[1]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,(09):806-811.[doi:0.3969/j.issn.1008-0589.201806026]
 XUE Yun,WANG Le,WANG Chen,et al.Establishment of the micro-agglutination test for detection of antibody against Haemophilus parasuis[J].Chinese journal of preventive veterinary medicine,2018,(09):806-811.[doi:0.3969/j.issn.1008-0589.201806026]
点击复制

副猪嗜血杆菌新型微量凝集试验抗体检测方法的建立()
分享到:

《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2018年09
页码:
806-811
栏目:
诊断和检测技术
出版日期:
2018-09-17

文章信息/Info

Title:
Establishment of the micro-agglutination test for  detection of antibody against Haemophilus parasuis
文章编号:
1008-0589(2018)9-0806-06
作者:
 

薛 云12王 乐2王 臣2赵朋超1司丽芳2赵战勤12*

 (1. 河南科技大学 医学技术与工程学院,微生物检验实验室,河南 洛阳 471023;
2. 河南科技大学 动物科技学院,兽医生物制品工程实验室,河南 洛阳 471023)
Author(s):
 

XUE Yun12 WANG Le2 WANG Chen2 ZHAO Peng-chao1 SI Li-fang2 ZHAO Zhan-qin12*

 (1. Laboratory of Veterinary biologics Engineering, College of Animal Science and Technology, Henan University of Science and Techology, Luoyang  471023, China; 2. Lab of Medical Microbiology Engineering, College of Medical Technology and Engineering, Henan University of Science and Techology, Luoyang 471023, China)
关键词:
副猪嗜血杆菌微量凝集试验抗体
Keywords:
pig  Haemophilus parasuis  micro-agglutination test  antibody
分类号:
S852.61
DOI:
0.3969/j.issn.1008-0589.201806026
文献标志码:
A
摘要:
为建立国内流行血清型副猪嗜血杆菌的新型微量凝集试验抗体检测方法(MAT),本研究以副猪嗜血杆菌4型、5型、12型和13型为抗原,分别对其MAT方法进行了最佳反应条件筛选、特异性、敏感性和重复性试验,以及疫苗免疫样品和临床样本检测。MAT试验结果显示,4型、5型、12型和13型凝集抗原的最佳反应浓度介于1.5×109 cfu/mL~2.0×109 cfu/mL,最佳反应温度37℃,最佳反应时间为6 h~8 h。4种型的MAT方法对猪巴氏杆菌病等6种细菌性疾病和猪瘟等5种病毒性疾病阳性血清的检测结果均为阴性,4种方法之间有轻微的交叉反应,但其抗体效价均不高于1∶4 (阴性临界值)。4种型的MAT方法比相应试管凝集方法的敏感性提高了2~4倍,重复性试验结果显示该方法检测副猪嗜血杆菌4种型变异系数均小于10 %,重复性良好。利用该方法检测临床样品,并与国家标准套式PCR方法对比,结果显示二者阳性符合率为82.1 %。疫苗免疫抗体检测结果显示,4种型的MAT方法均能检测到商品化疫苗免疫猪14 d的血清抗体,可用于疫苗免疫的抗体评价。对临床样品的检测结果显示,断奶仔猪、保育仔猪和经产母猪的总阳性率分别为12.3 %、49.3 %和69.8 %,提示保育期仔猪的合群可能导致了猪群的大量感染。本研究为副猪嗜血杆菌病的疫苗抗体评价和流行病学监测提供了一种简便经济可行的方法。
  
Abstract:
The objective of this study was to establish micro-agglutination test (MAT) for  detection of antibodies against Haemophilus parasuis. The 4 MATs were developed for detections of the antibodies against most prevalent H.parasuis serotypes of 4, 5, 12 and 13, respectively, and the reaction conditions were optimized with the standard positive and negative sera, which were determinated that antigen concentrations of inactivated whole-cell of H.parasuis serovar 4, 5, 12 and 13 were ranged from 1.5×109 to 2.0×109cfu/mL and the inclubation temperature was 37℃ for 6hrs to 8hrs. The 4 MATs were specificity for H.parasuis serovars of 4, 5, 12 and 13 detections, respectively, and the agglutination of the antibody titers were no more than 1:4 dilutions among the different H.parasuis serotypes, which was set as the cutoff value for the assays. However, the 4 assays had no cross-reactions to the positive sera of E.coli, Salmonella, P.multocida, B.bronchiseptia, A.pleuropneumoniae, S.suis2, classical swine fever virus,porcine circovirus, porcine reproductive and respiratory syndrome virus and Japanese encephalitis virus. The sensitivities of the four MATs were 2 to 4 times higher than the tube agglutination assay. The coincidence of the nested PCR and MATs were 82.1%. The antibodies in piglets could be detected at 14 days post vaccination with five commercial H.parasuis vaccines. The positive rates were 12.3%, 49.3% and 69.8% in the samples from weaned pigs, nursery piglets and sows, respectively, indicating that piglets were infected by H.parasuis during the nursery periods. The feasibility of the MATs for detecting antibodies of H.parasuis was confirmed in this study.

参考文献/References:

[1]Zhang Bin, Tang Cheng, Liao Ming, et al. Update on the pathogenesis of Haemophilus parasuis infection and virulence factors[J]. Vet Microbiol, 2014, 168(1): 1-7.
[2]Costa-Hurtado M, Aragon V. Advances in the quest for virulence factors of Haemophilus parasuis [J]. Vet J, 2013, 198(3):571-576.
[3]Cai Xu-wang, Chen Huan-chun, Blackall P J, et al. Serological characterization of Haemophilus parasuis isolates from China [J]. Vet Microbiol, 2005, 111(3-4): 231-236.
[4]Zhang Jian-min, Xu Cheng-gang, Guo Li-li, et al. Prevalence and characterization of genotypic diversity of Haemophilus parasuis isolates from southern China [J]. Can J Vet Res, 2012, 76(3): 224-229.
[5]Liu Hui-sheng, Xue Qiao, Zeng Qiao-ying, et al. Haemophilus parasuis vaccines [J]. Vet Immunol Immunopathol, 2016, 180:53-58.
[6]GB/T 34750-2017. 副猪嗜血杆菌检测方法[S].
[7]陈善真,李春玲,贾爱卿,等. 副猪嗜血杆菌OMP5基因的克隆、表达及间接ELISA检测方法的建立[J]. 中国农业科学,2011,44(14):3036-3044.
[8]贾爱卿,李春玲,王贵平,等. 副猪嗜血杆菌ompP2基因的克隆、表达及间接ELISA抗体检测方法的建立[J]. 中国兽医学报,2011,31(9):1266-1269.
[9]高艳. 副猪嗜血杆菌平板凝集抗原的研制与应用[D]. 西安:西北农林科技大学,2013.
[10]Zhang Bin, Tang Cheng, Liao Ming, et al. Update on the pathogenesis of Haemophilus parasuis infection and virulence factors[J]. Vet Microbiol, 2014, 168(1): 1-7.
[11]Mullins M A, Register K B, Bayles D O, et al. Characterization and comparative analysis of the genes encoding Haemophilus parasuis outer membrane proteins P2 and P5 [J]. J Bacteriol, 2009, 191(19): 5988-6002.
[12]中国兽医药品监察所,农业部兽药评审中心. 兽用生物制品质量标准汇编(2011) [M]. 北京:中国农业出版社,2011.
[13]徐惠娟,赵战勤,薛云,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究[J]. 中国兽医学报,2014,34(5):729-735.
[14]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.
[15]徐引弟,王治方,朱文豪,等. 副猪嗜血杆菌高免血清的制备与应用[J]. 中国农学通报,2010,26(13):14-16.
[16]赵战勤. 支气管败血波氏杆菌的重组沙门氏菌基因工程疫苗研究[D]. 武汉:华中农业大学,2008.
[17]赵战勤,卢顺,吴斌,等. 猪水肿毒素A亚基基因表达产物的生物学特性及抗体检测ELISA方法的建立[J]. 农业生物技术学报,2007,15(3):378-382.
[18]乔鹏芸. 猪源多杀性巴氏杆菌的分型及其毒力特性研究[D]. 洛阳:河南科技大学,2017.
[19]Zhao Zhan-qin, Liu Hui-sheng, Xue Yun, et al. Analysis of efficacy obtained with a trivalent inactivated Haemophilus parasuis serovars 4, 5, and 12 vaccine and commercial vaccines against Gl?覿sser’s disease in piglets [J]. Can J Vet Res, 2017, 81(1): 22-27.
[20]Zhao Zhan-qin, Liu Hui-sheng, Zhang Ke-shan, et al. Minimum dose, antigen content, and immunization duration of a trivalent vaccine of inactivated Haemophilus parasuis serovars 4, 5, and 12 against Gl?覿sser’s disease in pigs [J]. Can J Vet Res, 2016, 80(4): 287-293.
[21]Xue Qiao, Zhao Zhan-qin, Liu Hui-sheng, et al. First comparison of adjuvant for trivalent inactivated Haemophilus parasuis serovars 4, 5 and 12 vaccines against Gl?覿sser’s disease [J]. Vet Immunol Immunopathol, 2015, 168(3-4): 153-158.
[22]王乐. 副猪嗜血杆菌的分离鉴定及生物学特性研究[D]. 洛阳:河南科技大学,2014.
[23]Tang Xi-biao, Zhao Zhan-qin, Hu Jun-yong, et al. Isolation, antimicrobial resistance, and virulence genes of Pasteurella multocida strains from swine in China [J]. J Clin Microbiol, 2009, 47(4): 951-958.
[24]Ma Li-na, Wang Li-yan, Chu Yue-feng, et al. Characterization of Chinese Haemophilus parasuis isolates by traditional serotyping and molecular serotyping methods [J]. PLoS One, 2016, 11(12): e0168903.
                                      (本文编辑:彭永刚)

相似文献/References:

[1]蒋媛媛,郭军庆,颜焰,等.抗猪lgG单克隆抗体的制备与应用[J].中国预防兽医学报,2008,(09):727.
[2]李军星,姜 平*,李玉峰,等.副猪嗜血杆菌热休克蛋白70基因的序列分析及抗原性鉴定 [J].中国预防兽医学报,2009,(07):519.
 LI Jun-xing,JIANG Ping*,LI Yu-feng,et al.Sequencing and expression of the HSP70 gene of Haemophilus parasuis and antigenicity of heat shock protein 70 [J].Chinese journal of preventive veterinary medicine,2009,(09):519.
[3]高 明,魏 华*.重组猪IFN-α原核表达及其活性分析 [J].中国预防兽医学报,2009,(11):902.
 GAO Ming,WEI Hua*.Expression of recombinant porcine alpha-interferon in E.coli and the anti-virus activity analysis[J].Chinese journal of preventive veterinary medicine,2009,(09):902.
[4]施开创,李焕荣,杨汉春*,等.猪IL-4、IL-6和IL-10 SYBR Green I real-time PCR检测方法的建立及应用[J].中国预防兽医学报,2010,(02):102.
 SHI Kai-chuang,LI Huan-rong,YANG Han-chun*,et al.Development and application of a SYBR Green I-based real-time PCR assay for detection of porcine IL-4, IL-6 and IL-10[J].Chinese journal of preventive veterinary medicine,2010,(09):102.
[5]耿 屹,曹荣峰,高晓宇,等.副猪嗜血杆菌黏附基因的表达和免疫原性鉴定[J].中国预防兽医学报,2010,(05):398.
 GENG Yi,CAO Rong-feng,GAO Xiao-yu,et al.Expression and characterization of adhesin gene of Haemophilus parasuis[J].Chinese journal of preventive veterinary medicine,2010,(09):398.
[6]高鹏程,储岳峰,赵 萍,等.豚鼠副猪嗜血杆菌感染动物模型的建立[J].中国预防兽医学报,2009,(12):991.
 GAO Peng-cheng,CHU Yue-feng,ZHAO Ping,et al.Establishment of guinea pigs model for Haemophilus parasuis infection[J].Chinese journal of preventive veterinary medicine,2009,(09):991.
[7]张莉莉,白 娟,王先炜,等.猪Toll样受体3、7和8基因的克隆与序列分析[J].中国预防兽医学报,2010,(09):727.
 ZHANG Li-li,BAI Juan,WANG Xian-wei,et al.Cloning and sequencing analysis of porcine Toll-like receptor 3, 7 and 8 genes[J].Chinese journal of preventive veterinary medicine,2010,(09):727.
[8]何海健,陆国林,熊永忠.浙江地区副猪嗜血杆菌分离株的生物学特性[J].中国预防兽医学报,2010,(12):941.[doi:10.3969/j.issn.1008-0589.2010.12.07]
 HE Hai-jian,LU Guo-lin,XIONG Yong-zhong.Biological characterization of Haemophilus parasuis isolated in Zhejiang area[J].Chinese journal of preventive veterinary medicine,2010,(09):941.[doi:10.3969/j.issn.1008-0589.2010.12.07]
[9]王 新*,黄 山,周 婷,等.猪源耐甲氧西林金黄色葡萄球菌的耐药性及其SCCmec基因分型研究[J].中国预防兽医学报,2010,(12):975.[doi:10.3969/j.issn.1008-0589.2010.12.16]
 WANG Xin*,HUANG Shan,ZHOU Ting,et al.Antimicrobial susceptibility and SCCmec genetyping of methicillin-resistant Staphylococcus aureus isolates from swine[J].Chinese journal of preventive veterinary medicine,2010,(09):975.[doi:10.3969/j.issn.1008-0589.2010.12.16]
[10]胡明明,常月红,张艳禾,等.副猪嗜血杆菌菌影的制备[J].中国预防兽医学报,2011,(01):11.[doi:10.3969/j.issn.1008-0589.2011.01.03]
 HU Ming-ming,CHANG Yue-hong,ZHANG Yan-he,et al.Preparation of Haemophilus parasuis ghosts[J].Chinese journal of preventive veterinary medicine,2011,(09):11.[doi:10.3969/j.issn.1008-0589.2011.01.03]

更新日期/Last Update: 2018-09-28