& Infectious Diseases 27 (2004) 413-422 Acute phase response in reindeer after challenge T. Orroa,*, S. Sankaria, T. Pudasb, A. Oksanenc, T. Soveria aFaculty of Veterinary Medicine, Department of Clinical Veterinary Sciences, University of Helsinki, bDepartment of Biology, University of Oulu, P.O. BOX 333, 90571 Oulu, Finland cNational Veterinary and Food Research Institute, EELA, P.O. BOX 517, 60101, Oulu, Finland 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 femalereindeer received either 0.1 mg/kg Escherichia coli 0111:B4 lipopolysaccharide B or saline solutionintravenously. 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 causeda significant increase in SAA (peak 48 h) and a sharp decrease (8 - 12 h) of serum iron concentrationsin all animals. The mean Hp concentration increased at 8 h and remained elevated until 48 h, but nostatistically significant differences were found. This investigation demonstrates that challenge with asingle-bolus dose of E. coli endotoxin can activate the acute phase response (APR) and SAA appearsto 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) Les concentrations de deux prote'ines de l'inflammation, l'haptoglobine (Hp) et l'amyloi¨de A se'rique (SAA), la tempe'rature rectale et les parame`tres biochimiques sanguins ont e'te' suivis chezquatre rennes adultes femelles ayant rec¸u lors d'une premie`re inoculation intraveineuse soit0.1 mg/kg de lipopolysaccharide B d'Escherichia coli O111:B4, soit du se'rum physiologique, puislors d'une seconde inoculation l'inverse.
L'injection d'endotoxine a entraiˆne' une augmentation significative de la concentration de SAA (pic a` 48 h) et une diminution brutale (8 - 12 h) du fer se'rique chez tous les animaux. La concentration * Corresponding author. Tel.: þ358-19-529-5304; fax: þ358-19-685-1181.
E-mail address: (T. Orro).
0147-9571/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cimid.2004.01.005 T. Orro et al. / Comp. Immun. Microbiol. Infect. Dis. 27 (2004) 413-422 moyenne d'Hp a augmente' a` 8 h et est reste'e e'leve'e jusqu'a` 48 h, mais sans qu'aucune diffe'rencestatistiquement significative ne soit trouve'e. Ceci de'montre qu'une inoculation unique d'endotoxined'E. coli peut de'clencher une re'action inflammatoire chez le renne et qu'en cas d'infectionbacte'rienne la SAA apparaiˆt eˆtre plus sensible que l'Hp pour de'tecter cette re'action.
q 2004 Elsevier Ltd. All rights reserved.
Mots-cle': Re'action inflammatoire; Endotoxine; Haptoglobine; Amyloi¨de A se'rique; Renne 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 theinfecting agent, prevent further tissue damage and restore the homeostasis of the hostorganism. The early sets of reactions that occur immediately after tissue damage are knownas the acute phase response (APR). It is characterized by the presence of an inflammatoryreaction at the site of injury or infection and systemically by multiple changes throughoutthe organism such as fever, depression, leucocytosis, increased permeability of bloodvessels, stimulation of adrenocorticotropic hormone (ACTH) production, redistribution oftrace elements, etc. One of the main changes occurring during the APR is hepatic productionof various plasma proteins These so-called acute phase proteins (APPs) play a veryimportant role in the defense response of the host. For example, some APPs are involved innonspecific protection against infections caused by gram-negative bacteria , but in manycases 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 investigatedand 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 herdsby detection of subclinical diseases and identification of animals with inflammatorylesions at slaughter. Thus, APPs may also serve as valuable tools in reindeer husbandry aswell as in giving diagnostic information on the detection, prognosis and monitoring ofdiseases in reindeer.
Although the APR is highly conserved in nature, APP profiles show significant variability between different species Haptoglobin (Hp) has been the APP most commonly monitoredas a marker for inflammation in cattle and other domestic ruminants. The plasma Hpconcentration is very low in healthy bovines but during the APR it can increase over 100-fold. 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 infectionsand inflammatory conditions Evaluation of the concentrations of different APPsallows better estimates of the complex systemic effects of inflammatory mediators duringvarious inflammatory conditions, e.g. in distinguishing between acute and chronicinflammation 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 toevaluate 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 markers of infections. In addition to the APPs, other changes occurring in blood chemistryand in some clinical parameters were also monitored.
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 dividedinto 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 (about650 m2 each) in which they underwent a long adaptation period before the experiment wasinitiated. The animals were fed with lichen (Cladina spp.), commercial reindeer pelletsand fresh water ad libitum. The first group received 0.1 mg/kg E. coli 0111:B4lipopolysaccharide B ((LPS); 1 mg/ml Bactoq, Difco Laboratories, Inc., Detroit, MI,USA) into the jugular vein. The second group received the same volume of physiologicalsodium chloride solution (0.1 ml/kg). After 7 weeks, the same procedure was repeated viceversa. The dose was chosen based on published studies in small domestic ruminants suchas goat In these experiments, this endotoxin dose resulted in clear activation ofthe APR, as shown by significant decrease in serum iron concentrations and increase inrectal temperature and serum cortisol concentrations. This dose caused only modest andshort-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 firstday of the experiments. Clinical observations without manual handling were done everyhour during the first 12 h. After the experiments, the animals were killed and autopsiesperformed.
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 theendotoxin or physiological saline solution injections and after 1, 4, 8, 12, 24, 48, 96 and168 h into plain tubes. The serum was separated, frozen in portions and stored at 2 20 8Cfor further analysis.
Serum Hp was determined using the hemoglobin-binding assay described by Makimura and Suzuki with slight modifications, in which tetramethylbenzidine (0.06 mg/ml) wasused as substrate SAA was measured with a commercially available ELISA kit (PhaseSAA 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 T. Orro et al. / Comp. Immun. Microbiol. Infect. Dis. 27 (2004) 413-422 Chemistry and Clinical Physiology Spectrophotometric methods were used forthe determination of serum total protein , urea sorbitol dehydrogenase (SDH)albumin and g-glutamyl transferase (GGT) according to the InternationalFederation of Clinical Chemistry and Labarotory Medicine (IFCC) Expert Panel onEnzymes. Serum iron was determined with a colorimetric method. The analyseswere performed with an automatic chemistry analyzer (KONE Pro, Konelab, ThermoClinical Labsystems Oy, Vantaa, Finland). Cortisol was measured in 25-mL duplicatesusing radioimmunoassay with Coat-A-Count RIA kits obtained from Diagnostic ProductsCorporation (Los Angeles, CA, USA). All results are expressed as mean ^ standarddeviation (SD).
Carry-over and period effects were tested using the t-test procedures of Jones and Kenward These tests were run for selected time points, in cases of insignificant carry-over effects in the examined parameters, the data from both challenges were combined.
Differences between the E. coli LPS treatment and control groups were analyzed usingrepeated measures analyses of variance with treatment and time after challenge as withinfactors. Greenhouse - Geiser adjusted p-values were used for evaluation of results, whichwere significant at p , 0:05: 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 (over10-fold) concentrations in the 0-h sample, which remained high during the firstexperiment. Clear arthritic changes were found in the left tarsal joint of this animal atautopsy; in all other reindeer no pathological changes were found. The second excludedreindeer reacted very strongly to the restraint procedures used at the time of bloodsampling; its serum CK and ASAT activities increased markedly during the first 24 h ofexperiments, 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 andin the activities of ASAT ðp , 0:01Þ and GT ðp , 0:05Þ: In the first experiment, allanimals receiving LPS showed clear signs of general depression (they kept their headsdown and moved slowly) and two had tremor of the legs. These clinical signs wereobserved during the first 4 h after LPS administration. The same reindeer had loosedroppings on the following day. The signs of depression were less obvious during thesecond experiment.
Endotoxin administration caused a significant increase in the concentration of SAA (p , 0:001Þ: The rise in SAA in the LPS group was seen in the 12-h sample andpeaked at 48 h ð184:7 ^ 89:4 mg=lÞ: At this time, the LPS-treated animals displayed a 2 to29-fold increase of serum SAA from the baseline level. The mean SAA concentrations inthe 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 Fig. 1. Mean (^ SD) serum concentrations of SAA (a), Hp (b) and iron (c) in reindeer ðn ¼ 6Þ after IVadministration of E. coli LPS (S; 0.1 mg/kg) or saline (V) at 0 h. The data are combined from two successivechallenges. 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 attainedmaximum value at 24 h (0.87 ^ 0.25 g/l). The Hp concentrations remained relativelystable in the control group throughout the experiment (); however, these differencesbetween treatments were not statistically significant. Wide individual variation occurred inHp response to endotoxin administration; only one reindeer showed a near 2-fold increasein 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 (). The serum cortisol concentrationsshowed wide variability among animals, but no consistent increases in cortisol in the LPS T. Orro et al. / Comp. Immun. Microbiol. Infect. Dis. 27 (2004) 413-422 group were detected during the follow-up period ð0 - 96 hÞ at the time of both sets of theexperiment. The only exception was at the time of the first experiment when the LPS groupshowed values clearly elevated over those of the control animals in the 4-h sample. Thereindeer already had showed very high cortisol values in the pretreatment sample (mean102.9 nmol/l, range 9.8 - 240 nmol/l; 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 (time effectp , 0:001Þ: After one week, the mean urea concentration had nearly returned to thepretreatment 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 setof 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 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 thisdifference was not significant. The changes in concentrations of serum total protein,albumin, ASAT, CK, GT and SDH recorded during the follow-up period werenonsignificant between the endotoxin and control groups.
Bacterial endotoxins, which are LPSs from the cell wall of gram-negative bacteria, are considered to cause most pathophysiological reactions during bacterial infections. Thephysiological effects of LPS are based predominantly on activation of various molecularmediators such as the cytokines tumor necrosis factor (TNF)-a, interleukin (IL)-1band IL-6, which are released in response to LPS predominantly by monocytes andmacrophages These inflammatory mediators initiate host inflammatory response andthe production of hepatic APPs. Since cytokines are the main stimulators of excretion ofAPPs from the liver , LPS administration has been used in APP research as an acutephase stimulant in bovines In the present study, injection of E. coli LPS inducedactivation of cytokine release in reindeer as shown by significant decrease in serum ironconcentration and increase in serum SAA concentrations in all LPS-challenged animals.
These changes were similar to those previously reported in domestic ruminants. Aslight tendency for Hp concentrations to increase was seen earlier than SAA followingLPS challenge, but the Hp patterns differed widely between individuals and the relativeincreases were small. A similar, evident but nonsignificant elevation of Hp was reportedpreviously in pigs after a single-bolus LPS dose administration but the Hp responsewas clearly elevated when repeated injections were used Hp and SAA differ in theircytokine-controlled induction in the liver, and generally Hp needs stronger cytokinestimulation to be produced than SAA The cytokine response sequence is notaffected by LPS dose or administration route, but the amplitude of the response is dosedependent and a single-bolus dose of E. coli LPS may have been insufficient to initiatethe cytokine secretion needed for prolonged Hp increase. This is supported by other studiesin bovines in which Hp increased after experimental gram-negative infections when stronger and prolonged cytokine release occurs, or after higher E. coli LPS doseadministration 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 many-fold over the baseline level and appears to act as an APP in reindeer. This was observed inthe reindeer we had to remove from our study due to the presence of exceptionally highSAA 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 concen-trations occurring during the first blood samplings and subsequent decrease when animalsbecame used to handling, further confirm the presence of a strong stress response inreindeer. Increased concentrations of serum urea in stressed reindeer have been reportedand significantly higher urea concentrations have been seen in intensively handled T. Orro et al. / Comp. Immun. Microbiol. Infect. Dis. 27 (2004) 413-422 reindeer versus animals subjected to minimal handling The presence of a carry-overeffect in the cortisol results is very difficult to explain, especially when considering that wehad long wash-out period between experiment sets. However, the similarities in meancortisol concentration patterns within the animal groups during both experiments,regardless of LPS challenge and wide individual variability (), indicate that cortisolwas more affected by the experimental procedure than by the presence of endotoxaemiaand that the carry-over effect was at least partly the result of stress responses of individualanimals to handling. The elevated cortisol levels observed before LPS challenge in thisstudy probably affected the results. High endogenous glucocorticosteroid concentrationscan influence the APR and expression of APPs in many ways; e.g. it can directly stimulateAPP production in the liver, but this is not believed to be a very powerful response andadministration of dexamethasone failed to stimulate APP in cows. On the other hand,cortisol can inhibit APP increase by decreasing the cytokine response. Endogenous andexogenous glucocorticoids can suppress TNF-a production after endotoxin administrationand therefore the stress reaction induced by handling before endotoxinadministration may have an inhibiting effect on proinflammatory cytokine release andmay 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 isgiven by the fact that no fever response occurred, although the clinical, serum iron andSAA responses were clearly present. Cortisol and fever responses occurring duringendotoxaemia to some extent share a common mediating factor, namely prostaglandin,which is mediated by proinflammatory cytokines and the controlling mechanisms forfever and changes in serum iron concentrations are independent 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 thepathophysiology of inflammation and infection, although higher doses may be moreappropriate. However, in semidomesticated animals such as reindeer, stress reactions fromhandling are very strong and their possible effects on the results must be addressed. SAAappears to be a more sensitive indicator of the APR than Hp in reindeer after LPSchallenge. Since these proteins have different stimulation patterns, monitoring of bothproteins can provide more information on the ongoing APR of the host and thus mayprovide a useful tool in veterinary medical science in reindeer. Further investigation isneeded to increase our understanding of the expression of these proteins in cases of variousnaturally occurring and experimental infections or inflammations.
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