Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV-2)

Porcine circovirus (PCV)-2, a newly described single-stranded circular DNA virus pathogen of swine is the cause of postweaning multisystemic wasting syndrome (PMWS). In gnotobiotic piglets, PCV-2 infection alone produces asymptomatic infection without evidence of overt PMWS. Gnotobiotic piglets infected with PCV-2 were injected with keyhole limpet hemocyanin in incomplete Freund's adjuvant (KLH/ICFA), and the effects on virus production and development of PMWS were determined. In the first experiment, piglets were injected subcutaneously on the left hip and shoulder, and viral burden was assessed in regional lymph nodes draining the injection sites and in contralateral lymph nodes 13-14 days after infection. Immune activation increased the number of virus antigen-positive cells in draining lymph nodes and increased the amount of infectious virus recovered by 1-4 log10. In a second experiment, the effects of injections of KLH/ICFA with or without concurrent stimulation of peritoneal macrophages by intraperitoneal injections of thioglycollate broth on induction of PMWS was assessed. All immunized piglets developed moderate to severe PMWS, whereas none of the piglets infected with PCV-2 alone developed PMWS. In PMWS-affected piglets, extensive replication of PCV-2 was documented by both immunocytochemistry and quantitative viral titrations. Thus, immune activation is a key component of the pathogenesis of PCV-2-associated PMWS in swine.     

Porcine circovirus type 2 (PCV-2) coinfections in US field cases of postweaning multisystemic wasting syndrome (PMWS).

The prevalence of different pathogens detected in combination with porcine circovirus type 2 (PCV-2) was studied retrospectively in field cases of postweaning multisystemic wasting syndrome (PMWS) diagnosed at the Iowa State University Veterinary Diagnostic Laboratory, Ames, Iowa, between January 2000, and September 2001. The presence of PCV-2 antigen in lymphoid tissues and/or lung, demonstrated by immunohistochemistry, together with moderate to severe lymphoid depletion and/or granulomatous lymphadenitis, was used as the criteria for the diagnosis of PMWS. A total of 484 cases fulfilled these criteria. Most of the cases (294/369) of PMWS occurred in pigs between the ages of 8 and 18 weeks, with a peak at 10 weeks of age. Porcine reproductive and respiratory syndrome virus was detected in 51.9% of the cases, Mycoplasma hyopneumoniae in 35.5%, bacterial septicemia in 14.0%, bacterial pneumonia in 7.6%, swine influenza virus in 5.4%, and PCV-2 alone in 1.9%. In cases with bacterial septicemia the most frequently isolated pathogen was Streptococcus suis. In cases with bacterial pneumonia, Pasteurella multocida was the most prevalent.     

Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus

One-day-old gnotobiotic piglets were inoculated intranasally with in vitro passaged porcine circovirus 1 (PCV-1), PCV-2, and porcine parvovirus (PPV) alone or in combination (PCV-1/PCV-2, PCV-1/PPV, and PCV-2/PPV). Piglets were evaluated for 1) the development of porcine postweaning multisystemic wasting syndrome (PMWS), 2) distribution of viral antigens by immunochemistry, and 3) viremia and the presence of viral DNA in nasal and ocular secretions and feces. All single agent-infected piglets and piglets infected with PCV-1/PCV-2 or PCV-1/PPV were clinically asymptomatic. They were transiently viremic and seroconverted to homologous virus(es). At termination of the study on postinfection day (PID) 35, microscopic lesions were restricted to focal inflammatory cell infiltrates in livers and myocardia. One piglet given PCV-1/PPV was PPV viremic for 2 weeks after infection and had lymphangiectasia of the spiral and descending colon associated with granulomatous inflammation. All four PCV-2/PPV-inoculated piglets developed PMWS, characterized by sudden onset of depression and anorexia, icterus, and submucosal edema. One piglet became moribund on PID 27, and the remaining three piglets were euthanatized between PID 27 and PID 30 because of severe disease. Lymph nodes were small and the livers were mottled. Disseminated angiocentric granulomatous inflammation was present in all tissues examined except the brain. Multiple lightly basophilic intracytoplasmic inclusion bodies were identified in macrophages and histiocytes. PCV-2 antigen was widely distributed within macrophages; PPV antigen was sparse. Hepatocellular necrosis and bile retention were prominent. PCV-2 DNA was identified in ocular, fecal, and nasal secretions. Terminal sera contained antibodies to PPV (4/4) and PCV-2 (3/ 4). Production of PMWS in gnotobiotic swine appears to require PCV-2 and additional infectious agents such as PPV for full disease expression in gnotobiotic piglets.     

猪圆环病毒2型的全基因克隆和序列分析

参考GenBank中发表的猪圆环病毒2型(PCV-2)全基因序列,设计一对PCR引物,从病料中扩增获得4株PCV-2的全基因组序列,分别命名为SDNY-PCV-2、ShD-PCV-2、HBbd-PCV-2、GSLN-PCV-2,经测序确定长度均为1 767 bp。应用DNA Star序列分析表明,4个PCV-2分离株与国外部分分离株的核苷酸序列同源性达95.5%~99.8%,与PCV-1毒株的序列同源性为76.2%~77.6%。与国内外部分分离毒株序列进化树分析表明,4个PCV-2分离毒株处于不同分支中,存在一定差异。     

猪圆环病毒2型甘肃株全基因组克隆及序列分析

参照GenBank中登录的猪圆环病毒2型(PCV-2)全基因组序列,设计合成了两对特异性引物,从甘肃白银疑似断奶仔猪多系统衰竭综合征(PMWS)病料中提取病毒DNA,分步扩增全基因组。回收PCR产物,将其插入pGEM-TEasy载体,构建了重组质粒pGEM-PCV-2,转化后筛选、提取阳性重组质粒进行测序。结果表明,克隆到的PCV-2全基因组长度为1767bp。应用DNAStar序列分析软件,对所测PCV-2序列与GenBank中登录的包括甘肃在内的国内外PCV毒株进行同源性分析。结果显示,克隆的PCV-2与英国株(UK-EU656143)遗传关系最近,其核苷酸和推导的氨基酸序列同源性高达96.2%和91.0%,与已报道的2株甘肃毒株(GS03-EU547458,GSLZ-FJ447482)遗传关系较远,可能是流行于甘肃白银的一个变异毒株(GSBY)。     

Evaluation of a nested polymerase chain reaction assay to differentiate between two genotypes of Porcine circovirus-2.

Porcine circovirus-2 (PCV-2) is associated with several diseases in pigs, including postweaning multisystemic wasting syndrome (PMWS). A new genotype of PCV-2 was isolated from swine farms with and without clinical PMWS in North America. The new genotype was differentiated in a separate cluster by phylogenetic analyses and is now named PCV-2b compared with PCV-2a for the previously known genotype. The purpose of this study was to develop and evaluate a nested polymerase chain reaction (nPCR) assay to detect and differentiate between PCV-2a and PCV-2b. Genotype-specific primer sets were designed by using sequence data published for different PCV-2 strains. Specificity and sensitivity of the nPCR were examined by using PCV-2 isolates with known genotype. Nested PCR was found to be highly specific and sensitive for detecting and differentiating between the PCV-2 genotypes compared with the conventional 1-step PCR assay. Nested PCR was applied to detect PCV-2 and to identify the genotype in serum samples from swine farms with and without a clinical history of PMWS. Of 60 serum samples collected from 4 farms during clinical PMWS outbreaks, PCV-2a and PCV-2b were detected in 6 and 49 samples, respectively. Six of the 10 samples from one of the 4 farms had both PCV-2a and PCV-2b. Of 20 serum samples from 2 farms without PMWS, 11 were positive for PCV-2a only. These results suggest that the differential nPCR can be used to detect PCV-2 and to differentiate the 2 genotypes from field samples.     

The emergence of porcine circovirus 2b genotype (PCV-2b) in swine in Canada

Since late 2004, the swine industry in the province of Quebec has experienced a significant increase in death rate related to postweaning multisystemic wasting syndrome (PMWS). To explain this phenomenon, 2 hypotheses were formulated: 1) the presence of a 2nd pathogen could be exacerbating the porcine circovirus 2 (PCV-2) infection, or 2) a new and more virulent PCV-2 strain could be infecting swine. In 2005, 13 PMWS cases were submitted to the Quebec provincial diagnostic laboratory and PCV-2 was the only virus that could be found consistently by PCR in all 13 samples. The PCR detection results obtained for other viruses revealed the following: 61.5% were positive for porcine reproductive and respiratory syndrome virus, 30.8% for swine influenza virus, 15.4% for porcine parvovirus, 69.2% for swine torque teno virus (swTTV), 38.5% for swine hepatitis E virus (swHEV) and 84.6% for Mycoplasma hyorhinis; transmissible gastroenteritis virus and porcine respiratory coronavirus (TGEV/PRCV) was not detected. Sequences of the entire genome revealed that these PCV-2 strains belonged to a genotype (named PCV-2b) that has never been reported in Canada. Further sequence analyses on 83 other Canadian PCV-2 positive cases submitted to the provincial diagnostic laboratory during years 2005 and 2006 showed that 79.5% of the viral sequences obtained clustered in the PCV-2b genotype. The appearance of the PCV-2b genotype in Canada may explain the death rate increase related to PMWS, but this relationship has to be confirmed.     

Detection of Porcine Circovirus Type 2, Porcine Parvovirus and Porcine Pseudorabies Virus from Pigs with Postweaning Multisystemic Wasting Syndrome by Multiplex PCR

Multiplex PCR was established to detect porcine circovirus type 2 (PCV-2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV) and applied to samples from 137 piglets exhibiting clinical signs of postweaning multisystemic wasting syndrome (PMWS). PCV-2 DNA was detected from all samples. Moreover, 43 samples were positive for PPV but negative for PRV; 11 samples were positive for PRV but negative for PPV; and 35 samples were positive both for PPV and PRV. These results suggests that PCV-2 co-infection with PRV and PPV may play an important role in PMWS. Also, multiplex PCR is an appropriate candidate method for diagnosis of PCV-2, PRV and PPV simultaneously in field cases.     

Genotypic shift of porcine circovirus type 2 from PCV-2a to PCV-2b in Spain from 1985 to 2008

Changes in porcine circovirus type 2 (PCV-2) genotypes were evaluated before, during and after outbreaks of post-weaning multisystemic wasting syndrome (PMWS) in (1) a retrospective study using pig sera collected in Spain from 1985 to 2008 and (2) a longitudinal study using pig sera collected from two farms in Spain over periods of 7 and 14 years. In both studies, there was a rapid genotypic shift from PCV-2a to PCV-2b that was related to the peak of PMWS epizootics in Spain and the appearance of PMWS on the two farms studied longitudinally.     

Features of cell degeneration and death in hepatic failure and systemic lymphoid depletion characteristic of porcine circovirus-2-associated postweaning multisystemic wasting disease

Tissue section replicates from lymphoid tissues and livers of gnotobiotic swine were examined by immunohistochemistry for the colocalization of porcine circovirus-2 (PCV-2) nucleocapsid and terminal deoxynucleotidyl transferase (TdT)-mediated incorporation of biotinylated nucleotides (UTP) onto the 3'-exposed hydroxyl groups (nick end labeling) nuclear deoxyribonucleic acid (TUNEL), a marker for apoptosis. Single- and dually stained replicates from uninfected controls, subclinically affected PCV-2-infected gnotobiotic pigs, PCV-2-infected piglets immunosuppressed with cyclosporine (Cys), and PCV-2-infected piglets with post-weaning multisystemic wasting syndrome (PMWS) were evaluated. Thymuses were used as positive controls for apoptosis absent PCV-2, tissue sections from dogs given hyperthermic stress were examined as positive controls for induced TUNEL. Tissues from heat-stressed dogs contained TUNEL-positive cell nuclei in both lymphoid tissues and liver, TUNEL was greatest shortly after the delivery of the hyperthermic insult. In uninfected control and subclinically affected PCV-2-infected gnotobiotic pigs, rare hepatocytes and lymphoid cells were TUNEL positive, the frequency of these was similar to that seen in uninfected controls. In PMWS-affected and Cys-treated PCV-2 piglets, the only consistent strongly positive TUNEL signal was contained within the cytoplasm of virus-positive phagocytic mononuclear cells. In phagocytes, some PCV-2 inclusions were TUNEL positive. Collectively, these data indicate that apoptosis is not the primary mechanism of lymphoid depletion and hepatocyte loss in PMWS. Apoptosis associated with systemic viral diseases may be attributable to pyrexia rather than direct or indirect effects of viruses on target cells.     

Postweaning multisystemic wasting syndrome (PMWS) in pigs with particular emphasis on the causative agent, the mode of transmission, the diagnostic tools and the control measures. A review

Postweaning multisystemic wasting syndrome (PMWS) is a worldwide emerging disease of weaned piglets. The objective of this review is to summarize the current knowledge regarding PMWS, its causative agent, mode of transmission, diagnostic techniques to detect PCV-2, the possible control measures, and the association of PMWS and PCV-2 with porcine dermatitis and nephropathy syndrome (PDNS). The causative agent of PMWS is porcine circovirus type 2 (PCV-2), however, not all pigs infected with PCV-2 develop the syndrome. PCV-2 is consistently associated with PMWS and PMWS is considered not to occur without it. Both the syndrome and the virus are not regarded as new. Co-factors that could activate PCV-2 to cause PMWS are considered. This enigmatic nature of both the syndrome and the virus is triggering a concern towards uncertainties of the viral transmission, its introduction in to the herd, effective tools of diagnosis, and control strategies.     

Pathological investigations of pigs with porcine multisystemic wasting syndrome (PMWS)

Extract

Pathological changes and immunohistochemical (IHC) examination for porcine circovirus-2 (PCV-2) in nine weaner pigs from a property with PMWS are reported. The pigs were in moderate to poor body condition, and several had diarrhoea and noisy respiration. The outstanding pathological changes related to microscopic lesions in lymphoid tissues, including lymph nodes, palatine tonsils, Peyer's patches, peribronchial lymphoid aggregates and spleen. While there was variability of expression of change between and within cases, the main features of lesions could be summarised as depletion of lymphoid cells, infiltration of histiocytes (often syncytial), and the presence of botryoid intra-cytoplasmic inclusion bodies in macrophages. The changes in lymphoid tissue were typical of those described in PMWS. Also characteristic of this syndrome were histiocyte-dominated cellular inflammatory infiltrates in kidney, liver and lung. There was strong correlation between IHC positivity for PCV-2 and the lesions in lymphoid, pulmonary and renal tissues. Concurrent diseases that are probably promoted by immune suppression may complicate the diagnosis of PMWS. In the present cases, salmonellosis, attaching and effacing Escherichia coli infections, and secondary bacterial bronchopneumonia were identified.     

Features of porcine circovirus-2 disease: correlations between lesions, amount and distribution of virus, and clinical outcome.

Tissue sets from 36 snatch-farrowed colostrum-deprived (SF/CD) and 71 Caesarian-derived gnotobiotic swine infected with porcine circovirus type 2 (PCV-2) as neonates were examined and scored for the types and tissue distribution of histologic lesions associated with this viral infection. The occurrence and severity of these lesions were correlated with qualitative and quantitative determinations of viral burden in tissues by immunohistochemistry (IHC) and tissue titrations for infectious virus, respectively. These measures were, in turn, related to 1 of 3 categories of clinical disease expressed in PCV-2-infected swine as subclinical infection, preclinical postweaning multisystemic wasting syndrome (PMWS), and clinically evident PMWS, respectively. Statistically significant (P < 0.05 to 0.001) associations between both measures of viral burden, the severity of histologic lesions and the stage of disease were obtained. Discrimination between and among categories of disease was best accomplished by a combination of IHC and histopathology. The results of this study confirm that viral burden in PCV-2-infected tissues, specifically lymphoid tissues and liver, directly correlate with severity of clinical disease expression in PCV-2 infected swine.     

Viral distribution and lesions in Kunming mice experimentally infected with porcine circovirus type 2b

The viral distribution and lesions in Kunming mice experimentally infected with porcine circovirus type 2b (PCV-2b) were investigated. Seventy special pathogen free mice were divided into 2 groups with 35 mice in each group. The test group (TG) was infected with PCV-2b, the control group (CG) was inoculated with sterile cell cultures. Five mice in each group were sacrificed at 3, 7, 14, 21, 28, 35 and 42 dpi (day post infection), respectively. Necropsies were performed on all mice and tissues were collected for testing by histopathology, immunohistochemistry, transmission electron microscope (TEM) and polymerase chain reaction (PCR). Apoptosis and necrosis in lymphoid organs were observed in virus-infected mice, and became severe from 14 to 28 dpi. The proportion of PCV-2b antigen-positive cells was moderate in lung, heart, thymus, liver or kidney, and low in brain from TG. In spleen and cervical lymph node, the proportions of PCV-2b antigen-positive cells were low to high from 7 to 28 dpi, and moderate from 35 to 42 dpi. PCV-2b DNA was detected in all tissues examined in TG from 7 to 42 dpi. Viral inclusion bodies presented in the cytoplasm of lymphocytes, macrophages, hepatocytes, podocytes, neurocytes, spermatids and uterine epithelial cells in TG. In CG, no viruses and viral lesions were detected. PCV-2b could replicate in mice, and PCV-2b associated lesions in mice were similar to those observed in pigs. The present results indicate that it is possible to use Kunming mouse as an animal model for PMWS research.     

Porcine circovirus-2 and concurrent infections in the field

Porcine circovirus-2 (PCV-2) is the necessary cause of post-weaning multisystemic wasting syndrome (PMWS) in swine; however, a variety of co-factors, including other infectious agents, are thought to be necessary in the full expression of disease. Porcine parvovirus (PPV) was found in the inoculum used in the first experiments to reproduce PMWS in gnotobiotic swine. Retrospective and prospective studies in the field and laboratory have demonstrated PCV-2 can act synergistically with PPV to enhance the severity of PMWS. PCV-2 has been shown to play a role in the porcine infectious disease complex (PRDC). Other co-infecting agents with PCV-2 in the lung include, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV) and Mycoplasma hyopneumoniae. Exposure of pregnant sows to PPV, PRRSV, or encephalomyocarditis virus may interact with PCV-2 infected foetuses. The severity of hepatic lesions in PCV-2 infected pigs may be enhanced by co-infection with agents such as swine hepatitis E virus and Aujezsky's disease virus. Additional studies are required to determine the mechanistic basis for the interaction of PCV-2 with other agents in the pathogenesis of the various clinical syndromes that have been associated with PCV-2 infection.     

Prevalence of infection with porcine circovirus-2 (PCV-2) and porcine reproductive and respiratory syndrome virus (PRRSV) in an integrated swine production system experiencing postweaning multisystemic wasting syndrome

The objective of this study was to evaluate the prevalence of infection with porcine circovirus-2 (PCV-2) and porcine reproductive and respiratory syndrome virus (PRRSV) through a longitudinal study in an integrated swine production system (7 farms) experiencing postweaning multisystemic wasting syndrome (PMWS). Risk factors for PCV-2 infection and for PCV-2 and PRRSV coinfection were also evaluated. Fifteen sows from each herd and 4 non-cross-fostered piglets from each sow were randomly selected at farrowing and ear-tagged at birth. Serum samples were analyzed for antibodies to PCV-2 and for detection of the PCV-2 and PRRSV genomes. Statistical analyses involved 2 approaches. The 1st approach characterized the dynamics of PCV-2 infection and their relationship with PRRSV infection. The 2nd approach analyzed the probability of being infected by PCV-2 or by both PCV-2 and PRRSV through a generalized linear mixed model incorporating sow and farm characteristics. At the 1st sampling time (1 wk of age), there was a significant relationship between sow PCV-2 infection and piglet PCV-2 infection (P < 0.0001). The risk of PCV-2 and PRRSV coinfection was 1.85 times greater in piglets from a sow with low titers of PCV-2 antibodies than in piglets from sows with medium to high titers (P = 0.03) and was 2.54, 2.40, and 2.02 times greater, respectively, in piglets from primiparous sows, PCV-2-infected sows, and farms in an area of high pig density than in piglets from sows of higher parity (P = 0.004), noninfected sows (P = 0.04), and farms in a low-density area (P = 0.09).     

Limited susceptibility of three different mouse (Mus musculus) lines to Porcine circovirus-2 infection and associated lesions

Porcine circovirus associated disease (PCVAD), a major global problem for pork producers, is characterized microscopically by depletion and histiocytic replacement of follicles in the lymphoid tissues. The objectives of this study were to determine 1) if Porcine circovirus-2 (PCV-2) inoculated mice (Mus musculus) can develop PCV-2 associated lymphoid lesions and serve as a model for PCVAD, and 2) if differences in PCV-2 host susceptibility exist among mice lines. Three groups (n = 48/group) of 4-wk-old male mice were used: BALB/c, C57BL/6, and C3H/HeJ. A 2 × 2 factorial analysis was designed for each group using PCV-2 inoculation and keyhole limpet hemocyanin in incomplete Freund’s adjuvant injections on day 0 and 7 as factors. Necropsies were performed on days 12, 17, 22, 27, 32, and 37. Serum samples collected at each necropsy tested negative for anti-IgG PCV-2 antibodies in all mice at all time points by 2 different PCV-2 enzyme-linked immunosorbent assays (ELISA). The PCV-2 DNA was detected by polymerase chain reaction (PCR) in 93% (100/108) of tissues and 42.6% (46/108) of serum samples from PCV-2-inoculated mice from days 12 to 37. Microscopic lesions consistent with PCV-2 infection were not observed in any mice and PCV-2 DNA and PCV-2 antigen were not detected in tissues by in-situ-hybridization or immunohistochemistry assays, respectively. Based on incidence of PCV-2 DNA in serum samples, the C57BL/6 mouse line was more resistant to PCV-2 infection than the other lines. The results indicate the mouse model likely has limited utility to advance understanding of the pathogenesis of PCV-2 associated lesions, but mice could potentially be important in the epidemiology of PCV-2.     

猪圆环病毒2型山东分离株全基因组的克隆与序列分析

根据猪圆环病毒2型(PCV-2)全基因组序列,设计一对特异性引物,从疑似断奶仔猪多系统衰竭综合征(PMWS)病料中扩增出PCV-2基因组DNA,将基因片段克隆于pMD18-T载体,筛选获得含有相应片段的阳性重组质粒pMD18-T-PCV-2。对筛选出的阳性质粒进行测序。结果表明,PCV山东株的全基因组为1 767 bp,与国内外毒株核苷酸同源性高达99.6%。     

Modification of PCV-2 virulence by substitution of the genogroup motif of the capsid protein

ABSTRACT: Porcine circovirus type 2 (PCV-2) is the causal agent of the post-weaning multisystemic wasting syndrome (PMWS). PCV-2 are small single-stranded circular DNA viruses clustered into two main genogroups: PCV-2a and PCV-2b. Each genogroup present a specific highly-conserved motif of six amino acids (between amino acids 86 and 91) in the PCV-2 capsid protein. The aim of this study was to verify whether the motif located in the capsid protein and specific to each PCV-2 genogroup contributes to virulence. Two parental DNA clones, PCV-2a and PCV-2b, were constructed as well as two mutants DNA clones, PCV-2a/motif 2b and PCV-2b/motif 2a by exchanging the capsid motif of each genogroup. The four DNA clones were characterized in vitro as well as in vivo. Cells transfected by the four DNA clones produced infectious viruses. In specific-pathogen-free piglets transfected by the four infectious DNA clones, PCV-2b/motif 2a virulence was not attenuated while the PCV-2a/motif 2b virulence was drastically reduced compared to their parent virulence. These results suggest that the amino acids between positions 86 and 91 of the capsid protein are determinant for the virulence of isolates. However, the environment of this motif seems also involved.