J. Comp. Path. 1999 Vol.

120, 5978

Pathological, Immunohistochemical, and In-situ

Hybridization Studies of Natural Cases of
Postweaning Multisystemic Wasting Syndrome
(PMWS) in Pigs
C. Rosell, J. Segales, J. Plana-Duran, M. Balasch,
G. M. Rodrguez-Arrioja, S. Kennedy, G. M. Allan,
F. McNeilly, K. S. Latimer and M. Domingo
U.D. Anatomia Patolo`gica, Facultat de Veterina`ria (UAB), 08193 Bellaterra, Barcelona, Spain,
Fort Dodge Veterinaria, S.A., Crta. de Camprodon, s/n La Riba, 17813 Vall de Bianya,
Girona, Spain, Veterinary Sciences Division, Department of Agriculture for Northern Ireland,
Stormont, Belfast, UK and Department of Veterinary Pathology, College of Veterinary Medicine,
University of Georgia, Athens, Georgia, USA
Summary
Fifteen pigs from five farms on which there had been a previous clinical and
histopathological diagnosis of postweaning multisystemic wasting syndrome
(PMWS) were investigated. At necropsy, enlargement of lymph nodes was
the most obvious lesion; other lesions were non-collapsed lungs, ulceration
of the gastric pars oesophagica, and cranioventral pulmonary consolidation.
Microscopical lesions attributable to PMWS were found in lymphoid organs
(including lymph nodes, tonsil, Peyers patches and spleen), liver, kidney and
lungs. Varying degrees of lymphocellular depletion, affecting both lymphoid
follicles and parafollicular zones, and progressive multifocal to diffuse infiltration of lymphoid tissue by large histiocytic cells were the characteristic
lesions. Syncytial cells were seen frequently, especially in lymphoid organs.
A prominent finding was the presence of sharply demarcated, spherical,
basophilic, cytoplasmic inclusions in histiocytic cells. The lymphoid lesions
were suggestive of immunosuppression. Non-lymphoid lesions included interstitial pneumonia, periportal mononuclear inflammatory infiltration of the
liver in varying degrees, and interstitial nephritis. Porcine circovirus (PCV)
antigen and nucleic acid were regularly found in lymphoid organs, lung, liver
and, to a lesser degree, kidney. Target cells for PCV replication included
monocyte/macrophage lineage and antigen-presenting cells. To a lesser
extent, epithelial cells such as renal tubular, bronchial and bronchiolar cells,
endothelial cells, hepatocytes and lymphocytes were also labelled. One pig
did not show PCV nucleic acid; sequence differences among different viral
isolates are discussed as the probable cause of this lack of labelling by the
in-situ hybridization PCV-specific probe.
1999 W.B. Saunders Company Limited

Introduction
Postweaning multisystemic wasting syndrome (PMWS) is a recently described
disease that affects pigs shortly before weaning and fattening pigs (Harding,
1997; Segales et al., 1997). Pigs clinically affected by PMWS show poor
00219975/99/010059+20 $12.00/0

1999 W.B. Saunders Company Limited

60

C. Rosell et al.

body condition, skin pallor, dyspnoea and occasionally jaundice. Although

macroscopical examination is not diagnostically conclusive, the presence of
enlarged lymph nodes (particularly superficial inguinal and mesenteric), noncollapsed lungs with surface mottling, and a yellowish-orange liver point
towards the possibility of PMWS. The diagnosis is established microscopically
by detection of histiocytic infiltrates in many tissues, especially lymphoid
organs, liver and kidney (Clark, 1997; Segales et al., 1997).
The use of immunohistochemical methods and in-situ hybridization to
detect porcine circovirus (PCV) in formalin-fixed, paraffin wax-embedded
tissues from PMWS-affected pigs has shown abundant antigen and nucleic
acid of PCV in almost all affected organs (Harding and Clark, 1997; Segales
et al., 1997; Allan et al., 1998a; Ellis et al., 1998). These studies suggest that
macrophages and antigen-presenting cells from lymphoid tissues (including
lymph nodes, tonsil, Peyers patches and spleen) are the main target cells for
PCV. Although PCV has been regularly detected in the tissues of cases of
PMWS, no causal relationship has yet been demonstrated.
The objective of this work was to elucidate the pathological changes in the
tissues of pigs clinically affected with PMWS, and to investigate the tissue and
cellular distribution of PCV antigen and nucleic acid.
Materials and Methods
Animals and Farms
Fifteen pigs from five farms on which there had been a previous clinical and
histopathological diagnosis of PMWS were investigated. The pigs were selected, during
a single visit to each farm, on the basis of clinical signs, including loss of body
condition, skin pallor, and enlargement of superficial inguinal lymph nodes. Two of
the 15 pigs were found dead. The 13 live pigs were weighed and bled to provide
serum samples, which were tested for antibodies against porcine reproductive and
respiratory syndrome virus (PRRSV), Aujeszkys disease virus (ADV) and swine
influenza virus (SIV). Two of the farms were PRRSV-seropositive, but pigs from all
five farms were serologically negative for ADV and SIV. PCV serology was not done.
Serum samples were also inoculated on to primary porcine alveolar macrophage
cultures (Plana et al., 1992), and PRRSV was isolated from three sera obtained from
one farm. The 13 live pigs were killed on the farm by an intravenous injection of
sodium pentobarbital and necropsy was performed immediately. The farms were
widely separated and located in three different provinces of Spain. Affected pigs were
aged 715 weeks. Morbidity on the five farms ranged from 425%; mortality was at
least 80% of affected animals on all farms. Antibiotic treatment (colistin, enrofloxacin
and doxycycline) was unsuccessful in all cases. Two or three pigs were examined post
mortem on each farm, except for one farm on which six pigs were examined.
Necropsy and Histopathology
A complete necropsy was performed on all animals. Samples of brain, nasal turbinates,
lung, heart, tonsil, thymus, kidney, spleen, jejunum, ileum, colon, liver, adrenal
gland and lymph nodes (including superficial inguinal, mesenteric, mediastinal and
submandibular lymph nodes) were collected and fixed by immersion in 10% neutral
buffered formalin. Fixed samples were dehydrated, embedded in paraffin wax, sectioned at 4 lm, and stained with haematoxylin and eosin (HE). Lymphoid organs
were assessed for depletion of lymphoid cells, infiltration by histiocytic cells, presence

Postweaning Multisystemic Wasting Syndrome

61

of multinucleated cells (syncytia), and intracytoplasmic inclusions. The remainder of

the tissues were also assessed for lesions.
Immunohistochemical Detection of PCV
A hyperimmune polyclonal antibody to a PCV-like virus isolated from diseased pigs
in Canada (Ellis et al., 1998) was prepared in a rabbit. Briefly, a pre-immune
blood sample was taken from a rabbit and the animal was then immunized by the
subcutaneous and intramuscular routes with an equal mixture (v/v) of saponin solution
(Quil A, Superfos Biosector, Denmark) at a concentration of 1 mg/ml and a PK/15
cell lysate known to contain the PCV-like virus derived from pigs with PMWS. After
8 weeks, the rabbit was given an intravenous booster dose of 05 ml of the cell lysate
alone. After a further 4 weeks, the rabbit was bled and the serum was collected and
assayed for antibody to the PCV-like virus by indirect immunofluorescent (IIF) staining
on acetone-fixed cell culture preparations. This hyperimmune antibody had an IIF
titre of 20 000 and was used for immunohistochemical examination of formalin-fixed
tissue sections. For immunohistochemistry, serial sections (5 lm) were dewaxed in
xylene and rehydrated through graded alcohols. Endogenous peroxidase was blocked
with H2O2 05% in methanol, followed by a 10-min wash in running tap water.
Sections were then subjected to proteolytic enzyme digestion with protease XIV
(Sigma, Poole, UK) at a concentration of 05 mg/ml for 30 min at 37C. After
digestion, the sections were washed in tap water. The polyclonal rabbit antibody was
applied at a 1 in 2000 dilution in phosphate-buffered saline (PBS, pH 72) to tissue
sections for 1 h at 37C. The sections were then washed in PBS for 5 min and an
aliquot of biotinylated anti-rabbit Ig (Zymed, Cambridge Bioscience, Cambridge,
UK) was applied to each section for 30 min at 37C. The sections were then washed
again in PBS and an aliquot of a streptavidin peroxidase conjugate was applied for
10 min at room temperature. After a further 5-min wash in PBS, the sections
were incubated with 3-amino 9-ethyl-carbazole-hydrogen peroxide substrate solution
(Zymed, Cambridge Bioscience) for 5 min at room temperature, washed in running
tap water, and counterstained with haematoxylin for 1 min at room temperature. The
sections were then mounted with an aqueous mounting medium, and examined
microscopically. Lymph node sections from a pig from Northern Ireland that had
PMWS-like lesions (Kennedy et al., 1998) were used as a positive control. Negative
control procedures included omission of primary antiserum or substitution of an
inappropriate antiserum on selected sections.
Immunohistochemical Detection of PRRSV
The technique used was an avidinbiotinperoxidase method based on a previously
published procedure (Halbur et al., 1994). Briefly, tissue sections were placed on
silane-coated [3-(trietoxysilil)-propylamine] slides. Endogenous peroxidase activity was
inhibited by immersing the tissue sections in a hydrogen peroxide 3% in methanol
solution for 30 min. Antigen retrieval was performed with enzymatic treatment
(protease type XIV) in Tris-buffered saline (TBS, pH=74) for 10 min. Blocking was
carried out for 1 h with normal goat serum 10% in TBS. The primary monoclonal
antibody SDOW17 (Nelson et al., 1993) diluted 1 in 1000 in TBS was applied and
tissue sections were incubated overnight at 4C. Secondary antibody (biotinylated
goat anti-mouse linking antibody) and peroxidase-conjugated avidin were applied at
1 in 200 and 1 in 100 dilutions, respectively, for 1 h at room temperature. Sections
were finally incubated in diaminobenzidine (DAB)-hydrogen peroxide solution for
8 min, counterstained with Harriss haematoxylin, dehydrated, covered with a coverslip, and examined microscopically. Negative control procedures included omission
of primary antiserum.

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C. Rosell et al.

In-situ Hybridization (ISH) for the Detection of PCV

Tissue sections were placed on Probe On Plus glass microscope slides (Fisher Scientific,
Pittsburgh, USA). A work station was used to handle the slides, to control the
temperature of the hybridization reactions and various incubations, and to minimize
reagent consumption. Tissue sections were dewaxed in xylene and rehydrated in
graded alcohols to Automotion Buffer (Bimeda Corp., CA, USA). They were then
digested with 03% pepsin and incubated with 100% formamide for 5 min at 105C.
The sections were subsequently hybridized to a PCV-specific, single-stranded, 40base, oligonucleotide DNA probe that was end-labelled with digoxigenin. The design
of the probe was based on the sequence analysis of the PCV detected by Daft et al. (1996)
in a 6-week-old pig with interstitial pneumonia and generalized lymphadenopathy. This
probe detects PCV derived from PK-15 cells and has also detected PCV in tissues
from animals suffering from PMWS in the United States, Canada and Spain. Hybridization was performed for 5 min at 105C and then for 30 min at 37C. High
stringency washes were made with saline sodium citrate buffer to ensure a complete
match between the target nucleic acid and DNA probe. After the washes, an antidigoxigenin antibody conjugated to alkaline phosphatase was applied to the sections.
Colour was developed with nitroblue tetrazolium dye. Dye reduction to insoluble
blue-black formazan indicated areas of probe hybridization. The tissue sections were
counterstained with fast green, dehydrated, covered with coverslips, and examined.
Sections from a previous case of PMWS diagnosed in Spain were used as a positive
control for the ISH technique (Segales et al., 1997). Negative controls consisted of
lymph node tissues of a pig from an experimental farm with no history of PMWS or
PCV infection.

Results
Macroscopical Lesions
At necropsy, enlargement of inguinal and mesenteric ( jejunal) lymph nodes
was the most obvious lesion in 14 out of 15 pigs (Fig. 1). Other lesions
were non-collapsed lungs (14 out of 15 pigs), ulceration of the gastric pars
oesophagica (6/15), cranioventral pulmonary consolidation (3/15), cutaneous
haemorrhage and necrosis resembling porcine dermatitis and nephropathy
syndrome (2/15), and serofibrinous polyserositis (1/15).
Microscopical Lesions
Microscopical lesions attributable to PMWS were found in lymphoid organs
(including lymph nodes, tonsil, Peyers patches and spleen), liver, kidney and
lungs. The frequency of occurrence of lesions in the different organs is shown
in Table 1.
Varying degrees of lymphocellular depletion, affecting both lymphoid follicles and parafollicular zones, and progressive multifocal to diffuse infiltration
of lymphoid tissue by large histiocytic cells (Fig. 2) were the characteristic
lesions. Syncytial cells were seen frequently, especially in the lymph nodes,
Peyers patches, and lamina propria of the intestinal villi. Syncytial cells were
occasionally present within lymph vessels of the villi. A prominent finding
was the presence of sharply demarcated, spherical, basophilic, cytoplasmic
inclusions in histiocytic cells (Fig. 3). Inclusions were either large and single

Postweaning Multisystemic Wasting Syndrome

Fig. 1.

63

Enlarged superficial inguinal lymph nodes. Scale in cm.

or smaller and multiple (groups of up to 12 smaller inclusions). Varying degrees

and types of lymphoid tissue lesions were seen in the same animal.
In lymph nodes, depletion was observed within lymphoid follicles or in the
paracortical zones. In the follicles, large cells with abundant eosinophilic
cytoplasm, probably follicular dendritic cells, were prominent. Occasionally,
syncytial cells occupied the centre of the follicle. Lesions in the T-celldependent (paracortical) zone of the lymph node also included depletion of
small lymphocytes. In these areas, the fibrovascular stroma was more evident.
Large mononuclear cells and occasional mitotic figures were scattered between
stromal components. Large histiocytic cells infiltrated the cortical sinuses of
the lymph node. This infiltrate varied greatly in intensity, starting as small
cell aggregates beneath the trabecular sinuses and extending through wide
areas of the parafollicular zone in severe cases. Syncytial cells were mainly
observed in the cortical sinuses, but also appeared in the paracortical zones.
Lymphocellular depletion of the medullary cords and empty medullary sinuses
were also observed, to varying degrees.
Changes in Peyers patches and tonsil consisted of depletion of lymphoid
cells in the interfollicular area, depopulated follicles with stromal cells clearly
evident and, sporadically, syncytial cells within the follicles (Fig. 4). Infiltration
of the interfollicular area with histiocytic cells was also observed. Syncytia
were seen at the periphery of the lymphoid tissue and in the stroma of the

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C. Rosell et al.
Table 1
Frequency and type of lesions in organs from pigs with PMWS

Tissues

Number of pigs
from which tissue
was available

Lesion

Number of pigs
with lesion

Lymph nodes

15

Depletion of lymphoid cells

Histiocyte infiltration
Presence of syncytial cells
Presence of cytoplasmic inclusion bodies
Absence of follicles
Purulent lymphadenitis
Presence of granulomas

15/15
14/15
8/15
10/15
14/15
4/15
1/15

Peyers patches

14

Depletion of lymphoid cells

Histiocytic infiltration
Presence of syncytial cells
Presence of cytoplasmic inclusion bodies
Absence of follicles
Presence of granulomas

10/14
5/14
4/14
7/14
9/14
1/14

Tonsil

11

Depletion of lymphoid cells

Histiocytic infiltration
Presence of syncytial cells
Presence of cytoplasmic inclusion bodies
Absence of follicles

6/11
4/11
3/11
3/11
6/11

Depletion of lymphoid cells

Histiocytic infiltration
Presence of syncytial cells
Presence of cytoplasmic inclusion bodies

3/4
1/4
3/4
0/4

Spleen

13

Depletion of lymphoid cells

Histiocytic infiltration
Presence of syncytial cells
Presence of cytoplasmic inclusion bodies

7/13
3/13
2/13
0/13

Lung

12

Interstitial pneumonia
Bronchopneumonia
Presence of syncytial cells
Fibrinous pleuritis

11/12
2/12
2/12
1/12

Liver

14

Periportal hepatitis
Sinusoidal inflammation
Perilobular fibrosis, loss of hepatocytes

12/14
2/14
1/14

Kidney

12

Multifocal interstitial nephritis

Presence of syncytial cells
Glomerulonephritis
Renal fibrosis

Thymus

6/12
1/12
1/12
1/12

Lesions in at least one of the lymph nodes examined (superficial inguinal, mesenteric, mediastinal and
submandibular).

intestinal villi (Fig. 5). Cytoplasmic inclusions in histiocytic cells were also
observed in Peyers patches and tonsil.
The spleen showed depletion of lymphoid cells from the periarteriolar
lymphoid sheaths (PALS), with infiltration of histiocytic cells into the same area
(Fig. 6). Syncytial cells and intracytoplasmic inclusions were seen occasionally.

Postweaning Multisystemic Wasting Syndrome

65

Fig. 2.

Superficial inguinal lymph node. Histiocytic infiltration in the cortical zone, with depletion of
lymphoid cells. Lymph follicles are effaced, and syncytial cells occupy the centre of the follicle.
HE. 320.

Fig. 3.

Mesenteric lymph node. Note the many spherical, basophilic cytoplasmic inclusions in a histiocytic
cell in the centre of the figure. HE. 1600.
Peyers patch. Depletion of lymphoid cells, with infiltration of the follicle by many histiocytic cells.
Note the presence of a syncytial cell (arrow) in the follicle centre. HE. 160.

Fig. 4.

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C. Rosell et al.

Fig. 5.

Intestinal villi. Note syncytial cells in the stroma of an intestinal villus, in the lamina propria
(arrow), and also within a lymphatic vessel (arrowhead). HE. 640.

Fig. 6.

Spleen. Marked lymphoid depletion of a periarteriolar lymphoid sheath, with a syncytial cell
(arrow) among the histiocytic inflammatory infiltration surrounding an arteriole (arrowhead). HE.
320.

Postweaning Multisystemic Wasting Syndrome

67

The most common liver lesion was lymphohistiocytic infiltration of portal

zones, to variable degrees of intensity (Fig. 7). Groups of mononuclear
inflammatory cells were also observed in liver sinusoids. Multifocal necrosis
of single hepatocytes was observed in four cases. In one pig that died with
icterus, periportal fibrosis, loss of most hepatocytes, and mononuclear infiltrates
were observed throughout the liver acini (Fig. 8).
Lung lesions of PMWS-affected pigs were of variable severity. Mild multifocal
interstitial pneumonia, with some macrophage-like cells in the alveoli, was
observed in some lung sections. Other lung changes were characterized by
severe, diffuse interstitial pneumonia, with marked thickening of interalveolar
walls and inflammatory cells (mononuclear cells and degenerative polymorphonuclear neutrophils) in the alveoli. In some lung sections, peribronchial
and perivascular lymphohistiocytic infiltrates were present. Occasionally, syncytial cells were observed within the inflammatory infiltrates.
In the kidney, a mild to intense multifocal interstitial nephritis was present.
Lymphohistiocytic infiltrates were seen mainly within the renal cortex. In two
pigs, acute exudative glomerulitis, with fibrin casts in the Bowmans space
and proximal tubular lumina, and severe interstitial nephritis were found.
Systemic necrotizing vasculitis was present in these two pigs.
Other microscopical lesions included mononuclear meningoencephalitis
(2/11), lymphoplasmacytic myocarditis (2/11), and mild multifocal lymphoplasmacytic adrenalitis (4/12).
Immunohistochemical Detection of PCV Antigen
PCV antigen was detected in all 15 pigs (Table 2). In all tissues examined,
immunolabelling was most frequently seen in the cytoplasm of cells. Nuclear
labelling was less commonly seen but occasionally appeared to be almost as
abundant as cytoplasmic labelling. In lung tissue, PCV antigen was detected in
bronchial, bronchiolar and alveolar epithelial cells, in interstitial mononuclear
cells, and in inflammatory cell exudates in bronchial, bronchiolar and alveolar
lumina. Labelling was occasionally seen in bronchial and vascular smooth
muscle cells and in epithelial cells of bronchial submucosal glands. In liver
sections, PCV antigen was detected in hepatocytes, biliary epithelial cells,
Kupffer cells and infiltrating histiocytes. There was intense immunolabelling
of hepatocytic nuclei in the liver of several pigs. Immunolabelling of hepatocytes
was usually most intense in centroacinar regions. There was intense immunolabelling of PCV antigen in lymphoid tissues from all pigs examined.
The cells labelled in these tissues were mainly histiocytic and dendritic cells,
but cells with the morphology of lymphocytes were also labelled (Fig. 9). Weak
cytoplasmic labelling was seen frequently in syncytia. The smooth muscle
trabeculae of the spleen were occasionally labelled. PCV antigen was seen
in renal tubular epithelial cells, in renal mononuclear cell infiltrates, and
occasionally in arteriolar smooth muscle cells in the renal cortex. In small
and large intestinal tissue, antigen was frequently detected in gut-associated
lymphoid tissue and occasionally in villous epithelium. Cytoplasmic labelling
was seen in a large number of cells associated with capillaries in the adrenal

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C. Rosell et al.

Fig. 7.

Liver. Lymphohistiocytic inflammatory infiltrate in a periportal area. Note the presence of

inflammatory cells within the sinusoids. HE. 320.

Fig. 8.

Liver. Marked perilobular fibrosis, loss of most hepatocytes, and substitution by a diffuse mononuclear inflammatory infiltrate. HE. 160.

Pig 2
++

+++
++
++
+++
+++
NT

Pig 1

+
+
+
+
+
+
++
NT

Farm 1

++
+
+
+
+
+
+
NT

Pig 3

Pig 5

+++ NT
+++ +++
++
NT
+
NT

NT
+
NT
++
NT
NT
NT

Pig 4

Farm 2
Pig 7

NT
+
NT

+++ NT
+++

NT

NT

+++ +++
NT
NT

Pig 6

Farm 3
Pig 9

Pig 10

Pig 11

NT
+
+++
+
+
+
+
+
+++ +++ +++ NT
NT
NT
NT
+
NT
NT
NT
+
NT
NT
+
+
+++ +++ +++ +++
NT
NT
NT
NT

Pig 8

Farm 4

+
+
+++
+
+
+
+++
NT

Pig 12

+++
+
+
+
+
NT

Pig 13

NT
NT
NT
NT
NT
NT
NT
NT

NT
NT
NT
NT
NT
NT
NT
NT

Pig 14 Pig 15

Farm 5

Ln, Lymph node; NT, not tested; , absence of PCV antigen; +, slight presence of PCV antigen; ++, moderate presence of PCV antigen; +++, strong presence
of PCV antigen; pigs with porcine dermatitis and nephropathy syndrome.

Lung
Liver
Spleen
Ileum
Peyers patches
Tonsil
Superficial inguinal Ln
Mesenteric Ln

Tissues

Table 2
Immunohistochemical detection of PCV antigen in tissues from pigs with PMWS

Postweaning Multisystemic Wasting Syndrome

69

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C. Rosell et al.

Fig. 9.

Superficial inguinal lymph node. Abundant presence of porcine circovirus antigen in nuclei of
histiocytes and occasionally lymphocytes. Immunohistochemical technique with haematoxylin
counterstain. 320.

Fig. 10.

Mesenteric lymph node. Abundant presence of porcine circovirus nucleic acid within the
cytoplasm of histiocytic and syncytial cells in a lymphoid follicle. Note the labelled lymphocytes.
In-situ hybridization technique with fast green counterstain. 640.

Postweaning Multisystemic Wasting Syndrome

71

gland of the one pig in which this tissue was examined. Many of these cells
appeared to be capillary endothelial cells, but others could not be identified.
Cytoplasmic labelling was seen in many acinar and occasional ductular
epithelial cells in the pancreas from the same pig.

ISH for the Detection of PCV Nucleic Acid

PCV nucleic acid was detected in 14 of the 15 pigs (Table 3). Labelling was
found mostly in the cytoplasm of infected cells, and to a lesser extent in the
nucleus. The amount of PCV nucleic acid in tissues from the same pig was
highly variable. With the exception of pig no.3, which was negative for all
the tissues examined, all the lymphoid organs available for study yielded a
positive reaction. In most lymphoid tissues, large to intermediate amounts of
viral nucleic acid were detected primarily in the cytoplasm of cells with
dendritic morphology, histiocytic inflammatory cells, and syncytia (Fig. 10).
From the location of formazan deposition, several types of antigen-presenting
cells were identified in the lymph node. These cells included marginal-zone
macrophages, follicular dendritic cells, interdigitating dendritic cells and
medullary macrophages, all of which appeared to be infected by PCV. Small
round cells, which morphologically resembled lymphocytes, showed sporadic
cytoplasmic labelling.
In the lung, PCV nucleic acid was found mainly in the cytoplasm of alveolar,
interstitial and intravascular macrophages. In areas with very slight interstitial
pneumonia, infection was confined to these cells. As the severity of the
inflammatory lesions increased, other cells, such as alveolar, bronchial or
bronchiolar single epithelial cells were labelled.
In the liver, Kupffer cells (Fig. 11) and histiocytic infiltrates (Fig. 12) were
the usual targets of PCV infection. Hepatocytes located near the periportal
inflammatory infiltrates were sometimes labelled, mainly in the nucleus and
sporadically in the cytoplasm.
In the kidney, PCV nucleic acid was seen in the cytoplasm of macrophages
in interstitial inflammatory infiltrates, and occasionally in renal tubular epithelial and endothelial cells.
Pancreas, adrenal gland and bone marrow, although not extensively tested
by ISH, showed labelling in histiocytic inflammatory cells. Renal tubular
epithelial cells, usually associated with inflammatory infiltrates, were also
labelled. Sporadically, labelling of endothelial and smooth muscle vascular
cells was observed in many organs.

Immunohistochemical Detection of PRRSV Antigen

Pig no. 9 had PRRSV antigen in the cytoplasm of interstitial and alveolar
macrophages in the lung. These cells were scattered in a multifocal pattern.
All other pigs gave negative results.

Pig 2
++
+
++
+++
+++
+++
+++
++
+

Pig 1

+++
+++
+++
+++
+++
+++
+++
+++
+

Farm 1

Pig 3

Pig 5

+++
NT
+++ +++
+++ NT
+++ NT
+++ NT
+++ NT
+++ NT
+++ +++
+
+

Pig 4

Farm 2

+++
+++
+++
+
+++
+++
+++
+++
+

Pig 6
+

+
+
+
+
++
++
+

Pig 7

Farm 3

NT
+++
++
++
+++
NT
+++
+++
NT

Pig 8

Pig 10

Pig 11

+
+
+++
++

++
+
++
+++ ++
+
+++ ++
++
+++ ++ +++
+++ +++ +++
+++ +++ ++
+

Pig 9

Farm 4

+
+
+
+
+
++
++
++

Pig 12

+/
+
NT
NT
++
NT
+
+

Pig 13

+
+
NT
NT
+++ +
++ NT
+++ NT
NT
NT
NT +++
++++++
+
+

Pig 14 Pig 15

Farm 5

Ln, Lymph node; NT, not tested; , absence of PCV nucleic acid; +, slight presence of PCV nucleic acid; ++, moderate presence of PCV nucleic acid; +++,
strong presence of PCV nucleic acid; pigs with porcine dermatitis and nephropathy syndrome.

Lung
Liver
Spleen
Ileum
Peyers patches
Tonsil
Superficial inguinal Ln
Mesenteric Ln
Kidney

Tissues

Table 3
In-situ hybridization detection of PCV nucleic acid in tissues from pigs with PMWS

72
C. Rosell et al.

Postweaning Multisystemic Wasting Syndrome

73

Fig. 11.

Liver. Porcine circovirus nucleic acid in cytoplasm of Kupffer cells and mononuclear inflammatory
cells within the hepatic sinusoids. In-situ hybridization technique with fast green counterstain.
160.

Fig. 12.

Liver. Porcine circovirus nucleic acid in cytoplasm and nucleus of mononuclear inflammatory
cells present in periportal areas. Some hepatocytes near the inflammatory areas appear to be
labelled. In-situ hybridization technique with fast green counterstain. 64.

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C. Rosell et al.

Discussion
This study describes the pathological findings in 15 natural cases of PMWS,
together with the tissue distribution of PCV antigen and nucleic acid. Pigs
were classified as cases of PMWS on the basis of macroscopical and microscopical lesions previously documented, and the detection of PCV antigen or
nucleic acid (Daft et al., 1996; Harding and Clark, 1997; Segales et al., 1997).
The study confirmed that lymphoid tissues are the main target of PCV in
pigs with PMWS. Histiocytic infiltrates, formation of syncytia, and presence
of cytoplasmic inclusions in histiocytic cells were the most prominent microscopical lesions. The study also demonstrated that PCV was present in animals
with PMWS and was regularly associated with lymphoid lesions, as observed
by others (Harding and Clark, 1997). The use of DNA in-situ hybridization
and immunohistochemistry helped to establish the cell tropism of PCV in
cases of PMWS. PCV is unique in its tropism for a wide range of cells,
including monocyte-macrophage and antigen-presenting cell (APC) lineages.
Previous in-vitro studies failed to demonstrate infection of lymphocytes in
culture but showed PCV replication in monocytes and macrophages (Allan et
al., 1994; McNeilly et al., 1996). However, in the present study a few small
round cells resembling small lymphocytes were infected with PCV. Double
labelling studies with CD markers may be necessary to determine whether
lymphocyte subpopulations are preferentially infected.
Lung, liver and kidney have been described as major organs affected in
PMWS (Harding and Clark, 1997), but a definite causal relationship between
PCV and lesions has not been established. Whereas the lung appears to be
affected in nearly all pigs with PMWS, liver and kidney lesions are present in
only 75% of cases (Clark, 1997). In the present study, interstitial pneumonia
and lymphoplasmacytic periportal hepatitis of variable intensity were found
in almost all pigs; PCV antigen and nucleic acid were demonstrated in different
patterns in the lung and liver. Mild renal inflammation was found in about
half of the pigs.
Besides lymphoid organs, lung, and liver, many other organs contain PCV
in PMWS-affected pigs with concurrent histiocytic infiltrates. Normal traffic
of PCV target cells to many tissues may contribute to the spread of viral
infection to numerous organs. Some virus-infected tissues may be free of
discernible lesions. Haematogenous dissemination of the virus, with subsequent
infection of target cells in several organs, may also occur. Cells of epithelial
origin (such as hepatocytes, renal tubular epithelial cells, bronchiolar epithelial
cells and tonsillar epithelial cells) as well as endothelial cells and vascular
smooth muscle cells may also support PCV infection in natural cases of
PMWS. PCV nucleic acid was found not only in the cytoplasm of histiocytes
and macrophages, but also in nuclei of other cells, including hepatocytes.
Replication of PCV takes place in the cell nucleus (Tischer et al., 1995). When
the cell becomes infected, PCV DNA is by itself unable to penetrate the
nuclear envelope. The virus does not enter the nucleus until the end of mitosis,
when it is incorporated into the newly formed nuclei (Tischer et al., 1987).
Our results indicate that PCV nucleic acid and antigen accumulate to high

Postweaning Multisystemic Wasting Syndrome

75

concentrations in the cytoplasm of infected cells. The apparent absence

of nuclear labelling by ISH for PCV in most infected cells needs further
clarification.
Variation in intensity and distribution of lesions in lymphoid as well as
other target organs in cases of PMWS probably depends on the stage of the
disease in the affected pig. Most of our pigs were selected when showing initial
clinical signs of disease and were killed for the pathological study. These cases
probably represented acute or subacute forms of the disease.
In two dead pigs, one with jaundice, the presence of perilobular fibrosis in
the liver suggested an advanced or chronic form of the disease. These two
pigs also had severe lymphoid lesions, with marked lymphoid depletion, a
heavy concentration of PCV genome and antigen, and many cytoplasmic
inclusions in histiocytic cells. Based on these data, the suggested pathogenesis
of disease includes (1) entry of PCV by the oronasal route, (2) replication in
local lymphoid organs such as tonsils and regional lymph nodes, and (3)
systemic dissemination of PCV to other lymphoid tissues and organs, such
as lung, liver and kidney. Chronic effects in these organs may include
immunosuppression, enteritis, interstitial nephritis and liver failure. Experimental reproduction of PMWS and sequential pathological studies are
needed to clarify the pathogenesis of this disease.
PCV is a DNA virus included in the Circoviridae family (Studdert, 1993).
It was first described as a contaminant of the continuous porcine kidney (PK15) cell line ATCC-CCL-33 (Tischer et al., 1982). Previous experimental invivo and in-vitro studies failed to demonstrate pathogenicity of this virus for
pigs or other animal species (Allan et al., 1994, 1995). We and others have
provided evidence that links PCV to the newly described PMWS. On the
basis of restriction enzyme analysis of PCV fragments obtained by PCR
amplification from field cases of PMWS in Canada, some sequence differences
between PMWS-related PCV and PCV derived from PK-15 cells have been
detected (Nayar et al., 1997); the authors suggested that some specific strains
or variants of PCV are pathogenic to pigs and cause PMWS. Striking sequence
differences between PK-15-derived and PMWS-derived PCV have been published, with a sequence homology of 68% (Hamel et al., 1998). On the other
hand, the sequence of a French isolate of PCV (LeCann et al., 1997) apppears
to be more closely related to the original PK-15-derived PCV. On the basis
of these sequence differences, it has been suggested that isolates should be
designated as type I PCV (PK-15-derived) or type II PCV (PMWS-derived)
(Allan et al., 1998b).
Although the specificity of our reagents is not complete, it is suggested that
the PCV in the tissues of our pigs belongs to type II, for the following reasons:
the rabbit polyclonal serum used for immunohistochemical examination was
generated from a Canadian isolate of PCV from a case of PMWS (Ellis et al.,
1998); and at the dilution used in this study it does not recognize type I PCV
in acetone-fixed cell-culture coverslip preparations (Allan et al., 1998a). On
the other hand, the DNA probe used in ISH for PCV detection was designed
on the basis of sequence data from a PCV detected in a lymph node of a
PMWS-affected pig from the United States (Daft et al., 1996); however, it

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C. Rosell et al.

recognizes both type I and type II PCV. Pig no. 3 of our study did not have
PCV nucleic acid in any organ examined. This pig was considered to have
PMWS because of characteristic lesions and positive immunolabelling with a
rabbit anti-PCV polyclonal antibody. The reason for the negative ISH result
is unknown, but failure of the DNA probe due to absence of complementarity
is a possible explanation. A combination of techniques or broad-spectrum
reagents would seem desirable for the routine diagnosis of PCV infection.
A varying degree of lymphoid cell depletion was observed in the pigs.
Infection of macrophages and other antigen-presenting cells in lymphoid
organs may lead to depletion of lymphoid cells by an undetermined mechanism.
Follicular and parafollicular areas in lymph nodes, tonsil and Peyers patches
showed depletion, indicating that both T- and B-lymphocyte populations may
have been affected. Severe lymphoid depletion has recently been observed in
fatal cases of PMWS (data not shown). On this morphological evidence, we
suggest that PMWS causes immunosuppression, at least in the later stages of
the disease. A similar conclusion could be derived from the findings of Clark
(1997), who reported Pneumocystis carinii in the lungs of approximately 5% of
diseased pigs. We did not detect secondary infections by typical opportunistic
agents, but other concurrent diseases were found. Some pigs with PMWS were
co-infected with PRRSV, two pigs had porcine dermatitis and nephropathy
syndrome, and one pig had fibrinous polyserositis. Clinically, it may be difficult
to discriminate between the effects of these infections in the field. The
interrelationship between PRRSV and PCV, and their possible aetiological
role in the porcine dermatitis and nephropathy syndrome needs to be clarified.
The present study showed a close relationship between presence of lesions,
PCV nucleic acid and PCV antigen in PMWS. PCV has been successfully
transmitted to pigs by intranasal inoculation of tissue homogenates from
PMWS-affected pigs (LeCann et al., 1997; Balasch et al., 1998); however,
clinical disease has not been reproduced.

Acknowledgments
The authors thanks Miss Blanca Perez, Miss Silvia Usero and Mr Pere Losada for
technical assistance. This research was funded by a research contract with Fort Dodge
Veterinaria S.A. (Girona, Spain).

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Received, June 16th, 1998

Accepted, September 8th, 1998