Development of a polyclonal-antibody-based immunohistochemical method for the detection of type 2 porcine circovirus in formalin-fixed, paraffin-embedded tissue.

An immunohistochemical method for the detection of type 2 porcine circovirus (PCV2) in paraffin-embedded tissue was developed. Rabbits were inoculated with purified PCV2 to obtain a polyclonal antiserum. Antiserum was applied to sections of porcine tissue that contained lesions consistent with postweaning multisystemic wasting syndrome and in which PCV2 genome had been demonstrated by in situ hybridization. In all cases (18/18), the density and distribution of positive cells detected by in situ hybridization or immunohistochemistry were identical. The immunohistochemical method is more rapid and less expensive than in situ hybridization and is thus more suitable for routine diagnostic use.     

猪圆环病毒2型胶体金抗体检测方法的建立

为建立检测猪圆环病毒2型(PCV2)胶体金检测方法,本研究对SPA蛋白进行胶体金标记,并喷涂于玻璃纤维上制备金标垫,分别以重组PCV2 ORF2蛋白和猪IgG作为检测线和质控线,制作PCV2抗体检测胶体金免疫层析试纸条。检测结果表明,试纸条操作简单,肉眼于10 min内可以判定结果,对PCV2免疫血清具有高度特异性,与猪其它病毒免疫血清无交叉反应,检测灵敏度与ELISA相近。试纸条在室温保存6个月,其特异性及灵敏度无明显变化。对临床采集的324份血清样品进行检测,结果与ELISA试剂盒总符合率为98.77%。表明本研究建立的PCV2抗体免疫层析检测方法具有特异、敏感、稳定、操作简单快捷等特点,适合于PCV2抗体的现场检测。     

猪圆环病毒2型检测与免疫技术研究进展

对年近来国内外对猪圆环病毒2型(PCV2)的检测技术进展和疫苗研究进展作了简要回顾。PCV2实验室检验技术有间接免疫荧光(IFA)、间接酶联免疫吸附试验(ELISA)、免疫过氧化物酶单层细胞试验(IPMA)、单克隆抗体以及多种聚合酶链式反应(PCR)技术等。在疫苗研究方面,国外已研发多种PCV2灭活疫苗,国内亦有人正在研发PCV2灭活苗(SH株)。在PCV2基因工程疫苗研究方面主要有重组活载体疫苗和核酸疫苗两类。     

猪圆环病毒2型原位杂交检测方法的建立

参照GenBank发表的PCV2ORFl基因序列设计了1对引物,利用PCR地高辛探针合成的方法制备了长度为494bp的特异性探针,经检验具有良好的特异性和敏感性,可检测最低质粒DNA质量浓度为0．9728ug／L。用该探针建立了原位杂交组织切片检测方法,并用来检测PCV2感染猪的扁桃体和淋巴结组织,结果表明阳性信号主要存在于巨噬细胞胞浆中,信号强、背景良好,阴性对照无显色,说明该方法可作为PCV2实验室诊断和机理研究的一种有效检测方法。     

A comparison of immunohistochemistry and in situ hybridization for the detection of porcine circovirus type 2 in pigs

The aim of this study was to develop and to optimize an immunohistochemistry (IHC) method for PCV2 identification and to compare it with an in situ hybridization (ISH) technique. The results demonstrated that both ISH and IHC successfully detected PCV2 viral antigens or nucleic acid in the examined tissues. Most of the slides identified previously in ISH as PCV2-positive were also positive in IHC. In the case of nearly half of the slides the results of IHC examination revealed an increase in the intensity of staining. IHC presented higher sensitivity and specificity than ISH. No negative impact of the time of paraffin block storage on ISH detection results was observed. In addition, IHC results were easier to interpret due to better image quality after staining. Overall results confirmed IHC was a reliable and useful technique for PMWS diagnosis.     

猪圆环病毒2型环介导等温扩增(LAMP)检测方法的研究

本研究介绍了一种便捷、灵敏而又特异的环介导逆转录等温扩增(LAMP)基因检测技术,该技术分别使用特异对应于靶序列中2对特殊引物,并在Bst DNA聚合酶的作用下对靶序列进行等温核酸扩增反应.本研究对LAMP反应体系和反应条件的优化,结果表明PCV-2 LAMP在63℃1 h内能够成功的检测猪圆环病毒2型基因;敏感性和特异性试验表明,本研究能够特异的检测PCV-2并且其敏感度可以达到10个拷贝的DNA分子,初步研究LAMP的阳性检出率与PCR的阳性检出率比较结果为94%符合.以上结果证明,LAMP扩增技术是一种检测程序简便、灵敏度和特异性较高的基因检测手段,在猪圆环病毒2型的快速检测方面具有一定的开发潜力.     

猪圆环病毒2型感染性克隆的构建

利用PCR方法从临床疑似PMWS症状猪的脏器中扩增出1株猪圆环病毒2型(PCV-2)的全基因组(1 767bp),命名为SDZQ-1,对其基因进行克隆测序并与GenBank中收录的21个PCV-2毒株的全基因组序列进行比较,同源性结果为95.0%～99.7%.将2个SDZQ-1的全基因组序列正向串联连接入PUC19载体质粒中,成功构建了PCV-2双拷贝质粒.体外转染PK-15细胞,盲传5代后,用PCR和间接免疫荧光方法检测,结果构建的PCV-2克隆具有感染性.     

猪圆环病毒2型分离株优势基因型分析与检测

为探讨猪圆环病毒2型(PCV2)流行株基因型的变化,本研究对1999年～2009年NCBI中登录的556个PCV2分离株全基因序列进行分析。结果表明,1999年～2002年所登录的PCV2的优势流行毒株为基因A型(PCV2A);从2003年开始基因B型(PCV2B)逐渐为优势流行毒株,在PCV2分离株中基因B型所占比例,2004年为95.4%,2007年～2009年均超过76.0%。2003年～2009年国内的PCV2分离株有287个,其中250个为PCV2B,37个为PCV2A,表明PCV2B是国内猪群中的主要优势毒株。通过建立PCV2A和PCV2B PCR检测方法,对70份临床病料的检测显示,PCV2检出率为34.29%(24/70),基因型均为PCV2B,无PCV2A的检出,表明PCV2B为检测猪群中的主要优势毒株。     

Interlaboratory testing of porcine sera for antibodies to porcine circovirus type 2.

A panel of 20 porcine sera was distributed to 5 laboratories across Europe and Canada. Each center was requested to test the sera for the presence of porcine circovirus type 2 antibodies using the routine assays, indirect immunofluorescence assay (IFA) and indirect immunoperoxidase monolayer assay (IPMA), and to determine the titer of each serum. Results from all centers were then compiled and correlated. They demonstrate a wide variation in the titers obtained between laboratories. These differences were dependent on the assay used and the choice of fixative. In general, IPMA gave higher titers than did IFA, and paraformaldehyde gave higher titers than did acetone or ethyl alcohol. This report highlights the need for standardized procedures and biologicals for this virus.     

Simultaneous detection of porcine circovirus 2 and porcine parvovirus in naturally and experimentally coinfected pigs by double in situ hybridization.

A technique for double in situ hybridization to simultaneously detect porcine circovirus 2 (PCV2) and porcine parvovirus (PPV) in the same tissue section was developed and applied to lymph node and spleen from 8 pigs experimentally coinfected with PCV2 and PPV and 20 pigs with naturally occurring postweaning multisystemic wasting syndrome. For double labeling studies, the tissue samples were processed sequentially, first for PPV in situ hybridization using a digoxigenin-labeled probe and then for PCV2 in situ hybridization using a biotinylated probe. Positive cells contained reaction products for PCV2 and PPV, respectively. Both PCV2 DNA and PPV DNA were observed mainly in the cytoplasm but occasionally in the nucleus. With double in situ hybridization, both PCV2 DNA and PPV DNA were simultaneously detected in lymph node and spleen. This double labeling technique for the detection of PCV2 and PPV is suitable both for pathogenesis studies and for diagnostic applications.     

Infectivity of porcine circovirus type 2 DNA in semen from experimentally-infected boars

Porcine circovirus type 2 (PCV2) is an economically important pathogen. It has been demonstrated that PCV2 DNA can be detected in boar semen by PCR; however, the biological relevance of this is unknown. The objectives of this study were to determine if semen positive for PCV2 DNA is infectious (1) in a swine bioassay, or (2) when used for artificial insemination. For the first objective, 4-week-old pigs were inoculated intraperitoneally with PCV2 DNA-negative (bioassay-control; n = 3), PCV2a DNA-positive (bioassay-PCV2a; n = 3), or PCV2b DNA-positive (bioassay-PCV2b; n = 3) raw semen, or PCV2 live virus (bioassay-positive; n = 3), respectively. Pigs inoculated with PCV2 DNA-positive semen and PCV2 live virus became viremic and developed anti-PCV2 antibodies indicating that the PCV2 DNA present in semen was infectious. For the second objective, three Landrace gilts were inseminated with PCV2 DNA-negative semen (gilts-controls) from experimentally-infected boars, and six gilts were artificially inseminated with semen positive for PCV2a DNA (gilts-PCV2a; n = 3) or PCV2b DNA (gilts-PCV2b; n = 3). Serum samples collected from the gilts in all groups remained negative for anti-PCV2 antibodies for the duration of the experiment. In addition, fetal serum samples from all 105-day-gestation fetuses were negative for anti-PCV2 antibodies or PCV2 DNA. Under the conditions of this study, PCV2 DNA-positive semen was not infectious when used to artificially inseminate gilts; however, it was demonstrated to be infectious in a swine bioassay model and therefore is a potential means of PCV2 transmission amongst swine herds.     

PCR detection and characterization of type-2 porcine circovirus.

A polymerase chain reaction (PCR) assay was developed for detecting porcine circovirus (PCV). The assay readily detected type-2 PCV (PCV-2) and type-1 PCV (PCV-1). The PCR primers were designed based on DNA sequences conserved in all reported PCV genomes. Type 1 PCV and type 2 PCV both produced 438 bp amplification products, which were easily identified and differentiated from one another by restriction fragment length polymorphism (RFLP) analysis. Porcine circovirus was detected in 55% (931/1693) of randomly tested pigs with various clinical signs and lesions, most of which were difficult to differentiate from those associated with porcine reproductive and respiratory syndrome (PRRS). The PCR products from all positive clinical samples were identified by RFLP to be only PCV-2; DNA tested by PCR was extracted directly from one or more of lung, mesenteric or mediastinal lymph nodes, and tonsil. Type 2 PCV was also detected in 6% (2/34) of DNA extracted directly from semen of randomly chosen healthy boars. Positive PCR reactions from 554 diseased pigs were characterized by RFLP and categorized into 5 different profiles (A-E), of which 82.8% were PCV-2A (456/554), 3.0% were PCV-2B (17/554), 9.9% were PCV-2C (55/554), 1.1% were PCV-2D (6/554), and 3.2% were PCV-2E (18/554). The complete genomic nucleotide sequences of PCV-2A, B, C, D, and E were determined and found to have at least 95% homology compared with one another and with all other PCV-2 found in the GenBank database. All PCV-2 had less than 76% homology with PCV-1. This PCR assay will hopefully be useful to veterinary diagnostic laboratories for routine testing and surveillance of infection with PCV-2. The RFLP profiling system might be useful for preliminary characterization and identification of PCV isolates and might also benefit studies on the molecular epidemiology of PCV.     

Commercially produced spray-dried porcine plasma contains increased concentrations of porcine circovirus type 2 DNA but does not transmit porcine circovirus type 2 when fed to naive pigs

The porcine circovirus type 2 (PCV2) antibody and DNA status of porcine plasma products collected during the commercial spray-drying process were evaluated. Samples evaluated included 52 pooled liquid plasma (fresh) samples collected at 14 regional abattoirs before transport to 1 of 2 spray-drying facilities, 32 pooled liquid plasma (concentrated) samples collected after arrival at the spray-drying facilities at different stages before the spray-drying process, and 32 samples in powdered form (spray-dried) collected after spray drying. All 116 samples were positive for PCV2 antibody, with PCV2 ELISA sample-to-positive ratios ranging from 9.2 to 13.6 on a DM basis. Porcine circovirus type 2 DNA (4.5 to 7.9 log(10) PCV2 copies/mL, DM basis) was present in 82.7% (43/52) of the fresh plasma samples, 71.9% (23/32) of the concentrated plasma samples and 78.1% (25/32) of the spray-dried plasma samples, with a greater prevalence of PCV2b than PCV2a. To determine the infectivity of PCV2 DNA-positive commercial spray-dried plasma, nine 10-wk-old 68-kg PCV2-na?ve pigs were randomly assigned to 1 of 3 treatment groups and rooms: 1) a negative control (no plasma in the feed, not inoculated with PCV2); 2) a positive control (no plasma in the feed, inoculated with PCV2); and 3) plasma-fed pigs (4% porcine plasma in the feed for 42 d, not inoculated with PCV2). All positive control pigs became viremic by 7 d postinoculation and seroconverted by 42 d postinoculation, whereas pigs in the negative control group and in the spray-dried plasma group were PCV2 PCR negative and did not seroconvert to PCV2 for the duration of the study. The results indicate that PCV2 DNA and antibodies are commonly found in commercial spray-dried plasma. However, no evidence of infectivity of the PCV2 DNA was found in na?ve pigs when commercial spray-dried plasma was included in the diet under the conditions of this study.     

Characterization of porcine circovirus type 2 in Taiwan

In an effort to understand the genetic diversity of porcine circovirus type 2 (PCV2) and the prevalence of PCV2 infection in Taiwanese herds, we have sequenced the complete genomes from PCV2-infected specimens and individually measured the antibody titer against PCV2 from pigs reared in Taiwan between the years 2000 and 2002. A total of 623 specimens originating from pigs displaying varied clinical signs were screened with the polymerase chain reaction (PCR). Results showed that 309 pigs (49.6%) tested positive for PCV2. Eight of the positive specimens were used for the amplification of the complete viral genome. Sequence comparison of the complete genomes indicated that the 8 Taiwanese PCV2 isolates shared 95-99% similarity. Phylogenetic analysis of all 40 PCV2 isolates from North America, Europe, Asia and Taiwan revealed that those isolates were grouped together in one large group containing two minor subgroups. The Taiwanese PCV2 isolates were classified into the two minor subgroups. The prevalence of serum antibodies to PCV2 in pigs was investigated, and results showed that approximately 83.5% of the pigs in Taiwan were seropositive. Finishing pigs possess the highest titers of antibodies, while 9-week-old pigs contained the lowest titers for specific antibodies. Our results suggest that PCV2 infections have become common in Taiwanese pig farms.     

A DNA miniarray system for simultaneous visual detection of porcine circovirus type 1 (PCV1) and 2 (PCV2) in pigs

A miniarray system was developed for the simultaneous detection of porcine circovirus type 1 (PCV1) and type 2 (PCV2) in pigs. The system consists of a polymerase chain reaction (PCR) step to amplify target viral DNA, followed by detection of the amplified DNA using a membrane-anchored probe array and an avidin-alkaline phosphatase (Av-AP) indicator system. The lower limit of detection of PCV using the miniarray was 10^1.9 tissue culture infectious dose 50 (TCID₅₀)/ml and 10^2.08TCID₅₀/ml for PCV1 and PCV2, respectively, and 100 viral copies/μl for both PCV1 and PCV2. We validated the miniarray system using 141 lymph node specimens from pigs with suspected postweaning multisystemic wasting syndrome or porcine dermatitis and nephropathy syndrome. Of the 141 samples evaluated, 55 were identified as positive for PCV by the miniarray. Relative to in situ hybridization, the sensitivity and specificity of the miniarray was 100% and 98.9%, respectively. In contrast to other microarray systems, the miniarray does not require a DNA chip reader, since the results can be determined by visual inspection of colorized spots on a nylon membrane. This system represents an effective alternative method for the differential detection of PCV1 and PCV2 in pigs, as well as the maintenance of PCV-free cell lines and pre-screening of commercial vaccines for possible contamination.     

猪圆环病毒2型的分离与鉴定

利用PCR方法,从疑似断奶猪多系统衰竭综合征(PMWS)的自然病例的淋巴结和脾脏中扩增出了预期长度的猪圆环病毒2型(Porcine Circovirus type 2,PCV2)的DNA片段,并用限制性内切酶对PCR产物进行了酶切鉴定。将经PCR扩增和酶切鉴定为PCV2阳性的组织样品匀浆液接种到无PCV污染的PK15细胞中传代,经间接免疫荧光检查,在接毒细胞内观察到了特异性免疫荧光;用接毒细胞制备超薄切片,在电镜下观察到了直径大约为17nm的圆形病毒样粒子和大量不同形态的胞浆内包涵体。由此表明分离出的病毒为猪圆环病毒2型。     

猪圆环病毒2型毒株的分离鉴定

猪圆环病毒2型(PCV2)可引起断奶后仔猪多系统衰竭综合征(PMWS)、猪皮炎肾病综合征(PDNS)、繁殖障碍等相关疾病[1],其中以PMWS最为严重.该病主要侵害6～12周龄仔猪,引起渐进性消瘦、呼吸困难、腹泻和黄疸等,给世界养猪业造成了巨大的经济损失.     

猪圆环病毒2型及3型快速二重PCR检测方法的建立及其应用

针对猪圆环病毒2型(PCV2)和猪圆环病毒3型(PCV3)基因的保守序列设计了2对扩增PCV2和PCV3的特异性引物,通过优化各反应条件,建立同时检测PCV2和PCV3的二重PCR检测方法。敏感性和特异性结果显示,1.08×10~(-8)μg/μL是对PCV2和PCV3的最低检测限具有较好的灵敏度;而对猪细小病毒(PPV)、伪狂犬病病毒(PRV)、大肠杆菌、链球菌、副猪嗜血杆菌、胸膜肺炎放线杆菌的检测结果均无条带,表明所建立的方法具有较好的特异性。应用该方法检测了2017年9月—2018年8月从广西14个地市猪场采集的533份病猪死猪内脏组织,结果显示:PCV2阳性率为23.8%,PCV3阳性率为14.8%, PCV2和PCV3混合感染率为2.8%。广西14个地市中有12个地市的猪场送检样品检测出PCV2,有10个地市的猪场送检样品检测出PCV3,表明PCV2和PCV3在广西受检猪群中普遍存在,要重视和做好PCV2和PCV3感染的防控工作。     

猪圆环病毒2型ELISA抗体检测试剂盒的研制及应用

利用纯化的重组杆状病毒表达猪圆环病毒2型（PCV2）Cap蛋白作为检测抗原,建立一种间接ELISA方法用于血清抗体检测。昆虫细胞株（Sf-21）接种重组杆状病毒,对蛋白表达参数及纯化工艺进行了优化,制备了5批重组蛋白抗原,表达量占总蛋白的18.7%左右;Ni2＋亲和层析柱纯化获得重组蛋白纯度为90.1%;重组蛋白可被特异性抗体识别,具有良好的免疫活性。用建立的ELISA法对30份已知阴性血清样本检测临界OD405nm值为0.343;ELISA与IPMA对150份血清进行平行检测符合率为95.3%,特异性为91.2%,敏感性为96.5%;与其他几种常见猪病毒参考血清无交叉反应。组装的试剂盒批内和批间变异系数分别为4.0%～4.8%和8.9%～9.9%;置-20℃保存12个月稳定。试剂盒对人工感染猪血清检测结果,接种后第5周抗体阳转,第8周～9周抗体水平达到高峰;对来自黑龙江、吉林、河北、上海等地猪场1085份血清抗体检测,阳性率为73.6%。研制的ELISA试剂盒为临床PCV2血清抗体检测及疫苗免疫效果的评价提供了技术手段。