某猪场猪2型圆环病毒感染的诊断

猪2型圆环病毒(PCV2)是引起断奶仔猪多系统衰竭综合征(PMWS)的主要病原,此外,PCV2还可能与猪皮炎肾病综合征、猪呼吸系统综合征、仔猪先天性震颤等疾病有关.PMWS临床症状主要表现为进行性消瘦,生长发育受阻,皮肤苍白或有黄疸,体表淋巴结肿大,腹泻,贫血和呼吸道症状等.     

猪圆环病毒2型感染对猪CD80和CD86 mRNA表达的影响

通过构建猪共刺激分子CD80和CD86的缺失cDNA竞争分子,用竞争PCR技术定量检测了猪圆环病毒2型(PCV2)感染后猪肺泡巨噬细胞(PAM)中CD80和CD86的mRNA水平,分析了PCV2感染对猪共刺激分子CD80和CD86的mRNA表达的影响.结果显示,PCV2感染后,PAM中CD80和CD86的mRNA水平变化趋势一致,只是CD86的升降幅度更大.在PCV2感染后3d,CD80与CD86的mRNA水平下降至最低,随后迅速上升,至14d达到高峰,尔后快速下降,21d及以后恢复正常.本研究结果表明,PCV2初期可明显抑制猪共刺激分子CD80和CD86的基因转录.     

规模化猪场猪圆环病毒2型感染的实验室诊断

为了确诊贵州省某规模化猪场保育仔猪异常死亡原因,从怀孕母猪、产房母猪、后备母猪、种公猪、哺乳仔猪、保育仔猪6个猪群采集90份血清样本采用ELISA方法分别进行血清抗体检测,并对采集的90份血清样本和1份病死猪淋巴结组织采用荧光PCR方法进行病原学检测。结果:6个猪群综合的猪瘟病毒、猪蓝耳病病毒、伪狂犬病病毒g B蛋白、伪狂犬病病毒g E蛋白血清抗体阳性率分别为87.78%、70.00%、88.89%、4.44%;猪瘟病毒、猪蓝耳病病毒、伪狂犬病病毒病原核酸检测显示阴性,猪圆环病毒2型病原核酸检测淋巴结组织样本显示阳性。试验结果表明,引起该猪场保育仔猪死亡的原因为猪圆环病毒2型感染。同时,怀孕母猪群出现了伪狂犬病病毒g E蛋白抗体阳性,提示怀孕母猪群可能存在猪伪狂犬病野毒感染。     

仔猪人工感染猪圆环病毒2型后的病理学特征

自Harding首次报道1991年加拿大发现断奶仔猪多系统衰弱综合征（post-weaning multisystemic wasting syndrome，PMWS）以来，猪圆环病毒2型（porcine cirovirus type 2，PCV2）感染及其所致疾病已遍及世界各地，成为危害世界养猪生产的一大疫病。PCV2是引起PMWS的主要病原体。PMWS主要感染5～12周龄断奶仔猪，引起的临床症状包括体重下降、进行性消瘦、呼吸困难、贫血、腹泻和黄疸；组织病变包括淋巴结病，肉芽肿性间质性肺炎，肝炎和间质性肾炎，其发病率在5％～30％不等，死亡率可高达50％炉此外，由于PCV2感染引起的免疫抑制可造成二次感染，更增加了疾病的严重程度。     

某规模化猪场猪2型圆环病毒病流行病学调查

为了弄清猪2型圆环病毒感染在该猪场的感染状况、流行特点及优势基因型等情况。采用ELISA检测技术和PCR技术,对新疆某规模化猪场的不同生产阶段猪群进行猪2型圆环病毒(PCV2)抗体检测和基因型检测。结果猪圆环病毒病(PCVD)在该猪场猪群中的平均抗体阳性率为72.41%,不同生长阶段的猪群感染情况分别为哺乳仔猪69.74%、断奶仔猪62.75%、育肥猪77.88%、母猪81.67%以及种公猪83.33%;对采集的11份抗体阳性的断奶猪只的脏器病料PCR检测,其中5份为PCR阳性(45.45%),经克隆测序分析发现,该猪场5株PCV2毒株序列之间的相似性在99.3%⁹9.9%之间,且均属于PCV2b这一亚型。结果表明,该猪场的PCV2感染流行已非常严重,整体流行特征表现为猪只日龄越大,感染情况越严重,且流行的优势基因型为PCV2b亚型,应该引起养殖户的高度重视,需要立即采取针对有效的防治净化措施。     

广西猪圆环病毒2型感染的流行病学调查

结合流行病学、临床症状、病理变化，采用PCR技术，对2004年1月至2005年5月采自广西14个市97个疑似猪圆环病毒2型（PCV2）感染发病猪场的197份组织病料（脾、肺、淋巴结）进行了PCV2检测；同时，对鉴定为PCV2阳性的组织病料和猪场进行了猪生殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（CSFV）、猪流感病毒（SIV）和猪伪狂犬病病毒（PRV）的检测，另外，对6个地（市）11个生猪屠宰场采集的外观健康屠宰猪的295份组织样品（脾、肺、淋巴结）进行了PCV2检测。结果显示，在197份组织样品中检出PCV2阳性病料108份，平均阳性率为54．82％（108／197），阳性猪场62个，平均阳性率为63．92％（62／97）。PCV2与PRRSV、CSFV、SIV、PRV混合感染的组织病料总阳性率为42．13％（83／197），混合感染的猪场总阳性率为57．73%（56／97）。从21头外观健康屠宰猪的组织样品中检测到PCV2，阳性率为7．10％。由此可见，PCV2感染在广西猪群中已普遍存在，混合感染和健康带毒现象使病情更加复杂。     

猪圆环病毒2型感染对猪肺泡巨噬细胞生物学活性的影响

将猪圆环病毒2型（PCV2）BF株经口、鼻接种40日龄健康仔猪，在接种后不同时间宰杀，收集猪肺泡巨噬细胞（PAM），同时设立对照。用FITC—Annexin V／PI双染色流式细胞术和琼脂糖凝胶电泳检测PAM凋亡现象，通过EA花环试验测定Fc受体数目，通过吞噬鸡红细胞试验测定吞噬功能，分析PCV2感染对PAM生物学活性的影响。结果显示，在整个试验期内2种方法均没有检测到PAM凋亡，表明PCV2感染不会诱发PAM凋亡。PAM的Fc受体数和吞噬鸡红细胞数的变化规律一致，与对照组相比，两者数量在接种后第3d明显下降，第7d有所回升，之后基本恢复，表明PCV2感染后PAM吞噬和清除病原的功能出现短暂下降。     

2009年我国猪群中猪圆环病毒2型感染情况调查

为了解我国猪群中猪圆环病毒2型（Porcine circovirustype2,PCV2）的感染情况,分析感染与猪年龄的关系,本试验于2009年在我国28个省市的71个中小规模场、22个屠宰场和62家散养户采集了2905份猪血清样品,用PCR法对PCV2进行检测。结果显示,检出阳性血清429份,阳性率为14．8％。在被调查的场／P中,86．4％屠宰场、52．1％中小规模场和58．1％散养户为PCV2感染阳性,其中48．6％的PCV2阳性中小规模场的群阳性率低于10％。分析中小规模场PCV2感染情况和猪年龄之间的关系发现,在5个年龄组中,2～4周龄组未检出PCV2感染,5周龄以上各组均检出PCV2感染,其中11～14周龄组和15～26周龄组阳性率较高,高于5～7周龄组和8～10周龄组。     

Evidence of shedding of porcine circovirus type 2 in milk from experimentally infected sows

Detection of porcine circovirus type 2 (PCV2) was to evaluate the milk from experimentally infected sows using polymerase chain reaction (PCR) and virus isolation. Six pregnant sows were inoculated intranasally with PCV2 at 93 days of gestation, and milk samples were collected from all sows at 1, 3, 6, 9, 12, 15, 18, 21, 24, and 27 days of lactation. PCV2 was detected in milk as early as day 1 of lactation in all six sows. Thereafter, all infected sows remained positive by PCR for PCV2 in milk until 27 days of lactation. In addition, PCV2 itself was isolated from milk collected from a virus-infected sows. These results suggest that PCV2 may be shed in milk following infection of pregnant sows by the virus.     

免疫PCV2疫苗对生长猪增重与健康的影响

在猪圆环病毒2型(PCV2)阳性猪场仔猪免疫PCV2灭活疫苗,无论是小群试验还是大群试验,免疫均能提高猪的成活率,降低猪因PCV2感染引起相关疾病的发病程度,提高猪群平均增重速度。40日龄和56日龄免疫组的PCV2抗体极显著低于对照组(P0.01),而85日龄时,一次免疫组的抗体快速升高超过对照组,2次免疫仍较低,差异极显著(P0.01)。     

Effect of porcine circovirus type 2a or 2b on infection kinetics and pathogenicity of two genetically divergent strains of porcine reproductive and respiratory syndrome virus in the conventional pig model

To determine differences in infection kinetics of two temporally and genetically different type 2 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in vivo with and without concurrent porcine circovirus (PCV) type 2a or 2b infection, 62 pigs were randomly assigned to one of seven groups: negative controls (n=8); pigs coinfected with a 1992 PRRSV strain (VR-2385) and PCV2a (CoI-92-2a; n=9), pigs coinfected with VR-2385 and PCV2b (CoI-92-2b; n=9), pigs coinfected with a 2006 PRRSV strain (NC16845b) and PCV2a (CoI-06-2a; n=9), pigs coinfected with NC16845b and PCV2b (CoI-06-2b; n=9), pigs infected with VR-2385 (n=9), and pigs infected with NC16845b (n=9). Blood samples were collected before inoculation and at day post-inoculation (dpi) 3, 6, 9 and 12 and tested for the presence of PRRSV antibody and RNA, PCV2 antibody and DNA, complete blood counts, and interferon gamma (IFN-γ) levels. Regardless of concurrent PCV2 infection, VR-2385 initially replicated at higher levels and reached peak replication levels at dpi 6. Pigs infected with VR-2385 had significantly higher amounts of viral RNA in serum on both dpi 3 and dpi 6, compared to pigs infected with NC16845b. The peak of NC16845b virus replication occurred between dpi 9 and dpi 12 and was associated with a delayed anti-PRRSV antibody response in these pigs. PCV2 coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-PRRSV IgG response compared to pigs infected with PRRSV alone. This work further emphasizes in vivo replication differences among PRRSV strains and the importance of coinfecting pathogens.     

猪圆环病毒2型重组多肽抗原的免疫保护效果

为检验猪圆环病毒2型(PCV-2)重组多肽抗原对猪的免疫保护效果,以表达的重组大分子多肽作为抗原,断奶仔猪为实验动物,设置多肽疫苗组、常规市售Cap蛋白亚单位疫苗组和不同对照组进行免疫。用间接酶联免疫吸附法(ELISA)检测血清特异抗体(IgG),当抗体达到一定水平时进行攻毒试验,对试验各组的临床症状、体征变化及病毒血症进行观察和动态检测,对PCV-2易感组织进行病毒检测及组织病理学检查,最终评估该多肽疫苗的免疫效果及保护率。结果显示:表达的多肽疫苗能够快速产生体液免疫应答,具有显著的免疫效果;攻毒后采集的各项试验数据也表明该多肽疫苗的免疫保护效果理想。     

猪圆环病毒2型、猪巨细胞病毒和TTV-2混合感染的流行病学调查

本研究根据猪圆环病毒2型（PCV2）ORF2、猪巨细胞病毒（PCMV）gB基因和TTV2非编码区序列合成引物,利用PCR方法对2009年来自江苏、浙江、上海、广东和广西等5个省市76个患病猪群的仔猪病料进行了病原学检测。结果显示,PCV2阳性率64.5%（49/76）,TTV2阳性率47.4%（36/76）,PCMV阳...     

猪圆环病毒2型感染对猪瘟疫苗体液免疫应答的影响

采用ELISA方法对单独接种猪瘟疫苗组(CSFV组,n=3)、PCV2感染且出现病毒血症后接种猪瘟疫苗组(PCV2/CSFV组,n=3)及PCV2感染同时接种猪瘟疫苗组(CSFV/PCV2组,n=3)不同时相血清中的猪瘟抗体进行检测;并对PCV2感染对照组(PCV2组)及PCV2/CSFV和CSFV/PCV2组血清中PCV2特异的抗体和核酸分别进行ELISA和PCR检测.结果表明,在接种后52 d CSFV组血清中抗体的阻断值显著高于CSFV/PCV2组(P<0.05);接种后42 d和52 d CSFV组平均抗体效价明显高于PCV2/CSFV和CSFV/PCV2组,其中在52 d CSFV组抗体阳性率这100%(3/3)而PCV2/CSFV和CSFV/PCV2在相应时相抗体阳性率仅为67%(2/3).结果提示PCV2感染可在一定程度上抑制猪瘟疫苗特异性的抗体反应.     

A meta-analysis on experimental infections with porcine circovirus type 2 (PCV2)

A meta-analysis was performed with the aim to identify factors with a relevant influence on the expression of clinical postweaning multisystemic wasting syndrome (PMWS) under experimental conditions. Data from 44 studies were included in the analysis. Several variables were studied: number of pigs in the experiment, intake of colostrum, serological status against porcine circovirus type 2 (PCV2), strain of PCV2 used for inoculation, the route and dose of inoculation, and use of potential triggering factors (such as co-infections, vaccinations, or immunomodulator products). Multiple correspondence analysis and log-linear regression methods were used to establish the relationships between the studied variables and the number of PCV2 infected pigs that developed PMWS. Based on the results of the meta-analysis, the most successful animal experiment aimed to develop PMWS should include: (1) colostrum-deprived pigs, (2) age of inoculation below 3 weeks, (3) high doses of PCV2 inoculum, (4) PCV2 strain from genotype 1, and (5) co-infection with another swine pathogen as a triggering factor.     

PCV2感染对猪MCP-1和IL-8mRNA表达的影响

将猪圆环病毒2型（PCV2）BF株经口、鼻接种40日龄健康普通仔猪，于接种后不同时间宰杀，收集肺泡巨噬细胞（PAM），同时设立对照。用竞争PCR技术测定趋化性细胞因子IL-8和MCP-1 mRNA水平，分析PCV2感染对其mRNA表达的影响。结果显示，与对照组相比，在PCV2感染后第7d，IL-8mRNA水平下降至最低，随后迅速上升，至第14d时达到高峰，而后快速下降，但仍维持较高水平；MCP-1 mRNA水平在攻毒后第3d下降至最低，之后逐渐上升，至第14d时达到高峰，而后下降，第21d以后恢复正常。结果表明，PCV2感染初期可导致PAM趋化性细胞因子IL-8和MCP-1基因的转录明显下调。     

In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system

Porcine circovirus type 2 (PCV2) nucleic acid and/or antigens are consistently observed in cells of monocytic morphology in lesions of pigs affected by post-weaning multisystemic wasting syndrome (PMWS). In this study, PCV2 antigen was detected in the cytoplasm of monocytes, pulmonary macrophages (PMs) and monocyte-derived macrophages exposed to the virus in vitro, by immunofluorescence analysis (IFA) and the phenotype of these cells confirmed by detection of monocytic cell surface markers using flow cytometry. Viral antigen was not observed in lymphocytic cells. Replication of the virus in PMs was investigated further by comparison to that observed in the continuous pig kidney cell line (PK15A) using quantitative virus titration, quantitative PCR and by the detection of double stranded DNA intermediates of viral replication by Southern blotting analyses. Although increases in viral DNA and levels of infectious virus progeny and the presence of replicative intermediates, indicative of viral replication, were observed in PK15A cells, no such changes were observed in PMs in spite of the fact that infectious virus, viral antigen and viral DNA persisted in the cells for at least the duration of the experiment. These results suggest that in vivo, monocytic cells may not represent the primary target for PCV2 replication.     

猪圆环病毒2型感染对口蹄疫O型合成肽疫苗免疫应答的影响

采用阻断ELISA方法对单独接种口蹄疫O型(FMD)组(C组,n=3)及PCV2人工感染4周后接种FMDO型疫苗组(PC组,n=4)后不同时相血清中的FMDO型抗体进行检测;同时对不同时相前腔静脉血进行CD4/CD8流式细胞术及血常规分析。结果显示,除接种后4周C组白细胞及淋巴细胞含量与略低于PC组外,接种疫苗后2～8周C组白细胞及淋巴细胞含量均略高于CP组,且在接种疫苗后2周,C组淋巴细胞含量显著高于PC组(P0.05);在整个试验过程中PC组的幼稚型Th细胞含量均高于C组,且在接种疫苗后0周和8周2组间幼稚型Th细胞含量差异显著(P0.05);在接种疫苗后2～6周期间,除在接种疫苗后4周2组间Tc细胞含量较为接近外,C组的Tc细胞含量均高于PC组;除在8WPIPC组CD4+CD8+记忆/激活Th细胞含量略高于C组外,在接种疫苗后0～6周过程中,C组记忆/激活Th细胞含量均略高于PC组,且在接种疫苗后6周,2组间记忆/激活Th细胞含量差异显著(P0.05);在接种前疫苗4周和前2周(-4-2WPI)期间,C组的抗体阻断率均高于PC组,在接种疫苗后4～8周2组间抗体阻断率差异逐渐变小,且PC组抗体阻断率略高于C组。结果表明,PCV2感染对机体产生针对FMDO型特异性抗体早期有一定的抑制作用,但在后期这种抑制作用逐步减弱,甚至消失;PCV2感染确实可以在一定程度上抑制机体对猪FMDO型合成肽疫苗体液免疫应答,导致机体的淋巴细胞及白细胞含量降低,进而导致Tc细胞及记忆/激活Th细胞含量下降。     

猪圆环病毒2型感染的检测与流行病学调查

根据GenBank上发表的PCV2 ORF2和PRRSV ORF7序列设计合成引物,建立分别用于检测PCV2和PRRSV的PCR和RT-PCR方法,并对2003年9月至2005年5月采自189个可疑发病猪场的389份病料进行了PCV2和PRRSV的检测。结果显示,来自33个猪场的35份样品为PCV2阳性,猪场PCV2阳性率为17.1%;PCV2与PRRSV混合感染率为5.1%,PCV2阳性猪场中PRRSV感染率为57.6%。流行病学统计结果表明,安徽省猪场PCV2阳性率最高;安徽省和广西省猪场PCV2和PRRSV的混合感染最严重;春夏季节PCV2阳性率明显高于秋冬;消瘦、被毛粗乱是PCV2感染的主要特征;淋巴结水肿或出血,肾脏肿大、出血或呈灰白色以及脾脏病变是PCV2感染的主要病理变化;仔猪7～8周龄发病率最高。     

Viremia and effect of fetal infection with porcine viruses with special reference to porcine circovirus 2 infection

This publication reviews some pathogenetic features of the transplacental infection with porcine viruses in sows. Viremia either with virus freely circulating or associated to peripheral blood mononuclear cells (PBMC) is an essential part of such pathogenesis. Virus replication occurs either in fetal tissues only or both in fetal and maternal tissues and the outcome may be different.Since porcine circovirus 2 (PCV2) has been associated with reproductive failure in sows, the question was asked what type of viremia PCV2 causes and what the effect of PCV2 is on the pregnant uterus. Seronegative gilts were oronasally inoculated and plasma and PBMC were monitored for infectious virus and for quantity of viral DNA copies. Infectious virus was found in plasma only at 21 days post-inoculation (DPI). Virus associated to PBMC was detected between 14 and 49 DPI. Viral DNA was found in plasma between 14 and 49 DPI and associated to PBMC between 7 and 63 DPI (end of experiment). Direct intra-fetal inoculation at 57, 75 and 92 days of gestation and collection of fetuses 21 days later showed that the virus replicates highly in fetal tissues, particularly in the heart. Fetal death occurred in the 57 days sows while virus and antibodies were observed in the 75- and 92-day inoculated sows. Inoculation at 57 and 75 days of gestation and collection of the piglets at the end of pregnancy showed that intrauterine spread had occurred to fetuses adjacent to the inoculated ones and that fetal death occurred also in the presence of antibodies. The pregnancy was not interrupted.This study shows that PCV2 causes viremia which is largely cell-associated and that virus replication in fetuses causes fetal death with mummification. Whether such transplacental infection occurs in the immune sow population is questionable.