Overview of Porcine Circovirus Diseases
(Postweaning multisystemic wasting syndrome, Porcine dermatitis and nephropathy syndrome)
A novel, noncytopathogenic, picornavirus-like contaminant in the porcine kidney cell line PK-15 (ATCC-CCL33) was described in 1974. This agent was later shown to be a small, nonenveloped virus containing a single-stranded, circular DNA genome; it was named porcine circovirus (PCV). PCV antibodies in swine were found to be widespread, and experimental infections with this virus in pigs did not result in clinical disease, suggesting that PCV was nonpathogenic.
A new disease was described in Western Canada during the early and mid 1990s. The etiology was unknown, and the condition was named postweaning multisystemic wasting syndrome (PMWS). Affected pigs, mainly nursery pigs, showed primarily poor growth rate, ill thrift, and/or wasting, and they were histopathologically characterized by systemic inflammatory lesions. In the late 1990s, an apparently novel PCV-like virus was isolated from PMWS-affected pigs. The new virus was antigenically and genetically distinct from the PCV contaminant of PK-15 cell cultures. Subsequently, PCV isolates from diseased pigs were designated as porcine circovirus type 2 viruses (PCV2) and the original PCV from PK-15 cell cultures as porcine circovirus type 1.
PCV2 has been further associated with a number of disease syndromes in pigs, so the term porcine circovirus disease (PCVD) was proposed as a collective name. Recently, the terms PCV2-systemic disease (PCV2-SD) and PCV2-reproductive disease (PCV2-RD) have been proposed to replace PMWS and PCV2-associated reproductive failure, respectively. Moreover, it is currently thought that the most important PCVD is the PCV2-subclinical infection (PCV2-SI), which is linked with growth retardation without overt clinical signs. Porcine dermatitis and nephropathy syndrome (PDNS) is also included as a PCVD, although there is still no proof of PCV2 as the antigen linked with this immunocomplex disease. PCV2 has also been associated with porcine respiratory disease complex, although its impact is probably linked to the occurrence of PCV2-SD. PCV2-SD and PCV2-SI are the PCVDs considered to severely impact swine production worldwide, but the introduction of efficacious vaccines to the market has largely ameliorated their effects.
Circoviruses are small (17–22 nm in diameter), nonenveloped viruses that contain a single strand of circular DNA. There are two types of porcine circovirus, although only PCV2 is considered pathogenic. Phylogenetic studies have shown that at least three genotypes of PCV2 exist (PCV2 a, b, and c). Recent studies suggested a genotype shift (from a to b; PCV2c has been retrospectively detected in Denmark during the 1980s) coincidental with major outbreaks of PCV2-SD in North America, Japan, and some European countries. It is not clear whether differences in virulence exist among or within PCV2 genotypes.
Serologic surveys show that PCV2 is widespread in swine, independent of the PCV2-SD status of the farm. Results from retrospective serologic studies indicate that PCV2 has been infecting pigs for >50 years so far, and phylogenetic studies indicate that PCV2 has probably circulated in pigs during the past 100 years.
Initially, PCV2-SD was identified in high health herds that were free of most common swine pathogens. However, under field conditions, swine that show signs of PCV2-SD usually are infected with multiple agents, including porcine parvovirus, porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Pasteurella multocida, Haemophilus parasuis, Staphylococcus spp, and Streptococcus spp.
Accounts of multiple attempts to experimentally reproduce PCV2-SD have been published. Some early trials (using tissue homogenates from pigs affected with PCV2-SD or a PCV2 isolate) reproduced PCV2-SD–like histologic lesions but not the wasting condition. However, occasional studies subsequently reproduced clinical disease and lesions consistent with PCV2-SD using only PCV2, presumably, as inoculum. Consequently, it was suggested that PCV2 infection, linked to other cofactors, was necessary for the consistent development of full clinical disease. It appears that a number of factors, such as age and source of pigs, environmental conditions, genetics, the nature of the PCV2 inoculum used, and the immunologic status of the pig at PCV2 infection, play a significant role in the consistent experimental reproducibility of the disease. In fact, the more consistent and repeatable PCV2-SD disease models have been obtained using infectious and noninfectious cofactors as triggers. Also, the coinfection of PCV2a and b genotypes has been linked to reproduction of clinical disease under experimental conditions. The mechanisms by which other viruses or immunostimulation may trigger the development of wasting in PCV2-infected pigs is still unknown. High loads of PCV2 in blood, lymphoid, and other tissues and in potential excretion routes are associated with the expression of disease.
When multisystemic disease and wasting is apparent, damage to the immune system is the main feature suggesting that affected pigs have an acquired immunodeficiency. Lymphocyte depletion of lymphoid tissues, changes in peripheral blood mononuclear cell subpopulations, and altered cytokine expression patterns have all been demonstrated in pigs naturally and experimentally affected with PCV2-SD.
The identification of cells that support PCV2 replication has been a matter of controversy. The large amount of PCV2 virus antigen found in the macrophages and dendritic cells of diseased pigs appears to be the result of accumulation of viral particles. However, epithelial and endothelial cells seem to be the main target for PCV2 replication, as well as a small proportion of macrophages and lymphocytes.
Much less is known regarding the pathogenesis of other PCVDs. PCV2 is able to replicate in fetuses as well as in zona pellucida–free embryos. Moreover, an experiment with embryos exposed to PCV2 and then transferred to receptor sows suggested that infection can lead to embryonic death. Therefore, it is believed that one of the potential outcomes of PCV2 infection in sows could be return to estrus. Transplacental transmission of PCV2 has been demonstrated. However, experiments using pregnant sows inoculated intranasally have yielded variable results. When successful, those studies have shown that PCV2 may cause fetal death, similar to that of porcine parvovirosis, with live pigs together with dead piglets and mummies of different sizes.
PDNS is considered a type III hypersensitivity reaction in which the antigen present in the immune complexes is unknown. It has been speculated that PCV2 could be the antigen, but there is no definitive proof that PCV2 causes PDNS lesions. Indirect evidence exists, such as significantly higher serum antibody titers to PCV2 in affected pigs compared with healthy or PCV2-SD-affected pigs.
PCV2 is considered a ubiquitous virus in countries with and without PCVDs, including PCV2-SD. PCV2 infection and PCV2-SD have also been described in wild boars. The disease has been reported worldwide.
Transmission may be by direct contact with infected pigs. PCV2 has been detected in almost all potential excretion routes such as nasal, ocular, and bronchial secretions; saliva; urine; and feces. The virus can be found in semen, but the practical importance of this is probably negligible. Artificial insemination (AI) of sows with PCV2-infected semen from experimentally inoculated boars did not result in sow infection or fetal infection. However, when such AI was performed with PCV2-spiked semen, reproductive problems were developed. Therefore, it seems that reproductive disease linked to AI is possible, but only when semen has a high virus load, which is unlikely under natural conditions. Although not demonstrated, it is assumed that contact with contaminated fomites, exposure to contaminated feeds or biologic products, multiple use of hypodermic needles, or biting insects may play a role in transmission.
PCV2 may persist in swine for several months under either experimental or field conditions. Convalescent swine may carry virus for extended periods and be important in disease transmission. PCV2 is fairly resistant to commonly used disinfectants and to irradiation, probably allowing it to accumulate in the environment and be infective for new groups of susceptible pigs if rigorous sanitary measures are not followed. The decline of colostral antibody titer in pigs is associated with onset of PCV2-SD in late nursery or finishing pigs. Transplacental infection with PCV2 has been documented, but it is not known whether pigs infected in utero are able to subsequently develop clinical PCV2-SD.
Some reports have suggested that animals other than swine may be infected with PCV2 or PCV-like viruses. However, results of serologic studies for antibody against PCV in cattle and other livestock have been contradictory, and experimental induction of disease using PCV1 or PCV2 in species of livestock other than swine has not been successful. Mice may be able to replicate and harbor the virus.
PCV2-SD is characterized by overt weight loss. Disease often occurs in the fattening units in pigs 8–18 wk old, although the disease can be also seen in older or younger pigs. Morbidity is typically 5%–20% among cohorts in the late nursery or finishing stages. Mortality in swine with signs of PCV2-SD can occasionally be >50%. In addition to death loss, PCV2-SD in finishing pigs may cause a substantial increase in time to reach market weight, resulting in economic loss. Growth retardation, wasting, and dyspnea are the clinical signs seen most frequently in outbreaks. Pallor, anemia, jaundice, diarrhea, and palpable inguinal lymphadenopathy also are seen in some affected pigs. A low-grade fever (104°–106°F [40°–41°C]) that lasts several days may be seen as well. Overcrowding, poor air quality, insufficient air exchange, and commingled age groups seem to exacerbate the course of the disease. Usually, only a few pigs in a group show wasting. The onset of disease may be acute, leading to death within a few days in some pigs. Other pigs show a more chronic disease and fail to gain weight or thrive.
PCV2-SI is believed to occur in pigs that become infected with the virus and suffer from growth retardation (significantly lower average daily weight gain [ADWG]) but not overt clinical signs. In fact, in a farm affected by PCV2-SD, a variable proportion of pigs developed the systemic disease, while most had only subclinical infection. PCV2-SI had been unnoticed for many years until the advent of vaccines drew attention to this condition. Vaccinated pigs have an increased ADWG compared with nonvaccinated, apparently healthy counterparts. Such difference has been demonstrated to vary between 10−40 g/d, depending on the farm.
PCV2-RD characterized by late-term abortions and stillbirths in the absence or presence of other well-known reproductive pathogens seems to be the hallmark of clinical PCV2 infection in sows. Most of these descriptions come from North America and usually occur in start-up herds. Return to estrus due to embryonic death as a potential outcome of intrauterine PCV2 infection has been suggested based on experimental data. However, there are no field data unequivocally supporting this effect.
PDNS may affect nursery and growing pigs and, sporadically, adult animals. The prevalence of the syndrome in affected herds is relatively low (<1%), although higher prevalences (>20%) have been described occasionally. Pigs with severe acute disease die within a few days after the onset of clinical signs, due to acute renal failure with a significant increase in serum levels of creatinine and urea. Surviving pigs tend to recover and gain weight 7–10 days after the beginning of the syndrome. Affected pigs have anorexia, depression, prostration, stiff gait and/or reluctance to move, and normal temperatures or mild fever. The most obvious sign in the acute phase is the presence of irregular, red-to-purple macules and papules on the skin of the hindlimbs and perineal area, although distribution may be generalized in severely affected animals. With time, the lesions become covered by dark crusts and fade gradually (usually in 2–3 wk), sometimes leaving scars.
PCV2-SD is diagnosed by characteristic histopathologic findings in affected pigs. Grossly, lymph nodes may be substantially enlarged and pale on cut surface, the thymus atrophied, and the tonsils thinner than normal. Splenic infarcts also may be present in a low proportion of pigs affected with PCV2-SD. Histopathologic lymphoid lesions are characteristic, showing lymphocytic depletion and granulomatous inflammation, sometimes with the presence of multinucleate giant cells and amphophilic botryoid intracytoplasmic inclusion bodies of different sizes caused by accumulation of PCV2 particles.
Lesions in the lung are common in affected pigs; their severity is influenced by duration of disease and presence of concurrent infections. Gross lung lesions may include failure to collapse, firmness, diffuse pulmonary edema, mottling, and consolidation. Microscopically, a variable degree of lymphohistiocytic interstitial pneumonia to granulomatous bronchointerstitial pneumonia with bronchiolitis and bronchiolar fibrosis can be seen.
Grossly, the liver may appear icteric and/or atrophic in a low proportion of affected pigs. Interlobular connective tissue may be prominent. Microscopic lesions range from single cell necrosis (apoptosis) with mild lymphocytic infiltration of portal zones to extensive lymphohistiocytic periportal hepatitis with diffuse necrosis of hepatocytes. The kidneys may be enlarged and show scattered to diffuse white foci on the cortical surface. Microscopic lesions include interstitial lymphohistiocytic infiltration. Other lesions seen in affected pigs include gastric ulceration (probably due in part to a prolonged fattening period in chronically affected pigs) and occasional multifocal lymphohistiocytic myocarditis. In severely affected pigs, lymphohistiocytic infiltrates can be seen in virtually all tissues.
PCV2-SI pigs do not show gross lesions attributable to PCV2 infection. These animals may show microscopic lymphoid lesions similar to those seen in pigs with PCV2-SD, although only to a mild degree.
In PCV2-RD, stillborn and nonviable neonatal piglets show chronic passive congestion of the liver and cardiac hypertrophy with multifocal areas of myocardial discoloration. The key histopathologic feature is fibrosing and/or necrotizing myocarditis in fetuses.
PDNS is easy to detect from a clinical point of view because of the red-to-dark macules and papules, which correspond microscopically to necrosis and hemorrhage secondary to necrotizing vasculitis of dermal and hypodermal capillaries and arterioles. Necrotizing vasculitis is a systemic feature, but it is more prominent in the skin, renal pelvis, mesentery, and spleen (splenic infarcts may also be present as a result of necrotizing vasculitis of splenic arteries or arterioles). Apart from skin lesions, pigs that die acutely with PDNS have firm, bilaterally enlarged kidneys, with a fine granular cortical surface and edema of the renal pelvis. The renal cortex displays multiple, small, reddish pinpoint lesions, similar to petechial hemorrhages, which microscopically correspond to enlarged and inflamed glomeruli (fibrinonecrotizing glomerulitis). Histologically, a moderate to severe nonpurulent interstitial nephritis with dilation of renal tubules is also seen. Usually, both skin and renal lesions are present, but in some cases, skin or renal lesions may occur alone. Lymph nodes may be enlarged and red due to blood drainage from affected zones with hemorrhages (mainly skin). Histopathologically, PCV2-SD–like lesions such as lymphocyte depletion and histiocytic and/or multinucleate giant cell infiltration (although less severe) are usually found in lymphoid tissues of affected pigs, although to a milder degree.
The PCV2-SD case definition includes three main diagnostic criteria: 1) clinical signs of wasting or ill thrift, 2) presence of gross and microscopic (moderate and severe) lesions characteristic of the disease, and 3) presence of viral antigen or DNA (moderate to high amount) in the microscopic lymphoid lesions. Visualization of viral DNA or antigen in lesions is usually done using in situ hybridization or immunohistochemistry, respectively, and moderate to high amounts of virus are linked to the disease. A herd case definition has been proposed, which includes two main criteria: 1) significant increase of mortality and number of runt pigs or pigs failing to gain weight or thrive in comparison to previous values for the farm, and 2) fulfillment of the three individual criteria listed above in at least one of five examined pigs. Differential diagnoses include conditions causing increased mortality and growth retardation, such as PRRS, chronic respiratory disease, Glässer’s disease, salmonellosis, porcine intestinal adenomatosis, and many others.
Because PCV2 is ubiquitous and the virus replicates in individual pigs for weeks to months, isolation of virus, detection of PCV2 DNA in serum or tissues, or detection of PCV2 antibodies in serum is not sufficient to establish a diagnosis of PCV2-SD. Antibodies against PCV2 may be detected by ELISA, indirect fluorescent antibody, or immunoperoxidase staining of infected cell cultures. Viral isolation can be done on several porcine cell lines (mainly porcine kidney cells) using serum, bronchiolar lavage fluid, or tissue homogenates. Viral DNA can be detected using PCR in most tissues or in serum from affected pigs. Several tissue samples from multiple pigs may be required for detection of virus in cases of chronic disease. Virus quantification in serum by real time quantitative PCR (qRT-PCR) has been suggested as a potential diagnostic method in live pigs. Values of >107 PCV2 genome copies/mL of serum usually have been linked with PCV2-SD occurrence. However, PCV2 infection is extremely common in clinically healthy pigs, and interpretation of positive qRT-PCR results is not always straightforward.
The diagnostic approach of PCV2-SI is of less interest for clinicians, because lack of overt clinical signs plus demonstration of infection by PCR would be enough to establish such a diagnosis. Values of >105 or 106 PCV2 genome copies/mL of serum usually have been linked with the subclinical infection.
The diagnosis of PCV2-RD should include the following criteria: 1) late-term abortions and stillbirths, sometimes with hypertrophy of the fetal heart, 2) extensive fibrosing and/or necrotizing myocarditis, and 3) high concentrations of PCV2 in the myocardial lesions and other fetal tissues. Differential diagnoses for PCV2-RD include PRRS, porcine parvovirus, pseudorabies (Aujeszky disease), leptospirosis, and other diseases that cause late abortions, stillbirths, and weak piglets. So far, there are no formal criteria to diagnose a putative return to estrus associated with PCV2 infection. However, the occurrence of such signs together with evidence of viral circulation during the clinical episode should be demonstrated.
The case definition for PDNS is relatively simple and includes two main criteria: 1) presence of hemorrhagic and necrotizing skin lesions, mainly located on the hindlimbs and perineal area, and/or swollen and pale kidneys with generalized cortical petechiae, and 2) presence of systemic necrotizing vasculitis as well as necrotizing and fibrinous glomerulonephritis. From a diagnostic point of view, detection of PCV2 is not included in the diagnostic criteria.
Differential diagnosis of PDNS depends on the most significant pathologic outcome. Cutaneous manifestations may be confused with classical and African swine fever, swine erysipelas, septicemic salmonellosis, infection with Actinobacillus suis, porcine stress syndrome, transit erythema (urine-soaked floors, chemical burns, etc), and other bacterial septicemias. Differential diagnoses for kidney lesions include classical and African swine fever, swine erysipelas, and septicemic salmonellosis. Serum biochemical analyses may help differentiate PDNS from other diseases; urea and creatinine concentrations are markedly increased.
Because PCV2-SD is a multifactorial disease, effective control measures before the advent of PCV2 vaccines were focused on control or eradication of these triggers. The most widely used control measures were the use of antibiotics to prevent concurrent bacterial infections, improvement of biosecurity and sanitary measures such as isolation of affected pigs and disinfection of pens after their use, decreasing stressors (eg, high stocking density, inadequate ventilation, inadequate temperature control), and control of concomitant viral infections, especially PRRS. Other prevention and control measures used on young pigs before the anticipated time of onset include injection of vitamins, IP injection of serum harvested from finishing pigs, and vaccination against common pathogens.
Currently, control of PCV2-SD as well as PCV2-SI is based on use of PCV2 vaccines. There are four major commercial vaccines worldwide (plus a higher number with regional availability, mainly in southeast Asia). The first commercial vaccine was based on an inactivated PCV2 isolate and was licensed for use in sows and gilts. The same vaccine was later licensed for use in piglets. Subsequently, three more vaccines have been developed, all for use in piglets ~2–3 wk old or older. Two of these are subunit vaccines (PCV2 capsid protein produced in a baculovirus system), and the third is an inactivated virus constructed by replacing the capsid gene of the nonpathogenic PCV1 with that of PCV2. In addition to significantly reducing mortality and runting percentages, these vaccines seem to improve ADWG, batch uniformity, slaughter weight uniformity, and feed conversion rate.
All commercial PCV2 vaccines are based on PCV2a isolates, but cross-protection has been demonstrated against PCV2b. All PCV2 vaccines are able to generate both cellular and humoral immune responses, which are believed to be the key features to control the subsequent PCV2 infection that occurs under field conditions.
No treatment has proved successful for PDNS. Only those epizootic cases with moderate to high morbidity and mortality rates may be important in terms of economic losses. Treatment using a wide range of antimicrobial agents has been unsuccessful. Because the antigen responsible for triggering PDNS is not known, no preventive recommendations are indicated. Importantly, the use of PCV2 vaccines worldwide has significantly reduced occurrence of this condition, emphasizing the putative implication of PCV2 in its pathogenesis.