Comparative Immunology, Microbiology
& Infectious Diseases 27 (2004) 413–422
www.elsevier.com/locate/cimid
Acute phase response in reindeer after challenge
with Escherichia coli endotoxin
T. Orroa,*, S. Sankaria, T. Pudasb, A. Oksanenc, T. Soveria
a
Faculty of Veterinary Medicine, Department of Clinical Veterinary Sciences, University of Helsinki,
P.O. BOX 57, 00014 Helsinki, Finland
b
Department of Biology, University of Oulu, P.O. BOX 333, 90571 Oulu, Finland
c
National Veterinary and Food Research Institute, EELA, P.O. BOX 517, 60101, Oulu, Finland
Accepted 22 December 2003
Abstract
The serum concentrations of two acute phase proteins (APPs), haptoglobin (Hp) and serum
amyloid-A (SAA), were monitored in reindeer after challenge with endotoxin. Four adult female
reindeer received either 0.1 mg/kg Escherichia coli 0111:B4 lipopolysaccharide B or saline solution
intravenously. At the second challenge, the treatments were reversed. In addition to the APPs,
changes in blood chemistry and rectal temperature were monitored. The endotoxin challenge caused
a significant increase in SAA (peak 48 h) and a sharp decrease (8 –12 h) of serum iron concentrations
in all animals. The mean Hp concentration increased at 8 h and remained elevated until 48 h, but no
statistically significant differences were found. This investigation demonstrates that challenge with a
single-bolus dose of E. coli endotoxin can activate the acute phase response (APR) and SAA appears
to be a more sensitive indicator of the APR than Hp during bacterial infection in reindeer.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: Acute phase response (APR); Endotoxin; Haptoglobin (Hp); Reindeer; Serum amyloid-A (SAA)
Résumé
Les concentrations de deux protéines de l’inflammation, l’haptoglobine (Hp) et l’amyloı̈de A
sérique (SAA), la température rectale et les paramètres biochimiques sanguins ont été suivis chez
quatre rennes adultes femelles ayant reçu lors d’une première inoculation intraveineuse soit
0.1 mg/kg de lipopolysaccharide B d’Escherichia coli O111:B4, soit du sérum physiologique, puis
lors d’une seconde inoculation l’inverse.
L’injection d’endotoxine a entraı̂né une augmentation significative de la concentration de SAA
(pic à 48 h) et une diminution brutale (8– 12 h) du fer sérique chez tous les animaux. La concentration
* Corresponding author. Tel.: þ 358-19-529-5304; fax: þ358-19-685-1181.
E-mail address: [email protected] (T. Orro).
0147-9571/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cimid.2004.01.005
414
T. Orro et al. / Comp. Immun. Microbiol. Infect. Dis. 27 (2004) 413–422
moyenne d’Hp a augmenté à 8 h et est restée élevée jusqu’à 48 h, mais sans qu’aucune différence
statistiquement significative ne soit trouvée. Ceci démontre qu’une inoculation unique d’endotoxine
d’E. coli peut déclencher une réaction inflammatoire chez le renne et qu’en cas d’infection
bactérienne la SAA apparaı̂t être plus sensible que l’Hp pour détecter cette réaction.
q 2004 Elsevier Ltd. All rights reserved.
Mots-clé: Réaction inflammatoire; Endotoxine; Haptoglobine; Amyloı̈de A sérique; Renne
1. Introduction
The inflammatory reaction is a series of complex physiological events occurring in the
host after tissue injury or infection. The purposes of these events are to eliminate the
infecting agent, prevent further tissue damage and restore the homeostasis of the host
organism. The early sets of reactions that occur immediately after tissue damage are known
as the acute phase response (APR). It is characterized by the presence of an inflammatory
reaction at the site of injury or infection and systemically by multiple changes throughout
the organism such as fever, depression, leucocytosis, increased permeability of blood
vessels, stimulation of adrenocorticotropic hormone (ACTH) production, redistribution of
trace elements, etc. One of the main changes occurring during the APR is hepatic production
of various plasma proteins [1]. These so-called acute phase proteins (APPs) play a very
important role in the defense response of the host. For example, some APPs are involved in
nonspecific protection against infections caused by gram-negative bacteria [2], but in many
cases their exact physiological function is not clear.
Monitoring the plasma concentrations of APPs can provide information on progression
of the APR. Their potential use in veterinary medicine has been extensively investigated
and they are already widely used as markers of disease in veterinary and medical science.
Some of the many applications for APPs include evaluation of collective health of herds
by detection of subclinical diseases and identification of animals with inflammatory
lesions at slaughter. Thus, APPs may also serve as valuable tools in reindeer husbandry as
well as in giving diagnostic information on the detection, prognosis and monitoring of
diseases in reindeer.
Although the APR is highly conserved in nature, APP profiles show significant variability
between different species [3]. Haptoglobin (Hp) has been the APP most commonly monitored
as a marker for inflammation [4 –8] in cattle and other domestic ruminants. The plasma Hp
concentration is very low in healthy bovines [7] but during the APR it can increase over 100fold. Serum amyloid-A (SAA) is another major APP in many animal species, including cattle,
and has been shown to be a good diagnostic marker for determining the presence of infections
and inflammatory conditions [7,9 –11]. Evaluation of the concentrations of different APPs
allows better estimates of the complex systemic effects of inflammatory mediators during
various inflammatory conditions, e.g. in distinguishing between acute and chronic
inflammation and for evaluation of disease severity.
The main objective here was to investigate the host response of reindeer (Rangifer
tarandus tarandus) after bacterial endotoxin challenge as an acute phase stimulant and to
evaluate the changes occurring in serum concentrations of SAA and Hp as potential
T. Orro et al. / Comp. Immun. Microbiol. Infect. Dis. 27 (2004) 413–422
415
markers of infections. In addition to the APPs, other changes occurring in blood chemistry
and in some clinical parameters were also monitored.
2. Materials and methods
2.1. Animals and experimental procedures
This experiment was carried out at the Zoological Gardens of the Department of
Biology, University of Oulu, Finland. The first experiment was initiated on February 18,
2002 and the second on April 8, 2002. Eight adult female reindeer were randomly divided
into two equal groups. The mean weight of the animals was 76.7 kg (range 67.5 –90.5 kg)
and age was 5 years (3 –6 years). The groups were kept in two separate corrals (about
650 m2 each) in which they underwent a long adaptation period before the experiment was
initiated. The animals were fed with lichen (Cladina spp.), commercial reindeer pellets
and fresh water ad libitum. The first group received 0.1 mg/kg E. coli 0111:B4
lipopolysaccharide B ((LPS); 1 mg/ml Bactoq, Difco Laboratories, Inc., Detroit, MI,
USA) into the jugular vein. The second group received the same volume of physiological
sodium chloride solution (0.1 ml/kg). After 7 weeks, the same procedure was repeated vice
versa. The dose was chosen based on published studies in small domestic ruminants such
as goat [12 – 14]. In these experiments, this endotoxin dose resulted in clear activation of
the APR, as shown by significant decrease in serum iron concentrations and increase in
rectal temperature and serum cortisol concentrations. This dose caused only modest and
short-acting general signs of disease, e.g. depression and alteration of rumen contractility.
The rectal temperature was recorded at the same time that blood was taken during the first
day of the experiments. Clinical observations without manual handling were done every
hour during the first 12 h. After the experiments, the animals were killed and autopsies
performed.
2.2. Blood samples
The group of reindeer was herded into the small pen near the corrals and then caught
and manually restrained. Blood samples were taken from the jugular vein before the
endotoxin or physiological saline solution injections and after 1, 4, 8, 12, 24, 48, 96 and
168 h into plain tubes. The serum was separated, frozen in portions and stored at 2 20 8C
for further analysis.
2.3. Serum analysis
Serum Hp was determined using the hemoglobin-binding assay described by Makimura
and Suzuki [15] with slight modifications, in which tetramethylbenzidine (0.06 mg/ml) was
used as substrate [7]. SAA was measured with a commercially available ELISA kit (Phase
SAA kit, Tridelta Ltd, Ireland), according to the manufacturer’s instructions for cattle.
The activities of serum aspartate aminotransferase (ASAT) and creatine kinase (CK)
were determined following the recommendations of the Scandinavian Society for Clinical
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Chemistry and Clinical Physiology [16,17]. Spectrophotometric methods were used for
the determination of serum total protein [18], urea [19], sorbitol dehydrogenase (SDH)
[20], albumin [21] and g-glutamyl transferase (GGT) according to the International
Federation of Clinical Chemistry and Labarotory Medicine (IFCC) Expert Panel on
Enzymes [22]. Serum iron was determined with a colorimetric method [23]. The analyses
were performed with an automatic chemistry analyzer (KONE Pro, Konelab, Thermo
Clinical Labsystems Oy, Vantaa, Finland). Cortisol was measured in 25-mL duplicates
using radioimmunoassay with Coat-A-Count RIA kits obtained from Diagnostic Products
Corporation (Los Angeles, CA, USA). All results are expressed as mean ^ standard
deviation (SD).
2.4. Statistical analyses
Carry-over and period effects were tested using the t-test procedures of Jones and
Kenward [24]. These tests were run for selected time points, in cases of insignificant carryover effects in the examined parameters, the data from both challenges were combined.
Differences between the E. coli LPS treatment and control groups were analyzed using
repeated measures analyses of variance with treatment and time after challenge as within
factors. Greenhouse –Geiser adjusted p-values were used for evaluation of results, which
were significant at p , 0:05:
3. Results
After laboratory analysis, two reindeer were excluded from statistical analysis, one
from each group. One reindeer already showed increased Hp (over 3-fold) and SAA (over
10-fold) concentrations in the 0-h sample, which remained high during the first
experiment. Clear arthritic changes were found in the left tarsal joint of this animal at
autopsy; in all other reindeer no pathological changes were found. The second excluded
reindeer reacted very strongly to the restraint procedures used at the time of blood
sampling; its serum CK and ASAT activities increased markedly during the first 24 h of
experiments, indicating muscle injury.
A carry-over effect was found in cortisol and data analysis was carried out separately in
both experiments for that variable. Period differences could be seen in iron ðp , 0:05Þ and
in the activities of ASAT ðp , 0:01Þ and GT ðp , 0:05Þ: In the first experiment, all
animals receiving LPS showed clear signs of general depression (they kept their heads
down and moved slowly) and two had tremor of the legs. These clinical signs were
observed during the first 4 h after LPS administration. The same reindeer had loose
droppings on the following day. The signs of depression were less obvious during the
second experiment.
Endotoxin administration caused a significant increase in the concentration of SAA
(Fig. 1; p , 0:001Þ: The rise in SAA in the LPS group was seen in the 12-h sample and
peaked at 48 h ð184:7 ^ 89:4 mg=lÞ: At this time, the LPS-treated animals displayed a 2 to
29-fold increase of serum SAA from the baseline level. The mean SAA concentrations in
the 96-h sample were decreased below the pretreatment levels in both groups.
T. Orro et al. / Comp. Immun. Microbiol. Infect. Dis. 27 (2004) 413–422
417
Fig. 1. Mean (^ SD) serum concentrations of SAA (a), Hp (b) and iron (c) in reindeer ðn ¼ 6Þ after IV
administration of E. coli LPS (S; 0.1 mg/kg) or saline (V) at 0 h. The data are combined from two successive
challenges. Statistical differences are given in the text.
The mean serum Hp concentrations showed a small tendency to decrease during the
first 4-h period in the LPS group, increased slightly in the 8-h sample and attained
maximum value at 24 h (0.87 ^ 0.25 g/l). The Hp concentrations remained relatively
stable in the control group throughout the experiment (Fig. 1); however, these differences
between treatments were not statistically significant. Wide individual variation occurred in
Hp response to endotoxin administration; only one reindeer showed a near 2-fold increase
in the 48-h sample and one showed no increase at any time.
Serum iron concentrations decreased sharply in all endotoxin-treated animals and were
significantly lower ðp , 0:01Þ at 8 and 12 h (Fig. 1). The serum cortisol concentrations
showed wide variability among animals, but no consistent increases in cortisol in the LPS
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group were detected during the follow-up period ð0 – 96 hÞ at the time of both sets of the
experiment. The only exception was at the time of the first experiment when the LPS group
showed values clearly elevated over those of the control animals in the 4-h sample. The
reindeer already had showed very high cortisol values in the pretreatment sample (mean
102.9 nmol/l, range 9.8 –240 nmol/l; Fig. 2).
Serum urea values began to increase after treatment in both groups simultaneously and
after 4 h decreased slowly until 96 h to under the baseline level (Fig. 2; time effect
p , 0:001Þ: After one week, the mean urea concentration had nearly returned to the
pretreatment level. There were no differences between treatment groups at any time point.
Fig. 2. Mean (^ SD) serum concentrations of urea (a) n ¼ 6; cortisol from first set (b) n ¼ 3 and from second set
of the experiment (c) n ¼ 3 in reindeer after IV administration of E. coli LPS (S; 0.1 mg/kg) or saline (V) at 0 h.
The urea data are combined from two successive challenges. Statistical differences are given in the text.
T. Orro et al. / Comp. Immun. Microbiol. Infect. Dis. 27 (2004) 413–422
419
Mean rectal temperatures were already higher before treatment in the LPS group and
decreased steadily in both groups throughout the 24-h period, which was recorded, but this
difference was not significant. The changes in concentrations of serum total protein,
albumin, ASAT, CK, GT and SDH recorded during the follow-up period were
nonsignificant between the endotoxin and control groups.
4. Discussion
Bacterial endotoxins, which are LPSs from the cell wall of gram-negative bacteria, are
considered to cause most pathophysiological reactions during bacterial infections. The
physiological effects of LPS are based predominantly on activation of various molecular
mediators [25] such as the cytokines tumor necrosis factor (TNF)-a, interleukin (IL)-1b
and IL-6, which are released in response to LPS predominantly by monocytes and
macrophages [26]. These inflammatory mediators initiate host inflammatory response and
the production of hepatic APPs. Since cytokines are the main stimulators of excretion of
APPs from the liver [1], LPS administration has been used in APP research as an acute
phase stimulant in bovines [5,27,28]. In the present study, injection of E. coli LPS induced
activation of cytokine release in reindeer as shown by significant decrease in serum iron
concentration and increase in serum SAA concentrations in all LPS-challenged animals.
These changes were similar to those previously reported in domestic ruminants [27,29]. A
slight tendency for Hp concentrations to increase was seen earlier than SAA following
LPS challenge, but the Hp patterns differed widely between individuals and the relative
increases were small. A similar, evident but nonsignificant elevation of Hp was reported
previously in pigs after a single-bolus LPS dose administration [30], but the Hp response
was clearly elevated when repeated injections were used [31]. Hp and SAA differ in their
cytokine-controlled induction in the liver, and generally Hp needs stronger cytokine
stimulation to be produced than SAA [1,32]. The cytokine response sequence is not
affected by LPS dose or administration route, but the amplitude of the response is dose
dependent [33] and a single-bolus dose of E. coli LPS may have been insufficient to initiate
the cytokine secretion needed for prolonged Hp increase. This is supported by other studies
in bovines in which Hp increased after experimental gram-negative infections [34,35],
when stronger and prolonged cytokine release occurs, or after higher E. coli LPS dose
administration [5].
Variability in APP expression during the APR between animal species can also explain
the low Hp response observed. However, the serum Hp concentration can increase manyfold over the baseline level and appears to act as an APP in reindeer. This was observed in
the reindeer we had to remove from our study due to the presence of exceptionally high
SAA and Hp concentrations probably resulting from arthritis.
Another explanation for the mild APR to endotoxaemia may be the stress reactions of
reindeer. Although treatment did not affect the urea concentration, increased concentrations occurring during the first blood samplings and subsequent decrease when animals
became used to handling, further confirm the presence of a strong stress response in
reindeer. Increased concentrations of serum urea in stressed reindeer have been reported
[36,37] and significantly higher urea concentrations have been seen in intensively handled
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reindeer versus animals subjected to minimal handling [38]. The presence of a carry-over
effect in the cortisol results is very difficult to explain, especially when considering that we
had long wash-out period between experiment sets. However, the similarities in mean
cortisol concentration patterns within the animal groups during both experiments,
regardless of LPS challenge and wide individual variability (Fig. 2), indicate that cortisol
was more affected by the experimental procedure than by the presence of endotoxaemia
and that the carry-over effect was at least partly the result of stress responses of individual
animals to handling. The elevated cortisol levels observed before LPS challenge in this
study probably affected the results. High endogenous glucocorticosteroid concentrations
can influence the APR and expression of APPs in many ways; e.g. it can directly stimulate
APP production in the liver, but this is not believed to be a very powerful response [1] and
administration of dexamethasone failed to stimulate APP in cows [39]. On the other hand,
cortisol can inhibit APP increase by decreasing the cytokine response. Endogenous and
exogenous glucocorticoids can suppress TNF-a production after endotoxin administration
[40], and therefore the stress reaction induced by handling before endotoxin
administration may have an inhibiting effect on proinflammatory cytokine release and
may be one reason for the mild Hp response to the endotoxin dose used in this study.
Further support for the inhibitory effect of stress reactions before LPS administration is
given by the fact that no fever response occurred, although the clinical, serum iron and
SAA responses were clearly present. Cortisol and fever responses occurring during
endotoxaemia to some extent share a common mediating factor, namely prostaglandin,
which is mediated by proinflammatory cytokines [14] and the controlling mechanisms for
fever and changes in serum iron concentrations are independent [12].
In conclusion, we have demonstrated that single-dose E. coli endotoxin challenge
activates the APR and that this model can be used in reindeer for examining the
pathophysiology of inflammation and infection, although higher doses may be more
appropriate. However, in semidomesticated animals such as reindeer, stress reactions from
handling are very strong and their possible effects on the results must be addressed. SAA
appears to be a more sensitive indicator of the APR than Hp in reindeer after LPS
challenge. Since these proteins have different stimulation patterns, monitoring of both
proteins can provide more information on the ongoing APR of the host and thus may
provide a useful tool in veterinary medical science in reindeer. Further investigation is
needed to increase our understanding of the expression of these proteins in cases of various
naturally occurring and experimental infections or inflammations.
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