Investigating if the deer ked will influence

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The deer ked (Lipoptena cervi) is a haematophagous parasitic fly of cervids that spread to Finland in the early 1960s. Presently its northern distribution limit lies at approximately 65N and it is gradually spreading northwards. In Finland the principal host species has been the moose (Alces alces), but the deer ked is about to establish contact with another potential host, the semi-domesticated reindeer (Rangifer tarandus tarandus) causing possible threats to reindeer health and management. The aim of this study was to investigate if the deer ked would have an influence on the welfare of the reindeer. Eighteen adult reindeer were divided into three experimental groups: the control group and two infected groups with 300 deer keds per reindeer introduced in AugSep. One of the infected groups was treated with subcutaneous ivermectin in Nov. To gather comprehensive data on potential health hazards caused by the deer ked a wide array of physiological variables was measured during and at the end of the experiment in Dec. The keds caused no clear changes in the complete blood count, plasma clinical chemistry, amino acids, endocrinology, energy stores, enzyme activities or tissue fatty acid profiles of the host. The haematological, clinical chemical and endocrinological values displayed changes that could be related to the seasonal physiological adaptations of the species. In conclusion, at the duration and intensity of infection that were employed, the effects of the deer ked on the measured physiological variables of the reindeer were insignificant.

The deer ked (Lipoptena cervi L., Diptera, Hippoboscidae) is an ectoparasite of the moose (Alces alces) and other cervids. The deer ked is adapted to ectoparasitic life, it is dorsoventrally flattened with a hard exoskeleton (Metcalf and Metcalf, 1993) and has large claws enhancing attachment and preventing detachment (Haarlv, 1964). Both sexes live on the host and are haematophagous. The deer ked drops its wings upon attachment on the host making any subsequent host switch difficult or impossible (Hackman et al., 1983). The reproductive strategy is viviparous; the egg hatches in the reproductive tract and the developing larva is fed by maternal secretions (Meier et al., 1999). One female can produce 2032 pupae, which fall to the forest floor or snow and during the next autumn the imagines emerge (Popov, 1965; Ivanov, 1981). The deer ked does not fly actively searching for a host but stays close to its hatching site and waits for a potential host to arrive. Presumably, there is only one generation per year, which flies from the end of July till early Nov (Ivanov, 1981). After settling on a host the deer ked lives for 120180 d (Ivanov, 1981) and the total duration of parasitism may extend to next June (Arja Kaitala, personal communication, 2010).

The deer ked spread to Finland in the early 1960s from the South-East across the Soviet border (Hackman et al., 1983) and at present its northern distribution limit lies at approximately 65N (V�lim�ki et al., 2010). All moose in Eastern Finland are heavily parasitized (Paakkonen et al., 2010). The intensity of deer ked infection on the moose depends on the sex and age of the host: in autumn 2006, bulls had approximately 10,600, cows 3,500 and calves 1,700 keds. These figures are high compared to previous studies. For example, in the Leningrad region of the former Soviet Union the average intensity was 200�300 keds per moose (Popov, 1965). The deer ked can parasitize also other cervids, e.g., the red deer (Cervus elaphus), roe deer (Capreolus capreolus) and fallow deer (Dama dama; Kadulski, 1996; Szczurek and Kadulski, 2004), but on these species the infection intensities were lower than on the moose. The distribution limit of the deer ked has been spreading northwards in Finland at a rate of 11 km per year (V�lim�ki et al., 2010) and this parasite is thus establishing contact with another potential host, the semi-domesticated reindeer (Rangifer tarandus tarandus). It seems that pupal development would be possible north of the present distribution limit (H�rk�nen et al., 2010) and the species could perhaps use wild forest reindeer (R. t. fennicus) and semi-domesticated reindeer as hosts (Kaunisto et al., 2009; Kynk��nniemi et al., 2010). However, recent results indicate that reproductive success would be poor and survival of pupae low, as only one pupa of questionable viability was produced by 3,600 experimental deer keds on reindeer (Kynk��nniemi et al., 2010; Arja Kaitala, personal communication, 2010). Still, the deer ked may pose a potential threat to reindeer management in Finland and it is important to investigate, whether this parasite would have an influence on the health and welfare of the reindeer.

Ectoparasites may cause direct and indirect harm to their hosts (Balashov, 2007; Wall, 2007). Mild anaemia or decreased blood haemoglobin concentrations were occasionally documented in cattle (Bos taurus) infected with the Gulf Coast tick (Amblyomma maculatum; Williams et al., 1978) or the sheep scab mite (Psoroptes ovis; Stromberg et al., 1986). The latter caused also lowered haemoglobin concentrations in sheep (Ovis aries; O�Brien et al., 1995) and the sarcoptic mite (Sarcoptes scabiei) reduced the red blood cell counts of Spanish ibices (Capra pyrenaica; P�rez et al., 1999). The neutrophil and eosinophil counts increased in sheep (O�Brien et al., 1995) and the leukocyte counts in cattle infected with the sheep scab mite (Losson et al., 1988). Also leukopenia was observed due to Gulf Coast tick or sheep scab mite infection in cattle (Williams et al., 1977; Stromberg et al., 1986). The serum cortisol concentrations of cattle increased when infected with the horn fly (Haematobia irritans) and/or stable fly (Stomoxys calcitrans; Schwinghammer et al., 1986ab, 1987; Byford et al., 1992). These parasites increased also the heart and respiratory rates and rectal temperatures (Schwinghammer et al., 1986ab, 1987) and the sheep scab mite elicited dermatitis (Stromberg et al., 1986) and skin lesions for cattle (Losson et al., 1988). Indirect behavioural effects of ectoparasites include restlessness (Schwinghammer et al., 1986ab, 1987), rubbing (Corke and Broom, 1999) and grooming-related hair loss (Glines and Samuel, 1989). Energy-expending activities can be increased and the time allocated for feeding reduced (M�rschel and Klein, 1997; Hagemoen and Reimers, 2002). These effects may eventually lead to slower weight gain of infected animals (Williams et al., 1977; Stacey et al., 1978; Williams et al., 1978; Bianchin et al., 2007). Alopecia may also cause increased heat loss in winter (Glines and Samuel, 1989).

The aim of the present study was to assess in detail the possible manifestations of deer ked infection on reindeer health by measuring a wide array of physiological variables to gather comprehensive data on these potential effects. As ectoparasites induced previously many physiological responses in ruminants, it can be hypothesized that the deer ked would have negative effects on the health of the reindeer. In addition to answering this question, our multifaceted analytical approach also provided numerous indices of reindeer physiology, endocrinology and biochemistry valuable for the husbandry.

The experiment was performed between May 29 and Dec 13 2007 at the Zoological Gardens of the University of Oulu, Finland (65.062587�N, 25.456294�E) by the permission of the Committee on Animal Experiments of the University (STH378A; May 16 2007/ESLH-2007-03532/Ym-23). Adult reindeer (7 males, 11 females) were divided into three experimental groups (group I = control, group II = infection, group III = infection and medication) with an equal sex ratio and average age of 2.8 � 0.6 years. For identification all reindeer were provided with coloured collars and numbered ear tags. The males had been castrated to enable easier handling and to prevent the autumn rut. On May 29 and June 13 the reindeer were treated against any pre-existing endo- and ectoparasites with subcutaneous (sc) ivermectin (0.2 mg kg body mass (BM)�1; Vetpharma AB, Lund, Sweden) and on May 29 with topical deltametrin (75 mg reindeer�1; Schering Plough, Ballerup, Denmark). Ivermectin is commonly used against a wide range of endo- and ectoparasites of animals and humans (Dourmishev et al., 2005) and in Finnish Lapland, most reindeer are treated annually with ivermectin against eukaryotic parasites (Laaksonen et al., 2008). The half-life of ivermectin in ruminant plasma is 3 d and after approximately 3 weeks its plasma concentrations are very low or undetectable (Oksanen et al., 1995; Cerkvenik et al., 2002). Thus, the treatments in May�June would not have affected the survival of the keds in Aug. The reindeer were fed ad libitum with a commercial diet (Poron-Herkku, Rehuraisio, Espoo, Finland; 10.5% raw protein, 3.8% raw fat, 12.5% raw fiber, energy content 11.7 MJ ME kg dry matter�1) supplemented with lichen (Cladonia spp.), hay and dried birch (Betula spp.) and willow (Salix spp.) leaves. To prevent the possibility of parasite contamination each group was kept in its own outdoor enclosure (570 m2) at ambient temperature and photoperiod.

The deer keds were either reared at the University of Oulu, Department of Biology, from wild-collected pupae from various parts of Finland (60�65�N, n = 1260, 35%), or collected as imagines by hand in the communes of Rantsila (64�N) and Liperi (62�N) in Aug�Sep (n = 2340, 65%). They were housed in plastic containers with moist moss to retain humidity. The reindeer in groups II�III were infected on 6 occasions between Aug 16 and Sep 27 with an equal total number of parasites (300 per reindeer). All animals including group I were immobilized in a handling crib and the deer keds were placed on the anterior dorsum of the reindeer of groups II�III. The infection intensity used in this study was 6�35 times lower than that observed on wild moose (Paakkonen et al., 2010) but relatively similar to the intensities used in previous experimental infections of cattle with the horn fly and/or stable fly (Schwinghammer et al., 1986ab, 1987).

The reindeer were immobilized in the handling crib and blood samples were taken from the left jugular vein with aseptic needles into test tubes containing ethylenediaminetetraacetic acid on seven occasions (May 29, June 13, Aug 15, Oct 2 and 16, Nov 6, Dec 10). After the blood sampling on Nov 6, group III was treated with sc ivermectin (0.2 mg kg�1) and the other groups were given equivolume 0.85% saline injections. On Dec 10�13 the reindeer were stunned with a captive bolt pistol and killed by exsanguination. Some individuals from all experimental groups were selected for sampling each day. Blood samples were centrifuged at 2000 � g for 20 min to obtain plasma. One ml of whole blood was refrigerated at 4�C for the complete blood count analysis. The liver and adrenal glands were dissected and samples were taken from musculus rectus abdominis as well as from sc and intraabdominal (retroperitoneal, rp) adipose tissues. One of the adrenal glands was preserved in 5% formalin, processed conventionally into thin sections and stained with hematoxylin-eosin. The other tissue samples were immediately frozen with liquid nitrogen and stored at �80�C. The weights of the carcass, pelt, liver, kidneys, adrenal glands and omentum, the lengths of the carcass and pelt and the thickness of sc fat at an incision in the gluteal-sacral region (Finnish Game and Fisheries Research Institute, 2010) were also measured. After skinning and weighing the pelts were examined in detail by cutting the hair with scissors and calculating the numbers of live and dead deer keds and other visible ectoparasites�if present�as described by Paakkonen et al. (2010). The area of the skin was determined by drawing the outlines of each pelt on a paper and subsequently placing a metal grid (20 � 20 mm) on this and calculating the number of squares covering the area.

The complete blood count was determined with the Vet abc Animal Blood Counter calibrated for the equine blood profile (ABX Hematologie, Montpellier, France) at the Municipal Veterinary Clinic of Joensuu within 24 hours of collecting the blood. The plasma clinical chemistry was determined with reagents of Randox Laboratories Ltd. (Crumlin, UK) as described by Mustonen et al. (2006ab, 2009a). For the actual measurements, the Technicon RA-XTTM analyser (Swords, Dublin, Ireland) was used. The tissue glycogen, protein and lipid concentrations and enzyme activities were analyzed as outlined previously (Mustonen et al., 2006b; Rouvinen-Watt et al., 2010) and the plasma amino acid (AA), urea and ammonia levels were determined with ion-exchange chromatography (Biochrom 30 Amino Acid analyzer, Biochrom Ltd, Cambridge, UK).

The plasma leptin, ghrelin, adiponectin, insulin and glucagon concentrations were determined with commercial radioimmunoassay kits (Mustonen et al., 2005, 2009a). The plasma triiodothyronine (T3) and cortisol levels were measured according to Mustonen et al. (2009a) and the plasma thyroxine (T4) levels with the Coat-A-Count Total T4 kit (Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA). The hormone assays were validated such that serial dilutions of the reindeer plasma showed linear changes in BB0�1 values (B = standard or sample binding, B0 = maximum binding) that were parallel with the standard curves produced with the standards of the manufactures (Supplement 1). The determination of the adrenal catecholamine concentrations (noradrenaline NA, adrenaline A, dopamine DA) was also described previously (Nieminen et al., 2004).

For the fatty acid (FA) analyses, samples of the commercial diet, adipose tissues (sc, rp), liver, muscle and plasma were transmethylated according to Christie (1993). The formed FA methyl esters were extracted with hexane and analyzed by a gas-liquid chromatograph (6890N network GC system with a flame ionization detector (FID) and 5973 mass selective detector (MSD), Agilent Technologies, Santa Clara, CA; equipped with DB-wax capillary columns, 30 m, ID 0.25 mm, film thickness 0.25 ?m, J&W Scientific, Folsom, CA) using the MSD for FA identification and FID for quantification (Mustonen et al., 2007ab). The peak areas of the FID chromatograms were converted to mol% by using the theoretical response factors (Ackman, 1992) and calibrations with quantitative authentic standards. The results represent the FA composition of tissue total lipids as structural phospholipids and storage neutral lipids were not separated before analysis. Fractionation coefficients were calculated as follows: (mol% in tissue)/(average mol% in diet). The dietary FA profiles included the commercial diet but not the feed supplements.

Comparisons between the study groups were performed with the one-way analysis of variance (ANOVA) and the Duncan�s post hoc test and with the Student�s t-test for independent samples using the SPSS-program (v. 17.0, SPSS Inc, Chicago, IL, USA). For nonparametric data, the Kruskal-Wallis ANOVA followed by the Dunn�s post hoc test was performed with the SigmaPlot-program (v. 11.0, Systat Software Inc, Chicago). Significant differences in the time-series were analyzed with the general linear model for repeated measures (repeated measures ANOVA). To analyze the relationships in the FA composition of the different study groups and tissues, the data were also subjected to the multivariate principal component analysis (PCA) using the SIRIUS 6.5 software package (Pattern Recognition Systems AS, Bergen, Norway; Kvalheim and Karstang, 1987). The data were standardized and the relative positions of the samples and variables were plotted using 2 new coordinates, the principal components PC1 and PC2, describing the largest and the second largest variance among the samples. A p-value < 0.05 was considered statistically significant. The results are presented as the mean � SE.

At the end of the experiment, group I had no deer keds on their pelts, group II had 6 � 3 live and 5 � 1 dead deer keds, while group III had only dead keds (17 � 3; Table 1). The survival of the keds on group II was 2.1 � 0.9% and the average recovery of the dead and live parasites on groups II�III was 4.7 � 0.8%. There was a single deer ked pupa in group II. The deer ked densities (live and dead keds) in groups II�III were close to equal. No other visible ectoparasites could be detected in any of the study groups. The efficacy of ivermectin could not be determined with accuracy based on the present results, as the survival-% of the deer keds was very low and the numbers of live parasites could not be determined at the time of ivermectin treatment.

The BM, organ masses, weight of the omentum or thickness of the sc fat layer did not differ according to the treatments at the end of the study (Supplement 2). There were no differences between the study groups in the absolute or relative thicknesses of the layers of the adrenal cortex (zona glomerulosa 11.1 � 0.7%, z. fasciculata 63.3 � 1.2%, z. reticularis 25.6 � 0.9%, groups I�III pooled). The blood haemoglobin and haematocrit values were higher in groups I�II compared to group III already prior to the infections and these values remained higher also on several subsequent occasions (Table 2). On Dec 10, group III had a lower platelet count than groups I and II, but there were no consistent differences in the other haematological variables between the experimental groups during the study (Fig. 1; Table 2). The plasma clinical chemistry values (Supplement 3), the plasma and adrenal endocrinological variables (Supplement 4), the tissue glycogen, protein and lipid concentrations or enzyme activities (Supplement 5) did not show any consistent differences between the groups. At the end of the experiment, group II had higher plasma valine concentrations than group I with no differences in the other AA (Supplement 6).

Generally, the differences in the FA proportions between the experimental groups were minor, although in some rare cases statistically significant. The livers of group II had higher percentages of total saturated FA (SFA) and lower unsaturated FA (UFA)/SFA ratios than the other groups (Supplement 7a). Group II displayed also higher proportions of hepatic i16:0 but lower proportions of 18:1n-9 and 18:2c9t11 than group I, which had less 19:1n-10 than groups II�III and less 20:3n-6 than group III. The muscle tissue of group III contained more i15:0 and less 14:1n-5 than group I and more i17:0 than group II (Supplement 7b). There were no differences in the FA profiles of rp fat (Supplement 7c) but sc fat of group I had more 22:0 than group II and less 20:5n-3 than group III (Supplement 7d). The proportion of 20:3n-6 in plasma was the highest in group III (Supplement 7e).

One of the infected reindeer groomed itself more than the others and had also hair loss indicating skin irritation possibly caused by the deer ked infection. Based on this type of disturbed behaviour observed by the caretakers, the consulting veterinarian decided to euthanize the individual on Oct 24. The reindeer had six live deer keds on its pelt, four on the neck region, one on the chest and one on the head. In addition, there were two dead keds on the neck region. There were also six hairless patches on the posterior back, 3�15 cm in diameter. Despite of this, the health indices of this reindeer were approximately within the minimum and maximum values of the other animals, e.g., plasma glucose (8.55 vs. 5.90�8.12 mmol L�1), creatine kinase (36 vs. 92�1737 U L�1), cortisol (153 vs. 5�167 nmol L�1) and adrenal catecholamines (NA: 8229 vs. 544�11832 ng mg�1; A: 1735 vs. 107�29074 ng mg�1; DA: 44.8 vs. 32.4�86.3 ng mg�1). The absolute and relative weights of the adrenal glands were also within the range of values of the other reindeer (5.2 vs. 3.8�9.8 g; 0.073 vs. 0.053�0.090 �, respectively) as was the change in the BM during the study (+14.5 vs. �3.5 to +19.0 %). In addition, the PCA indicated that this individual did not differ from the other reindeer in its experimental group.

According to the PCA (Supplement 8), the FA profiles differed by the various tissues, while the deer ked infection did not have any clear effects. The percentages of total SFA increased according to the sequence: muscle ? plasma ? liver ? sc fat ? rp fat (Supplement 9). Liver contained the lowest proportions of 16:0 while the highest percentages were found in adipose tissues. Rp fat and liver contained more 18:0 than the other tissues. The proportions of 18:1n-9 and total monounsaturated FA (MUFA) were lower in liver and plasma than in the other tissues. The polyunsaturated FA (PUFA) sums increased as follows: sc fat ? rp fat ? muscle ? plasma ? liver. The percentages of total n-3 PUFA, 22:5n-3 and 22:6n-3 were the highest in liver and plasma. Muscle and plasma contained more 18:3n-3, and sc and rp fats less 20:5n-3 than the other tissues. The n-6 PUFA sums were the highest in liver and plasma and the percentages of the family precursor 18:2n-6 increased according to the sequence: sc and rp fat < muscle and liver < plasma. With 20:4n-6, the sequence was as follows: rp and sc fat < muscle and plasma < liver. Muscle contained less trans FA than the other tissues.

Compared to the dietary profile, the tissues contained more C14�19 SFA. An exception was liver with slightly less 16:0 than in the commercial diet (Fig. 2). The diet contained more C20�24 SFA than most tissues but liver had higher percentages of C22�24 SFA than the diet. Generally, the tissues had more C14�19 MUFA than the feed, except of 18:1n-9 in all tissues, especially liver, 14:1n-5 in liver and 18:1n-5 in liver and muscle. Liver contained more 24:1n-9 than the diet, while the proportions of C20�22 MUFA were higher in the feed. The percentages of 18:3n-3 were higher in the diet than in the tissues and the same was observed in total n-3 PUFA, except of liver. Liver and muscle had more C20�22 n-3 PUFA than the feed except of 20:3n-3 in muscle. The diet contained more 18:2n-6 and total n-6 PUFA than the tissues, which for their part had higher proportions of 20:4n-6, and the other tissues except of rp fat contained also more 20:3n-6 than the feed. The diet had more total PUFA than the tissues. The proportions of total trans FA were higher in the tissues compared to the dietary profile.

Several seasonal changes could be observed in the measured variables (for details see Fig. 1; Table 2; Supplements 3�4). For instance, the blood haemoglobin, haematocrit and mean corpuscular haemoglobin (MCH) values increased towards the winter, while the response of lymphocytes was an increase with a peak in Oct�Nov followed by a decrease (Fig. 1; Table 2). The concentrations of plasma ghrelin and T3 decreased during the autumn (Supplement 4). Also the plasma total cholesterol and urea concentrations, creatine kinase activities and urea/creatinine ratios decreased in the autumn, while the creatinine concentrations increased (Supplement 3).

Ectoparasites have occasionally influenced the red blood cell variables of their hosts (Williams et al., 1978; Stromberg et al., 1986; O�Brien et al., 1995; P�rez et al., 1999). In the present study, however, this could not be observed during the deer ked infection�similar to many previous experiments showing no statistically significant or physiologically relevant effects of parasitism on haematological values (Williams et al., 1977; Schwinghammer et al., 1986ab, 1987). The survival-% of the deer keds was very low and, due to this, the blood loss of the reindeer was probably too minor to cause anaemia. In fact, the blood haemoglobin, haematocrit and MCH values increased towards the winter in all experimental groups (see also Nieminen, 1980; DelGiudice et al., 1992). In addition to effects on red blood cells, parasitism may cause leukocytosis on the hosts (Losson et al., 1988), which could imply, e.g., microbial infection, but in the present study there were no differences in the white blood cell counts of the reindeer. However, the platelet count was lower in group III after it had been treated with ivermectin. The previously described side-effects of ivermectin on humans include a sporadic case of reduced neutrophil count (Bagheri et al., 2004) and the observed decrease in the platelet count might be a novel side-effect as, to our knowledge, it has not been reported previously. However, the material of the present study is insufficient to determine this with certainty.

Infections caused by the horn fly and/or stable fly increased the serum cortisol levels of cattle indicating that these parasites could induce stress to their hosts (Schwinghammer et al., 1986ab, 1987). In the reindeer, there were no differences in the plasma cortisol or adrenal catecholamine concentrations, nor in the histological findings of the adrenal glands between the groups at the end of the study. Cortisol is excreted in a pulsatile manner (Genuth, 1998) causing fluctuations in its plasma concentrations, and sampling may cause handling stress for reindeer inducing higher cortisol secretion (S�kkinen et al., 2004). The decreased cortisol levels of group II in Nov may thus be associated with interindividual differences in the rhythms of cortisol excretion or in the responses to handling. One reindeer displaying behavioural disturbance was euthanized on Oct 24 based on the veterinary evaluation but its physiological values did not differ significantly from the other animals. Therefore, the results of the present study indicate that the deer keds did not cause measurable changes in the biochemical indicators of stress at this intensity of infection.

Harassment of reindeer subspecies by oestrid flies increased previously the time allocated to standing, walking and running, which could affect their energy budgets in a deleterious manner (M�rschel and Klein, 1997; Hagemoen and Reimers, 2002). Ectoparasites have also induced restlessness to their hosts (Schwinghammer et al., 1986ab, 1987) causing reductions in the eating time (M�rschel and Klein, 1997; Hagemoen and Reimers, 2002) and nitrogen retention (Schwinghammer et al., 1986ab, 1987). These data fit to the observations of ectoparasites reducing the weight gain of their hosts (Williams et al., 1977; Stacey et al., 1978). In the present experiment, there were no major differences in the clinical chemical variables, AA or weight-regulatory hormones between the study groups and the infection did not cause significant BM loss, either. This indicates that the deer keds did not have any significant effects on the nitrogen metabolism or weight-regulation at this intensity of infection in captive animals with ad libitum feeding.

The plasma creatinine concentrations increased and the urea concentrations decreased towards the winter leading to a lowered urea/creatinine ratio, as documented also previously for the reindeer (S�kkinen et al., 2001; S�kkinen, 2005). The plasma total protein concentrations were relatively stable in the autumn and, thus, these findings do not suggest increased levels of protein catabolism in the reindeer. The autumnal reduction in the plasma ghrelin levels may play a role in the regulation of appetite of the animals (Tsch�p et al., 2000). The plasma T3 concentrations also decreased towards the winter, which probably represents seasonal adaptation observed in the reindeer and other northern mammals. The decreased thyroid hormone levels participate in reducing the metabolic rate and thus assist in surviving over the harsh winter with scarcity of food (Ringberg et al., 1978; Ryg and Jacobsen, 1982ab). These seasonal changes�as well as those observed in haematology�could perhaps partly mask some of the physiological effects of parasitism. In addition, the high intensity of feeding in our experiment could have attenuated physiological responses caused by the deer keds as the nutritional state of the host can affect the responses to parasitism (Nelson, 1984).

Nutritional scarcity induces fat mobilization, which is selective leading to modifications in tissue FA profiles (Mustonen et al., 2007b, 2009b). Generally, the mobilization of n-3 PUFA is more efficient than that of n-6 PUFA, which may lead to an unfavourable n-3/n-6 PUFA ratio (Rouvinen-Watt et al., 2010). During undernutrition the tissue proportions of, e.g., 18:3n-3 and 18:2n-6 of reindeer decrease due to their preferable mobilization (Soppela, 2000; Soppela and Nieminen, 2002). These essential FA (EFA) are precursors of longer-chain n-3 and n-6 PUFA, which can be further metabolized into eicosanoids, important lipid derivatives mediating, e.g., the immune response (Chapkin, 2008; Nelson and Cox, 2008). There is also evidence that 20:4n-6, 20:5n-3 and 22:6n-3 could enhance host resistance against endoparasites (Kumaratilake et al., 1997; Kumar and Das, 1999) and the same was observed for an increase in the n-3/n-6 PUFA ratio (Muturi et al., 2005). In the present study, however, the differences in the FA proportions and sums between the experimental groups were minor and inconsistent. Thus, there were no indications of FA alterations caused by the deer ked infection that could have elicited effects on the immune response to eukaryotic parasites.

As expected, the reindeer tissues contained less EFA than the diet, which is partly due to the biohydrogenation of dietary EFA by rumen microbes, a process that leads to SFA via trans FA intermediates (Jenkins, 1993; Bessa et al., 2007). The levels of EFA were low in adipose tissues (Soppela and Nieminen, 2002) and they were mainly incorporated into liver and muscle. The high SFA sums in the reindeer tissues mostly comprised of 16:0 (16.9�31.4%) and 18:0 (16.6�31.7%; see also Wiklund et al., 2001; Soppela and Nieminen, 2002). In general, the tissues contained plenty of shorter C14�19 SFA while the proportions of C20�24 SFA were mostly lower than in the feed. The major MUFA was 18:1n-9 (13.6�32.7%), the proportions of which in adipose and muscle tissues confirmed previous results (Wiklund et al., 2001; Soppela and Nieminen, 2002). MUFA are present in adipose tissues of several homeothermic species in levels superior to those of the diets (Cochet et al., 1999; Mustonen et al., 2007a, 2009b), which could not be observed in the reindeer. Generally, the percentages of FA in tissues depend on their concentrations in the diet, although the final proportions are also partly determined by postabsorptive modifications (). However, in ruminants 18:1n-9 is largely biohydrogenated into 18:0 in the rumen and its percentages in tissues depend on the activity of ?9-desaturase (Mosley et al., 2002; Smith et al., 2006). In the reindeer the proportions of 18:1n-9 in muscle and adipose tissues were quite similar to those of the diet, while the percentages in liver were lower.

Considering the PUFA incorporation into the lipids of reindeer tissues in detail, the percentages of the other n-6 PUFA except of 18:2n-6 (1.7�9.8%) were mostly higher in the tissues than in the feed, yet the total n-6 PUFA content was higher in the diet. In addition to biohydrogenation, this indicates metabolic conversion of 18:2n-6 to longer and more unsaturated successors. The longer-chain n-6 PUFA accumulated in the muscle and especially in the hepatic lipids. The proportions of total n-3 PUFA and several individual n-3 PUFA, except of 18:3n-3 (0.2�0.4%), were higher in liver than in the feed and the adipose tissue n-3 PUFA percentage was at approximately the same low level as documented earlier (Soppela and Nieminen, 2002). The relative enrichment of both n-6 and n-3 PUFA in muscle and liver is due to their high contents of glycerophospholipids that preferably incorporate PUFA into the sn-2 position of the glycerol trunk. Biohydrogenation of UFA in the digestive tract prior to absorption explains the relatively high levels of trans FA in the tissues of ruminants and other ruminant-like species harbouring large microbial communities for fermentation in their gastrointestinal tracts (Hartman et al., 1955).

The present study describes the physiological responses of the reindeer to a novel parasite, the deer ked. There were no consistent effects of the deer ked parasitism on the measured physiological variables of the reindeer at the intensity of infection employed. As the survival-% of the deer keds was very low in this experiment, it is unlikely that a longer follow-up period would have produced significant effects later in the winter.