Received: 8 June 2021 Revised: 22 July 2021 Accepted: 23 July 2021 DOI: 10.1111/tbed.14260 SPECIAL ISSUE ARTICLE Multisystemic lymphoplasmacytic inflammation associated with PCV-3 in wasting pigs Jaume Alomar1 Viviane Saporiti2,3 Mònica Pérez2,3 Diogo Gonçalvez4 Marina Sibila2,3 Joaquim Segalés1,3,5 1 Servei de Diagnòstic de Patologia Veterinària, Departament de Sanitat i Abstract Anatomia Animals, Facultat de Veterinària, Porcine circovirus 3 (PCV-3) has been detected in diseased and healthy pigs of dif- Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain ferent ages. Several reports have associated the agent with reproductive failure and 2 IRTA, Centre de Recerca en Sanitat Animal mummified and stillborn piglets. One report from North America has proposed a con- (CReSA, IRTA- UAB), Campus de la Universitat sistent potential association with postweaning disorders. Thus, the present case report Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain aimed to describe the histopathological lesions and their association with the presence 3 OIE Collaborating Centre for the Research of PCV-3 genome in postweaning pigs showing growth-retardation and thrown-back and Control of Emerging and Re-emerging Swine Diseases in Europe (IRTA-CReSA), ears. All affected animals displayed multi-organic lymphoplasmacytic periarteritis, Bellaterra, Barcelona, Spain lymphocytic myocarditis and/or lymphoplasmacytic meningoencephalitis. PCV-3 4 Raporal, SA, Brejo do Lobo, Montijo genetic material was detected by in situ hybridization within the lesions and confirmed 2870-683, Portugal 5 by PCV-3 real-time quantitative PCR detection in tissues. This study represents the UAB, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat first report of PCV-3 associated with clinical disease in postweaning pigs in Europe. Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès 08193, Spain KEYWORDS Correspondence in situ hybridization, myocarditis, periarteritis, pig, porcine circovirus 3, postweaning Joaquim Segalés, Servei de Diagnòstic de Patologia Veterinària, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Email: joaquim.segales@irta.cat Funding information E-RTA2017-00007-00-00 INIA Project from the Instituto Nacional de Investigación y Tec- nologia Agraria y Alimentaria (Spanish Govern- ment) 1 INTRODUCTION known as a major pathogen for swine as the cause of porcine circovirus diseases (PCVD) (Segalés, 2012). A third PCV, named Porcine circovirus Porcine circoviruses (PCVs) are circular, non-enveloped, single- 3 (PCV-3), was discovered in 2015 in the United States by means of stranded DNA viruses included in the family Circoviridae, genus metagenomics (Palinski et al., 2017; Phan et al., 2016). It is known that Circovirus (Biagini et al., 2012). PCVs have worldwide distribution in PCV-3 is distributed worldwide and has been found in samples from swine herds and, until few years ago, only two species were known sick and healthy pigs (Klaumann et al., 2018; Saporiti et al., 2020) as to infect pigs: Porcine circovirus 1 (PCV-1), which is considered non- well as wild boar (Franzo et al., 2018; Klaumann et al., 2019). Recently, pathogenic to pigs, and Porcine circovirus 2 (PCV-2), which is widely a fourth PCV was found in several Chinese farms (Zhang et al., 2020), This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH Transbound Emerg Dis. 2021;68:2969–2974. wileyonlinelibrary.com/journal/tbed 2969 2970 ALOMAR ET AL . but not in Italy and Spain (Franzo et al., 2020), which has been named Porcine circovirus 4 (PCV-4) (Opriessnig et al., 2020). PCV-4 genetic material has been detected in 7- to 12-week-old pigs suffering from porcine dermatitis and nephropathy syndrome, and respiratory and enteric clinical signs, as well as in healthy animals of all age groups (Opriessnig et al., 2020; Zhang et al., 2020). PCV-2 is a well-established pig pathogen, but very demanding cri- teria were established to diagnose PCVDs (Grau-Roma et al., 2012). For instance, the diagnosis of PCV-2-systemic disease (PCV-2-SD) needs the assessment of three major criteria, namely clinical signs, histopathological lesions in lymphoid tissues and presence of the virus within these lesions (Segalés, 2012). Disease causality demonstration for a ubiquitous agent is not an easy task, and most of the reports published to date on PCV-3 (Opriessnig et al., 2020) are based on the detection of viral genome in sick animals. However, the mere pres- ence of nucleic acid in a sample does not constitute evidence of dis- ease causality (Arruda et al., 2019). Among all published studies, only few of them showed the presence of PCV-3 within lesions, which may account for this virus as a cause of the observed clinical condition (Arruda et al., 2019; Kim et al., 2018; Phan et al., 2016). Moreover, only F I G U R E 1 Postweaning pig suffering from the so-called ‘flying pig syndrome’ with wasting and thrown-back ears one has demonstrated a significant amount of PCV-3 nucleic acid in the damaged tissues of postweaning pigs, further suggesting the role of this virus in causality (Arruda et al., 2019). and stained with hematoxylin and eosin (HE) for routine histology To date, there are no peer-reviewed published reports of PCV-3 assessment. associated disease in pigs from Europe, although cases with high viral The same fixed tissues were used to assess the presence of PCV-3 loads in stillborn and lactating piglets from United Kingdom and Spain genetic material by in situ hybridization (ISH) using RNAscope® tech- have been described (Saporiti et al., 2021; Williamson et al., 2019). nology (Saporiti et al., 2021). Additionally, a pool of paraffin-embedded Here, we report the first case of postweaning disease associated with tissues (Table 1) was used for DNA extraction to detect PCV-3 genome PCV-3 described in Europe. by real-time quantitative PCR (qPCR) (Saporiti et al., 2021). For the DNA extraction, four tissue slices were treated firstly with 1 ml of xylol and afterwards with 1 ml of absolute ethanol. Then, the obtained tissue 2 MATERIALS AND METHODS pellet was dried and resuspended according to the instructions of the QiAamp® DNA FFPE Tissue kit. Since January 2020, a Portuguese 200 sow-farm with a 3-week farrow- Since the condition was clinically characterized by growth retar- ing batch production system weaning 30.5 piglets per sow/year suf- dation, two major pathogens able to cause a similar clinical picture fered from a clinical disorder in nursery piglets (4-10 weeks of age). It were considered (PCV-2 and porcine reproductive and respiratory syn- consisted of growth-retarded animals showing thrown-back ears (the drome virus, PRRSV). Specifically, PCV-2 and PRRSV antigens were condition has been vulgarly named as ‘aviator’ or ‘flying pig syndrome’), investigated by immunohistochemical (IHC) techniques (Rosell et al., wasting and rough hair (Figure 1). The initial incidence of the condi- 1999; Segalés et al., 2002) on formalin-fixed, paraffin embedded tis- tion was 1.25%, increasing up to 3.85% in April 2020 and decreasing sues (lung, tonsil and superficial inguinal and submandibular lymph to 0.90% in November of the same year. The overall incidence for year nodes). 2020 was 1.41% (range 0.31–3.85%). By October and November 2020, the Veterinary Pathology Diag- nostic Service (SDPV) of the Universitat Autònoma de Barcelona received 3 RESULTS four affected animals and two non-affected ones from the same batch to be used as controls. All animals were subjected to an exhaustive Macroscopic evaluation of clinically affected pigs showed poor body necropsy. condition with rough hair but no evidence of gross lesions in internal Tissue samples (superficial inguinal and submandibular lymph organs. Non-diseased animals were of smaller size but were clinically nodes, Peyer’s patches, tonsil, heart, central nervous system [CNS] – healthy and did not show gross lesions as well. cerebrum, cerebellum and pons –, kidneys, lungs, spleen, large and Microscopically, diseased pigs had multi-organic, moderate to small bowel, liver and/or nasal turbinate) were collected and fixed by severe lymphoplasmacytic periarteritis and arteritis in mesenteric immersion in a 10% buffered formaldehyde solution for 48 h. Tissues arteries (4/4), heart (4/4), kidneys (4/4), spleen (4/4), portal arterioles were subsequently embedded in paraffin wax, sectioned at 3 µm, (3/4), meninges (2/4), lungs (2/4) and/or stomach (2/4). The histological ALOMAR ET AL . 2971 TA B L E 1 Real time quantitative PCR results expressed as PCV-3 copies/ml of resuspended formalin-fixed, paraffin-embedded tissues obtained for each studied pig Viral load (copies of Ref. Pool of formalin-fixed, paraffin-embedded tissues PCV-3/ml) Pig No. 1 Lymph node, lung, tonsil, brain, liver, heart, kidney, nasal turbinate. 1.20 × 107 Pig No. 2 Lymph node, lung, tonsil, brain, liver, heart, spleen, kidney, nasal turbinate. 1.45 × 107 Pig No. 3 Lymph node, lung, tonsil, brain, liver, heart, spleen, kidney. 9.38 × 107 Pig No. 4 Lymph node, lung, tonsil, brain, liver, heart, spleen, kidney. 4.10 × 107 Pig No. 5a Lymph node, lung, tonsil, brain, liver, heart, spleen, kidney, small bowel. 1.56 × 103 Pig No. 6a Lymph node, lung, tonsil, brain liver, heart, spleen, kidney, small bowel. 1.39 × 103 a Non-diseased pigs. features of affected arteries were circumferential or segmental nasal turbinate inflammatory infiltrate (2/2) were also observed. Over- lymphoplasmacytic inflammatory infiltration (Figure 2A). These all, the number of labeled cells were directly proportional to the sever- inflammatory infiltrates expanded and disrupted the periarteriolar ity of lesion detected, although in lung and brain the amount of viral connective tissue and tunica adventitia, only affecting the tunica media genome was high in comparison with the severity of lesions (high viral and intima in the most severe cases. Smooth muscle cells of the tunica amount but mild lesions). Control animals had no evidence of PCV-3 media showed a mild-to-intense vacuolization with loss of cytoplasm genome detection by ISH (Figure 2H). borders definition. Occasionally, endothelial cells were plumped to the All sick and control pigs were positive for PCV-3 qPCR (Table 1). The lumen and leukocytes attached to the endothelium displaying rolling four clinically affected animals with histological lesions and ISH label- and exocytosis. Besides the blood vessel lesions, the CNS of the four ing had high viral loads in tissues (from 1.20 to 9.38 × 107 copies/ml sick pigs had mild lymphoplasmacytic meningoencephalitis (Figure 2B), of the resuspended pellet from deparaffined tissues). The two control characterized by diffuse gliosis and multifocal discrete perivascular animals had low viral load in the pooled tissues (from 1.39 to 1.56 × 103 cuffing. Other observed lesions in diseased animals were generalized copies/ml of the resuspended pellet from deparaffined tissues). lymphoplasmacytic myocarditis (4/4), mild lymphoplasmacytic and Furthermore, all animals were evaluated for PCV-2 and PRRSV anti- histiocytic interstitial pneumonia (1/4), mild lymphoplasmacytic gen detection by IHC techniques, yielding negative results by both nephritis (Figure 2C) (3/4) and mild lymphoplasmacytic periportal techniques. Noteworthy, none of the animals suffered from lesions con- hepatitis (3/4). Nasal turbinates were examined in only two pigs, which sistently attributable to those viruses. displayed mild lymphoplasmacytic rhinitis and mild lymphoplasmacytic periarteritis. Control animals did not show gross or microscopic lesions (Figure 2D). 4 DISCUSSION In all sick animals, the detected PCV-3 nucleic acid by in situ hybridization (ISH) was found in association of the abovementioned The present report described a wasting disease in postweaning pigs lesions. In the evaluated samples of the four sick animals, all arteries showing multisystemic inflammatory lesions associated to PCV-3 with consistent histologic lesions showed a segmental or circumferen- infection. To date, most of the descriptions of PCV-3 detection in sick tial PCV-3 labeling of smooth muscle-like cells, lymphocytes, plasma animals have been based on qPCR (Opriessnig et al., 2020), which cells and endothelial cells (Figure 2E). Viral genome detection in lym- makes it difficult to establish a sound causality association of the virus phoid tissues was observed as stellate-shape cells in germinal centers with the clinical-pathological condition. Therefore, and considering the of lymph nodes (2/3), tonsils (2/3), Peyer’s patches (1/3) and white widespread nature of PCV-3 over the world, it is urgently needed to pulp of the spleen (3/4). In the heart, widespread PCV-3 labelling was establish criteria for a formal case definition of PCV-3 associated dis- present in myocardiocyte-like cells (4/4) (Figure 2E, inset). CNS showed ease/s (PCV-3-AD). This aspect is even more important considering extensive labelling in spotted and stellate-like cells, mainly in the white that both sick and healthy pigs in a farm may be infected with this virus, matter but also in multiple foci in the grey matter in neuronal body- as highlighted by the qPCR results in the present study. like cells (4/4) from cerebellum and cerebrum (Figure 2F and inset). A similar situation occurred more than two decades ago with a Besides arterial labelling, viral RNA was detected multifocally in the genetically related circovirus, PCV-2, also ubiquitous. The sole pres- cytoplasm of renal tubular epithelial cells (2/4) (Figure 2G) in the lung, ence of the virus in a pig does not constitute a diagnosis of PCVDs, type I pneumocyte-like cells and interstitial macrophage-like cells were and the triad (1) clinical signs, (2) moderate to severe histological labeled (2/4). Scant positivity in the Kupffer’s cells (3/4), scattered lesions and (3) moderate to high amount of PCV-2 in damaged tissues, labelling in intestinal smooth muscle-like cells of the small bowel and is required to substantiate a definitive diagnosis (Segalés, 2012). spiral colon (1/4) and scant positivity in macrophage-like cells in the Equivalent criteria have been demonstrated in very few reports for 2972 ALOMAR ET AL . F I G U R E 2 Histology (H&E stain, A, B, C, D) and PCV-3 in situ hybridization (E, F, G, H) results. (A) Heart; intense lymphoplasmacytic periarteritis from pig No. 3. (B) Brain; segmental periarteritis in meningeal artery and cortical section without lesions. (C) Kidney; mild interstitial lymphoplasmacytic nephritis from pig No. 4. (D) Mesenteric arteries; histological normal arteries from control pig, No. 5. (E) Heart; PCV-3 genome detection in arterial wall and endothelial cells from pig No. 3. (Inset) PCV-3 nucleic acid detection in myocardiocyte-like cells of the same animal. (F) High amount of PCV-3 genome in cerebrum white matter and mild-to-moderate in grey matter from pig No. 3. (Inset) PCV-3 labelling in neuron-like cells of the same case. (G) PCV-3 nucleic acid detection in renal tubular epithelial cells of the same animal. (H) Mesenteric arteries; lack of PCV-3 detection by ISH in control pig, No. 5 ALOMAR ET AL . 2973 PCV-3, mainly regarding reproductive failure (Arruda et al., 2019; ACKNOWLEDGMENTS Palinski et al., 2017; Saporiti et al., 2021; Williamson et al., 2019) and This research was funded by E-RTA2017-00007-00-00 INIA Project myocarditis and periarteritis in postweaning pigs (Arruda et al., 2019; from the Instituto Nacional de Investigación y Tecnologia Agraria y Ali- Phan et al., 2016). Some descriptions have used ISH to detect PCV-3 mentaria (Spanish Government) and CERCA Programme/Generalitat genome in different tissues from sick animals, but the amount of viral de Catalunya. The authors thank the technical support of Blanca Pérez material detected was low (Kim et al., 2018; Phan et al., 2016), which and Aida Neira, from the Servei de Diagnòstic de Patologia Veterinària poses uncertainty on the real causality of the condition. (SDPV) at the Universitat Autònoma de Barcelona and Eva Huerta from PCV-3 genome was found to high loads by qPCR in the four diseased the Institut de Recerca i Tecnologia Agroalimentàries. pigs, coinciding with the high viral nucleic acid amount detected by ISH in damaged tissues. Importantly, PCV-3 DNA was also detected in the CONFLICT OF INTEREST non-diseased animals by qPCR, even to a low amount, which coincided The authors declare no conflict of interest. with the lack of detection by the ISH. These results further mirror PCV- 2 in the sense that overt disease is described when high viral loads are DATA AVAILABILITY STATEMENT detected within lesions (Segalés, 2012). Data published in this report are available upon request. Arruda et al. (2019) described myocarditis and multi-organic peri- arteritis associated with a significant amount of PCV-3 genome in 12 ORCID cases of postweaning pigs suffering from different clinical conditions Joaquim Segalés https://orcid.org/0000-0002-1539-7261 in the United States. However, to the authors’ knowledge, no further cases have been reported around the world to date. Therefore, the cases described here represent the first description of PCV-3-AD in REFERENCES Arruda, B., Piñeyro, P., Derscheid, R., Hause, B., Byers, E., Dion, K., Long, postweaning pigs in Europe. D., Sievers, C., Tangen, J., Williams, T., & Schwartz, K. (2019). PCV3- Importantly, an abundant amount of PCV-3 genome was found in all associated disease in the United States swine herd. Emerging Microbes four diseased pigs, including the brain (mainly cerebrum and cerebel- & Infections, 8(1), 684–698. https://doi.org/10.1080/22221751.2019. lum), a fact that has not been previously reported in postweaning pigs 1613176 (Arruda et al., 2019). However, these animals did not show CNS clin- Biagini, P., Bendinelli, M., Hino, S., Kakkola, L., Mankertz, A., Niel, C., Okamoto, H., Raidal, S., Teo, C. G., & Todd, D. (2012). Circoviridae. In A. ical signs despite the mild meningoencephalitis associated with PCV- M. Q. King, M. J. Adams, E. B. Carstens, E. J. Lefkowitz (Eds.), Virus taxon- 3. Interestingly, presence of viral genome in brain has been found in omy: classification and nomenclature of viruses. Ninth report of the Interna- cases of perinatal disease in several reports (Arruda et al., 2019; Sapor- tional Committee on Taxonomy of Viruses (pp. 343–349). Elsevier Academic iti et al., 2021; Williamson et al., 2019). Press. Franzo, G., Legnardi, M., Hjulsager, C. K., Klaumann, F., Larsen, L. E., Segales, Obtained data suggest that PCV-3 may replicate in epithelial cells J., & Drigo, M. (2018). Full-genome sequencing of porcine circovirus from the endothelia, as well as renal tubular cells, pneumocytes, 3 field strains from Denmark, Italy and Spain demonstrates a high smooth muscle cells from blood vessels, myocardial cells, dendritic within-Europe genetic heterogeneity. Transboundary and Emerging Dis- cells/macrophages and glial cells. Some of these cell types have already eases, 65(3), 602–606. https://doi.org/10.1111/tbed.12836 Franzo, G., Ruiz, A., Grassi, L., Sibila, M., Drigo, M., & Segalés, J. (2020). Lack been suggested as supporting viral replication by means of experimen- of Porcine circovirus 4 genome detection in pig samples from Italy and tal settings (Mora-Díaz et al., 2020). In any case, much more research is Spain. Pathogens (Basel, Switzerland), 9(6), 433. https://doi.org/10.3390/ needed to establish the pathogenesis of PCV-3 infection in pigs and to pathogens9060433 elucidate the cells supporting viral replication. Grau-Roma, L., Baekbo, P., Rose, N., Wallgren, P., Fraile, L., Larsen, L., & Segalés, J. (2012). Clinical and laboratory studies on herds affected To date, minimal information does exist regarding the pathogenesis with postweaning multisystemic wasting syndrome in Denmark, France, of PCV-3 infection (Mora-Díaz et al., 2020). It could be speculated that Spain, and Sweden: Disease progression and a proposal for herd case this novel virus may require the presence of other infectious and/or definition. Journal of Swine Health and Production, 20, 129–136. non-infectious co-factors to trigger disease, in a similar manner as hap- Kim, S. C., Nazki, S., Kwon, S., Juhng, J. H., Mun, K. H., Jeon, D. Y., Jeong, C. G., Khatun, A., Kang, S. J., & Kim, W. I. (2018). The prevalence and genetic pen with PCV-2 (Grau-Roma et al., 2012). Therefore, more extensive characteristics of porcine circovirus type 2 and 3 in Korea. BMC Veteri- experimental and field data is necessary to ascertain the importance of nary Research, 14(1), 294. https://doi.org/10.1186/s12917-018-1614-x PCV-3 as a cause of disease in swine. Klaumann, F., Correa-Fiz, F., Franzo, G., Sibila, M., Núñez, J. I., & Segalés, J. Although two important diseases as those caused by PCV-2 and (2018). Current Knowledge on Porcine circovirus 3 (PCV-3): A novel virus PRRSV were ruled out, the potential causality of the condition by PCV- with a yet unknown impact on the swine industry. Frontiers in Veterinary Science, 5, 315. https://doi.org/10.3389/fvets.2018.00315 3 should be further confirmed in a wider set of animals displaying Klaumann, F., Dias-Alves, A., Cabezón, O., Mentaberre, G., Castillo- described lesions. PCV-3 nucleic acid was definitively located within Contreras, R., López-Béjar, M., Casas-Díaz, E., Sibila, M., Correa-Fiz, F., the inflammatory lesions, but the concomitance of other pathogens or & Segalés, J. (2019). Porcine circovirus 3 is highly prevalent in serum and conditions in triggering the clinical condition cannot be excluded. tissues and may persistently infect wild boar (Sus scrofa scrofa). Trans- boundary and Emerging Diseases, 66(1), 91–101. https://doi.org/10.1111/ In summary, the present report describes the first documented case tbed.12988 of postweaning wasting disease showing multisystemic angiocentric Mora-Díaz, J., Piñeyro, P., Shen, H., Schwartz, K., Vannucci, F., Li, G., Arruda, inflammatory lesions associated with PCV-3 in pigs in Europe. B., & Giménez-Lirola, L. (2020). Isolation of PCV3 from perinatal and 2974 ALOMAR ET AL . reproductive cases of PCV3-associated disease and in vivo characteri- stillborn piglets from swine reproductive failure cases. Viruses, 13(2), zation of PCV3 replication in CD/CD growing pigs. Viruses, 12(2), 219. 264. https://doi.org/10.3390/v13020264 https://doi.org/10.3390/v12020219 Segalés J. (2012). Porcine circovirus type 2 (PCV2) infections: clinical signs, Opriessnig, T., Karuppannan, A. K., Castro, A., & Xiao, C. T. (2020). Porcine pathology and laboratory diagnosis. Virus Research, 164(1-2), 10–19. circoviruses: current status, knowledge gaps and challenges. Virus Segalés, J., Calsamiglia, M., Rosell, C., Soler, M., Maldonado, J., Research, 286, 198044. https://doi.org/10.1016/j.virusres.2020.198044 Martín, M., & Domingo, M. (2002). Porcine reproductive and res- Palinski, R., Piñeyro, P., Shang, P., Yuan, F., Guo, R., Fang, Y., Byers, E., & Hause, piratory syndrome virus (PRRSV) infection status in pigs nat- B. M. (2017. A novel porcine circovirus distantly related to known cir- urally affected with post-weaning multisystemic wasting syn- coviruses is associated with porcine dermatitis and nephropathy syn- drome (PMWS) in Spain. Veterinary Microbiology, 85(1), 23–30. drome and reproductive failure. Journal of Virology, 91(1), e01879-16. https://doi.org/10.1016/s0378-1135(01)00474-6 https://doi.org/10.1128/JVI.01879-16 Williamson, S., Wilson, L., Collins, R., Floyd, T., Grierson, S., & Dastjerdi, A. Phan, T. G., Giannitti, F., Rossow, S., Marthaler, D., Knutson, T. P., Li, L., Deng, (2019). Multisystemic inflammation in piglets in a herd with congeni- X., Resende, T., Vannucci, F., & Delwart, E. (2016). Detection of a novel cir- tal tremor and concurrent deformities. 11th European Symposium of covirus PCV3 in pigs with cardiac and multi-systemic inflammation. Virol- Porcine Health Management, Utrecht, The Netherlands. ogy Journal, 13(1), 184. https://doi.org/10.1186/s12985-016-0642-z Zhang, H. H., Hu, W. Q., Li, J. Y., Liu, T. N., Zhou, J. Y., Opriessnig, T., & Xiao, C. Rosell, C., Segalés, J., Plana-Durán, J., Balasch, M., Rodríguez-Arrioja, G. T. (2020). Novel circovirus species identified in farmed pigs designated as M., Kennedy, S., Allan, G. M., McNeilly, F., Latimer, K. S., & Domingo, Porcine circovirus 4, Hunan province, China. Transboundary and Emerging M. (1999). Pathological, immunohistochemical, and in-situ hybridization Diseases, 67(3), 1057–1061. https://doi.org/10.1111/tbed.13446 studies of natural cases of postweaning multisystemic wasting syn- drome (PMWS) in pigs. Journal of Comparative Pathology, 120(1), 59–78. https://doi.org/10.1053/jcpa.1998.0258 Saporiti, V., Cruz, T. F., Correa-Fiz, F., Núñez, J. I., Sibila, M., & Segalés, J. How to cite this article: Alomar, J., Saporiti, V., Pérez, M., (2020). Similar frequency of Porcine circovirus 3 (PCV-3) detection in Gonçalvez, D., Sibila, M., & Segalés, J. (2021). Multisystemic serum samples of pigs affected by digestive or respiratory disorders and age-matched clinically healthy pigs. Transboundary and Emerging Diseases, lymphoplasmacytic inflammation associated with PCV-3 in 67(1), 199–205. https://doi.org/10.1111/tbed.13341 wasting pigs. Transboundary and Emerging Diseases, 68, Saporiti, V., Valls, L., Maldonado, J., Perez, M., Correa-Fiz, F., Segalés, J., & 2969–2974. https://doi.org/10.1111/tbed.14260 Sibila, M. (2021). Porcine circovirus 3 detection in aborted fetuses and