DISTINGUISHING DISEASE EFFECTS FROM ENVIRONMENTAL EFFECTS IN A MOUNTAIN UNGULATE: SEASONAL VARIATION IN BODY WEIGHT, HEMATOLOGY, AND SERUM CHEMISTRY AMONG IBERIAN IBEX (CAPRA PYRENAICA) AFFECTED BY SARCOPTIC MANGE

Abstract Our study focuses on the Iberian ibex (Capra pyrenaica) from the Sierra Nevada Natural Space (southern Spain), where sarcoptic mange is an endemic disease and animals are affected by a highly seasonal environment. Our aim was to distinguish between disease and environmental influences on seasonal variation in body weight, hematology, and serum biochemistry in Iberian ibex. We sampled 136 chemically immobilized male ibexes. The single effect of mange influenced hemoglobin, hematocrit, mean corpuscular volume, leukocytes, band neutrophils, monocytes, cholesterol, urea, creatine, and aspartate aminotransferase. Both mange and the period of the year also affected values of mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, neutrophils, glucose, and serum proteins. Scabietic animals showed a marked reduction in body weight (21.4 kg on average), which was more pronounced in winter. These results reveal that 1) infested animals are anemic, 2) secondary infections likely occur, and 3) sarcoptic mange is catabolic.


INTRODUCTION
Sarcoptic mange caused by Sarcoptes scabiei is responsible for epizootic disease in free-ranging populations of a broad range of wild mammals around the globe (Pence and Ueckermann 2002).Hematologic and biochemical responses to mange are relatively well known in domestic and wild mammals (Sarasa et al. 2010;Akomas et al. 2011;Kido et al. 2011).However, physiologic response of animals affected by mange in highly seasonal mountain ecosystems has not been evaluated.Using a sample of 136 male Iberian ibexes (Capra pyrenaica) from the Sierra Nevada Natural Space (southern Spain), we assessed whether mange affects body weight, which shows markedly seasonal variations, but also examined the effects of mange on hematologic and serum chemistry values of affected animals.Because ibexes in the Sierra Nevada experience strong seasonal changes in body reserves (Serrano et al. 2011) and such body stores are correlated to some hematologic parameters (Serrano et al. 2008), we expected that mange may drive the seasonal hematologic profile of this mountain ungulate.

Study area
We studied the Iberian ibex population of the Sierra Nevada Natural southern Spain).This area comprises the alpine massif, including the highest peaks in the Iberian Peninsula (e.g., the Mulhace ´n, 3,481 m above sea level).This area is characterized by strong seasonal climatic variations; snow cover is present for 6 mo (December to May), and most plant growth occurs from June to August (Peinado and Rivas-Martı ´nez 1987).

Ibex capture and blood collection
Between 2005 and 2012 blood samples from 136 male Iberian ibexes 3-11 yr old (81 clinically healthy and 55 scabietic animals) were collected.Animals were chemically immobilized with a mixture of xylazine (3 mg/ kg) and ketamine (3 mg/kg; Casas-Dı ´az et al. 2011) and aged by growth segments on the horns (Fandos 1991).During immobilization we collected blood into tubes containing ethylenediaminetetraacetic acid tripotassium and serum tubes.Time required for blood collection was similar for all animals with a mean of 5 min (range, 3-15 min).
Scabietic animals were visually assigned to four categories according to the percentage of skin surface affected by mites (Pe ´rez et al. 2011: 0 5 ibexes without skin lesions; 1 5 skin surface affected ,25%; 2 5 skin surface affected 25-50%; and 3 5 skin surface affected .50%).Skin scrapings were analyzed at the laboratory to confirm the diagnoses.

Hematology
Whole blood samples were stored at 4 C until arrival at the laboratory.Hematologic analyses were performed within 24 hr of collection.Clotted tubes were centrifuged at 4,750 3 G for 10 min, and sera were stored at 220 C until analysis.Red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count, and differential leukocyte count were determined with a Cell-Dyn 3500H autoanalyzer (Abbott Cientı ´fica, S.A., Madrid, Spain).

Statistical analyses
We evaluated whether the interaction between mange infection and seasons influenced body weight of ibexes using linear models (LM).Four categories of mange infection were considered.The effects of seasons were evaluated in two periods of the year that represent the main seasonal changes of body stores (Serrano et al. 2011): winter-spring (15 December-15 June) and summer-autumn (16 June-14 December).To control for potential We evaluated the physiologic response of ibexes to mange using multivariate analysis of variance (MANOVA).Our sampling design included a balanced proportion of sick and healthy ibexes in both periods of the year (Kruskal-Wallis53.6,P50.06), minimizing the possibility of spurious results.
For both LM and MANOVA, a model selection procedure based on the Akaike Information Criterion corrected for small sample size (AICc) was performed (Burnham and Anderson 2002).The model with the lowest AICc was retained, and the remaining competing models were ordered according to their Akaike differences (Di) with respect to the best model (lowest AICc).The Akaike weight (W i, ) and the percentage of explained variability (R 2 ) for the best model were also estimated.Statistical analyses were carried out in the statistical software R version 3.0.3(R Development Core Team 2014).

RESULTS
Descriptive statistics for body weight and hematologic and serum biochemical parameters studied are summarized in Tables 1-3.Our model selection suggested a seasonal-dependent effect of mange on ibex weight (w Mange status*Period of the year 50.52;Table 4).However, and according to the principle of parsimony, the second competing model (D i 50.17), which suggested the additive effects of both factors, would have more support (w Mange status+Period of the year 50.47;Table 4).The model suggests that 40% of the observed body weight variability relied on mange infection and period of the year.Reduced body weight in scabietic ibexes appeared in the early stages of infection (b 2nd mange category 5210.19,SE52.5, t524.01) but peaked when more than half of the skin surface was affected (b 3rd mange category 5221.47,SE52.9, t527.21).During the course of infection, weight loss in ibexes ranged between 32% (17 kg) in winter and 39.9% (25.8 kg) in summer (  1).The effects of mange on both hematologic and serum biochemical parameters depended on the period of the year (w Mange status*Period of the year 51 for both parameters; Table 4).This model explained between 97.8% (F 7,128 5848.4) to 82.6% (F 7,128 551.44) of the observed hematologic and serum biochemical changes of ibexes, respectively.Nevertheless, all hematologic parameters were not affected by mange or seasons in the same way (Tables 5, 6).The single effect of mange influenced RBC, Hb, hematocrit, MCV, leukocytes, band neutrophils, and monocytes, but MCH, MCHC, and neutrophils were affected by both mange and the period of the year (Table 5).Similarly, cholesterol, urea, and AST were solely influenced by the severity of mange infection, whereas glucose and proteins were also influenced by the period of the year.The interaction of effects of mange and the period of the year influenced values of creatine, LDH and albumin (Table 6), and CK was exclusively affected by the period of the year.

DISCUSSION
Ibexes in our study were chemically immobilized in a way similar to that described elsewhere (Casas-Dı ´az et al. 2011), so confounding effects due to different capture methods were not expected.Anemia detected by reduced RBC, Hb, and hematocrit in scabietic ibexes was in agreement with previous studies (Arlian et al. 1988;Kido et al. 2011).Anemia in scabietic ibexes might be due to the inflammatory process associated with mange (Pence and Ueckermann 2002).In response to inflammatory cytokines, iron may be sequestered in bone marrow macrophages as a protective mechanism to deny this key element to potential pathogens that require it for growth and multiplication (Carlson 1990;Weiss and Goodnough 2005).Iron-related measurements were not carried out in our study.Iron may also have a nutritional origin due to combined protein, vitamin, and mineral deficiencies (Watson and Canfield 2000).Neutrophils were higher in ibexes affected by sarcoptic mange than in clinically normal animals, in agreement with previous studies on infected mammals (Kido et al. 2011).Season-related neutrophilia has been reported in wild ungulates (Cross et al. 1994).In winter, the additive effect of season and disease could lead to a rise in neutrophils and, therefore, in total leukocytes.Ibexes showing more extensive and chronic lesions (category 3) showed the lowest values of blood eosinophils throughout the year.The number of eosinophils in the dermis was positively correlated with the density of mites (Skerrat 2003); the fact that mites decrease in lesions as the hypersensitivity reaction progresses (Arlian 1996) may explain our finding.However, we did not count mites.
The increased blood urea, particularly in winter-spring, coupled with a decrease in creatine concentration suggest an accelerating net catabolism of body protein storage consistent with the progressive weight loss previously reported in scabietic Alpine chamois (Tataruch et al. 1985).Scabietic animals had higher values of total serum proteins than nonaffected animals, which is expected in chronic infections (Jain 1993;Lastras et al. 2000).Values of In conclusion, sarcoptic mange produced a marked reduction of body weight in infested ibexes with a clear seasonal trend.Scabietic animals exhibited anemia and poor nutritional condition.Most of the hematologic parameters studied were related to mange status and leukograms, suggesting secondary infection was involved.Some of the biochemical parameters sampled depended on both the disease stage and the period of the year (e.g., glucose, creatine, LDH, proteins, and albumin); others (e.g., cholesterol, urea, and AST), depended exclusively on mange status; finally, CK depended solely on the period of the year.
Continued studies of how food shortages, due to changes in feeding behavior, primary productivity, or overpopulation of wild and domestic herbivores, influence the response of ibexes to mange infection will provide new strategies and opportunities for controlling this disease in the wild.

ACKNOWLEDGMENTS
Thanks to the staff of the Sierra Nevada Natural Space for their help.Our study was funded by the Ministerio de Economı ´a y Competitividad of the Spanish Government (project CGL2012-40043-C02-01).The authors' research activities are partially funded by the Plan Andaluz de Investigacio ´n (RNM-118 group), and E.S. was supported by the postdoctoral program (SFRH/BPD/96637/2013) of the Fundac ¸a ˜o para a Cie ˆncia e a Tecnologia, Portugal.This study complied with current Andalusian and Spanish laws.

TABLE 1 .
Mean6SE, sample size (applicable to all tables), and range (in parentheses) of body weights (kg) of 136 male Iberian ibex, Capra pyrenaica, .3yr old, captured in the Sierra Nevada Natural Space, southern Spain, 2005-12.WS indicates ibexes captured in winter and spring and SA in summer and autumn.Numbers in columns represent four categories based on the percentage of skin surface affected by mange.

Table 1 )
. Winter conditions also

TABLE 3 .
Mean6SE (range)of values of serum biochemical parameters in 136 male Iberian ibex, Capra pyrenaica, over 3 yr of age, captured in the Sierra Nevada Natural Space, southern Spain, 2005-12.WS indicates ibexes captured in winter and spring and SA in summer and autumn.

TABLE 4 .
Model selection for the seasonal effects of sarcoptic mange on body weight and hematologic and serum biochemical parameters in 136 male Iberian ibex, Capra pyrenaica, .3yr old, captured in the Sierra Nevada Natural Space, southern Spain, 2005-12.Models with substantial support are in bold.a a K 5 number of parameters; AICc 5 Akaike Information Criterion corrected for small samples sizes; Di 5 difference of AICc with respect to the best model; W i 5 Akaike weight; Mo 5 null model.

TABLE 5 .
Analysis of variance table showing those hematologic parameters influenced by the period of year, mange infection, or their interaction in 136 male Iberian ibex, Capra pyrenaica, .3yr old, captured in the Sierra Nevada Natural Space, southern Spain, 2005-12.For both eosinophils and lymphocytes, neither mange infection, period of year, nor their interaction were statistically significant.

TABLE 6 .
Analysis of variance table showing those serum biochemical parameters influenced by period of the year, mange infection, or their interaction in 136 male Iberian ibex, Capra pyrenaica, .3yrold, captured in the Sierra Nevada Natural Space, southernSpain, 2005-12.