Apoptosis and proliferation during seasonal testis regression in the brown hare (Lepus europaeus L.)

Testes samples of 52 brown hares (Lepus europaeus L.), sacrificed between July and January, were subjected to immuno histochemical analysis. The terminal deoxynucleotidyl transferase-mediated d'UTP nick end labelling (TUNEL) method was applied to detect apoptosis; and antibodies to proliferating cell nuclear antigen (PCNA) were used to evaluate cell proliferation in the testes. In the seminiferous epithelium, the apoptotic processes were evident from August to early November with maximal values in September. Cell death in germ cells occurs predominantly during the prophase of the first meiotic division. In July, and from mid-November onwards, only the occasional TUNEL-positive cells can be seen. The proliferation of germ cells continues during the testis regression phase. The average number of PCNA-positive cells decreases slightly from September onwards and rises again in mid-November.     

Tuberculosis in a free-living brown hare (Lepus europaeus occidentalis)

A free-living brown hare (Lepus europaeus occidentalis) trapped during a survey of tuberculous possums in the Ahaura Valley, Westland, was found to have both microbiological and histopathological evidence of infection with Mycobacterium bovis. The mesenteric lymph nodes were enlarged and showed extensive caseation, tuberculous nodules were present in the liver and kidney, and there was a proliferative pleurisy. Histologically, characteristic tuberculous granulomatous foci were seen in the mesenteric lymph nodes, liver, kidney, pleura and lungs, often in association with small numbers of acid-fast organisms. This is the first report of naturally occurring tuberculosis due to M. bovis in the brown hare.     

Tuberculosis in a free-living brown hare (Lepus europaeus occidentalis)

A free-living brown hare (Lepus europaeus occidentalis) trapped during a survey of tuberculous possums in the Ahaura Valley, Westland, was found to have both microbiological and histopathological evidence of infection with Mycobacterium bovis. The mesenteric lymph nodes were enlarged and showed extensive caseation, tuberculous nodules were present in the liver and kidney, and there was a proliferative pleurisy. Histologically, characteristic tuberculous granulomatous foci were seen in the mesenteric lymph nodes, liver, kidney, pleura and lungs, often in association with small numbers of acid-fast organisms. This is the first report of naturally occurring tuberculosis due to M. bovis in the brown hare.     

Blood coagulation times in the European brown hare (Lepus europaeus)

Background:  Many causes of mortality in the European brown hare, such as bacterial and viral infections, anticoagulant poisoning, and trauma, may result in hemorrhage. There are, however, no reference values concerning blood clotting in this species.  Objectives:  The aim of this study was to determine reference values for blood coagulation times and related parameters in healthy European brown hares.  Methods:  Blood samples from 30 clinically healthy adult hares (15 males and 15 females) were obtained. Hares were physically restrained for blood collection from the cephalic vein into tubes containing citrate and EDTA.  Results:  Mean ± SD were obtained for thrombin time (TT) (13.97 ± 1.37 seconds), prothrombin time (PT) (13.32 ± 2.15 seconds), activated partial thromboplastin time (APTT) (16.73 ± 1.86 seconds), fibrinogen concentration (2.98 ± 1.06 g/L), and platelet count (355.28 ± 128.73 × 10⁹/L). Conclusions: Reference values for blood coagulation times and other parameters associated with blood clotting will be useful in the laboratory evaluation of hemorrhage in the European brown hare.     

Antibodies to Neospora caninum in European brown hare (Lepus europaeus)

Neospora caninum antibodies in European brown hares (Lepus europaeus) were reported for the first time in sera collected from hares imported in Italy from East Europe. Sera from 93 hares coming from Hungary and 44 hares coming from Slovakia were tested using a direct agglutination test (DAT). Seroprevalence was 8.0% at the 1:40 dilution and 1.5% at 1:320 dilution. Seroprevalence in females and males and in different countries of origin did not differ significantly. Our results indicate that European brown hares are exposed to N. caninum. The low prevalence observed suggests a hares minimal exposure, but, considering that hares are common prey for foxes, it could be supposed that, also with a low prevalence, hares can be an important source of N. caninum infection in foxes.     

Spontaneous lesions in the aorta of the common brown hare (Lepus europaeus).

The intimal surface of the aortic arch of 16 of 20 male common brown hares and 10 of 14 females had raised pale yellow to grey-white plaques up to 6 mm in diameter. These lesions were characterised by necrosis and calcification mainly in the tunica media and were not related to the age of the hares. In the hare this vascular disease may develop after injury to the connective tissue during obliteration of the ductus arteriosus. Although these aortic lesions were not the 'spontaneous' atherosclerotic variety they were morphologically similar to those in rabbits.     

Comparison of acetamide and dimethylsulfoxide as cryoprotectants of European brown hare (Lepus europaeus) spermatozoa

The aim of the present study was to assess the effect of dimethylsulfoxide (DMSO) and acetamide on the post-thaw properties of hare semen and to perform an AI trial with frozen-thawed semen. Semen was collected under general anaesthesia by the electroejaculation method from 6 males. Immediately after collection, the semen was diluted with an extender containing the following components: 250 mM Tris, 80 mM citric acid, 70 mM glucose, 1.0 M DMSO, egg yolk (17% v/v) and kanamycin (80 mg/l); this extender was used for Protocol I (n=17). In Protocol II (n=15), the DMSO was replaced with 1.0 M acetamide. Immediately after thawing and after incubation for 90 and 180 min at 37 C, the properties of semen were evaluated by computer-assisted semen analysis, and the percentage of viable, acrosome intact spermatozoa was evaluated using flow cytometry. During the 3-h incubation, the percentages of motile spermatozoa and spermatozoa with progressive motility were significantly higher in Protocol I (P<0.01). Immediately after thawing, path and straight velocity were significantly higher in Protocol I (P<0.01), as was the curvilinear velocity (P<0.05). The amplitude of lateral head displacement was higher after 3-h incubation in Protocol I (P<0.05), and no differences in beat cross frequency were found between Protocol I and II at any incubation time. The percentage of viable, acrosome intact spermatozoa determined with flow cytometry was higher in Protocol I (P<0.01) at all incubation times. As a result of artificial insemination with the semen frozen with DMSO as a cryoprotectant, two out of three inseminated females delivered two healthy young each. Following artificial insemination with the semen frozen with acetamide as a cryoprotectant, two out of three inseminated females delivered one healthy young each. On the basis of the results, it should be stated that DMSO ensures better post-thaw properties of hare spermatozoa than acetamide.     

Preliminary reference values for electrocardiography,echocardiography and myocardial morphometry in the European brown hare (Lepus europaeus)

The study aimed at defining reference values for electrocardiographic (ECG) and echocardiographic parameters as well as macroscopic dimensions of the heart and microscopic dimensions of cardiomyocytes in the European brown hare. The studies were conducted on 30 adult, clinically healthy hares of either sex caught in Poland. ECG and echocardiography were performed supravitally on anaesthetized hares. After euthanasia, gross and microscopic myocardial and cardiomyocyte dimensions were determined. Heart rate amounted to 140 ± 37.5 beats/min, the leading rhythm involved the sinus rhythm. P wave time was 26 ± 5 ms, PQ time was 80 ms, QRS time was 29 ± 3.5 ms, and ST was 97.5 ± 7 ms. Echocardiography determined a left ventricular wall end-diastolic diameter of 8.6 ± 2.0 mm and an intraventricular septum end-diastolic diameter of 5.75 ± 1.0 mm. The thickness of the interventricular septum corresponded to that of the free wall of the left ventricle, a finding consistent with physiological hypertrophy. Preliminary reference values were established for echocardiography. The findings were similar to those obtained at necropsy. The ECG and echocardiographic studies represent the first supravital examination of cardiac function in the hare. The obtained results illustrate adaptation of hare''s myocardium to its mode of life. The cardiac findings resemble the athlete''s heart syndrome described in humans. The findings may prove useful in further studies on the physiology of the cardio-vascular system in the hare.     

Detection of Francisella tularensis subsp. holarctica in a European brown hare (Lepus europaeus) in Thuringia, Germany

The isolation of Francisella tularensis subsp. holarctica biovar II (strain 06T0001) from a European brown hare (Lepus europaeus) from Thuringia, Germany, is described for the first time. Identification of the microorganism was carried out by phenotypic characterisation, partial sequencing of the 16S rRNA gene and specific PCR using the primers TUL4-435/TUL4-863 and FtC1/FtC4. The epidemiology of tularemia in Germany is discussed and a risk assessment for humans is made.     

The etiology of liver dystrophy in the field hare (Lepus europaeus pallas)

The liver dystrophy of the European Brown Hare is widely distributed in Europe and predominantly called European Brown Hare Syndrome (EBHS) or Hemorrhagic Septicemia Syndrome. The disease could be reproduced by injection of sterile filtered organ material of two field cases in two hares held in captivity. The clinical symptoms and the pathologic alterations are described.     

Contents of selected heavy metals in the liver, kidneys and abdominal muscle of the brown hare (Lepus europaeus Pallas, 1778) in Central Pomerania, Poland

The content of iron, zinc, copper, manganese, lead, and cadmium was determined in the kidney, liver, and abdominal muscle of the brown hare (Lepus europaeus), a species known for its bioindicative potential in environmental quality assessment. The animals assayed were divided into immature (in their first year of life; n=25) and adult (n=39). The hares were acquired in an area situated far away from major cities and direct impacts of industrial and transport pollution. The zinc content found in the assayed hare's muscles (25.9 microg/g w.w.) and that of manganese in the kidneys, liver, and muscles (2, 2.51 i 0.85 microg/g w.w., respectively) tended to be higher than those reported from the brown hare elsewhere in Europe. The content of cadmium (particularly in the kidneys) and lead proved substantial and close to those typical of the hare inhabiting industry-affected areas. Analysis of the data pooled for all the individuals showed Zn and Cd contents to increase with age in the kidneys, the liver Cd content increasing with age as well; on the other hand, the age-Cu content was negative. In addition, a number of significant correlations between the metals themselves were revealed, particularly with respect to Zn-Cd correlation in kidneys (r(s)=0.47), Fe-Cd, Zn-Cu, Mn-Zn, Mn-Cu, and Mn-Pb in the liver (r(s) of 0.42, 0.86, 0.72, 0.79, and 0.42, respectively), and Zn-Cu in the muscle (r(s)=0.56). The kidney cadmium content was higher by 81% in the adult than in the immature hare, the adult hare muscle copper content being lower by 15.5%.     

Health screening of free-ranging European brown hares (Lepus europaeus) on the German North-Sea island Pellworm

Background

A sudden decline of the European brown hare (Lepus europaeus) population in one of the best hunting districts for small game species in northern Germany, the German North-Sea island Pellworm, in the years 2007/08 following marked habitat changes led to the implementation of a thorough health assessment program of the population. 110 animals were collected during the normal hunting season in the years 2010 and 2011. A post-mortem examination and histopathological investigation was performed on all animals. Additionally, routine bacteriology of the small intestine and parasitology were carried out. Sera of hares were tested for European Brown Hare Syndrome (EBHS) by enzyme linked immunosorbent assay, and for Treponema sp. by indirect immunofluorescent test. Additional testing was performed when deemed necessary.

Results

The most striking result was a shift in the intestinal bacterial flora towards Gram-negative Enterobacteriaceae with a predominance of either Escherichia coli, or Aeromonas sp., or a high-grade double-infection with these two pathogens with subsequent catarrhal enteritis. Additionally, a marked coccidiosis, and varying infestations with the nematode Trichostrongylus retortaeformis were found. The sero-prevalence for EBHS was 78.1%, and for Treponema 43.9%.

Conclusions

The shift and decrease in diversity of the intestinal flora was the main and most consistent result found. In the authors’ opinion the change of the habitat combined with other stressors increased the animals’ sensitivity to ubiquitous bacterial species and parasites which usually would not have such fatal effects.     

Fatal toxoplasmosis in brown hares (Lepus europaeus): possible reasons of their high susceptibility to the infection

Brown hares (Lepus europaeus) trapped in the countryside and domestic rabbits were experimentally infected with Toxoplasma gondii (K7 strain) oocysts. Hares (n=12) were divided into groups of 4 and infected with 10, 10(3) and 10(5) oocysts. Rabbits (n=12) were infected in the same way. The experimentally infected animals were monitored for 33 days after infection (p.i.). Most of the infected hares demonstrated behavioural changes, and all of them died between 8 and 19 days p.i. Three of the rabbits demonstrated only clinical changes related to the concurrent pasteurellosis. The typical pathological finding in the hares were haemorrhagic enteritis, enlargement and hyperaemia of mesenteric lymph nodes, splenomegaly and multiple miliary necrotic lesions in the parenchyma of the liver and other organs. Pathological changes in the rabbits were less pronounced than in the hares. In rabbit brains, tissue cysts of the T. gondii were found. The incidence of T. gondii antibodies both in the hares and the rabbits was first ascertained on day 7 p.i. On day 12 p.i., antibodies were already found in all the animals infected. Antibody titres in indirect fluorescence antibody test (IFAT) using the anti-rabbit conjugate were markedly higher in rabbits than in hares. In all hares, T. gondii was isolated post mortem from the liver, brain, spleen, kidney, lung, heart and skeletal muscles. Although T. gondii was also isolated in all rabbits, it was not always isolated in all their organs. In all hares, parasitemia was demonstrated on days 7 and 12 p.i. The percentage of rabbits with detected parasitemia was lower. In hares, a decrease in the numbers of leukocytes during the infection was observed. No such decrease was observed in the rabbits. The lymphocyte activity after the stimulation with non-specific mitogens showed significant differences between the hares and the rabbits even before the infection. After the infection, the hares infected with 10(3) and 10(5) doses and in rabbits infected with a 10(5) dose showed a decrease of lymphocyte activity. Rabbits infected with a 10(3) dose showed an increase of the lymphocyte activity. While in hares toxoplasmosis was an acute and fatal disease, the infection in rabbits had subclinical manifestations only and easily passed to a latent stage. The different courses of toxoplasmosis in the hare and the rabbit may be due to the differences in the natural sensitivity of the two species to the T. gondii infection or a negative impact of stress to the immune status of hares.     

Acepromazine‐dexmedetomidine‐ketamine for injectable anaesthesia in captive European brown hares (Lepus europaeus)

ObjectiveTo evaluate a combination of acepromazine, dexmedetomidine and ketamine (ADK) on induction and recovery from anaesthesia, and on physiological parameters in hares undergoing non‐invasive procedures.Study designProspective clinical study.AnimalsSixteen European hares (Lepus europaeus), seven males and nine females, aged (mean ± SD) 3.25 ± 0.9 months and weight 2.1 ± 0.6 kg.MethodsAcepromazine 1% (A), dexmedetomidine 0.05% (D) and ketamine 5% (K) were mixed and given intramuscularly (IM) at 0.25 mL kg^?1, representing 10 mg kg^?1 K, 0.25 mg kg^?1 A, 12.5 μg kg^?1 D. If the righting reflex was present after four minutes, a second injection of 0.15 mL kg^?1 (6 mg kg^?1 K, 0.15 mg kg^?1 A, 7.5 μg kg^?1 D) was administered IM. Surgical anaesthesia was judged as present when righting, palpebral, ear‐pinch and pedal withdrawal reflexes were absent. Anaesthetized hares were tagged, and underwent blood sampling and ocular ultrasound examination. Physiological parameters were recorded every ten minutes, and were compared by Kruskal‐Wallis tests.ResultsA single dose induced loss of righting reflex in 11/16 (69%) hares within four minutes; the second dose was effective in the remaining hares. Ten minutes after the loss of the righting reflex, a surgical plane of anaesthesia was present in all hares. Sleep time to regaining righting reflex was 34 ± 11 (range 21–62) minutes and recovery was calm. Although there were some statistical differences over time, cardiovascular parameters remained within an acceptable range but there was respiratory depression and hares were hypoxemic.Conclusions and clinical relevanceThe ADK mixture produced a smooth and rapid induction of anaesthesia, a low incidence of untoward side effects and full recovery after four hours. Supplementary oxygen might be advisable if a deeper plane of anaesthesia was required. Chemical restraint was adequate to perform non‐invasive procedures.