[1]胡耀方,吴昊,江昌盛,等.猪胸膜肺炎放线杆菌ApxI 毒素中和试验的建立与初步应用[J].中国预防兽医学报,2021,(02):158-164.[doi::10.3969/j.issn.1008-0589.202005027]
 HU Yao-fang,WU Hao,JIANG Chang-sheng,et al.The development and preliminary application of a neutralization test ofApxI toxin of Actinobacillus pleuropneumoniae[J].Chinese journal of preventive veterinary medicine,2021,(02):158-164.[doi::10.3969/j.issn.1008-0589.202005027]
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猪胸膜肺炎放线杆菌ApxI 毒素中和试验的建立与初步应用
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2021年02
页码:
158-164
栏目:
诊断和检测技术
出版日期:
2021-03-16

文章信息/Info

Title:
The development and preliminary application of a neutralization test of
ApxI toxin of Actinobacillus pleuropneumoniae
文章编号:
:1008-0589(2021)02-0158-07
作者:
 胡耀方123吴昊123江昌盛1库旭钢23孙琪123于学祥123何启盖123*
 (1. 华中农业大学动物医学院,湖北武汉430070;2. 生猪健康养殖协同创新中心,湖北武汉430070;
3. 华中农业大学动物疫病诊断中心,湖北武汉430070)
Author(s):
 HU Yao-fang123 WU Hao123 JIANG Chang-sheng1 KU Xu-gang23 SUN Qi123 YU Xue-xiang123 HE Qi-gai123*
 (1. College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China; 2. The Cooperative Innovation
Center for Sustainable Pig Production, Wuhan 430070, China; 3. The Diagnostic Center for Animal Disease of Huazhong Agricultural University,
Wuhan 430070, China)
关键词:
 :猪胸膜肺炎放线杆菌ApxI毒素中和试验
Keywords:
: Actinobacillus pleuropneumoniae ApxI neutralization test
分类号:
S852.61
DOI:
:10.3969/j.issn.1008-0589.202005027
文献标志码:
A
摘要:
为建立测定血清中猪胸膜肺炎放线杆菌(APP)外毒素ApxI中和抗体的方法,本研究采用(NH4)2SO4
沉淀法从血清10型APP培养液中经盐析浓缩提取具有溶血活性的天然毒素ApxI,将毒素与待检血清在37 ℃孵育
2 h,加入4%猪红细胞悬液并反应0.5 h,利用酶标仪测定反应混合物上清液的OD540nm值判断溶血程度,计算出能
够保护50%红细胞不发生溶血的最低血清稀释倍数,即为该血清的中和效价。利用该方法进一步检测了副猪嗜
血杆菌和多杀性巴氏杆菌等阳性血清,无交叉反应,表明该方法特异性较强;利用该方法最高能够检测到中和
效价为1:1 580的APP ApxI毒素抗体阳性的猪血清,表明建立的中和试验具有较高的敏感性;以BALB/c小鼠为
模型的感染试验结果表明,当其血清中ApxI毒素抗体的中和效价达到1:20时能够为小鼠提供有效保护。采用建
立的方法检测临床采集的103份猪血清样品,能够有效检测其中和抗体,表明该方法可以应用于临床检测可分泌
ApxI毒素的APP的感染和含ApxI毒素的亚单位疫苗免疫效果评估,为评价亚单位疫苗的免疫效果和猪传染性胸
膜肺炎的诊断提供了技术支持。
Abstract:
In this study, a method was established to determine the titer of neutralizing antibody against ApxI toxin of
Actinobacillus pleuropneumoniae. In this method, ApxI, a natural toxin with hemolytic activity, was extracted via (NH4)2SO4
precipitation and further concentrated by salting out. The extracted toxin, mixed with the tested serum, was incubated at 37 ℃ for
2 hours, then a 4% porcine red blood cells suspension was added and allowed to react for 0.5 h. Finally, the OD540nm value of the
supernatant was measured by ELISA reader to determine the degree of hemolysis. Then the minimum dilution of serum which can
protect 50% of red blood cells from hemolysis is calculated as the neutralization titer of the serum. The specificity of this method
was identified by testing positive serum from pigs infected with Haemophilus parasuis and Pasteurella multocida, and the results showed no cross-reactivity, indicating high specificity. Further detection of the positive pig serum against ApxI toxin can detect the
pig serum with different titers of 1:1 587, indicating that the neutralization test established has good sensitivity. The results of
challenge experiments in BALB/c mice model showed that the 1:20 titer of neutralization antibody could effectively protect mice
from challenge. The neutralization antibody in 103 clinical serum samples could be effectively detected by this in-house method,
showing that this method can be applied to detect neutralization antibody elicited by ApxI- positive strains and to evaluate the
potency of ApxI-based subunit vaccine of Actinobacillus pleuropneumoniae, which provides a new tool for diagnosis and vaccine
evaluation. It also provides technical support for the evaluation of immune effect of subunit vaccine, as well as disease diagnosis of
porcine contagious pleuropneumonia.

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更新日期/Last Update: 2021-03-17