Abstract
The potential of a capture enzyme-linked immunosorbent assay (ELISA) specific for chicken interferon-γ (ChIFN-γ) has been evaluated as a tool to assess cell-mediated immunity (CMI) in the chicken. In a first step, ChIFN-γ production and cell proliferation of mitogen-activated chicken splenocytes have been compared. In general, for each of the stimulation conditions where significant proliferation was observed, production of ChIFN-γ could be measured by ELISA. In our hands, the combination of ionomycin and phorbol-12-myristate 13-acetate or the use of recombinant chicken interleukin-2 gave the most satisfactory results. Then, the CMI response induced by live or killed Newcastle disease virus (NDV) vaccines has been evaluated sequentially by ex vivo antigen-specific ChIFN-γ production and cell proliferation of splenocytes from immune chickens. The ex vivo data showed that both types of NDV vaccines are capable of stimulating CMI responses to NDV in chickens as measured by the ChIFN-γ ELISA. However, most of the chickens vaccinated with the live vaccine produced ChIFN-γ after antigen recall stimulation, from 2 to 4 weeks after vaccination, when only some chickens vaccinated with the inactivated vaccine showed a specific response 4 weeks after vaccination. No significant proliferative responses to either NDV vaccine were detectable during the 4 weeks of the study. From our results, it appears that antigen-specific ChIFN-γ production can be used as a good indicator of actively acquired immunity to NDV and that the sensitivity range of the capture ELISA test is well adequate to measure ex vivo release of ChIFN-γ.
Introduction
Birds can respond to vaccination by developing humoral and cellular immune responses (Sharma, Citation1999). Often, the success of vaccination in a flock is only monitored by demonstrating a rising antibody titer within a few days after vaccination, although cell-mediated immune (CMI) responses are also important in vaccine-induced protective immunity. Assay procedures for CMI assessment are cumbersome and not routinely used in the chicken. As in mammalian systems, several studies in poultry have shown that the measurement of chicken interferon-γ (ChIFN-γ) released by T cells after in vitro stimulation might be a good evaluation of CMI in the chicken after infection and vaccination (Prowse & Pallister, Citation1989; Martin et al., Citation1994; Karaca et al., Citation1996; Breed et al., Citation1997, Citation1999). Until now, the measurement of ChIFN-γ remained, however, limited by the lack of an adequate assay. This forced the use of biological assays mainly based on the ability of ChIFN-γ to activate the HD11 macrophage cell line, as measured by nitric oxide production (Karaca et al., Citation1996). This bioassay is time consuming and susceptible to the activity of other cytokines present in tested samples.
In other species, capture enzyme-linked immunosorbent assays (ELISAs) have been developed and have proven to be very reliable and simple ways to measure cytokine release after activation, and are therefore considered good indicators of CMI (Whiteside, Citation1994; Mateu de Antonio et al., Citation1998; Rothel et al., Citation1990). Moreover, this kind of test is polyvalent and should allow the measurement of CMI after mitogen or recall stimulation in a variety of diseases as well as after immunization, as it is specific for the released cytokine and not for the stimulating agent.
During recent years, several cytokines associated with CMI have been identified in the chicken, allowing the development of new immunological assays for this species (Digby & Lowenthal, Citation1995; Sundick & Gill-Dixon, Citation1997; Schneider et al., Citation2000). In this context, we have recently described the development of a monoclonal antibody (mAb)-based capture ELISA specific for ChIFN-γ with a sensitivity estimated to be down to 0.1 ng/ml ChIFN-γ (Lambrecht et al., Citation2000). The purpose of the study is to evaluate the use of this ChIFN-γ capture ELISA for the measurement of CMI in the chicken in response to ex vivo stimulation of splenocytes by mitogens or recall antigen after vaccination with attenuated or inactivated Newcastle disease virus (NDV).
Materials and Methods
Chickens
Specific pathogen free (SPF) chickens used in this study were hatched from eggs provided by Lohmann Valo (Cuxhaven, Germany) and were kept in isolators.
Vaccines
The lentogenic NDV strain La Sota was used for both vaccinating chickens and preparing the antigen (Goldhaft, Citation1980). Chickens received 106.3 median embryo infective doses/per dose of live vaccine via the oculo-nasal route. The oil–emulsion-inactivated Newcavac vaccine (Intervet) was administrated intramuscularly according to the manufacturer's instructions.
Mitogens, NDV recall antigen production and purification of chicken interleukin-2 produced in Escherichia coli
The mitogens Concanavalin A (ConA), phytohaemagglutinin (PHA), Pokeweed mitogen (PWM), phorbol 12-myristate 13-acetate (PMA) and Ionomycin were purchased from Sigma.
For the preparation of the recall antigen, a virus stock of the La Sota NDV strain was produced in 10-day-old embryonated eggs from SPF chickens and the allantoic fluid was harvested 5 days post-inoculation. The virus was purified by sucrose density gradient centrifugation (20 to 40% sucrose in 0.01 M Tris–HCl, 0.1 M NaCl, 0.001 M ethylenediamine tetraacetic acid, pH 7.4) at 50,000×g for 3 h. The virus band was collected, extensively dialyzed against phosphate buffer and the protein concentration determined by a protein assay reagent (Sigma). The NDV used as the recall antigen was heat-inactivated at 60°C for 1 h. For solubilization, the virus preparation (1 mg protein/ml) was disrupted in phosphate buffer containing octylglucoside (Sigma) at a final concentration of 2%. After a further 1 h incubation at room temperature, it was dialyzed against phosphate buffer. To purify the NDV envelope glycoproteins (gpNDV), the solubilized virus was centrifuged at 200,000×g for 45 min and the supernatant was dialyzed against phosphate buffer. The protein concentration of the solubilized virus and the purified glycoproteins were determined by protein assay reagent (Sigma).
The cDNA of mature chicken interleukin-2 (ChIL-2) was amplified by reverse transcriptase-polymerase chain reaction on total RNA extracted from ConA-stimulated chicken splenocytes, using specific primers based on the published sequence of cloned ChIL-2 (Sundick & Gill-Dixon, Citation1997). The cDNA was ligated to the prokaryotic expression vector ProEx HTb (Life Technologies) to give the ProEx HTb-ChIL-2 construct. The resulting recombinant protein contained six N-terminal histidine residues (His-ChIL-2) and was purified by affinity chromatography on a nickel-chelate agarose column followed by exchange chromatography, following the techniques described in Lambrecht et al. (Citation1999).
Lymphocyte proliferation assay
Spleen cell suspensions were prepared as described previously (Lambrecht et al., Citation1999). Splenocytes were adjusted to 107 cells/ml in RPMI 1640 (Invitrogen) and 100 μl cells per well were transferred into flat-bottomed 96-well plates. Equal volumes of medium with 2% of inactivated foetal calf serum containing mitogens, recombinant ChIL-2 (10 μg/ml) or specific NDV recall antigens (10, 1 or 0.1 μg/ml) were added in triplicate and cultures were incubated for 2 or 3 days. Negative controls received 100 μl RPMI 1640 medium only. After 60 h or 84 h of incubation, 100 μl cell supernatant were removed from each well for the measurement of ChIFN-γ production. Subsequently, to each well, 100 μl fresh RPMI 1640 medium containing 1 μCi [3H]thymidine (specific activity, 2.0 Ci/mmol; ICN) was added for a final incubation of 18 h. Cell proliferation was quantified by measuring [3H]TdR incorporation by scintillation counting. Proliferation responses were calculated as the mean of triplicate wells and expressed as a stimulation index that was calculated for each bird by dividing the counts per minute values of mitogen-activated splenocytes by the counts per minute values of non-activated splenocytes. The stimulation indices (SIs) per group were calculated as the arithmetic average stimulation index. A SI equal to or greater than 2 was considered evidence of significant proliferation (Emery et al., Citation1988).
ChIFN-γ ELISA assay
The ELISA was performed as described previously (Lambrecht et al., Citation2000) with several modifications. Briefly, microtiter plates were coated with mAb 1E12 diluted at 2 μg/ml in phosphate-buffered saline (PBS) for 1 h at 37°C. Plates were then blocked with PBS containing 2.5% Casein (Sigma) and incubated with non-diluted supernatant of stimulated splenocytes for 1 h at 37°C. The use of biotin-labeled mAb 7C4 (0.5 μg/ml) with horseradish peroxidase-labeled streptavidin (Biosource) as the detection reagent increased the sensitivity of the ELISA to picogram levels. This improved ELISA is now commercially available from Biosource Europe (catalog number #CAC1233).
Measurement of the humoral response by a haemagglutination inhibition test
The haemagglutination inhibition (HI) test was performed essentially as described elsewhere (Council of the European Communities, Citation1992). Briefly, 50 μl two-fold dilutions of sera in PBS were prepared in round-bottomed 96-well plates. Then, 50 μl of 4 haemagglutinin units of NDV were added to each well and incubated for 20 min at room temperature. Chicken red blood cells (0.75% in PBS) were added and incubated at 4°C for 1 h. The reciprocal of the last serum dilution showing an inhibition of haemagglutination was considered as the HI antibody titer of the serum. The HI geometric mean titers were expressed as reciprocal log2 .
Experimental design of the Ag recall trials
Experiment 1.
Groups of four 4-week-old SPF chickens were either vaccinated with the La Sota strain or used as unvaccinated controls. Three weeks after vaccination, splenocytes from each chicken were isolated for stimulation ex vivo. Different mitogen and NDV antigen concentrations were tested for their ability to stimulate the cells as measured by lymphocyte proliferation and ChIFN-γ production in the supernatant.
Experiment 2.
Groups of 16 4-week-old SPF chickens were immunized with either La Sota vaccine or inactivated NDV vaccine. One week after vaccination and on a weekly basis, four chickens per group were killed and their splenocytes were either grown in culture medium alone (negative control) or stimulated with PMA/ionomycin (0.1 μg/ml), ChIL-2 (10 μg/ml) (positive controls) or with different NDV antigen preparations. Stimulation of splenocytes was measured by lymphocyte proliferation and ChIFN-γ production. This experiment was repeated twice and each gave similar results.
Statistical analysis
Statistical analysis of the lymphocyte proliferation data was performed using the analysis of variance one-way test on log10-transformed SIs. For the ChIFN-γ production, a similar transformation was performed. Differences were considered as significant at P<0.05.
Results
Comparison of ChIFN-γ production and proliferation of splenocytes in response to mitogen stimulation
Different concentrations of mitogens (ConA, PHA, PWM or PMA/ionomycin in combination) were tested for their ability to activate splenocytes from 28 4-week-old SPF chickens. The same cultures were evaluated for proliferation and for ChIFN-γ production. The mean SIs and ChIFN-γ levels, expressed as optical density values, are presented in . The highest and consistent response after mitogen stimulation was observed with the combination of PMA and ionomycin (0.1 μg/ml), whereas stimulation with ConA, PHA and PWM were lower and/or showed individual variations in the responses regarding both proliferation and ChIFN-γ levels, as shown by high standard deviation values. Recombinant ChIL-2 (10 μg/ml) was also tested for its potential as an immune stimulator and was able to activate splenocytes from all chickens tested. In a preliminary experiment, a dose response for ChIL-2 had been determined with a dose range of 50, 10, 1 and 0.1 μg/ml. A concentration of 10 μg/ml appeared to be the optimal concentration for the highest ChIFN-γ production and proliferation without non-specific response, as demonstrated by the absence of splenocyte stimulation in the presence of an irrelevant E. coli recombinant protein, VPx (data not shown).
Table 1. Mitogenic response of splenocytes of 4-week-old SPF chickens (n=28) stimulated with different concentrations of several mitogens and ChIL-2
In general, there was no significant correlation between proliferation and ChIFN-γ levels measured after splenocyte stimulation with the different mitogens. The best example is the pokeweed mitogen stimulation, which induced a good ChIFN-γ production but a lower proliferation of splenocytes than the other mitogen stimulations. When the data of individual chickens were analyzed, the splenocyte cultures with the highest lymphoproliferative responses did not necessarily show the highest level of ChIFN-γ production and vice versa, except following activation with ChIL-2 where a good correlation could be observed. The R 2 value of ChIL-2 activation was 0.56.
Determination of the optimal conditions for ChIFN-γ production by immune cells after specific NDV recall stimulation
As a first step, splenocytes from 4-week-old NDV-immunized chickens and negative chickens were examined 3 weeks after live vaccination for their ability to produce ChIFN-γ upon ex vivo stimulation (). Splenocytes from vaccinated and negative chickens produced ChIFN-γ 48 or 72 h after PMA/ionomycin and ChIL-2 stimulation as detected by specific ELISA. Only splenocytes from vaccinated chickens produced ChIFN-γ after NDV-specific antigen recall stimulation with dissociated NDV proteins as well as with purified gpNDV. This production was more pronounced after 72 h although the increase was not statistically significant. No stimulating effect was observed with the purified/inactivated virus used as recall antigen. In non-immunized control chickens, there was no significant difference in ChIFN-γ production between the antigen-stimulated and unstimulated cultures.
Figure 1. Optimization of the type of recall antigen (at 1 μg/ml) and the time of the harvest for antigen-specific ChIFN-γ production of splenocytes from immune and negative chickens. Splenocytes were stimulated with a combination of PMA/ionomycin (PMA/Iono, 0.1 μg/ml), recombinant ChIL-2 (rChIL2, 10 μg/ml), inactivated and purified NDV (inactivated NDV), dissociated NDV proteins (All prot. NDV) or purified NDV envelope glycoproteins (gpNDV), and supernatants of stimulated splenocytes were harvested after 48 or 72 h of activation. ChIFN-γ production was determined by the ChIFN-γ capture ELISA. O.D., optical density.
In a parallel experiment, the mixture of all purified NDV proteins as well as the purified gpNDV were tested at 0.1, 1 and 10 μg/ml in order to determine the optimal concentration of NDV antigen necessary for ChIFN-γ production ex vivo (). A concentration of 1 μg/ml of the mixture of NDV proteins or gpNDV appeared the optimal concentration for the highest ChIFN-γ production and did not yield non-specific response. The magnitude of NDV-specific ChIFN-γ production by splenocytes stimulated with both NDV antigens was comparable. Therefore, 1 μg/ml purified NDV glycoproteins was used as recall antigen throughout this study, and the future timing for stimulation was fixed as 72 h.
Figure 2. Determination of the optimal concentration of antigen for the specific recall stimulation. Splenocytes were stimulated with dissociated NDV proteins (All prot. NDV) or purified NDV glycoproteins (gpNDV) used at different concentrations (μg/ml), and supernatants of stimulated splenocytes were harvested after 72 h of activation. O.D., optical density.
Comparison of the CMI responses after attenuated or inactivated NDV vaccines
Two groups of chickens were primed with either live or inactivated NDV vaccine. A third group was used as unvaccinated control. One week after immunization, and on a weekly basis, four chickens per group were killed. Splenocytes from all three experimental groups exhibited high reactivity without significant differences upon stimulation with PMA/ionomycin and with ChIL-2, in terms of both splenocyte proliferation and ChIFN-γ production during the 4 weeks of the experiment (data not shown). After NDV recall stimulation, no specific proliferation could be observed in the NDV vaccinated groups. The mean stimulation indices are presented in . Only splenocytes from two birds vaccinated with the attenuated vaccine presented proliferative responses in the presence of recall antigen, and this after 3 and 4 weeks of vaccination. However, these responses were not statistically significant ().
Table 2. NDV-specific cell-mediated and humoral immune responses following a vaccination with live or killed NDV
ChIFN-γ production was measured from supernatants of NDV antigen-stimulated splenocytes by ELISA (). The levels of ChIFN-γ observed in NDV-stimulated splenocytes from negative chickens were as low as those of unstimulated controls over the 4 weeks of the study. The ex vivo data showed that both type of vaccines are able to induce the production of significant levels of ChIFN-γ after NDV recall stimulation. Moreover, earlier and stronger primary responses were observed after live vaccination compared with the inactivated vaccine. Indeed, from 2 weeks after live vaccination, splenocytes from three out of four chickens produced more ChIFN-γ than the background level. On the contrary, splenocytes from two out of four chickens vaccinated with the inactivated NDV showed a NDV-specific ChIFN-γ production, only 4 weeks after vaccination. There was a significant difference (P<0.05) in NDV-specific ChIFN-γ production between the live and killed vaccinated groups. A correlation between proliferation and ChIFN-γ production was impossible to establish due to the very low proliferative responses.
During the 4 weeks of this experiment, sera from NDV-vaccinated and negative chickens were also taken on a weekly basis and specific NDV antibody titers were determined by HI test in order to monitor the humoral response against NDV (). A humoral response was detected as early as 1 week after live vaccination but not until 2 weeks after inactivated vaccination. After 4 weeks, however, live vaccine elicited lower titers than those observed in the group receiving inactivated vaccine. There was no close correlation between CMI values (ChIFN-γ production) and HI titers.
Discussion
CMI is a major protective mechanism against viral infection (Zinkernagel, Citation1996). A classical tool for the evaluation of CMI consists of measuring lymphocyte proliferation upon recall antigen stimulation (Giambrone et al., Citation1980; Karaca et al., Citation1996; Thiagarajan et al., Citation1999; Reynolds & Maraqa, Citation2000b; Alvarez et al., Citation2003). During this recall stimulation, cytokines such as IFN-γ are produced by activated responder T cells. The applicability of this system to chickens has been investigated in coccidiosis, where the functional activity of chicken Peripheral Blood Lymphocytes (PBL) upon recall stimulation with Eimeria tenella sporozoites was demonstrated by determining both their potential to proliferate and to produce ChIFN-γ, as measured by bioassay (Breed et al., Citation1997, Citation1999). We have developed a ChIFN-γ capture ELISA that presents a higher specificity and sensitivity than the available bioassay (Lambrecht et al., Citation2000). This ELISA allows the detection of as little as 50 pg/ml ChIFN-γ, a range that should be adequate to measure the production of ChIFN-γ in response to viral recall antigens or mitogen stimulation.
As a first step, the usefulness of the ChIFN-γ ELISA as an alternative to the proliferation test was examined after mitogen stimulation of chicken splenocytes. For each condition where significant proliferation was observed, significant production of ChIFN-γ could be observed. However, we also observed a lack of close correlation between the intensity of the proliferative responses to most mitogens and the levels of ChIFN-γ in respective cultures. These results are not really surprising considering that proliferative T cells do not necessarily produce ChIFN-γ and vice versa. In addition, in cases where very high concentrations of mitogens are used, the cells are probably dead by using up all the culture medium, when the cell proliferation is measured. This could explain the low SI at ConA concentrations of 40 μg/ml despite high ChIFN-γ production.
In our hands, the combination of PMA with ionomycin gave the most consistent cell activation, confirming previously results showing that PMA is mitogenic for T cells as well as for B cells in birds (McNeilly et al., Citation1999). In mammalian systems, PWM is reported to be mainly a B-cell mitogen (Peacock et al., Citation1990). We have showed that PWM is able to induce IFN-γ production by chicken splenocytes and appears as a mixture of both T-cell and B-cell mitogen, as already observed (Vainio & Ratcliffe, Citation1984; Lawson et al., Citation2001). Further research into identifying the phenotype of lymphocytes, which produce specifically ChIFN-γ following PWM and other mitogen stimulations, should be undertaken by double labeling and FACS analysis.
We have also observed that recombinant ChIL-2 stimulates T cells in the spleen, albeit less efficiently, suggesting that post-translational modifications (such as N-linked glycosylation) are not essential for bioactivity in contrast to earlier observations reported by Lillehoj et al. (Citation2001). As it is less damaging for T cells than activation with PMA/ionomycin (data not shown) and induces well-correlated activation (ChIFN-γ) and proliferation, this type of physiologic activation might prove very suitable in the future as a positive control for activation.
NDV is an avian paramyxovirus that causes an economically important and highly contagious disease of poultry (reviewed by Seal et al., Citation2000; Al-Garib et al., Citation2003). Numerous commercial NDV vaccines, live attenuated or inactivated, provide satisfactory protection against the disease. The presence of NDV neutralizing and HI antibodies is necessary to provide long-term protection against Newcastle disease (Reynolds & Maraqa, Citation2000a). But currently little is known about the nature and role of NDV-specific cell-mediated responses to NDV in the chicken, especially with regards to the ability of different types of vaccines to stimulate such responses. Although specific CMI to NDV by itself is not sufficient to protect against virulent NDV challenge (Reynolds & Maraqa, Citation2000b), T cells but not B cells may therefore be essential for virus clearance, and principally CD8+ cytotoxic T cells may be key players in vaccinal immunity to NDV (Russell et al., Citation1997). Cytotoxic T cells were detected in the spleen of vaccinated chickens after in vitro re-stimulation (Cannon & Russell, Citation1986) or in chickens vaccinated twice or vaccinated and challenged with virulent virus (Jeurissen et al., Citation2000).
To our knowledge, the primary CMI response after vaccination with Newcastle disease vaccine has not been assessed previously by the measurement of ChIFN-γ by ELISA after antigen recall stimulation of immune splenocytes. Since NDV is known to cause agglutination and lysis of lymphocytes (Thiagarajan et al., Citation1999), the La Sota strain used as recall antigen was heat-inactivated prior to the stimulation experiments. However, no specific stimulation could be observed with this type of recall antigen. In the presence of gpNDV or a mixture of all NDV proteins, however, splenocytes from vaccinated chickens produced ChIFN-γ. The F and HN surface glycoproteins, the principal antigens eliciting a protective immune response (Meulemans et al., Citation1986; Alexander, Citation1997), thus seem sufficient to induce specific recall stimulation. This ChIFN-γ production was specific, as demonstrated by the lack of splenocyte stimulation in the presence of an irrelevant antigen, namely inactivated and purified infectious bursal disease virus (data not shown).
Most of the chickens vaccinated with the live NDV vaccine produced ChIFN-γ after recall stimulation, and this from 2 to 4 weeks after vaccination. With the inactivated NDV vaccine, however, one-half of the chickens showed a significant response and this only at 4 weeks after vaccination. These results are in agreement with numerous data demonstrating that vaccines containing live agents are generally superior to those with inactivated agents with respect to the induction of cell-mediated responses to disease-causing agents (Timms & Bracewell, Citation1983; Tizard, Citation1987). However, we cannot exclude that the difference between CMI induced by killed or live vaccine may be the result of distinct kinetics; in other words, the CMI induced by killed vaccine could increase more slowly.
Although the proliferation response to polyclonal T-cell activation by mitogens was satisfactory, NDV-specific proliferation upon recall stimulation of the vaccinated groups was low and unreliable, as already observed (Giambrone et al., Citation1980; Karaca et al., Citation1996). The discrepancy between proliferation and ChIFN-γ production after an ex vivo NDV stimulation might be attributed to the functional status of the responder cells or to the timing of the proliferation test. The majority of splenocytes from the vaccinated and control animals responded vigorously to PMA/ionomycin. The NDV-primed cell population did not allow further ex vivo proliferation following a specific recall stimulation, but might still contribute to the observed ChIFN-γ levels. Using duck hepatitis B virus for recall stimulation, Vickery et al. (Citation1997) reported a maximal antigen-specific proliferation of duck splenocytes between 4 and 7 days after establishment of the culture. We have tested different incubation timings in the cell proliferation assay (2, 3, 4 and 7 days) and no significant proliferation was observed after ex vivo NDV stimulation (data not shown). NDV-specific proliferative responses of PBLs from NDV-vaccinated chickens, using an inactivated NDV as recall antigen, have been reported (Katz et al., Citation1993; Reynolds & Maraqa, Citation2000b). These different results might be explained by differences in the vaccination schedule. In our experimental design CMI was evaluated after a single vaccination, whereas in the other study the proliferation tests were performed on PBLs from NDV-vaccinated and revaccinated chickens. This revaccination could perhaps boost the expansion of antigen-specific T-cell clones allowing CMI measurement after recall stimulation by the proliferation test.
Finally, no close correlation between CMI values (ChIFN-γ production) and humoral response (HI titers) could be demonstrated after NDV vaccination, corroborating results obtained by other workers after NDV vaccination where the CMI was determined by the leukocyte-migration inhibition technique (Timms & Alexander, Citation1977; Agrawal & Reynolds, Citation1991) or after La Sota live vaccination of turkeys (Ghumman & Bankowski, Citation1976). However, these studies indicated that CMI developed more rapidly than the serum antibody response, a feature not confirmed in the present study.
In conclusion, our results indicate that the sensitivity range of the capture ELISA appears adequate to measure the ex vivo release of ChIFN-γ by T cells in response to mitogens and specific recall antigen. The ChIFN-γ test can thus be considered as an effective alternative for the measurement of CMI in birds. The ChIFN-γ ELISA has great potential for measuring the role of CMI in protection against avian infectious diseases in the future and will facilitate the study of the role of ChIFN-γ in various immune mechanisms in the chicken.
Acknowledgements
The authors would like to thank Jan Mast for advice and a critical reading of the manuscript, Jean-François Toussaint for the statistical analysis and Roel Schuurmans for technical assistance.
Related Research Data
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