Journal of Veterinary Diagnostic Investigation Vol. 19 Issue 6, 591-615
Copyright © 2007 by the American Association of Veterinary Laboratory Diagnosticians
Porcine Circovirus Type 2–associated disease: Update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies
Tanja Opriessnig1,
Xiang-Jin Meng and
Patrick G. Halbur
Correspondence: 1Corresponding Author: Tanja Opriessnig, Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, e-mail: tanjaopr{at}iastate.edu
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Abstract
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Porcine circovirus type 2 (PCV2)–associated disease (PCVAD) continues to be an important differential diagnosis on pig farms in the United States and worldwide. Case trend analyses indicate that the incidence of PCVAD is on the rise in the United States. Accurate diagnosis is important in order to implement appropriate intervention strategies. PCVAD can manifest as a systemic disease, as part of the respiratory disease complex, as an enteric disease, as porcine dermatitis and nephropathy syndrome, or as reproductive problems. PCVAD may be only a sporadic individual animal diagnosis; however, PCVAD may also manifest as a severe herd problem accelerated and enhanced by concurrent virus or bacterial infections. This article is intended to discuss the most common disease manifestations, pathogenesis, diagnostic approaches, and intervention strategies associated with PCVAD in North America.
Key Words: Diagnosis • porcine circovirus type 2 (PCV2)–associated disease (PCVAD) • swine
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Introduction
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Porcine circovirus type 2 (PCV2) is now considered one of the most important viral pathogens in the U.S. pig population. Case trend analysis based on submissions to the Iowa State University Veterinary Diagnostic Laboratory indicates a marked increase in PCV2-associated disease (PCVAD) cases in 2006 (Fig. 1). This may be the combined result of increased awareness of the disease, increased case submissions, and true increased incidence of PCVAD in the Midwest.
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Figure 1 Overall trend of porcine circovirus type 2–associated disease (PCVAD) cases submitted to the Veterinary Diagnostic Laboratory at Iowa State University.
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Historical Background
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Porcine circovirus (PCV), a small, nonenveloped, single-stranded DNA virus with a circular genome,133 was first recognized as a contaminant of the continuous porcine kidney cell line PK-15 (ATCC-CCL31) in 1974.136 Under experimental conditions, the PK-15–derived PCV isolate did not produce disease in pigs.5,134 In the late 1990s, a variant strain of PCV was associated with a newly emerged disease syndrome in pigs, which became known as postweaning multisystemic wasting syndrome (PMWS).7 Sequence analyses of the PMWS-associated PCV revealed significant genetic differences compared with the PK-15 cell-derived PCV.10,29,42,88,92 To distinguish the two, the pathogenic PMWS-associated PCV was designated as porcine circovirus type 2 (PCV2) and the nonpathogenic PCV as porcine circovirus type 1 (PCV1).88
The presence of PCV2 can be traced back to 1969 in Belgium (Sanchez R, Nauwynck H, Pensaert M: 2001, Proc Conference of ssDNA Viruses, Plants, Birds, Pigs, and Primates, p. 122), 1970 in the United Kingdom,40 1973 in Ireland,139 1985 in Canada,83 and 1985 in Spain.115 Archived serum samples obtained from slaughterhouses in Belgium were tested for PCV2-specific antibodies by indirect immunoperoxidase monolayer assay (IPMA), and it was found that all samples (50 from 1969, 50 from 1975, and 50 from 2000) were positive for PCV2-specific antibodies (Sanchez R, Nauwynck H, Pensaert M: 2001, Proc Conference of ssDNA Viruses, Plants, Birds, Pigs, and Primates, p. 122).
Sporadic cases of PMWS were retrospectively identified in archived tissues, before the emergence of PMWS in the 1990s, in formalin-fixed tissues from 68 porcine cases that had been submitted to a laboratory in England between 1970 and 1997.40 PCV2-specific nucleic acids were found in 41% (9/22) of the submissions from the 1990s, in 31% (4/13) of the submissions from the 1980s, and in 32% (8/25) of the submissions from the 1970s. Sequence analyses of PCV2 sequences from 5 archived tissues revealed a high sequence identity to PCV2 isolates obtained from a 2000 porcine dermatitis and nephropathy syndrome (PDNS) case, implying that a similar PCV2 isolate has been present in the UK pig population for more than 30 years.40
Archived tissues from 189 pigs and archived sera from 388 pigs collected from 1985 to 1997 in Spain were tested for the presence of PCV2 DNA by in situ hybridization (ISH) and for the presence of PCV2-specific antibodies by IPMA.115 Approximately 41.3% (78/189) of the tissues were found to be ISH positive for PCV2, and 72.7% (282/388) of the sera were found to be IPMA positive, which is indicative of enzootic infection in Spain since 1985.115
PCV2 antibodies were detected in most pig serum samples collected in Northern Ireland from 1973 to 1999.139 The percentage of PCV2-seropositive sera showed an increased incidence in the samples collected in 1988 (100%; 80/80) and 1999 (92.1%; 129/140) when compared with 1973 (69.1%; 56/80), 1981 (61.3%; 49/80), and 1984 (55%; 44/80).139
Archived serum samples obtained from Canadian slaughterhouses were tested by indirect fluorescent assay (IFA) for antibodies to PCV1 and PCV2.83 In 1985, 8% (14/177) of the sera were positive for PCV1 and 13.6% (24/177) were positive for PCV2. In 1989, 41.4% (60/145) of the sera were positive for PCV1 and 72.4% (105/145) were positive for PCV2. In 1997, 38.1% (56/147) of the sera were positive for PCV1 and 66.7% (98/147) were positive for PCV2.83
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Taxonomy
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Both PCV1 and PCV2 are members of the Circoviridae family.137 The Circoviridae family is divided into the genera Circovirus (Circo indicates that the viral genome has a circular conformation) and Gyrovirus (Gyro is a derivation from the Greek work gyrus, meaning "ring" or "circuit"). The genus Circovirus contains the following species: beak and feather disease virus (BFDV), canary circovirus, goose circovirus, pigeon circovirus, PCV1, and PCV2, and tentatively duck circovirus, finch circovirus, and gull circovirus. The genus Gyrovirus contains only chicken anemia virus (CAV).137
Viruses that belong to the Circoviridae family have characteristic virions that exhibit icosahedral symmetry and lack an envelope. The genomes are covalently closed, circular, single-strand DNA molecules, which range in size from 1.8 to 2.3 kb. The genome polarity of CAV is negative sense, whereas those of the other circoviruses are ambisense.137 CAV, PCV2, and BFDV were found to have an icosahedral structure containing 60 capsid protein molecules arranged in 12 pentamer-clustered units.25 Circoviruses are host specific or exhibit a narrow host range, and most of the known circoviruses infect avian species.137 Subclinical infections are common; however, singular circovirus infections are associated with clinical disease in some cases, such as with infectious chicken anemia, psittacine beak and feather disease, and circovirus disease of pigeons. Circovirus infections in several species cause varying degrees of lymphoid depletion and are thought to be immunosuppressive.137
Phylogenetic analyses of PCV1, avian circovirus, plant geminiviruses, and nanoviruses classified PCV1 as most closely related to BFDV and were intermediate between the 2 plant viral groups.95 Furthermore, it has been proposed that a predecessor to PCV1 and BFDV may have originated from a plant nanovirus that infected a vertebrate host and recombined with a vertebrate-infecting RNA virus, most likely a calicivirus.37
Recent research has shown that PCV2 isolates can be grouped into 2 major groups, PCV2 group 1 and PCV2 group 2, which can be further divided into clusters: PCV2 group 1 viruses can be divided into 3 clusters (1A–1C), and the PCV2 group 2 can be divided into 5 clusters (2A–2E).97 Simultaneously with the introduction of the terms PCV2 group 1 and PCV2 group 2, North American laboratories started to group PCV2 field isolates into European-like isolates or PCV2b (falls into PCV2 group 1) and into North American–like isolates or PCV2a (falls into PCV2 group 2; Gagnon CA et al.: 2007, Proc Am Assoc Swine Practitioners 38:535–540). In addition, some North American laboratories report the predicted restriction fragment length polymorphism (RFLP) patterns rather than sequence information, and most isolates fall into 1 of 2 RFLP patterns designated 422 and 321. Isolates with the RFLP pattern 422 typically cluster into PCV2 group 2 (or PCV2a or North American–like isolates), whereas isolates with a 321 RFLP pattern can be either PCV2 group 2 (PCV2a or North American–like isolates) or PCV2 group 1 (PCV2b or European-like isolates). This has led to the use of the term old 321, which is associated with PCV2 group 2, and new 321, which is associated with PCV group 1 that can be verified only by sequencing. Proposed nomenclatures of PCV2 subgroupings are summarized in Table 1.
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Biological and Physical Properties
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The buoyant density of PCV1 in CsCl has been reported to be 1.37 g/cm3 by Tischer et al.136 and 1.36 to 1.37 g/ml by Allan et al.12 The sedimentation coefficient (S) was determined to be 57S when compared with the sedimentation coefficient of a bovine enterovirus.12 PCV1 was found to be stable at pH 3, at 56°C and at 70°C for 15 minutes, and was resistant to inactivation after exposure to chloroform.12 The infectivity of PCV2 was reduced by 1.6 log by pasteurization for 10 hours at 60°C, by 0.75 log by dry heat treatment for 72 hours at 80°C, and by 1.25 log by extreme dry heat treatment for 30 minutes at 120°C.140 PCV2 is readily isolated from tissues that have been stored at –70°C.29
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Transmission
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Transmission of PCV2 is thought to occur through direct contact via oronasal, fecal, and urinary routes.16,82 Direct contact with pigs inoculated with PCV2 42 days previously resulted in the transmission of virus to 3 of 3 control cesarean-derived, colostrum-deprived pigs.16 PCV2 shedding in experimentally infected cesarean-derived, colostrum-deprived pigs was determined by polymerase chain reaction (PCR) on oropharyngeal swabs, nasal swabs, feces, whole blood, and serum.125 All pigs tested were PCV2 DNA positive on nasal swabs, feces, and oropharyngeal swabs 1 day after inoculation, and PCV2 DNA was detected in all samples, with the exception of oropharyngeal swabs (1 of 2 pigs positive) up to 70 days post–PCV2 inoculation. Serum and whole-blood samples were first tested at 7 days postinoculation, at which time they were positive by PCR.125 Tonsillar, nasal, tracheobronchial, urinary, and fecal swabs of pigs with and without severe systemic PCVAD were tested by quantitative real-time PCR, and PCV2 DNA was detected in a high percentage of the samples, leading to the conclusion that PCV2 is most likely excreted through respiratory secretions, oral secretions, urine, and feces of both PCVAD-affected and clinically healthy pigs, with higher viral loads in the PCVAD-affected pigs.124 PCV2 nucleic acids were detected in the digestive tract of pigs with (14/54 intestines and 4/9 feces) and without (3/14 intestines and 16/20 feces) enteric disease by PCR, suggesting fecal-oral transmission of PCV2 via feces.145
Vertical transmission has been demonstrated to occur in individual sows in the field73,96 and experimentally.52 There are reports of vertical intrauterine transfer of PCV2 resulting in viremic or persistently infected piglets at birth.144 PCV2 can also be demonstrated in semen samples. Seven-month-old boars were inoculated intranasally with PCV2.75 PCV2 DNA was detected at the first serum collection day at 4 days postinoculation in serum samples of 3 of the 4 boars, and serum samples were positive up to 35 days postinoculation but negative by 90 days postinoculation. PCV2 DNA was detected as soon as 5 days postinoculation in semen of 2 of the boars and intermittently through 47 days postinoculation in all 4 boars.75 Semen from 98 one-year-old boars from 49 herds in Korea were tested by PCR, and 13 of 98 semen samples were found to be positive for PCV2 by conventional PCR, 26 of 98 semen samples were positive by seminested PCR, and 11 of 98 semen samples were positive by virus isolation.66 The same study also investigated the prevalence of PCV2 in seminal fluid, nonsperm cells, and sperm heads, and it detected the greatest amount of PCV2 DNA in the seminal fluid and nonsperm fraction.66 The frequency of PCV2 DNA in semen from naturally infected boars was found to be low and sporadic, and boars seropositive for PCV2 may have persistent shedding of the virus in semen.84 PCV2 DNA in semen did not appear to affect the percentage of morphologically normal or live sperm cells in PCV2-shedding boars, and boars older than 17.5 months of age did not appear to shed PCV2 DNA in semen.84 Although PCV2 DNA has been demonstrated in semen, experimental confirmation that PCV2 can be transmitted via artificial insemination is lacking to date.
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Disease Terminology
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The first report of disease associated with PCV2 was described as postweaning multisystemic wasting syndrome, or PMWS.44 Since PCV2 is ubiquitous in the pig population and infection does not necessarily equate to disease, a case definition for PMWS was proposed by Sorden.128 Based on this definition, a diagnosis of PMWS required 1) the presence of clinical signs such as wasting, weight loss, and respiratory disease; 2) the presence of the hallmark PCV2-associated microscopic lesions (lymphoid depletion and/or histiocytic replacement of follicles in lymphoid tissues), and 3) PCV2 antigen or nucleic acids associated with the microscopic lesions as determined by immunohistochemistry or ISH.128
Soon it became evident that PMWS described only a portion of the PCV2-associated diseases. For example, large amounts of PCV2 antigen can be found in older pigs or even fetuses and neonates that do not manifest wasting, or PCV2 antigen may be found solely in an organ system other than lymphoid tissues such as lungs or intestines. In 2002, it was proposed that existing abbreviations, including PMWS, should be replaced with porcine circovirus disease, or PCVD.1 PCVD is now used in Europe to summarize diseases associated with PCV2.123
In North America, it was felt that any new term used in connection with PCV2 should include the word associated, which led to the creation and introduction of the term porcine circovirus–associated disease (PCVAD) in March 2006 by the American Association of Swine Veterinarians (AASV). There are several reasons why the term wasting has been eliminated from the case definition and categorizations: 1) wasting is a subjective clinical term typically used in reference to weight loss, 2) weight loss or failure to gain weight is common in animals with a wide variety of different infectious and noninfectious diseases, and 3) the term wasting may incorrectly associate PCVAD with transmissible spongiform encephalopathies observed in cervids. The AASV PCVAD case definition was posted in October 2006 at http://www.aasp.org/aasv/position-PCVAD.htm (accessed February 4, 2007). Based on this case definition, PCVAD can be subclinical or include 1 or more clinical manifestations such as multisystemic disease with weight loss, high mortality (doubling of the mortality rate without introduction of a new known pathogen), respiratory signs, PDNS, enteric signs including diarrhea, and reproductive disorders individually or in combination in a herd or group of pigs.
In the diagnostic database of the Veterinary Diagnostic Laboratory at Iowa State University (ISU VDL) in Ames, Iowa, PCVAD now includes systemic infection (the category in which PMWS now fits), PCV2-associated pneumonia, PCV2-associated enteritis, PCV2-associated reproductive failure, and PCV2-associated PDNS (Table 2). Cases that are categorized in this way may have PCV2-associated lesions that vary in severity from mild to severe, and such descriptions are added to the case reports. Diagnosticians at the ISU VDL generally feel that this classification scheme allows for better and more uniform classification of the different manifestations of PCVAD.
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Table 2 Case trends in porcine circovirus type 2–associated disease based on submissions to the Veterinary Diagnostic Laboratory at Iowa State University.
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Pathogenesis of Pcvad
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The pathogenesis of PCV2 infection and the major cell types that support PCV2 replication are still not fully understood. Lymphoid depletion and lymphopenia in peripheral blood is a consistent feature in pigs that develop clinical PCVAD. Immunohistochemistry (IHC) or ISH techniques demonstrate large amounts of PCV2 antigen or nucleic acids in the cytoplasm of macrophages and dendritic cells replacing lymphocytes in the depleted follicles in lymphoid tissues.3,20,128 However, PCV2 antigen in lymphocytes was only sporadically detected,20 and it is still unknown whether the reduction of lymphocytes in PCVAD-affected pigs is due to reduced production in the bone marrow, reduced proliferation in secondary lymphoid tissues, or increased loss of lymphocytes in the bone marrow, peripheral blood, or secondary lymphoid tissues via virus-induced necrosis or apoptosis.
Despite the presence of PCV2 in macrophages and dendritic cells, recent in vitro studies suggest that monocytic cells may not represent the primary target for PCV2 replication.38 Monocytes and macrophages were tested for the ability to support PCV2 replication in vitro. PCV2 replication in these cell types was not observed; however, PCV2 was not degraded in the cytoplasm of the cells.38 Similarly, no evidence of in vitro virus replication in dendritic cells was found by Vincent et al.138; however, PCV2 did persist in dendritic cells without loss of infectivity or the induction of cell death. It has been speculated that because of their migratory capacity, dendritic cells can provide a vehicle for transport of the virus throughout the host.138
To clarify the role of PCV2 in systemic PCVAD and other clinical manifestations solely attributed to PCV2 infection, an infectious DNA clone of PCV2 was developed.32 Using an infectious DNA clone has ensured the purity and homogenicity of the inoculum in in vivo animal studies and has allowed for genetic manipulation of the virus at the molecular level to evaluate the biological effects of the genetic changes.32,35,36,105 Specific pathogen-free (SPF) pigs infected with the cloned genomic PCV2 DNA developed PCV2-associated lymphoid lesions.32 Evidence of wasting was not observed in the 35-day duration of the study; however, the PCV2 infectious DNA clone research clearly established PCV2 as the cause of the hallmark lymphoid lesions of PCVAD.32 In 2005, a French group similarly demonstrated that SPF pigs inoculated with a cloned PCV2 DNA developed lesions consistent with systemic PCVAD.39 Taken together, the data from the infectious DNA clone work support the hypothesis that PCV2 is essential for the development of PCVAD; however, most strains of PCV2 likely require other cofactors or building blocks to induce the full spectrum of clinical signs and lesions associated with advanced cases of PCVAD.
Evidence from the field supports the thought that PCVAD is multifactorial in causality and that not all pigs that are infected with PCV2 will develop clinical PCVAD (Fig. 2). Factors that are currently thought to influence the outcome of PCV2 infection can be broken down into 4 main areas of focus: virus, host, coinfections, and immune modulation (Fig. 2). Experimental work has further confirmed that at least these 4 components are the key building blocks, identified to date, in the PCVAD models.
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Figure 2 A current model of the understanding of the progression of porcine circovirus type 2 (PCV2) infection toward porcine circovirus–associated disease (PCVAD). Adapted from Dr M. Fenaux, personal communication, 2004.
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Virus-dependent Factors
A high percentage of clinically healthy pigs are known to be infected with PCV2, whereas others develop severe disease. Genetic analyses and sequence comparison of PCV2 isolates to date have failed to fully explain differences in clinical manifestations. The complete genomes of 10 Dutch PCV2 isolates from PCVAD-affected and -unaffected farms were sequenced and when compared were found to have 95.6% to 100% sequence identity.41 No consistent pattern was evident between PCV2 isolates from affected and unaffected pigs, leading the authors to conclude that the differences in clinical manifestations were likely due to something other than the virus.41 Thirty-four PCV2 isolates from eastern Canadian herds with varying clinical manifestations of disease were sequenced and found to be closely related to each other and to Canadian, U.S., European, and Asian strains.76 The complete sequences of 38 PCV2 isolates from PCVAD-affected and -unaffected herds in France were determined, but molecular markers of virulence could not be identified.26 It was concluded that all of the farms studied in Brittany are infected with closely related yet distinct PCV2 isolates and that recent PCVAD outbreaks are most likely not due to the emergence of a new genotype of PCV2.26
It was also demonstrated that a virus strain that persisted for 10 years in an SPF herd in Sweden without causing clinical PCVAD was able to induce systemic disease under experimental conditions in pigs.2,48 When compared with a recent Canadian reference isolate of PCV2 (PCV2-1010), it was found that both isolates were highly virulent in the PCV2-porcine parvovirus (PPV) coinfection model, which led to the overall conclusion that the virulence of the Swedish isolate was indistinguishable from that of the more recent reference Canadian strain.48
Research on the construction and characterization of 2 chimeric infectious DNA clones of PCV1 and PCV2 has provided further insight into the virus-dependent factors.33 The chimeric PCV1–2 DNA clone contained the PCV2 capsid gene cloned in the backbone of the nonpathogenic PCV1. The chimeric PCV1–2 virus induced a strong and specific antibody response to the pathogenic PCV2 capsid antigen and was attenuated (minimal to no lesions, low level and reduced length of viremia, low or nondetectable levels of viral antigen in lymphoid tissues) when inoculated into pigs.33
Two amino acid mutations occurred in the capsid protein of PCV2 after 120 serial passages in cell culture and resulted in attenuation of the virus in vivo.35 Significant differences were observed in the PCV2 genomic copy numbers in serum, and the gross and microscopic lesions in pigs inoculated with the wild-type PCV isolate were more severe than those inoculated with the passage 120 PCV2 isolate.35 This study confirmed that minimal changes in the genome of a PCV2 isolate can markedly alter the virulence of PCV2 viruses.
Possible differences in virulence among Midwestern US field isolates of PCV2 with 98.9% nucleic acid and 96.7% amino acid sequence identities in ORF2 were investigated in an in vivo study reported in 2006.105 PCV2 isolate ISU-40895 was isolated from a case with severe lymphoid depletion and inflammation associated with high amounts of PCV2 antigen consistent with PCVAD. In contrast, PCV2 isolate ISU-4838 was isolated from a case with no PCV2-associated lesions. The in vivo study using SPF pigs confirmed that PCV2 isolates with minimal genomic differences can differ significantly in virulence as measured by levels of virus load in serum and tissues and severity of PCV2-associated lesions.105
Since late 2004, an increase in the incidence and severity and a change in the clinical manifestation of PCV2-associated diseases have been observed in Ontario and Quebec, Canada. Practitioners, diagnosticians, and researchers reported their observation of pathological lesions new to Ontario, including pulmonary edema, granulomatous enteritis, more severe lymphoid depletion with large numbers of circoviral inclusion bodies, and lymphoid necrosis associated with PCV2 antigen. Analysis of PCV2 isolates demonstrated a change in the type.27 Similar observations have been reported in Quebec, Canada (L. Batista, personal communication, 2005; Gagnon CA et al.: 2007, Proc Am Assoc Swine Practitioners 38:535–540). Similar reports of increased incidence and severity of PCVAD associated with a newly emerging genotype were subsequently reported in the United States.19 PCV2 type 1 isolates had not been reported in the United States prior to 2005 but appear to be associated with the 2006 outbreaks of PCVAD in Kansas, North Carolina, and Iowa.19 It is of interest to note that, to date, the PCV2 isolates compared in experimental models or field studies have compared isolates only within but not across types 1 or 2 in the same model.
It is unknown if the increased finding of PCV2 type 1 (PCV2b) is due to a change (increase) in virulence, due to new introduction into the area (i.e., via semen, etc.), or if some other factor X might support increased replication of a previously insignificant PCV2 genotype. The factor X theory is supported by a retrospective cohort study done on 116 British farms between 2003 and 2004 (Green LE, Woodbine KA, Turner MJ: 2005, Proc Intern Conf Animal Circoviruses and Associated Disease, pp. 23–24). The main conclusions from this work were that PMWS behaved as an epidemic moving through a naïve population without obvious causal association between PMWS and PCV2 antigen and antibody and indicative of a novel infectious agent. Similarly, a New Zealand research group found that PMWS can be transmitted via direct and indirect contact from PMWS-affected pigs to healthy pigs, but exposure of naïve pigs to PCV2 alone did not cause disease indicative of a transmissible agent other than PCV2 as the necessary cause of PMWS (Jaros P, McIntyre LH, Morris RS, et al.: 2006, Proc Intern Pig Vet Soc, 19:168).
Host-dependent Factors
Pigs of all breeds seem to be susceptible to PCV2 infection, and clinical PCVAD has been observed in a wide variety of purebred and crossbred pigs submitted to a U.S. diagnostic laboratory (PG Halbur, unpublished data). A cohort study was conducted to investigate a suspected decreased susceptibility to PCV2-associated disease in Pietrain pigs by manipulating the genetics via artificial insemination on 4 PMWS-affected farms.116 Half of the sows were inseminated with Pietrain semen, whereas the remaining sows received the semen that was typically used on these farms. The PCV2-associated disease in the Pietrain offspring did not differ from that observed in other pigs on these farms in terms of PCV2 seroconversion, morbidity, and mortality.116 In contrast, a field study using 2 identical 5000-sow herds with 3 different paternal genetic lines (100% Pietrain, 50% Large White/50% Pietrain, 25% Large White/75% Duroc) demonstrated that, under the circumstances of that field study, host genetics influenced the expression of PCVAD manifest as increased mortality in the offspring of the Large White/Duroc paternal line compared with the offspring of the 2 other lines.79
Host susceptibility and its effect on the outcome of PCV2 infection were recently investigated in a controlled pilot project.99 Three breeds were compared in this study: Duroc, Landrace, and Large White. The incidence of systemic PCVAD based on gross and microscopic lesions was 0% (0/23) in Durocs, 15.8% (3/19) in Landrace, and 0% (0/21) in Large White.99 The purebred Landrace pigs used in this experiment were clearly more susceptible to PCV2-associated diseases as measured by severity of clinical signs and microscopic lesions associated with PCV2.99 Singular experimental infection using the same PCV2 isolate at a similar cell culture passage and dose had failed to induce clinical PCVAD in crossbred SPF pigs.32–36,49,100,101,104,106,107 Another research group experimentally induced systemic PCVAD in colostrum-deprived Landrace/Large White crossbred pigs inoculated with a PCV2 isolate recovered from a subclinically infected Yorkshire pig, implying host- and/or environment-dependant factors associated with the development of PMWS.2
A correlation between the type of adaptive immune response against PCV2 and the level of virus replication provided evidence of host variations in the onset of the adaptive immune response, which may account for the differences in PCV2 replication and clinical manifestation and outcomes of PCV2-associated disease among pigs.90 A recent in vitro study investigating the replication patterns of PCV2 in pulmonary alveolar macrophages found clear differences between macrophages derived from different conventional crossbred pigs suggestive of differences in susceptibility to PCVAD.89 Although clinical and experimental evidence is minimal, further investigation of differences in host susceptibility to PCVAD is warranted.
Effect of Coinfections
Experimental coinfection of pigs with PCV2 and other viruses such as PPV,4,11,55,70,100 porcine reproductive and respiratory virus (PRRSV),6,46,120 or bacteria such as Mycoplasma hyopneumoniae106 has been shown to enhance the amount of PCV2 viral load and PCV2-associated lesions and to increase the incidence of PCVAD. In fact, the enhancing effect of coinfections on PCV2 replication and disease was accidentally detected when gnotobiotic pigs were inoculated with filtered cell culture material and filtered lymphoid tissues from pigs with naturally acquired PCVAD.30 The experimentally inoculated pigs developed clinical PCVAD; however, both PCV2 and PPV were retrospectively detected in the inoculum and in the pigs.
Common coinfections that occurred in 484 US pigs with PCV2-associated systemic disease were summarized in 2002.110 PRRSV was detected in 52% (251/484) of the cases, M. hyopneumoniae in 36% (172/484) of the cases, bacterial septicemia and/or pneumonia in 22% (105/484), swine influenza virus (SIV) in 5.4% (26/484), and singular PCV2 infection in only 2% (9/484) of the cases.110 Aujeszky disease virus infection concurrent with systemic PCVAD was demonstrated in pigs associated with multifocal necrotizing tonsillitis and lymphadenitis.114 Pogranichniy et al.112 performed a case-control study on pigs with a clinical history of wasting and microscopic lesions characteristic of PCVAD and on control pigs without clinical signs or microscopic lesions typical of PCVAD. Among all viruses tested, PCV2, PRRSV, PPV, porcine enterovirus types 1 to 3, SIV, porcine respiratory coronavirus, transmissible gastroenteritis coronavirus (TGEV), porcine endogenous retrovirus, porcine lymphotropic herpesvirus type 1, and bovine virus diarrhea virus, PCV2 had the strongest association with PCVAD. The risk for clinical PCVAD was much higher if the pig was coinfected with PCV2/PRRSV.112
In the authors' opinion, there is no single other copathogen (i.e., agent X) that can be solely attributed to enhancing the severity of PCV2-associated disease and incidence. At this time, it appears more likely that several known and unknown (agent X) pathogenic and nonpathogenic organisms that vary from region to region may be able to trigger progression of PCV2 infection to PCVAD.
Effect of Immune Modulation
Immunostimulation
Studies have demonstrated that immunostimulation may trigger progression of PCV2 infection to disease and lesions characteristic of PCVAD. Krakowka et al.68 reproduced clinical PCVAD in gnotobiotic pigs stimulated with keyhole limpet hemocyanin in incomplete Freund adjuvant and inoculated with PCV2. Based on this initial work, there has been considerable interest and concern about the effect of adjuvanted vaccines on the enhancement of PCV2-induced disease, and there is a growing body of evidence in the literature to support the hypothesis that common vaccine regimens, under the right set of circumstances, may actually enhance PCV2-associated disease.9,71,107 In contrast, others have demonstrated that PCVAD could be induced by PCV2 infection without coinfection or immunostimulation, implying that PCV2 may be a primary pathogen in some cases.13,16,72
In addition to the type of vaccine used, other factors such as the timing of injection of adjuvanted vaccines and the age of the pig at the time of PCV2 infection may also influence the outcome of PCV2 infection. A recent study to determine if the timing of vaccination with a commercially available M. hyopneumoniae vaccine had an effect on PCV2 replication and PCV2-associated lesion severity confirmed differences in PCV2-associated lesions among the treatment groups and concluded that no or minimal PCV2-associated lesions were observed when pigs were vaccinated 2 to 4 weeks prior to expected PCV2 exposure.101
A study was conducted to determine if the adjuvants (as opposed to the antigen) in commercial swine vaccines induce increased replication of PCV2 and increased incidence of PCV2-associated disease and lesions and if there is a difference among adjuvants in this effect.49 The pigs were vaccinated at 4 and 6 weeks of age and were inoculated with PCV2 at 6 weeks of age. Under the conditions of the study, it was found that by the later stages of infection (35 days postinoculation), the pigs vaccinated with the oil-in-water adjuvants had an increased length of PCV2 viremia, increased amount of PCV2 in serum and tissue, and increased severity of lymphoid depletion compared to pigs vaccinated with the aqueous and aluminum hydroxide products.49
Immunosuppression
Twelve gnotobiotic pigs were inoculated with PCV2 at 1 day of age.69 In addition, 4 pigs received cyclosporine orally on a daily basis, and 4 pigs received corticosteroid (triamcinolone aceonide suspension) intramuscularly twice a week. The cyclosporine treatment, but not the corticosteroid treatment, resulted in increased PCV2 replication in tissues and promoted the spread of PCV2 to hepatocytes. Inflammatory reactions typical of PCVAD were absent, although tissues contained a high titer of virus, which led to the conclusion that granulomatous inflammatory lesions are immune mediated.69 In another study, 7 cesarean-derived, colostrum-deprived pigs were inoculated with PCV2 intransally and intraperitoneally.54 Three of the 7 pigs were treated with dexamethasone at 8 days of age and developed granulomatous lyamphadenitis, which was not observed in pigs inoculated with PCV2 alone. It was concluded that dexamethasone treatment influenced PCV2 infection of lymphoid tissues and that suppression of the cell-mediated immunity may play a role in the etiology of PCVAD.54
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Manifestations of Pcvad
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PCVAD in an individual pig is diagnosed by the presence of characteristic microscopic lesions that are associated with a moderate-to-abundant amount of PCV2 antigen. To distinguish the different manifestations of PCVAD, it is important to evaluate intestines, lungs, and lymphoid tissues by immunohistochemistry for the presence of PCV2 antigen. Furthermore, on a herd basis, there is a distinction between sporadic PCVAD and PCVAD manifest at a level that it is considered a herd problem. It has been proposed that PCVAD is a herd-level problem if there is an increase in mortality of equal to or more than the mean of historic levels plus 1.66 times the standard deviation (Segalés J: 2006, Proc Am Assoc Swine Veterinarians: PCV2/PMWS seminar 12, 37:1–7). Alternatively, the chi-square test may be used to determine if the current mortality is higher than that of previous periods. If there is no historical herd mortality data available, an increase in mortality that exceeds the national or regional level by 50% is thought to be indicative of PCVAD at the herd level. In summary and for simplification, PCVAD diagnosis on a herd basis takes into account 1) the percentage of pigs diagnosed with PCVAD from all submitted pigs and 2) an increase of mortality on the particular farm investigated. In general, if PCVAD is diagnosed in 50% or more of the pigs from a representative sample of the farm and if there is a significant increase in mortality compared to previous herd parameters, PCVAD is considered to be a herd problem. If PCVAD is diagnosed in less than 50% of the pigs in a representative sample of the farm with a concurrent increase of mortality or if PCVAD is diagnosed in more than 50% of the pigs in a representative sample of the farm without concurrent increase in morality, then this is considered as sporadic PCVAD.
Subclinical Pcv2 Infection
A diagnosis of subclinical PCV2 infection implies that although PCV2 is present, it is not responsible for the disease observed in the pig (i.e., low amounts of PCV2 antigen associated with no to minimal lesions). Based on experimentally PCV2-inoculated pigs, it is known that PCV2 infection and lesions can be limited to 1 or 2 lymph nodes in a pig without causing any apparent clinical problems.102,106 However, it also has been demonstrated experimentally that subclinical PCV2 infection may be associated with decreased vaccine efficacy.104
PCV2-associated necrotizing lymphadenitis in individual lymph nodes in clinically healthy pigs has also been described.61,102 The main lesion is follicular necrosis in the center of prominent lymphoid follicles and is usually restricted to 1 or 2 lymph nodes. The significance of this is unknown, other than in cases in which carcasses with large lymph nodes are condemned or deemed unfit for human consumption at the slaughterhouse and submission of samples to diagnostic laboratories confirms necrotizing lymphadenitis.
Pcv2-associated Systemic Infection
The primary clinical signs include weight loss or decreased rate of weight gain, paleness or icterus, and gauntness and ill thrift (Fig. 3). The infected pigs may also experience labored respiration with coughing and dark-colored diarrhea. Macroscopic lesions include but are not limited to enlarged lymph nodes. Lungs often fail to collapse and are mottled tan, and in chronic cases, the kidneys may have white streaks or spots. Less commonly, gastric ulceration can also be observed. As the name implies, systemic PCV2 infection is characterized by lymphohistiocytic to granulomatous inflammatory lesions in lymphoid tissues (Fig. 4) and/or lung, liver, kidney, heart, and intestines. Scoring systems for lesion severity and virus antigen estimation have been described (Table 3; Fig. 5).106 One scoring system uses 7 lymphoid tissues and accounts for severity of lesions, amount of PCV2 antigen, and distribution of lesions. In brief, each lymphoid tissue (tracheobronchial lymph node, mesenteric lymph node, mediastinal lymph node, superficial inguinal lymph node, external iliac lymph node, tonsil, and spleen) is given a score ranging from 0 to 9 (lymphoid depletion score 0–3, granulomatous inflammation score 0–3, PCV2 IHC score 0–3). The individual scores of the 7 lymphoid tissues are added together and divided by 7. Depending on the final number, a pig is categorized as having no PCV2-associated lesions (score = 0), mild PCV2-associated lesions (score = 1–3), moderate PCV2-associated lesions (score = 4–6), or severe PCV2-associated lesions (score = 7–9). This scoring system has been useful for classification of the severity of experimentally induced PCVAD; however, it is unrealistic to expect this to be done on field cases, as an incomplete set of lymphoid tissues is typically submitted. The authors propose that to diagnose systemic PCV2 infection, it is necessary to at least demonstrate PCV2 antigen in more than 1 lymphoid tissue (lymph node, tonsil, spleen) or in 1 lymphoid tissue (lymph node, tonsil, spleen) and at least 1 other organ system (i.e., lung, liver, kidney, intestines) or in 2 two organ systems such as lung, liver, kidney, intestines. If abundant PCV2 antigen is associated with only 1 specific organ system, it should be referred to as PCV2-associated respiratory disease, PCV2-associated enteritis, or PCV2-associated reproductive failure rather than PCV2-associated systemic infection. If there is a limited amount of PCV2 antigen present but the lesions are severe, this is consistent with severe chronic PCVAD. With combined scoring of lesions and the amount of PCV2 antigen, it may also be possible to draw conclusions on the stage of infection (Table 4).
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Figure 3 Eight-week-old pig experimentally coinfected with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) showing icterus and a poor body condition typical of systemic porcine circovirus–associated disease (PCVAD).
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Figure 4 Lymph node, pig, field case. Porcine circovirus type 2 (PCV2)–associated lymphoid depletion and histiocytic-to-granulomatous replacement of the follicle with multinucleated giant cells in the center. Hematoxylin and eosin. Inset, abundant PCV2 antigen (brown staining), immunohistochemistry. Streptavidin-biotin peroxidase complex method, hematoxylin counterstain.
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Table 3 Scoring system for the severity of porcine circovirus type 2 (PCV2)–associated lymphoid lesions and amount of PCV2 antigen.
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Figure 5 Grading of porcine circovirus type 2 (PCV2) antigen (brown staining) associated with PCV2-induced lymphoid depletion in the tonsils. Immunohistochemistry (IHC). Streptavidin-biotin peroxidase complex method, hematoxylin counterstain. The severity of lesions and amount of antigen ranges from none (IHC score of 0) to severe (IHC score of 3).
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Table 4 Relative timing of porcine circovirus type 2 (PCV2)–associated lymphoid lesions (depletion, histiocytic-to-granulomatous inflammation, and amount of PCV2-antigen ranging from 0 to 3) based on observations in pigs experimentally infected with PCV2.
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Pcv2-associated Respiratory Disease
Recent field investigations45,63 and case trend analysis at U.S. diagnostic laboratories (Table 2) suggest that PCV2 may play an important role in the porcine respiratory disease complex (PRDC). PRDC is a condition observed mainly in 8- to 26-week-old pigs and is associated with multiple respiratory pathogens including PRRSV, SIV, and M. hyopneumoniae. PRDC is characterized by a decreased rate of growth, decreased feed efficiency, anorexia, fever, cough, and dyspnea. There may be diagnostic overlap between PCV2-associated systemic infection and PCV2-associated pneumonia. The presence of prolonged and unusually severe clinical respiratory disease, granulomatous bronchointerstitial pneumonia with bronchiolitis and bronchiolar fibrosis, and abundant PCV2 antigen associated with the lesions is suggestive that PCV2 may play a role in the PRDC problem.
Interstitial pneumonia with bronchiolitis was reported in the first cases of PMWS (Clark EG: 1997, Proc Am Assoc Swine Practitioners 28:499–501).29 PCV2-associated pneumonia is characterized by lymphohistiocytic to granulomatous interstitial pneumonia, peribronchiolar fibroplasia, and mild-to-severe necrotizing and ulcerative bronchiolitis (Fig. 6). The PCV2-associated bronchiolitis lesions can resemble those induced by swine influenza virus or porcine respiratory coronavirus.
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Figure 6 Lung, pig, experimentally infected with PCV2. A, moderate peribronchiolar lymphohistiocytic infiltration and mild necrotizing and ulcerative bronchiolitis. Hematoxylin and eosin. B, PCV2 antigen (brown staining) within the cytoplasm of macrophage-like cells, immunohistochemistry. Streptavidin-biotin peroxidase complex method, hematoxylin counterstain.
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Bronchointerstitial pneumonia was reproduced in addition to the hallmark lymphoid lesions of PMWS in conventional pigs inoculated with PCV2.82 Mild respiratory disease and multifocal interstitial pneumonia were induced in cesarean-derived, colostrum-deprived pigs inoculated with PCV2.16 Mild interstitial pneumonia and lymphoplasmacytic rhinitis was induced in conventional pigs inoculated with PCV2.120 Mild granulomatous bronchointerstitial pneumonia was reproduced in conventional pigs using an infectious DNA clone of PCV2.32
Pcv2-associated Enteritis
Cases of PCV2-associated enteritis are increasingly common. Most of the PCV2-associated enteritis field cases are from 8- to 16-week-old pigs. PCV2-associated enteritis cases often clinically and grossly resemble subacute or chronic ileitis associated with Lawsonia intracellularis. The intestinal mucosa is grossly thickened, and mesenteric lymph nodes are enlarged.51 Microscopic examination confirms the presence of granulomatous enteritis, which is usually associated with abundant PCV2 antigen by IHC staining (Fig. 7).
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Figure 7 PCV2-associated enteritis. A, PCV2 antigen (brown staining) within lymphocytes and macrophage-like cells in the lamina propria and Peyers patches of the ileum. Immunohistochemistry. Streptavidin-biotin peroxidase complex method, hematoxylin counterstain. B, Thickened intestinal mucosa and a markedly enlarged mesenteric lymph node. C, Grow-finish pig with mild diarrhea.
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PCV2-associated enteritis was diagnosed in 6 weanling pigs by histopathology, PCV2 isolation, and PCV2 ISH64 from a herd that was apparently free of PMWS and PDNS. The affected pigs had no lymphoid depletion or histiocytic replacement of follicles in lymphoid tissues. The authors proposed that diagnosis PCV2-associated enteritis occurs only 1) if diarrhea is present, 2) if characteristic lesions are present in Peyer patches but not in other lymph nodes, and 3) if PCV2 antigen or nucleic acids are present within the lesions.64
Pcv2-associated Reproductive Failure
There have been several reports of PCV2-associated reproductive failure73,96 since the original report of West et al.144 in western Canada in 1999. Clinical manifestations on affected farms include more abortions, stillbirths, and fetal mummification (Fig. 8) and increased preweaning mortalities. Affected herds are typically gilt startups or new populations. Nonsuppurative to necrotizing or fibrosing myocarditis associated with abundant PCV2 antigen (Fig. 8) is the hallmark lesion in stillborn and neonatal pigs from field cases.91
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Figure 8 Porcine circovirus-associated reproductive failure. A, Myocardium, fetus. Separation of myocardiocytes by edema and low numbers of inflammatory cells. Hematoxylin and eosin. B, PCV2 antigen (brown staining) within the cytoplasm of myocardiocytes, immunohistochemistry. Streptavidin-biotin peroxidase complex method, hematoxylin counterstain. C, Affected litter showing fetuses at different stages of mummification and maceration.
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Experimental intrauterine infection of fetuses with PCV2 resulted in virus replication in the fetuses and supports the heart as the primary site of PCV2 replication in fetuses.122 When fetuses were inoculated in utero on 57, 75, and 92 days of gestation, it was found that virus replication was significantly higher in fetuses inoculated at 57 days of gestation compared to those inoculated at 75 and 92 days of gestation. All fetuses were killed at 21 days postinoculation and, lesions (edema, enlarged livers, congestion) were observed only in the fetuses inoculated at 57 days of gestation.122 In another study, 37 fetuses from 3 sows were inoculated at 86, 92, and 93 days of gestation intramuscularly, and at parturition, 24 normal pigs and 13 mummified, stillborn, or weak-born pigs were observed, confirming that PCV2 can infect late-term fetuses and cause reproductive abnormalities.52 Although evidence is convincing that PCV2 is a reproductive pathogen, data from field cases suggest that most breeding herds are apparently immune and PCV2-associated reproductive failure is relatively rare.
Pcv2-associated Pdns
PDNS is characterized clinically by acute onset of skin lesions (raised purple skin lesions progressing to multifocal raised red-purple scabs with black centers most prominent on the rear legs; Fig. 9), fever, and lethargy. PDNS is often fatal. Other characteristic macroscopic lesions include enlarged tan, waxy-appearing kidneys with petechial hemorrhages. Microscopically, there is systemic vasculitis with dermal and epidermal necrosis and necrotizing and fibrinous glomerulonephritis. The hallmark microscopic lesions of PDNS, generalized vasculitis and glomerulonephritis, are suggestive of a type 3 hypersensitivity reaction, which is characterized by deposition of antigen-antibody aggregates or immune complexes at certain tissue sites. Several pathogens including viruses (PRRSV)22,130 and bacteria (Pasteurella multocida, Streptococcus suis type 1 and 2, Escherichia coli, Proteus sp., Haemophilus parasuis, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Arcanobacterium pyogenes, Staphyloccoccus aureus, or Salmonella sp.)74,131 have been incriminated as possible etiologies for PDNS.
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Figure 9 Twelve-week-old pig suffering from porcine dermatitis and nephropathy syndrome (PDNS). The perineal region, ventral abdomen, and legs are covered by raised coalescing red-purple lesions.
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The association of PCV2 with PDNS was first reported in 2000.119 Investigation of PDNS cases observed in Northern Ireland in 1990, which at the time was PRRSV free, demonstrated the presence of PCV2 antigen associated with granulomatous lymphadenitis.8 A recent case-control study investigating PDNS in the Netherlands found that there was a significant association of high anti-PCV2 antibody titers to PCV2 and the development of PDNS.141 The authors were not able to show PCV2 antigen by IHC in all of the PDNS cases, but they were able to confirm the presence of PCV2 DNA by PCR in all cases of PDNS. Importantly, the authors were able to show that porcine parvovirus or PRRSV nucleic acids were not present in many of the PDNS cases as determined by PCR.141 A study comparing PCV2 serum viral load in PMWS and PDNS cases found that PDNS cases had significantly lower numbers of PCV2 DNA in serum compared to healthy, subclinically PCV2-infected pigs.98 This study further confirms that PDNS pigs are infected with PCV2. However, to the authors' knowledge, no one has yet experimentally reproduced PDNS.
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Diagnostic Approaches and Diagnostic Tools Available for Pcvad
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PCV2 infection is ubiquitous in the global pig population. PCV2 can be found in healthy pigs as well as in diseased pigs.3 This makes the choice and interpretation of diagnostic tests important for confirmation of PCVAD. PCVAD (systemic infection, enteritis, pneumonia, PDNS, abortion) is diagnosed by demonstration of characteristic lesions associated with PCV2 antigen or nucleic acids in the respective organs. Currently, IHC or ISH are considered the gold standard for detecting PCV2 as part of the diagnosis of PCVAD.128
Detection of Anti-pcv2 Antibodies by Indirect Fluorescent Antibody, Ipma, Enzyme-linked Immunosorbent Assay, and Serum Virus Neutralization Assays
Serology is best used on a herd basis to determine the time of PCV2 infection by sequential or cross-sectional analyses of the population. Serological studies have found that PCV2 antibodies are present globally in almost all swine herds tested and in up to 100% of individual pigs within herds.83,108,139 Most US breeding herds and most of the sows within those herds were found to be seropositive for PCV2.108 The mean PCV2 antibody half-life in weanlings was estimated to be 19.0 days, and the window for PCV2-passive antibody decay within a population is relatively wide. Passively acquired anti-PCV2 antibodies present at 10 to 12 days of age were found to decay below enzyme-linked immunosorbent assay (ELISA) cutoff levels by approximately 4.9 ± 1.2 weeks of age in piglets with low levels of antibodies at weaning, by approximately 8.1 ± 1.9 weeks of age in piglets with moderate levels of antibodies at weaning, and by approximately 11.1 ± 2.5 weeks of age in piglets with high levels of passive antibodies at weaning.108 Although PCV2 is widespread, PCV2-naïve populations and subpopulations such as young boars within boar studs exist and have been found to be susceptible to PCVAD.103 Early identification of such naïve populations by serology may be useful to determine appropriate strategies to reduce the risk of subsequent PCV2 exposure.
Indirect Fluorescent Antibody Assay
This assay is not automated and is subjective. Indirect fluorescent antibody (IFA) assays for PCV2 have been described in the literature.7,111,132 Recently, an IFA assay based on an open reading frame (ORF) 2 protein has been described,113 and it was found that the regular whole PCV2-based IFA assay had only a 57.1% relative sensitivity compared to the ORF2 protein-based IFA assay.113 Immunofluorescence assays have also been described for antibodies against nonpathogenic PCV1 and appear to be specific.36 Studies have shown that there is a low level of cross-reactivity between PCV1 and PCV2 on the IFA test.7,111
Ipma
This assay is also not automated, and end points are determined subjectively. IPMA for PCV2 is widely used.14,29 Interlaboratory testing comparing IFA and IPMA results on the same 20 serum samples performed in different laboratories in Europe and Canada found a wide variation in titers among laboratories.85 In general, IPMA gave higher titers than IFA, and paraformaldehyde used as fixative gave higher titers than did acetone or ethyl alcohol.85
Elisa
The ELISA is a sensitive technique for detecting and measuring serum antibodies. There are several publications describing PCV2 ELISAs.14,77,93 Recently, commercially available IgG (Ingezim PCV IgGa) and IgM PCV2 ELISAs (Ingezim PCV IgMa) have been introduced in Europe. A comparison of IgG and IgM values might be useful for determining the timing of PCV2 infection: IgM value 
IgG value: early active infection (within the first 21 days postinoculation); IgM value < IgG value: active infection (approximately between 20 and 50 days postinoculation); high IgG value and negative IgM value: late or resolving infection of convalescent (approximately 2 months after infection; Segalés J, Rodríguez J, Resendes A, et al.: 2005, Proc Intern Conf Animal Circoviruses and Associated Disease, p. 61). A variant of the regular ELISA is the competitive (blocking) ELISA.139 A competitive ELISA specific for PCV2 antibodies is available in Europe (SERELISA PCV2 Ab Mono Blockingb). This ELISA can also be used to detect PCV2-specific antibodies in feces (Lopez P, Guillossou S, Deshaies E, et al.: 2005, Proc Intern Conf Animal Circoviruses and Associated Disease, p. 91).
Serum-virus Neutralization Assay
Neutralizing antibody assays for PCV2 have been described in the literature.90,111 Neutralizing antibodies were detected between 1590 and 28111 days after PCV2 infection and were correlated with protection or clearance of PCV2 infection in gnotobiotic pigs.90 Since PCV2 does not induce a visible cytopathic effect in infected cells, the serum-virus neutralization assay requires either fluorescent antibody (FA) or immunoperoxidase staining at the end of testing to determine the presence or absence of virus replication. Requirements for pretreatment of cells to synchronize the cell cycles make serum virus neutralization tests hard to perform, and end-point titers may be less accurate. Typically, the percentage reduction is used to assess the neutralizing activit
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Abstract
Introduction
