Cobos et al. Porcine Health Management (2022) 8:51 https://doi.org/10.1186/s40813-022-00293-8 RESEARCH Open Access Retrospective assessment of porcine circovirus 3 (PCV‑3) in formalin‑fixed, paraffin‑embedded tissues from pigs affected by different clinical‑pathological conditions Àlex Cobos1,2,4, Marina Sibila1,3,4, Jaume Alomar2, Mónica Pérez1,3,4, Eva Huerta1,3,4 and Joaquim Segalés1,2,3* Abstract Background: Porcine circovirus 3 (PCV-3) is a recently discovered pathogen of swine that has been associated with several conditions. However, many questions remain unanswered regarding its infection, especially in terms of pathogenesis and disease impact. The aim of the present study was to retrospectively investigate the presence of PCV-3 genome by real time quantitative PCR (qPCR) and in situ hybridization (ISH) on selected formalin-fixed paraffin- embedded tissues of pigs affected by different clinical conditions and histological lesions. Materials and methods: Conditions investigated included porcine dermatitis and nephropathy syndrome (PDNS), periweaning failure-to-thrive syndrome (PFTS), congenital tremors type AII, reproductive disorders, and pigs affected by systemic periarteritis/arteritis, myocarditis, or encephalitis. Studied cases (n = 587) were investigated from a diagnostic database (n = 4162) that comprised samples collected within the period 1998–2021. From each condition/ lesion, 10 to 12 cases were subsequently selected and tested by qPCR and ISH (72 cases total). Results: A total of 587 cases fulfilled inclusion criteria of the different studied conditions and were distributed among the seven groups. For the further selected cases, PCV-3 genome was found by qPCR in 12/12 periarteritis, 5/10 reproductive disease, 5/10 PFTS, 3/10 myocarditis, 1/10 encephalitis and 1/10 congenital tremor cases. PCV-3 was not found in any of the PDNS cases assessed. In periarteritis cases, tissues more commonly affected were mesenteric arteries and kidney. Reproductive disease cases associated to PCV-3 genome consistently displayed myocarditis. The lesions and labelling distribution of PFTS cases with presence of PCV-3 genome were comparable to those of the periarteritis group. qPCR and ISH yielded similar results within each studied case and were statistically comparable. Conclusion: Our results suggest that periarteritis is the hallmark lesion of PCV-3-SD, and that mesenteric lymph node and kidney appeared to be the most reliable organs to confirm the presence of PCV-3 genome in cases with periarteritis. Keyword: Porcine circovirus 3 (PCV-3), PCV-3 systemic disease (PCV-3-SD), PCV-3 reproductive disease (PCV-3-RD), Periarteritis, In situ hybridization, Real time quantitative polymerase chain reaction (qPCR) Background *Correspondence: joaquim.segales@irta.cat Porcine circovirus 3 (PCV-3) was first identified in 2015 1 Unitat Mixta d’Investigació IRTA‑UAB en Sanitat Animal, Centre de Recerca [1] and retrospective studies have dated evidence of en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona infection as soon as 1967 in Brazil [2], 1993 in Sweden (UAB), 08193, Bellaterra, Barcelona, Catalonia, Spain [3] and 1996 in Spain and China [4, 5]. Ever since, this Full list of author information is available at the end of the article © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Cobos et al. Porcine Health Management (2022) 8:51 Page 2 of 9 virus has been found in pigs affected by different clini- de Barcelona (UAB), Barcelona, Spain, between 1998 cal conditions and histological lesions, both under natu- and 2021 were investigated. The database was initially ral and experimental conditions: porcine dermatitis and filtered and only porcine circovirus 2 (PCV-2) negative nephropathy syndrome (PDNS) [1], reproductive failure pigs were included in this study (n = 4,162); such nega- [1, 6, 7], multisystemic inflammation associated to wast- tivity was determined through either ISH or immunohis- ing piglets with or without anorexia [8, 9] and congenital tochemistry (IHC), depending on the year of submission tremors (CT) [10]. Interestingly, an exploratory metagen- [15]. Animals were firstly classified as perinatal (aborted, omic study of pigs revealed significant higher abundance stillborn and weak-born piglets less than 3-day-old pig- of PCV-3 genome in piglets affected by periweaning lets) and postnatal (more than 3-day-old piglets). Perina- failure-to-thrive syndrome (PFTS) compared to healthy tal piglets were classified as CT or reproductive disease matched ones [11]. When histopathological assessment (RD) according to its clinical history. Postnatal piglets has been available, periarteritis has been the most com- were classified as PDNS based on presence of charac- monly described lesion in these reports, but PCV-3 has teristic histological lesions, or as periarterititis/arteritis also been found in cases of non-suppurative encephalitis (periarteritis from now on), myocarditis or encephalitis [6] and myocarditis [6, 8]. Based on existing information, based on presence of such histological lesions. Within two separate conditions have been proposed as being the postnatal group, a priority of lesions was established causally associated with PCV-3, namely PCV-3-systemic to distribute cases with more than one lesion as follows: disease (PCV-3-SD) and PCV-3-reproductive disease periarteritis > myocarditis > encephalitis. All encephalitis (PCV-3-RD) [12]. cases were tested against Aujesky’s disease virus by IHC Although the diagnostic criteria of these two PCV-3 [18], being negative. Cases previously diagnosed as PFTS associated diseases are well defined, their final diagno- were included in this study regardless of the presence of sis is still challenging for several reasons: (1) laboratory any of the histological lesions mentioned in Table 1. Since techniques able to detect the virus within lesions are porcine reproductive and respiratory syndrome virus restricted to in situ hybridization (ISH) [7], since no relia- (PRRSV) has also been described as causing vascular ble antibodies to detect PCV-3 antigen in formalin-fixed, lesions, an IHC study to detect this viral antigen was per- paraffin-embedded tissues do exist commercially to date. formed as previously described [16] for cases classified in Importantly, the cost of ISH is also a major constraint the periarteritis and PFTS groups. for being offered as a routinary laboratory technique; (2) clinical outcomes associated to PCV-3 are rather unspe- Histopathology cific, and many other pathogens should be considered; All selected cases (n = 587, Table 1) were re-assessed (3), histopathological assessment experience is a funda- histopathologically to describe the presence and sever- mental asset, since associated lesions both in PCV-3-SD ity of microscopic lesions such as non-suppurative myo- and PCV-3-RD may be relatively subtle and acquaintance carditis, interstitial pneumonia, interstitial nephritis and is needed to suspect those conditions. On the other hand, non-suppurative encephalitis (glial foci and perivascu- viral circulation among swine populations without evi- lar cuffing). These lesions were graded as absence (−), dence of disease has also been widely reported, suggest- mild (+), moderate (++) and severe (+++) accounting ing that subclinical infection may be the most common for the intensity of the inflammatory infiltrates and the outcome, without evidence regarding the extent of the extension of the lesions. impact on productive parameters [13, 14]. Periarteritis/arteritis lesions were evaluated in a The objective of the present study was to analyse the blinded fashion by two independent pathologists using a presence of PCV-3 infection (by qPCR and ISH) in for- newly defined score (Table 2, Fig. 1a and 1b). Such scor- malin-fixed paraffin-embedded (FFPE) samples from ing considered the number of affected small and medium retrospective diagnostic cases of pigs showing clinical sized arteries in tissues, thus it is mainly suitable for tis- conditions and histological lesions previously associated sues with large numbers of arteries (such as kidney and to this virus. Additionally, the present study aimed to mesentery). In the other studied organs, only presence compare results of both genome-detecting techniques on (P) or absence (−) of periarterial lesions was recorded. FFPE tissues. Subset selection for PCV‑3 genome detection Materials and methods A subset of 10 to 12 cases from each group (Table 1) Retrospective histopathological assessment of lesions were selected to determine the presence of PCV-3 compatible with PCV‑3 associated diseases genome in FFPE samples. These cases were selected Pig cases submitted to the Servei de Diagnostic en Pato- taking into consideration the age of the animals, their logia Veterinaria (SDPV) at the Universitat Autònoma farm origin, the year of submission and the lesion type Cobos et al. Porcine Health Management (2022) 8:51 Page 3 of 9 Table 1 Inclusion criteria and number of cases found for each clinical-pathological group Age classification Group Number Inclusion criteria Investigated target tissue/s by of cases ISH and qPCR Perinatal Reproductive 149 History of reproductive failure (abortion, mummified, stillborn) with Heart disease or without myocarditis and/or arteritis/periarteritis Congenital 84 Newborn piglets clinically affected by this condition Central nervous system tremors Postnatal PDNS 80 Presence of characteristic systemic necrotizing vasculitis and fibri- Kidney nosuppurative glomerulitis [17] Arteritis/Peri- 40 Presence of systemic periarteritis and/or arteritis with or without Any tissue displaying periarteritis arteritis other lesions in postnatal pigs (mononuclear myocarditis, non- suppurative encephalitis, interstitial pneumonia) Myocarditis 22 Presence of mononuclear myocarditis in > 3-day-old piglets and Heart absence of systemic arteritis/periarteritis. Some cases had also other lesions (non-suppurative encephalitis, interstitial pneumonia and/or interstitial nephritis) Encephalitis 101 Presence of non-suppurative encephalitis in absence of myocarditis Central nervous system and/or systemic periarteritis. All cases were negative for Aujeszky’s disease virus detection by IHC [18] PFTS 111 Clinical signs compatible with PFTS and presence of thymic atro- Pooled tissues phy, superficial gastritis and villous atrophy in small intestine [19]. These cases featured or not any of the lesions mentioned above Perinatal: aborted, stillbirth or weak born < 3-day-old piglets; Postnatal: ≥ 3-day-old pigs PDNS Porcine dermatitis and nephropathy syndrome, PFTS Periweaning failure-to-thrive syndrome Table 2 Histopathological score for assessing arteritis/periarteritis selected for PFTS cases, containing at least lymphoid in well-vascularized tissues such as kidney and mesentery tissues, lung, heart, kidney, liver, digestive tract (stom- ach and/or intestine) and CNS. Score/tissue Description qPCR in FFPE samples − Absence of inflammatory infiltrates in the arterial A qPCR to quantify the amount of viral DNA was per- walls or around them formed on FFPE material. Four tissue slices measuring + Less than 10% of arteries affected 8 µm thick each were obtained and DNA was extracted ++ Equal or more than 10% and less than 25% of arteries as previously described [9]. In PFTS cases a single tis- affected sue slice was obtained for each one of the four selected +++ Equal or more than 25% of arteries affected blocs and pooled together. The qPCR was performed as described elsewhere [9]. The results obtained by qPCR were expressed in viral copies per mL of re-suspended and severity to maximize variability within groups (data pellet from deparaffined tissues. Samples with a viral load not shown). In the RD group, cases featuring myocardi- higher than ­ 102copies/mL of PCV-3 were considered tis were selected, which meant that half of them (n = 5) positive and quantifiable. Samples with a viral load lower displayed this lesion. In the PFTS group, four cases than ­102 copies/mL were considered positive but below were selected with periarteritis, and 6 cases without. quantification limit (BQL). One to six FFPE tissue blocks were available from each case. Each one had been initially ensembled for diagnostic purposes and contained multiple tissues, PCV3 in situ hibridization (ISH) PCV-3 ISH using RNAscope® technology was performed pooled in similar but not always identical distribution. Additional 4 µm thick cuts were obtained and then a One FFPE block was selected for each case for PCV-3 genome detection, containing heart in myocarditis and as described previously [7] using tissues matching the RD cases, central nervous system (CNS) in encephali- ones elected for qPCR assay. The presence of labelling was tis and CT groups, kidney in PDNS cases, and for peri- recorded semi quantitatively as described as mild ( +), mod- arteritis cases, blocks containing any tissue displaying erate (++) or marked (+++), as described previously [20]. these lesions were selected. Four FFPE tissue blocs were Cobos et al. Porcine Health Management (2022) 8:51 Page 4 of 9 Fig. 1 Examples of arteritis and periarteritis score, as well as other lesions: a Periarteritis in mesenteric arteries, H&E. Score 1 (<10% arteries affected); b Periarteritis in mesenteric arteries, H&E. Score 3 (>25% arteries affected); c Case of periarteritis within bronchial artery; note that bronchial artery (arrow) is adjacent to principal bronchi. H&E stain; d Moderate mononuclear lymphohistiocytic myocarditis. H&E stain; e Mild multifocal lymphoplasmacytic interstitial nephritis. H&E stain; f Periarteritis in mesenteric arteries, PCV-3 ISH. This case received a score of 1; g Periarteritis in mesenteric arteries, PCV-3 ISH. This case received a score of 3; h Bronchial artery with periarteritis where positive labelling is seen within bronchial artery muscular wall and inflammatory infiltrates, PCV-3 ISH; i Strong PCV-3 positive labelling (score 3) of myocardial fibres in case of moderate mononuclear myocarditis, PCV-3 ISH; j Positive viral genome labelling (score 1) within tubular epithelial cells of the kidney, PCV-3 ISH Statistical analyses Results from histopathological score and ISH labelling were given for each tissue assessed. qPCR results from FFPE material were expressed as viral load for the pool of tissues contained in the same paraffin block. In order to compare the results obtained with the different tech- niques, the highest value for ISH scoring per paraffin block was considered. Statistical analyses were performed with cases that resulted positive for both qPCR and ISH using GraphPad Prism 9. Viral quantifications were grouped by maximum ISH score (scores 1, 2 or 3). Saphiro-Wilk test was used to assess distribution normality of qPCR results within each ISH score. The Kruskal–Wallis test with multiple com- parisons (Dunn’s multiple comparisons test) was used to compare the mean PCV-3 viral load in tissues with differ- ent ISH score. Results Collection of studied cases The arteritis/periarteritis group contained 40 cases, from which 8 corresponded to lactating piglets and 32 post- weaning piglets. The main clinical concern at farm level for those cases was wasting and, to a lesser extent, ano- rexia, and dyspnoea. The IHC against PRRSV yielded positive results in 7 out of 40 (17.5%) cases (Additional file 1: Table S1). In these cases, positivity was restricted to macrophages in pulmonary parenchyma and spared arterial lesions in all cases. For myocarditis, 22 cases were found, all in postwean- ing piglets. The concern at farm level was rather hetero- geneous for this group of animals (respiratory problems, weaning, nervous signs…). Cobos et al. Porcine Health Management (2022) 8:51 Page 5 of 9 Table 3 Frequencies of histopathological results in different tissues for the cases within the periarteritis/arteritis group (n = 40) Lesion Organ/tissue Total affected Total submitted Percentage of affected (%) Arteritis/Periarteritis Heart 11 35 31.4 Spleen 16 34 47.1 Kidney 30 35 85.7 Liver 15 36 41.7 CNS (meninges) 15 26 57.7 Lymph node 16 34 47.1 Lung 6 33 18.2 Nasal turbinate 2 14 14.3 Tonsil 5 31 16.1 Stomach 7 17 41.2 Intestine 16 35 45.7 Mesenteric arteries 17 18 94.4 Thymus 0 2 0.0 Skeletal muscle 3 7 42.9 Adrenal gland 1 4 25.0 Non-suppurative myocarditis Heart 20 35 57.1 Interstitial nephritis Kidney 8 35 22.9 Interstitial pneumonia Lung 16 33 48.5 Gliosis CNS 11 26 42.3 Perivascular cuffing 6 26 23.1 For RD, 149 cases were collected. Main concerns at Some cases additionally presented non-suppurative myo- farm level were abortion, mummified foetuses, and still- carditis (21 out of 41, 51.2%) (Fig. 1d), interstitial nephri- born and weak born piglets. tis (8 out of 41, 19.5%) (Fig. 1e), interstitial pneumonia (16 For the rest of conditions, a total of 80 PDNS diagnosed out of 39, 41.0%) and non-suppurative encephalitis featur- pigs, 101 cases with en-cephalitis, 111 with PFTS and 84 ing gliosis (14 out of 32, 43.8%) and perivascular cuffing with CT were histopathologically re-assessed. At farm level, (9 out of 32, 28.1%). Periarteritis/arteritis in the CNS was the clinical signs in piglets diagnosed as PFTS and CT were recorded in the meningeal arteries. Within neuroparen- compatible for these conditions. For PDNS and encephalitis chyma only perivascular cuffing was recorded. A summary the clinical history was rather heterogeny. The IHC against of organs affected within the periarteritis/arteritis group PRRSV in the PFTS cases always yielded negative results. can be found in Table 3. Results for each specific case can be found in Additional file 1: Table S1. Histopathological results Myocarditis cases showed mild to severe lymphop- The cases selected displaying systemic arteritis/periarteri- lasmacytic, non-necrotizing myocarditis. Concomitant tis (n = 40 of the periarteritis group and n = 7 cases with lesions recorded in this group were non-suppurative en- PFTS and arteritis/periarteritis), showed mainly perivas- cephalitis (1/22, 4.5%), interstitial nephritis (7/22, 31.8%) cular and less frequently vascular lymphoplasmacytic to and interstitial pneumonia (2/22, 9.1%). lymphohistiocitic infiltrates in media and adventitia walls RD cases showed no significative histological lesions of small and medium caliber arteries, found in all tis- (144/149, 96,7%) or mild to moderate non-suppurative sue types available except for the thymus (Fig. 1a and b). myocarditis (5/149, 3,3%). Only one of them had charac- The more frequently affected vessels were the mesenteric teristic multisystemic periarteritis. arteries adjacent to the mesenteric lymph node (22 out of PDNS cases (n = 80) had characteristic histological 23, 95.7%) and kidney (32 out of 41, 78.0%). Nevertheless, lesions featuring systemic fibrinoid necrosis of arteries’ the lung was an exception for periarteritis, as pulmonary walls and fibrino-necrotizing glomerulitis. arteries were always spared. Periarteritis was present in a Encephalitis cases (n = 101) displayed mild-to-severe subset of lungs (6 out of 39, 15.4%) only affecting bronchial non-suppurative, non-necrotizing encephalitis character- arteries, which were identified as small-sized arteries adja- ized by multifocal to generalized glial foci and perivascu- cent to bronchial cartilage in large-sized bronchi (Fig. 1c). lar cuffing. Cobos et al. Porcine Health Management (2022) 8:51 Page 6 of 9 Table 4 Summary of qPCR and ISH results for the different studied groups qPCR Maximum ISH score (number of cases) Group positive cases Mean viral load [Range]* − + ++ +++ Arteritis/Periarteritis 12/12 9.82 × ­106 [1 × ­102–6.05 × ­107] 1 1 7 3 Myocarditis 3/10 1.03 × ­106 [1 × ­102–3.08 × ­106] 9 0 1 0 RD 5/10 2.97 × ­107 [1 × ­102–8.10 × ­107] 6 0 1 3 PDNS 0/10 – 10 0 0 0 Encephalitis 1/10 6.53 × ­102 10 0 0 0 PFTS 5/10 2.07 × ­106 [1 × ­102—1.21 × ­107] 5 3 1 1 Congenital tremors 1/10 1 × ­102 10 0 0 0 * Results are given in PCV-3 copies/mL of resuspended pellet from deparaffined tissue PFTS cases (n = 111) showed mild histological lesions in the smooth muscle in the gastrointestinal tract was of thymus atrophy, superficial lymphoplasmacytic gastri- seen. tis and enteric villus blunting, in absence of other causes Three cases of the myocarditis group tested positive by of wasting (PFTS is diagnosed by exclusion criteria [21]). qPCR, two of them being BQL. The remaining sample Periarteritis was found in a subset of cases (7 out of 111, showed moderate ISH labelling within myocardiocytes. 6,3%), accompanied with mild non-suppurative encepha- For RD cases, five were positive by qPCR although litis in 3 of these cases. one of them was BQL. Four cases (matching the ones CT cases (n = 84) had no histological lesions or mild with highest viral load by qPCR) were positive by vacuolization of cerebellar white matter. PCV-3 ISH, with the highest scores found in the heart (labeling within myocardiocytes), spleen (viral genome qPCR and ISH results located in PALS), and lymph node (follicular distribu- A summary of the qPCR results and ISH scoring can be tion of PCV-3 labelling). A lower ISH score (1 or 2) was found in Table 4. Results for each case are included in observed in the lung and kidney. Labelling on arter- Additional file 2: Table S2. ies was scant, fitting well with the lack of periarteritis, All cases in the arteritis/periarteritis group resulted except for one case that showed prominent periarterial positive by qPCR, with one of them being BQL; this lat- labelling and evident periarteritis (the one with ISH ter case was negative by ISH. The labelling in the other score (3). When lung was available, pulmonary inter- nine was prominent in association to the arterial lesions, stitial labelling was recorded as mild to moderate, and with scores ranging from 1 to 3, mostly in agreement renal labelling was mild to moderate and distributed with the histological score. All tissues assessed with arte- interstitially, in contrast to what was recorded in the rial lesions had positive labelling within those damaged periarteritis group. arteries (Fig. 1f and g); the only histological location that PDNS group cases were all negative for both ISH and did not show such labelling was pulmonary arteries. qPCR. Nevertheless, bronchial arteries had viral nucleic acid The encephalitis group revealed no labelling by ISH, detected by ISH (Fig. 1h). Myocardial labelling (intersti- also in the only positive case by qPCR (with very low tial) matched the presence of myocarditis and was posi- viral load). tive with high scores (2 or 3), with viral genome located Within the PFTS group, four cases with and six cases both diffuse and periarterial (Fig. 1i). In lungs, PCV-3 without periarteritis were tested. All cases with peri- labelling (score 1 or 2) was confined to alveolar septa and arteritis tested positive by both qPCR and ISH, but bronchial arteries, always sparing pulmonary arteries. only one out of the six cases without vascular lesions Also, follicular centers in lymph nodes and periarteri- was positive also by both techniques, with follicu- olar lymphoid sheets (PALS) of spleen were ISH positive lar ISH labelling mainly in the lymph nodes (score 1). with variable scores (1 to 3). Kidney also had high scores The 4 cases with periarteritis displayed labelling within (2 or 3) due to its arterial involvement, with tubular such lesions similarly to the cases in periarteritis group. labeling being seldom observed and matching the pres- PCV-3 labelling in CNS was focal and very mild and ence of interstitial nephritis (Fig. 1j). Positive ISH results only present in two cases. were also recorded in mesenteric arteries, almost always Congenital tremors group yielded no positivity by matching the histopathological score given. Rare labelling ISH, and only one case was positive by qPCR but BQL. Cobos et al. Porcine Health Management (2022) 8:51 Page 7 of 9 and myocarditis without presence of periarteritis in wasting piglets is not as characteristic as periarteritis for PCV-3-SD. The interstitial pneumonia seen in cases of PCV-3-SD was of mild severity and accompanied by low to moderate amounts of genome through ISH, which matches previous descriptions of PCV-3-SD. Its pres- ence in cases in which PCV-2 and PRRSV infections were also excluded further supports the potential of PCV-3 in inducing interstitial pneumonia. In the case of RD, only four cases had significant viral loads and ISH positivity, and three of them had myocar- ditis. On the contrary, four out of the five cases without myocarditis tested negative to both techniques. Although not fully conclusive, it appears that cases of reproductive failure with myocarditis are more prone to be associated with the presence of PCV-3 than those without myocar- Fig. 2 Boxplot of PCV-3 load detected by qPCR versus ISH score ditis. Periarteritis was only present in one case. Notewor- thy, other causes of foetal myocarditis cannot be ruled out since they were not specifically investigated. Findings in the PFTS group suggest again an associa- qPCR and ISH result comparison tion with PCV-3 infection when periarteritis lesions are Kruskal–Wallis and Dunn’s multiple comparisons tests present. Presence of PCV-3 infection in absence of peri- revealed statistically significant differences between the arteritis was noted in only one piglet, which showed qPCR results of cases with different ISH labelling score exclusively lymph node follicular labelling, which may (Fig. 2). indicate a potential subclinical infection. In cases of PFTS, all tissues were tested systematically by PCR (by Discussion pooling) and ISH. Moreover, the PFTS compatible lesions The present study contributes to the current knowledge (thymic atrophy, superficial gastritis and intestinal villus of PCV-3 infection, and expands existing data related blunting [19]) showed no positivity for PCV-3 by ISH. with lesions apparently caused by this virus. As has been This fact suggests that PCV-3 seems not to be the direct already described, arteritis and periarteritis are promi- cause of the lesions commonly described in PFTS; how- nent lesions associated to PCV-3 infection in postwean- ever, these described lesions are rather unspecific and ing pigs affected by wasting [9, 20]. In this retrospective could be derived from anorexia and wasting. Since half of study, all selected periarteritis cases were positive by the tested PFTS affected animals were positive for PCV- qPCR and all but one also for ISH. This latter case was 3, it cannot be ruled out whether this virus may play a surprising since the lesions were equivalent to those role in the pathogenesis of this condition, as has been observed in the rest of submissions with periarteritis; suggested [11]. however, the case was from 1998 and it can be specu- None of the cases from the congenital tremors group lated that time-related degradation of genetic material in resulted positive for PCV-3, except one having very low paraffin embedded material may have happened; alterna- viral load measured by qPCR. Therefore, obtained results tively, since this was 6-month-old pig (the oldest in the do not point out PCV-3 as a potential cause of congenital series of periarteritis cases), clearance of viral particles by tremors. Since most of the studied cases corresponded to immune system due to already chronic lesions may have congenital tremors type AII, presence of atypical porcine occurred. However, the obtained results strongly suggest pestivirus should be investigated as the most probable that finding periarteritis in PCV-2-negative diagnostic cause [22]. cases is rather suggestive of PCV-3 infection. Overall obtained results are consistent with the pro- A significant number of the cases with arteritis/peri- posed diagnostic criteria for PCV-3 associated diseases arteritis also featured other lesions in certain organs, [12] and get insights on the potential best samples to use such as mild to severe non-suppurative myocarditis and for diagnostic purposes. Mesenteric arteries and, to a mild to moderate interstitial nephritis, non-suppurative lesser extent, kidney, are the most commonly affected tis- encephalitis, and interstitial pneumonia. Nevertheless, sues with periarteritis, which definitively seems to be the the low qPCR and ISH detection of PCV-3 in myocardi- best “marker” of PCV-3 infection associated with lesions. tis and encephalitis groups may indicate that encephalitis Besides, other organs and tissues rich in small and Cobos et al. Porcine Health Management (2022) 8:51 Page 8 of 9 medium-sized vessels (such as heart, meninges, spleen, However, the specific cell types that unequivocally sup- lymph nodes, gastro-intestinal tract, and liver) may be of port PCV-3 replication are still to be defined. interest to be examined since 30 to 50% of them may also Lastly, this study strongly provides retrospective his- be positive in those cases displaying systemic arteritis/ topathological and laboratory evidence of PCV-3 infec- periarteritis. Heart was an interesting organ to be evalu- tion on field cases of naturally occurring disease, further ated because although periarteritis in this organ are rare, supporting the recently proposed diagnostic criteria of interstitial myocarditis was observed in approximately PCV-3-SD and PCV-3-RD [12]. Cases fulfilling the PCV- half of studied cases. Existence of periarteritis in lung 3-SD and PCV-3-RD case definitions in this retrospective is rarely observed (due to the low number of bronchial study dated as early as 2002, confirming that PCV-3 has arteries) but its submission to a diagnostic pathology ser- not only been circulating for decades but also has been vice can be interesting to rule out interstitial pneumonia associated with reproductive disease and wasting piglets, related to PCV-3, which in studied cases has been always reinforcing its putative importance on swine industry. mild. However, in most of these cases the limited clinical his- Both qPCR and ISH yielded similar results, with ISH tory could not give clearer insights on the specific clinical being unable to detect positivity on some cases with very profile of PCV-3 associated diseases. low viral loads. Nonetheless, presence of lesion and viral genome detection in the FFPE material do not necessar- Conclusion ily reflect overall infection severity (i.e., presence of focal This study gathered retrospective data of PCV-3-SD periarteritis in the intestine in a paraffin block containing and PCV-3-RD in diagnostic cases. Periarteritis seemed only an intestine section), yielding low qPCR viral load to be consistently related to PCV-3-SD, with high- but consistent ISH labelling within the lesions. In these est amount of lesion and ISH positivity located within cases, qPCR results may not necessarily correlate with mesenteric arteries and kidney, which makes them disease severity or other organs affection. To solve this excellent options for submitting for diagnosis. In this issue, appropriate tissues (mesenteric lymph node and/ retrospective series, presence of PCV-3-SD without or kidney) should be assessed for qPCR. Importantly, the periarteritis was only found in one case with myocar- availability of serum or fresh tissues may help correlating ditis and encephalitis, suggesting that periarteritis is a the amount of virus detected by qPCR with the presence hallmark lesion of PCV-3-SD. Participation of PCV3 in of histological lesions compatible with PCV-3-SD and PFTS cases was also assessed, resulting in some posi- PCV-3-RD, especially when no ISH expertise is available. tive cases with characteristic features of PCV-3-SD, One of the limitations of this study is that viral quanti- which could imply that some cases diagnosed as PFTS fications were performed in a pool of FFPE tissues, which could be reclassified and diagnosed as PCV-3-SD. contained variable quantities of different combinations Regarding PCV-3-RD, our cases were mainly charac- of tissues. The lack of fresh tissue individual samples or terized by mononuclear myocarditis with only one of serum from studied animals prevented the possibility to them presenting multisystemic periarteritis. establish a potential viral load threshold for discriminat- ing between PCV-3 subclinical infection and associated Supplementary Information diseases. Future studies quantifying the PCV-3 load indi- The online version contains supplementary material available at https://​doi.​ vidual tissue types would be more specific and compara- org/​10.​1186/​s40813-​022-​00293-8. ble to the ISH results. Although the state of knowledge regarding PCV-3 is Additional file 1: Table S1. Histopathological results for cases displaying arteritis/periarteritis (n=40) and cases of PFTS featuring periarteritis/arteri- progressing rapidly, it is still far from reaching the exist- tis (n=7), in total 47 cases. ing understanding of the most well-known porcine cir- Additional file 2: Table S2. Tissue availability per each studied case, covirus, PCV-2 [23]. Based on a very limited number of including histological results and PCV-3 genome detection results (qPCR experimental inoculations and natural infection data, and ISH results). proposed mechanisms of PCV-3 pathogenesis are (1) virus-induced injury, (2) type 3 hypersensitivity and (3) Acknowledgements type 4 hypersensitivity [24], although it is yet to be deter- Not applicable. mined to what extent each of these might play a role. Pre- Author contributions vious natural infection reports have shown viral genome Retrospective investigation, A.C.; case selection, A.C.; Histopathological evalu- presence in different tissues and, therefore, suggested ation, A.C. and J.A.; histopathological supervision, J.S.; qPCR, E.H. and M.S.; ISH, A.C. and M.P.; data analysis, A.C.; writing-original draft preparation, A.C., writing- viral replication sites in the parenchyma and in arteries review and editing, M.S. and J.S. Financial support acquisition, M.S. 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