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.     

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.     

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.     

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.     

Genetic typing of porcine circovirus type 2 (PCV-2) isolates from Slovakia

Of 120 clinical specimens obtained from pigs bred on 28 PMWS-affected farms in Slovakia, porcine circovirus type 2 (PCV-2) was detected by single PCR in 77 samples. A short 224 bp fragment of ORF2 was used for preliminary grouping of isolates by phylogenetic analysis. Nucleotide sequences of the entire ORF2 region provided more precise genetic typing and segregation of preselected isolates (n = 10) into two known genotypes, PCV-2a (n = 1) and PCV-2b (n = 9). Complete genome sequencing of three selected isolates allowed their definitive grouping into genotype PCV-2b, cluster 1A or genotype PCV-2a, cluster 2D. No correlation between the mutations and the geographic origin of isolates was observed. Results confirmed that many PCV-2 isolates are genetically very stable since similar viruses circulate in Central and Western Europe.     

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。     

First report of porcine circovirus type 2 infections in Cuba

To obtain information about the porcine circovirus type 2 (PCV2) infection status of pigs in Cuba and the probable association of PCV2 with other porcine viruses, tissue samples collected from ill pigs were evaluated using polymerase chain reaction (PCR). The PCR analysis showed that 67.7% of the samples (23/34) from seven swine herds of six different geographic regions were detected to be positive for PCV2. Ten of the 23 PCV2 positive samples (43.5%) shown a concurrent infection with porcine parvovirus (PPV) and 17 of 23 PCV2 positive samples (73.9%) exhibited a concomitant infection with classical swine fever virus (CSFV). This study is the first report of PCV2 infecting pigs with different clinical conditions in Cuban swine herds and provides evidence of PCV2 co-infection with PPV and CSFV in the field.     

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.     

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.     

猪圆环病毒2型研究进展

猪圆环病毒2型（PCV2）已成为家猪和野猪常见的一种病毒病,由其引起的疾病称为猪圆环病毒相关疾病（PCVAD）,自猪圆环病毒相关疾病（PCVAD）暴发以来,已成为近年来阻碍养猪业发展的重要传染病。为了进一步了解该病,以便更好地预防和控制该病,本文从病原学、流行病学、致病机理、临床症状、防控措施等方面进行了综述。     

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。     

猪圆环病毒2型灭活疫苗免疫效果试验

本试验将10窝同期出生的共105头仔猪,随机分为试验组和对照组,试验组于14日龄、45日龄分别进行猪圆环病毒2型灭活疫苗免疫,对照组不免疫。通过临床跟踪观测和定期采血进行猪圆环病毒2型ELISA抗体和PCR病毒核酸检测。结果表明,临床免疫猪群成活率及生长指标明显好于不免疫猪群;试验组及对照组病毒核酸阳性检出率和抗体阳性率差异不显著,且可同时检出圆环病毒核酸及抗体。     

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.     

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.     

Proinflammatory cytokine expression in the lung of pigs with porcine circovirus type 2-associated respiratory disease

Porcine circovirus type 2 (PCV2) causes a significant health problem for the swine industry worldwide. In this study, we investigated the cytokine expression profiles (IFN-γ, IL-1α, IL-8, and IL-10) in the lungs of pigs with PCV2-associated respiratory disease. The mRNA expressions of IL-1α and IL-8 were significantly up-regulated in pigs with PCV2-associated respiratory disease, while IL-10 expression was not detected. These results suggest that the increased expressions of proinflammatory cytokines in the lungs may play an important role in the immunopathologic response in pigs with PCV2-associated respiratory disease.     

Efficacy of bivalent vaccines of porcine circovirus type 2 and Mycoplasma hyopneumoniae in specific pathogen-free pigs challenged with porcine circovirus type 2d

BackgroundPorcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (MHP) are economically significant pathogens in the pig industry. The use of combined vaccines against PCV2 and MHP is one of the most effective ways of protecting pigs from both diseases, and it has become a part of general management.ObjectivesThis study evaluated the efficacy of two new bivalent vaccines of PCV2 and MHP (Myco-X and Myco-XD) in SPF pigs. Myco-X and Myco-XD are a combined vaccine of MHP with PCV2b and PCV2d, respectively.MethodsSixteen pigs were divided into four groups: Myco-X-vaccinated challenged, Myco-XD-vaccinated challenged, unvaccinated challenged, and unvaccinated unchallenged. Two milliliters of Myco-X were administered intramuscularly, and 0.5 mL of Myco-XD was injected intradermally at 3 wk of age. The pigs were challenged with virulent PCV2d via the intramuscular and intranasal route 4 wk post-vaccination.ResultsAll vaccinated pigs showed effective reduction of the clinical signs, the PCV2d load in the blood and nasal swab samples, as well as lung and lymphoid tissue lesions in the challenge test. Compared to unvaccinated challenged animals, the vaccinated challenged animals showed significantly higher (p < 0.05) levels of anti-PCV2 IgG, PCV2d-specific interferon-γ (IFN-γ), and anti-MHP IgG.ConclusionsBased on clinical, microbiological, serological, and pathological assessments, this study confirmed that both combined vaccines could protect pigs against PCV2 infection caused by PCV2d. On the other hand, further research on the efficacy evaluation of these new vaccines against the MHP challenge and PCV2d/MHP co-challenge is needed.     

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.     

Multiply-primed rolling-circle amplification (MPRCA) of PCV2 genomes: Applications on detection, sequencing and virus isolation

Multiply-primed rolling-circle amplification (MPRCA) was used to amplify porcine circovirus type 2 (PCV2) genomes isolated from tissues of pigs with signs of post-weaning multisystemic wasting syndrome (PMWS). Two of the amplified PCV2 genomes were cloned in prokaryotic plasmids and sequenced. Both were nearly identical (1767 nt) except for one silent substitution in the region coding for the capsid protein (ORF2). In addition, they showed high nucleotide sequence similarity with PCV2 isolates from others countries (93–99%). To investigate whether the MPRCA amplified PCV2 genomes could be used to produce infectious virus, the cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 10^5.55 TCID₅₀/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes aiming at sequencing and virus isolation strategies, where particularly useful is the fact that it allows straightforward construction of PCV2 infectious clones from amplified genomes. However, it was less sensitive than PCR for diagnostic purposes.     

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。