Veterinary Microbiology 98 (2004) 137–149

Pathological findings associated with naturally acquiredporcine circovirus type 2 associated disease

Joaquim Segalés∗, Carles Rosell, Mariano DomingoCentre de Recerca en Sanitat Animal (CReSA), Departament de Sanitat i d’Anatomia Animals, Facultat de Veterinària,

Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

Abstract

Porcine circovirus type 2 (PCV2) is a novel virus of the Circoviridae family which is considered the cause of postweaningmultisystemic wasting syndrome (PMWS). PCV2 has also been associated to a number of pathological conditions of pigs,including porcine dermatitis and nephropathy syndrome, reproductive failure, porcine respiratory disease complex, proliferativeand necrotising pneumonia and congenital tremor type AII. Pathological studies have been used to describe and characterisePMWS and these emerging conditions associated with PCV2. The objective of this review is to concentrate on the gross,microscopic and ultrastructural pathology associatedwith natural cases of PCV2 associated disease, alongwith some speculationson the pathogenesis of naturally occurring PMWS.© 2003 Elsevier B.V. All rights reserved.

Keywords: Porcine circovirus type 2; Necropsy; Histopathology; Immunohistochemistry; In situ hybridisation; Postweaning multisystemicwasting syndrome

1. Introduction

Since the first description of postweaning multisys-temic wasting syndrome (PMWS) in Saskatchewan(Canada) in 1991 (Harding, 1996), this syndrome hasnow been described world-wide but Oceania (Allanand Ellis, 2000). The acronym PMWS was initiallyused to describe an apparently new disease which wasclinically characterised by wasting, paleness of theskin and, occasionally, icterus in weaned pigs; affectedpigs also had characteristic lesions in multiple tissues(multisystemic), mainly in lymphoid organs (Harding

∗ Corresponding author. Tel.: +34-93-5811599;fax: +34-93-5813142.E-mail address: joaquim.segales@uab.es (J. Segales).

et al., 1998). In 1997, the presence of porcine cir-covirus (PCV) antigen was demonstrated in lesions ofanimals affected by PMWS (Clark, 1997). Nucleotidesequence analysis of the PCV associated with PMWSrevealed important differences compared to the previ-ously known PCV derived from PK-15 cells (ATCCCCL-33) (Hamel et al., 1998), and it was suggestedthese viruses be named PCV type 1 (PCV1) for thecell culture-derived virus, and PCV type 2 (PCV2) forthe virus associated with the new disease (Allan et al.,1999a).The clinical and pathological scope of PCV2 infec-

tion has been expanded since 1991. Although PCV2is a virus mainly associated with PMWS, it has alsobeen implicated in other clinical and pathologicalconditions. Since 1999, PCV2 has been suggested

0378-1135/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.vetmic.2003.10.006

138 J. Segales et al. / Veterinary Microbiology 98 (2004) 137–149

to play a role in reproductive disorders (West et al.,1999; O’connor et al., 2001), porcine dermatitis andnephropathy syndrome (PDNS) (Rosell et al., 2000a),the so-called porcine respiratory disease complex(PRDC) (Thacker and Thacker, 2000), proliferativeand necrotising pneumonia (PNP) (Pesch et al., 2000),and congenital tremor (Stevenson et al., 2001). Therole of PCV2 in these conditions has not been fullyclarified and, in some of these conditions, it remainsa controversial issue.Experimental studies with PCV2 have mainly been

focused on PMWS reproduction, and, although PCV2is considered a necessary but not sufficient factor todevelop PMWS, little doubt exists on the causal rela-tionship between the virus and the wasting syndromeat present (Kennedy et al., 2000; Krakowka et al.,2001). The pathogenic basis by which PCV2 is associ-ated with conditions other than PMWS is still not un-derstood. In fact, the relationship between PCV2 andsome of these conditions has only been establishedbased on the presence of lesions associated with detec-tion of the presence of the virus, but no cause–effectrelationship has been demonstrated. Therefore, patho-logical studies have been essential in the descriptionand characterisation of PMWS and these apparentlyemerging conditions associated with PCV2. This re-view concentrates on the gross, microscopic and ul-trastructural pathology associated with natural casesof PCV2 associated disease, along with some spec-ulations on the pathogenesis of naturally occurringPMWS cases.

2. Postweaning multisystemic wasting syndrome

The final diagnosis of PMWS is established basedon three criteria (Sorden, 2000; Quintana et al., 2001):(1) presence of compatible clinical signs (basically,wasting), (2) presence of characteristic histopatholog-ical lesions in lymphoid tissues, and (3) presence ofPCV2 (antigen and/or nucleic acid) within the micro-scopic lesions. However, in a PMWS-affected farm, itis possible to find healthy pigs showing mild to moder-ate microscopic lesions typical of PMWS in lymphoidtissues, associated with low to moderate amounts ofPCV2 nucleic acid (Quintana et al., 2001). Further-more, a percentage of pigs that suffer from PMWS areable to recover and, despite the emaciated aspect of

animals in this chronic stage, histopathological exam-ination of them reveals only mild or no lesions com-patible with PMWS together with minimal amounts ofPCV2 in lymphoid tissues. Therefore, the best sam-ples to look for PMWS gross and microscopic lesions,and PCV2 within these lesions, are lymphoid tissuesof pigs in the first week of clinical disease.

2.1. Gross pathology

Although wasting and respiratory alterations in aproportion of late nursery and early fattening pigsfavour a diagnosis of PMWS, most of the clinical signsof this disease are non-specific. There are many otherdiseases or conditions that may include wasting or res-piratory distress. Similarly, non-specific macroscopiclesions may occur in PMWS cases, so a strong sus-picion of PMWS based on gross lesions can only beestablished in these cases where the full spectrum oflesions is present.At necropsy, the most striking changes are non-

collapsed, tan-mottled lungs (Fig. 1A) and enlarge-ment of lymph nodes (mainly inguinal, submandibular,mesenteric (Fig. 1B) and mediastinal) (Rosell et al.,1999). However, these lesions are not always presentand they cannot be used as the only marker for PMWSin a farm. In fact, lymph nodes of normal size to at-rophic have been found with very severe microscopiclesions of PMWS (personal observations). In a lowproportion of cases, lymph nodes may have multifocalto coalescent areas of necrosis that are visible macro-scopically (Fig. 1C) (Segalés et al., 2000a). Othergross lesions associated with PMWS include reductionor increase in liver size, with orange–yellow discol-oration in those cases where icterus is present (Fig. 1D)(Rosell et al., 2000b), and kidneys with multiple palefoci of variable diameter (Fig. 1E).Moderate to high numbers of PMWS-affected pigs

have cranio-ventral pulmonary consolidation (bron-chopneumonia) and gastric ulceration of the pars oe-sophagea, which are usually associated to bacterialinfections and multifactorial origin, respectively, andnot to the direct effect of PCV2 infection. Gastric ul-ceration may cause internal haemorrhage, being thecause of death in a number of pigs with PMWS andable to worse the paleness of skin (Segalés et al.,2000b), which has been frequently related to the dis-ease. Anaemia without gastric ulceration has also been

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Fig. 1. PMWS gross findings. (A) The lung of the left shows lack of pulmonary collapse and interstitial oedema; the lung of the rightshows a normal collapse together with cranio-ventral pulmonary consolidation of bacterial origin. Both from cases of PMWS. (B) Markedincrease in size of the mesenteric lymph node. (C) Multifocal to coalescent areas of coagulation necrosis (asterisks) in a mediastinal lymphnode. (D) The liver in the top of the figure shows slight increase in size and marked discoloration; it showed a microscopic stage IV inthe classification of Rosell et al. (2000b). The liver in the bottom of the figure corresponds to a normal liver of age-matched pig. Bothfrom cases of PMWS. (E) Kidney; multiple white pale foci, typical of multifocal interstitial nephritis.

described as a feature of severely PMWS-affected pigs(Darwich et al., 2003). Pigs with chronic wasting dis-ease may develop cachexia, with marked muscle wast-ing and serous atrophy of fat.Table 1 summarises the frequencies of the gross

findings in a study involving 396 pigs infected withPCV2 detected by an in situ hybridisation technique(Rosell et al., 1999) at the Diagnostic Service fromthe Veterinary School of Barcelona, Spain.

2.2. Histopathological lesions

The microscopic lesions of PMWS in lymphoidtissues are almost unique for pigs. A variable de-gree of lymphocyte depletion with loss of the fol-licular architecture (Fig. 2A and B) is present inalmost all pigs having PMWS. This finding is usu-ally combined with a multifocal to diffuse, slight tovery intense histiocytic and/or multinucleate giant

140 J. Segales et al. / Veterinary Microbiology 98 (2004) 137–149

Table 1Frequency of macroscopic lesions observed in 396 pigs with PMWS

Macroscopic findings Frequency (n = 396) %

Wasting (emaciation, marked spine)a 318 80.3Non-collapsed, tan-mottled lungsa 255 64.4Pulmonary consolidation (bronchopneumonia) 235 59.3Enlargement of at least one lymph node (lymphadenopathy)a 209 52.8Gastric ulceration of pars oesophagea 113 28.5Serositis (mono or polyserositis) 99 25.0Serous atrophy of fat 90 22.7Kidneys with white focia 73 18.4Soft rectal feces (catarrhal colitis associated with diarrhoea) 44 11.1Hepatic atrophya 13 3.3Fibrino-necrotising colitis 13 3.3Jaundicea 12 3.0Lymph node necrosisa 9 2.3Necrotising pneumonia 8 2.0Hepatomegaly 2 0.5

a Most suggestive findings of PMWS.

cell infiltration (Fig. 2C). Another key finding isthe presence of sharply demarcated, spherical, ba-sophilic cytoplasmic inclusions (Fig. 2D) of PCV2in histiocytic cells (Clark, 1997; Rosell et al., 1999).

Fig. 2. PMWS microscopic features. (A and B) Inguinal superficial lymph nodes; normal structure (A) and marked depletion of lymphocytesin a severe PMWS case (B), Haematoxylin and Eosin (HE), 50×. (C) Mesenteric lymph node; histiocytic and multinucleate giant cellinfiltration in a medullary sinus, HE, 100×. (D) Mediastinal lymph node; presence of multiple, grape-like intracytoplasmic inclusion bodiescharacteristic of PCV2 in macrophages, HE, 500×. (E) Mediastinal lymph node; multifocal to coalescent coagulative necrosis of the lymphnode parenchyma, HE, 50×.

Occasionally, necrotising lesions have been detectedin lymph nodes associated with PCV2 infectionin severely PMWS-affected pigs (Segalés et al.,2000a); multifocal to coalescent areas of coagulative

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necrosis (Fig. 2E) (in some cases, affecting the wholeparenchyma of the lymph node) are usually associatedwith vascular thrombosis, but the pathogenic mecha-nism of the latter lesion in PMWS has not been yetelucidated. No evidence of necrotising vasculitis hasbeen observed in the affected lymph nodes or in othertissues of these animals showing coagulative necrosisin lymph nodes. Necrotising lesions in lymph nodeshave also been described in PMWS-affected pigsconcomitantly infected with Aujeszky’s disease virus(Rodrıguez-Arrioja et al., 1999).Subacute interstitial pneumonia is the most usual

lung lesion found in PMWS-affected pigs whichmacroscopically show non-collapsed, tan-mottledlungs (Rosell et al., 1999). In some cases it is pos-sible to observe large histiocytic and multinucleategiant cells in the thickened interalveolar walls and/orwithin alveoli. In chronic cases, bronchiolitis fibrosaobliterans may be present (Clark, 1997; Segalés et al.,2000a). In some animals, lymphocyte depletion andhistiocytic infiltration in bronchus-associated lym-phoid tissue (BALT) can be observed. While lymphoidlesions can be very suggestive of PMWS, many infec-tious agents are able to produce interstitial pneumoniain the pig and a wide differential diagnosis list has tobe established when lung is the only available sample.Hepatic lesions have been described as lympho-

histiocytic inflammatory infiltration in portal zones,single cell necrosis of hepatocytes, swelling and vac-uolation of hepatocyte cytoplasm and karyomegaly(Clark, 1997). However, in some cases there are verysevere lesions with generalised perilobular fibrosis,disorganisation of liver plates and massive loss of hep-atocytes. These lesions are associated with icterus andmacroscopic lesions in the liver. Four stages of hepaticdamage in PMWS-affected pigs have been establishedbased on intensity and distribution of lesions (Rosellet al., 2000b).Other microscopic lesions detected in severely

PMWS-affected pigs may include mild to moderatelympho-histiocytic inflammatory infiltrates in virtu-ally all tissues (Clark, 1997; Rosell et al., 1999).Sporadically, very severe subacute to chronic intersti-tial nephritis associated with high amounts of PCV2can be observed in some PMWS-affected pigs.Table 2 summarises the most frequent histopatho-

logical lesions found in the study dealing with 396pigs infected with PCV2.

Table 2Frequency of microscopic lesions observed in 396 pigs with PMWS

Microscopic findings Frequency(n = 396)

%

Lymphoid tissuesLymphocyte depletion 353 89.1Histiocytic inflammatory infiltration 305 77.0Intracytoplasmic inclusion bodies 137 34.6Syncytial (multinucleate giant) cells 113 28.5Multifocal coagulative necrosis 39 9.9

LungInterstitial pneumonia 304 76.8Catarrhal-purulentbronchopneumoniaa

218 55.1

Necrotising pneumoniaa 26 4.0

LiverbStage I 131 33.1Stage II 34 8.6Stage III 8 2.0Stage IV 5 1.3Total 178 45.0

KidneyInterstitial nephritis 149 37.6

ColonLymphoplasmacytic colitis 68 15.0Fibrino-necrotising colitisa 17 3.8a Lesions usually associated with bacterial infections.b Based on the classification of Rosell et al. (2000b).

2.3. Ultrastructural findings

A chronological characterisation of the ultrastruc-tural changes associated with PCV2 infection in tis-sues of naturally and/or experimentally infected pigsor cell cultures has not been published. Therefore,knowledge on the cellular stages of infection is stilllacking for this virus.In a few studies of PMWS, there has been some

ultrastructural work on affected lymph nodes. Thesestudies have shown the intracytoplasmic inclusionsin macrophages to be electron dense, round to ovoidbodies with sharp margins. The matrix of the bod-ies was heterogeneous, with different areas beinggranular, crystalline in a herringbone pattern or crys-talline in cross-sectional arrays of non-enveloped,small, icosahedral-viral particles, approximately17 nm in diameter (Kiupel et al., 1998; Sato et al.,2000).

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3. Gross and microscopic lesions in non-PMWSconditions associated with PCV2

3.1. Porcine dermatitis and nephropathy syndrome(PDNS)

PDNS was firstly described in the UK in 1993(Smith et al., 1993). Since then, countries from Eu-rope, North and South America, Oceania and Africahave described cases of this condition, suggestinga world-wide distribution of PDNS at present. Theclinical detection of PDNS is relatively easy becauseof the presence of haemorrhagic cutaneous infarcts(Fig. 3A), mainly located on the hindlimbs and per-ineal area (Hélie et al., 1995; Segalés et al., 1998). Atnecropsy in most animals both kidneys are swollen,pale and have generalised cortical petechiae (Fig. 3B).Not all affected pigs show macroscopic kidney andskin lesions. A few pigs which are also considered asPDNS cases do not have skin lesions, and others havevery slight or no kidney lesions. The latter cases are

Fig. 3. PDNS pathological findings. (A) Skin; necrotising cutaneous lesions of generalised distribution, which tend to coalesce on thehindlimbs. (B) Kidney; generalised cortical petechiae and increase in size of the perirenal lymph node. (C) Kidney; exudative fibrinousglomerulonephritis, HE, 250×. (D) Skin; leukocytoclastic necrotising vasculitis, HE, 200×.

considered atypical cases of the syndrome; and arestill considered PDNS because of the regular presenceof systemic necrotising vasculitis.Major histopathological findings include necrotis-

ing and fibrinous glomerulonephritis (Fig. 3C) andsystemic necrotising vasculitis (Fig. 3D) (Segaléset al., 1998; Thibault et al., 1998). These microscopicfeatures, together with the presence of immunoglobu-lin and complement components in the damaged ves-sels and glomeruli, suggest a type III hypersensitivityreaction as the possible pathogenic mechanism forthe disease (Hélie et al., 1995; Sierra et al., 1997). Onthe other hand, mild to massive lymphocyte depletionin lymph nodes has been regularly observed in casesof PDNS. In about 50% of the affected pigs, gran-ulomatous inflammatory infiltrates with histiocytesand/or multinucleate giant cells are seen in lymphnodes, mainly within follicular areas (Segalés et al.,1998; Rosell et al., 2000a). Another usual lesion inPDNS-affected pigs is interstitial pneumonia (Rosellet al., 2000a).

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The responsible antigen or antigens involved inthis immune complex-mediated disorder is currentlyunknown, and theoretically a wide spectrum of fac-tors including drugs, chemicals, food allergens, en-dogenous antigens, and infectious agents may beinvolved (Drolet et al., 1999). At present, the roleof infectious agents has been suspected more thanother factors. Among them, porcine reproductiveand respiratory syndrome virus (PRRSV) and PCV2have been strongly suggested as the possible antigen(Thibault et al., 1998; Rosell et al., 2000a). However,neither viral nucleic acid nor antigen of either agenthas been detected in the vascular and glomerularlesions of PDNS-affected pigs. A particular variantof Pasteurella multocida (ApaI pulsed-field gel elec-trophoresis (PFGE) restriction pattern) has also beensuggested as the potential antigen associated withPDNS (Lainson et al., 2002).

3.2. Reproductive disorders

Porcine circovirus type 2 can infect foetuses andcause reproductive failure in absence (West et al.,1999) or presence (O’connor et al., 2001) of other wellestablished reproductive pathogens. PCV2 has beendetected in stillborn and non-viable neonatal pigletspresenting hepatic chronic passive congestion, cardiachypertrophy and severe diffuse myocarditis (O’connoret al., 2001). The presence of the virus in the heart,liver, and sporadically in lung and kidney was con-firmed by polymerase chain reaction, immunohisto-chemistry, and virus isolation techniques (West et al.,1999; O’connor et al., 2001). Almost all of these de-scriptions of reproductive disorders are from Canada,and very few cases have been reported in the rest ofthe world. Some data also point out an associationbetween PCV2 and reproductive disorders in Europe(Ohlinger et al., 2000), but no description of patho-logic findings, if any, were reported from these cases.Although the natural infection of adult boars with

PCV2 has been reported (Hamel et al., 2000), nopathologic studies have been performed on this sub-ject.

3.3. Porcine respiratory disease complex (PRDC)

PRDC is characterised by slow and uneven growth,reduced feed intake, higher feed to gain conversion

rates, cough and clinical pneumonia (Thacker andThacker, 2000). This condition often appears in aherd on a regular basis when pigs are 16–20 weeksold. Major pathogens involved in PRDC are PRRSVand Mycoplasma hyopneumoniae. However, a num-ber of other bacterial and viral pathogens have alsobeen detected in PRDC outbreaks, PCV2 being oneof these agents (Thacker and Thacker, 2000).The final gross and microscopic pathological pic-

ture will mostly depend on the number of pathogensaffecting the pig in each case. Moreover, in mostof the cases, no clear-cut differences exist betweenPRDC and PMWS cases, since the clinical picture canbe very similar. Gross lung lesions caused by PCV2are virtually indistinguishable from those induced byPRRSV or even systemic bacterial infections suchas salmonellosis (Harms and Sorden, 2000). Eventhe microscopic lesions of PCV2-infected pigs in thelung (interstitial pneumonia) are not exclusive. How-ever, the presence of airway epithelial sloughing andmucosal or submucosal replacement by fibroplasiaand lympho-histiocytic cell infiltrations throughoutall lung lobes are considered quite characteristic ofPCV2 infection (Clark, 1997).

3.4. Proliferative and necrotising pneumonia (PNP)

PNP was initially described in 1990 in Canadaas a condition associated with respiratory problemsin nurseries and finishing units (Morin et al., 1990).The diagnosis of PNP was based on histopathologi-cal criteria, including the presence of necrotic cells,large macrophages and proteinaceous material withinthe alveoli, proliferation of type II pneumocytes andnecrotising bronchiolitis; occasionally, presence ofhyaline membranes and multinucleate giant cells inalveoli, and thickening of alveolar septa by mononu-clear cells were also observed (Larochelle et al., 1994).The aetiology of PNP was initially unknown, but

a new antigenic variant of swine influenza virus(SIV) was reported as the causal agent in the early90s (Girard et al., 1992). Some years later it wassuggested that the frequent detection of PRRSV inlungs with PNP was something more than just a for-tuitous association, and this virus was considered asa primary or a predisposing agent in the aetiology ofPNP (Larochelle et al., 1994). More recently, it wassuggested that this condition is a result of a PRRSV

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and PCV2 co-infection (Pesch et al., 2000). In thisstudy, 192 lungs with PNP-like lesions were studiedand PCR was used as a method to detect PRRSV,PCV2 and SIV; 164 of them (85.4%) showed theconcomitant presence of PRRSV and PCV2.In fact, PNP has been observed in PMWS-affected

pigs. Since PMWS frequently occurs with other vi-ral and bacterial infections (Quintana et al., 2001), itmake sense to include PNP within the scope of PCV2infection and PMWS. Of course, this fact cannot ex-clude the occurrence of PNP without the presence ofPCV2, which has already been described (Pesch et al.,2000).

3.5. Congenital tremor (CT)

CT in pigs (or myoclonia congenita) is characterisedby a tremor of the head and limbs of the new-bornpigs (Edwards and Mulley, 1999). Two types of CThave been described based on the presence (type A)or absence (type B) of myelin deficiency in the centraland peripheral nervous system. In addition, CT type Ahas been divided in five different subtypes (AI–AV),which include genetic abnormalities, in utero trichlor-fon toxicity and in utero infection by some viruses(Edwards and Mulley, 1999).CT subtype AII has been traditionally associ-

ated with an unidentified virus, but several reportsfrom the US have suggested that porcine circovirus(Hines, 1994) or, more precisely, PCV2 (Stevensonet al., 2001) is the causal agent of this CT subtype.In the latter work, no gross or microscopic lesionswere recorded in any CT-affected, nor in any normalpiglets from four different farms experiencing out-

Fig. 4. In situ hybridisation to detect PCV2. (A) Mesenteric lymph node; presence of PCV2 nucleic acid in the cytoplasm of multinucleategiant cells and macrophages, fast green counterstain, 75×. (B) Lung; PCV2 genome located in the cytoplasm of alveolar and interstitialmacrophages, fast green counterstain, 75×.

breaks of CT were recorded. However, PCV nucleicacid was detected in neural tissues and liver of bothCT-affected and normal pigs (Stevenson et al., 2001).On the other hand, recent work performed in Europe(Kennedy et al., 2003) on cases of CT also classi-fied as AII did not show any association with PCV2.Therefore, the association between PCV2 and CT is,at present, a controversial issue and further work isneeded to elucidate the role of PCV2 in CT.

4. Detection methods for PCV2 in tissues, and celltypes infected

Several methods have been developed to detectPCV2 in tissues and to correlate its detection withthe presence of lesions. Among them, in situ hybridi-sation (ISH) and immunohistochemistry (IHC) arethe most routinely used tests (McNeilly et al., 1999;Rosell et al., 1999). PCV2 nucleic acid or antigen inPMWS-affected pigs is usually found in the cytoplasmof histiocytes, multinucleate giant cells (Fig. 4A)and other monocyte/macrophage lineage cells suchas alveolar macrophages (Fig. 4B), Kupffer cells andfollicular dendritic cells of lymphoid tissues (Rosellet al., 1999; Allan and Ellis, 2000). Sporadically, it isalso possible to detect virus in the cytoplasm of re-nal and respiratory epithelium, vascular endothelium,lymphocytes, pancreatic acinar and ductular cells andnuclei of monocyte/macrophage lineage cells, smoothmuscular cells, hepatocytes and enterocytes (McNeillyet al., 1999; Rosell et al., 1999, 2000b; Sirinarumitret al., 2000). Although no lesions were detected inthe reported PCV2-associated cases of CT and their

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Table 3Distribution and frequency of detection of PCV2 nucleic acid byin situ hybridisation in different tissues from 50 PMWS-affectedpigs

Tissue Frequencya %

Tonsil 33/40 82.5Mesenteric lymph node 37/45 82.2Inguinal lymph node 33/41 80.5Peyer’s patches 31/39 79.5Lung 33/45 73.3Mediastinal lymph node 19/26 73.0Submandibular lymph node 21/29 72.4Spleen 30/43 69.8Intestinal mucosa 25/39 64.1Liver 25/45 55.6Kidney 17/31 54.8Pancreas 5/11 45.5Gastric mucosa 2/5 40.0Bone marrow 4/10 40.0Perirenal lymph node 6/17 35.3Nasal turbinates 1/3 33.3Adrenal gland 3/13 23.1

a Number of pigs with PCV2 nucleic acid/number ofPMWS-affected pigs tested for that particular tissue.

clinically normal pen-mates, the virus was detected byin situ hybridisation in large and small neurons, Purk-inje cells and a few oligodendrocytes in the centralnervous system (Stevenson et al., 2001). The presenceof PCV2 nucleic acid detected by in situ hybridisa-tion in a number of tissues from PMWS-affected pigs(n = 50) is summarised in Table 3.A strong correlation has been observed between the

amount of PCV2 nucleic acid or antigen and the sever-ity of microscopic lymphoid lesions (Rosell et al.,1999; Quintana et al., 2001). However, it is also pos-sible to find PCV2 nucleic acid or antigen in tissuesof clinically healthy pigs. In these cases, the amountof virus and the intensity of histopathological lesionsare very low (Quintana et al., 2001). Positive resultsin clinically healthy pigs or diseased pigs withoutPMWS should be interpreted with caution since PCV2infection may remain subclinical. Moreover, recentlyinfected pigs or pigs in a convalescent phase fromPMWS may have very mild or no microscopic le-sions in lymphoid tissues, and PCV2 antigen or nu-cleic acid in lymphoid tissues can be detected only insmall amounts (Quintana et al., 2001). These findingshave important implications when selecting the mostappropriate submissions for diagnosis.

ISH and IHC have been also useful to retrospec-tively study tissues from pigs prior to 1996–1997 yearsin which PMWS started to be detected in most of theimportant pig rearing areas in the world (Allan andEllis, 2000). Retrospective studies using in situ hy-bridisation have shown the presence of PCV2 nucleicacid associated with PMWS microscopic lesions atleast in 1986 in Spain (Rosell et al., 2000c) and in 1989in Japan (Mori et al., 2000), indicating that PCV2 in-fection and PMWS-like lesions were present aroundthe world at least since the 80s. However, it is sur-prising that the clinicopathological picture of what weknow now as PMWS has eluded recognition by clin-icians and pathologists for so long. Low prevalenceof the problem in earlier times, non-specific clinicalsigns, and/or concurrent presence of other infectiousagents have all tended to obscure the infection formany years (Rosell et al., 2000c).

5. Discussion

The significance of natural infection by PCV2 hasundoubtedly been expanded since its initial associationwith PMWS. However, the relationship between PCV2and other non-PMWS conditions have only been es-tablished by means of retrospective studies (PDNS,PNP) and clinical cases (reproductive disease, PRDC,CT), and no experimental background is supportingthis putative association at present. Further studiesare needed to confirm or extend the knowledge onthe cause–effect relationship of PCV2 with conditionsother than PMWS. These studies should be focusedon the experimental reproduction of such conditions,but the immune-mediated nature of PDNS and themulti-aetiologic origin of PRDC and PNPmake it verydifficult.Experimental studies on PCV2 have mainly aimed

at reproducing PMWS. In most of the published stud-ies, PMWS has been reproduced by co-inoculationof PCV2 with other aetiologic agents (PRRSV andporcine parvovirus) (Allan et al., 1999b; Krakowkaet al., 2000; Harms et al., 2001; Rovira et al., 2002) orimmunostimulation of PCV2-infected pigs (Krakowkaet al., 2001). However, in some of these and otherstudies (Bolin et al., 2001), PMWS has been repro-duced using PCV2 alone. Therefore, it is generallyagreed that Koch’s postulates have been fulfilled for

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PMWS by the inoculation of pigs with PCV2 as theonly pathogen.The pathogenesis of naturally acquired PCV2 infec-

tion has not been fully characterised, but an increas-ing amount of data is being generated on this subject.Viral transmission may occur vertically, or by direct(oro-nasal) or sexual contact, since PCV2 has beendetected in nasal cavities (Sibila et al., 2001), semen(Hamel et al., 2000), and in foetuses between 32 and114 days of gestation (Ohlinger et al., 2000). In nat-ural PMWS, initial PCV2 replication probably takesplace in macrophages and antigen-presenting cells oflymphoid tissues such as tonsil and regional lymphnodes (Clark, 1997; Rosell et al., 1999), or, alterna-tively, Peyer’s patches (Rosell et al., 1999) if virusis swallowed and is able to survive the low pH ofthe stomach (Royer et al., 2001). After infection andreplication in resident mucosal macrophages and otherantigen-presenting cells, PCV2 might be transportedeither intracellularly or free in lymph and/or blood.The normal traffic of PCV2 target cells to many tis-sues may contribute to the spread of viral infectionto numerous organs (Rosell et al., 1999). Althoughthe underlying mechanism by which histiocytic infil-tration occurs in tissues (particularly lymph nodes)is not known, it seems that histiocytic infiltration isone of the initial events during PCV2 infection, andcoincides with macroscopic lymphadenopathy (Clark,1997; Rosell et al., 1999). More chronic cases tendto show less severe lymphocyte depletion with lesspronounced histiocytic/multinucleate giant cell infil-tration (Quintana et al., 2001). In addition, mild tomoderate PMWS-like lesions can be observed in clin-ically normal pigs (Quintana et al., 2001), indicatingthat PCV2 subclinical infection in pigs frequently oc-curs in both PMWS and non-PMWS-affected farms(Rodrıguez-Arrioja et al., 2000).Some virus-infected tissues may be free of dis-

cernible lesions (Rosell et al., 1999), but lesions can befound in almost all tissues in severely PMWS-affectedpigs. As a general rule, the more severe the lymphoidlesions, the higher the number of PCV2 infected tis-sues for a particular pig. However, no evident corre-lation has been found between the severity of PMWSlymphoid lesions and the severity of lesions in othertissues such as lung, liver or kidney (Rosell et al.,1999, 2000b; Quintana et al., 2001). It is difficult tosuggest a pathogenesis model for PMWS based on

naturally-occurring disease, but current data indicatethat, while histiocytic/multinucleate giant cell infil-tration in tissues is an early event in PMWS, severelymphocyte depletion of lymphoid tissues, and severehepatic and kidney lesions might be a late event in thewasting disease (Rosell et al., 1999). Therefore, po-tential outcomes such as immunosuppression and liverfailure would be effects of subacute to chronic PMWS(Rosell et al., 1999). Taking the data altogether, asuggested pathogenetic scheme for PCV2 infection innursery and fattening pigs is displayed in Fig. 5.The pathogenesis of naturally occurring PCV2 in-

fection in adult pigs is highly speculative at the presentmoment (Fig. 5). Some preliminary experimental datahave suggested that intra-uterine infection at the timeof mating would play a major role in the develop-ment of future reproductive disorders in the last thirdof gestation (Cariolet et al., 2001a). In contrast, thesame researchers found that PCV2 was not able tocross the placental barrier when the virus was inoc-ulated intratracheally and intramuscularly in gestat-ing sows (Cariolet et al., 2001b). Therefore, furtherexperimental and on-field studies on PCV2 transmis-sion in adult pigs are needed to clarify transmissionroutes, replication sites and tissue distribution of thevirus.Abortions have been sporadically associated with

PCV2 infection, especially in start-up herds (Westet al., 1999; Sanford, 2002). Although very few dataon natural PCV2 infection of foetuses are available,it seems that the heart is the target organ for both vi-ral replication and lesion generation in the foetal stage(West et al., 1999). Experimental studies of PCV2 in-fection in foetuses also support the role of the heart inthe pathogenesis of this viral infection (Pensaert et al.,2001). This fact is in contrast with naturally occurringPMWS in which heart lesions are very sporadic andrarely of clinical importance (Dr. Edward G. Clark,personal communication).On the other hand, although subclinical, natu-

ral PCV2 infection of sows has been described(Calsamiglia et al., 2002), the outcome of this infec-tion on the offspring is not yet known. SubclinicalPCV2 infection in boars has also been reported inexperimental and on-field studies (Larochelle et al.,2000; Le Tallec et al., 2001) but its relevance andclinical implications for a future offspring are alsonot known.

J. Segales et al. / Veterinary Microbiology 98 (2004) 137–149 147

Fig. 5. Suggested pathogenesis of natural PCV2 infection.

Continued research on PCV2 infection, PMWS andother PCV2-associated diseases is required to betterunderstand these conditions. Specifically, additionalstudies are needed to clarify the pathogenesis of PCV2infection in PMWS and in utero infections, to com-pare the effect of different PCV2 isolates, to assessif PMWS is really an immunosuppressive disease andto characterise the humoral and cellular immune re-sponse in naturally and experimentally infected pigswith this virus.

Acknowledgements

We would like to thank all researchers and techni-cians from the pathology department at the VeterinarySchool of Barcelona for their help and assistance onthe study of PMWS and PCV2 infection in the last4 years: M. Calsamiglia, F. Chianini, P. Losada, N.Majó, B. Pérez, M. Pérez, J. Quintana, A. Resendes,G.M. Rodrıguez-Arrioja, A. Rovira, M. Sibila and S.Usero. We also thank the concession of the projectsQLRT-PL-199900307 from the Fifth Framework Pro-gramme 1998–2002 of the European Commission, and

2-FEDER-1997-1341 from the I + D National Plan(Spain).

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