CASE III: 265/0912 (JPC 4165419)
A two- month-old, male, Canary Black Pig (Sus scrofa domesticus).
A litter of pigs exhibited weight loss and severe dyspnea. All animals died and one was submitted to our diagnostic laboratory for a complete necropsy. The animals belonged to a swine farm with no vaccination plan.
The animal was in poor body condition. The cerebellum showed abundant petechial and ecchymotic hemorrhages in the meninges and parenchyma, affecting mainly the white matter.
The lungs were diffusely firm with moderate interlobular septal edema. Cranioventral portions were patchy dark red. There was generalized lymphoadenomegaly. Additionally, mild hydropericardium, ascites, fibrinous pleuritis and multifocal hepatic fibrosis (consistent with Ascaris suum larvae migration) were observed.
Immunohistochemistry against PCV2 and PRRSV antigens was performed on brain, lung and lymph node tissue sections.
In the cerebellum, immunoreaction against PCV2 was observed in the cytoplasm and nuclei from intralesional perivascular macrophages and endothelial-like cells.
Immuno-labelling was also seen in alveolar macrophages, lymphocytes and syncytial cells in the lung, and in histiocytes, lymphocytes and occasional multinucleated giant cells in lymph nodes.
No PRRSV antigen was detected in any sample.
Cerebellum: affecting the leptomeninges and both the grey and white matter there are numerous multifocal to coalescing and variably sized areas of hemorrhage, frequently centered on small caliber blood vessels. The wall of these vessels shows loss of cellular detail with karyorrhexis and karyolysis, and is replaced by eosinophilic amorphous material and cellular debris (fibrinoid necrosis). Occasionally, fibrin thrombi occlude the lumen. Around less affected blood vessels, the Virchow-Robin space is expanded by a mild infiltrate of lymphocytes, plasma cells, macrophages and rare eosinophils (perivascular cuffing), which occasionally invade the wall (vasculitis) and extend into the adjacent neuroparenchyma. Endothelial cells are hypertrophied and have plump and vacuolated nuclei (reactive). Multifocally, the neuropil adjacent to affected areas shows moderate rarefaction. Diffusely, glial cells are mildly increased in number (gliosis).
Contributor's Morphologic Diagnoses:
Cerebellum and leptomeninges: Vasculitis, lymphohistiocytic, multifocal, moderate, with fibrinoid necrosis, perivascular cuffing, hemorrhage, thrombosis and mild gliosis.
In addition to the lesions observed in the cerebellum, other significant histological findings were severe lymphoid depletion with histiocytic infiltrate in lymph nodes, Peyer's patches, spleen and thymus, severe lymphohistiocytic interstitial pneumonia, and mild to moderate lymphohistiocytic interstitial nephritis. The postmortem and histological findings in combination with immunohistochemistry were consistent with PCV2-systemic disease.
Circoviruses (family Circoviridae) are small, nonenveloped, single-stranded DNA viruses, whose genome is circularly arranged (hence the name). They are worldwide distributed and have been identified in mammals, birds, fishes and even insects.2, 29 Four porcine circoviruses (PCVs 1-4) have been recognized so far, named in the order of discovery. Only PCV1, a contaminant in PK-15 cells, is considered non-pathogenic.27,28
In the early 90's a new disease, named postweaning multisystemic wasting syndrome (PMWS), was described in pigs in Canada.8 The disease was characterized by poor weight gain, wasting, dyspnea, pallor, diarrhea and jaundice. Few years later, a new porcine circovirus, different from the one of PK-15 cells (PCV1), was isolated from pigs with PMWS.1,5,10,13 Since then, PCV2 has been associated with many other syndromes in pigs, and is now one of the most widespread viral infections in swine husbandry, causing significant economic losses.
PCV3 and PCV4 have been discovered and isolated just recently, and the available information on their distribution, prevalence, and pathogenicity is still limited and sometimes controversial.15,16,19,20,33
PCV2 is characterized by a high nucleotide substitution rate, thus having the highest genetic variability among single-stranded-DNA viruses. At present, eight different PCV2 genotypes have been identified (PCV-2a to PCV-2h) and more are likely to appear in the future.16 PCV2a, PCV2b and PCV2d are the most common ones. Recently, genotype prevalence has switched from PCV2b to PCV2d. 9
All PCVs share two main open reading frames: ORF1 (rep gene) encoding proteins associated with replication, and ORF2 (cap gene) which encodes the capsid protein, the main antigenic determinant of the virus.16 The ORF3 gene, present on PCV2 genome, encodes a protein which is thought to induce apoptosis of infected cells.9
Infection with PCV2 occurs mainly in domestic pigs, but it also happens in feral pigs and wild boars.4,6,11,30 The virus is transmitted both vertically and horizontally via inhalation/ingestion of fomites from oronasal-pharyngeal body fluids, feces, and urine.14, 18 PCV2 uses a viral capsid attachment protein to bind to heparin sulfate and chondroitin sulfate B glycosaminoglycans on host cells. It enters the cell by several pathways including clathrin-mediated endocytosis, caveolin-mediated endocytosis, actin and Rho-GTPase 21, 32. Viral DNA replication relies mainly on the DNA replication machinery of the host cells, since the small viral genome has a very limited coding capacity.21 Cellular tropism seems to change depending on the age of the animal: in fetuses, PCV2 is found in cardiomyocytes, hepatocytes and cells of the monocytic lineage, whereas after birth it shows predilection for lymphoblasts and macrophages.24 In vitro studies demonstrated that B lymphocytes are one of the most important cell population for viral replication.31 PCV2 is able to persist but not replicate in dendritic cells, which are probably used as a vehicle for spreading.7 As PCV2 directly targets the cells of the immune system, pigs develop severe immunosuppression, which increases the risk of coinfections.
In most cases, infection is subclinical. Clinical forms manifest as a variety of syndromes, like systemic disease (which includes PMWS), respiratory disease, enteric disease, porcine dermatitis and nephropathy syndrome (PDNS), reproductive disease, encephalitis and congenital tremors.3,17,18,26 Recently, severe granulomatous and necrotizing myositis has been described for the first time in pigs with natural PCV2 infection.12 All these syndromes have been grouped together under the name "Porcine Circovirus Associated Disease, PCVAD".
In systemic forms, necrotizing arteritis, periarteritis, and fibrinoid vasculitis are seen in different organs, including the brain.22 Neurological disease has rarely been reported
with PCV2 systemic infection.23,25 Symptoms include lethargy, ataxia, paddling, opisthotonos, nystagmus, and seizures. Gross lesions in the central nervous system consist mainly of hemorrhage of the leptomeninges and the grey and white matter of the cerebellum. Histologically, in addition to the fibrinoid degeneration and vasculitis, other reported lesions are apoptosis of endothelial cells, gliosis, lymphohistiocytic coroiditis and meningitis, spongiosis, and neuronal degeneration and necrosis.23,25 Immunohistochemistry demonstrates cytoplasmic and nuclear labelling in intralesional perivascular macrophages and endothelial cells.
The presence of PCV2 within endothelial cells of the affected vessels suggests that the virus plays a role in the pathogenesis. However, as mentioned before, PCV2 is often associated with other agents, which could also contribute to the lesions. The presence of PCV2-labelled endothelial cells with increased activity of cleaved caspase-3 and apoptotic figures, suggests that a direct cytopathic effect of PCV2 may be possible.22, 25 Vascular damage could also result from cytokine secretion by infected cells and recruited inflammatory cells or by deposition of immune complexes in the vessels wall.22 In the study published by Seeliger and colleagues, no immune complexes were detected in the affected vessels.25
The reason why only a small percentage of animal develops disease, mechanisms beyond immunopathogenesis and the cause of persisting viremia are largely unknown. Both clinical and subclinical infections have an impact on porcine health and production. It is believed that factors related with the host (like age and genetics) as well as environmental factors play a role in the development of clinical forms.18 The high viral load of infected pigs, the long-term shedding, and the resistance of the virus in the environment make PCV2 one of the most highly prevalent viruses in swine farms worldwide.
PCV vaccines are now the single most-selling prophylactic agents in porcine farming and appear to confer cross protection against genotypes 2a and 2b. Although efforts in vaccination have remarkably decreased the impact of clinical forms and improved production performances, the infection is still widespread among the vaccinated population. Furthermore, vaccines may not confer protection under conditions of repeated exposure and the influence of other cofactors.9 It has been shown that epitope variations, which have been identified between genotypes, can enable the virus to escape pre-existing immunity and have been implicated in apparent vaccine failure cases involving genotype 2d.9
Unit of Veterinary Histology and Pathology. University Institute of Animal Health and Food Safety (IUSA), Veterinary School, University of Las Palmas de Gran Canaria.
Cerebellum: Vasculitis, fibrinonecrotizing, multifocal, severe, with multifocal hemorrhage, focally extensive necrosis, thrombosis, and mild lymphohistiocytic meningitis.
The contributor provides an outstanding review of PCV2. First identified in 1974 by Tischer et al., circoviruses are some of the smallest viruses known, measuring as little as 17 nm in diameter. Interestingly, evidence of PCV-2 has retrospectively been detected in archived tissues from as early as 1962 using in-situ hybridization and PCR.16
As previously mentioned by the contributor, PCV-2 specifically targets lymphoblasts and macrophages, with coinfection a common and expected feature as a result of immunosuppression. Furthermore, coinfection with porcine parvovirus, porcine reproductive and respiratory syndrome virus, and Mycoplasma hyopneumonia results in upregulation of PCV-2 replication, suggesting synergistic relationships between these infectious agents.16
Although circoviruses are typically regarded as host specific, PCV-2 and PCV-3 have both been detected in non-porcine hosts, indicating potential for cross-species infection. Natural infection with PCV-3 has been identified in domestic and wild pigs, as well as dogs, cattle, wild ungulates, and laboratory mice. Furthermore, PCV-3 was detected for prolonged periods of time in experimentally infected baboons.16
In addition to pigs, multiple avian species are affected by pathogenic members of genus Circovirus in the Circoviridae family, including psittacine beak and feather disease virus (BFDV), pigeon circovirus (PiCV), and goose circovirus (GoCV). Histopathologic features include intracytoplasmic botryoid inclusions of macrophages and depletion of both T and B lymphocytes. Chicken anemia virus, the only member of the closely related genus Gyrovirus, specifically targets hemocytoblasts and T lymphocytes, resulting in the T-lymphocytes depletion.29 Clinical signs associated with infection bear resemblance to Postweaning Multisystemic Syndrome (PMWS) in pigs and include ill-thrift, lethargy, anorexia, poor performance, and increased mortality as the result of secondary infection.29
Although the majority of species affected by the Circovirus genera are avian, they do not include those of commercial significance. Particularly when compared to PCV-2, research interest toward these entities has been historically limited. However, additional discovery of disease mechanisms across these species may prove to be useful toward enhanced understanding and control of PCV-2.
1. Allan GM, McNeilly F, Kennedy S, Daft B, Clarke EG, Ellis JA, Haines DM, Meehan BM, Adair BM. Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. J Vet Diagn Invest. 1998, 10, 3?10.
2. Blunt R, McOrist S, McKillen J, McNair I, Jiang T, Mellits K. House fly vector for porcine circovirus 2b on commercial pig farms. Vet Microbiol. 2011, 149, 452?455.
3. Choi J, Stevenson GW, Kiupel M, Harrach B, Anothayanontha L, Kanitz CL, Mittal SK. Sequence analysis of old and new strains of porcine circovirus associated with congenital tremors in pigs and their comparison with strains involved with postweaning multisystemic wasting syndrome. Can J Vet Res. 2002, 66, 217?224.
4. Dei Giudici S, Lo Presti A, Bonelli P, Angioi PP, Sanna G, Zinellu S, Balzano F, Salis F, Ciccozzi M, Oggiano A. Phylogenetic analysis of porcine circovirus type 2 in Sardinia, Italy, shows genotype 2d circulation among domestic pigs and wild boars. Infect Genet Evol. 2019, 71:189-196.
5. Ellis J, Hassard L, Clark E, Harding J, Allan G, Willson P, Strokappe J, Martin K, McNeilly F, Meehan B, Todd D, Haines D. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. The Canadian veterinary journal = La revue veterinaire canadienne. 1998, 39, 44?51.
6. Franzo G, Cortey M, de Castro AM, Piovezan U, Szabo MP, Drigo M, Segales J, Richtzenhain LJ. Genetic characterisation of Porcine circovirus type 2 (PCV2) strains from feral pigs in the Brazilian Pantanal: An opportunity to reconstruct the history of PCV2 evolution. Vet Microbiol. 2015, 178, 158?162.
7. Franzoni G, Graham S, Dei Giudici S, Oggiano A. Porcine Dendritic Cells and Viruses: An Update. Viruses. 2019, 11(5): 445.
8. Harding JC. The clinical expression and emergence of porcine circovirus 2. Vet Microbiol. 2004, 98, 131?135.
9. Karuppannan A and Opriessnig T. Porcine Circovirus Type 2 (PCV2) Vaccines in the Context of Current Molecular Epidemiology. Viruses. 2017, 9(5)
10. Kiupel M, Stevenson GW, Mittal SK, Clark EG, Haines DM. Circovirus-like viral associated disease in weaned pigs in Indiana. Vet Pathol. 1998, 35, 303?307.
11. Knell S, Willems H, Hertrampf B, Reiner G. Comparative genetic characterization of porcine circovirus type 2 samples from German wild boar populations. Vet. Microbiol. 2005,109, pp. 169-177.
12. Konradt G, Cruz RAS, Bassuino DM, et al.Granulomatous Necrotizing Myositis in Swine Affected by Porcine Circovirus Disease. Vet Pathol. 2018, 55(2):268-272.
13. Morozov I, Sirinarumitr T, Sorden SD, Halbur PG, Morgan MK, Yoon KJ, Paul PS. Detection of a novel train of porcine circovirus in pigs with postweaning multisystemic wasting syndrome. J Clin Microbiol. 1998, 36, 2535?2541.
14. O'Connor B, Gauvreau H, West K. Multiple porcine circovirus 2-associated abortions and reproductive failure in a multisite swine production unit. Can Vet J. 2001, 42:551?553.
15. Oh T, Chae C. First isolation and genetic characterization of porcine circovirus type 3 using primary porcine kidney cells. Vet Microbiol. 2020, 241, 108576.
16. Opriessnig T, Karuppannanc A, Castrod A, Xiaoe C. Porcine circoviruses: current status, knowledge gaps and challenges. Virus Res. 2020, 286:198044.
17. Opriessnig T, Langohr I. Current state of knowledge on porcine circovirus type 2- associated lesions. Vet Pathol. 2013, 50, 23?38.
18. Opriessnig T, Meng XJ, Halbur PG. Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies. J Vet Diagn Invest. 2007, 19, 591?615.
19. Palinski R, Pineyro P, Shang P, Yuan F, Guo R, Fang Y, Byers E, Hause BM. A Novel Porcine Circovirus Distantly Related to Known Circoviruses Is Associated with Porcine Dermatitis and Nephropathy Syndrome and Reproductive Failure. J Virol. 2017, 91.
20. Phan TG, Giannitti F, Rossow S, Marthaler D, Knutson TP, Li L, Deng X, Resende T, Vannucci F, Delwart E. Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation. Virology journal. 2016, 13, 184.
21. Ren L, Chen X, Ouyang H. Interactions of porcine circovirus 2 with its hosts. Virus Genes. 2016, 52:437?444.
22. Resendes AR, and Segalés J. Characterization of Vascular Lesions in Pigs Affected by Porcine Circovirus Type 2?Systemic Disease. Vet Pathol. 2015, Vol. 52(3) 497-504.
23. Ribeiro Correa AM, Zlotowski P, Santos Neves de Barcellos DE, Farias da Cruz CE, Driemeier D. Brain lesions pigs affected with postweaing multisystemic wasting syndrome. J Vet Diagn Invest. 2007, 19:109-12.
24. Sanchez Jr RE, Meerts P, Nauwynck HJ, Pensaert MB. Change of porcine circovirus 2 target cells in pigs during development from fetal to early postnatal life. Vet Microbiol. 2003, 95, 15?25.
25. Seeliger F, Brügmann M, Krüger L, Greiser-Wilke I, Verspohl J, Segalés J, Baumgärtner W. Porcine Circovirus Type 2-Associated Cerebellar Vasculitis in Postweaning Multisystemic Wasting Syndrome (PMWS)-Affected Pigs. Vet Pathol. 2007, 44:621?634.
26. Segales J. Porcine circovirus type 2 (PCV2) infections: clinical signs, pathology and laboratory diagnosis. Virus Res. 2012, 164, 10?19.
27. Tischer I, Rasch R, Tochtermann G. Characterization of papovavirus-and picornavirus-like particles in permanent pig kidney cell lines. Zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene. Erste Abteilung Originale. Reihe A: Medizinische Mikrobiologie und Parasitologie, 1974, 226, 153?167.
28. Tischer I, Mields W, Wolff D, Vagt M, Griem, W. Studies on epidemiology and pathogenicity of porcine circovirus. Arch Virol. 1986, 91, 271?276.
29. Todd D. Avian circovirus diseases: lessons for the study of PMWS. Vet Microbiol. 2004, 33:525?529.
30. Vicente J, Segales J, Hofle U, Balasch M, Plana-Duran J, Domingo M., Gortazar C. Epidemiological study on porcine circovirus type 2 (PCV2) infection in the European wild boar (sus scrofa). Vet. Res.2004, 35, pp. 243-253
31. Yu S, Opriessnig T, Kitikoon P, Nilubol D, Halbur PG, Thacker E. Porcine circovirus type 2 (PCV2) distribution and replication in tissues and immune cells in early infected pigs. Veterinary immunology and immunopathology. 2007,115, 261?272.
32. Zachary JF. Mechanisms of microbial infections. In: Pathologic Basis of Veterinary Disease, 6th ed. Zachary JF (ed) Elsevier, St. Louis, Missori, 2016, 219-220.
33. Zhang HH, Hu WQ, Li JY, Liu TN, Zhou JY, Opriessnig T, Xiao CT. Novel circovirus species identified in farmed pigs designated as Porcine circovirus 4, Hunan province. China. Transbound Emerg Dis. 2020, 67(3):1057-1061.