of Haemophilus parasuis[J]. J Clin Microbiol, 2015, 53(12):3812-3821.
[31]	薛云, 王乐, 王臣, 等. 副猪嗜血杆菌新型微量凝集试验抗体检测方法的建立[J]. 中国预防兽医学报, 2018, 40(9):806-811.XUE Y, WANG L, WANG C, et al. Establishment of the micro-agglutination test for detection of antibody against Haemophilus parasuis[J]. Chinese Journal of Preventive Veterinary Medicine, 2018, 40(9):806-811. (in Chinese)
[32]	REED L J, MUENCH H. A simple method of estimating fifty per cent endpoints[J]. Am J Hyg, 1938, 27(3):493-497.
[33]	丁文文. 副猪嗜血杆菌的病原流行病学及生物学特性研究[D]. 洛阳:河南科技大学, 2019.DING W W. Study on the pathogenic epidemiology and biological characteristics of Haemophilus parasuis[D]. Luoyang:Henan University of Science and Technology, 2019. (in Chinese)
[34]	王乐, 赵战勤, 薛云, 等. 副猪嗜血杆菌国内流行血清型菌株的致病性比较研究[J]. 中国兽医学报, 2014, 34(11):1748-1752.WANG L, ZHAO Z Q, XUE Y, et al. Comparative study on pathogenicity of the most prevalent serovars of Haemophilus parasuis isolates in China[J]. Chinese Journal of Veterinary Science, 2014, 34(11):1748-1752. (in Chinese)
[35]	徐惠娟, 薛云, 赵战勤, 等. 副猪嗜血杆菌国内流行血清型菌株的生物学特性比较研[J]. 中国兽医学报, 2014, 34(5):729-735.XU H J, XUE Y, ZHAO Z Q, et al. Comparative study on biological characterization of the epidemic serotypes Haemophilus suis in China[J]. Chinese Journal of Veterinary Science, 2014, 34(5):729-735. (in Chinese)
[36]	MOROZUMI T, HIRAMUNE T. Effect of temperature on the survival of Haemophilus parasuis in physiological saline[J]. Natl Inst Anim Health Q (Tokyo), 1982, 22(2):90-91.
[37]	OLIVEIRA S, PIJOAN C. Haemophilus parasuis:new trends on diagnosis, epidemiology and control[J]. Vet Microbiol, 2004, 99(1):1-12.
[38]	ARAGON V, CERDÀ-CUÉLLAR M, FRAILE L, et al. Correlation between clinico-pathological outcome and typing of Haemophilus parasuis field strains[J]. Vet Microbiol, 2010, 142(3-4):387-393.
[39]	李昂, 徐成刚, 张建民, 等. 华南地区两株副猪嗜血杆菌的生物学特性与致病性研究[J]. 中国畜牧兽医, 2011, 38(7):179-183.LI A, XU C G, ZHANG J M, et al. Biological characteristics and pathogenicity of two Haemophilus parasuis strains collected from South China[J]. China Animal Husbandry & Veterinary Medicine, 2011, 38(7):179-183. (in Chinese)
[40]	林标声, 刘建奎, 李晓华, 等. 副猪嗜血杆菌血清型4、5的生物学特性及致病力[J]. 龙岩学院学报, 2013, 31(5):55-58, 88.LIN B S, LIU J K, LI X H, et al. Study on biological characteristics and pathogenicity of Haemophilus[J]. Journal of Longyan University, 2013, 31(5):55-58, 88. (in Chinese)