Laboratório de Doenças Infecciosas, CIISA, Faculdade de Medicina Veterinária, Rua Professor Cid dos Santos, 1300-477 Lisbon, Portugal1
Centro de Veterinária e Zootecnia, CIISA, Instituto de Investigação Científica Tropical, Rua Professor Cid dos Santos, 1300-477 Lisbon, Portugal2
Departamento de Virologia, Laboratório Nacional de Investigação Veterinária, Estrada de Benfica 701, 1549-011 Lisbon, Portugal3
Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal4
Author for correspondence: Carlos L. V. Martins. Fax +351 21 365 28 21. e-mail cmartins{at}fmv.utl.pt
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Abstract |
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Introduction |
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In this work, we have focused on an interesting ASFV isolate, the non-fatal, non-haemadsorbing ASFV/NH/P68 (NHV), isolated from a chronically infected pig and used to obtain anti-ASFV sera for diagnostic purposes (Vigário et al., 1974 ). Our observations extend previous studies on cellular immune responses in experimentally infected pigs (Martins et al., 1993
; Leitão et al., 1998
, 2000
), and provide the basis for a useful and relevant infection model for studies on the mechanisms of protective immunity; in particular, the fact that exposure to NHV induces significantly high levels of NK cell activity and protects against subsequent infection with the highly virulent ASFV/L60 (L60). Thus, pigs inoculated with NHV and subsequently challenged with L60 were studied for the development of clinical signs, occurrence of viraemia and the development of cellular and serological immunity.
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Methods |
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Animals and animal inoculation.
Large WhitexLandrace cross-bred pigs, weighing 2545 kg, were used for experimental inoculations. Pigs from the Lisbon slaughterhouse were used as blood donors to prepare macrophage cultures for virus propagation.
Pigs were inoculated with 5x106 CPE50 NHV either by oronasal (o.n.) or intramuscular (i.m.) routes and monitored daily for body temperature and development of clinical signs. In each experiment there was a control group of two non-inoculated pigs. Blood samples were taken at different days post-inoculation (p.i.) from the anterior vena cava and collected in heparinized syringes (20 IU/ml blood). The clinical course and immunological parameters studied are summarized in Table 1. Animals developing clinical signs of ASF (ASF chronic type lesions) were slaughtered and necropsied. Animals not developing apparent clinical signs of ASF were challenged by i.m. inoculation of 5x106 CPE50 of the highly virulent L60 isolate.
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Collection of peripheral blood mononuclear cells (PBMC) and plasma.
Heparinized blood samples collected from the anterior vena cava were layered on a FicollHypaque gradient (density=1·077 g/cm3; Seromed) and centrifuged (room temperature, 400 g, 30 min). Cells from the interface were collected and washed with HBSS three times by centrifugation (4 °C, 200 g, 10 min). The final pellet was resuspended in culture medium supplemented with 10% heat inactivated foetal calf serum (FCS). Cell viability, determined by trypan blue dye exclusion, was always higher than 95%. Plasma samples were clarified by centrifugation (4 °C, 1000 g, 30 min), stored at -20 °C and used to evaluate specific titres of anti-ASFV antibodies and total immunoglobulin (Ig) concentrations.
Estimation of viraemia in experimentally inoculated pigs.
Quadruplicate cultures of macrophages in 96-well microplates were inoculated with 20 µl blood per well and incubated (37 °C, 7 days in 5% CO2 and >80% humidity). Individual cultures were resuspended, transferred to cytospin slides, fixed with acetone (-20 °C, 5 min) and screened with FITC-conjugated swine anti-ASFV serum. Positive samples were titrated as described above.
Lymphoproliferative responses to mitogens [phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM)], and to ASFV (NHV and L60).
Lymphoproliferative responses to mitogens and ASFV were studied using PBMC in triplicate microplate cultures (200 µl; 2·5x106 cells/ml in culture medium supplemented with 0·01 mM 2-mercaptoethanol and 10% FCS) and stimulating with PHA (50 and 100 µg/ml; Wellcome PHA HA15), ConA (0·25 and 0·5 µg/ml; Sigma type IV C-2010) and PWM (0·5 and 1%, v/v; Gibco 670-5360) and ASFV isolates L60 and NHV (m.o.i. 0·01). Cells without mitogens or virus were used as controls. Microplates were incubated (37 °C, 72 h in 5% CO2 and >80% humidity) and then [3H]thymidine (1 µCi, 5 Ci/mmol) incorporation was allowed to proceed for 4 h. Cultures were harvested onto nitrocellulose filters, which were counted in scintillation fluid (Optiphase HiSafe 3, LKB 1200-437) using a scintillation counter. Incorporation of [3H]thymidine was measured as c.p.m. with coefficient of variation within triplicate cultures always less than 10%. The data from longitudinal samples were calculated for individual pigs as difference ratios at each time-point sampled, by dividing the observed c.p.m. in stimulated cultures by the observed c.p.m. of similarly stimulated PBMC cultures of the same pig at day p.i. 0. The final data were then expressed as the average ratio in each experimental infected group (infected with NHV symptomatic; infected with NHV healthy; infected with NHV healthy then challenged with L60) on a given day after commencement of the experiment minus the average ratio in the control uninfected group studied over the same time-scale. Thus values of zero indicate that infection with ASFV neither inhibits nor augments the subsequent response of PBMC to stimulation in vitro. The variation between ratios obtained on a given day within a given group of animals was always less than 20% and so, for clarity of presentation, this information is omitted from the graphical representation of the results. Levels of proliferation in the absence of antigen or mitogen were similarly low in all the samples and mitogen stimulation gave stimulation indices ranging from 30 to 100 while virus stimulation gave stimulation indices up to 10.
Measurement of anti-ASFV antibodies.
Plasma samples were tested by indirect ELISA using a crude preparation of ASFV proteins as antigen. The protein extract was obtained from ASFV-infected Vero cells by treatment with hypotonic buffer solution (67 mM sucrose, 5 mM TrisHCl pH 8, 1% Nonidet P40) followed by centrifugation (4 °C, 800 g, 20 min). The supernatant was adjusted to contain 50 mM 2-mercaptoethanol, 2 mM EDTA, 5 mM TrisHCl pH 8, and then centrifuged through a 2060% sucrose gradient. The antigen collected at the 2060% sucrose interface was suspended in 50 mM 2-mercaptoethanol, 2 mM EDTA, 0·5 M NaCl, 0·5% Nonidet P40, centrifuged (4 °C, 100000 g, 10 min), and resuspended in carbonatebicarbonate buffer (1·59 g Na2CO3, 2·93 g NaHCO3 per litre, pH 9·6) at a concentration empirically determined for each antigen preparation for sensitizing ELISA 96-well microplates (Dynatech M129B) overnight at 4 °C.
Serial twofold dilutions of plasma samples in PBS were added to the antigen-coated microplate wells and, after 1 h at 37 °C, bound antibody was detected using protein Aperoxidase conjugate, and revealed with o-phenylenediamine in the presence of hydrogen peroxide. The reaction was stopped with 1 M H2SO4 and the absorbance at 492 nm was read using a spectrophometer. Sample titres were calculated by comparison with a reference anti-ASFV swine serum previously titrated by indirect immunofluorescence.
Measurement of total IgG1, IgG2, IgM and IgA concentrations by ELISA.
Serial twofold dilutions of plasma samples and reference serum were dispensed in ELISA microplates and incubated overnight at 4 °C. After coating, plates were incubated (37 °C, 23 h) with PBS containing 0·5% (v/v) Tween 20. Differential detection of swine Ig classes was achieved by separate development with mouse monoclonal antibodies (MAbs) (diluted 1/1000 in PBS) to swine-IgG1 (Serotec MCA635), -IgG2 (Serotec MCA636), -IgM (Serotec MCA637) and -IgA (Serotec MCA638). For negative controls, PBS replaced the anti-immunoglobulin MAbs. After incubation (37 °C, 2 h), peroxidase-conjugated rabbit anti-mouse antibody (Dako P0260) was added (1/2000 dilution in PBS) and incubation was continued (37 °C, 1h). After washing the substrate ABTS [2,2'-azino-bis(3-ethylbenzothiazoline 6-sulfonic acid)] solution (Boehringer Mannheim) was added, and after 40 minutes the reaction was stopped by addition of 2·5% (w/v) sodium fluoride, 5% (w/v) SDS. The plates were read (=405 nm) in a spectrophotometer as above. The linear portion of the absorbance versus dilution plot was used to determine concentrations, after subtracting the non-specific background linear regression from the sample linear regression.
Concentrations of IgM and IgA were calculated with reference to a standard serum (kindly supplied by Dilip Patel, Department of Animal Husbandry, School of Veterinary Medicine, University of Bristol, UK) with concentrations of IgM and IgA of 2·9 g/l and 3·2 g/l respectively. Because a reference serum with known IgG1 and IgG2 concentrations was not available, relative concentrations of these two subclasses in the plasma samples were calculated arbitrarily, taking the absorbance for IgG1 plus the absorbance for IgG2 in the reference serum as 100 units, and using the following formula:(Asample-Asample backgr)x100/[(Ast IgG1-Ast backgr)+(Ast IgG2-Ast backgr)]
where st stands for standard and backgr refers to the background observed in wells tested without anti-immunoglobulin MAb.
NK activity assays.
Target cells (K562, ATCC CCL 243) were harvested at exponential growth phase by centrifugation (4 °C, 200 g, 10 min) and 107 cells were labelled by incubation (1 h, 37 °C) with 100 µCi of Na251CrO4 isotonic solution (Amersham). The cells were washed three times in cold HBSS, and finally suspended in culture medium at 105 cells/ml. Microcytotoxicity assays were performed in triplicate 200 µl cultures in round-bottom 96-well tissue culture microplates, adding aliquots of effector cells and target cells at different volumes, to obtain different effector to target (E:T) ratios (10:1, 25:1, 50:1 and 100:1). As controls, target cells added with medium or with 10% Triton-X100 solution were included to measure spontaneous and total 51Cr release, respectively. Microplates were incubated (37 °C, 18 h, >80% humidity and 5% CO2), supernatants collected and sample radioactivity was counted in a -radiation counter. The percentage of specific lysis was calculated from the mean of the three replicate wells using the following formula: specific lysis=(mean c.p.m. wells with effector cells-mean c.p.m. spontaneous release)/(mean c.p.m. total release-mean c.p.m. spontaneous release)x100. Spontaneous radioisotope release by target cells was always below 30% of total released radioactivity.
Statistical analysis.
Statistical analysis of the results was performed using multifactor analysis of variance taking the time (day p.i.) as covariate. Differences between counts were considered significant at P<0·05. Standard errors were below 20% of the mean, and thus for clarity are omitted from the graphs.
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Results |
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The occurrence of such chronic type lesions tended to be correlated with later bouts of viraemia and fever, the latter being defined as body temperatures above 40 °C for at least two consecutive days. Thus most animals with early (before day 14 p.i.) or no fever remained asymptomatic throughout the experiments, while the development of lesions was, with one exception, associated with fever after day 14 p.i., with or without an earlier (before day 14 p.i.) phase of fever (Table 1). Similarly, the majority of the pigs with late viraemia developed chronic type lesions. In contrast, those animals without a late viraemia, even those which had a early phase of viraemia, never developed chronic lesions (Table 1
).
Of 240 blood samples collected at different times post-virus-inoculation, virus was detected, albeit at low titre (<103), in 36 samples. The presence of virus was not necessarily correlated with fever. Thus only 16 positive samples were collected when the animals body temperatures were above 40 °C. The remaining 20 samples were collected when the body temperatures were equal to or below 40 °C. Viraemia was not detected in 9 out of 12 pigs inoculated o.n. and in 3 out of 19 pigs inoculated i.m. Two of these animals (one inoculated by each route) developed chronic ASF. Early viraemia in pigs not showing lesions was mainly detected in pigs inoculated i.m. (Table 1).
Lymphoproliferative responses to mitogens
Lymphoproliferative responses of PBMC to mitogens after NHV inoculation were studied in two experiments on a total of 15 animals (Table 1, groups 3 and 4). Four normal, non-inoculated animals were used as negative controls. Of the eight animals inoculated by the o.n. route only one developed chronic type lesions, whereas four of the seven inoculated by the i.m. route developed chronic type lesions. Lymphoproliferative responses to ConA, PHA or PWM were not depressed in any of the 15 inoculated animals, a consistent observation, independent of either the inoculation route or the presence or absence of chronic lesions. Indeed, in one of the two experiments (Fig. 1
), the proliferative responses to mitogens of lymphocytes taken from days 7 to 28 p.i. were higher in the inoculated animals than in the control animals, with statistical significance (P<0·05) for responses to ConA (0·5 µg/ml) and PHA (50 µg/ml). Moreover, lymphoproliferative responses to NHV and L60 virus antigens were also demonstrable with PBMC collected from day 7 p.i. onwards (Fig. 1
).
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For the study of total Ig concentrations in plasma samples, 15 NHV-inoculated pigs were divided into two groups for independent experiments (groups 3 and 4, Table 1). In group 3, two groups of four pigs were infected i.m. or o.n. In group 4, four and three pigs were infected o.n. and i.m. respectively. Two control pigs were used in each experiment. There were no major oscillations in the levels of plasma IgG1 and IgG2 concentrations in the control animals during the time-course of the experiments. Asymptomatic inoculated animals (n=10) had IgG1 and IgG2 levels similar to control animals (Fig. 4
). In contrast, inoculated animals with lesions (n=5) progressively developed increased levels of both IgG1 and IgG2. Thus, at day 39 p.i. these levels were nearly three times higher than those observed at day 0. The ratio of IgG2/IgG1 for each group of animals was nearly constant throughout the experiments (Fig. 4
), although a slight increase in IgG2/IgG1 ratio can be identified in animals remaining asymptomatic after inoculation. At the same time, the IgG2/IgG1 ratio was consistently higher in the animals developing lesions.
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Five pigs infected with NH virus and then challenged with L60 (group 4, Table 1) had normal lymphoproliferative responses to mitogens (Fig. 6
). This is in marked contrast to the inhibition of mitogen-stimulated proliferation observed in the three pigs inoculated with L60 by the o.n. route. The latter animals died from acute ASF between days 6 to 9 p.i. and their PBMC taken on day 4 p.i. exhibited a profound reduction in lymphoproliferative response to ConA and PHA (data not shown). In fact, the levels of proliferation observed in response to both concentrations of ConA and PHA in the animals exposed to NHV and L60 were significantly (P<0·05) above the response levels in the two controls. Responses of control and inoculated pigs to PWM, on the other hand, were not so clearly differentiated (P>0·05) (Fig. 6
). Proliferative responses of PBMC from four animals taken at 0, 2 and 4 days post-i.m. inoculation with L60 were normal, relative to responses obtained with two control, uninfected pigs (data not shown).
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Discussion |
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Inoculation (i.m. or o.n.) of pigs with NHV established two clinical groups: pigs developing chronic type ASF lesions and pigs remaining asymptomatic. Those developing lesions showed viraemia and fever in a late phase of infection (after day 14 p.i.), NK activity levels similar to control animals, high levels of anti-ASFV specific antibodies and increasing concentrations of total IgG1, IgG2, IgM and IgA. Pigs remaining asymptomatic after infection, on the other hand, were neither viraemic nor febrile after day 14 p.i., had normal plasma immunoglobulin concentrations throughout the duration of the experiments, relatively low levels of anti-virus antibody titres, and markedly high levels of NK activity. The appearance of anti-ASFV antibodies and specific sensitized lymphocytes in the circulation was first detected from days 7 to 18 p.i., in agreement with previous observations (reviewed by Wardley et al., 1987 ).
The correlation of high levels of NK cell activity with the development of resistance is particularly interesting as it implies a functional role for this lymphocyte subset in protection. Thus, there was a clearly enhanced level of NK at day 7 p.i. in those animals remaining healthy after inoculation with NHV and developing resistance to the L60 strain of ASFV. In some of these pigs high levels of NK cell activity were observed throughout the duration of the experiments. In striking contrast, the animals that developed chronic lesions of ASF showed NK levels similar to or only slightly above the control animals. In contrast, a moderately virulent ASFV apparently depresses NK activity in pigs (Norley & Wardley, 1983 ), and in vitro the NK activity of porcine mononuclear cells was inhibited by both a low and a highly virulent ASFV (Mendonza et al., 1991
). Although the phenotype of porcine NK cells has been described (Saalmuller et al., 1994
; Yang & Parkhouse, 1996
), there is little information on their role against viral diseases of pigs. Indeed, the foregoing and potential role of NK cells in the activation of immune responses in both mouse and man (review by Biron et al., 1999
) provide an urgent argument for further work on these cells as possible modulators of porcine immune responses against ASFV, particularly as INF-
and IFN-
are known to inhibit ASFV replication in a synergistic manner in vitro (Esparza et al., 1988
; Paez et al., 1990
). The other obvious arm of cellular immunity, the cytotoxic T cell, is also a candidate for future investigation in this experimental model, in which the generation of CD8+ T-cells able to lyse ASFV-infected macrophages has already been demonstrated (Martins et al., 1993
).
In negative correlation with the NK activity results, hypergammaglobulinaemia was only found in animals developing ASF chronic type lesions, and not in animals remaining asymptomatic and developing resistance after virus inoculation. Thus, Ig concentrations in inoculated animals remaining asymptomatic showed identical profiles to control animals, independent of the route of inoculation used. In contrast, the development of chronic ASF type lesions was associated with a significant increase in IgG levels (data not shown) in agreement with the across-the-board increased levels of IgG1, IgG2, IgM and IgA in the same animals. Consistent with the increased total Ig levels, anti-ASFV specific antibody titres observed in pigs developing chronic infection were much higher than those of asymptomatic animals. Although the IgG2/IgG1 ratio largely remained constant throughout the experiment in all the animal groups (controls, chronically infected and asymptomatic), this ratio was considerably higher at the beginning of the experiment in the group of animals that developed lesions, perhaps suggesting a predisposition for the establishment of chronic ASF in animals with high IgG2/IgG1 ratios. Further work is required to confirm this possible correlation, but this work does not provide positive evidence for a role for antibodies in protective immunity to ASFV.
The demonstration of hypergammaglobulinaemia in pigs developing chronic ASF confirms earlier work (Pan et al., 1970 ; Pan, 1987
) and may be related to the systemic immune activation and associated increase in macrophages, B- and CD8+ T-cells that occurs in pigs with chronic persistent infection (Ramiro-Ibañez et al., 1997
). Other authors have demonstrated that infection with ASFV isolates of different virulence may result in over-stimulation of B-lymphocytes in vitro and in vivo (Wardley, 1982
; Takamatsu et al., 1999
). Finally, a specific immunosuppression due to overstimulation of CD4+ and CD8+ T-cells, and mediated by IL-4 and IL-10, has been described in mice inoculated with ASFV protein p36 (Arala-Chaves et al., 1988
; Ribeiro et al., 1991
; Vilanova et al., 1999
).
A variety of results has been obtained when mitogen responses of lymphocytes from pigs infected with ASFV have been investigated. In contrast to some previous work (Sanchez-Vizcaiño et al., 1981 ; Childerstone et al., 1998
), but in agreement with other studies (Wardley & Wilkinson 1980
; Knudsen & Genovesi, 1987
; Scholl et al., 1989
) there was no depression of lymphoproliferative responses to virus antigens or mitogens (ConA, PHA and PWM) in any of the animals infected with NHV. It is possible that this variation is due to differences in the systems investigated, e.g. PBMC versus spleen, strain of virus. Furthermore, the mitogen responses of PBMC were not depressed after a subsequent inoculation with the highly virulent L60 isolate, although, PBMC from pigs infected with only the virulent L60 isolate had reduced lymphoproliferative responses. An inhibitory effect of ASFV on mitogenic responses of normal lymphocytes in vitro has also been noted (Wardley, 1982
; Gonzalez et al., 1990
), and should be distinguished from the effect of ASFV infection upon mitogenic responses of lymphocytes subsequently taken from the infected animals. Relevant to this work, Borca et al. (1998)
similarly demonstrated normal mitogen responses in PBMC from pigs infected with a virulent ASFV isolate with the CD2 homologue deleted.
Finally, the question of protective serological immunity to ASFV remains controversial. These data, with a negative correlation between antibodies and pathology, on the one hand, versus the positive correlation between increased NK cell activity and the development of protective immunity in the absence of pathological symptoms, on the other, support the notion that immunity to ASFV depends, at least in part, on cellular mechanisms, in particular NK cells. Thus, pigs inoculated with the NHV isolate developed significantly high levels of NK cell activity in the absence of clinical symptoms, and survived subsequent infection with the fatal highly virulent L60. Further studies with this infection model, particularly future work on NK cells and cytotoxic T cells, may provide new insights on mechanisms of protective immunity to ASFV.
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Acknowledgments |
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Footnotes |
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c This paper is dedicated to the memory of José D. Vigário.
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References |
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Received 15 August 2000;
accepted 24 November 2000.