1起猪细小病毒病的PCR诊断与防制

2005年10月,郑州市某猪场发生了一种以初产母猪流产、死产为特征的疾病,经PCR检测猪细小病毒阳性,结合病史和临床症状,诊断为猪细小病毒病。对该猪场采取了综合防制措施,取得了较好的效果。     

多重PCR检测猪细小病毒和猪伪狂犬病病毒的研究

根据GenBank上已发表的猪细小病毒（Porcine parvovirus，PPV）的VP2基因序列和猪伪狂犬病病毒（Porcine pseudorabies virus，PRV）的gH基因序列，设计合成了两对特异引物，分别建立了PPV和PRV的单项PCR诊断方法，通过对扩增条件的筛选，最终成功地建立了PPV和PRV的复合PCR诊断方法，即利用一次PCR反应，可同时扩增PPV的751bp和PRV355bp的特异性片段，而扩增猪圆环病毒Ⅱ型（PCV．2）及相应的培养细胞（PK-15）核酸结果均为阴性，对PPV和PRV的最低检出量分别为100Pg和10Pg的DNA。该方法适合对PPV和PRV的联合检测和鉴别诊断。     

猪细小病毒与猪伪狂犬病毒二重SYBR GreenⅠ实时荧光PCR方法的建立与检测

本研究旨在建立一种能快速、灵敏、同时检测出猪细小病毒与猪伪狂犬病毒的基于SYBR GreenⅠ实时荧光定量PCR方法。参照GenBank中登录的相关基因序列,设计了2对引物分别用于扩增PRV gH基因与PPV NS1基因的部分片段。将测序正确的PRV gH基因与PPV NS1基因片段克隆入pGEM-T Easy载体,转化大肠杆菌DH5α,经测序鉴定后得到阳性重组质粒,作为标准品模板建立SYBR GreenⅠ荧光定量PCR标准曲线和熔解曲线,并对其灵敏性、特异性和重复性进行验证。结果表明,猪细小病毒与猪伪狂犬病毒荧光定量PCR的标准曲线的Tm值分别为80.9℃和86.5℃,熔解曲线特异,灵敏度分别可达248拷贝/μL和160拷贝/μL,是普通PCR检测方法的100倍。本次建立的猪细小病毒与猪伪狂犬病毒荧光定量PCR检测方法实现了2种病毒的同时检测,能够对PRV、PPV混合感染的临床病料进行快速诊断。     

Pathogenesis of in utero infection: experimental infection of eight- and ten-week-old porcine fetuses with porcine parvovirus.

Selected numbers of fetuses in each of 4 pregnant gilts were exposed to a porcine parvovirus by injecting the virus into the allantoic fluid at gestation day 56 or 70. The fetuses were examined on postexposure day 7 or 14. When pregnancy was terminated, 2 of 15 exposed fetuses were dead. Several fetuses tested at post-exposure day 14 had hemagglutination-inhibiting antibodies to porcine parvovirus. Virus was detected most frequently and in highest concentration in the parenchymatous organs of thorax and abdomen of fetuses exposed on gestation day 56. A small amount of antigen was in neurons and capillary endothelium of cerebral and cerebellar cortexes.     

A sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: immunostaining of cryostat sections and virus isolation

The sequential tissue distribution of virus was investigated using virus isolation and immunofluorescence tests in 1-day-old piglets inoculated with porcine circovirus 2 (PCV2) and/or porcine parvovirus (PPV). Enlarged mesenteric lymph nodes were seen in the pig inoculated with PCV2 alone and killed at 26 days post-inoculation (PI). One of the pigs inoculated with PCV2 and PPV and killed at 21 days PI had an enlarged liver. The pig killed at 26 days PI in this group had enlarged liver, kidneys and heart. Histopathological changes were seen in lymphoid tissues of the pigs inoculated with PCV2 alone and killed at 14 and 26 days PI. Similar, but more severe, lesions were observed in the pigs infected with PCV2 and PPV and killed from 10 days PI onwards. Histological lesions of nephritis, pneumonia and hepatitis were also apparent in these animals. Mild nephritis was also seen in the pigs infected with PPV alone and killed at 14 and 26 days PI. Moderate amounts of PPV antigen were detected in tissues from the pigs inoculated with PPV alone and killed at 14 days PI. Low levels of PCV antigen were detected, mainly in lymphoid tissues, in the pigs inoculated with PCV alone and killed at 14 days PI. Low to moderate amounts of PCV antigen were detected in a wider range of tissues in the pig in this group killed at 26 days PI. In the pigs inoculated with both viruses, PPV antigen was detected in tissues of pigs killed from 3 to 26 days PI with maximal amounts detected between 6 and 14 days PI. PCV2 antigen was detected in low to moderate amounts in the tissues of pigs killed at 14 days PI. Large amounts of PCV2 antigen were detected in most of the tissues from pigs in this group killed between 17 and 26 days PI. Virus isolation results for PCV2 generally correlated well with the results for immunofluorescent staining. PPV was isolated from almost all tissues from pigs inoculated with PCV2 and PPV, a much higher incidence of positive tissues than observed for immunofluorescent staining.     

Multiplex PCR for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses

A multiplex PCR (mPCR) assay was developed and subsequently evaluated for its effectiveness as a means to simultaneously detect multiple viral infections of swine. Specific primers for each of four common DNA viruses, namely, pseudorabies virus (PRV), porcine circovirus type I (PCV1), porcine circovirus type II (PCV2), and porcine parvovirus (PPV), were used for testing procedure. The assay was shown to be highly sensitive in that as little as 10(-4) ng of each of the respective amplicons (approximately equal to 10,000 molecules) was detected when a composite of all four viruses (including both field and gene-deleted permutations of PRV) was tested as a single sample. It was also effective for detecting one or more of these same viruses in various combinations in specimens including lymph nodes, lungs, spleens, and tonsils collected from clinically ill pigs, and in specimens in spleen collected from aborted fetuses. The relative efficiency (compared to performing separate assays for each virus) and apparent sensitivity of mPCR suggest its potential application for routine molecular diagnostic purposes.     

Effects of a porcine reproductive and respiratory syndrome virus infection on the development of the immune response against pseudorabies virus

The aim of this study was to investigate the effects of a porcine reproductive and respiratory syndrome virus (PRRSV) infection on the development of the immune response after pseudorabies virus (PRV) vaccination in pigs. Pigs were intranasally inoculated with the European PRRSV strain, Lelystad virus ter Huurne, and were vaccinated intramuscularly with PRV 2 weeks later (LV-PRV group). Control pigs were vaccinated with PRV only (PRV group). Eight weeks after PRV vaccination, pigs from both groups were challenged intranasally with wild-type PRV. We measured the lymphoproliferative, and the cytolytic responses to PRV of peripheral blood mononuclear cells (PBMC), isolated from blood samples. In addition, serum samples were examined for antibodies against PRV and LV. One week after PRV vaccination, PBMC proliferated abundantly to PRV in both groups. However, in the LV-PRV group the lymphoproliferative response declined after 1 week, whereas, in the PRV group, the lymphoproliferative response was high for 3 weeks and declined thereafter (P<0.05). After challenge, the lymphoproliferative response was 1 week earlier and was consistently and significantly higher in the PRV group than in the LV-PRV group. The PRV-specific killing was higher at 3 weeks after PRV vaccination and 5 weeks after PRV challenge 19+/-3 and 24+/-6%, respectively, in the PRV group, compared to 7+/-4 and 6+/-9%, respectively, in the LV-PRV group (P<0.05). However, later after vaccination and challenge the cytolytic response was identical in both groups. The antibody titre against PRV developed equally in both groups. After challenge, no PRV virus was isolated from both groups. From these results we conclude that, although PRRSV infection did cause changes in the time course of the T-lymphocyte response after PRV vaccination, PRRSV infection did not inhibit the development of vaccine-induced protection after PRV.     

Prenatal and preweaning deaths caused by pseudorabies virus and porcine parvovirus in a swine herd

Sequential outbreaks of pseudorabies virus and porcine parvovirus infections were documented at a swine farm in southern Minnesota. Data for the prevalence of mummified fetuses born and the preweaning mortality were recorded over a 3-year-period. The farm was a farrow-to-finish facility, with breeding females housed in 4 groups according to their stage of pregnancy. The herd consisted of approximately 130 breeding females in December 1981, and expanded to 220 females during the 12 months of 1982. Excluding the outbreaks, the mean preweaning mortality was 20.43% (SE 1.59) and the number of mummified fetuses per litter was 0.19 (SE 0.01). An outbreak of porcine parvovirus infection caused the preweaning mortality and number of mummified fetuses to increase to 50% and 4.10 per litter, respectively. Two outbreaks of pseudorabies 27 months apart, caused the preweaning mortality to increase to 95% and 82%, and the number of mummified fetuses to increase to 0.96 and 1.25 mummified fetuses per litter, respectively. The increase in mummification was observed 1 month after the increase in preweaning mortality caused by pseudorabies virus infections, whereas the increase in mummification and preweaning mortality was simultaneous with porcine parvovirus infections.     

The effect on reproductive performance of porcine parvovirus infection in a susceptible pig herd

An acute episode of reproductive failure occurred following natural introduction of porcine parvovirus to a susceptible herd of 48 breeding sows. Serological data gave a close estimate of the time that infection spread through the herd, and enabled a correct forecast of the reproductive failure that followed. Severe fetal mummification was seen over a three-week period. Epidemiological data is presented strongly linking in utero parvovirus infection with the mummification that occurred, and the significance of this data is discussed in connection with the present knowledge of transplacental porcine parvovirus infection.     

Characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection

The present study characterized the homologous and heterologous immune response in type-I porcine reproductive and respiratory syndrome virus (PRRSV) infection. Two experiments were conducted: in experiment 1, eight pigs were inoculated with PRRSV strain 3262 and 84 days post-inoculation (dpi) they were challenged with either strain 3262 or strain 3267 and followed for the next 14 days (98 dpi). In experiment 2, eight pigs were inoculated with strain 3267 and challenged at 84 dpi as above. Clinical course, viremia, humoral response (neutralizing and non-neutralizing antibodies, NA) and virus-specific IFN-γ responses (ELISPOT) were evaluated all throughout the study. Serum levels of IL-1, IL-6, IL-8, TNF-α and TGF-β were determined (ELISA) after the second challenge. In experiment 1 primo-inoculation with strain 3262 induced viremia of ≤ 28 days, low titres of homologous NA but strong IFN-γ responses. In contrast, strain 3267 induced longer viremias (up to 56 days), higher NA titres (≤ 6 log₂) and lower IFN-γ responses. Inoculation with 3267 produced higher serum IL-8 levels. After the re-challenge at 84 dpi, pigs in experiment 1 developed mostly a one week viremia regardless of the strain used. In experiment 2, neither the homologous nor the heterologous challenge resulted in detectable viremia although PRRSV was present in tonsils of some animals. Homologous re-inoculation with 3267 produced elevated TGF-β levels in serum for 7–14 days but this did not occur with the heterologous re-inoculation. In conclusion, inoculation with different PRRSV strains result in different virological and immunological outcomes and in different degrees of homologous and heterologous protection.     

表达猪细小病毒VP2蛋白和猪IL-18重组伪狂犬病毒的构建

为了获得表达猪细小病毒(PPV)VP2蛋白和细胞因子猪白细胞介素-18(IL-18)的重组猪伪狂犬病毒(PRV)。将PPV VP2基因和猪IL-18基因分别插入到PRV转移质粒PG中,得到重组质粒PG18-VP2。利用脂质体转染法将重组转移质粒PG18-VP2与猪PRV弱毒株DNA共转染猪睾丸(ST)细胞,以EGFP荧光标记,通过5轮蚀斑筛选纯化,成功获得表达PPV VP2蛋白和猪IL-18且带EGFP标记的重组伪狂犬病毒IL18-VP2-rPRV。用重组病毒感染ST细胞,12h后在荧光显微镜下可见明亮的绿色荧光;通过RT-PCR证实感染细胞中含有PPV VP2和猪IL-18mRNA;Western-blot试验结果显示,重组病毒能表达具有生物活性的PPV VP2蛋白和猪IL-18。重组病毒经连续20次传代后感染细胞仍能发出绿色荧光,PCR检测表明VP2及IL-18基因在重组病毒中能稳定遗传。为进一步研究表达PPV VP2蛋白和猪IL-18的重组猪伪狂犬病毒的免疫效力奠定了基础。     

猪瘟病毒猪细小病毒猪伪狂犬病病毒 PCR检测方法的建立

猪瘟、猪细小病毒感染和猪伪狂犬病是3种最为常见的、引起猪繁殖障碍的传染性疾病，在世界范围内广泛流行，给养猪业带来严重的经济损失。在兽医临床上这3种疾病有时呈混合感染，且临床表现相似，给临床诊断造成很大困难。目前对这3种疾病的确诊方法都存在着繁琐、耗时、特异性差等缺点，因而有必要建立一种特异、快速、灵敏的诊断方法用于这些疾病的临床诊断和检测。     

Pseudorabies virus, porcine parvovirus, and porcine enterovirus interactions with the zona pellucida of the porcine embryo

Porcine embryos (n = 93) were incubated on cell monolayers that had been previously inoculated with pseudorabies virus, porcine parvovirus (PPV), or each of 2 porcine enteroviruses. After 2, 24, or 48 hours of incubation, the embryos were fixed in glutaraldehyde and examined by electron microscopic procedures. It was found that pseudorabies virus adsorbed to the zona pellucida (ZP) and entered sperm tracks in the ZP. The PPV and both enteroviruses entered pores in the ZP and were associated with sperm that were at or near the outer surface of the ZP. In addition, PPV was seen enmeshed in cellular debris on the outer surface of the ZP. Evidence of a productive viral infection of the blastomeres of the embryos was not found.     

应用PCR方法对广西公猪精液的伪狂犬病、圆环病毒2型和细小病毒混合感染情况的检测

应用PCR技术,2006年对广西11个猪场的公猪精液235头份进行猪伪狂犬病(PRV)、圆环病毒2型(PCV2)和猪细小病毒(PPV)混合感染情况的检测。结果,235份样品中PRV阳性率为5.53%,PCV2阳性率为17.02%,PPV阳性率为13.62%;PRV与PCV2、PPV均存在不同的混合感染现象。结果表明,我区公猪精液PRV、PCV2和PRV带毒情况比较严重,成为当前造成母猪繁殖障碍和规模场猪群发病的主要原因,这将为今后种猪场对PRV、PCV2和PRV的控制与净化工作提供了依据。     

Reproduction of postweaning multisystemic wasting syndrome in pigs by prenatal porcine circovirus 2 infection and postnatal porcine parvovirus infection or immunostimulation

Postweaning multisystemic wasting syndrome (PMWS) was reproduced in prenatally porcine circovirus 2 (PCV2)-infected pigs by either postnatal infection with porcine parvovirus (PPV) or by immunostimulation. Twenty-four randomly selected piglets from 3 sows, which had been experimentally infected during gestation with PCV2, were randomly divided into 3 groups; group 1 (prenatal PCV2 infection, with postnatal PPV infection), group 2 (prenatal PCV2 infection, with postnatal keyhole limpet hemocyanin, emulsified in incomplete Freund's adjuvant [KLH/ICFA] injection), and group 3 (prenatal PCV2 infection only). Twenty-four randomly selected piglets from 3 uninfected sows were randomly divided into 3 groups; group 4 (no prenatal infection, with postnatal PCV2 and PPV infection), group 5 (no prenatal infection, with postnatal PCV2 infection), and group 6 (negative control pigs). Body weight in negative control pigs (group 6) was increased significantly compared with pigs in groups 1, 2, and 4 at 49, 52, 56, 59, and 63 days of age. The granulomatous inflammatory reaction and lymphoid depletion that are typical lesions in pigs with PMWS were observed in the lymph node of piglets in groups 1, 2, and 4 at 63 days of age. Pigs in group 3 had significantly fewer PCV2-positive cells than those from groups 1, 2, 4, or 5. When the prenatally PCV2-infected pigs were infected with PPV or injected with immunostimulant in the postnatal period, they developed PMWS. Thus, factors that potentiate the progression of prenatal PCV2 infection to PMWS are postnatal infection with PPV or immune stimulation.     

多重PCR／RT—PCR技术检测PRRSV、PPV、PRV和PCV-2

根椐GenBank中已发表的猪蓝耳病毒(PRRSV)、猪细小病毒(PPV)、伪狂犬病毒(PRV)和猪圆环病毒Ⅱ型(PCV-2)等4种病毒基因序列,对各病毒基因区进行同源性分析,确定PRRSV M和N、PPV VP2、PRV gD、PCV-2ORF2基因的保守区为各病毒的诊断靶序列.在建立各病毒单项PCR技术的基础上,优化多重PCR反应条件,建立了4种病毒的四重PCR技术,可同时扩增PRRSV的660 bp(北美株),PPV的313 bp,PRV的217 bp,PCV-2的447 bp的特异性片段.用82份临床病料对本研究多重PCR技术和单项PCR/RT-PCR技术进行对比验证,结果显示,两者的总符合率为93%以上.表明建立的多重PCR检测方法,具有特异、快速、准确的特点,可用于对这4种病毒的同时检测和鉴别诊断.     

Experimental infection of 3-week-old conventional colostrum-fed pigs with porcine circovirus type 2 and porcine parvovirus

This report describes an experimental infection with porcine circovirus type 2 (PCV2) in combination with porcine parvovirus (PPV) in 3-week-old conventional colostrum-fed pigs with maternal antibodies to both viruses. Two groups of four pigs each were inoculated with PCV2 and PPV. One of the groups received also a commercial inactivated vaccine against porcine pleuropneumonia to evaluate possible effects of the stimulation of the immune system of pigs on the infection. Another group of four pigs was kept as uninfected control. Clinical signs, rectal temperatures and body weights were recorded. Serum antibody titers to PCV2 and PPV were determined at weekly intervals. Pigs were killed 42 days after inoculation and tissue samples were examined for the presence of gross and microscopic lesions. Tissues were also analyzed for the presence of PCV2 and PPV DNA by PCR, and for the presence of PCV2 antigen by immunohistochemistry (IHC). All the pigs had serum antibodies to PCV2 and PPV at the beginning of the trial. None of them developed clinical symptoms or pathological lesions typical of post-weaning multisystemic wasting syndrome (PMWS), a disease associated to PCV2 infection. However, IHC and/or PCR analyses showed that clinically silent PCV2 infection developed in five of the eight inoculated pigs, regardless of the administration of the vaccine. In particular, PCV2 DNA and/or antigen were detected in most of the tissues examined in the two pigs with the lowest titer of maternal PCV2 antibodies at the beginning of the trial. PPV DNA was not detected in any of the samples examined. The five pigs with PCR and/or IHC evidence of PCV2 infection had a mean weight gain during the experiment lower than that of the inoculated PCR-negative pigs considered together and that of the control pigs. In conclusion, it would appear that passive immunity against PCV2 can play a role in preventing the development of PMWS, but is not able to prevent the establishing of clinically silent PCV2 infections. The dissemination and persistence of the virus in the tissues may depend on the level of PCV2 antibodies at the time of inoculation.