Epidemiological study on porcine circovirus type 2 (PCV2) infection in the European wild boar (Sus scrofa)

Porcine circovirus type 2 (PCV2) is considered as the causative agent of postweaning multisystemic wasting syndrome (PMWS) in domestic pigs, where the virus is ubiquitous as evidenced by serological surveys. We present the results of the first nationwide sero-survey on the presence of PCV2 antibodies in European wild boars, and report the first PMWS case in a wild boar from Spain. Sera from 656 hunter harvested wild boars from 45 different geographical sites and 22 additional imported animals were analysed by means of an immunoperoxidase monolayer assay (IPMA). We also examined the tissues from 55 healthy and one diseased wild boars for the presence of PCV2 nucleic acid and PMWS lesions by in situ hybridisation and histopathology, respectively. Additionally, abundance estimates of wild boars and field interviews were carried out on 30 sampling sites. The prevalence of medium to high PCV2 serological titres among the examined wild boars was 47.89 +/- 1.9%. Seropositive wild boars appeared in all but one of the geographical regions analysed. Seroprevalence and titre of PCV2 antibodies were closely related to the management of the wild boar populations. Wild boars from intensively managed, farm-like populations had higher prevalence than wild boars living in more natural situations. The effect of wild boar abundance and management on PCV2 antibody prevalence was further evidenced by the high correlation existing between the relative abundance estimates of animals and the percentage of wild boars with medium to high levels of PCV2 antibodies. PCV2 nucleic acid was detected in the tissues of three wild boars. One of these was diagnosed as PMWS. The results, in addition to information on piglet mortalities, suggest a potential role of PMWS in piglet mortality in intensively managed wild boar populations.     

Reproduction of PMWS in immunostimulated SPF piglets transfected with infectious cloned genomic DNA of type 2 porcine circovirus

Postweaning multisystemic wasting syndrome (PMWS) is a recently emerged disease affecting pigs. Type 2 porcine circovirus (PCV2) has been associated with this syndrome although other factors are required in association with this virus for PMWS expression. The aim of this study was to investigate whether general immunostimulation (injections of keyhole limpet hemocyanin emulsified in incomplete Freund adjuvant and of thioglycollate medium) could strengthen the severity of PMWS in six-week-old specific-pathogen-free (SPF) piglets transfected with pure tandem-cloned PCV2 DNA by the intramuscular route. Non-immunostimulated piglets transfected with the viral clone did not present clinical signs but only mild pathological microlesions characteristic of PMWS. These piglets seroconverted and high viral genome loads and infectious titers were detected in the lymphoid organs at the end of the trial. Mild-to-moderate forms of PMWS were generally observed in the immunostimulated transfected piglets, as well as one severe form for a piglet (8003) which died. These piglets with mild-to-moderate forms had higher DNA loads than the transfected-only animals. Thus, viral replication was enhanced by immunostimulation. This is the first time that clinical PMWS has been reported in an SPF immunostimulated piglet infected with a pure inoculum consisting of tandem-cloned PCV2 DNA. This result confirms that PCV2 is the agent of PMWS and that immunostimulation could enhance PMWS in SPF piglets transfected with a PCV2 DNA clone.     

Experimental reproduction of postweaning multisystemic wasting syndrome in cesarean-derived, colostrum-deprived piglets inoculated with porcine circovirus type 2 (PCV2): investigation of quantitative PCV2 distribution and antibody responses.

Sixteen cesarean-derived, colostrum-deprived piglets were inoculated intranasally with porcine circovirus type 2 (PCV2), originally isolated from a pig affected with postweaning multisystemic wasting syndrome (PMWS). At 1 day postinoculation (PI), 3 of the 5 piglets in the uninoculated control group were moved to the room of inoculated piglets for contact exposure. Porcine circovirus type 2 was detected by polymerase chain reaction (PCR) in swabs from inoculated piglets from 1 day PI and from contact piglets from 2 days after cohabitation. Porcine circovirus type 2 was also detected in all serum samples but not in control piglets 7 days PI. Until the end of study, PCV2 was detected in swabs and serum samples by PCR but not in the control piglets. One inoculated piglet died suddenly without clinical signs 19 days PI. Beginning at 14 days PI, 5 piglets, including 1 contact piglet, had clinical signs of depression, anorexia, and icterus, and 1 inoculated piglet died 21 days PI. Most of the piglets exhibiting the above clinical signs became moribund and were necropsied 21 and 28 days PI. In the piglets that showed clinical signs, gross lesions, including icterus of liver and hemorrhage in stomach, and typical histopathological lesions of PMWS, such as lymphoid depletion and basophilic intracytoplasmic inclusion bodies in lymph nodes and other tissues, were observed. Porcine circovirus type 2 was detected by PCR in all tissue samples except in those of the control piglets. Porcine circovirus type 2 was recovered from several tissue samples of the piglets necropsied until 35 days PI. In particular, PCV2 was recovered in high titer from most of the tissue samples of the piglets exhibiting clinical signs. Serum antibody against PCV2 was mostly detected in inoculated piglets and in contact piglets 14 and 21 days PI by an indirect fluorescence antibody test but was not detected in the piglets exhibiting clinical signs until 28 days PI. These results indicate that PCV2 was able to induce clinical PMWS in the absence of other swine pathogens and that there were significant differences in both the quantitative PCV2 distribution in tissues and the antibody response between the piglets that were infected and developed PMWS and those that were infected but remained healthy.     

Brain lesions in pigs affected with postweaning multisystemic wasting syndrome.

Anti-porcine circovirus type 2 (anti-PCV2) immunostaining was associated with cerebellar lymphohistiocytic vasculitis combined with hemorrhages (50 pigs) or with lymphohistiocytic meningitis (23 pigs) in pigs naturally affected with postweaning multisystemic wasting syndrome (PMWS). The animals originated from 12 farms in Rio Grande do Sul, Brazil. In total, 456 unthrifty 3- to 5-month-old postweaning pigs confirmed as PMWS cases were necropsied. Although most findings mimicked those extensively reported in PMWS-affected pigs, there were distinctive brain lesions that included multiple hemorrhages in the cerebellar leptomeninges associated with lymphohistiocytic vasculitis and fibrinoid degeneration in vessels of the cerebellum and periventricular areas (69 pigs). These vascular lesions were also seen in conjunction with lymphohistiocytic meningitis (38 additional pigs). PCV2 antigen was immunohistochemically demonstrated in the cytoplasm and nuclei from intralesional perivascular macrophages and endothelial-like cells in brain tissues. Together these findings suggest that these lesions were caused by PCV2.     

Evidence of breed-dependent differences in susceptibility to porcine circovirus type-2-associated disease and lesions

Porcine circovirus type 2 (PCV2) has been confirmed as the primary cause of postweaning multisystemic wasting syndrome (PMWS). However, in the field, PMWS is seen only in a small percentage of pigs infected with PCV2. The overall objective of the study reported here was to determine whether host genetic differences in the susceptibility to PCV2-associated disease exist among selected breeds of pigs. This study included Duroc (n = 23), Landrace (n = 19), and Large White (n = 21) pigs. The pigs were infected intranasally and intramuscularly at 5-7 weeks of age with PCV2. A portion of the pigs (31/63; 30.2%) had low passively acquired PCV2 antibodies at the time of infection. There were no differences in mean weight gain, rectal temperature, or respiratory score. Clinical disease compatible with PMWS was observed only in the Landrace pigs. Most of the PCV2-infected pigs had enlarged lymph nodes, and individual Duroc and Landrace pigs had mottled tan lungs. PCV2-associated lymphoid depletion and granulomatous inflammation were observed in pigs of all breeds. Three of 19 Landrace pigs and none of the Duroc or Large White pigs developed severe lymphoid lesions associated with large amounts of intralesional PCV2 antigen typical of PMWS. Compared with seronegative Landrace pigs, Landrace pigs that had low maternal antibodies at the time of PCV2 inoculation had significantly (P < 0.05) less-severe PCV2-associated lesions. The results suggest a predisposition of the Landrace pigs of this study to PCV2-induced disease and lesions, and that low levels of passively acquired antibodies are protective.     

Myocarditis and abortion associated with intrauterine infection of sows with porcine circovirus 2.

Porcine circovirus 2 (PCV2) is a recently identified agent that has been associated with postweaning multisystemic wasting syndrome (PMWS) in swine populations. In this report, the potential spectrum of disease associated with PCV2 is expanded by evidence of vertical transmission and associated reproductive failure. PCV2 was isolated from a litter of aborted piglets from a farm experiencing late-term abortions and stillbirths. Severe, diffuse myocarditis was present in 1 piglet associated with extensive immunohistochemical staining for PCV2 antigen. Variable amounts of PCV2 antigen were also present in liver, lung, and kidney of multiple fetuses. The presence of other agents that have been associated with fetal lesions and abortion in swine, including porcine parvovirus, porcine reproductive respiratory syndrome virus, encephalomyocarditis virus, and enterovirus, could not be established.     

Postweaning multisystemic wasting syndrome (PMWS) in pigs. A review

Postweaning multisystemic wasting syndrome, caused by porcine circovirus type 2 (PCV2), is a recently described clinical condition which affects nursery and growing pigs. PMWS was initially recognized in Canada in 1991 and nowadays is considered to be worldwide distributed. Clinically, PMWS most representative symptoms include wasting, unthriftness, paleness of the skin, respiratory distress, diarrhea and sometimes icterus. PCV2 infection occurs in both PMWS affected and non-affected farms, and viral seroconversion shows a typical pattern, with declining of colostral antibodies during the lactating and nursery periods, with the lowest levels at the end of the nursery period, and active seroconversion of almost all pigs during the grower period. Although antibodies to PCV2 have been detected as early as 1969, no explanation for the emergence of this disease in the 90s has been established. Macroscopic lesions associated with PMWS are quite unspecific, but histopathological lesions in lymphoid tissues (lymphocyte depletion with histiocytic infiltration) are almost unique for this disease. These lesions together with other clinical and laboratorial findings suggest that severely affected pigs may be immunosuppressed. The criteria used for the diagnosis of PMWS include the existence of compatible clinical signs, presence of characteristic microscopic lesions and detection of PCV2 within these lesions. Because of the lack of appropriate treatment or vaccination against PCV2, zootechnical changes have been proposed in affected farms to reduce the so-called "infection pressure" due to PCV2 as well as to any other pathogen.     

Reproduction of postweaning multisystemic wasting syndrome in pigs experimentally inoculated with a Swedish porcine circovirus 2 isolate.

In recent years, porcine circovirus type 2 (PCV2)-associated postweaning multisystemic wasting syndrome (PMWS) has been reported worldwide. However, to date, PMWS has not been reported in Sweden despite the demonstration of serum antibodies to a PCV2-like virus in Swedish pigs. This communication reports the experimental reproduction of clinical PMWS after inoculation of colostrum-deprived (CD) pigs, derived from a Northern Ireland herd, with an isolate of PCV2 virus recovered from a clinically normal Swedish pig that was necropsied in 1993. The clinical disease and histological lesions observed in CD pigs inoculated with this virus were indistinguishable from those observed in previous studies on CD pigs inoculated with a PCV2 virus isolate recovered from pigs with PMWS. These results highlight the disease potential of PCV2 isolated from regions apparently free of PMWS and suggest that the status of the host and its environment is an important factor in the development of clinical 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.     

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.     

猪圆环病毒感染及动物模型研究进展

1974年德国学者Tischer从多株连续传代的猪肾细胞(PK-15)中发现了猪圆环病毒(PCV),其致病性及其疾病直到1991年才由Clark报道,称为断奶仔猪多系统衰竭综合征(PMWS).随后,许多学者对PCV-2感染以及PMWS的动物模型建立进行了广泛研究,证实了免疫激发及PCV-2与其他病原混合感染对PMWS的...     

Case-control study on the association of porcine circovirus type 2 and other swine viral pathogens with postweaning multisystemic wasting syndrome.

A field-based case-control study was conducted to assess the strength of association of porcine circovirus type 2 (PCV2) and some major swine viruses with postweaning multisystemic wasting syndrome (PMWS). Cases were defined as individual pigs with a clinical history of progressive weight loss and histopathological lesions characteristic of PMWS. Controls were pigs without clinical signs and histopathological lesions typical of PMWS. A total of 31 cases and 56 controls was identified from diagnostic submissions. Serum and various tissues were collected from all animals and assayed for PCV, porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus, porcine enterovirus types 1-3, swine influenza virus, porcine respiratory coronavirus, transmissible gastroenteritis virus, porcine endogenous retrovirus, porcine lymphotropic herpesvirus type 1, and bovine viral diarrhea virus. The proportion of case and control pigs positive for each virus was determined and statistically compared for determining the strength of the association that each virus had with PMWS individually or in combinations. Porcine circovirus type 2 had the strongest association (OR = 9.3, P = 0.006) with PMWS among the viruses tested for. Risk for PWMS was much higher (OR = 31.2, P = 0.0009) if the animal was concurrently infected with PCV2 and PRRSV, suggesting that development of PMWS may be enhanced by cofactor(s). Because PCV2 was also found in 62.5% of the controls, PCV2 from 5 cases and 4 controls were selected and genetically compared. No significant genetic difference was observed between PCV2 from PMWS and control pigs.     

Postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology

This review paper concentrates on the aetiology, diagnosis, and pathological aspects of postweaning multisystemic wasting syndrome (PMWS). PMWS was first recognized in Canada in 1996 as a new emerging disease which caused wasting in postweaned pigs. Since then, PMWS has been recognized in pigs in many countries. The syndrome is caused by a DNA virus referred to as porcine circovirus 2 (PCV2), which is classified in the family Circoviridae. PMWS primarily occurs in pigs between 25 and 120 days of age with the highest number of cases occurring between 60 and 80 days of age. The diagnosis of PMWS must meet three criteria: (i) the presence of compatible clinical signs, (ii) the presence of characteristic microscopic lesions, and (iii) the presence of PCV2 within these lesions. In order to establish the diagnosis, techniques are required that link virus and tissue lesions, such as immunohistochemistry and in situ hybridization, but not polymerase chain reaction or virus isolation. The three criteria considered separately are not diagnostic of PMWS. For example, the detection of PCV2 alone does not indicate PMWS but merely PCV2 infection. A hallmark of microscopic lesions of PMWS is granulomatous inflammation in the lymph nodes, liver, spleen, tonsil, thymus, and Peyer's patches. Large, multiple, basophilic or amphophilic grape-like intracytoplasmic inclusion bodies are often seen in the cytoplasm of macrophages and multinucleated giant cells.     

PCV2感染对仔猪NO-NOS系统的动态影响

将19头PCV2和PRRSV血清抗体阴性的普通断奶仔猪分为对照组(4头)和攻毒组(15头),攻毒组每头仔猪滴鼻接种PCV2病毒悬液4mL(5×105 TCID50/mL)并用KLH刺激,分别在攻毒后14、21、35d各扑杀5头仔猪采集血清和脾脏、淋巴结、胸腺(对照组4头在攻毒当天扑杀)。检测各种组织中NO、TNOS和iNOS的含量。结果显示,攻毒猪各组织中NO含量均在攻毒后14d显著升高(P<0.05),然后缓慢下降;血清和3种免疫器官中NOS(包括TNOS和iNOS)活性的变化趋势比较一致,均表现出先升高然后再逐渐降低的特点。此外,iNOS活性变化与其相应的组织中NO含量呈显著正相关(P<0.01)。结果表明,PCV2在感染早期(1～14d)可激活仔猪多种组织中NO-NOS系统,中后期逐渐恢复,这种变化与PCV2所致的组织损伤密切相关,因此,NO-NOS系统可能是PCV2相关疾病发生发展的重要信号。     

Post-weaning multisystemic wasting syndrome (PMWS) clinical expression under field conditions is modulated by the pig genetic background

Post-weaning multisystemic wasting syndrome (PMWS) is a worldwide distributed disease of multifactorial origin and porcine circovirus type 2 (PCV2) has been identified as its essential infectious aetiology. Pig genetic background has been pointed to influence disease expression. In the present study, three different boar lines, namely A (100% Pietrain), B (50% Large White × 50% Pietrain) and C (25% Large White × 75% Duroc), were used to inseminate sows from the same genetic line (37.5% Large White × 37.5% Duroc × 25% Landrace) located on two PMWS-affected farms (farm-1 and farm-2). The PMWS clinical expression of their offspring was studied from weaning to slaughter, evaluating three parameters: total post-weaning mortality (PWM), PWM associated to PMWS (PMWS-PWM) and body weight (BW) evolution. The effect of other variables potentially related with PMWS, including sow and piglet PCV2 exposure, sow parity, piglet gender and piglet BW at weaning, were also considered in the study design. Overall, a total of 6.5% PWM and 4.3% PMWS-PWM occurred in the monitored farms. Pigs from boar line C showed the highest PWM (16.3%) and PMWS-PWM (12.4%), and the lowest BW; pigs from boar line A showed the lowest PWM (1.8%) and the highest BW. Furthermore, PWM was also higher in piglets from farm-2 and from multiparous sows. In farm-2, PMWS-PWM was higher in piglets from multiparous sows. Finally, BW was influenced by interactions between genetics and both farm and pig age, and was lower in piglets from farm-2. This study represents a consistent observation of the genetic background effect on PMWS clinical expression under field conditions.     

Quantification of porcine circovirus type 2 (PCV2) DNA in serum and tonsillar, nasal, tracheo-bronchial, urinary and faecal swabs of pigs with and without postweaning multisystemic wasting syndrome (PMWS)

The present study focused on PCV2 quantification by TaqMan PCR in nasal (n=99), tonsillar (n=108), tracheo-bronchial (n=72), urinary (n=91) and faecal (n=42) swabs, as well as in serum (n=57), from a total of 146 pigs received at the Pathological Diagnostic Service at the Veterinary School of Barcelona (Spain). Animals were classified into three categories based on histopathological and in situ hybridisation (ISH) results: PMWS affected pigs (Group A, n=42), PCV2 subclinically infected pigs (Group B, n=29), and non-PMWS with PCV2 ISH negative pigs (Group C, n=75). Overall, tracheo-bronchial swabs had the higher PCV2 load followed by serum, tonsillar, nasal, faecal and, finally, urinary swabs. PCV2 genome was also detected in different proportions in all three categories of pigs; in all tested sites, viral load means were significantly higher (P0.05) were observed among tested specimens when age-groups (pigs younger than 1.5 months, and equal or older than 1.5 months of age) were compared. In summary, PCV2 is presumably excreted through respiratory (nasal and tracheo-bronchial) and oral (tonsillar) secretions, urine and faeces of both PMWS and non-PMWS affected pigs, with higher viral loads being associated with the presence of PMWS lesions.     

Postweaning multisystemic wasting syndrome (PMWS) in pigs in Croatia: detection and characterisation of porcine circovirus type 2 (PCV2)

The objective of this study was to characterise porcine circovirus type 2 (PCV2) from pigs with naturally occurring postweaning multisystemic wasting syndrome (PMWS) in Croatia, and to determine the epizootiological, clinical and pathomorphological features of the disease. During a systematic health monitoring programme conducted in the period from January 2002 to June 2003, PMWS was suspected on eight different pig-producing farms in Croatia. The diagnosis of PMWS met all three key criteria: the presence of compatible clinical signs, the presence of the characteristic microscopic lymphoid lesions, and the detection of PCV2 within the lesions by polymerase chain reaction (PCR) and by in situ hybridisation (ISH). Moreover, PCV2 DNA from swine tissues was extracted and sequenced. The phylogenetic analysis of 4 Croatian PCV2 strains showed close relationship to PCV2 strains isolated in Slovenia, France, the Netherlands, the United Kingdom, China and Hungary. PCV2 was also demonstrated by electron microscopy in the lymph node of an affected animal. This is the first demonstration of PMWS in Croatia based on all scientifically accepted diagnostic criteria.     

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 proposal on porcine circovirus type 2 (PCV2) genotype definition and their relation with postweaning multisystemic wasting syndrome (PMWS) occurrence

Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS). Despite first sequencing studies did not find any association between PCV2 sequences and PMWS occurrence, recent works have suggested the opposite. In the present study, 87 open reading frame 2 (ORF2) sequences obtained from pigs with different clinical conditions and coming from farms with different PMWS status were analyzed. Results further confirmed the existence of two genogroups and the definition of two PCV2 genotypes (1 and 2) is proposed. All sequences included in genotype 1 came from pigs from PMWS affected farms, while all sequences obtained from non-PMWS affected farms corresponded to genotype 2. Moreover, infection of single pigs from PMWS affected farms harbouring both genotypes is described. Present results suggest that PCV2 genotype 1 may potentially be more pathogenic than PCV2 genotype 2.