Apoptosis and proliferative activity in lymph node reaction in postweaning multisystemic wasting syndrome (PMWS)

Apoptosis and proliferative activity in lymph node reaction inpostweaning multisystemic wasting syndrome (PMWS)

L. Mandrioli*, G. Sarli, S. Panarese, S. Baldoni, P.S. MarcatoDepartment of Veterinary Public Health and Animal Pathology, Section of General Pathology and Anatomic Pathology,

Faculty of Veterinary Medicine, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy

Received 22 November 2002; received in revised form 14 July 2003; accepted 4 August 2003

Abstract

Postweaning multisystemic wasting syndrome (PMWS) affects nursery and growing pigs, and is characterized by wasting,

failure to thrive, pale skin, respiratory distress, diarrhoea and sometimes jaundice. Macroscopic findings are aspecific, but

lymphocyte depletion in lymphoid tissues is one of the histological hallmarks [Vet. Q. 24 (2002) 109]. Spontaneous cases of

PMWS were studied to evaluate proliferative activity and apoptosis as mechanisms involved in the pathogenesis of cell

depletion in lymph nodes. The presence of Porcine Circovirus type 2 (PCV2) genome in the processed material was confirmed

by in situ hybridization (ISH). The lymph node pattern of depletion was graded as initial, intermediate or final stage according

to histological criteria in 10 superficial inguinal nodes from piglets with PMWS which died spontaneously or were

slaughtered by euthanasia. The apoptotic and proliferative fraction were investigated by monoclonal antibody MIB1

immunohistochemistry and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end labeling)

methods, respectively, and compared to three normal cases. One-way analysis of variance (ANOVA) comparison between

the MIB1 index (number of positive cells per 100 nuclei) in controls and PMWS cases revealed a decrease of proliferation in

both lymphoid and medulla-like tissues in the initial group (respectively, P ¼ 0:0017 and 0.024) but not in the intermediate

(respectively, P ¼ 0:25 and 0.88) or final (respectively, P ¼ 0:47 and 0.81) cohorts. The apoptotic index (number of

apoptosis/apoptotic bodies in 100 cells) revealed a statistically significant decrease only in the initial group (one-way

ANOVA P ¼ 0:05). The proliferation/apoptosis ratio (MIB1/APO ratio) assessed to determine cell turnover disclosed a

significant decrease of cell turnover from initial to final PMWS cases (Spearman’s rank test: P ¼ 0:027). Decreased cell

proliferation and not increased apoptosis seems to be the most important variable leading to cell depletion in PMWS

lymphoid tissues.

# 2003 Elsevier B.V. All rights reserved.

Keywords: Pig; PCV2; Lymph node; Lymphoid depletion; Apoptosis; Cell proliferation

1. Introduction

PMWS is a multifactorial disease caused by PCV2

infection. Diagnostic criteria include the detection

of PCV2 by immunohistochemistry or in situ hybri-

dization (ISH) among characteristic microscopic

lesions (Segales and Domingo, 2002). Lymphoid

depletion is the histological hallmark of PMWS.

Veterinary Immunology and Immunopathology 97 (2004) 25–37

* Corresponding author. Tel.: þ39-051-792973;

fax: þ39-051-792970.

E-mail address: [email protected] (L. Mandrioli).

Abbreviations: PMWS, postweaning multisystemic wasting

syndrome; PCV2, porcine circovirus type 2; MIB1, monoclonal

antibody that identifies a nuclear antigen expressed in G2/M phases

of the cell cycle; TUNEL, terminal deoxynucleotidyl transferase

(TdT)-mediated dUTP-nick end labeling; ISH, in situ hybridization

0165-2427/$ – see front matter # 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetimm.2003.08.017

Swine have two main lymph node zones: the so called

medulla-like tissue considered an intranodal broad

filter bed of not lymphoid tissue and lymphoid aggre-

gates organized in follicles and paracortex-like

(or interfollicular) areas (Charles, 1996). Lymphoid

depletion in PMWS is characterized by a relative

increase in the medulla-like tissue due to a pro-

gressive loss of lymphocytes in both follicular and

interfollicular areas (Done et al., 2002). Severe lym-

phocytic depletion, like that seen in final stages of

PMWS, impairs lymph node function because the

nodes lack effector immune cells (Segales and

Domingo, 2002).

Lymphoid depletion may occur as a consequence of

genetically determined primary immunodeficiency

disorders (i.e. X-linked agammaglobulinemia of Bru-

ton, X-linked SCID) or as secondary immunodefi-

ciency states resulting from infections, the side

effects of immunosuppression, irradiation or che-

motherapy for cancer (Cotran et al., 1999; Brown

et al., 2002).

Apoptosis, cytokine imbalance, and altered migra-

tion pathways have been implicated in indirect

lymphocyte depletion (Domingo et al., proceedings

of ssDNA viruses of plants, birds, pigs and primates,

2001). Apoptosis of thymocytes (chicken anemia

virus), mainly CD4þ but also other lymphocytes

(HIV) and T lymphocytes positive for antigen 4

(TCLA4þ) in feline immunodeficiency virus (FIV)

occurs in viral infection (Jeurissen et al., 1992; Muro-

Cacho et al., 1995; Tompkins et al., 2002). PCV2 was

recently shown to induce apoptosis in B lymphocytes

leading to dramatic B-lymphocyte depletion and sys-

temic immunosuppression in pigs (Shibahara et al.,

2000). In a mice model of PCV2 infection, apoptosis

occurred in histiocytes of germinal centers in several

lymphoid tissues (Kiupel et al., 2001). Histopatholo-

gical lesions in PMWS are well described, but the

mechanisms responsible for lymphoid depletion

remain unsettled. In addition to apoptosis, cell pro-

liferation may lead to cell depletion in the lymph node,

an organ in which cell numbers are maintained both by

blood recruitment and cell proliferation (activation)

of lymphocytes.

Spontaneous PCV2 ISH positive cases were studied

to investigate the mechanism involved in lymphoid

cell depletion and the role of apoptosis and/or pro-

liferative activity lymph node lymphocyte depletion.

2. Materials and methods

2.1. Cases

Cases were selected among formalin-fixed and

paraffin-embedded tissues collected during necropsies

performed in a farm in northern Italy. One-third of

cases comprised piglets which had died less than 2 h

before necropsy, while the remainder were moribund

piglets sedated with Azaperone (Stresnil1, Jansen,

Belgium) and slaughtered by euthanasia with Euthal

(Merial, Italy). A complete necropsy was done on

these pigs. Tissue fixation started less than 2 h after

death and lasted not longer than 48 h at room tem-

perature, then samples were embedded in paraffin. In

all cases, PMWS was diagnosed by the presence of

characteristic histologic lesions in lungs and lymphoid

tissues, in situ hybridization positive stain in lymph

node lesions and PCR identification of PCV2 genome

in lung, blood and lymph node samples (Sarli et al.,

2001).

Ten superficial inguinal nodes with hallmark histo-

logic PMWS lesions were selected and compared to

three normal tissue samples to investigate the apoptotic

and proliferative fraction. In PMWS cases, the lymph

node reaction was graded as initial, intermediate and

final stage (Sarli et al., 2001) according to the following

histologic criteria: initial stage shows remnants of

follicles, cell depletion in the lymphoid interfollicular

tissue and prevalence of the lymphoid over the medulla-

like tissue; intermediate stage shows absence of folli-

cles, more extensive cell depletion in the lymphoid

interfollicular tissue, but lymphoid tissue still prevails

over medulla-like tissue; final stage is characterized

by absence of follicles, lymphoid cell depletion and

medulla-like tissue prevalence over lymphoid tissue.

For each case, haematoxylin-eosin and in situ hybri-

dization stained sections were available, and two

consecutive four micron thick sections were cut from

the same paraffin block of formalin-fixed tissue to

assess proliferative activity by the MIB1 monoclonal

antibody and the apoptotic fraction by the TUNEL

method.

2.2. In situ hybridization

Four micron thick sections were placed on Superfrost

slides (Menzel-Glaser1, Braunschweig, Germany),

26 L. Mandrioli et al. / Veterinary Immunology and Immunopathology 97 (2004) 25–37

dewaxed in xylene, rehydrated in graded alcohols and

finally immersed in a TBS-based buffer. Digestion was

performed with 0.3% pepsin in the TBS-based buffer,

pH 2 for 10 min at 37 8C. Slides were then washed twice

in the buffer and incubated with 100% formamide (J.T.

Baker, Phillipsburg, NJ, USA) for 5 min at 105 8C in a

thermal-cycler (Primus 96, MWG Bio-Tech, Ebersberg,

Germany).

The sections were subsequently hybridized to a

PCV2 specific, single-stranded, 41 base, oligonucleo-

tide DNA probe labeled at the 30 end with digoxigenin

(MWG Bio-Tech, Ebersberg, Germany). The probe

sequence is: 50-CCT TCC TCA TTA CCC TCC TCG

CCA ACA ATA AAATAATCA AA-30. The slides were

incubated first at 105 8C for 5 min (pre-hybridization)

and then at 37 8C for 60 min. High stringency washes

were performed with saline sodium citrate buffers, to

guarantee complete coupling between the target nucleic

acid and the probe.

The digoxigenin-labeled samples were subsequently

detected in an enzyme assay using an antidigoxigenin

antibody conjugated to alkaline phosphatase. All

the reagents were contained in the ‘‘Digoxigenin detec-

tion kit’’ supplied by Roche Diagnostics (Mannheim,

Germany). Nitroblue tetrazolium dye (NBT) was

used for color development (incubation time: 5 min

at room temperature, RT). Dye reduction to insoluble

blue–black formazan indicated areas of probe hybri-

dization.

Fast Green 1.5% (Sigma, St. Louis, MO, USA) was

used to counterstain the sections that were further

dehydrated in graded alcohols, then in acetone–xylene

and mounted with DPX (Fluka, Biochemica, Buchs).

2.3. MIB1 immunohistochemistry

Four-micron thick sections of formalin-fixed and

paraffin-embedded lymph node samples were

dewaxed in toluene and rehydrated in a graded acetone

series (acetone 100: two immersions of 10 min each;

acetone 70: 10 min; acetone 50: 10 min). Endogenous

peroxidase was blocked by means of 3% hydrogen

peroxide for 30 min. Sections were then rinsed in

Tris buffer, immersed in citrate buffer (2.1 g citric

acid monohydrate/l distilled water), pH 6.0, and incu-

bated for four periods of 5 min each in a microwave

oven at 750 W. After microwave irradiation, sections

were allowed to cool to RT (approximately 20 min).

The primary antibody, known as clone MIB1 diluted

1:30 (Immunotech Int., Marseilles, France) is a

mouse anti-human proliferation antigen expressed

in G2/M phases of the cell cycle, and recognizes

on formalin-fixed and paraffin-embedded sections

the same antigen as Ki67 on frozen sections. After

the primary antibody a highly sensitive streptavidin-

biotin-peroxidase kit (BIO SPA, Milan, Italy) was

employed to reveal the reaction. In control sec-

tions, the primary antibody was substituted by PBS

buffer.

2.4. TUNEL for apoptosis

Four-micron thick sections of formalin-fixed and

paraffin-embedded lymph node samples were stained

with the ApopTag kit (ONCOR). The kit utilizes

reagents for non-isotopic DNA end-extension in situ

(digoxigenin-11-dUTP), and other reagents for immu-

nohistochemical staining (anti-digoxigenin-peroxi-

date antibody) of the extended DNA. Residues of

digoxigenin-nucleotide are catalytically added to the

DNA by terminal deoxynucleotidyl transferase (TdT)

which, to the 30-carboxylic ends of double or single-

stranded DNA, generates tails of digoxigenin dUTP

revealed immunohistochemically by the anti-digoxi-

genin antibody.

Four-micron thick sections were dewaxed, rehy-

drated and pre-treated according to Labat-Moleur

et al. (1998) with irradiation in 50 ml of citrate buffer

pH 3 at 750 W up to boiling point, rapidly cooled, and

then incubated for 15 min at RT with Proteinase K

(ONCOR): 0.002 g in 100 ml Tris/HCl buffer (0.15 M,

pH 7.6) for 15 min at RT. The sections were washed

in distilled water and endogenous peroxidase was

blocked by means of 3% hydrogen peroxide in

Tris/HCl buffer for 5 min at RT. After these washes,

sections were allowed to react with the equilibration

buffer for 30 min at RT, with terminal TdT-enzyme

solution for 60 min at 37 8C and then with stop/wash

solution for 30 min at 37 8C, followed by washes

in Tris buffer. Sections were finally allowed to react

with the anti-digoxigenin-peroxidate antibody for

30 min at RT in a humid chamber. After the washes

in the Tris/HCl buffer, the reaction was developed

in a solution of 0.04% diaminobenzidine (DAB)

and 0.04% hydrogen peroxide for 6 min at RT. The

sections were counterstained with Papanicolau

L. Mandrioli et al. / Veterinary Immunology and Immunopathology 97 (2004) 25–37 27

hematoxylin for 10 s, dehydrated and mounted in

DPX. For each case, a control slide was obtained

by substituting TdT-enzyme solution with distilled

water.

2.5. Scoring method

Quantitative analysis was performed with a Cyto-

metrica image cytometer (Byk Gulden, Milan, Italy).

2.5.1. MIB1 immunohistochemistry quantitation

Ten areas of lymphoid tissue (follicles if present and

interfollicular zones) and five areas in the medulla-like

zones were selected from each superficial inguinal

lymph node using a 25� lens and a 10� eyepiece,

yielding a final magnification of 250�. In each field,

a first image was mapped with a green (575/10 nm)

bandpass filter to obtain the total nuclear area; a

second area was mapped with a blue (490/10 nm)

bandpass filter to obtain the total area of the positive

nuclei. Capsular and trabecular cells were erased if

present in the field. The MIB1 index was calculated

as the percentage of labeled nuclei in the total area.

The final result of measurements for each case was

the mean value of MIB1 index in lymphoid and

medulla-like zones.

2.5.2. TUNEL quantitation

Ten areas of lymphoid tissue (follicles if present and

interfollicular zones) and five areas in the medulla-like

zones were selected from each superficial inguinal

lymph node, using a 40� lens and a 10� eyepiece,

yielding a final magnification of 400�. The number of

apoptotic nuclei and/or apoptotic bodies, the total

nuclear area and the mean area of a nucleus were

measured in each field. To compare apoptosis quanti-

tation among the histoarchitectural zones character-

ized by different cellularities, apoptosis was expressed

as the number of positive nuclei per 100 cells, named

apoptotic index and calculated as follows:

total nuclear area of the fields

mean area of a nucleus¼ N

where N is the number of cells present in the total

nuclear area of the chosen fields

apoptotic index ¼ total number of apoptosis

N� 100

The final result of measurements for each case was the

mean apoptotic index in lymphoid and medulla-like

zones.

2.5.3. The MIB1/APO ratio

Defined as the balance between cytoproliferation

and apoptosis (Farinati et al., 2001), was used to

quantitate cell turnover and calculated as follows:

mean MIB1 index between lymphoid

and medulla-like zones of each casemean apoptotic index between lymphoid

and medulla-like zones of each case

2.6. Statistical analysis

Statistical analysis was performed with the Com-

plete Statistical System (CSS Statistic, Statsoft, Tulsa,

USA). One-way ANOVA was employed to compare

the proliferative activity and apoptotic expression

among the groups. Cell turnover among groups was

compared by Spearman’s rank test.

3. Results

The ISH reaction in controls was negative. In

PMWS cases, the cytoplasm of histiocytic cells in

interfollicular lymphoid tissue and medulla-like tis-

sue, and follicular dendritic cells and lymphocytes in

follicles (Fig. 1a) were positive by ISH. The presence

of PCV2 genome was expressed multifocal to diffuse

more in the follicles than in interfollicular areas

(Fig. 1b) and to a lesser extent in the medulla-like

tissue in initial cases. With respect to the initial group,

intermediate cases had an intense diffuse positivity in

all lymphoid compartments and in some histiocytic

cells filling medulla-like tissue (Fig. 1c). In the final

stage, only sparse positive spots were found in the

remaining lymphoid tissue (Fig. 1d).

At low magnification, MIB1 immunohistochemis-

try in control pig nodes showed nuclear positivity

multifocally displaced at their germinal centers in

the follicles, while in the interfollicular areas and in

the medulla-like tissue single positive nuclei were

detectable (Fig. 2a). Initial PMWS cases showed a

decrease of MIB1 positive cells in follicular and

interfollicular areas and in the medulla-like tissue that


showed only few positive nuclei (Fig. 2b). Intermedi-

ate and final PMWS cases had only sparse, single

positive nuclei in the remnants of the lymphoid tissue

(Fig. 2c and d).

ANOVA comparison of the MIB1 index in lym-

phoid tissue in controls and PMWS cases grouped

according to histological stage showed a significant

difference among all four groups (P ¼ 0:043). Com-

parison of each PMWS group with controls revealed a

decrease of proliferative activity in the initial group

(P ¼ 0:0017) but not in intermediate (P ¼ 0:25) or

final (P ¼ 0:47) cases (Fig. 3). The same trend was

revealed in medulla-like tissue, with a significant

decrease in the initial group (P ¼ 0:024) with respect

to controls but not in intermediate (P ¼ 0:88) or final

cases (P ¼ 0:81) (Fig. 4).

By the TUNEL method, apoptotic nuclei appeared

brown with various morphological phases such as

condensation and margination of chromatin, nuclear

fragmentation and apoptotic body formation (Fig. 5).

Fig. 1. Swine. ISH to PCV2 genome in superficial inguinal lymph node graded as initial (a and b); intermediate (c); and final (d). The initial

case shows a positive reaction mainly in follicles (F) (a and b) and to a lesser extent in interfollicular lymphoid tissue (I) (b). In the follicle in

(a) both follicular dendritic cells and lymphocytes are positive. A diffuse positive ISH stain in the lymphoid tissue (LT) and a lower staining in

the medulla-like (ML) tissue characterize the intermediate stage (c), while few positive clusters of cells are evident in the final case

(bar ¼ 50 mm).


At medium magnification, many apoptotic bodies

were present at the center of the lymphoid follicles

in controls. Initial PMWS cases had few apoptotic

cells, mainly detected in the follicles (Fig. 6). Both

intermediate and final stage cases had apoptotic

bodies uniformly and constantly evident in all remnant

areas.

In lymphoid tissue, the apoptotic index was lower

in PMWS groups than in controls but the difference

was not significant (P ¼ 0:067). Only the comparison

between controls and initials revealed a statistically

significant decrease of apoptotic index (P ¼ 0:05)

(Fig. 7). In medulla-like tissue, apoptosis in the

PMWS group was lower than controls, but the differ-

ence was not significant (P ¼ 0:49) (Fig. 8).

The MIB1/APO ratio revealed a significant

decrease of cell turnover from initial to final PMWS

stages (Spearman’s rank test: P ¼ 0:027) (Fig. 9).

Fig. 2. Swine. MIB1 immunohistochemistry in superficial inguinal lymph node of a control (a) and in PMWS cases graded as initial (b);

intermediate (c); and final (d). Proliferating cells (MIB1 positive) show brown stained nuclei. In control (a) MIB1 positive cells are present in

both follicles (F) (higher amounts) and interfollicular lymphoid tissue (I). In initial case (b) an evident reduction of proliferating cells appears

both in the follicle (F) and interfollicular lymphoid tissue (I). Few MIB1 positive cells are present in the lymphoid tissue in the intermediate (c)

case, while the number of MIB1 positive cells increases in the final stage (d) (bar ¼ 50 mm).


4. Discussion

Several mechanisms have been implicated in virus-

induced lymphocyte depletion, including direct viral

cytopathic ability, immune hyperactivation and

exhaustion, immune suppression mediated by viral

and regulatory gene products, and inappropriate

immune killing of uninfected cells (Tompkins et al.,

2002), but the true mechanism remains unsettled

(Cloyd et al., 2001). Although widespread PCV2

Fig. 3. Box and whiskers plot (mean � S.E. � S.D.) of MIB1 index in lymph node lymphoid tissue of controls and PMWS cases grouped as

initial, intermediate and final. Comparison by one-way ANOVA of the MIB1 index between controls and PMWS cases shows a significant

difference among the four groups (P ¼ 0:043). Comparison of each PMWS group with controls reveals a decrease of proliferative activity in

initials (P ¼ 0:0017) and not in intermediates (P ¼ 0:25) and finals (P ¼ 0:47).

Fig. 4. Box and whiskers plot (mean � S.E. � S.D.) of MIB1 index in lymph node medulla-like tissue of controls and PMWS cases grouped

as initial, intermediate and final. MIB1 index in medulla-like tissue shows the same trend reported in Fig. 3 for lymphoid tissue. A significant

decrease in initials (P ¼ 0:024), but not in intermediates (P ¼ 0:88) and finals (P ¼ 0:81), with respect to controls by one-way ANOVA.


infection of follicular dendritic cells and other anti-

gen-presenting cells is the major feature in PMWS, it

is not known how far cell function is impaired

by PCV2 or its mediating effect on lymphoid deple-

tion (Domingo et al., proceedings of ssDNA viruses of

plants, birds, pigs and primates, 2001). It is, however,

known that antigen-presenting function is a prere-

quisite for T (mainly TCD4þ) and even B (as for

T-dependent B cell response) activation (Tizard, 2000),

and proliferation occurs following lymphocyte activa-

tion and cytokine production.

The relationship between PCV2 and cells involved

in the immune response differs in the origin and

outcome of the disease. In several experimental mod-

els of PMWS (Allan et al., 2000; Krakowka et al.,

2002), the availability of activated macrophages (i.e.

immunostimulation) is a triggering factor for the

development of the syndrome, whereas immuno-

suppression seems to be the outcome of severely

affected pigs (Segales and Domingo, 2002). Data on

immunodeficiency during PCV2 infection are avail-

able from the literature on B- and T-cell blood levels

and their determination in tissues. In peripheral blood,

circulating monocytes are increased in PMWS

affected piglets, but T cells (mainly CD4þ) and B

lymphocytes are decreased (Segales et al., 2001;

Nielsen et al., 2003), while a significant decrease of

T cells (mainly CD8þ and CD4þ/CD8þ) and B

lymphocytes was detected comparing PMWS and

healthy pigs (Darwich et al., 2002).

By immunohistochemistry, there was a reduction or

complete loss of B cells in lymphoid tissues of PMWS

pigs, associated with loss of lymphoid follicles

(Shibahara et al., 2000; Sarli et al., 2001). T-cell

population and histiocytic cell quantitation in cortical

and paracortical zones of lymphoid tissues revealed a

reduction of T cells (CD3þ) and an increase in cells

expressing lysozyme (mononuclear-phagocytic cells)

(Chianini et al., 2001). Depletion of T (CD2 positive)

cells has been related to a decrease of CD4þ cells, and

Fig. 5. Swine. TUNEL in superficial inguinal lymph node of a control case. Apoptotic cells in the center of a follicle are appreciable in various

morphologic phases: chromatin margination (arrow and thin arrow) and apoptotic bodies (thin double arrow head and arrow head)

(bar ¼ 50 mm).


Fig. 6. Swine. TUNEL in superficial inguinal lymph node of an initial case. Compared to the follicle of the control in Fig. 5, an evident

reduction of apoptosis in the follicle of this initial case appears (bar ¼ 25 mm).

Fig. 7. Box and whiskers plot (mean � S.E. � S.D.) of apoptotic index in lymph node lymphoid tissue of controls and PMWS cases grouped

as initial, intermediate and final. Apoptotic index in PMWS groups shows a lower value than in controls, even if the difference by one-way

ANOVA did not result statistically significant (P ¼ 0:067). Only the comparison between controls and initials revealed a statistically

significant decrease of apoptotic index in the latter (P ¼ 0:05).


to a minor extent of CD8þ, in lymph node lymphoid

tissue of PMWS cases (Sarli et al., 2001). Histopatho-

logic lesions and virus titrations in lymphoid tissues

following PCV2 infection were more severe in cyclos-

porine treated piglets (Krakowka et al., 2002), empha-

sizing the role of immunosuppression as a key factor

in severe disease.

The poor lymphocyte activation (proliferation)

found during PCV2 infection has been attributed

to the limited production of immunomodulatory

cytokines, reflecting an aberrant expression of class

I and II MHC antigens which could impair immune

function (McNeilly et al., 1996). This hypothesis was

recently confirmed by the down-regulation of MHC-II

Fig. 8. Box and whiskers plot (mean � S.E. � S.D.) of apoptotic index in lymph node medulla-like tissue of controls and PMWS cases

grouped as initial, intermediate and final. No significant difference among groups resulted by one-way ANOVA (P ¼ 0:49), even if apoptotic

index in PMWS groups appears lower than controls.

Fig. 9. Scatterplot of MIB1/APO ratio in lymph node of PMWS cases grouped as initial, intermediate and final. MIB1/APO ratio shows a

progressive significant decrease of cell turnover from initial to final PMWS stages (Spearman’s rank test: P ¼ 0:027).


expression found in PCV2 infected macrophages

(Done et al., 2002).

A transient decrease was reported in the ability of

PCV-infected alveolar macrophages (AMs) to recon-

stitute the proliferation response of monocyte/macro-

phage depleted peripheral blood mononuclear cells

(PBMNCs) compared to controls. It is likely that PCV

infection of alveolar macrophages somehow affects

their ability to interact with B and T cells and stimulate

proliferation (McNeilly et al., 1996).

The MIB1 index in PMWS groups compared to

controls offers an objective indication of the scant

amount of lymphoid cell proliferation. The TUNEL

method and MIB1 immunohistochemistry are widely

employed in formalin-fixed and paraffin-embedded

material to quantitate apoptosis and cell proliferation

in neoplastic and other conditions (Dervan et al., 1992;

Hall and Levison, 1992; Kerr et al., 1994; Negoescu

et al., 1996; Schafer, 1998; Woosley and Hart, 1983).

The results of this study were obtained after fixation,

that started less than 2 h after death and lasted not

longer than 48 h at room temperature. After these

steps, both the expression of cell cycle-related anti-

gens and DNA modification after apoptosis are con-

sidered not liable to further change. A reference on

delayed fixation (after 1, 2, 4, 6, 24 h) on formalin-

fixed and paraffin-embedded human samples, on

which the detection of apoptosis in situ was applied,

showed that elapsed times do not adversely affect the

result of the assay (Bardales et al., 1997).

The number of ISH PCV2 positive cells, mainly in

initial and intermediate cases, indicates that PCV2 is

responsible for the changes in MIB1 and apoptosis

expression in PMWS, as reported by Kiupel et al.

(2001) for apoptosis in PCV2-infected mice lymph

nodes. A comparison of Figs. 3 and 4 shows that, after

the significantly decreased MIB1 index in initial cases

compared to controls, proliferation renewal was

slower in lymphoid than in medulla-like tissue. Resi-

dent cells (reticulum cells and scant lymphocytes,

macrophages and plasma cells (Hoshi et al., 1988))

fill the medulla-like compartment more than the lym-

phoid part in which the number of lymphocytes is

maintained both by blood recruitment across the walls

of high endothelial venules and local cell (activation)

proliferation (Charles, 1996).

Two recent papers emphasized the ability of PCV2

to trigger apoptosis. Shibahara et al. (2000) showed

that PCV2 in swine induces apoptosis in B lympho-

cytes and leads to selective B-lymphocyte depletion.

They suggested that the virus infects dividing cells,

including B lymphocytes and macrophages, inducing

apoptosis directly in individual B lymphocytes, but not

in macrophages. Instead, Kiupel et al. (2001) revealed

the ability of the virus to induce apoptosis in histio-

cytic cells in lymphoid tissues in a mouse model of

PCV2 infection. The ability of African swine fever

virus (ASF) and porcine respiratory reproductive syn-

drome virus (PRRSV) to trigger apoptosis in lymphoid

tissues is also known in the pig (Carrasco et al., 1996;

Sur et al., 1998). A typical lymphoid hyperplasia

follows the initial lymphoid depletion in PRRSV

infection. Apart from PCV2 infection, lymphadeno-

pathies of the pig, in which lymphoid depletion is

characterized by disappearance of follicles, interfolli-

cular-lymphocyte reduction, histiocytic infiltration

and increased medulla-like tissue are not known.

In our investigation, apoptosis did not seem impor-

tant in the pathogenesis of cell depletion in PMWS,

contrary to what is known for FIV and HIV lympha-

denopathy (Sarli et al., 1998; Tompkins et al., 2002).

Except for the comparison between controls and

initial cases revealing a significantly lower value in

lymphoid tissue, the apoptotic index of our PMWS

cases was lower than controls both in lymphoid and

medulla-like tissue, an obvious consequence of scant

lymphocyte activation. This was an expected result of

long-standing non activated nodes, since a high rate

of apoptosis is commonly found after antigen stimula-

tion (Charles, 1996) and one of the mechanisms

preventing uncontrolled T-cell activation during a

normal immune response is apoptotic death of acti-

vated lymphocytes by the Fas–Fas ligand system

(Cotran et al., 1999).

The MIB1/APO ratio employed to quantitate cell

turnover revealed a decreasing turnover from initial to

final cohorts, objectifying the reduced cellularity

observed during transition from initial to final PMWS

stages. Given the role attributed to proliferation and

apoptosis, the reduced turnover appears more strongly

influenced by proliferation than by apoptosis.

These results confirm the previous hypothesis on

the failure to trigger an immune response by PCV2

infected piglets: lymphoid tissue depletion is mainly

related to decreased proliferative activity in lymphoid

tissue, and is caused by a long-standing absence


of lymph node positive growth factors (mainly

cytokines) produced by lymphocyte activation (Sarli

et al., 2001). The prolonged inactivation of the

lymphocytes may result from the altered interaction

between lymphocytes and macrophages, whose ability

to stimulate B- and T-cell proliferation is impaired by

PCV2 (McNeilly et al., 1996).

References

Allan, G.M., McNeilly, F., Kennedy, S., Meehan, B., Ellis, J.,

Krakowka, S., 2000. Immunostimulation, PCV-2 and PMWS.

Vet. Rec. 147, 170–171.

Bardales, R.H., Xie, S.S., Hsu, S.-M., 1997. In situ DNA

fragmentation assay for detection of apoptosis in paraffin-

embedded tissue sections. Technical considerations. Am. J.

Clin. Pathol. 107, 332–336.

Brown Jr., T.T., Sutter, M.M., Slauson, D.O., 2002. Immunopathol-

ogy. In: Slauson, D.O., Cooper, B.J. (Eds.), Mechanisms of

Disease. A Textbook of Comparative Pathology, third ed.

Mosby, Missouri, pp. 289–291.

Carrasco, L., De Lara, F.C., Martin de Las Mulas, J., Gomez-

Villamandos, J.C., Perez, J., Wilkinson, P.J., Sierra, M.A.,

1996. Apoptosis in lymph nodes in acute African swine fever. J.

Comp. Pathol. 115, 415–428.

Charles, J.A., 1996. Lymph nodes and thymus. In: Sims, L.D.,

Glastonbury, J.R.W. (Eds.), Pathology of the Pig. A Diagnostic

Guide. D.G. Walker Pty Ltd., Victoria, Australia, pp. 185–

210.

Chianini, F., Majo, N., Segales, J., Dominguez, J., Domingo, M.,

2001. Immunohistological study of the immune system of cells

in paraffin-embedded tissues of conventional pigs. Vet.

Immunol. Immunopathol. 82, 245–255.

Cloyd, M.W., Chen, J.J., Adeqboyega, P., Wang, L., 2001. How

does HIV cause depletion of CD4 lymphocytes? A mechanism

involving virus signalling through its cellular receptors. Curr.

Mol. Med. 1, 545–550.

Cotran, R.S., Kumar, V., Collins, T., 1999. In: Robbins Pathologic

Basis of Disease, sixth ed. W.B. Saunders, Oxford.

Darwich, L., Segales, J., Domingo, M., Mateu, E., 2002. Changes

in CD4(þ), CD8(þ), CD4(þ), CD8(þ), and immunoglobulin

M-positive peripheral blood mononuclear cells of postweaning

multisystemic wasting syndrome-affected pigs and age-

matched uninfected wasted and healthy pigs correlate with

lesions and porcine circovirus type 2 load in lymphoid tissues.

Clin. Diagn. Lab. Immunol. 9, 236–242.

Dervan, P.A., Magee, M.H., Buckley, C., Carney, D.N., 1992.

Proliferating cell nuclear antigen counts in formalin-fixed

paraffin-embedded tissue correlate with ki-67 in fresh tissue.

Am. J. Clin. Pathol. 97, S21–S28.

Done, S.H., Bailey, M., Gresham, A.C.J., White, M.E.C., Potter,

R.A., Chennels, D., Thomson, J., 2002. Post-weaning multi-

systemic wasting syndrome (PMWS). The current European

situation. Pig J. 49, 215–232.

Farinati, F., Cardin, R., Fiorentino, M., D’Errico, A., Grigioni, W.,

Cecchetto, A., 2001. Imbalance between cytoproliferation and

apoptosis in hepatitis C virus related chronic liver disease. J.

Viral Hepat. 8, 34–40.

Hall, P.A., Levison, D.A., 1992. Review: assessment of cell

proliferation in histological matherial. J. Clin. Pathol. 43,

184–192.

Hoshi, N., Hasimoto, Y., Kitagawa, H., Kon, Y., Kudo, N., 1988.

Fine structures of the medulla-like tissues and the lymph

sinuses in the lymph node of pig. Jpn. J. Vet. Sci. 50, 83–92.

Jeurissen, S.H., Wagenaar, F., Pol, J.M., van der Eb, A.J., Noteborn,

M.H., 1992. Chicken anemia virus causes apoptosis of

thymocytes after in vivo infection and of cell lines after in

vivo infection. J. Virol. 66, 7383–7388.

Kerr, J.F.R., Winterford, C.M., Harmon, B.V., 1994. Apoptosis: its

significance in cancer and cancer therapy. Cancer 73, 2013–

2026.

Kiupel, M., Stevenson, G.W., Choi, J., Latimer, K.S., Kanitz, C.L.,

Mittal, S.K., 2001. Viral replication and lesions in BALB/c

mice experimentally inoculated with porcine circovirus isolated

from a pig with postweaning multisystemic wasting disease.

Vet. Pathol. 38, 74–82.

Krakowka, S., Ellis, J.A., McNeilly, F., Gilpin, D., Meehan, B.,

McKullough, K., Allan, G., 2002. Immunologic feature

of porcine circovirus type 2 infection. Viral. Immunol. 15,

567–582.

Labat-Moleur, F., Guillermet, C., Lorimier, P., Robert, C.,

Lantuejoul, S., Brambilla, E., Negoescu, A., 1998. TUNEL

apoptotic cell detection in tissue sections: critical evaluation

and improvement. J. Histochem. Cytochem. 46, 327–334.

McNeilly, F., Allan, G.M., Foster, J.C., Adair, B.M., McNulty,

M.S., Pollock, J., 1996. Effect of porcine circovirus infection

on porcine alveolar macrophage function. Vet. Immunol.

Immunopathol. 49, 295–306.

Muro-Cacho, C.A., Pantaleo, G., Fauci, A.S., 1995. Analysis of

apoptosis in lymph nodes of HIV-infected persons. J. Immunol.

154, 5555–5565.

Nielsen, J., Vincent, I.E., Botner, A., Ladekaer-Mikkelsen, A.S.,

Allan, G., Summerfield, A., Mc Cullough, K.C., 2003.

Association of lymphopenia with porcine circovirus type 2

induced postweaning multisystemic wasting sindrome

(PMWS). Vet. Immunol. Immunopathol. 92, 92–111.

Negoescu, A., Lorimier, P., Labat-Moleur, F. et al., 1996. In situ

apoptotic cell labeling by the TUNEL method: improvement

and evaluation on cell preparations. J. Histochem. Cytochem.

44, 959–968.

Sarli, G., Della Salda, L., Zaccaro, L., Bendinelli, M., Piedimonte,

G., Marcato, P.S., 1998. Apoptotic fraction in lymphoid tissue

of FIV-infected SPF cats. Vet. Immunol. Immunopathol. 64,

33–44.

Sarli, G., Mandrioli, L., Laurenti, M., Sidoli, L., Cerati, C., Rolla,

G., Marcato, P.S., 2001. Immunohistochemical characterisation

of the lymph node reaction in pig post-weaning multisystemic

wasting syndrome (PMWS). Vet. Immunol. Immunopathol. 83,

53–67.

Schafer, F.A., 1998. The cell cycle: a review. Vet. Pathol. 35,

461–478.


Segales, J., Domingo, M., 2002. Postweaning multisystemic

wasting sindrome (PMWS) in pigs. A review. Vet. Q. 24,

109–124.

Segales, J., Alonso, F., Rosell, C., Pastor, J., Chianini, F., Campos,

E., Lopez-Fuertes, L., Quintana, J., Rodrıguez-Arrioja,

G., Calsamiglia, M., Pujols, J., Domınguez, J., Domingo,

M., 2001. Changes in peripheral blood leukocyte popula-

tions in pigs with natural postweaning multisystemic wasting

syndrome (PMWS). Vet. Immunol. Immunopathol. 81, 37–

44.

Shibahara, T., Sato, K., Ishikawa, Y., Kadota, K., 2000. Porcine

circovirus induces B lymphocyte depletion in pigs with wasting

disease syndrome. J. Vet. Med. Sci. 62, 1125–1131.

Sur, J.H., Doster, A.R., Osorio, F.A., 1998. Apoptosis induced in

vivo during acute infection by porcine reproductive and

respiratory syndrome virus. Vet. Pathol. 35, 506–514.

Tizard, I.R., 2000. Dendritic cells and antigen processing. In:

Veterinary Immunology: An Introduction. W.B. Saunders

Company. Pennsylvania, pp. 50–68.

Tompkins, M.B., Bull, M.E., Dow, J.L., Ball, J.M., Collisson, E.W.,

Winslow, B.J., Phadke, A.P., Vahlenkamp, T.W., Tompkins,

W.A., 2002. Feline immunodeficiency virus infection is

characterized by B7þCTLA4þ T cell apoptosis. J. Infect.

Dis. 185, 1077–1083.

Woosley, K., Hart, A.A.M., 1983. Measuring cell proliferation.

Arch. Pathol. Lab. Med. 115, 555–557.


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Apoptosis and proliferative activity in lymph node reaction in postweaning multisystemic wasting syndrome (PMWS)