Hematologic and biochemical variables of hedgehogs (Erinaceus europaeus) after overwintering in rehabilitation centers

Introduction

The European Hedgehog (Erinaceus europaeus) is a widespread wild insectivorous mammal in Italy and the rest of Europe. The absence of natural predators and the protection by law contributed to a wide distribution of these animals. Nevertheless, due to environmental changes such as the extensive use of pesticides resulting in a reduced availability of insects or other natural food resources for hedgehogs, together with a continuing elimination of rural areas, many hedgehogs are now living in human residential areas where they are exposed to risks such as respiratory and neoplastic diseases probably associated with environmental pollution, and traumas due to getting hit by cars. The breeding and detention of hedgehogs is prohibited by law in Italy, thus rescued sick hedgehogs must be rehabilitated in officially recognized centers for wild animal rescue (eg, WWF oasis or veterinary clinics), and, after hospitalization and overwintering in such centers, the animals must be released back into the wild.

One of the main causes for hospitalization are weak and underweight young animals born in autumn, which will probably not survive hibernation.1, 2 Other common problems at admission are heavy infestation by ecto- and endoparasites, particularly fleas and ticks,3 potentially causing blood loss anemia,4 and parasitic bronchopneumonia due to Capillaria aerophila and/or Crenosoma striatum. Moreover, hedgehogs can harbor zoonotic agents such as Salmonella spp., Yersinia pseudotubercolosis, Rhabdovirus, and Trychophyton mentagrophytes.5 In general, any deviation from normal and healthy can result in hematologic and biochemical changes. However, despite the high number of hedgehogs that are hospitalized in private clinics or in rehabilitation centers, it can be challenging for practicing veterinarians to assess the basic health status, and to diagnose or monitor specific diseases of rescued hedgehogs, not last to assess whether previously hospitalized animals can be safely released. These problems are in part due to the lack of information about hematologic and biochemical reference intervals (RIs) of normal healthy hedgehogs.

In the literature, the majority of studies published on hedgehogs refers to parasitic disease,3, 5-7 and only 2 studies report laboratory variables.1, 8 The goal of this study was to establish RIs for European hedgehogs following the guidelines published by the American Society of Veterinary Clinical Pathology (ASVCP).9 These RIs should be useful for future assessment of laboratory results of sick and hospitalized hedgehogs. In addition, we investigated whether sex or management of the rehabilitation centers show an influence on hematologic and biochemical variables in hedgehogs overwintering there.

Materials and methods

Results

Thirty-eight hedgehogs fulfilled the inclusion criteria described above. For 4 animals, EDTA blood was unavailable or inadequate; therefore, hematologic RIs were determined for 34 samples. In 2 cases, there was no blood smear available, thus WBC differential counts were performed on 32 samples. Biochemical analyses were performed in 30 hedgehogs, as 8 samples were excluded due to insufficient sample size or preanalytical factors such as hemolysis or lipemia. Calcium and phosphate were measured in the 26 hedgehogs from centers 1 and 2, but not in the animals at center 3. Among the 12 excluded hedgehogs (8 from center 2 and 4 from center 3), 3 animals were not underweight at admission and went into hibernation, and 9 animals were sick after overwintering (eg, cranial trauma, bone fracture, respiratory disease, neurologic deficit, cachexia, and bleeding disorders), had para-physiologic conditions such as obesity (2 cases) or were pregnant.

Precision data of the Sysmex variables are reported in the supplemental Table S1. Blood smear examination revealed that almost all hedgehogs had up to 6% nRBCs, and between 1 and 14 Howell–Jolly (H–J) bodies per 20 high power fields (HPF, Table 1, Figure S1). PLT-O counts were higher than PLT-I counts (data not shown). In this study, > 8–10 platelets per HPF were considered adequate. In addition, large pleomorphic hypergranular platelets (compared to platelets in common domestic species) were noticed on smears (Figure 1), while actual platelet clumps were rare (0–2/smear). The differential WBC counts revealed a prevalence of lymphocytes, which occasionally appeared as large granular lymphocytes (LGL) or as lymphocytes with hyperbasophilic cytoplasm (activated lymphocytes). Up to 10% basophils were counted (Figure 2), and up to 10% of neutrophils and eosinophils had bilobed nuclei (Figure 3).

Precision data of biochemical variables are reported in the supplemental Table S1. They were characterized by high activities for ALP and GGT, and high concentrations of creatinine and urea, when compared with common domestic species (Table 2, supplemental Figure S1).

There were no significant differences between males and females, with the exception of a higher MCV in females than in males (Figure 4). Conversely, significant differences related to the management were observed (Figure 5). Specifically, the MCH was significantly higher in hedgehogs at center 3 than at center 1, and nRBCs were more numerous in animals at center 3 than at centers 1 and 2. Relative eosinophil counts were lower in animals at center 3 than at centers 1 and 2.

Among biochemical analytes, cholesterol concentrations were significantly lower in hedgehogs at centers 2 and 3 compared with animals at center 1, urea concentration was significantly higher in animals at center 3 than at center 1, and glucose concentration was significantly higher in hedgehogs at center 3 compared with animals at centers 1 and 2 (Figure 5). Creatinine concentration was higher in animals at center 2 than at the other 2 centers, although only the difference in comparison with animals at center 3 was statistically significant. The concentrations of total protein and total globulin were higher and the A/G ratio was lower in animals at center 1 compared with hedgehogs at the other 2 centers (Figure 5).

Discussion

The establishment of RIs in wild animals like hedgehogs is difficult, due to the potential influence of poorly controlled parameters even in captivity. Often, exact age determination is impossible. In this study, all hedgehogs were likely juveniles (probably less than 3 months old when captured and then approximately less than 9 months old when sampled before release into the wild) and the age differences between animals at the 3 centers were balanced by the deferred blood collection. Conversely, circadian fluctuations should not have affected the results as blood was collected approximately at the same time of day in all 3 centers.

Stress associated with captivity and subclinical diseases can potentially affect the results. Therefore, in this study, samples were only collected from clinically healthy animals with normal physiologic parameters such as body weight and BI. Finally, a variable that could have influenced the results is the effect of general anesthesia. However, as anesthesia was performed in all groups, possible artifacts due to anesthesia (hypoglycemia, increased RBC mass) are unlikely to explain the differences reported in Figures 4 and 5.

Only a few reports about RIs of hedgehogs are available.1, 8 However, published hematology data were obtained using instruments and sampling sites (medial saphenous vein) different from that of our study; the only report on clinical chemistry does not provide information about the reference population or about the method employed to calculate the RI. Due to these limitations, it is difficult to statistically compare our results with those reported in literature. Nevertheless, hematologic RIs recorded in this study were similar to those of previous reports.1, 8

The most interesting findings were the high number of nRBCs, H–J bodies, and reticulocytes. In most species, these findings are suggestive of erythroid regeneration in response to erythropoietin release or of marrow-blood barrier damage and inappropriate erythroblastosis in the case of nonregenerative anemia.13-15 Hedgehogs have a nonsinusoidal spleen16 and this may explain the presence of H–J bodies, as demonstrated for horses and cats.13 Although these findings may depend on the juvenile age of the animals included in the study (in most species young animals have an intense erythropoiesis) or on splenic contraction due to stress, it is possible that RBCs in hedgehogs are released to blood earlier than in other species. Additional studies on mechanisms of erythropoiesis in hedgehogs are needed for a better understanding of these observations.

The automated PLT-I count, used in many veterinary diagnostic laboratories, underestimates the real number of platelets, likely due to the overlapping between small RBC and large PLT, a well-described phenomenon in cats.17 This hypothesis is confirmed by the detection of large hypergranular, polymoprhic platelets on blood smears, in the absence of excessive platelet clumping, and by the higher PLT-O (that differentiate large PLT from small RBCs), which provided counts similar to those reported for other species.18-20 The platelecrit (PCT) would probably provide a more accurate estimate of platelet mass in hedgehogs, as demonstrated in dogs.21 However, automated instruments do not accurately count large PLTs.22 In our hedgehogs, the instrument reported a flag of “abnormal” platelet morphology instead of the actual mean platelet volume or PCT in almost all cases; therefore calculation of an RI for this variable was not possible. Consequently, in the absence of PLT-O counts, the microscopic assessment of platelets on blood smears may represent the best method to estimate PLT numbers in hedgehogs.

WBC counts were similar to those previously reported1 and the differential count revealed a relative prevalence of lymphocytes, as established in other species, such as rats, mice,23 and ruminants.24 There were some LGL, which have been considered cytotoxic lymphocytes, at least in cattle;24 alternatively, the granulation could also indicate immune stimulation.25

An interesting finding were the relatively high relative and absolute basophil counts, which, in other species, have been associated with allergic conditions26 or helminthiasis.27 Whether this is a reasonable explanation in these study animals is questionable in the face of negative fecal examinations and regular antiparasitic treatments.28

In most species, the number of nuclear lobes increases with the maturation stage of neutrophils and eosinophils.29 Cells with bilobed nuclei could thus be erroneously interpreted as immature cells. Nevertheless, their morphology in hedgehogs was different from that of nonsegmented immature granulocytes. Therefore, they could be considered as normal mature cells with an atypical nuclear shape as it occurs in Pelger–Huet anomaly.30

The MCV was higher in females than in males, as already reported,1 but the RIs from both genders were largely overlapping, suggesting that this difference is significant from a statistical standpoint but not biologically relevant, as it does not affect the interpretation of the hemogram.

The RIs for biochemical values were generally wider than those of previous reports8 and had higher upper reference limits. In other species, high creatinine and urea concentrations are consistent with renal insufficiency.31 Nevertheless, based on the lack of symptoms or of other laboratory changes, the high values recorded in hedgehogs may depend on prerenal azotemia due to the protein overload associated with the use of commercial food for carnivores instead of a species-specific diet. Similarly, in spite of the high ALP and GGT activity, cholestasis is unlikely, as no other clinical or laboratory abnormalities were found. No information about ALP isoenzymes in hedgehogs is available. The bone isoenzyme, which is usually high in young animals due to bone remodeling,32 may be responsible for the high ALP activity. Alternatively, this may depend on the intestinal isoenzyme that mostly contributes to total ALP activity in rodents, especially in the case of fasting or damage.33

Significant differences were determined between animals managed in the 3 different centers, such as highest eosinophil counts in centers that did not practice anti-parasitic treatments, and lower total protein and globulin concentrations in center 3, which may depend on an overall cleaner environment, and consequently, less immune stimulation. Similarly, the dietary regimen may have influenced some metabolites, such as urea, glucose, and cholesterol concentrations induced by lipomobilization by a non-species-specific diet. Other differences were slight and probably biologically irrelevant (eg, nRBC, MCH) or attributable to the high individual variability (eg, creatinine).

In conclusion, the RIs determined in this study should be helpful in the monitoring and assessment of the health status of non-hibernized male and female hedgehogs in rehabilitation centers. Future studies on a larger number of animals with selected diseases are needed to assess the clinical diagnostic usefulness in routine practice of the RIs established in this study.