Journal of Zoology
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Journal of Zoology. Print ISSN 0952-8369
Acute infection of avian malaria impairs concentration of
haemoglobin and survival in juvenile altricial birds
I. A. Krams1,2, V. Suraka2,3, M. J. Rantala4, T. Sepp1, P. Mierauskas5, J. Vrublevska2 & T. Krama2
1
2
3
4
5
Institute of Ecology and Earth Sciences, University of Tartu, Tartu, Estonia
Institute of Systematic Biology, University of Daugavpils, Daugavpils, Latvia
Rı̄ga Stradins University, Rı̄ga, Latvia
Department of Biology, University of Turku, Turku, Finland
Department of Environment Policy, Mykolas Romeris University, Vilnius, Lithuania
Keywords
avian malaria; immunity; parasite vectors;
haemoglobin; survival; Parus major.
Correspondence
Indrikis A. Krams, Institute of Ecology and
Earth Sciences, University of Tartu, 51014
Tartu, Estonia. Tel: 371 29465273; Fax: 372
7375830
Email: [email protected]
Editor: Robert Knell
Received 28 December 2012; accepted 27
March 2013
Abstract
Blood parasites are often considered as indicators of immunity in birds, and data
on parasite prevalence and intensity of infections are essential to reveal information about the condition of both individuals and populations. We prevented
parasite vectors from biting and infecting nestling great tit Parus major by using
insect repellent inside nest boxes. We found that in the absence of blood parasites,
great tit nestlings had higher concentrations of haemoglobin, and they survived at
significantly higher rates through the nestling phase and also during the first weeks
of their fledgling period. This is the first demonstration so far of the impact these
parasites have on haemoglobin levels of the hosts, which reveals one mechanism of
adverse impact by blood parasites. This study shows that the effects of blood
parasites can be assessed without using anti-malaria drugs, which can cause additional risk of oxidative stress.
doi:10.1111/jzo.12043
Introduction
The general health and immune function of an organism are
important determinants of its resistance against pathogens
and parasites, and therefore its longevity and reproductive
output (e.g. Schmid-Hempel, 2011). Life-history theory
assumes that an individual cannot invest equal amounts of
resources into all its needs, and allocation of limited resources
to different functions of an organism causes numerous tradeoffs between competing needs (Stearns, 1989; Sheldon &
Verhulst, 1996). Hence, individual condition can be defined as
the amount of resources available for allocation to fitnessenhancing traits such as survival and reproduction.
Both veterinary medicine and ecological research have a
long history of using various haemato-serological parameters
for diagnostic purposes to assess quality in poultry. Haemoglobin is one of the traits strongly linked to health condition.
It is the iron containing oxygen metalloprotein in the red
blood cells of nearly all vertebrates (Sidell & O’Brien, 2006).
Haemoglobin in the blood carries oxygen from the respiratory
organs to the tissues where it releases the oxygen to provide
energy to power the functions of the organism. It has been
recently shown that the concentration of haemoglobin is
strongly positively related to the physiological condition of
nestling altricial birds, which depend on the quantity and
quality of food delivered to them by parents (Kilgas et al.,
Journal of Zoology •• (2013) ••–•• © 2013 The Zoological Society of London
2006; Słomczyński et al., 2006; Bańbura et al., 2007). Moreover, it has been shown that survival of nestlings from hatching
to fledging is linked to haemoglobin level in the blue tit
Cyanistes caeruleus (Bańbura et al., 2007).
Haemoglobin concentration may also be affected by the
presence of blood parasites of birds via depletion of red blood
cells (Metcalf et al., 2011). Avian malaria is caused by parasites of the genera Leucocytozoon, Plasmodium and Haemoproteus (Valkiūnas, 2005; Outlaw & Ricklefs, 2011). These
parasites are commonly detected in a variety of reptiles, birds
and mammals being transmitted via bloodsucking insects
(Atkinson et al., 2005; Pérez-Tris & Bensch, 2005; Hutchings,
2009; Krams et al., 2010, 2012a,b). In birds, malaria has been
shown to have direct pathogenic effects on the host, and these
effects in turn result in reductions in parental care (Merino
et al., 2000) and reduction of fledgling success (Merino et al.,
2000; Yorinks & Atkinson, 2000; Garvin, Homer & Greiner,
2003; Sol, Jovani & Torres, 2003; but see Szöllősi et al., 2009;
Knutie, Waite & Clayton, 2013). These effects furthermore
lead to decreased survival in infected hosts (Warner, 1968;
Dawson & Bortolotti, 2000; Merino et al., 2000; Sol et al.,
2003; Marzal et al., 2005; Martinez-de la Puente et al., 2010),
and so these effects have important consequences for fitness.
The use of anti-malaria drugs is becoming increasingly
popular in ecological studies, allowing experimental reductions of avian malaria and investigation of subsequent
1
Costs of malaria infection
physiological and population effects of avian malaria, for
example (Merino et al., 2000; Marzal et al., 2005). The detrimental effects of avian malaria are caused by the stimulation
of immune response (Atkinson, Forrester & Greiner, 1988;
Ots & Hõrak, 1998; Ots, Murumagi & Hõrak, 1998; Garvin &
Greiner, 2003; Garvin et al., 2003). During an immune
response against parasites, the generation of free oxidative
radicals (ROS) takes place (Alonso-Alvarez et al., 2004;
Costantini & Møller, 2009; Dowling & Simmons, 2009). Leukocytes are the major source of ROS, and in the case of
inflammation the production of ROS is considerably elevated
and can negatively impact the anti-oxidative protection
system. It is believed that the use of anti-malaria drugs protects the organism against parasite-generated ROS and oxidative stress. However, the therapeutic effects of anti-malaria
drugs are based on the ability of these chemicals to produce
ROS, which then attack intracellular malaria parasites
(Farombi, Shyntum & Emerole, 2003). Such anti-malaria
chemicals as primaquine and chloroquine phosphate increase
the lipid peroxidation level, while decreasing plasma antioxidant levels, leading to elevated ROS levels and a number of
adverse side effects (Norris & Evans, 2000). Because the activation of the immune system to fight malaria parasites and the
most popular anti-malaria drugs have the same mechanisms
to achieve anti-parasite protection, anti-malaria drugs should
be used with caution in ecological research.
In this study, we tested whether experimental removal of
parasite vectors, and therefore prevention of infections by
avian malaria, improves condition and survival of nestlings of
the great tit Parus major, a cavity-nesting passerine bird with
altricial young. Incidence and prevalence of blood parasites
may be dependent on abundance and activity of parasite
vectors (Allander & Bennett, 1994; Ruiz, Oro &
Ganzalez-Solis, 1995). We prevented the parasite vectors from
biting nestlings by using repellents against bloodsucking
insects in nest boxes. The condition of nestlings was assessed
as concentration of white blood cells (WBCs) and haemoglobin in the peripheral blood, and survival was assessed to
the end of the nestling phase.
I. A. Krams et al.
Vector manipulation
All nest boxes were placed in 50- to 60-year-old pine forest,
and all nests of great tits were located within 0.3 km of small
streams, lakes or bogs, where most bird blood parasites reproduce. Pairs of great tits often bred 100-150 m from another
pair. By flipping a coin, we randomly divided these naturally
occurring pairs of nest boxes occupied by great tits into two
groups. One of the neighbouring nest boxes was included in
the experimental group and the second one was assigned to the
control group.
In the experimental group, 1–2 days before the onset of
hatching, we attached plastic canisters (30 mm diameter,
50 mm height) filled with bloodsucking insect repellent. The
canister was attached 3 cm away from the entrance to the
inner wall of the nest box. The repellent consisted of citronella
oil (Triple J LLC, Palatka, FL, USA), carrageenan and water.
Each canister had 20 small holes (2.0 mm) on its walls to
ensure the repellent activity of citronella oil. The repellent was
changed once every 2 days until the nestlings fledged. Empty
canisters were also attached inside the nest boxes in the
control group. To check the efficacy of the repellent, we
attached sticky traps to the ceilings of the nest boxes. A square
(15 ¥ 15 cm) of thick paper covered by a layer of nonhardening epoxide resin was attached using pins. To prevent
birds from sticking themselves to the trap’s sticky surface, we
attached a wire mesh 1 cm above the trap (mesh size 1 ¥ 1 cm),
which did not constrain the movements of insects. These traps
were placed in all of the nest boxes (n = 12 experimental group,
n = 12 control group). All experimental and control nest boxes
were checked every other day, and all of the bloodsucking
insects trapped were removed, identified and counted, and the
relative number of trapped bloodsucking insects per day
across the experimental and control nest boxes was estimated.
One important inclusion criterion was that all the nests contained 10 eggs. Furthermore, incubation in all of the nests was
initiated within 5 days. This approach allowed us to consider
the reproductive effort to be equal across the pairs.
Body condition and blood samples
Materials and methods
Study site and birds
The study was conducted in the vicinity of Krāslava, southeastern Latvia (56°N, 27°E), in 2009. The study area is covered
by coniferous and mixed forests of different age, with Scots
pine Pinus sylvestris, Norway spruce Picea abies, and birch
Betula pendula being the most common tree species (Rytkönen
& Krams, 2003). Great tits bred in wooden nest boxes and this
population has been studied as part of a long-term project of
the ecology of hole-nesting birds carried out by the University
of Daugavpils (Rytkönen & Krams, 2003). Nest boxes were
checked to record basic breeding parameters such as clutch
size, brood size and the number of fledglings, reflecting reproductive success. We studied the first breeding attempt of great
tits. No nests were deserted due to our activities.
2
Fourteen days after hatching, the nestlings were individually
ringed with numbered leg rings. We took blood samples from
all the nestlings. The samples (50 ml) were taken from the
tarsal vein of nestlings with HemoCue cuvettes. Samples were
analysed in the field using a portable HemoCue Hb 201 +
photometer (HemoCue AB, Angelholm, Sweden) to measure
haemoglobin concentration (g L–1). In total, we measured 120
nestlings in the experimental group and 110 in the control
group.
As avian malaria parasites cannot be detected in the blood
until 3 weeks after infection, we captured fledglings at the age
of 1 month by mist nets at feeders with sunflower seeds. We
captured 12 fledglings of the control group and 25 fledglings of
the experimental group. At capture, blood samples (c. 150 mg)
were taken from the tarsal vein. For identification of blood
parasites and leukocytes, a drop of blood was smeared on
three individually marked microscope slides, air dried, fixed in
Journal of Zoology •• (2013) ••–•• © 2013 The Zoological Society of London
I. A. Krams et al.
absolute methanol and stained with Gimsa stain (Bennett,
1970; Valkiūnas, 2005).
Provisioning behaviour
Because citronella repellent potentially may affect parental
behaviour, we estimated the feeding rate of adult great tits
(Wilkin, King & Sheldon, 2009). We collected data on parental behaviour with the use of videos at six pairs of nest boxes
within a long-term study on the diet of great tit nestlings. The
camcoders (Canon MV890, Canon, Inc., Tokyo, Japan) were
placed 5 m apart from the nest boxes. We covered them with
a large piece of pine bark and adult great tits did not pay any
attention to the recording equipment. Recording took place
when the young were 11 days old and duration of the recordings was 60 min. To compare the parental effort, we counted
the number of visits recorded per 1 h.
Blood parasites
Smears were screened with a light microscope under oil
immersion at 1000¥ magnification for Haemoproteus and
Plasmodium and at 500¥ magnification for Leucocytozoon,
Trypanosoma and Microfilaria. Parasites were enumerated
from 100 fields by moving the slide to areas where blood cells
formed a monolayer for Leucocytozoon, and from more than
200 fields for Haemoproteus and Plasmodium. Slides were
screened by T. K. Individuals were classified as infected when
smears were positive for at least one haemoparasite taxon.
Plasmodium is a genus of parasitic protists transmitted by
mosquitoes. Infection by these organisms is known as malaria.
During the erythrocyte stage, Plasmodium enters erythrocytes
where the parasite feeds by ingesting haemoglobin and other
materials from red blood cells and serum (Valkiūnas, 2005).
This feeding process damages the erythrocytes. Leucocytozoon
is a genus of parasitic protozoa transmitted by Simuliidae
blackflies as their definitive host. The newly released merozoites infect mainly leukocytes. Haemoproteus is an intracellular, protozoan parasite that infects erythrocytes. Like
Plasmodium and Leucocytozoon, Haemoproteus belongs to the
family Haemosporidia. Like other Haemosporidia, Haemoproteus is transmitted by bloodsucking insects including louse
flies (hippoboscid flies) and biting midges (Culicoides sp.).
Haematological parameters
After being scanned for blood parasites, slides were used to
obtain the total number and proportion of different types of
leukocytes such as heterophils and lymphocytes. The proportions of leukocyte types were assessed on the basis of examination of a total of 100 leukocytes from each of the three slides
under oil immersion under 1000¥. The total WBC count was
estimated by counting the number of leukocytes per c. 10 000
erythrocytes (Ots et al., 1998). For this purpose, all leukocytes
were counted in 100 microscope fields, including both leukocytes and erythrocytes. Concentration of leukocytes per
number of red blood cells was obtained by multiplying proportions of different leukocytes with WBC. The repeatabilities
Journal of Zoology •• (2013) ••–•• © 2013 The Zoological Society of London
Costs of malaria infection
of leukocyte concentrations obtained from repeated scanning
of the same blood smear by D. C. and T. K. were high [r =
0.90, 0.93, 0.90 and 0.92 for heterophil concentration,
heterophil/lymphocyte (H/L) ratio, WBC and lymphocyte
concentration, respectively; all P < 0.001] (Saino, Calza &
Møller, 1997).
Total WBC count is the number of leukocytes per c. 10 000
erythrocytes. Elevated leukocyte number is symptomatic of a
stress syndrome, inflammatory processes and oxidative stress.
Usually, leukocytosis is caused by an elevated concentration
of heterophils and/or lymphocytes (Dein, 1986; Ots et al.,
1998). Lymphocytes are immune cells that assist in the recognition and destruction of many types of pathogens. Although
sometimes difficult to interpret, decreased lymphocyte concentrations may signal stress-induced immunosuppression
(Hõrak et al., 1999), or may indicate a lack of parasite infections (Ots & Hõrak, 1998). Heterophils are non-specific
phagocytosing cells that enter the tissues during inflammatory
processes. The production of reactive oxidants by heterophils
during inflammation can be harmful to host tissues. Leukocytes are the major source of ROS, and in the case of inflammation the production of ROS is very much elevated and can
deprive the anti-oxidative protection system. Heterophil concentrations increase during inflammatory processes, stress and
infections (Ots et al., 1998). H/L ratio is a widely used indicator of stress as it increases in response to various stressors such
as infectious diseases, starvation and reproductive effort (Ots
& Hõrak, 1998; Moreno et al., 2002).
Statistics
All traits except blood parasite counts in the experimental
nest boxes and total parasite counts in the control and experimental nest boxes were normally distributed (one-sample
Kolmogorov–Smirnov test). For these non-normally distributed traits, we used non-parametric methods to compare total
parasite counts between the two types of nest boxes. All tests
performed were two tailed. To avoid pseudoreplication in the
analysis of haemoglobin concentrations, we analyzed the data
with a mixed-effect model design with treatment as fixed
factor and nest identity as random factor.
Results
We trapped only four biting midges in two experimental nest
boxes with the repellent during 15 days of the nestling phase.
In contrast, we trapped 4.75 ⫾ 1.77 (mean ⫾ sd) bloodsucking
insects per day per nest box in the control group of nest boxes.
This resulted in 285 bloodsucking insects trapped (215 biting
midges, 46 mosquitoes and 24 blackflies) in the control nest
boxes, likely reflecting only a proportion of bloodsucking
insects visiting nests of great tits and attacking their nestlings.
The total number of bloodsucking insects trapped per day was
significantly lower in the group of nest boxes with repellent
than in the control group [one-way analysis of variance
(ANOVA), F1, 238 = 86.259, P < 0.0001, Fig. 1].
Males and females provisioned their nestlings at equal
frequencies ranging from 16 to 34 times per hour in the
3
Costs of malaria infection
Figure 1 The total number of bloodsucking insects trapped per day
(⫾SE) in the experimental group of nest boxes with repellent and in the
control group of nest boxes without repellent.
I. A. Krams et al.
Figure 3 Estimated nestling survival (⫾SE) in experimental nest boxes
with repellent and in control nests without repellent.
Table 1 Heterophils, lymphocytes and H/L of control and experimental
fledgling great tits Parus major at the age of 30–33 days
Group
Sex
n
Heterophils
Lymphocytes
HL ratio
Mean SD
Mean SD
Mean SD
Control
Male
6 11.33 7.15 20.83 7.78
Female 6 15.33 10.21 23.32 10.46
Experimental Male
12 27.58 15.83 57.35 18.52
Female 13 26.00 14.22 55.85 18.43
0.55
0.93
0.51
0.49
0.37
1.09
0.29
0.27
H/L, heterophil/lymphocyte ratio.
Figure 2 Concentration of haemoglobin (⫾SE) in the blood of nestlings
in the experimental group with the repellent and in nestlings of control
group.
experimental nest boxes (paired t-test male vs. female, t =
-0.277, d.f. = 5; P = 0.793) and in the control nest boxes (paired
t-test male vs. female, t = -0.229; d.f. = 5, P = 0.828). Therefore,
we combined data from males and females by using the average
parental provisioning rate for all 12 nests, and we did not find
any significant difference in parental effort of adults between
experimental and control groups (t = 0.132, d.f. = 10, P = 0.897).
However, at the age of 14 days, the nestlings from the nests with
the repellent had on average 17 g L-1 higher haemoglobin levels
than the control nestlings (mixed-effect model ANOVA, F1,238 =
67.428, P < 0.0001, Fig. 2). The survival of nestlings at the nests
with the repellent was significantly greater than survival of the
control nestlings (one-way ANOVA, F1,238 = 13.778, P = 0.01,
Fig. 3) estimated at day 19 when the nestlings fledged.
4
At the age of 30–33 days, only 2 out of 25 fledglings from
nest boxes with the repellent were infected with Haemoproteus
parasites, and these infections were of low intensity (1–2 Haemoproteus gametocytes per 10 000 erythrocytes). The control
fledglings (n = 12) were infected with all of the three major
groups of blood parasites (Haemoproteus, Plasmodium and
Leycocytozoon). On average, each control fledgling had 10.33
⫾ 4.86 Haemoproteus gametocytes per 10 000 erythrocytes,
2.00 ⫾ 1.18 Plasmodium per 10 000 erythrocytes, and 2.89 ⫾
1.45 Leucocytozoon per 10 000 erythrocytes. The intensity of
infection by blood parasites (the total number of parasites/
10 000 erythrocytes) was significantly greater in the control
fledglings than in the experimental fledglings at roughly
1 month of age (Mann–Whitney test: z = 9.011, n1 = n2 = 120,
P = 0.0001). Sex of fledglings did not have a significant effect
on the intensity of infection (one-way ANOVA, F1,238 = 0.994,
P = 0.142).
We did not find any significant difference between males
and females in heterophil and lymphocyte concentrations, and
H/L ratio within the control and experimental fledgling
groups (Tables 1 and 2). We also did not detect a significant
difference in H/L ratio between the groups. However,
heterophil and lymphocyte concentrations of the control
group and experimental group differed significantly (Tables 1
and 2).
Journal of Zoology •• (2013) ••–•• © 2013 The Zoological Society of London
I. A. Krams et al.
Costs of malaria infection
Table 2 The effect of treatment (control or experimental group) and sex on heterophil and lymphocyte concentrations, and H/L ratio
Heterophils
Group
Sex
Group ¥ sex
Lymphocytes
HL ratio
d.f.
F
P
d.f.
F
P
d.f.
F
P
1
1
1
8.131
0.066
0.350
0.007
0.80
0.56
1
1
1
40.489
0.009
0.135
0.001
0.926
0.716
1
1
1
1.847
1.004
1.254
0.183
0.324
0.271
H/L, heterophil/lymphocyte ratio.
We captured significantly more fledglings of the experimental group (n = 25) than fledglings of the control group (n = 12)
(two tailed, Fisher’s exact test, P = 0.031). This may suggest
better survival in fledglings protected against vectors of blood
parasite infections during their nestling phase.
Discussion
The use of repellent appeared to be an effective method to
prevent access of bloodsucking insects to nestling of great tits.
Experimental prevention of infection had a positive effect on
haemoglobin concentration of nestlings and improved survival of nestlings and fledglings. This supports some previous
studies showing that avian malaria has pathogenic effects on
body condition of the host (Ots & Hõrak, 1998; Ots et al.,
1998; Apanius et al., 2000; Merino et al., 2000; Garvin et al.,
2003; van Oers et al., 2010; van de Crommenacker et al., 2012)
and that reduction of parasite burden improves adult survival
(Martinez-de la Puente et al., 2010). Previous studies have
shown that natal dispersal distances are shorter in great tits
attacked by parasites (Heeb et al., 1996, 1999). It is also
known that immigrant individuals mount a significantly
stronger immune response towards a novel antigen than do
local recruits in great tits (Snoeijs et al., 2004). This suggests
that the observed decrease in the number of infected fledglings
was due to their higher mortality rather than dispersal.
Our results suggest that acute avian malaria infections, consisting of Haemoproteus, Plasmodium and Leucocytozoon,
may have adverse effects on haemoglobin concentration of
blood of birds. Because parasites mostly infect erythrocytes,
cells containing haemoglobin, our results suggest a mechanism of lower survival rates of both nestlings and fledglings.
However, we do not know what species of blood parasites
were responsible for the decreased concentration of haemoglobin and reduced survival. Recent experimental evidence
shows that chronic infections of Haemoproteus have longterm survival costs in wild birds (Martinez-de la Puente et al.,
2010). However, studies on the effects of parasites are still in
their infancy, awaiting more experimental research both in
wild birds and poultry.
The absence or reduction of parasite burden should be
beneficial in terms of the ability to invest more energy in needs
other than the reduction of the direct adverse effects of parasitism, the resources used by the parasites themselves, and the
energy used to mount the immune response (de Lope, Møller
& de la Cruz, 1998; Martinez-Abrain, Esparza & Oro, 2004;
Martínez, Merino & Rodríguez-Caabeiro, 2004). The availJournal of Zoology •• (2013) ••–•• © 2013 The Zoological Society of London
ability of more resources in the experimental group may
explain significantly better survival of the repellent-protected
nestlings and fledglings of the great tit. Increased survival of
the experimental fledglings supposedly can be also explained
by their higher quality and ability to invest more time in their
anti-predator behaviour, reducing the probability of being
captured by predators (Møller & Nielsen, 2007).
We found significant differences in heterophil and lymphocyte concentrations between the control group and the
experimental birds protected by repellent. Higher concentration of leukocytes in experimental group may indicate a better
developed adaptive immune system. Although the avian
humoral immune system is thought to require at least 4–6
weeks to begin production of adequate numbers of peripheral B-cell lineages that express functionally different
immunoglobulin specificities (Klasing & Leshchinsky, 1999;
Ratcliffe, 2006), a recent finding shows that adaptive immune
system may mature much earlier in altricial birds (Killpack &
Karasov, 2012). However, age-related change in antibody
response and its possible dependence on parasite loads still
need to be confirmed in vaccination experiments (Krams
et al., 2012a,b, 2013).
While blood parasites can be considered to be a powerful
stressor, we did not find any increase in the H/L ratio in the
control group, which is considered to be positively associated
to the magnitude of the stressor (Davis, Maney & Maerz,
2008; Cı̄rule et al., 2012). Although concentrations of lymphocytes and heterophils significantly differed between the
control and experimental groups, these differences did not
affect the rates of H/L, suggesting that changes in the H/L
ratio should be interpreted with caution.
Our study shows the importance of alternative methods of
parasite removal. Medication of birds by using anti-malaria
drugs may have important negative consequences on bird
health by increasing oxidative stress during treatment. The use
of anti-malarial drugs may affect host body condition, making
effects by blood parasites hard to interpret especially in the
conservation context. One more problem is a sex-specific
effect of medication on the intensity of blood parasite infections (Møller, Sorci & Erritzøe, 1998; Klein, 2004), causing
different efficacy of anti-malaria treatment between sexes.
This can be avoided by preventing vectors of blood parasite
infections to attack experimental animals in the first place.
Finally, the repellents may be more widely used in the future
research because of their potential effect also on other
ectoparasites such as flees and, perhaps, even microbes living
in the nest structure.
5
Costs of malaria infection
Acknowledgements
We thank Todd M. Freeberg for improving the English. The
authors were supported by the Science Council of Latvia
(T.K.) and the Academy of Finland (M.J.R., I.A.K.). The
European Social Fund within the project ‘Support for the
implementation of doctoral studies at Daugavpils University’
No. 2009/0140/1DP/1.1.2.1.2/09/IPIA/VIAA/015 supported
J.V. T.S. was supported by the European Union through the
European Regional Development Fund (Centre of Excellence
FIBIR).
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