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.     

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.     

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.     

A review of porcine circovirus 2-associated syndromes and diseases

Clinical expression of porcine circovirus 2 (PCV2) infection in swine may result in several distinct syndromes and diseases including post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, porcine respiratory disease complex, granulomatous enteritis, necrotizing lymphadenitis, and possibly exudative epidermitis. Association of PCV2 with congenital tremor in piglets is still controversial. The extent of the involvement of PCV2 in swine disease other than PMWS is currently poorly understood. This review concentrates on PCV-2-associated syndromes and diseases other than PMWS.     

Sow porcine circovirus type 2 (PCV2) status effect on litter mortality in postweaning multisystemic wasting syndrome (PMWS)

Previous studies have described a "litter effect" associated with mortality in postweaning multisystemic wasting syndrome (PMWS) affected farms. The main objective of this study was to evaluate litter mortality in different PMWS affected farms and to characterize it in relation to three variables of the sow: parity, porcine circovirus type 2 (PCV2) infectious status and PCV2 antibody titres. The study was performed in seven farms that experienced PMWS in nurseries and/or fattening areas. Fifteen sows from each farm were randomly selected from the same farrowing batch. Serum samples were analyzed for antibodies to PCV2 and for genomic detection of PCV2. Four piglets from each sow (60 piglets per farm) were selected and ear-tagged at birth. Out of 420 initial piglets, 104 (25%) died. Sixty three of them (60%) were necropsied, and 40 (63%) diagnosed as PMWS based on case definition criteria. Our results show that sow PCV2 viremia was significantly related to piglet mortality since more piglets per litter died from viremic than from non-viremic sows. Additionally, a significantly greater proportion of animals died from sows that had low antibody titres against PCV2 (39% vs. 18% from sows with medium to high antibody titres). The present study, of exploratory nature, confirms previous results and further characterizes the so called "litter effect" by establishing that the sow PCV2 status had a significant effect on litter mortality in PMWS affected farms.     

Genetic characterization of porcine circovirus type 2 in Republic of Korea

Porcine circovirus type 2 (PCV2) is a major causative agent of postweaning multisystemic wasting syndrome (PMWS). Sequence and phylogenetic analyses based on the ORF2 capsid protein gene fragment showed that field isolate in Republic of Korea (ROK), PCV2 YJK 0703, was closely related with the PCV2 Fh18 isolate. PCV2 YJK 0703 was genetically distinct and not related to previously reported ROK isolates. Therefore, genotypic variation exists among prevailing PCV2 in ROK. This result suggests that several PCV2 genotypes exist in Korean pig farms.     

Genetic diversity of porcine circovirus type 2 from pigs in Republic of Korea

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS). In this study, 38 PCV2 cases obtained from different pig farms with different health conditions in Republic of Korea were sequenced and analyzed. Phylogenetic analysis showed that our cases had a greater variation and the existence of two PCV2 groups with at least four subgroups (1A, 1C, 2D, and 2E). Most cases obtained from PMWS-affected herds were in group 1, whereas cases with no clinical signs compatible with PMWS (wasted non-PMWS) were included within group 2. Moreover, four cases from the wasted non-PMWS belonged to PCV2-group 1. Therefore, our results suggest that PCV2-group 1 is more related to PMWS than group 2.     

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.     

猪圆环病毒检测与分型寡核苷酸芯片的建立及应用

本研究应用寡聚核苷酸基因杂交技术建立了一种快速可靠的检测猪圆环病毒(PCV)基因型的方法.在PCV保守序列复制酶基因内设计了2对特异性引物,在此物之间设计了2种基因型特异的核苷酸探针(22～30 mer).通过PCR扩增Cy5标记的DNA片段与固定在玻片表面的探针杂交进行PCV基因分型.该技术结合了PCR方法的高度敏感性和DNA-DNA杂交技术的选择特异性.利用该方法对58倒临床样品进行了检测鉴定.结果显示,该技术能准确鉴别PCV病毒基因型.且其灵敏度是凝胶电泳的5倍.试验结果表明寡核苷酸基因芯片技术可有效地应用于PCV临床诊断和分子流行病学调查.     

Reproduction in porcine circovirus type 2 (PCV2) seropositive gilts inseminated with PCV2b spiked semen

Background

Since 1999, field evidence of transplacental infection by porcine circovirus type 2 (PCV2) and reproductive failure has been reported in pigs. The objective of this study was to evaluate the clinical and pathological consequences of PCV2 infection in conventional PCV2-seropositive gilts by insemination with PCV2b-spiked semen.

Results

Six PCV2 seropositive gilts were inseminated with PCV2b-supplemented semen (infected) and three animals with semen and cell culture medium (controls). Only three out of the six infected animals were pregnant by ultrasonography on day 29 after insemination, while two out of the three controls were pregnant. One control gilt aborted on day 23 after insemination but not due to PVC2. Viraemia was demonstrated in four out of six infected and in one control gilt that became infected with PCV2a. Anti-PCV2 antibody titres showed dynamic variations in the infected group throughout the study. Among infected gilts, the animal with the lowest anti-PCV2 titre (1/100) at the beginning of the experiment and another that reached a similar low value during the experiment showed evident seroconversion over time and had also PCV2 positive foetuses. One placenta displayed mild focal necrosis of the chorionic epithelium positively stained by immunohistochemistry for PCV2 antigen.

Conclusions

PCV2-seropositive gilts can be infected with PCV2 after intrauterine exposure and low maternal antibody titre may increase the probability of a foetal infection.     

Detection and genetic characterization of porcine circovirus type 2 (PCV2) in pigs from Croatia

Porcine circovirus type 2 (PCV2) from the Circoviridae family has recently been associated with two serious diseases of swine, post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). During 2002, several outbreaks of clinical disease in pigs with weights ranging from 10 to 70 kg occurred on four farms in different locations in Croatia. The signs were consistent with PMWS and PDNS. Apart from progressive weight loss, pneumonia and/or diarrhoea, multifocal erythematous skin lesions and dermal necrosis were also observed. The PCR results obtained from PCV2 specific oligonucleotide primers confirmed a PCV2 infection. In addition, archive samples that were classical swine fever virus positive and derived from domestic pigs during an outbreak in 1997 were included in this study and one out of the three isolates was found to be positive for PCV2. For a better epizootiological understanding, genetic typing of representative isolates was carried out and compared with available isolates reported in the GenBank databases.     

Characterization of porcine circovirus type 2 (PCV2) infection in swine lymphocytes using mitogen-stimulated peripheral blood lymphocytes from healthy PCV2-carrier pigs

Information regarding the susceptibility of swine lymphocytes to PCV2 is rather limited. To further explore and characterize the PCV2 infection in swine lymphocytes, an in vitro model using concanavalin A (Con A)-stimulated peripheral blood lymphocytes (PBLs) obtained from clinically healthy PCV2-carrier pigs was introduced. It was found that the PCV2 antigen-containing rate was below 2% in PBLs from healthy PCV2-free pigs following treated simultaneously with Con A and PCV2. However, significantly higher PCV2 antigen- and nucleic acid-containing rates could be seen in Con A-stimulated PBLs from clinically healthy PCV2-carrier pigs. Prior to Con A treatment, both of the PCV2 antigen- and nucleic acid-containing rates in PBLs from healthy PCV2-carrier pigs were less than 1%; however, they reached 22.1+/-5.7% by flow cytometry and 27.1+/-6.5% by in situ hybridization, respectively, at 4-day post-incubation with Con A. Phenotyping of PCV2 antigen-containing cells revealed that PCV2-positive cells could be detected in both T and B lymphocyte populations within which IgM-positive B lymphocytes appeared to have a relatively higher positive rate. The Con A-stimulated PBLs also displayed a significantly higher viral load by the measurement of either PCV2 DNA copy number or viral titer when compared with the non-treated PBLs from healthy PCV2-carrier pigs. The results indicate that PBLs, especially IgM-bearing B lymphocytes, are indeed susceptible to PCV2 infection and PCV2 is capable of replicating in dividing lymphocytes. This activation-induced replication may explain in part the pathogenesis of lymphoid depletion in PMWS-affected pigs.     

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。     

Assessing PCV2 antibodies in field pigs vaccinated with different porcine circovirus 2 vaccines using two commercial ELISA systems

Porcine circovirus type 2 (PCV2) is the primary causative agent for post-weaning, multisystemic, wasting syndrome. Consequently, serologic detection of and vaccination against PCV2 are important for the swine industry. Among several serological tests, the enzyme-linked immunosorbent assay (ELISA) is commonly used to measure anti-PCV2 antibody levels. In the present study, we used two commercial ELISA systems to comparatively evaluate anti-PCV2 antibodies in field pigs treated with three different PCV2 vaccines. Among a total of 517 serum samples, the results of the two ELISAs were fully concordant for 365 positive and 42 negative samples, indicating 78.7% agreement. In addition, the Pearson coefficient (0.636) indicated a moderate correlation between data from the two ELISAs. Results from the farms with pigs vaccinated with the three different PCV2 vaccines demonstrated that most of the vaccinated animals underwent seroconversion. However, the increase and duration of antibody titers varied depending on the vaccine, the presence of maternal antibodies, and the vaccination program. PCV2 serologic status and anti-PCV2 antibody levels of herds from this study could be utilized to determine the best timing for vaccination and assessing vaccination compliance.     

ORF9基因缺失突变PCV2的构建与部分生物学特性

设计1对针对猪圆环病毒2型（PCV2）全基因组的特异性引物,从疑似断乳仔猪多系统衰竭综合征（PMWS）的病料中经PCR直接扩增出PCV2全基因组,再与载体pMD18-T Simple连接后构建重组质粒pMD18-T Simple-PCV2（命名为P-S-PCV2）。用ORF9特异的限制性内切酶Bpu10Ⅰ对P-S-PCV2进行酶切、补平连接反应,构建了ORF9基因缺失突变的重组质粒（命名为P-S-PCV2-J）。用SacⅡ对基因缺失突变的重组质粒进行酶切,获得的线性化基因突变PCV2基因组在体外进行自身环化,形成了相应缺失基因DNA（命名为PCV2-J）。用PCV2-J缺失突变株进行细胞转染、动物致病性和免疫原性、T淋巴细胞亚群的动态变化等部分生物学特性的研究。结果显示：PCV2-J转染IBRS-2细胞后,电镜观察可见病毒颗粒,PCR-RFLP检测有突变株生长;PCV2-J接种仔猪后无临床典型大体病变,PCR-RFLP检测淋巴结中有PCV2-J突变病毒的感染;PCV2-J免疫仔猪后的抗体水平在第2周开始上升,与对照组相比差异显著,CD3＋下降,与对照组无差异,CD4＋下降,第1周与对照组差异显著,CD8＋与对照组差异不显著。结果表明,ORF9基因缺失突变的PCV2仍具有复制感染能力,但免疫原性减弱。     

Porcine circovirus 2 replication in colostrum-deprived piglets following experimental infection and immune stimulation using a modified live vaccine against porcine respiratory and reproductive syndrome virus

Porcine circovirus type 2 (PCV2) infection is now recognized as the major factor in the development of post-weaning multisystemic wasting syndrome (PMWS). Although Koch's postulates have been fulfilled for PCV2 and PMWS, the severe clinical expression of the disease observed in field cases has been difficult to reproduce experimentally. Some studies have demonstrated that immune stimulation associated with the use of some commercially available swine vaccines may trigger progression of PCV2 infection to disease and lesions characteristic of PMWS. Here we describe the effects on PCV2 infection in an experimental model following the use of a commercially available modified live vaccine to porcine respiratory and reproductive syndrome virus (PRRSV). Although none of the piglets infected with PCV2 developed clinical PMWS, the severity of microscopical lesions and the PCV2 antigen load associated with these lesions were higher in the PRRSV-vaccinated piglets compared with those detected in the PCV2 only infected animals.     

Immunopathological effects of porcine circovirus type 2 (PCV2) on swine alveolar macrophages by in vitro inoculation

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), a multifactorial disease, in pigs. Monocyte/macrophage lineage cells, including alveolar macrophages (AMs), are the major target cells for PCV2. Swine AMs are essential for the pulmonary defense system against various pathogens. Concurrent infection of lung with opportunistic pathogens in pigs suffered from PMWS is speculated as a feature of immunosuppression. The present study was conducted to characterize the effects of PCV2 inoculation on swine AMs in the in vitro system. The parameters selected for evaluation included PCV2 antigen- and nucleic acid-containing rate, viability, TUNEL-positive rate, phagocytosis, microbicidal capability, and capacity for production of reactive oxygen species (superoxide anion, O₂⁻, and hydrogen peroxide, H₂O₂), cytokines, and chemokines. High intracytoplasmic PCV2 antigen- and nucleic acid-containing rate, absence of intranuclear signals for PCV2 antigen and nucleic acid, and lack of noticeable cell death were seen in PCV2-inoculated AMs. The PCV2-inoculated AMs displayed a transient as well as persistent reduction in the up-take and destruction of Candida albicans, respectively, accompanied by decrease in the production of O₂⁻ and H₂O₂. In PCV2-inoculated AMs, the levels of tumor necrosis- (TNF-) and interleukin-8 (IL-8) were significantly increased; the mRNA expression levels of alveolar macrophage-derived neutrophil chemotactic factors-II (AMCF-II), granulocyte colony-stimulating factor (G-CSF), monocyte chemotactic protein-1 (MCP-1), and IL-8 were strongly up-regulated. The reduced phagocytosis and microbicidal capability in conjunction with decreased production of reactive oxygen species in PCV2-inoculated AMs suggest that PCV2-containing AMs may favor the survival and spread of PCV2. It is speculated that the functional alterations observed in PCV2-containing AMs may be potentially harmful to the lung tissue and local pulmonary defense system, especially in those PCV2-infected pigs conditioned by various PMWS development-dependent co-factors.     

Porcine circovirus type 2 (PCV2) vaccination reduces PCV2 in a PCV2 and Salmonella enterica serovar Choleraesuis coinfection model

We previously reported that prior porcine circovirus type 2 (PCV2) infection potentiates the severity of clinical signs, lung lesions, and fecal shedding and tissue dissemination of Salmonella enterica serovar Choleraesuis in infected pigs. Here, we evaluated whether PCV2 vaccination is effective in reducing fecal shedding and tissue dissemination of S. Choleraesuis and improving clinical signs associated with PCV2 and S. Choleraesuis infection in 15 Cesarean-derived, colostrum-deprived pigs randomly assigned to 3 groups (n = 5/group). The vaccinated and co-infected (VAC-COINF) group received 2 ml of a commercial PCV2 vaccine at age 3 weeks. The VAC-COINF and co-infected (COINF) groups were inoculated intranasally with PCV2 and S. Choleraesuis at 5 and 7 weeks of age, respectively. The CONTROL group pigs received a similar volume of PBS for sham-vaccination and sham-inoculation. PCV2 vaccination clearly reduced PCV2 DNA load in the serum and postmortem tissue samples and decreased PCV2 antigen levels in tissue samples of the VAC-COINF group. After S. Choleraesuis infection, the incidence of several clinical signs increased in the VAC-COINF group compared to that in the COINF group. The microscopic lung lesions and weight gain, fecal shedding and tissue dissemination of S. Choleraesuis except in the spleen were not significantly different in the VAC-COINF and COINF groups. Thus, PCV2 vaccination reduced PCV2 in the S. Choleraesuis and PCV2 coinfection model and the effects on S. Choleraesuis were minimal.     

猪圆环病毒2型细胞培养适应毒株的培育和鉴定

从临床表现为仔猪断奶后多系统衰竭综合征（PMWS）淋巴组织病料，经聚合酶链式反应（PCR）证实为猪圆环病毒2型（PCV2）感染，采用无污染的猪肾细胞系（PK15）分离培养，并连续传代培育成一株细胞培养适应毒，命名为PCV2／LG株。分离毒株经细胞培养，于第25代后毒价显著升高，于第35代毒价可达10^5.6TCID 50/mL。采用免疫过氧化物酶单层细胞染色法（IPMA）、免疫电镜技术、分子克隆及核酸序列分析等鉴定表明，分离株感染细胞后病毒抗原主要分布在细胞核及细胞质中；病毒感染的阳性细胞呈散在分布，阳性细胞数可达50％以上；免疫电镜观察到与PCV2特异抗体结合形成的病毒免疫复合物呈实心小颗粒样粒子团，病毒粒子直径约为17nm；病毒抗原基因组由1768个核苷酸组成，与GenBank登录的8个PCV2基因组序列同源性达96．2％以上。用2mL的病毒细胞培养物（10^5.6TCID 50/mL）接种30日龄PCV2抗体阴性仔猪3头，可引起典型PMWS临床症状。本研究为进一步开展该病毒的致病性、疫苗免疫、诊断及分子生物学等研究奠定了基础。