Departament de Sanitat i d'Anatomia Animals and Centre de Recerca en Sanitat Animal (CReSA), Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
Correspondence
Ana R. Resendes
ana.resendes{at}uab.es
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histological and haematological findings and cytokine profiles found in PMWS-affected pigs suggest that an immune system dysfunction, modulated by PCV2, underlies the pathogenesis of PMWS (Darwich et al., 2004). The main features of PMWS are T- and B-lymphocyte depletion in lymphoid tissues, peripheral blood lymphopenia and a specific downshift of B cells, CD4+CD8+, CD4+CD8, CD4CD8+,
TCR+ T cells and natural killer cells (Darwich et al., 2002
, 2003b
; Chianini et al., 2003
; Nielsen et al., 2003
). Also, cytokine mRNA expression is altered in several lymphoid organs (Darwich et al., 2003a
). It is speculated that viral load could be associated with the intensity of lymphocyte depletion and PMWS outcome (Liu et al., 2000
; Rovira et al., 2002
). Nevertheless, the mechanisms that lead to these alterations are not yet known.
Virus-induced apoptosis can contribute significantly to cytotoxicity, with the advantage of minimizing the host immune response (O'Brien, 1998); thus, this mechanism can contribute to a successful viral host infection. Lymphocyte depletion can be induced in viral infections through apoptosis. For example, human immunodeficiency virus (HIV) uses apoptosis specifically for CD4+ T-cell depletion (O'Brien, 1998
; Hay & Kannourakis, 2002
). Apoptosis has been proposed as the mechanism that is responsible for B-cell depletion in naturally PMWS-affected pigs (Shibahara et al., 2000
). However, this is not clear, as a recent report gave contradictory results (Mandrioli et al., 2004
).
One of the best approaches to detecting apoptosis in paraffin-embedded material, particularly when tissues are from a variety of sources and have been subjected to different processing treatments, is an immunoperoxidase method for active detection of effector caspases (Huppertz et al., 1999; Dukers et al., 2002
); this is also valid for porcine lymphoid tissue (Resendes et al., 2004
). Caspase-3 is an effector caspase and its activation represents a point of no return in the apoptotic pathway; therefore, its activation can be considered as a good marker for apoptosis (Hengartner, 2000
).
The objective of this study was to determine rates of apoptosis in the thymus and several peripheral lymphoid organs of naturally PCV2-infected pigs with different lesional stages, and to correlate these rates with the level of viraemia in infected pigs.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Categorization of animals in lesional stages.
Shortly after euthanasia by intravenous pentobarbital overdose, lymphoid tissue samples (spleen, thymus, tonsil, ileum and superficial inguinal lymph node) were collected from each pig and fixed in 10 % neutral-buffered formalin for approximately 24 h. Afterwards, samples were dehydrated, paraffin-embedded, sectioned at 4 µm and stained with haematoxylin and eosin for histopathological examination. In situ hybridization to detect the PCV2 genome was performed in lymphoid tissues as described previously (Rosell et al., 1999; Kennedy et al., 2000
). The positive control was a lymph node with PMWS histological lesions that was positive for PCV2 by PCR detection; the negative control was a histologically normal lymph node obtained from a pig that was negative for PCV2 by PCR. In lymphoid tissues from the study cases, the grade of lymphocyte depletion, histiocytic infiltration and amount of PCV2 DNA were scored according to Chianini et al. (2003)
. Pigs were classified into three lesional stages by considering the overall lymphoid depletion grade and the amount of PCV2 genome in lymphoid tissues. Lesional stage 1 (S1) included pigs with absent to mild PMWS lesions and low amounts of PCV2 in tissues (n=5); S2 included pigs with moderate lymphoid lesions and moderate amounts of PCV2 in most tissues (n=7); and S3 included pigs with severe lymphoid lesions and high amounts of PCV2 in most tissues (n=9). Stage S0 comprised the pigs that were selected as controls, which had no lesions or PCV2 genome in lymphoid tissues.
TaqMan real-time PCR.
PCV2 genomic load was quantified in serum samples by using the methodology described previously (Olvera et al., 2004).
Cleaved caspase-3 (CCasp3) immunohistochemistry to detect apoptosis.
CCasp3 was investigated in paraffin wax-embedded tissues by the avidinbiotin peroxidase (ABC) method, as reported previously (Resendes et al., 2004). Briefly, 4 µm correlative tissue sections were placed on silane-coated slides [3-(trietoxysilyl)-propylamine]; then, they were dewaxed in xylene, rehydrated in graded alcohols and placed in dH2O. Afterwards, antigen retrieval was done by immersion of sections in 0·01 M citrate buffer (pH 6·0) in a steam bath at 98 °C for 25 min, followed by rapid cooling over 20 min. After blocking endogenous peroxidase with 3 % H2O2 in dH2O (30 min) and washing with 0·1 M Tris-buffered saline (TBS, pH 7·4), non-specific binding was blocked with 2 % BSA (Sigma) for 30 min at room temperature. The anti-CCasp3 antibody used was an activation-specific polyclonal antibody (anti-Asp175; Cell Signalling), which does not recognize the caspase-3 precursor form (procaspase-3), but only the active form (CCasp3). The antibody was diluted 1/50 in 2 % BSA and incubated overnight at 4 °C. After washing in TBS, sections were incubated with a biotinylated goat anti-rabbit antibody (1/200 in TBS) (Dako) for 60 min at room temperature and subsequently treated with the ABC complex (1/100 in TBS) (Pierce) for 1 h at room temperature. Finally, sections were incubated in 0·05 % diaminobenzidine plus 3 % H2O2 in TBS for 10 min, rinsed in dH2O, counterstained with haematoxylin, dehydrated and mounted with DPX (Panreac). A porcine liver section with apoptotic hepatocytes was used as the positive control; as a negative control, the primary antibody was replaced by an irrelevant antibody (isotype-matched control antibody).
Apoptotic cell quantification.
Morphometrical analysis was carried out by taking the histological compartments of each lymphoid organ into account, as described previously (Resendes et al., 2004). In tonsil and superficial inguinal lymph node compartments, follicular and interfollicular areas were considered; in the thymus, cortex and medulla; and in the spleen, white and red pulp were considered. In Peyer's patches, only follicles were quantified, as dome areas were scant and interfollicular areas were small and difficult to outline. CCasp3 immunopositivity was quantified automatically by using a microscope equipped with an image analyser program (VISILOG 5.3; Noesis 2000). The software was programmed to quantify positivity with approximate lymphocyte size and to exclude labelled nuclear dust (free apoptotic bodies). Ten random fields of 0·035 mm2 (approximately one field of x400 magnification) of each compartment were quantified per tissue and pig. When tissues lost morphological compartmentalization due to lymphocyte depletion, only 10 random fields were counted per tissue and pig. Morphometric results from each compartment were grouped into three categories, according to the mean number of apoptotic cells per field (0·035 mm2): low (
5 cells); moderate (>5 to
10 cells); and high (>10 cells) (Resendes et al., 2004
).
Statistical analysis.
For each organ, mean apoptotic rates were compared between different compartments and lesional stages (S0S3) by the Poisson regression model (Kleinbaum & Klein, 2002). Mean PCV2 loads were compared between the three PMWS lesional groups (S1S3) by the KruskalWallis test. In addition, overall numerical apoptotic rates were correlated with viral load by a simple linear regression model, and with lesional stages by the TukeyKramer test. For each organ, numerical apoptotic rates were also correlated with viral load by using Pearson's correlation model. In addition, categorized apoptotic rates were correlated with lesional stages by using the
2 test and rxc tables, and lesional stages were correlated with viral load by the TukeyKramer test. Statistical analysis was performed with the Stats Direct and SAS programs. Differences were considered to be significant when P<0·05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Apoptosis in pigs in S2
In this lesional stage, the labelled cells were of the same type as that found in S1; however, the pattern of labelling distribution in tonsils, Peyer's patches and spleen was different. In these tissues, low to moderate rates of CCasp3-positive cells had a scattered distribution in a tissue that had lost compartmentalization as a consequence of lymphocyte depletion. Occasionally, when follicular structures were present, moderate rates of apoptosis were found in follicles and low rates were found in interfollicular areas. Necrotizing lymphadenitis was found in the inguinal superficial lymph node, the tonsils and Peyer's patches from two pigs. In these tissues, focal areas with high rates of positivity were detected (>30 cells per field; data not shown), whereas areas that were adjacent to necrotic foci had low rates of labelled cells. In the spleen, low rates of scattered positive cells were detected in the red pulp; white pulp was not present. In the thymus, low to moderate rates of immunolabelled cells had a scattered distribution in the cortex, whilst low rates were found in the medulla. Overall, apoptotic rates for each organ were significantly lower than those in the control group. Moreover, apoptotic rates for tonsils and Peyer's patches were significantly lower than those from S1 pigs. In general, S2 lymphoid lesions were associated mainly with low rates of apoptosis. CCasp3 quantification and statistical results are displayed in Fig. 2.
Apoptosis in pigs in S3
The cell types labelled and the pattern of labelling distribution in this lesional stage were as noted for S2. In all pigs, tonsils, superficial inguinal lymph nodes (Fig. 1d) and Peyer's patches had low rates of positive cells with a scattered distribution in tissue that had lost compartmentalization. In the spleen and thymus (Fig. 1b
), CCasp3 labelling distribution and rates were similar to those found in S2. One pig presented with necrotizing lymphadenitis, where high amounts of apoptotic cells were observed focally, as described previously, and purulent splenitis with high rates of positive cells (>30 cells per field; data not included in statistical analysis) that were distributed diffusely. Apoptotic rates from all tissues were significantly lower than those from the control group. When compared with S1, significantly lower rates of apoptosis were found for tonsils and Peyer's patches. When compared with S2, significantly lower rates were found only for Peyer's patches. Overall, in most lymphoid tissues, S3 lesions were mainly associated with low rates of apoptosis. CCasp3 quantification and statistical results are displayed in Fig. 2
.
PCV2 DNA load in serum
Viral load from each animal is shown in Fig. 3. In pigs, the mean±SD from S1 (n=5/5) were 1·6x105±1·2x105 genomes ml1; from S2 pigs (n=6/7), 3·3x107±5·1x107 genomes ml1; and from S3 pigs (n=7/9), 1·3x108±9·9x107 genomes ml1. Significant differences among viral loads were found only between groups S3 and S1.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Taking into account other viral infections (Summerfield et al., 1998; Sato et al., 2000
; Sánchez-Cordón et al., 2002
), it appears that when apoptosis is implicated in the pathogenesis of lymphocyte-depletion lesions, its incidence is usually increased and can be demonstrated by immunohistochemical methods in lymphoid tissues. Moreover, it appears that higher rates of apoptosis are found during the acute phase of infection, when active virus replication occurs. For example, this is the case in classical swine fever virus (Summerfield et al., 1998
; Sato et al., 2000
; Sánchez-Cordón et al., 2002
), African swine fever virus (Oura et al., 1998
; Salguero et al., 2004
) and chicken anemia virus (Jeurissen et al., 1992
) infections. Nevertheless, in our study, no increased apoptotic rates were found in any lymphocyte-depletion lesional stage, and they were inversely proportional to the viral load.
The present study included pigs with the full spectrum of lymphoid-depletion lesions and a range of PCV2 load in serum, which is likely to be found during PMWS (Rosell et al., 1999; Olvera et al., 2004
). Thus, we believe that the acute phase of PMWS infection was well-represented within the study cases. If apoptosis was massive or increased in PMWS, it should, at least, have been found in S1 and S2 pigs, which could be considered to be in the initial or early phases of disease. On the other hand, the control pigs had the expected apoptotic rates for healthy conventional pigs at this age (Resendes et al., 2004
), which, in all cases, were higher than for diseased pigs. Our observations contrast with those of Shibahara et al. (2000)
, who found increased apoptotic rates in B-cell areas from PMWS-affected pigs with respect to control pigs. Compared with what are described as normal apoptotic rates for healthy conventional pigs (Resendes et al., 2004
), the apoptotic rates observed in control pigs in the study of Shibahara et al. (2000)
could be categorized as low. In view of these facts, the high apoptotic rates found in PMWS-affected pigs from that study should be revised, considering the full range of apoptotic rates that can be found in normal lymphoid tissues from healthy conventional pigs. In agreement with Mandrioli et al. (2004)
, who used the TUNEL assay for detection of apoptosis, we found similar results by using CCasp3 immunolabelling: an inverse correlation between apoptotic rates and PMWS lesional severity. Furthermore, the scarce cellularity we found in S2 and S3 cases must, in part, account for the low apoptotic rates observed. Nevertheless, interpreting the apoptotic rates from S1 thymic cortex, which was only mildly depleted, it seems feasible that in addition to cell reduction, inhibited apoptosis may also coexist, accounting for low apoptotic rates. It is important to understand that this study does not exclude partial involvement of apoptosis in the pathogenesis of lymphocyte depletion in PMWS, as this phenomenon could affect small regulator lymphocyte subpopulations or precursor cells, as can happen in HIV infection (O'Brien, 1998
).
In normal lymphoid tissues, apoptosis and proliferation are known to be related phenomena through which the total lymphocyte population is kept at a fairly constant level (Janeway, 2001; Rathmell & Thompson, 2002
). Thus, in an indirect way, apoptosis can be interpreted as an indicator of proliferative activity. Our results suggest that lymphocyte proliferation could be inhibited in several lymphoid tissues of PMWS-affected pigs. This hypothesis was also suggested by Mandrioli et al. (2004)
, who found low ratios of a proliferation marker in the lymph nodes of PMWS-affected pigs. In addition, we believe that thymocyte proliferation may also be decreased in early stages of the disease. Apoptotic rates in B-cell follicles apparently tend to decrease when compared with those from healthy pigs. It is possible that the loss of B-cell areas in S2 and S3 may also be associated with decreased proliferation of those cells. Inhibited lymphocyte proliferation in PMWS pathogenesis is supported by other studies. For example, PMWS-affected pigs show thymic interleukin 10 (IL10) mRNA overexpression (Darwich et al., 2003b
), which could inhibit thymocyte proliferation (Rouleau et al., 1999
). It is remarkable that, in vitro, PCV2 is able to induce IL10 overproduction in infected peripheral blood mononuclear cells (Darwich et al., 2003a
); thus, it is not surprising that PCV2 may inhibit thymocyte proliferation in vivo. In addition, low IL4 mRNA expression is found in peripheral lymphoid tissues from PMWS-affected pigs, which may induce B cells to cease proliferation (Darwich et al., 2003b
). The relationship between apoptosis and inhibition of lymphocyte proliferation should be investigated further, as it may be relevant to the development of PMWS.
The high variability in apoptotic rates found in some tissues from S2 pigs could be explained by considering the wide variability in lesion severity that can exist in this lesional category. This stage is also characterized by high variability in the loads of PCV2 in serum (Olvera et al., 2004). The areas with high apoptotic rates that are found in necrotizing lymphadenitis and splenitis, which are respectively associated with necrotic foci and diffuse neutrophilic infiltration, are probably a consequence of an undetermined bacterial infection. Most of the pigs used in this study suffered from secondary bacterial infections, which occur frequently in PMWS (Segalés et al., 2004b
).
In conclusion, considering the relationship found between PCV2 serum load, apoptotic rates and severity of lymphocyte depletion, it appears that apoptosis is not a remarkable feature in PMWS lesion development. Further investigation is needed in order to evaluate the implication of apoptosis and defective production of lymphocyte subpopulations and precursor cells, and to exclude the potential of PCV2-infected antigen-presenting cells to induce defective stimulation of lymphocytes. The implications for defective lymphocyte homing should also be assessed.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allan, G. M., Kennedy, S., McNeilly, F., Foster, J. C., Ellis, J. A., Krakowka, S. J., Meehan, B. M. & Adair, B. M. (1999). Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus. J Comp Pathol 121, 111.[CrossRef][Medline]
Bolin, S. R., Stoffregen, W. C., Nayar, G. P. & Hamel, A. L. (2001). Postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum-deprived piglets with type 2 porcine circovirus. J Vet Diagn Invest 13, 185194.[Medline]
Chianini, F., Majó, N., Segalés, J., Domínguez, J. & Domingo, M. (2003). Immunohistochemical characterisation of PCV2 associate lesions in lymphoid and non-lymphoid tissues of pigs with natural postweaning multisystemic wasting syndrome (PMWS). Vet Immunol Immunopathol 94, 6375.[CrossRef][Medline]
Darwich, L., Segalés, J., Domingo, M. & Mateu, E. (2002). Changes in CD4+, CD8+, CD4+ CD8+, 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, 236242.
Darwich, L., Balasch, M., Plana-Durán, J., Segalés, J., Domingo, M. & Mateu, E. (2003a). Cytokine profiles of peripheral blood mononuclear cells from pigs with postweaning multisystemic wasting syndrome in response to mitogen, superantigen or recall viral antigens. J Gen Virol 84, 34533457.
Darwich, L., Pié, S., Rovira, A., Segalés, J., Domingo, M., Oswald, I. P. & Mateu, E. (2003b). Cytokine mRNA expression profiles in lymphoid tissues of pigs naturally affected by postweaning multisystemic wasting syndrome. J Gen Virol 84, 21172125.
Darwich, L., Segalés, J. & Mateu, E. (2004). Pathogenesis of postweaning multisystemic wasting syndrome caused by Porcine circovirus 2: an immune riddle. Arch Virol 149, 857874.[CrossRef][Medline]
Dukers, D. F., Oudejans, J. J., Vos, W., ten Berge, R. L. & Meijer, C. J. L. M. (2002). Apoptosis in B-cell lymphomas and reactive lymphoid tissues always involves activation of caspase 3 as determined by a new in situ detection method. J Pathol 196, 307315.[CrossRef][Medline]
Hay, S. & Kannourakis, G. (2002). A time to kill: viral manipulation of the cell death program. J Gen Virol 83, 15471564.
Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature 407, 770776.[CrossRef][Medline]
Huppertz, B., Frank, H.-G. & Kaufmann, P. (1999). The apoptosis cascade morphological and immunohistochemical methods for its visualization. Anat Embryol 200, 118.[CrossRef][Medline]
Janeway, C. A. (2001). The development and survival of lymphocytes. In Immunobiology: the Immune System in Health and Disease, pp. 221294. Edited by C. A. Janeway, P. Travers, M. Walport & M. Shlomchik. New York: Garland Publishing.
Jeurissen, S. H. M., Wagenaar, F., Pol, J. M. A., van der Eb, A. J. & Noteborn, M. H. M. (1992). Chicken anemia virus causes apoptosis of thymocytes after in vivo infection and of cell lines after in vitro infection. J Virol 66, 73837388.[Abstract]
Kennedy, S., Moffett, D., McNeilly, F., Meehan, B., Ellis, J., Krakowka, S. & Allan, G. M. (2000). Reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus. J Comp Pathol 122, 924.[CrossRef][Medline]
Kim, J. & Chae, C. (2003). A comparison of the lymphocyte subpopulations of pigs experimentally infected with porcine circovirus 2 and/or parvovirus. Vet J 165, 325329.[CrossRef][Medline]
Kleinbaum, D. G. & Klein, M. (2002). Logistic Regression, 2nd edn. New York: Springer.
Krakowka, S., Ellis, J. A., McNeilly, F., Ringler, S., Rings, D. M. & Allan, G. (2001). Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV-2). Vet Pathol 38, 3142.
Liu, Q., Wang, L., Willson, P. & Babiuk, L. A. (2000). Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome. J Clin Microbiol 38, 34743477.
Mandrioli, L., Sarli, G., Panarese, S., Baldoni, S. & Marcato, P. S. (2004). Apoptosis and proliferative activity in lymph node reaction in postweaning multisystemic wasting syndrome (PMWS). Vet Immunol Immunopathol 97, 2537.[CrossRef][Medline]
Nielsen, J., Vincent, I. E., Bøtner, A., Ladekjær-Mikkelsen, A.-S., Allan, G., Summerfield, A. & McCullough, K. C. (2003). Association of lymphopenia with porcine circovirus type 2 induced postweaning multisystemic wasting syndrome (PMWS). Vet Immunol Immunopathol 92, 97111.[CrossRef][Medline]
O'Brien, V. (1998). Viruses and apoptosis. J Gen Virol 79, 18331845.
Olvera, A., Sibila, M., Calsamiglia, M., Segalés, J. & Domingo, M. (2004). Comparision of porcine circovirus type 2 load in serum quantified by a real time PCR in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs. J Virol Methods 117, 7580.[CrossRef][Medline]
Oura, C. A. L., Powell, P. P. & Parkhouse, R. M. E. (1998). African swine fever: a disease characterized by apoptosis. J Gen Virol 79, 14271438.[Abstract]
Quintana, J., Balasch, M., Segalés, J., Calsamiglia, M., Rodríguez-Arrioja, G. M., Plana-Durán, J. & Domingo, M. (2002). Experimental inoculation of porcine circoviruses type 1 (PCV1) and type 2 (PCV2) in rabbits and mice. Vet Res 33, 229237.[CrossRef][Medline]
Rathmell, J. C. & Thompson, C. B. (2002). Pathways of apoptosis in lymphocyte development, homeostasis, and disease. Cell 109, S97S107.[Medline]
Resendes, A. R., Majó, N., Segalés, J., Espadamala, J., Mateu, E., Chianini, F., Nofrarias, M. & Domingo, M. (2004). Apoptosis in normal lymphoid organs from healthy normal, conventional pigs at different ages detected by TUNEL and cleaved caspase-3 immunohistochemistry in paraffin-embedded tissues. Vet Immunol Immunopathol 99, 203213.[Medline]
Rodríguez-Arrioja, G. M., Segalés, J., Balasch, M., Rosell, C., Quintant, J., Folch, J. M., Plana-Durán, J., Mankertz, A. & Domingo, M. (2000). Serum antibodies to porcine circovirus type 1 and type 2 in pigs with and without PMWS. Vet Rec 146, 762764.[Medline]
Rosell, C., Segalés, J., Plana-Durán, J. & 7 other authors (1999). Pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs. J Comp Pathol 120, 5978.[CrossRef][Medline]
Rosell, C., Segalés, J. & Domingo, M. (2000). Hepatitis and staging of hepatic damage in pigs naturally infected with porcine circovirus type 2. Vet Pathol 37, 687692.
Rouleau, M., Cottrez, F., Bigler, M., Antonenko, S., Carballido, J. M., Zlotnik, A., Roncarolo, M.-G. & Groux, H. (1999). IL-10 transgenic mice present a defect in T cell development reminiscent to SCID patients. J Immunol 163, 14201427.
Rovira, A., Balasch, M., Segalés, J., García, L., Plana-Durán, J., Rosell, C., Ellerbrok, H., Mankertz, A. & Domingo, M. (2002). Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2. J Virol 76, 32323239.
Salguero, F. J., Sánchez-Cordón, P. J., Sierra, M. A., Jover, A., Núñez, A. & Gómez-Villamandos, J. C. (2004). Apoptosis of thymocytes in experimental African swine fever virus infection. Histol Histopathol 19, 7784.[Medline]
Sánchez-Cordón, P. J., Romanini, S., Salguero, F. J., Núñez, A., Bautista, M. J., Jover, A. & Gómez-Villamos, J. C. (2002). Apoptosis of thymocytes related to cytokine expression in experimental classical swine fever. J Comp Pathol 127, 239248.[CrossRef][Medline]
Sato, M., Mikami, O., Kobayashi, M. & Nakajima, Y. (2000). Apoptosis in the lymphatic organs of piglets inoculated with classical swine fever virus. Vet Microbiol 75, 19.[CrossRef][Medline]
Segalés, J. & Domingo, M. (2002). Postweaning multisystemic wasting syndrome (PMWS) in pigs. A review. Vet Q 24, 109124.[Medline]
Segalés, J., Domingo, M., Chianini, F., Majó, N., Domínguez, J., Darwich, L. & Mateu, E. (2004a). Immunosuppression in postweaning multisystemic wasting syndrome affected pigs. Vet Microbiol 98, 151158.[CrossRef][Medline]
Segalés, J., Rosell, C. & Domingo, M. (2004b). Pathological findings associated with naturally acquired porcine circovirus type 2 associated disease. Vet Microbiol 98, 137149.[CrossRef][Medline]
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, 11251131.[CrossRef][Medline]
Summerfield, A., Knötig, S. M. & McCullough, K. C. (1998). Lymphocyte apoptosis during classical swine fever: implication of activation-induced cell death. J Virol 72, 18531861.
Received 23 April 2004;
accepted 16 June 2004.