Abstract
Nineteen pigs with clinical signs of porcine circovirus associated diseases (PCVAD) on 5 Alberta pig farms were examined pathologically, including gross pathology, histopathology, and immunohistochemistry. Polymerase chain reaction (PCR) for porcine circovirus type-2 (PCV-2) and sequence analysis was performed on tissue samples of 12 animals. Results showed that new strains of porcine circovirus type-2 genogroup b were present in most pigs that were positive for PCV-2. Furthermore, a mixed infection with PCV-2a and PCV-2b occurred in the liver and lungs of 1 pig. Comparison of whole genome sequences representing known viruses and the newly discovered Alberta viruses revealed mutations distributed over the entire genome of PCV-2; however, sequences encoding for immunodominant epitopes of PCV-2 appear to be unaffected by these mutations.
Résumé
Dix-neuf porcs présentant des signes cliniques de maladie associée au circovirus (PCVAD) dans 5 fermes de l’Alberta ont été soumis à un examen pathologique, incluant un examen macroscopique, histopathologique et immuno-histochimique. Une réaction d’amplification en chaîne par la polymérase (PCR) pour le circovirus porcin de type 2 (PCV-2) et une analyse de séquence ont été effectuées sur des échantillons de tissus provenant de 12 animaux. Les résultats ont montré que de nouvelles souches de PCV-2 appartenant au génogroupe b étaient présentes chez la majorité des porcs qui étaient positifs pour PCV-2. De plus, une infection mixte avec PCV-2a et PCV-2b s’est produite dans le foie et les poumons de 1 porc. Une comparaison des séquences du génome entier représentant des virus connus et les nouveaux virus découverts en Alberta ont révélé des mutations distribuées sur l’ensemble du génome de PCV-2; toutefois, les séquences codant pour des épitopes immunodominants de PCV-2 ne semblaient pas être affectées par ces mutations.
(Traduit par Docteur Serge Messier)
Porcine circovirus type-2 (PCV-2) was discovered in the 1990s in association with a new wasting syndrome in growing pigs (1). This post-weaning multisystemic wasting syndrome (PMWS) and other porcine circovirus associated diseases (PCVAD) have caused severe economical losses for swine farmers and the pig industry worldwide. The exact mechanisms of how PCV-2 contributes to the clinical manifestation of PCVAD are poorly understood and might include host and viral genetic determinants (2,3), the presence of other infectious agents (4), or environmental factors.
Analysis of genome sequences led to the classification of 2 subgroups within PCV-2, based on a sequence motif in the capsid protein: PCV-2a and PCV-2b (5,6). Porcine circovirus type-2a (PCV-2a) was the predominant strain in North America (7) until recent studies revealed that PCV-2b is now present on a large number of pig farms throughout Canada (5,8,9).
Identification and characterization of PCV-2 in Alberta, was accomplished by analyzing PCV-2 genomes obtained from pigs with clinical signs consistent with PCVAD. Tissue samples of 19 pigs from 5 Alberta farms affected by PCVAD were collected during necropsy. Organs sampled included tonsils, inguinal, mesenteric, and bronchial lymph nodes, spleen, lung, liver, small intestine (jejunum and/or ileum), kidney, and skin. For histopathological and immunohistochemical (IHC) analysis, the samples were fixed in 10% neutral buffered formalin (NBF); for PCR analysis they were stored at −20°C until use. After fixation in 10% NBF, tissue samples were embedded in paraffin for routine hematoxylin & eosin staining and IHC processing. Immunohistochemical detection of PCV-2 antigens was performed using a streptavidin-biotin complex technique adapted for an automated slide stainer (CodeOn Histomatic slide stainer; Fisher Scientific, Edmonton, Alberta) as previously described (1), with the exception that the primary antibody was a chicken anti-PCV-2 antiserum (from Dr. G. Allan, Belfast, N. Ireland) used at a 1:1000 dilution. All sections were examined by light microscopy in a non-blinded manner applying the scoring system described by Opriessing et al (6) (Table I).
Table I.
Severity and distribution of gross and histological lesions and PCV-2 staining intensity in PCVAD affected pigs from 5 farms
| Presence of clinical signs: — wasting — weight loss — dyspnea — enteritis |
Presence of histopathological lesions in lymphoid tissues | Percentage of lymphoid follicles that have cells with staining for PCV-2 antigen (determined by immunohistochemistry) | ||
|---|---|---|---|---|
| Farm A | Pig 1 | + | 1 | < 10% |
| Pig 2 | + | 3 | > 50% | |
| Pig 3 | + | 3 | 10% to 50% | |
| Farm B | Pig 1 | + | 2 | 10% to 50% |
| Pig 2 | + | 1 | < 10% | |
| Pig 3 | + | 2 | > 50% | |
| Pig 4 | + | 2 | > 50% | |
| Pig 5 | + | 2 | > 50% | |
| Farm C | Pig 1 | + | 2 | 10% to 50% |
| Pig 2 | + | 3 | > 50% | |
| Pig 3 | + | 3 | > 50% | |
| Pig 4 | + | 3 | > 50% | |
| Farm D | Pig 1 | + | 3 | > 50% |
| Pig 2 | + | 1 | < 10% | |
| Pig 3 | + | 0 | 0 | |
| Farm E | Pig 1 | + | 3 | > 50% |
+ — present; 0 — not present; 1 — mild lymphoid depletion with loss of overall cellularity and mild histiocytic/granulomatous inflammation; 2 — moderate lymphoid depletion and moderate histiocytic/granulomatous inflammation; 3 — severe lymphoid depletion with loss of lymphoid follicle structure/replacement of follicles by histiocytic-granulomatous infiltration. Diagnosis of PCVAD based on the definition by Sorden (14) and on a scoring system by Opriessnig et al (6).
Amplification and cloning of viral genomic sequences, was accomplished by isolating DNA from tissue samples using QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s protocol. Muscle tissue from a healthy pig mixed with distilled water was used as negative control and a synthesized PCV-2 genome from DNA2.0 (GenBank accession number EF394779) served as a positive control. Viral DNA was amplified through PCR using 4 different primer pairs as previously described (10,11), with 2 modifications: 1) A new primer with an identical nucleotide position was based on the genomic sequence of a strain with GenBank accession number AY094619 instead of AF201897; and 2) the nucleotide position of primer no. P6 was changed to 1569–1588 to cover a more conserved sequence among PCV-2 strains. Amplification of complete PCV genomes from tissue samples, was accomplished by cloning amplicons into pEGFP-N1 vector (Clontech). At least 10 positive clones were selected for plasmid DNA isolation from DH5α competent cells (Invitrogen), using QIAprep Spin Miniprep Kit (Qiagen) and were sent for sequencing (Genome Quebec, McGill University, Montreal, Quebec). Complete genome sequences were submitted to NCBI under GenBank accession numbers FJ233905 and FJ233908.
Using Blast, ClustalW2 (EMBL-EBI), and GeneDoc, fragments resulting from the same PCR primer pairs were aligned and geno-groups were verified by the signature motif sequence in the cap gene as described by Cheung et al (12).
From all selected animals (2 to 5 pigs per farm with typical clinical signs of PCVAD), necropsies revealed the following gross pathological findings typical for PCVAD: the lungs failed to collapse and showed interstitial edemas which led to puffy, sponge-like consistency of the organ. Bronchial and mediastinal lymph nodes were enlarged. The gastro-intestinal system showed thickened intestinal walls with prominent Peyer’s patches, enlarged mesenteric lymph nodes and an edematous mesentery. In some animals, livers and kidneys exhibited alterations consistent with hepatitis and focal glomerulonephritis, respectively. The inguinal lymph nodes were enlarged in most pigs that were examined. One animal showed multifocal cutaneous lesions, which are characteristic of the porcine dermatitis and nephropathy syndrome (PDNS) (13). Histopathological lesions included depletion of lymph follicles in the ileal Peyer’s patches, tonsils, spleen, and lymph nodes with infiltration of histiocytic cells and occasional giant cell formation. The lung showed diffuse granulomatous pneumonia. Multifocal granulomatous nephritis was observed in the kidneys. The liver was affected by non-suppurative inflammation of the triads. The small intestine showed granulomatous enteritis and the mesenteric arteries were affected by non-suppurative arteritis. Not all animals clinically affected by PCVAD appeared to exhibit overt histological changes and/or positive immunostaining for PCV-2 (Table I).
Results from PCR analyses indicated that PCV-2b was present on farms A, C, D, and E; PCV-2a on farms A and B (Table II). One pig on farm A showed an infection with both PCV-2a and PCV-2b in the liver and lungs. Tissue samples from 3 pigs (farm C: pigs 5, 6; farm E: pig 2) were found PCR-negative for PCV-2. For further characterization, full genome sequencing was performed on a subset of isolates. Two different PCV-2 strains were found in pig 1 on farm A: a PCV-2a strain, which is closely related to a South African strain (GenBank accession number AY325495.1) but displaying differences in 3 bases (nucleotide [nt] position 536, 1355, and 1369). The first mutation (A > G) at nt 536 is located within the overlapping portion of the genome which encodes for both open reading frames (ORF) 1 and 3. The mutation would result in 1 amino acid substitution (phenylalanine to leucine) in the ORF 3 protein. Both mutations at nt 1355 and nt 1369 are located within ORF 2 which encodes for the capsid protein (Cap). While the substitution of A > G at nt 1355 did not result in a change of an amino acid, replacing C > T at nt 1369 led to a substitution of valine by isoleucine. The second PCV-2 strain found in pig 1 on farm A is a PCV-2b strain, which is — with 99% sequence identity — closest related to the recently emergent PCV-2b strains from the USA (GenBank accession numbers EU594440, EU594438, and DQ629115) and to 1 strain from Quebec (GenBank accession number EF394777). This PCV-2b strain found in Alberta differs from the above-mentioned strains in 2 nucleotides: T > A (nt 236) which does not result in an amino acid change in the replication associated protein Rep; and A > C (nt 1735) which is located in a non-coding region immediately before the translation start of Cap.
Table II.
Genotype of PCV-2 DNA detected by PCR and sequencing in different pig tissues
| Organ | Genogroup | ||
|---|---|---|---|
| Farm A | Pig 1 | Lymph node | PCV-2b |
| Liver | PCV-2b/PCV-2a | ||
| Lung | PCV-2b/PCV-2a | ||
| Small intestine | PCV-2b | ||
| Pig 2 | Tonsil | PCV-2b | |
| Lymph node | PCV-2b | ||
| Liver | PCV-2b | ||
| Lung | PCV-2b | ||
| Small intestine | PCV-2b | ||
| Farm B | Pig 1 | Tonsil | PCV-2a |
| Lymph node | PCV-2a | ||
| Pig 2 | Lymph node | PCV-2a | |
| Farm C | Pig 1 | Lung | PCV-2b |
| Lymph node | PCV-2b | ||
| Pig 2 | Tonsil | PCV-2b | |
| Lymph node | PCV-2b | ||
| Pig 3 | Tonsil | PCV-2b | |
| Lung | PCV-2b | ||
| Pig 4 | Tonsil | PCV-2b | |
| Lymph node | PCV-2b | ||
| Farm D | Pig 1 | Lymph node | PCV-2b |
| Kidney | PCV-2b | ||
| Tonsil | PCV-2b | ||
| Heart | PCV-2b | ||
| Pig 2 | Lymph node | PCV-2b | |
| Kidney | PCV-2b | ||
| Spleen | PCV-2b | ||
| Lung | PCV-2b | ||
| Pig 3 | Lymph node | PCV-2b | |
| Kidney | PCV-2b | ||
| Lung | PCV-2b | ||
| Skin | — | ||
| Farm E | Pig 1 | Lymph node | PCV-2b |
| Kidney | PCV-2b | ||
| Lung | PCV-2b |
The PCV-2b strain found on farm C showed 1 mutation compared to the reference strains: C > T (nt 1706), which leads to an amino acid substitution (arginine to lysine) in the nuclear localization signal region of Cap. Three mutations were found in the PCV-2b strain on farm D, 2 of which were silent (C > T): 1 occurred immediately before the translational start of the Rep protein (nt 50); the other was located in the encoding region of the Cap protein (nt 1132). The 3rd mutation led to an amino acid mutation (arginine to lysine) in the nuclear localization signal (NLS) of Cap (nt 1703).
In summary, we found mostly PCV-2 strains belonging to the previously described PCV-2b subtype (8). Before 2004, PCV-2b was only found in animals in Europe and Asia and the dominant genogroup in North America was PCV-2a (7). The severity of the PCVAD outbreaks caused by the PCV-2b subtype, as well as the distinct clinical and histopathological lesions of these cases, lead to the hypothesis that the newly recognized PCV-2b strains display a higher virulence compared with those of genogroup 2a (5). However, this assumption still needs to be verified, as a recent study showed that PCV-2b was also present in healthy or unaffected animals, as well as in diseased pigs (9).
The capsid protein is the immunodominant component of all PCV-2 piglet-vaccines. The mutations in Cap detected here are located in the NLS region, which is not expected to contain immunodominant epitopes. Hence, these mutations are unlikely to influence the immunity acquired through current PCV-2 vaccines.
One of the detected PCV-2b strains had a mutation located in the ORF 3 which led to an amino acid exchange in the ORF 3-protein. Whether or not this protein is expressed in PCV-2-infected pigs and plays a role in the pathogenesis remains to be elucidated.
One pig had a mixed infection of the liver and lungs, where strains of both PCV-2 genogroups were present. A co-infection of pig tissue with both genotypes of PCV-2 has been reported before (7); however, no data are available to test whether co-infection would lead to an increase of virulence.
Acknowledgments
The authors thank Pranav Patel and Karin Orsel for reviewing the manuscript and Prairie Diagnostic Services, Saskatoon for processing the histopathological and IHC samples, as well as Genome Quebec at McGill University, Montreal for sequencing services. This study was supported by the Alberta Agriculture Funding Consortium and the Faculty of Veterinary Medicine, University of Calgary.
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