猪繁殖-呼吸综合征活疫苗对仔猪的安全性试验

本试验用猪繁殖-呼吸综合征（PRRS）活疫苗和国内分离的PRRS强毒CH—1a株接种PRRS阴性的断奶仔猪，分别在接种后的3、7、14d各剖杀1头，取各脏器分别做冰冻切片和病理切片观察。用间接免疫荧光法检测各脏器PRRS病毒的分布。结果表明，PRRS活疫苗在免疫初期，抗原主要分布在脾脏、淋巴结，其次是肾脏和肺脏，少见于肝脏和心脏，第14d时在脾、淋巴结和肾脏有一定量的抗原，而肺脏相比则数量很少，肝脏和心脏未检到PRRS病毒抗原的存在，表明接种PRRS活疫苗随着时间的推移抗原分布呈下降趋势。而强毒抗原分布以脾脏最多，依次是肾脏、肺脏、淋巴结、肝脏、心脏，接种后第14d仍能在各脏器检到PRRS病毒抗原。病理组织学检测结果表明，活疫苗产生以下颌淋巴结、脾脏增生为特征的免疫应答，组织损伤轻微，对肺的病变较少，且仔猪生长良好。强毒则引起以大面积的肺泡隔增宽为特点的间质性肺炎和微循环障碍的病理变化，淋巴小结、脾脏滤泡发生崩解与周围界限不清，个别淋巴细胞核浓缩，组织损伤严重。本试验表明弱毒疫苗对仔猪是安全的。     

Efficacy of single dose of an inactivated porcine circovirus type 2 (PCV2) whole-virus vaccine with oil adjuvant in piglets

Background

Post-weaning multisystemic wasting syndrome (PMWS) associated with PCV2 is one of the most costly diseases currently faced by the swine industry. The development of effective vaccines against PCV2 infection has been accepted as an important strategy in the prophylaxis of PMWS.

Methods

In the present study, a PK-15 cell-adapted formalin-inactivated prototype vaccine candidate was prepared using a strain of PCV2 from China. Inactivation of the virus was accomplished using a standard formalin inactivation protocol. The protective properties of the inactivated PCV2 vaccine were evaluated in piglets. Ten 28-day-old pigs were randomly assigned to two groups, each with five. Group 1 was vaccinated intramuscularly with the inactivated virus preparation; Group 2 received sterile PBS as a placebo. By 28 days post-vaccination (DPV), Groups 1 and 2 were challenged intranasally and intramuscularly with 5 × 10⁷ TCID₅₀ of a virulent PCV2 isolate.

Results

The vaccinated pigs seroconverted to PCV2 and had high levels of serum antibodies to PCV2 at 28 days after vaccination, whereas the control pigs remained seronegative. No significant signs of clinical disease were recorded following the challenge with PCV2, but moderate amounts of PCV2 antigen were detected in most lymphoid organs of the control pigs. PCV2 was detected in two out of the five vaccinated pigs. Furthermore, pathological lesions and viremia were milder in the vaccinated group.

Conclusions

The obtained results indicate that the inactivated PCV2 virus vaccine with an oil adjuvant induce an immunological response in pigs that appears to provide protection from infection with PCV2. The vaccine, therefore, may have the potential to serve as a vaccine aimed to protect pigs from developing PMWS.     

猪生殖与呼吸综合征病毒和猪圆环病毒2型共感染对猪外周血天然免疫细胞含量的影响

将猪生殖与呼吸综合征病毒（PRRSV）和猪圆环病毒2型（PCV2）单感染和共感染的6周龄仔猪，于感染前和感染后第3、7和10d，采用血常规法、SWC3a单色流式细胞术以及CD3／CD4／CD8三色流式细胞术分析了感染猪外周血中白细胞、粒细胞、单核细胞、NK细胞、弦T细胞的含量。结果，感染后第3d和第7d，PRRSV感染组、PCV2感染组以及PRRSV＋PCV2共感染组猪的白细胞、单核细胞、粒细胞、NK细胞、弦T细胞总数显著下降，并且共感染组比单感染组下降更加严重。结果表明，PRRSV＋PCV2共感染对仔猪外周血天然免疫细胞的影响具有协同效应，意味著在感染旱期可导致更加严重的先天免疫抑制。     

Prevalence of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in piglets after weaning on a commercial pig farm in Japan

To investigate the transition in concentration of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) and antibody for these viruses in serum, serum samples were collected from 29 pigs on weaning day and at 7, 14, 21, 28, 53, 84, and 120 days after weaning. The concentration of circulated PRRSV and PCV2 in serum was measured by real-time RT-PCR and real-time PCR, respectively. The specific antibody for PRRSV and PCV2 was measured using ELISA. PRRSV was not detected on 0 days post-weaning (dpw). The specific antibody for PRRSV began to increase as the concentration of PRRSV in serum increased, and the level of PRRSV then tended to decrease. PCV2 was detected in 12 of 28 pigs on 0 dpw. The concentration of PCV2 and the specific antibody for PCV2 showed a similar tendency to those of PRRSV. The correlation analysis suggests that a decline in the daily weight gain coincided with an increase in the PRRSV concentration. Pigs with a higher antibody titer against PRRSV or PCV2 on 0 dpw showed the lower level of PRRSV or PCV2, respectively.     

猪GM-CSF基因对猪圆环病毒2型DNA疫苗诱导仔猪免疫反应的调节作用

粒细胞-巨噬细胞集落刺激因子（GM-CSF）能够诱导树突状细胞的成熟和存活，并提高其抗原递呈功能，是非常有潜力的分子佐剂之一。为了探讨猪GM-CSF对猪圆环病毒2型（PCV2）DNA疫苗诱导免疫反应的调节作用，本研究将表达猪GM-CSF和PCV2衣壳蛋白的重组质粒混合物（pcDNA3．1-gm-csf和pcDNA3．1-orf2）及在同一载体上表达PCV2衣壳蛋白与猪GM-CSF的双表达重组质粒（pIRES-orf2／gm-csf），分别接种于7日龄健康仔猪，共肌肉注射3次，每次间隔2周。结果表明猪GM-CSF基因能够明显增强PCV2DNA疫苗诱导的特异性体液免疫反应，提高外周血淋巴细胞（PBLC）对ConA刺激的增殖活性，增强NK细胞的杀伤功能，影响细胞因子IL-12、IFN-γ、IL-4和IL-10的表达水平。但GM-CSF基因单独表达和与PCV2衣壳蛋白基因在同一载体上表达，对上述4个细胞因子表达的影响有一定差异。     

Experimental reproduction of porcine respiratory disease complex in pigs inoculated porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae and followed by inoculation with porcine circovirus type 2

The aim of this study was to reproduce severe pneumonic lesions, similar to those during naturally-occurring porcine respiratory disease complex, in pigs dually inoculated with porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae at 6 weeks of age, followed by inoculation with porcine circovirus type 2 at two weeks after. Time and sequence of infection with three pathogens mirror Asian field conditions. Microscopically, interstitial pneumonia and peribronchiolar lymphoid hyperplasia are considered the most characteristic lung lesions in infected pigs. The results of the present study demonstrate that inoculation of pigs with these three pathogens can lead to severe interstitial pneumonia with peribronchial or peribronchiolar lymphoid hyperplasia and fibrosis.     

Changes in peripheral blood leukocyte subpopulations in piglets co-infected experimentally with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are pathogens, which can significantly affect the swine industry worldwide. Field surveys suggest that simultaneous PRRSV and PCV2 infection is common in pigs. The objective of this study was to measure the changes in peripheral blood leukocyte subpopulations in piglets co-infected experimentally with PRRSV and PCV2, in order to analyze the synergistic influence of co-infection on the immune system. Changes in peripheral blood leukocyte subpopulations were systematically measured by flow cytometry (FCM). The levels of antibodies to PRRSV and PCV2 were detected by indirect Enzyme-Linked ImmunoSorbent Assay (ELISA) and the indirect fluorescent antibody test (IFA), respectively. Serum viral loads were measured using real-time PCR. The results showed that piglets co-infected with PRRSV and PCV2 exhibited slower generation and lower levels of antibodies to PRRSV and PCV2, and increased amounts and a prolonged presence of both PRRSV and PCV2 in serum, in comparison to the piglets infected with either virus alone. The major finding in our study was that the total and differential leukocyte counts, including white blood cells (WBCs), monocytes, granulocytes and lymphocytes (T, B and NK cells, as well as T-cell subpopulations), dramatically decreased early during co-infection with PRRSV and PCV2 for about two weeks, in contrast with animals singly infected with either PRRSV or PCV2. These results suggest that PRRSV and PCV2 co-infection results in a synergistic decrease in immune cells in the peripheral blood of piglets. These data contribute to the understanding of the immunosuppressive effects resulting from PRRSV and PCV2 co-infection in pigs.     

Comparative analyses of humoral and cell-mediated immune responses upon vaccination with different commercially available single-dose porcine circovirus type 2 vaccines

The objective of this study was to compare the induction of humoral and cell-mediated immune responses by four commercially available single-dose porcine circovirus type 2 (PCV-2) vaccines. A total of 50 3-week-old piglets were assigned to five groups (10 pigs per group). Four commercial PCV-2 vaccines were administered according to the manufacturer's instructions and the piglets were observed for 154 days post vaccination (dpv). Inactivated chimeric PCV-1-2 vaccines induced higher levels of PCV-2-specific neutralizing antibodies (NA) and interferon-γ-secreting cells (IFN-γ-SC) in pigs than did the other three commercial PCV-2 vaccines. The proportions of CD4⁺ cells were significantly higher in animals vaccinated with inactivated chimeric PCV-1-2 and PCV-2 vaccines than in animals vaccinated with the two subunit vaccines. To our knowledge, this is the first comparison of humoral and cell-mediated immunity induced by four commercial single-dose PCV-2 vaccines under the same conditions. The results of this study demonstrated quantitative differences in the induction of humoral and cell-mediated immunity following vaccination.     

猪圆环病毒和猪繁殖与呼吸综合征病毒混合感染对仔猪致病性的评估

本研究通过猪圆环病毒2型(Porcine circovirus type 2,PCV2)和猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome virus,PRRSV)强毒共感染3周龄健康仔猪来评价其致病性。试验动物随机分为3组,空白对照组(n=3头),PRRSV单独感染组(n=3头),PCV2和PRRSV共感染组(n=6头),从而比较相互之间的差异。通过临床症状、病理学变化、病原学和血清学检查,对二者混合感染仔猪的致病性进行了研究。结果表明PCV2和PRRSV共同感染能引起仔猪断奶后多系统消耗性综合征,表现为淋巴组织肿大、出血,肉芽肿性炎症,坏死性肝炎,仔猪消瘦、生长缓慢等特征性病变;混合感染能加重PRRSV对仔猪引起的间质性肺炎的严重程度。混合感染可以出现支气管肺炎和明显的肝病变,淋巴结多呈界限明显的块状出血等典型病变。     

猪圆环病毒2型感染对伪狂犬疫苗免疫应答的影响

为明确PCV2感染对伪狂犬(PR)疫苗免疫应答的影响,本研究采用阻断ELISA方法对单独接种猪PR疫苗组(A组)及PCV2人工感染3周后接种猪PR疫苗组(PA组)不同时相血清中的猪PR病毒gB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。结果表明,在PCV2感染后2周至5周间,A组白细胞含量均高于PA组,随后PA组白细胞恢复至与A组略高的正常水平;在整个实验中,除接种猪PR疫苗后1周(WPI)和9周(WPI)外的所有时相PA组的淋巴细胞含量均略高于A组;PCV2感染后可使记忆/激活Th细胞数量略有升高,幼稚型Th细胞含量的下降;PCV2感染后2周～7周PA组Tc细胞均高于A组,在9WPIPA组Tc细胞数量显著下降(p0.05);除9WPI外,A组的S/N值均低于PA组。结果表明,PCV2感染看降低机体产生针对PRVgB特异性抗体水平,而且在一定程度上降低了幼稚型Th细胞及Tc细胞含量。     

Immunogenicity of Empty Capsids of Porcine Circovius Type 2 Produced in Insect Cells

Porcine circovirus type 2 (PCV2), a single-stranded DNA virus, is associated with postweaning multisystemic wasting syndrome (PMWS). ORF2 protein (capsid) of PCV2 was recently demonstrated to be a major immunogenable to induce protection in pigs with a prime–boost protocol. In this study, the ORF2 gene of PCV2 was expressed in insect cells. The product self-assembled into particles that were structurally and antigenically indistinguishable from regular PCV2 capsids. To evaluated the immunogenicity of these virus-like particles, PCV2-free piglets were vaccinated with the crude lysate from recombinant baculovirus (Ac.ORF2)-infected insect cells, at doses of 0.1 ml (10⁶ cells), 0.5 ml (5 × 10⁶ cells) or 1.0 ml (10⁷ cells). The immune response was monitored by an indirect enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and lymphocyte proliferation assay. The ELISA results indicated that primary immune response was elicited with 0.5 ml or 1.0 ml of crude lysate from Ac.ORF2. After boost immunization, relatively higher levels of PCV2 antibody were elicited in 0.5-ml or 1.0-ml vaccinated groups, compared to the 0.1-ml group. In addition, higher PCV2 specific lymphocyte proliferation response was developed in piglets vaccinated with 0.5 ml or 1.0 ml of crude lysate, especially in those vaccinated with with 1.0 ml of crude lysate. Thus, the expressed ORF2 protein has significant potential as a subunit vaccine against PCV2 infection.     

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.     

猪瘟、猪圆环病毒病和猪繁殖与呼吸综合征寡核苷酸芯片诊断方法的建立与初步应用

猪繁殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（CSFV）和猪圆环病毒Ⅱ型（PCV-2）是引发猪繁殖障碍的主要病原,建立其基因芯片快速检测体系,对开发猪繁殖障碍快速检测试剂盒具有重要意义。根据GenBank中已发表的PRRSV、CSFV和PCV-2的病毒基因组序列,设计合成特异性引物和特异性较强的60mer的寡核苷酸探针,并将探针按所设计阵列固定于表面经氨基化修饰的玻片上,制备出寡核苷酸芯片。通过引物标记及特异性验证,建立了带标记引物的多重PCR检测体系,从而产生大量可与寡核苷酸探针特异性互补的带标记的DNA片段。将标有荧光染料的扩增产物与芯片上寡核苷酸探针杂交,扫描、分析芯片上荧光信号。结果表明,芯片上各样本对应探针位点呈现阳性荧光信号,而阴性对照和空白对照则基本不能检测到荧光信号。分别用基因芯片检测方法和PCR/RT-PCR检测60份临床病料,两者符合率高达92%,表明该检测技术能够用于临床病料的检测及快速诊断PRRSV、CSFV和PCV-2。     

An attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) protects piglets against HP-PRRS

Porcine infections with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) cause significant morbidity and mortality and currently there are no effective vaccines for disease prevention. An attenuated strain, HuN4-F112, was obtained by passaging the HP-PRRSV HuN4 on Marc-145 cells (112th-passage). PRRSV-free pigs were inoculated intramuscularly with HuN4-F112 (10^2.0, 10^3.0, 10^4.0, 10^5.0 and 10^6.0 TCID₅₀ for groups 1–5, respectively). The groups 3–5 could resist the lethal challenge and did not show any obvious changes in body temperature nor clinical signs throughout the experiment, the pathological lesions were milder and the gained weight at a greater rate (P < 0.05), compared to group 1 and control. Sequence analysis of the HuN4 passages showed a conserved epitope in GP5 protein was mutated (¹⁹⁶QWGRL/P²⁰⁰ → ¹⁹⁶RWGRL/P²⁰⁰), as a result the monoclonal antibody could not recognize the HuN4-F112 any more. These results suggested that the HuN4-F112 could protect piglets from lethal challenge and might be a candidate vaccine against the HP-PRRSV.     

多重PCR同时检测猪圆环病毒2型,猪细小病毒,猪繁殖与呼吸综合征病毒和猪瘟病毒

本试验建立一种可同时检测猪圆环病毒2型(PCV-2),猪细小病毒(PPV),猪繁殖与呼吸综合征病毒(PRRSV),猪瘟病毒(CSFV)4种病毒的多重PCR方法.对于每一种特定的病毒,用4对寡核苷酸引物均能特异扩增其目的片段.以含有病毒目的片段的质粒为模板,测定了多重PCR的检测灵敏度,PRRSV和CSFV检测最低限是48 pg,而PPV和PCV-2为0.48 pg.利用建立的多重PCR方法对具有产自有繁殖障碍母猪的仔猪或具有呼吸障碍症状的76个仔猪样本进行检测.检出了4种病毒的存在,其中26个样本(34.2%)同时感染了2种以上病毒.结果表明多重PCR方法检测猪混合感染的病毒,是一种快速、灵敏、低成本、高效率的病原学诊断工具.     

Immunisation of pigs with a major envelope protein sub-unit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) results in enhanced clinical disease following experimental challenge

Disease exacerbation was observed in pigs challenged with virulent porcine reproductive and respiratory syndrome virus (PRRSV) following immunisation with a recombinant GP5 sub-unit PRRSV vaccine (rGP5) produced in E. coli. Eighteen animals were divided into three experimental groups: group A were immunised twice IM with rGP5, 21 days apart; group B acted as positive controls (challenged but not immunised); and group C were negative controls. Pigs in groups A and B were challenged 21 days after the second immunisation of the group A animals. Following challenge, three pigs given rGP5 exhibited more severe clinical signs than the positive controls, including respiratory distress and progressive weight-loss. Although not statistically significant, the more severe disease exhibited by group A animals may suggest previous immunisation as a contributory factor. The mechanisms of these findings remain unclear and no association could be established between the severity of disease, non-neutralising antibody concentrations and tissue viral loads.     

Double in situ hybridization for simultaneous detection and differentiation of porcine circovirus 1 and 2 in pigs with postweaning multisystemic wasting syndrome

Double in situ hybridization using a digoxigenin-labelled porcine circovirus 1 (PCV1) and biotinylated PCV2 probe, was developed for the simultaneous detection and differentiation of PCV1 and PCV2 in formalin-fixed, paraffin-embedded tissues from pigs with postweaning multisystemic wasting syndrome. The combination of an alkaline phosphatase conjugated antidigoxigenin system with alkaline phosphatase conjugated streptavidin-biotin system allowed identification of PCV1 and/or PCV2. No evidence of cross-reaction was observed. Positive cells exhibited a red or dark brown reaction product for PCV1 and PCV2, respectively. Both PCV DNAs were observed mainly in the cytoplasm but occasionally in the nucleus. Co-localization of hybridization signal for both PCV1 and PCV2 was present in macrophages and multinucleated giant cells of the lymph node and spleen. This double-labelling technique for the differentiation between PCV1 and PCV2 is suitable for pathogenesis studies and diagnostic applications.     

Distribution of antibodies against porcine circovirus type-2 (PCV2) in single site and multi-site farrow-to-finish farms in Brazil

A comparative serologic study was performed in seven single site (SS) farrow-to-finish farms and four multi-site (MS) farrow-to-finish farms, with or without post-weaning multisystemic wasting syndrome (PMWS). In each farm, 30 blood samples were collected for each category of the production cycle: sows, farrowing crate, nursery, grower pigs, and finishing pigs. Sera were evaluated for the presence of antibodies to porcine circovirus type-2 (PCV2) via immunoperoxidase monolayer assay. Serologic profiles for PCV2 were different between SS and MS farms. Seroconversion following the decline in maternal antibodies occurred at a later stage on SS farms (grower pigs) than MS farms (nursery pigs). MS farms tended to have lower antibody titers than SS farms in the categories of sow, piglet, and nursery, while higher antibody titers were found in grower pigs. Characterization of serologic profiles for different farms may provide important information for the adoption of vaccination programs.     

Porcine circovirus type 2 decreases the infection and replication of attenuated classical swine fever virus in porcine alveolar macrophages

Recently, it has been noted that porcine circovirus type 2 (PCV2) infection adversely affects the protective efficacy of Lapinized Philippines Coronel (LPC) vaccine, an attenuated strain of classical swine fever virus (CSFV), in pigs. In order to investigate the possible mechanisms of the PCV2-derived interference, an in vitro model was established to study the interaction of LPC virus (LPCV) and PCV2 in porcine alveolar macrophages (AMs). The results showed that PCV2 reduced the LPCV infection in AMs and the levels of PCV2-derived interference were dose-dependent. The PCV2-derived interference also reduced the replication level of LPCV in AMs. The full-length PCV2 DNA and its fragment DNA C9 CpG-ODN were involved in the reduction of LPCV infection in AMs, whereas UV-inactivated PCV2 was not. In addition, a moderate negative correlation between the LPCV antigen-containing rate and IFN-γ production was observed, and had a dose-dependent trend with the level of PCV2-inoculation. The results of the present study may partially explain how PCV2 infection interferes with the efficacy of LPC vaccine.     

PCR detection of porcine circovirus type 2 (PCV2) DNA in blood, tonsillar and faecal swabs from experimentally infected pigs

PCV2 infection is now recognized as the major factor in the development of post-weaning multisystemic wasting syndrome (PMWS). In this study we evaluated the use of PCR to detect the presence of PCV2 DNA in blood, faecal and tonsillar swabs collected from 12 pigs experimentally infected with PCV2 and sampled at selected time points post-infection. The PCR results were evaluated together with the presence of PMWS typical histopathological lesions and the presence of PCV2 antigen. PCV2 DNA was present in the blood of all 12 infected pigs at the end of the experiment and faecal and tonsillar swabs of 11 of the 12 pigs. The rate of PCR-positive serum and plasma samples was significantly higher in four pigs that showed virological and pathological evidence of PMWS, than in infected pigs without evidence of disease. In conclusion this study confirms that PCR cannot substitute for the traditional methods used for diagnosis of PMWS, however, PCR amplification of PCV2 DNA from serum or plasma could be a useful tool to support an early diagnosis of PMWS in live animals.