PCV4 Cap抗体ELISA检测方法的建立及血清流行病学调查

doi:10.11843/j.issn.0366-6964.2024.05.025

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (5): 2072-2079.doi: 10.11843/j.issn.0366-6964.2024.05.025

PCV4 Cap抗体ELISA检测方法的建立及血清流行病学调查

宋晓晴¹, 邓瑞德¹, 李欣¹, 李姣², 李润成¹, 杜丽飞³, 董伟¹, 葛猛^1*

1. 湖南农业大学动物医学院, 长沙 410128;
2. 湖南美神育种有限公司, 株洲 412000;
3. 湖南省畜牧兽医研究所, 长沙 410131

收稿日期:2023-08-15 出版日期:2024-05-23 发布日期:2024-05-27
通讯作者: 董伟，主要从事兽药新制剂研发及动物传染病防控研究，E-mail：dongwei@hunau.edu.cn；葛猛，主要从事动物传染病诊断及防控研究，E-mail：gmg02@126.com
作者简介:宋晓晴(1999-)，女，湖南浏阳人，硕士生，主要从事动物传染病流行病学研究，E-mail：2459653794@qq.com
基金资助:
国家自然科学基金面上项目(32072871)；湖南省重点研发计划项目(2023NK2017)；云南省重大科技专项计划(202202AE090032)

Establishment of ELISA for Detection of PCV4-Cap Antibody and Sero-epidemiological Survey

SONG Xiaoqing¹, DENG Ruide¹, LI Xin¹, LI Jiao², LI Runcheng¹, DU Lifei³, DONG Wei¹, GE Meng^1*

1. College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China;
2. Hunan Meishen Breeding Co., Ltd, Zhuzhou 412000, China;
3. Hunan Animal and Veterinary Science, Changsha 410131, China

Received:2023-08-15 Online:2024-05-23 Published:2024-05-27

摘要/Abstract

摘要： 旨在建立一种猪圆环病毒4(porcine circovirus type 4, PCV4)Cap蛋白间接ELISA抗体检测方法，对猪群中PCV4抗体进行检测，并与以往的关于PCV4血清流行病学的调查结果进行对比研究，从而更全面地了解PCV4在猪群中的感染和流行情况。本研究利用原核表达系统成功表达PCV4 Cap蛋白，对反应条件进行优化，建立了基于PCV4 Cap蛋白的间接ELISA方法。采用建立的ELISA方法对中国18个省份的不同阶段的2 298份猪血清样本进行检测，以调查中国猪群中PCV4的血清学流行情况。同时将Cap-ELISA检测方法与之前建立的Rep-ELISA进行对比，平行检测845份血清样本。结果显示，在18个省份中，17个省的血清样本中检测到PCV4抗体。PCV4总血清阳性率为34.94%，母猪和育肥猪阳性率分别为59.95%和31.64%，仔猪阳性率为21.14%，保育猪中检出的阳性率最低，为4.20%。两种方法共同检测845份血清样本，符合率为82.84%，其中Cap-ELISA检测总阳性率为31.83%，Rep-ELISA检测总阳性率为35.03%。研究表明，PCV4在中国广泛传播，不同年龄阶段的猪只均能感染。Cap-ELISA和Rep-ELISA两种方法一致性高，从抗体消长规律来分析，两种方法都说明猪群感染PCV4主要在育肥猪和母猪阶段。本研究提供了中国PCV4的最新血清流行病学特征和PCV4在不同阶段猪群中的感染情况，进一步证实了PCV4在我国猪群中有一定的感染率，其危害值得进一步研究和关注。

关键词: 猪圆环病毒4, 原核表达, 间接ELISA, 血清流行病学调查

Abstract: The present study aimed to develop an indirect ELISA to detect antibodies against the Cap protein of porcine circovirus type 4 (PCV4), and to detect PCV4 antibodies in pigs, then compare the results with previous investigations on PCV4 seroepidemiology, so as to understand the prevalence and infection of PCV4 in swine populations more comprehensively. PCV4 Cap protein was successfully expressed in a prokaryotic expression system. By optimizing the reaction conditions, an indirect ELISA method based on PCV4 Cap protein was established. To investigate the serological prevalence of PCV4 in Chinese pig herds, 2 298 serum samples from pigs at different growth stages from 18 provinces in China were analyzed using the established ELISA method. Concurrently, the Cap-ELISA method was compared with Rep-ELISA, and 845 serum samples were tested in parallel. The results showed that PCV4 antibodies were detected in serum samples from 17 out of the 18 provinces. The total seroprevalence of PCV4 was 34.94%, and the positive rates of sows and finishing pigs were 59.95% and 31.64%, respectively. The positive rate of piglets was 21.14%, while nursery pigs had the lowest positive rate at 4.20%. A total of 845 serum samples were tested using both methods, and the coincidence rate was 82.84%. The Cap-ELISA test yielded an overall positive rate of 31.83%, while the Rep-ELISA test had an overall positive rate of 35.03%. Studies have demonstrated the widespread prevalence of PCV4 across the country and its ability to infect pigs of all ages. The Cap-ELISA and Rep-ELISA techniques exhibited a high degree of concordance, and based on the antibody growth and decline trends, both methods indicated that PCV4 infection primarily occurs in finishing pigs and sows. The present study provides the most recent data on the seroepidemiological characteristics of PCV4 in China and PCV4 infection prevalence in pigs at different growth stages, which could facilitate further research on PCV4 and its management.

Key words: PCV4, prokaryotic expression, indirect ELISA, seroepidemiological survey

中图分类号:

S852.659.2

宋晓晴, 邓瑞德, 李欣, 李姣, 李润成, 杜丽飞, 董伟, 葛猛. PCV4 Cap抗体ELISA检测方法的建立及血清流行病学调查[J]. 畜牧兽医学报, 2024, 55(5): 2072-2079.

SONG Xiaoqing, DENG Ruide, LI Xin, LI Jiao, LI Runcheng, DU Lifei, DONG Wei, GE Meng. Establishment of ELISA for Detection of PCV4-Cap Antibody and Sero-epidemiological Survey[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2072-2079.

参考文献

[1] ZHANG H H, HU W Q, LI J Y, et al. Novel circovirus species identified in farmed pigs designated as Porcine circovirus 4, Hunan province, China[J]. Transbound Emerg Dis, 2020, 67(3):1057-1061.
[2] TISCHER I, GELDERBLOM H, VETTERMANN W, et al. A very small porcine virus with circular single-stranded DNA[J]. Nature, 1982, 295(5844):64-66.
[3] TISCHER I, MIELDS W, WOLFF D, et al. Studies on epidemiology and pathogenicity of porcine circovirus[J]. Arch Virol, 1986, 91(3-4):271-276.
[4] ELLIS J, HASSARD L, CLARK E, et al. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome[J]. Can Vet J, 1998, 39(1):44-51.
[5] SAOULIDIS K, KYRIAKIS S C, KENNEDY S, et al. First report of post-weaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome in pigs in Greece[J]. J Vet Med B, 2002, 49(4):202-205.
[6] JIANG H J, WANG D, WANG J, et al. Induction of porcine dermatitis and nephropathy syndrome in piglets by infection with porcine circovirus type 3[J]. J Virol, 2019, 93(4):e02045-18.
[7] PALINSKI R, PIÑEYRO P, SHANG P C, et al. A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure[J]. J Virol, 2016, 91(1):e01879-16.
[8] 唐宁, 吕艳丽, 郭鑫, 等. 免疫组化法检测猪圆环病毒2型在人工感染猪体内的分布[J]. 畜牧兽医学报, 2008, 39(3):372-375.
TANG N, LÜ Y L, GUO X, et al. Detection of distribution of porcine circovirus type 2 in experimentally infected piglets with immunohistochemical method[J]. Acta Veterinaria et Zootechnica Sinica, 2008, 39(3):372-375. (in Chinese)
[9] 库旭钢, 于学祥, 孙琪, 等. 猪圆环病毒3型的分离及致病性分析[J]. 畜牧兽医学报, 2023, 54(4):1568-1578.
KU X G, YU X X, SUN Q, et al. Isolation and pathogenicity analysis of porcine circovirus type 3[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4):1568-1578. (in Chinese)
[10] NGUYEN V G, DO H Q, HUYNH T M L, et al. Molecular-based detection, genetic characterization and phylogenetic analysis of porcine circovirus 4 from Korean domestic swine farms[J]. Transbound Emerg Dis, 2022, 69(2):538-548.
[11] SIRISEREEWAN C, NGUYEN T C, PIEWBANG C, et al. Molecular detection and genetic characterization of porcine circovirus 4 (PCV4) in Thailand during 2019-2020[J]. Sci Rep, 2023, 13(1):5168.
[12] XU T, CHEN L, HUANG B Z, et al. The first dog-origin porcine circovirus type 4 complete genomic sequence have high homology with that of pig-derived strains[J]. Front Microbiol, 2023, 14:1121177.
[13] ZHANG L H, WANG T X, FU P F, et al. First molecular detection and genetic analysis of a novel porcine circovirus (porcine circovirus 4) in dogs in the world[J]. Microbiol Spectr, 2023, 11(2):e0433322.
[14] XU T, CHEN X M, FU Y, et al. Cross-species transmission of an emerging porcine circovirus (PCV4):first molecular detection and retrospective investigation in dairy cows[J]. Vet Microbiol, 2022, 273:109528.
[15] WANG Y J, YAN S J, JI Y T, et al. First identification and phylogenetic analysis of porcine circovirus type 4 in fur animals in Hebei, China[J]. Animals, 2022, 12(23):3325.
[16] HOU C Y, ZHANG L H, ZHANG Y H, et al. Phylogenetic analysis of porcine circovirus 4 in Henan Province of China:a retrospective study from 2011 to 2021[J]. Transbound Emerg Dis, 2022, 69(4):1890-1901.
[17] SUN W C, DU Q, HAN Z X, et al. Detection and genetic characterization of porcine circovirus 4 (PCV4) in Guangxi, China[J]. Gene, 2021, 773:145384.
[18] TIAN R B, ZHAO Y, CUI J T, et al. Molecular detection and phylogenetic analysis of Porcine circovirus 4 in Henan and Shanxi Provinces of China[J]. Transbound Emerg Dis, 2021, 68(2):276-282.
[19] NIU G Y, ZHANG X W, JI W L, et al. Porcine circovirus 4 rescued from an infectious clone is replicable and pathogenic in vivo[J]. Transbound Emerg Dis, 2022, 69(5):e1632-e1641.
[20] GE M, HU W Q, NING K M, et al. The seroprevalence of the newly identified porcine circovirus type 4 in China investigated by an enzymed-linked immunosorbent assay[J]. Transbound Emerg Dis, 2021, 68(6):2910-2914.
[21] LIAN Z M, LIU J B, LIU P R, et al. Development and application of an indirect ELISA for the detection of antibody to porcine circovirus 4 in pigs[J]. Transbound Emerg Dis, 2021, 68(6):2975-2979.
[22] HU X F, DING Z, LI Y, et al. Serum investigation of antibodies against porcine circovirus 4 Rep and Cap protein in Jiangxi Province, China[J]. Front Microbiol, 2022, 13:944679.

PCV4 Cap抗体ELISA检测方法的建立及血清流行病学调查

Establishment of ELISA for Detection of PCV4-Cap Antibody and Sero-epidemiological Survey

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