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Link to information on (Jan 2003)

National Pork Board
PMWS Fact Sheet

A PMWS/PDNS fact sheet produced by the National Pork Board and the AASV (October 2002)

Link to information on both diseases on (Oct. 2002)
About PDNS
Porcine Dermatitis and Nephropathy Syndrome - By Jake Waddilive MA, VetMB, MRCVS - March 2001

PMWS & Porcine Circovirus
Dr Geoff Gard, CAHNet Update: Information on the disease plus results of a survey of veterinarians - Spring 1999 (Canada).

PMWS: A New Condition Affecting Pigs
CAHNet - A useful, if rather old article on the disease. Provides a good background to the disease - November 1997

Porcine Circovirus type 2
Dr. Igor Morozov, Iowa State University, Veterinary Medi- cal Research Institute
Emerging Disease Notice
PMWS: Centre for Emerging Issues (CEI)

Emerging Disease Notice
PDNS: Centre for Emerging Issues (CEI), March 2001

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Published Information & Scientific Data
(Most recent first)

Published: Veterinary Research Communications, Volume 30, Number 2, February 2006 (Abstract)
Serological Investigation and Genomic Characterization of PCV2 Isolates from Different Geographic Regions of Zhejiang Province in China
Zhou JY, Chen QX, Ye JX, Shen HG, Chen TF, Shang SB.
Sera collected from 46 swine farms in Zhejiang province were evaluated for the presence of antibodies to PCV2 using an indirect-fluorescent antibody procedure. In addition PCV2 isolated from superficial inguinal lymph node samples collected from 40-to 90-day-old pigs with clinical signs of post-weaning multisystemic wasting syndrome (PMWS) using the PK-15 cell line were sequenced and compared. Overall seroprevalence of PCV2 antibody averaged 58.34% for all samples. Breakdown of serology by groups was as follows: 59.38% for sows, 57.41% for post-weaning piglets, 44.83% for Landrace sows and 64.28% for Landrace piglets. The seroprevalence of Landrace sows was higher than that of Yorkshire and Duroc sows, but non-significant (p > 0.05). Serological analysis also showed that seroprevalence of PCV2 antibody was a negative correlation to that of PRRSV antibody. The complete genomes of five PCV2 isolates identified in the herds with PMWS consisted of 1767nt, containing the 11 potential ORFs. Genome of the virus isolates shared 93.8% to 99.8% identity with PCV2 reference strains from GenBank, 76.6% to 77.9% identity with PCV1. Phylogenetic analysis indicated that there were two subgenotypes within PCV2: subgenotype I (1767 nt) and subgenotype II (1768 nt).

Published: Virology - January 2006 (Abstract)
Inhibition of porcine circovirus type 2 replication in mice by RNA interference.
Liu J, Chen I, Chua H, Du Q, Kwang J.
Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome (PMWS) for which no antiviral treatment is available. To exploit the possibility of using RNA interference (RNAi) as a therapeutic approach against the disease, plasmid-borne short hairpin RNAs (shRNAs) were generated to target the PCV2 genome. Transfection of these shRNAs into cultured PK15 cells caused a significant reduction in viral RNA production that was accompanied by inhibiting viral DNA replication and protein synthesis in infected cells. The effect was further tested in vivo in a mouse model that has been developed for PCV2 infection. Mice injected with shRNA before PCV2 infection showed substantially decreased microscopic lesions in inguinal lymph nodes compared to controls. In situ hybridization and immunohistochemical analyses showed that shRNA caused a significant inhibition in the level of viral DNA and protein synthesis detected in the lymph nodes of the treated mice relative to the controls. Taken together, these results indicate that shRNAs are capable of inhibiting PCV2 infection in vitro as well as in vivo and thus may constitute an effective therapeutic strategy for PCV2 infection

Published: Vet Journal 171(1):166-8. - January 2006 (Abstract)
Identification of porcine circovirus type 2 in retrospective cases of pigs naturally infected with porcine epidemic diarrhoea virus.
Jung K, Ha Y, Ha SK, Kim J, Choi C, Park HK, Kim SH, Chae C.
The identification of porcine circovirus type 2 (PCV2) was studied in fresh intestinal tissues by polymerase chain reaction (PCR) and in formalin-fixed, paraffin-wax-embedded intestinal tissues by in situ hybridisation. The tissues came from pigs naturally infected with porcine epidemic diarrhoea virus (PEDV). A total of 35 (32.7%) of 107 small intestinal samples from pigs naturally infected with PEDV were found to be positive using PCR. Positive signals for PCV2 were detected in 32 (29.9%) of 107 small intestinal samples from pigs naturally infected with PEDV by in situ hybridisation. The distribution of positive cells in the jejunum and ileum was multifocal or patchy. Distinct positive labelling was found throughout the lamina propria in the small intestines. The results of this study indicate that PCV2 is highly prevalent in pigs naturally infected with PEDV.

Published: J Comp Pathol January 2006 (Abstract)
Cardiovascular lesions in pigs naturally or experimentally infected with porcine circovirus type 2.
Opriessnig T, Janke BH, Halbur PG.
Abundant intracytoplasmic porcine circovirus type 2 (PCV2) was associated with myocardiocyte swelling or necrosis, or myocardial fibrosis (or both) in three naturally infected pigs aged 4-7 weeks from three different farms. One 6 week old pig from a fourth farm had severe diffuse segmental to circumferential lymphohistiocytic and plasmacytic periarteritis and endarteritis in several organs, PCV2 antigen was demonstrated in endothelial cells, and inflammatory cells in the arterial walls. In three pigs experimentally infected with PCV2, viral antigen was also associated with obliterated blood vessels in areas of granulomatous and necrotizing lymphadenitis. Together these findings suggest that the cardiovascular system in general and endothelial cells in particular play an important role in the pathogenesis of PCV2-associated diseases.

Published: Acta Vet Hung. - 53(3):385-96. September 2005 (Abstract)
Postweaning multisystemic wasting syndrome (PMWS) in pigs in Croatia: detection and characterisation of porcine circovirus type 2 (PCV2).
Lipej Z, Segales J, Toplak I, Sostaric B, Roic B, Lojkic M, Hostnik P, Grom J, Barlic-Maganja D, Zarkovic K, Oraic D.
Croatian Veterinary Institute, Zagreb, Savska c 143, Croatia.
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.

Published: Veterinary Immunology and Immunopathology - Volume 107, Issues 3-4, Pages 303-313 - 15 September 2005 (Abstract)
Porcine dermatitis and nephropathy syndrome (PDNS) is associated with a systemic cytokine expression profile indicative of proinflammation and a Th1 bias
Sipos W, Duvigneau JC, Pietschmann P, Schilcher F, Hofbauer G, Hartl RT, Schmoll F.
Porcine dermatitis and nephropathy syndrome (PDNS) is broadly discussed as a porcine circovirus type 2 (PCV2)-associated disease, although PCV2, in contrast to postweaning multisystemic wasting syndrome (PMWS), has to date not been proven to be the aetiologic agent. In order to better understand the complex immunopathology of PDNS, the systemic cytokine expression profiles of (i) five pigs suffering from PDNS, (ii) five animals suffering from naturally acquired PMWS and (iii) five controls were investigated at mRNA and protein levels by means of multiplex real-time RT-PCR and flow cytometric intracellular cytokine detection, respectively. IL-1a, IL-6 and IFN-? mRNA expressions were found to be elevated in PDNS pigs. At the protein level, an increased capacity of peripheral blood mononuclear cells to produce IL-2, IL-4, IL-6, IL-12, TNF-a and IFN-? was evident. Hematological investigations revealed a hypochromic anemia while basophils and monocytes were relatively and neutrophils absolutely increased in PDNS pigs. PCV2 antibody levels did not differ significantly between PDNS and PMWS affected animals. Taken results together, the cytokine profile of the PDNS affected animals together with hematological data pointed towards a proinflammatory condition supporting a Th1 bias. Cytokine data of PMWS affected animals exhibited only minor non-significant differences when compared to controls, only IL-10 was significantly decreased at the mRNA level.

Published: Journal of Biotechnology - Volume 118, Issue 2, Pages 201-211, 4 August 2005 (Abstract)
In vitro expression, monoclonal antibody and bioactivity for capsid protein of porcine circovirus type II without nuclear localization signal
Ji-Yong Zhou, Shao-Bin Shang, Hui Gong, Qing-Xin Chen, Jian-Xiang Wu, Hui-Gang Shen, Ting-Fei Chen and Jun-Qing Guo
We expressed firstly the Capsid protein gene defecting the nuclear localization signal (NLS) of Porcine circovirus type II (PCV2) in Escherichia coli as a fusion protein with glutathione S-transferase (rGST-dCap protein). The purified rGST-dCap protein and the recombinant NLS-defected Cap protein of PCV2 (rdCap protein) from the purified rGST-dCap protein reacted specifically with swine antiserum to PCV2. Furthermore, the obtained monoclonal antibodies (mAbs) to rdCap protein were shown to bind to PCV2 particles replicated in PK15 cell and capsid protein (Cap protein) of PCV2 expressed in PK15 cells, respectively. mAbs to rdCap protein also revealed the neutralizing ability to PCV2 particles. These results demonstrated that rGST-dCap protein expressed in E. coli was folded correctly or at least partly, and mAbs to rdCap protein possessed the binding epitopes of PCV2 particles whereas mAbs 4C4 and 3F6 to rdCap protein remained the neutralization epitope of PCV2 particle, showing a possibility of neutralizing mAb to rdCap protein as an immnuotherapeutic agent and a potential of rGST-dCap protein as a vaccine antigen or serodiagnostic reagent.

Published: Veterinary Microbiology - Volume 109, Issues 3-4, Pages 179-190 0 August 2005 (Abstract)
Immunogenicity of a recombinant pseudorabies virus expressing ORF1-ORF2 fusion protein of porcine circovirus type 2.
Ju C, Fan H, Tan Y, Liu Z, Xi X, Cao S, Wu B, Chen H.
Laboratory of Animal Virology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, PR China.
Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS). Pseudorabies (PR) is also an important infectious disease in swine and sometimes co-infect with PCV2. An attenuated pseudorabies virus (PRV) has been successfully used as a vector for live viral vaccines. In this study, a recombinant PRV expressing ORF1-ORF2 fusion protein of PCV2 was constructed and its immunogenicity was tested in mice and pigs. The ORF1 and partial ORF2 gene of PCV2 Yu-A strain were amplified by PCR and inserted into a transfer vector. The recombinant transfer plasmid was co-transfected with the EcoRI digested genome of vector virus (PRV TK(-)/gE(-)/LacZ(+)) into IBRS-2 cells. The recombinant pseudorabies virus PRV-PCV2 was purified by plaque purification and identified by PCR and Southern blotting. Expression of the ORF1-ORF2 fusion protein by the recombinant PRV-PCV2 virus was demonstrated by Western blotting analysis. The growth properties of the recombinant virus in cells were similar to that of the parent vector virus. In animal experiments, PRV-PCV2 elicited strong anti-PRV and anti-PCV2 antibodies in Balb/c mice as indicated by PRV-neutralizing assay, anti-PCV2 ELISA and PCV2 specific lymphocyte proliferation assay, respectively. And PRV-PCV2 immunization protected mice against a lethal challenge of a virulent PRV Ea strain. In pigs, PRV-PCV2 elicited significant immune response towards PRV and PCV2 as indicated by PRV-ELISA, PRV neutralizing assay and PCV2 specific lymphocyte proliferation assay, respectively. This is a first step toward the development of a potential candidate divalent vaccine against PRV and PCV2 infections.

Published: Vet Microbiol - Volume 109, Issues 3-4, Pages 169-177, August 2005 (Abstract)
Comparative genetic characterization of Porcine Circovirus type 2 samples from German wild boar populations.
Knell S, Willems H, Hertrampf B, Reiner G.
Department of Swine Diseases, University of Giessen, Frankfurter Strasse 112, 35392 Giessen, Germany.
PCV-2 is involved in "postweaning multisystemic wasting syndrome" (PMWS), "porcine dermatitis and nephropathy syndrome" (PDNS), respiratory and reproductive disorders, and thereby plays a crucial role in today's swine production world-wide. The virus is apparently ubiquitous in domestic pigs and has also been demonstrated in wild pigs. Up to now, a characterization of PCV-2 samples from wild pigs, which might help to estimate the possible role of wild pigs as sources of domestic pig infection, has not been carried out. Spleen samples from 16 PCV-2-positive wild pigs from hunting grounds of four regions in Germany were used for the analysis of the viral genome. In one sample, the complete sequence of the genome was determined. In the other, a 742 nucleotide fragment from the highly variable capsid sequence of the ORF2 was sequenced. Analysis of the sequences led to the identification of three PCV-2 strains. One strain, representing 14 of the 16 samples, was closely related with Chinese, but not with German strains. The genome of this strain was shortened by one nucleotide by a deletion close to the end of ORF2. The deletion led to a shift of the stop-codon and to the insertion of a further codon. Two further strains differed in up to 4.7% of nucleotides and up to 10.5% of amino acids (aa). These strains were aligned with clusters of PCV-2 samples from mainly French and German domestic origin.

Published: The Veterinary Journal - Volume 170, Issue 1, Pages 132-134 - July 2005 (Abstract)
PMWS: an emerging disease identified in archived porcine tissues
S. Staebler, T. Sydler, E. Buergi, K. McCullough, F. McNeilly, G. Allan and A. Pospischil
Postweaning multisystemic wasting syndrome (PMWS) is a disease caused by porcine circovirus type 2 (PCV-2). The disease was present as early as 1986 in Spain, 1989 in Japan and 1993 in Thailand. In view of this, we considered it possible that the disease may also have been present in Switzerland prior to its first description in 2001. A retrospective investigation was performed on paraffin-embedded lymphoid organs and ileum from 496 pigs aged 5?13 weeks collected between 1976 and mid-2001. The sections were investigated immunohistochemically using a monoclonal antibody specific for PCV-2 capsid antigen encoded by ORF2. Virus antigen was detected in tissue samples of 39 pigs from 28 farms. The earliest positive sample originated from 1986. After 1989, positive pigs were found almost every year among the 20?40 cases investigated annually. These results indicate that PCV-2 has been present in Switzerland for some time, and at least since 1986.

Published: American Association of Swine Veterinarians - Proceedings, 23-25 - 2005 )
Effect of PCV2 passive antibody levels on immunization with chimeric PCV1-2 vaccine and challenge with wild-type PCV2.
In a previous study (see Bibliographical Bulletin of July 2004), the ORF2 capsid protein of PCV2 expressed by a chimeric PCV1-2 resulted immunogenic and the infection with the chimeric PCV1-2 induced only a limited infection with mild pathological lesions. In this study, the interference of different levels (negative/low/high) of passively-acquired antibodies with vaccine-induced protective immunity was investigated. The experimental design was based on 7 groups of 3-week old piglets consisting of 2 groups of pigs considered negative for antibody to PCV2 (ELISA S/P ratio <0.2), 2 groups of pigs with low antibody titers (ELISA S/P ratio of 0.2-0.5), 2 groups of pigs with high antibody titers (ELISA S/P ratio >0.5) and one group with variable titers. Each group with negative/low/high titers was either vaccinated with PCV1-2 chimera or inoculated a wild type PCV2. All groups were subsequently challenged with a wild type PCV2 6 weeks following vaccination/inoculation (at 9 weeks of age). A protective immunity against wild type PCV2 challenge was achieved in PCV1-2 chimera-vaccinated pigs with negative and low (S/P<0.5) passive antibody levels. In conclusion, a clear interference between vaccination and passively-acquired antibodies has been evidenced. Therefore, a vaccination of piglets with the chimeric PCV1-2 that takes place 6 weeks prior to the expected exposure to PCV2 may give good results if piglets have low PCV2 antibody titers.

Published: Società Italiana di Patologia ed Allevamento dei Suini - Proceedings, 321-330 - 2005 (Abstract)
PCR detection of PCV2 in blood, tonsil and feces swabs for diagnosis of PCV2 infection and postweaning multisystemic wasting syndrome (PMWS) in live pigs
Twelve pigs were inoculated with PCV2 at 5 weeks of age. Following infection, blood samples, feces and tonsil swabs were collected every 3 days and submitted to detection of PCV2 by qualitative PCR. At 21 days post-infection, all animals were euthanized and tissue samples were submitted to histopathological examination for PMWS lesions and indirect immunofluorescence for PCV2 antigen. At the end of the study (21 days PI), PCV2 was detected in the blood samples of all animals and in the feces and tonsil swabs of 11 animals. PMWS lesions and PCV2 antigen were present in only four pigs which, in fact, showed a significantly higher number of PCR-positive blood samples than the other pigs. These results confirm that PCV2 DNA can be detected in samples from pigs with or without pathological and virological evidence of PMWS but also that PCR alone is not sufficient to establish a diagnosis of PMWS. However, the fact that PCV2 DNA was detected more frequently in pigs with PMWS-typical lesions suggests that PCV2 DNA detection if associated to PCV2 DNA quantification could be a useful diagnostic tool for PMWS diagnosis in live pigs.

Published: Vet Microbiol - 2005 Jul 1;108(3-4):179-86. - July 2005 (Abstract)
Experimental infection of 3-week-old conventional colostrum-fed pigs with porcine circovirus type 2 and porcine parvovirus.
Ostanello F, Caprioli A, Di Francesco A, Battilani M, Sala G, Sarli G, Mandrioli L, McNeilly F, Allan GM, Prosperi S.
Department of Veterinary Public Health and Animal Pathology, Faculty of Veterinary Medicine, Bologna University, Via Tolara di Sopra 50, 40064 Ozzano Emilia (BO), Italy.
This report describes an experimental infection with porcine circovirus type 2 (PCV2) in combination with porcine parvovirus (PPV) in 3-week-old conventional colostrum-fed pigs with maternal antibodies to both viruses. Two groups of four pigs each were inoculated with PCV2 and PPV. One of the groups received also a commercial inactivated vaccine against porcine pleuropneumonia to evaluate possible effects of the stimulation of the immune system of pigs on the infection. Another group of four pigs was kept as uninfected control. Clinical signs, rectal temperatures and body weights were recorded. Serum antibody titers to PCV2 and PPV were determined at weekly intervals. Pigs were killed 42 days after inoculation and tissue samples were examined for the presence of gross and microscopic lesions. Tissues were also analyzed for the presence of PCV2 and PPV DNA by PCR, and for the presence of PCV2 antigen by immunohistochemistry (IHC). All the pigs had serum antibodies to PCV2 and PPV at the beginning of the trial. None of them developed clinical symptoms or pathological lesions typical of post-weaning multisystemic wasting syndrome (PMWS), a disease associated to PCV2 infection. However, IHC and/or PCR analyses showed that clinically silent PCV2 infection developed in five of the eight inoculated pigs, regardless of the administration of the vaccine. In particular, PCV2 DNA and/or antigen were detected in most of the tissues examined in the two pigs with the lowest titer of maternal PCV2 antibodies at the beginning of the trial. PPV DNA was not detected in any of the samples examined. The five pigs with PCR and/or IHC evidence of PCV2 infection had a mean weight gain during the experiment lower than that of the inoculated PCR-negative pigs considered together and that of the control pigs. In conclusion, it would appear that passive immunity against PCV2 can play a role in preventing the development of PMWS, but is not able to prevent the establishing of clinically silent PCV2 infections. The dissemination and persistence of the virus in the tissues may depend on the level of PCV2 antibodies at the time of inoculation.

Published: Journal of Virology - p.8262-8274, Vol. 79, No. 13 - July 2005 (Abstract)
Characterization of a Previously Unidentified Viral Protein in Porcine Circovirus Type 2-Infected Cells and Its Role in Virus-Induced Apoptosis
Jue Liu, Isabelle Chen, and Jimmy Kwang
Received 9 December 2004/ Accepted 14 February 2005
Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome in pigs. In this study, transcription and translation of a novel viral gene (termed ORF3 here) was detected during productive infection of PCV2 in PK15 cells. The results of infection with ORF3-deficient PCV2 by site-directed mutagenesis indicated that the protein is not essential for viral replication. To investigate the underlying mechanism of cell death caused by replication of PCV2, apoptosis characterized by chromosomal condensation and fragmentation, formation of apoptotic bodies, and significant increase in hypodiploids were detected in infected cells. We further demonstrated that PCV2-induced apoptosis required the activation of caspase-8 but not caspase-9. The activation of caspase-8 results in the activation of caspase-3 as shown by an increase in the cleavage of the caspase substrate in the infected cells. To determine whether ORF3 protein could trigger apoptosis, ORF3 as well as ORF1 and ORF2 genes were transiently expressed in PK15 and Cos-7 cells for apoptotic activity assay. Transfection of cells with the ORF3 alone induced apoptosis using a pathway similar to that described in the context of viral infection. This is further confirmed by a significant decrease in apoptotic activity of infected cells in the absence of the ORF3 expression, suggesting that the protein plays a major role in the induction of virus-induced apoptosis. Altogether, these results indicate that ORF3 is a novel PCV2 protein that is not essential for viral replication in cultured cells but is involved in PCV2-induced apoptosis by activating caspase-8 and caspase-3 pathways

Published: J. Gen. Virol - 86: 2057 - 2068. - July 2005 (Abstract)
Binding and entry characteristics of porcine circovirus 2 in cells of the porcine monocytic line 3D4/31
G. Misinzo¹, P. Meerts¹, M. Bublot², J. Mast³, H. M. Weingartl&sup4; and H. J. Nauwynck¹
1 Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
2 Merial, Biological Research, Lyon, France
3 Veterinary and Agrochemical Research Centre, Brussels, Belgium
4 Canadian Science Centre for Human and Animal Health, Winnipeg, Manitoba, Canada
Porcine circovirus 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome and reproductive problems in pigs. Cells of the monocyte/macrophage lineage are important target cells in PCV2-infected pigs, but the method of binding and entry of PCV2 into these cells is unknown. Therefore, binding and entry of PCV2 to the porcine monocytic cell line 3D4/31 were studied by visualization of binding and internalization of PCV2 virus-like particles (VLPs) by confocal microscopy and chemical inhibition of endocytic pathways (clathrin- and caveolae-mediated endocytosis and macropinocytosis), followed by evaluation of the level of PCV2 infection. It was shown that PCV2 VLPs bound to all cells, with maximal binding starting from 30 min post-incubation. Bound PCV2 VLPs were internalized in 47?5?0 % of cells. Internalization was continuous, with 70?5?9?7 % of bound PCV2 VLPs internalized at 360 min post-incubation. Internalizing PCV2 VLPs co-localized with clathrin. PCV2 infection was decreased significantly by chemical inhibitors that specifically blocked (i) actin-dependent processes, including cytochalasin D (75?5?7?0 % reduction) and latrunculin B (71?0?3?0 % reduction), and (ii) clathrin-mediated endocytosis, including potassium depletion combined with hypotonic shock (50?2?6?3 % reduction), hypertonic medium (56?4?5?7 % reduction), cytosol acidification (59?1?7?1 % reduction) and amantadine (52?6?6?7 % reduction). Inhibiting macropinocytosis with amiloride and caveolae-dependent endocytosis with nystatin did not decrease PCV2 infection significantly. PCV2 infection was reduced by the lysosomotropic weak bases ammonium chloride (47?0?7?9 % reduction) and chloroquine diphosphate (49?0?5?6 % reduction). Together, these data demonstrate that PCV2 enters 3D4/31 cells predominantly via clathrin-mediated endocytosis and requires an acidic environment for infection.

Published: Immunology - Volume 115, Issue 3, Page 388-398 - July 2005 (Abstract)
Subset-dependent modulation of dendritic cell activity by circovirus type 2
Isabelle E. Vincent, Carlos P. Carrasco, Laurence Guzylack-Piriou, Brigitte Herrmann, Francis McNeilly, Gordon M. Allan, Artur Summerfield and Kenneth C. McCullough
Viral interactions with dendritic cells (DCs) have important consequences for immune defence function. Certain single-stranded DNA viruses that associate with a number of species, including humans and pigs, exhibit interesting characteristics in this context. Porcine circovirus type 2 (PCV2) can persist within myeloid DCs in the absence of virus replication. Internalization was observed with both conventional blood DCs and plasmacytoid DCs [natural interferon-producing cells (NIPCs)], as well as DC precursors. This PCV2DC interaction neither induced nor inhibited DC differentiation. The maturation of myeloid DCs induced by a cocktail of interferon-α/tumour necrosis factor-α (IFN-α/TNF-α), and the ability to process and present antigen to T lymphocytes, remained intact in the presence of PCV2. The virus was clearly internalized by the DCs, a process noted with both mature and immature cells. This suggested a non-macropinocytic uptake, confirmed by an insensitivity to wortmannin but sensitivity to cytochalasin D, chlorpromazine and bafilomycin. Nevertheless, PCV2 was immunomodulatory, being effected through the reaction of NIPC to danger signals. When NIPCs responded to the CpG-oligonucleotide (CpG-ODN), their costimulatory function which induces myeloid DC maturation was clearly impaired by the presence of PCV2. This was caused by a PCV2-induced inhibition of the IFN-α and TNF-α normally produced following interaction with CpG-ODN. Thus, the immunomodulatory activity of PCV2 is mediated through the disruption of NIPC function. This would impair the maturation of associated myeloid DC and have major implications for the efficient recognition of viral and bacterial danger signals, favouring the establishment of infections additional to that of PCV2.

Published: Vet J. Jun 11; [Epub ahead of print] - June 2005 (Abstract)
Muco-cutaneous candidiasis in two pigs with postweaning multisystemic wasting syndrome.
Zlotowski P, Rozza DB, Pescador CA, Barcellos DE, Ferreiro L, Sanches EM, Driemeier D.
Laboratory of Veterinary Pathology, College of Veterinary Medicine, Federal University of Rio Grande do Sul (UFRGS), Av. Bento Goncalves 9090, 91540-000 Porto Alegre, RS, Brazil.
In two distinct commercial swine herds, poor weight gain and an increased number of animals showing wasting were observed among nursery and growing pigs. Cases of postweaning multisystemic wasting syndrome (PMWS) and infection with Haemophilus parasuis had been previously diagnosed in these herds. One growing wasted pig from each herd was necropsied and showed enlarged lymph nodes. Pseudomembranous material adhered to the dorsum of the tongue, soft and hard palate in case 1, and in case 2, fibrinous material was seen as whitish plaques on the oesophageal surface with hyperkeratosis of the non-glandular stomach. The main histological lesions in both cases were found in lymphoid tissues with a multifocal accentuated lymphohistiocytic infiltrate, areas of lymphoid depletion and intracytoplasmic inclusions in histiocytic cells in lymph nodes and Payer's patches. Focally, extensive ulceration was found in the stratified pavement epithelium of the tongue with necrosis and necrosuppurative infiltrate in case 1; in case 2, there was ulceration in the stomach with lymphohistiocytic infiltrate in the submucosa and ulceration in the mucosa of the oesophagus associated with yeast cells and pseudo-hyphae. Candida albicans was isolated from the oral cavity lesions. Immunohistochemistry of the lymph nodes was positive for porcine circovirus 2 (PCV2). The association between PMWS and mucocutaneous candidiasis reported here supports the potential immunosuppressive state of PMWS infected pigs.

Published: Journal of Comparative Pathology, Article in Press, Corrected Proof - Received 14 June 2004; accepted 3 January 2005. Available online 17 June 2005. - June 2005 (Abstract)
Synthetic Peptide-derived Antibody-based Immunohistochemistry for the Detection of Porcine Circovirus 2 in Pigs with Postweaning Multisystemic Wasting Syndrome
Y. Ha, K. Jung, C. Choi, K.-K. Hwang and C. Chae
Porcine circovirus 2 (PCV2) was detected consistently in formalin-fixed paraffin wax-embedded lymph node and spleen from experimentally and naturally infected pigs by synthetic peptide-derived polyclonal antibody-based immunohistochemistry. Synthetic peptides were generated from open reading frame 2 of PCV2 by solid-phase peptide synthesis, purified by high performance liquid chromatography, and injected into rabbits to produce polyclonal antibody. Positive cells had large nuclei with abundant cytoplasm, and resembled macrophages. In serial sections, a similar distribution of PCV2 antigen and DNA was confirmed in virus-infected cells by immunohistochemistry and in-situ hybridization, respectively. The immunohistochemical method described was successfully applied to formalin-fixed, paraffin wax-embedded tissues and should prove helpful in diagnosing postweaning multisystemic wasting syndrome.

Published: Preventive Veterinary Medicine - June 2005 (Abstract)
An exploratory study on risk factors for postweaning multisystemic wasting syndrome (PMWS) in Spain
S. López-Soria, J. Segals, N. Rose, M.J. Viñas, P. Blanchard, F. Madec, A. Jestin, J. Casal and M. Domingo
An exploratory case?control study was carried out in Spain in 2002/2003, involving 62 pig farms of different production systems to assess risk factors that, in association with PCV2 infection, induce postweaning multisystemic wasting syndrome (PMWS) expression. To achieve this objective two groups of farms selected according to their PMWS status were compared: ?cases? (farms with clinical PMWS, n = 32) and ?controls? (farms without clinical PMWS, n = 30). A filled-in questionnaire and 45 blood samples (15 sows, and two groups of 15 pigs of 12 and 20 weeks of age, respectively) were obtained from each farm. Additionally, two to three diseased pigs were necropsied and relevant tissues to diagnose PMWS collected when PMWS was clinically suspected (?case? farms). A statistical analysis to compare ?case? versus ?control? farms was performed with the variables obtained from the questionnaire (191 variables) and the serologic test results (20 variables). Data were analysed using conditional logistic regression with a nested n:m matched design taking into account the farm size. Three variables were found significant in the final model: two related to vaccination scheme and one to PCV2 seroprevalence in growing pigs. Vaccination of gilts against PRRSV increased the odds of PMWS expression and vaccination of sows against atrophic rhinitis was related to decreased odds of the disease; however, the possibility that those two factors could be spurious effects (due to the small sample size) or confounding variables cannot be ruled out. On the other hand, a higher prevalence of antibodies to PCV2 at 12 weeks of age was observed in pigs from ?case? farms than in pigs from ?control? farms. This result suggests that an earlier infection with PCV2 might be a risk factor for PMWS expression.

Published: In Practice, Volume 27, Number 5, 31, pp. 230-237(8) May 2005 (Abstract)
Biosecurity: reducing disease risks to pig breeding herds
Pritchard, Geoff; Dennis, Ian; Waddilove, Jake
Disease imposes considerable constraints on the productivity and profitability of the livestock industry. Pig producers have probably suffered more than other sectors from the devastating effects of a succession of infectious disease outbreaks over the past 30 years. Many of these have been highly contagious viral diseases, including transmissible gastroenteritis (TGE), swine influenza (SI), Aujeszky's disease (AD), and porcine respiratory and reproductive syndrome (PRRS). Postweaning multisystemic wasting syndrome (PMWS), which is linked to porcine circovirus (PCV-2) infection, has challenged the very survival of the pig industry in Britain and elsewhere. Even long established endemic diseases, such as enzootic pneumonia (EP) and swine dysentery (SD), still cause significant losses if introduced into naive herds. The recent reappearances of classical swine fever (CSF) and foot-and-mouth disease (FMD) in Britain were timely reminders that there is no place for complacency in disease prevention programmes at both national and herd level. The concept of biosecurity, which gained prominence in Britain during the FMD epidemic, encompasses the full range of measures aimed at disease prevention. In fact, it is one of the cornerstones of the Government's Animal Health and Welfare Strategy for Great Britain, which aims to promote biosecurity to a livestock industry that, in the past, has not always given it high priority. This article reviews the most important sources of disease on pig units - and, thus, the key areas for consideration in terms of farm biosecurity.

Published: Aust Vet J. 2005 May;83(5):300-4 - May 2005 (Abstract)
The detection of porcine circovirus in the Australian pig herd.
Raye W, Muhling J, Warfe L, Buddle JR, Palmer C, Wilcox GE.
School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia 6150.
OBJECTIVE: To determine if porcine circovirus (PCV) type 1 (PCV1) or type 2 (PCV2) is present in the Australian pig herd, to conduct preliminary genetic characterisation of any viruses detected, and to determine if there is any obvious virological reason why post-weaning multisystemic wasting disease (PMWS), associated with PCV infection in other countries, has not been detected in Australia. DESIGN: Serum samples were collected from 14 randomly selected pig farms in Western Australia and used for detection of PCV antibody. Additional samples from one farm were obtained at 2-week intervals from pigs between 2 and 12 weeks of age to detect any age-associated variations in prevalence of infection. Veterinary practitioners from four Australian states submitted tissues of dead or unthrifty weaned pigs, and these were examined for evidence of PCV1 and PCV2 infection. PROCEDURE: Sera were tested for antibody to PCV using an indirect immunofluorescence assay (IFA). Tissues were tested for PCV1 and PCV2 genomic material using a multiplex PCR. RESULTS: PCV antibody was detected in approximately 30% of Western Australian pigs tested. PCV1 DNA was detected in tissue samples from Western Australia, South Australia and New South Wales and PCV2 DNA was detected in tissue samples from Western Australia, New South Wales and Queensland. Sequence analysis of the PCR products indicated the PCV1 and PCV2 present in Australia were very similar to strains in other countries where PMWS is endemic. CONCLUSION: Both PCV1 and PCV2 are present in Australia and the viruses present appear similar to those in countries with PMWS. The absence of PCV2-associated PMWS in Australia may be due to absence of essential secondary factors required for PCV2 to produce PMWS.

Published: J Vet Diagn Invest. 2005 May;17(3):213-22. - May 2005 (Abstract)
Features of porcine circovirus-2 disease: correlations between lesions, amount and distribution of virus, and clinical outcome.
Krakowka S, Ellis J, McNeilly F, Waldner C, Allan G.
Department of Veterinary Biosciences, College of Veterinary Medicine, Ohio State University, Columbus, OH 43210, USA.
Tissue sets from 36 snatch-farrowed colostrum-deprived (SF/CD) and 71 Caesarian-derived gnotobiotic swine infected with porcine circovirus type 2 (PCV-2) as neonates were examined and scored for the types and tissue distribution of histologic lesions associated with this viral infection. The occurrence and severity of these lesions were correlated with qualitative and quantitative determinations of viral burden in tissues by immunohistochemistry (IHC) and tissue titrations for infectious virus, respectively. These measures were, in turn, related to 1 of 3 categories of clinical disease expressed in PCV-2-infected swine as subclinical infection, preclinical postweaning multisystemic wasting syndrome (PMWS), and clinically evident PMWS, respectively. Statistically significant (P < 0.05 to 0.001) associations between both measures of viral burden, the severity of histologic lesions and the stage of disease were obtained. Discrimination between and among categories of disease was best accomplished by a combination of IHC and histopathology. The results of this study confirm that viral burden in PCV-2-infected tissues, specifically lymphoid tissues and liver, directly correlate with severity of clinical disease expression in PCV-2 infected swine.

Published: Vet J. 2005 May;169(3):326-36. - May 2005 (Abstract)
A review of porcine circovirus 2-associated syndromes and diseases
C. Chae
Clinical expression of porcine circovirus 2 (PCV2) infection in swine may result in several distinct syndromes and diseases including post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, porcine respiratory disease complex, granulomatous enteritis, necrotizing lymphadenitis, and possibly exudative epidermitis. Association of PCV2 with congenital tremor in piglets is still controversial. The extent of the involvement of PCV2 in swine disease other than PMWS is currently poorly understood. This review concentrates on PCV-2-associated syndromes and diseases other than PMWS.

Published: Vet Res Commun. Apr;29(3):263-9. - April 2005 (Abstract)
Detection of porcine circovirus type 2, porcine parvovirus and porcine pseudorabies virus from pigs with postweaning multisystemic wasting syndrome by multiplex PCR
Cao S, Chen H, Zhao J, Lu J, Xiao S, Jin M, Guo A, Wu B, He Q.
Multiplex PCR was established to detect porcine circovirus type 2 (PCV-2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV) and applied to samples from 137 piglets exhibiting clinical signs of postweaning multisystemic wasting syndrome (PMWS). PCV-2 DNA was detected from all samples. Moreover, 43 samples were positive for PPV but negative for PRV; 11 samples were positive for PRV but negative for PPV; and 35 samples were positive both for PPV and PRV. These results suggests that PCV-2 co-infection with PRV and PPV may play an important role in PMWS. Also, multiplex PCR is an appropriate candidate method for diagnosis of PCV-2, PRV and PPV simultaneously in field cases.

Published: Vet Rec., 156: 177 - 178. - February 2005
Necrotising lymphadenitis associated with porcine circovirus type 2 in pigs
J Kim and C Chae
PORCINE circovirus type 2 (PCV-2), a single-stranded DNA virus within the family Circoviridae, is now recognised as the aetiological agent of postweaning multisystemic wasting syndrome (PMWS) (Mankertz and others 1997, Allan and Ellis 2000, Chae 2004). In addition to PMWS, PCV-2 has convincingly been linked to porcine dermatitis and nephropathy syndrome, porcine reproductive disorders and porcine respiratory disease complex (Choi and Chae 2001, Kim and others 2003, 2004, Chae 2005). This short communication describes PCV-2-associated necrotising lymphadenitis, which may be a new clinical manifestation of PCV-2 infection in pigs.

Published: Livestock Production Science - February 2005 (Abstract)
Effect of the Pietrain breed used as terminal boar on Post-weaning Multisystemic Wasting Syndrome (PMWS) in the offspring in four PMWS-affected farms
Nicolas Rose, Alain Abherv-Guguen, Grald Le Diguerher, Eric Eveno, Jean-Pierre Jolly, Philippe Blanchard, Aurélie Oger, Andr Jestin and François Madec
The aim of this study was to determine the potential risk factors for PMWS at the individual pig level and assess the effect of the Pietrain paternal genetic background of the animals in a cohort study. The survey was set up in four PMWS-affected farms with 2 repetitions (batches) per farm. A representative sample of 60 pigs per batch, stratified according to the paternal genetic background (Pietrain: yes vs. no), was randomly selected after farrowing. The representative cohort was divided into 8 batches and the pigs were individually monitored from birth to slaughter. Survival analysis was used to determine the factors related to the time to PMWS. The litter-cluster effect was taken into account using the marginal Cox model (robust estimation of the covariance matrix) and the gamma shared frailty model which were compared.

No protective effect of the Pietrain breed on the time to PMWS and the proportion of affected pigs in the offspring was found in this study. Piglets showing low circovirus type 2 (PCV2) titres at 7 weeks-old with no subsequent seroconversion and piglets from PCV2 negative sows were most likely to be affected by PMWS (HR=7.0 and 2.8, respectively). Active infection of the pregnant dams with parvovirus was related to an increased risk of PMWS in the offspring (HR=2.3). Neck injuries due to poorly performed injections in the dams were associated with an increased risk of PMWS with the marginal model (HR=2.1). Oxytocin injection (dams) during farrowing was protective against PMWS in the offspring (HR=0.6).

Published: International Journal of Experimental Pathology, vol. 86, no. 1, pp. 33-43(11) - February 2005 (Abstract)
Effect of granulocyte-macrophage colony-stimulating factor on post-weaning multisystemic wasting syndrome in porcine circovirus type-2-transfected piglets
Christophe Loizel; Philippe Blanchard; Béatrice Grasland; Daniel Dory; Aurélie Oger; Anne-Cécile Nignol; Roland Cariolet; André Jestin
Post-weaning multisystemic wasting syndrome (PMWS) is a complex disease syndrome in swine, affecting nursery and fattening pigs. Although ongoing evidence suggests that porcine circovirus type-2 (PCV2) is the causal agent of PMWS, the host immune system appears to have a crucial role in the PMWS pathogenesis of PCV2-affected pigs. Owing to difficulties in producing a biologically pure form of PCV2 devoid of the other viral agents commonly present in swine tissues, we decided to use a tandem-cloned PCV2 DNA providing highly pure grade reagent in order to monitor the virulence of PCV2 alone or with an immunostimulating co-factor, granulocyte-macrophage colony-stimulating factor (GM-CSF). A single intramuscular injection of tandem-cloned PCV2 DNA into 5-week-old piglets produced plasmid to viral genome progeny and infectious particles as early as 8 days post-injection in all the organs tested (the lung, the tonsil and the inguinal, mesenteric, bronchial and upper-right axial lymph nodes). The initial plasmid load was not detected with the help of primers designed to specifically detect the acceptor plasmid, thus confirming the replication of the viral genome. Despite the presence of a high level of PCV2 genome copies in the lymphoid organs ? the tonsil and the lung ? and the presence of infectious particles, no detectable clinical manifestations or pathological lesions were observed in the transfected pigs over the period of observation, regardless of whether they had been co-injected with plasmid containing GM-CSF DNA or had received plasmid containing PCV2 DNA alone. GM-CSF encoding DNA injection had no significant effect on viral replication or on the production of viral particles and appearance of the disease.

Published: Veterinary Microbiology - Received 1 July 2004; revised 19 November 2004; accepted 1 December 2004. Available online 3 February 2005. (Abstract)
Experimental reproduction of postweaning multisystemic wasting syndrome (PMWS) in pigs in Sweden and Denmark with a Swedish isolate of porcine circovirus type 2
Hasslunga, P. Wallgrenb, A.-S. Ladekj?r-Hansenc, A. B?tnerc, J. Nielsenc, E. Wattranga, G.M. Alland, F. McNeillyd, J. Ellise, S. Timmuska, K. Bel?kb, T. Segallb, L. Melinb, M. Berga and C. Fossuma
An experimental model using 3-day-old snatch-farrowed colostrum-deprived piglets co-infected with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) is at present one of the best methods to study factors affecting development of postweaning multisystemic wasting syndrome (PMWS). A Swedish isolate of PCV2 (S-PCV2) retrieved in 1993 from a healthy pig has been used in this model to reproduce PMWS in pigs from Northern Ireland. This virus has been present in the Swedish pig population for at least a decade without causing any known PMWS disease problems, despite its potential pathogenicity. The reasons for this are unknown, but

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