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First published online April 17, 2009
Journal of Experimental Biology 212, 1336-1343 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.028449
Dynamics of PHA-induced immune response and plasma carotenoids in birds: should we have a closer look?
1 Equipe Ecologie Evolutive, UMR 5561 Biogéosciences, Université
de Bourgogne, 6 Bd Gabriel, F-21000 Dijon, France
2 Konrad Lorenz Institute for Ethology, Austrian Academy of Sciences,
Savoyenstraße 1a, A-1160 Wien, Austria
* Author for correspondence (e-mail: c.biard{at}klivv.oeaw.ac.at)
Accepted 22 February 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: bill colour, blackbird, carotenoid-based signals, cell-mediated immune response, immunocompetence
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Schmid-Hempel, 2003). One such
trade-off based on the allocation of limited resources to immunity or other
competing functions is that involving carotenoids. Carotenoids are important
immune-modulators and part of the antioxidant system (reviewed by
Bendich, 1993;
Edge et al., 1997;
Møller et al., 2000;
Surai, 2002). These pigments
are also potentially limiting, so that carotenoids allocated to immune
function might be drawn out of the pool available for developing
carotenoid-based signals honestly revealing the quality of their bearer
(Lozano, 1994;
Shykoff and Widmer, 1996)
(reviewed by Møller et al.,
2000). Indeed, immune activation with various antigens eliciting
inflammatory and/or humoral immune response has generally been reported to
have negative effects on circulating carotenoids, quickly reducing their
levels (Koutsos et al., 2003;
McGraw and Ardia, 2003;
Alonso-Alvarez et al., 2004;
Peters et al., 2004) [but see
Costantini and Dell'Omo and Hõrak et al.
(Costantini and Dell'Omo, 2006;
Hõrak et al., 2006)].
In addition, carotenoid-based bill colour relying on constant pigment
deposition was shown to be negatively affected by immune responses induced by
controlled immune challenge (Faivre et
al., 2003a; Alonso-Alvarez et
al., 2004; Peters et al.,
2004; Gautier et al.,
2008).
One widely used immune challenge in studies of immunocompetence in birds is
the phytohemagglutinin (PHA)-induced skin swelling test, in which this plant
lectin is injected subcutaneously. PHA first induces an acute response 4 h
after injection, primarily characterised by oedema. Then a delayed-type
hypersensitivity response is induced through stimulating heterophil, basophil,
eosinophil, macrophage and thrombocyte cell infiltration in the dermis and
dense perivascular infiltration of T-lymphocytes at the site of injection
(Sharma, 1990;
Parmentier et al., 1998;
Martin et al., 2006). This
late response generally peaks 18 h after injection and may last up to 36 h.
This test therefore reflects the combined responses of T-cells, cytokines and
inflammatory cells (Davison et al.,
1996) and involves both innate and adaptive components of the
immune system (Martin et al.,
2006; Tella et al.,
2008). An index of the immune reaction is given by the resulting
skin swelling at the site of injection, which is generally measured after 24 h
(Martin et al., 2006).
PHA-induced immune response has been shown to trade-off with other functions
and to correlate with individual condition and quality (reviewed by
Martin et al., 2006). However,
the use of the PHA test and its interpretation in terms of immunocompetence is
currently being questioned, at least until we have gained a better knowledge
of its relationship with parasite resistance and also of the induced immune
response and physiological consequences
(Kennedy and Nager, 2006;
Owen and Clayton, 2007).
Accordingly, recent studies in immuno-ecology have not only measured PHA
response but at the same time several physiological parameters that might
reveal its functional significance and potential costs, such as oxidative
stress and associated damages (Costantini
and Dell'Omo, 2006;
Hõrak et al., 2007;
Pérez-Rodríguez et al.,
2008).
However, another aspect of the immune response to PHA that has been
relatively neglected until now, despite its biological relevance, is its
temporal dynamics. Individuals may not only vary in the maximum response
attained (peak response) but also in the latency to reach it and/or in how
long they maintain the immune response. This may have consequences for the
individual in terms of parasite resistance and fitness. All three components
of the immune response might trade-off against each other, be subjected to
different constraints and induce different benefits and costs for the
individual. Another source of variation among individuals in immune response
is senescence (Haussmann et al.,
2005; Palacios et al.,
2007). For example, in house sparrows Passer domesticus,
sex and age influenced the number of immune cells recruited at the site of PHA
injection (Martin et al.,
2006), and stronger immune responses took longer to develop than
weaker ones (Navarro et al.,
2003). Experimentally increased levels of testosterone compromised
the ability to maintain PHA response and humoral immune response to sheep red
blood cells compared with controls in house finches Carpodacus
mexicanus (Deviche and Cortez,
2005).
In line with the recent calls for a more detailed description of the
PHA-induced immune response in order to refine its interpretation
(Kennedy and Nager, 2006;
Owen and Clayton, 2007), we
investigated the temporal dynamics of PHA-induced immune response and a
potential associated change in blood carotenoids, body mass and a
carotenoid-based coloured signal. The underlying question being whether there
was individual variation in response patterns and whether this variation might
be related to physiological or morphological parameters. We chose the
blackbird Turdus merula Linnaeus 1758 as a model species, as males in
this sexually dimorphic species exhibit yellow to orange carotenoid-based bill
colour, which has been previously shown to positively correlate with the
intensity of PHA response and to reveal the current state of activation of the
immune system (Faivre et al.,
2003a; Faivre et al.,
2003b). We first wanted to follow in detail the dynamics of the
immune response to determine whether there would be more information to be
gained from using indices of immune response later than 24 h after injection.
Another aim of this study was to investigate the relationship between plasma
carotenoids and immune response to PHA. In particular we wanted to determine
whether basal carotenoid levels measured before immune challenge influenced
the immune response. We might indeed expect a positive effect as increased
carotenoid levels after dietary supplementation have been shown to result in
higher immune response to PHA (Blount et
al., 2003; McGraw and Ardia,
2003). In addition, if carotenoid levels were found to change
following the immune challenge, we wanted to test whether relative changes in
carotenoids were associated with the dynamics of the immune response. Lastly,
we monitored changes in bill colour to assess its potential as a dynamic
signal of quality as reflected by immune status. We expected bill colour to be
correlated to plasma carotenoids and to predict the immune response. If bill
colour were a rapidly variable trait we would also expect a parallel change in
colour with any change in plasma carotenoids. Bill colour has been shown to
decrease as quickly as within one week of immune challenge, probably following
a decrease in plasma carotenoids (Faivre
et al., 2003a; Peters et al.,
2004). However, an increase in bill colour following an increase
in plasma carotenoids has been shown after long-term (four weeks) dietary
supplementation experiments, although detailed timing of these colour changes
were not reported (Blount et al.,
2003; McGraw and Ardia,
2003; Alonso-Alvarez et al.,
2004).
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Immune-challenged blackbirds were injected with 100 µl of 10 mg
ml–1 PHA (PHA-P, Sigma-Aldrich, Lyon, France) dissolved in
PBS in the centre of right wing web (patagium)
(Smits et al., 1999;
Faivre et al., 2003b).
Blackbirds of the control group were injected with 100 µl of PBS. All
individuals were injected on 17 July in the morning between 08:00 h and 12:00
h.
We measured the thickness of the patagium at the injection site just before
injection (thereafter day 0) and 1, 2, 4, 7, 9 and 11 days after injection,
using a pressure-sensitive spessimeter with an accuracy of 0.01 mm (Teclock
SM-112, Alpa SpA, Milano, Italy). All captures and bird manipulations were
performed at the same time of the day (i.e. between 08:00 h and 12:00 h) and
in the same order between aviaries. This ensured that the same time elapsed
between subsequent captures for all birds and that measurements for each bird
were spaced by 24 h (or a multiple of 24 h) ±1 h. In order to limit the
number of captures and associated stress, we planned not to measure skin
thickness after 12 h and to measure thickness every two days after the
measurements at 24 h and 48 h. However, on day 4 birds showed signs of stress
(reduced activity) and measurements planned on day 6 were then delayed until
day 7 (as birds then behaved normally). An assistant restrained the bird while
the micrometer was placed over the injection site. Each wing was measured in
duplicate. The repeatability of measurements was high and significant
(intra-class correlation coefficient r=0.99, P<0.0001)
(Lessells and Boag, 1987), and
we subsequently used mean swelling in the analyses. Body mass was recorded
using an electronic balance (Scout Pro SPU 202, Ohaus Corp., Pine Brook, NJ,
USA) to the nearest 0.1 g on all occasions. After measuring the wing web
thickness, a blood sample was collected from the brachial vein in a
heparinised capillary. Blood was immediately centrifuged
(1073g, 4°C, 15 min) and the plasma was stored in 1.5 ml
Eppendorf tubes at –80°C for later analysis.
Bill colour was scored on the first and last capture. Colour was assessed
by visual comparison with a Roche Yolk Colour Fan (Roche, Neuilly-sur-Seine,
France). The colour scores, ranging from index 1 (pale yellow) to index 15
(orange), are characterised by tristimulus values of the
CIE–1931-standard colorimetric system and have been shown to be
consistently correlated to colour parameters obtained with a spectroradiometer
(Faivre et al., 2003b). Scores
were always given by the same observer (J.M.) under the same light conditions
blindly with respect to treatment group. Scorings were highly repeatable (see
Baeta et al., 2008). Observed
bill colour scores ranged from index 8 to index 11 with 0.5 intervals.
Blackbirds were immediately placed back in their aviary after measurements and blood sampling, i.e. within 5 min.
Determination of plasma carotenoid concentration
Plasma samples (30µl) were first mixed with 60µl ethanol, followed by
antioxidants being extracted twice with 500 µl hexane. Hexane extracts were
pooled and evaporated at ambient temperature under nitrogen flow and the
residue was dissolved in 150 µl ethanol and kept on ice. The optical
density of 100µl of the re-suspended samples was read in 96-wells
Microtitration plates (Sterilin, London, UK) at 450 nm with a
spectrophotometer (Versamax, Molecular Devices, Sunnyvale, CA, USA). A
standard solution of lutein, starting at 20 ng µl–1 and
subsequently serially diluted in ethanol six times to 0.31 ng
µl–1, with pure ethanol as the blank standard was run in
duplicate in each plate. Lutein standard was obtained from
Extrasynthèse (Genay, France). Solvents used for extraction and
colorimetry contained 0.01% of 2,6-di-tert-butyl-p-cresol (Fluka Chemika,
Buchs, Switzerland) as an antioxidant. Repeatability of optical density
readings was assessed from the standards; mean intra-plate coefficient of
variation ± s.e.m. was 2.28±0.45% and mean inter-plate
coefficient of variation ± s.e.m. was 2.77±0.53% (N=5
plates). Carotenoid concentration in the samples was determined using the mean
curve of the ten standard curves. Repeatability of concentrations was assessed
from the standards; mean intra-plate coefficient of variation ± s.e.m.
was 3.78±0.95% and mean inter-plate coefficient of variation ±
s.e.m. was 4.72±1.23%.
Statistical analysis
All statistical analyses were conducted using SAS v.8.2 (SAS Institute
1999–2001, Cary, NC, USA). Tests of residuals for normality
(Shapiro–Wilk) and homoscedasticity were used to check the validity of
the model. Repeated-measures analysis of variance (ANOVA) was used to test for
differences in changes in wing web thickness, body mass and plasma carotenoids
between immune-challenged and control birds over the course of the experiment,
i.e. on the seven capture occasions. Repeated-measures analysis of covariance
(ANCOVA) was also used to investigate the sources of variation in immune
response on day 1, using wing web thickness on days 0 and 1 as dependent
variables. In the same way, variation in the pattern of decrease in immune
response was analysed with repeated-measures ANCOVA, using wing web thickness
on days 1, 2 and 4 as dependent variables. When investigating variation in
wing web thickness with repeated-measure models, we specified a contrast with
the first (initial or earliest) measurement in order to control for initial
wing web thickness. Maximum variation in plasma carotenoid concentration
between day 0 and day 2 was calculated as the minimum value attained on day 1
or day 2 minus the initial value on day 0. Preliminary analyses showed no
significant aviary effect and these were therefore not included in the models.
Tarsus length was first entered as a covariate in all models to control for
any confounding effect of body size. Tarsus length was never statistically
significant and was thus not retained in the final versions of the analyses.
Values are given as means ± s.e.m.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Body mass
All birds showed a transient mass loss during the course of the experiment,
decreasing after injection until day 2 then increasing back to initial levels
by day 7 (Fig. 1B). Compared
with day 0, birds weigh significantly less on days 1, 2 and 4 (all contrasts
with day 0: F1,49>12.91, P<0.001) but not
on day 7 (contrast with day 0: F1,49=0.14,
P=0.71). This variation in body mass was similar in PHA-injected and
control birds (Table 1).
Immune response on day 1 was not significantly related to initial body mass (repeated-measures ANCOVA, interaction time x initial body mass, F1,29=0.04, P=0.84).
The decrease in swelling between days 1 and 4 was not significantly related to initial body mass (repeated-measures ANCOVA, interaction time x initial body mass, F2,28=1.80, P=0.18).
Plasma carotenoids
Immune-challenged birds showed a significant decrease in plasma carotenoid
concentration compared with control birds on days 1 and 2
(Table 1;
Fig. 1C). Plasma carotenoid
levels of immune-challenged birds increased back to initial levels from day 2
to day 4, when they were not significantly different from that of control
birds anymore (Table 1;
Fig. 1C). Among PHA-injected
birds, mean (±s.e.m.) maximum change in circulating carotenoids was
–34.2±4.3% of the initial carotenoid levels (range –96.0%
to +18.6%). The maximum change in plasma carotenoid concentration between day
0 and day 2 was negatively related to initial carotenoid concentration; the
higher the initial carotenoid levels on day 0, the greater the decrease in
plasma carotenoid concentration (Fig.
3). An index of relative maximum change in carotenoid
concentration independent of initial level was calculated as the residuals of
this regression. We used this index to test whether changes in circulating
carotenoids were associated with the immune response.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Hõrak et al., 2007;
Pérez-Rodríguez et al.,
2008; Tella et al.,
2008). In this study, we investigated the temporal dynamics of
PHA-induced immune response and potential associated changes in blood
carotenoids, body mass and a carotenoid-based coloured signal.
The peak of the immune response to PHA was reached within 24 h after
injection, except in two cases (see also
Ewenson et al., 2003;
Martin et al., 2003;
Navarro et al., 2003). Two
contrasting temporal patterns in the subsequent decrease in wing web swelling
were detected. The majority of birds showed a steep decrease back to initial
thickness within the next 24 h, while for ca. 30% of birds, wing web
thickness decreased slowly until day 7, suggesting that in this last case
immune response and/or inflammation at the site of injection was maintained
for longer. Wing web swelling after PHA injection has indeed been found to
reflect the local inflammatory response
(Martin et al., 2006;
Tella et al., 2008) as well as
the activation of the T-cell-mediated immune system through changes in the
amount of circulating T-lymphocyte subsets
(Tella et al., 2008). To our
knowledge, such individual variation in the duration of the swelling has not
been studied previously, although it is relevant to the issue of
immunocompetence and associated trade-offs. However, Navarro and colleagues
(Navarro et al., 2003)
investigated individual variation in latency to maximum immune response in the
house sparrow and showed that stronger immune responses took longer to
develop. They focused on the first 72 h after injection during which responses
remained relatively stable after the peak was reached. An immune response
maintained for longer might be adaptive if more efficient but might also be
more costly in terms of energy or self-damage
(Schmid-Hempel, 2003).
Alternatively, it might not be adaptive and might result from a deficient
control of the immune system, for example, in the case of unrestrained
inflammatory activity. The type of immune response developed by an individual,
i.e. fast or long-lasting, may be linked to the current trade-offs it faces
[e.g. reproductive state and moult
(Greenman et al., 2005);
hormonal status (Deviche and Cortez,
2005)], and/or to constraints linked to the development of the
immune system and environmental effects (see
Martin et al., 2004).
Understanding the proximate causes of individual variation in immune response
patterns would probably be insightful if we are to interpret immune response
to PHA in terms of immunocompetence and resistance to parasites. We therefore
attempted to explain both peak intensity and decrease pattern of the swelling
in relation to body mass, as an index of general body condition, and plasma
carotenoids because of their role as immunostimulants and in the antioxidant
system.
All birds experienced a transient body mass loss over the course of the
experiment. This variation in body mass probably reflected the stress of the
first captures and handling. However, the birds relatively quickly overcame
this stress as body mass increased again between days 4 and 7 back to initial
values. The absence of a significant difference between PHA and control birds
suggests that the immune challenge did not cause any detectable energetic
stress [consistent with Martin et al. and Greenman et al.
(Martin et al., 2003;
Greenman et al., 2005)].
Initial body mass did not influence the intensity of the immune response,
which seems to be generally the case in adult birds
(Alonso-Alvarez and Tella,
2001; Martin et al.,
2003; Haussmann et al.,
2005; Tella et al.,
2008) (but see Navarro et al.,
2003), or the pattern of decrease in wing web swelling. The birds
were maintained for several months in captivity before this experiment and
were provided with food ad libidum. Differences in body condition
among birds may therefore have been too small to induce any differences in
immune response (Alonso-Alvarez and Tella,
2001) and, conversely, PHA-induced immune response was not costly
in terms of body mass.
Plasma carotenoid levels decreased in immune-challenged birds and this
decrease followed a pattern that closely matched that of wing web swelling.
This is the first study showing such inversely matched dynamics of circulating
carotenoids with that of the immune response, indicative of a clear causal
relationship. The two previous studies monitoring plasma carotenoid levels
after PHA injection in captive birds reported contrasting results
(Pérez-Rodríguez et al.,
2008; Hõrak et al.,
2007). A decrease in plasma carotenoids, although proportionally
less important, was also observed in red-legged partridges Alectoris
rufa one day after injection
(Pérez-Rodríguez et al.,
2008). No change in plasma carotenoids was detected in
greenfinches Carduelis chloris three days after injection
(Hõrak et al., 2007) at
a time when, in our experiment, circulating carotenoids were increasing back
to their initial level again. This suggests that in order to draw reliable
conclusions on variation in carotenoid levels following immune challenge we
should explore a temporal window rather than relying on single
measurements.
Contrary to our expectations, there was a negative effect of initial plasma
carotenoid concentration on the magnitude (peak) of the immune response; wing
web swelling on day 1 was less important in individuals with high plasma
carotenoid levels before injection (Fig.
4A). However, previous studies on the relationship between basal
carotenoid levels and the intensity of the PHA-induced immune response
reported different results; no relationship was found in greenfinches
(Hõrak et al., 2007)
and a positive result was found in partridges
(Pérez-Rodríguez et al.,
2008) (see also Blas et al.,
2006). These mixed results might suggest either a
species-dependent or condition-dependent effect of carotenoids on immune
response or differences in food nutritional quality among studies. Plasma
carotenoid levels may exert a dose-dependent effect on the immune response,
which might vary according to species and dietary availability of carotenoids
in natural conditions.
By contrast, we found a positive effect of the relative decrease in plasma
carotenoids on the strength of the immune response. To our knowledge, only one
previous study explicitly tested for a relationship between a change in plasma
carotenoids and the intensity of PHA-induced immune response; in zebra finches
Taeniopygia guttata, carotenoid levels decreased in experimental,
testosterone-implanted birds, mounting a stronger immune response whereas this
relationship was reversed in control birds
(McGraw and Ardia, 2007).
Likewise, in our study, individuals whose plasma carotenoid levels decreased
relatively more after injection showed a greater wing web swelling on day 1.
It still remains to be determined however whether this relationship is direct
or indirect, i.e. whether carotenoids were involved in the development of the
immune response or in other physiological processes activated as a consequence
of the immune response. The capacity to mobilise plasma carotenoids may
positively affect the magnitude of the immune response or a strong immune
response may induce a more important decrease in plasma carotenoids. These two
hypotheses linking variation in plasma carotenoids and the immune response in
a direct and indirect way are not mutually exclusive. Carotenoids exert
important immunomodulatory functions that might be especially important in a
PHA response, such as stimulating the production of cytokines and the activity
of immune cells (reviewed by Chew,
1993; Møller et al.,
2000), as well as regulating inflammation
(Rafi and Shafaie, 2007;
Kim et al., 2008). Changes in
plasma carotenoid levels might therefore directly reflect their rapid
re-allocation to immune tissues or cells
(Koutsos et al., 2003).
Carotenoids are part of the integrated antioxidant system in birds
(Surai, 2002;
Ewen et al., 2006;
Costantini, 2008). Indeed,
although the relative contribution of carotenoids to total antioxidant
activity has been recently shown to be probably low and might vary among
species (Costantini and Møller,
2008), carotenoids are involved in the antioxidant system of birds
in many ways, not necessarily implying a direct contribution to antioxidant
capacity (e.g. Costantini,
2008). For example, carotenoids exert a protective and recycling
role for antioxidants like vitamins E and A (e.g.
Edge et al., 1997;
Surai and Speake, 1998;
Surai et al., 2001;
Surai, 2002). Changes in
carotenoid levels might therefore directly or indirectly reflect short-term
changes in oxidative status
(Pérez-Rodríguez et al.,
2008) due to the immune response that induces an increase in
oxidative stress and associated damages
(Costantini and Dell'Omo, 2006;
Hõrak et al., 2007) and
may also increase metabolic rate (Martin
et al., 2003; Lee et al.,
2005; Nilsson et al.,
2007).
Individual differences in the maintenance of the skin swelling could also
be partly explained by plasma carotenoids. The relative decrease in plasma
carotenoids during the early immune response did not affect swelling duration.
However, high initial carotenoid levels were associated with a slower decrease
in wing web swelling in the late part of the PHA skin response: between days 2
and 4. Previous studies investigating the variation in physiological
parameters associated with the immune response to PHA have yielded complex
results on changes in oxidative stress, antioxidant status and lipid
peroxidation and on when such changes occur during the response
(Costantini and Dell'Omo, 2006;
Hõrak et al., 2007;
Pérez-Rodríguez et al.,
2008). Plasma carotenoids, antioxidant capacity, oxidative stress
and consecutive damages might influence the immune and inflammatory responses
and, in particular, their dynamics might be linked. It would therefore be
insightful to examine their potential simultaneous influence on the pattern of
decrease of the swelling.
There was thus a contradictory dual effect of carotenoids. On the one hand, high plasma levels were detrimental to the development of the immune response. On the other hand, the decrease in circulating carotenoids was positively related to the strength of the immune response, and high initial carotenoid levels were associated with a slower decrease of the swelling. This suggests that carotenoid levels might have a complex effect on the immune and/or inflammatory response. More detailed investigations are thus needed to improve our understanding of the role of carotenoids in PHA-induced immune response and to assess how an allocation trade-off between immune function and carotenoid-based colour might ensure honesty in these signals.
The immune challenge did not induce a decrease in bill colour, suggesting
that the immune response due to PHA was not sufficiently intense or long
lasting to induce the re-allocation of carotenoids from the bill to
circulation as suggested for humoral immune response
(Faivre et al., 2003a).
Although bill colour was correlated to initial plasma carotenoid levels (see
also McGraw and Ardia, 2003),
it did not predict the immune response either in its magnitude [contrary to
Faivre et al. (Faivre et al.,
2003b)] or duration. This suggests that bill colour reflects
medium- or long-term quality rather than immediate quality. Accordingly, in
the partridge, only the carotenoid-based colour of soft skin tissues was
positively correlated to the strength of PHA response and reflected short-term
changes in body condition and not that of the more keratinised bill
(Pérez-Rodríguez et al.,
2008;
Pérez-Rodríguez and
Viñuela, 2008).
In this study, we showed that individuals might strongly differ in the
dynamics of the maintenance of the swelling response to PHA. Plasma
carotenoids were closely associated with the development of the immune
response and partly explained duration of the swelling. We could not explain
the main variation in the decrease pattern in swelling observed here (i.e.
within 24 h of peak response) with the variables we measured in this
experiment. However, we would like to emphasise that interpreting PHA-induced
immune responses in terms of immunocompetence would certainly gain from taking
into account individual variation in response patterns as well as the
proximate causes and consequences of these variations. The simultaneous
assessment of antioxidant capacity and of oxidative-stress-induced damages has
been underlined as a necessary approach to improve our understanding of the
costs of immune reactions (Hõrak et
al., 2007). We would suggest taking into account the dynamics of
physiological parameters linked to, or influenced by, the immune response as
important. Future studies in captive birds should ideally combine measuring
several physiological parameters potentially associated with the immune
response to that of its detailed dynamics.
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Acknowledgments |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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