Sexing European rabbits (Oryctolagus cuniculus), European brown hares (Lepus europaeus) and mountain hares (Lepus timidus) with ZFX and ZFY loci

We describe a new method for the sex determination of tissue originating from Oryctolagus cuniculus (European rabbit), Lepus europaeus (European brown hare) and Lepus timidus (mountain hare) based on PCR-RFLP analysis of point mutations that differentiate the ZFX and ZFY gene sequences. Among several applications, this PCR-RFLP method could be used to investigate gender ratio and evaluate the population dynamics of these species using samples collected when sex cannot be identified.     

Temporal dynamics of European brown hare syndrome infection in Northern Italian brown hares (Lepus europaeus)

The progressive decline in the hare population across Europe has been associated with the occurrence of European brown hare syndrome (EBHS), a highly contagious disease considered endemic in all European countries. This study aimed to evaluate the in-field temporal dynamics of European brown hare syndrome virus (EBHSV) infection in wild European brown hares (Lepus europaeus) and to test the influence of population density on EBHS seroprevalence. A total of 512 blood samples were collected from free ranging hares captured for restocking in seven different areas of the province of Brescia (Northern Italy) during seven consecutive years (2006–2013) and tested using a competitive ELISA. A generalized linear mixed model estimated the yearly effects of population density on EBHS prevalence. Of the 512 tested, 344 (67.2 %) tested positive for EBHSV antibodies, with the annual seroprevalence ranging from 94.3 to 35.8 %. The prevalence was 3.303 times higher in areas with a density of over 15 hares/km² and declined over the years. The results indicate the ongoing transmission of the virus in the tested brown hare population. Since the eradication of EBHS in a wild population is not feasible, a strategy aimed at promoting the endemic stability of the virus through density-dependent mechanisms could be applied; however, this seems more difficult in practice than in theory and would most likely require a very high density of brown hares.     

A single multiplex of twelve microsatellite markers for the simultaneous study of the brown hare (Lepus europaeus) and the mountain hare (Lepus timidus)

The management of hunted species is challenging, as it must conciliate the conservation of species and their sustainable exploitation. Nongenetic tools are widely used in this context but they may present limitations notably when species can hybridize or when large‐scale spatial monitoring is required to establish optimal management actions. This is why genetic tools have been more and more integrated in wildlife management practices. However, the markers proposed are often amplified in small multiplexes when larger ones could allow to better cope with the small quantities of DNA obtained with noninvasive sampling methods. Here, we propose a unique multiplex of 12 autosomal microsatellite markers for the study of two hare species that exist in sympatry in some areas in Europe and are hunted notably in France: the brown hare Lepus europaeus and the mountain hare L. timidus. We tested 17 markers previously used in these two species or other lagomorph species, from which 12 were included in this single multiplex. Diversity was between 4 and 30 alleles per locus totaling 126 alleles, and we showed that these markers possess appropriate genetic resolution for individual and species identification for the populations under study. This multiplex panel represents the largest number of microsatellites amplified in one reaction proposed for these two hare species and provides a cost‐effective and valuable tool for further hybridization studies and the management of hares.     

The distribution of mountain hares Lepus timidus in Europe: a challenge from brown hares L. europaeus?

1. Throughout the most recent glacial period (Weichsel), the mountain hare Lepus timidus had a continuous distribution in the tundra habitat south of the ice‐rim. When the ice retreated, mountain hares colonized deglaciated land, and spread over northern Europe. 2. Since the Weichsel, the mountain hare's distribution in Europe has been gradually reduced and at present comprises Ireland and the Scottish Highlands, high altitudes in the Alps, isolated forests in eastern Poland, most of Fennoscandia and from the Baltic countries eastwards through Russia. Declines during the last century have been observed in Sweden and Russia. 3. This review defines and evaluates causes for this gradual reduction and fragmentation of the mountain hare's distribution, with special focus on interactions with brown hares Lepus europaeus. The relative importance of diseases, predation, cultivation and interactions with other herbivores than brown hares are discussed. 4. A plausible cause of the possible permanent disappearance of mountain hares in Europe appears to be exclusion by interspecific competition and hybridization with, and/or epidemic diseases mediated by, the congeneric brown hare.     

Factors related to the occurrence of hybrids between brown hares Lepus europaeus and mountain hares L. timidus in Sweden

Hybridization occurs among many species, and may have implications for conservation as well as for evolution. Interspecific gene flow between brown hares Lepus europaeus and mountain hares L. timidus has been documented in Sweden and in continental Europe, and has probably to some extent occurred throughout history in sympatric areas. What local factors or ecological relationships that correlate with or trigger hybridization between these species has however been unclear. We studied spatial distribution of hybrids between brown hares and mountain hares in Sweden in relation to characteristics of the sampled localities (hunting grounds). In a sample of 70 brown hares collected from 39 populations in south‐central Sweden during 2003–2005, 11 (16%) showed introgressed mtDNA from mountain hares. Among the brown hares from their northern range, i.e. in general the most recent establishments, the corresponding figure was 75% (9/12). The frequency of samples with hybrid ancestry increased significantly with latitude, altitude and hilliness, and were higher (p<0.1) in recently established populations and/or where the proportion of arable land was low. Several site‐specific parameters were correlated, e.g. latitude as expected to hilliness, and no parameter explained the occurrence of hybrids exclusively. Instead, the appearance of mountain hare mtDNA among brown hares was associated with a conglomerate of parameters reflecting landscapes atypical for the brown hare, e.g. forest dominated and steep areas where the species quite recently was established. We suggest that these abiotic factors mirror the main aspect influencing hybridization frequency, namely the density or relative frequency of the two species. In atypical brown hare landscapes with recent establishment, mountain hares are probably relatively more common. When one species dominate in numbers, or when both species display low densities, increased frequency of hybridization is expected due to low availability of conspecific partners, a phenomenon referred to as Hubbs’ principle.     

Outbreak of Cronobacter turicensis in European brown hares (Lepus europaeus)

This is the first report of acute deaths in five European brown hares (Lepus europaeus) attributed to mucoid and necrotizing typhlocolitis caused by genetically different Cronobacter (C.) turicensis strains in northeastern Austria. As this opportunistic pathogen is mainly known for causing disease in immunocompromised humans and neonates, this previously unrecognized potential for a spill over from a wildlife reservoir to humans warrants further attention.     

Unilateral hydrocephalus in a wild European brown hare (Lepus europaeus)

Macroscopic and histological study of a case of unilateral hydrocephalus in a young wild European brown hare (Lepus europaeus) is reported here. Morphological changes were mostly massive dilatation of the left lateral ventricle, atrophy of the cortex and a striking reduction of the corresponding white matter. Lymphocytic cell infiltrates and hemosiderin-laden macrophages in the choroid plexus and meninges also were observed. Although the exact cause of the lesions was not found all findings suggested a hydrocephalus of possible traumatic, infectious or congenital hypoplasia origin.     

European brown hare syndrome in free-ranging European brown and mountain hares from Switzerland.

From 1997 to 2000, complete necropsy and histopathologic investigations were performed on 157 free-ranging European brown hares (Lepus europaeus) found dead throughout Switzerland. Organ samples of all these individuals (157 livers and 107 spleens available) were tested for European brown hare syndrome virus (EBHSV)-antigen by enzyme-linked immunosorbent assay (ELISA) test kit. Furthermore, 60 additional blood samples were tested for antibodies against EBHSV by ELISA. In addition, liver samples of 87 free-ranging mountain hares (Lepus timidus) hunted in 1996 were tested for EBHSV-antigen. In two European brown hares from southern Switzerland lesions suggestive of changes induced by EBHSV were present, and high titers of EBHSV-antigen were detected in both liver and spleen samples of these animals. Based on negative staining electron microscopy investigations of liver and spleen homogenates, we observed calicivirus in one antigen-positive hare. Low EBHSV-antigen titers were found in three additional European brown hares from central and western Switzerland, but EBHS-lesions were absent. Antibodies against EBHSV were not detected in any of the sera of European brown hares, and EBHSV-antigen was not found in the samples of mountain hares. This is the first report of EBHS in European brown hares from Switzerland.     

European hare (Lepus europaeus) invasion ecology: implication for the conservation of the endemic Irish hare (Lepus timidus hibernicus)

European hare Lepus europaeus populations have undergone recent declines but the species has successfully naturalised in many countries outside its native range. It was introduced to Ireland during the mid-late nineteenth century for field sport and is now well established in Northern Ireland. The native Irish hare Lepus timidus hibernicus is an endemic subspecies of mountain hare L. timidus and has attracted major conservation concern following a long-term population decline during the twentieth century and is one of the highest priority species for conservation action in Ireland. Little is known about the European hare in Ireland or whether it poses a significant threat to the native mountain hare subspecies by compromising its ecological security or genetic integrity. We review the invasion ecology of the European hare and examine evidence for interspecific competition with the mountain hare for habitat space and food resources, interspecific hybridisation, disease and parasite transmission and possible impacts of climate change. We also examine the impact that introduced hares can have on native non-lagomorph species. We conclude that the European hare is an emerging and significant threat to the conservation status of the native Irish hare. Invasive mammal species have been successfully eradicated from Ireland before and immediate action is often the only opportunity for cost-effective eradication. An urgent call is issued for further research whilst the need for a European hare invasive Species Action Plan (iSAP) and Eradication strategy are discussed.     

Cladogenesis of the European brown hare (Lepus europaeus Pallas, 1778)

A substantial portion of today’s biodiversity is attributed to the climatic oscillations of the Pleistocene Ice Ages. Gradual but dramatic climate changes were accompanied by expansion, contraction, and isolation of populations, promoting the accumulation of genome differences and adaptations in refugial populations and resulting in allopatric differentiation in a variety of taxa. In the present study, partial mitochondrial DNA sequences of the widely distributed European brown hare (Lepus europaeus) were analyzed to test whether the species’ present genetic structure is the result of postglacial re-colonization of Europe from Asia Minor (clade A) and the Balkans (clade B) only, as suggested previously, or if additional refugia are likely. Analyses indicated the presence of an additional refugium (Italy, clade I). The genealogic network of Italian hares displayed the tree-like structure expected from refugial populations, whereas central European brown hare haplotypes revealed a clear star-phylogeny indicative of past-bottleneck population growth. This population size expansion, which was confirmed by mismatch analysis, was estimated to have occurred ∼50–55 thousand years ago (kya). The divergence of clade A* from the remaining matrilines is estimated at 239 kya, whereas the divergence of the ancestors of clades B* and I from A* occurred about 128 kya. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Acute hepatosis in the European brown hare (Lepus europaeus) in Italy.

Since October 1986 an unusually high mortality has been observed both in wild European brown hares (Lepus europaeus) and in hare farms in Italy. Pathological alterations, including severe hepatosis, nephrosis, congestion and hemorrhages of tracheal mucosa and lungs, and splenic vascular congestion, were observed in 179 of 381 hares necropsied from 1986 to 1988. Jaundice also was seen in 30% of these hares. Histologically, the liver damage was characterized by coagulative necrosis, mainly located around the portal areas, or by degenerative changes. Hyperemia, focal hemorrhages and periportal mononuclear cell infiltration were also present. The epithelium of renal tubules showed the presence of various degrees of vacuolar degeneration and necrosis, and eosinophilic granular hyaline casts or homogenous proteinaceous material were found within the lumen of tubules. Only the adult hares were affected. In wild hare populations night counts revealed a reduction of the number of observed wild hares during the winter period which ranged from 27 to 40%, whereas in hare farms the mortality ranged from 30 to 90%. Bacteriological, parasitological, and toxicological investigations were unable to confirm the primary cause of these deaths. Negative stain electron microscopy and immunoelectronmicroscopy conducted since October 1988 on liver and spleen homogenates from hares with acute hepatosis revealed the presence of viral particles with morphological aspects resembling those of calicivirus, antigenically related to the etiological agent of viral haemorrhagic disease of rabbits.     

Infections with Francisella tularensis biovar palaearctica in hares (Lepus timidus, Lepus europaeus) from Sweden

The occurrence of tularemia was studied in 1,500 hares submitted to the National Veterinary Institute, Uppsala, Sweden for postmortem examination during 1973 through 1985. A total of 109 tularemia cases was recorded based on the fluorescent antibody (FA) test for Francisella tularensis and on the gross and microscopic pathology. Tularemia was diagnosed only in the varying hare (Lepus timidus) and not in the European brown hare (Lepus europaeus). The geographical distribution of the 109 cases indicates that tularemia has not spread in Sweden during the last 45 yr, with the exception of an endemic occurrence of the disease on the island of Stora Karls? in the Baltic sea. The disease was most frequent in the autumn and only a few cases were recorded during winter. Cases were not seen in the spring. The annual prevalence varied, with several cases in 1974 and 1981, but there were no cases in 1976 and 1980. The postmortem findings in hares dying of tularemia in the autumn were characterized by focal coagulative necrosis in liver, spleen and bone marrow, with high numbers of bacteria FA-positive for F. tularensis. In hares dying during winter months, the most characteristic findings were hemorrhagic enteritis and typhlitis, although necrotic lesions could occur in liver, spleen and bone marrow. Diseased hares on the island of Stora Karls? were demonstrated to be infected with ticks, while hares on the mainland of Sweden generally were fed upon by mosquitoes. Twenty-six of the 109 hares with tularemia were examined bacteriologically and F. tularensis biovar palaearctica was isolated from eight. The lung extract antibody test for F. tularensis was performed in 18 of the 109 hares. All were negative. In addition to the field study, an experimental study with F. tularensis biovar palaearctica was performed. Four varying hares and three European brown hares were inoculated. None of the hares died from tularemia, and generalized infection was not demonstrated.     

Taxon 20 (Fam. Pasteurellaceae) infections in European brown hares (Lepus europaeus).

Hemolytic bacteria, phenotypically related to organisms previously identified as Pasteurella haemolytica and tentatively named Taxon 20, were isolated from cases of purulent bronchopneumonia and from conjunctivitis in European brown hares (Lepus europaeus). The bronchopneumonia, sometimes accompanied by lesions in other organs, occurred without other concomitant disease. The conjunctivitis was found mainly in animals suffering from the European brown hare syndrome.     

Biochemical genetic variability in brown hares (Lepus europaeus) from Greece

Allozyme variability of 91 brown hares (Lepus europaeus) from seven regions in Greece was compared to existing data of Bulgarian populations to test the hypothesis of the occurrence of specific alleles in Greece, likely stemming from an isolated Late Pleistocene refugial population in the southern Balkans. This hypothesis is particularly suggested by some subfossil Late Pleistocene hare remains in Greece and the reported high mtDNA diversity in Greek hares. Allozymic diversity could be higher in Greek hares than in hares from neighboring regions as a result of the accumulation of variants in a long-lasting Pleistocene refugium. Conversely, Greek hares could exhibit reduced genetic diversity because of long-lasting low effective population sizes during the Late Glacial Maximum and a lower chance of postglacial gene flow from other populations into this rather marginal part in the southern Balkans. Horizontal starch gel electrophoresis of proteins from 35~loci revealed three alleles (Es-1 ^–162, Pep-2 ¹¹⁴, Mpi ⁸⁸) at low frequencies, which were not found in Bulgarian or any other brown hare population. In contrast, some alleles from the populations from Bulgaria and other regions of Europe were absent in the Greek samples. Population genetic statistics indicated only a slight tendency of increased gene pool diversity in Greek hares, little substructuring in Greek and Bulgarian populations, respectively, as well as an only slightly lower level of gene flow between the two neighboring regions, as compared to the gene flow within each region. The results conform to the hypothesis of a Late Pleistocene refugial population in the southern Balkans, with some few specific nuclear gene pool characteristics, but little effect on the overall genetic differentiation between Greek and Bulgarian hares.     

Habitat selection and activity patterns in Alpine mountain hare (Lepus timidus varronis)

We investigated activity patterns and habitat use of 34 radio-tracked mountain hares (Lepus timidus) in the Italian Alps. We first showed that hares were nocturnal and that activity patterns changed seasonally in parallel with circadian rhythms. We predicted that day home ranges will include suitable resting (shelter) habitats, and night home ranges will primarily include suitable foraging habitats. A hare's night-range was larger than its day-range. On average, night and day ranges overlapped by 36%, suggesting that selective pressures affecting space use were, at least partly, different at night than day. Dwarf mountain-pine was the most preferred habitat in all seasons and was selected both for active behaviour (night) and resting (day) and hares avoided the most open habitats. Exploring the effects of season, time of day (day vs. night) and site, we found that habitat use by mountain hares did not differ between seasons or between the active (night) and resting (day) period of circadian cycle. Also, we found no effects of differences in landscape structure (habitat patchiness and heterogeneity) on the patterns of habitat selection. Hares always preferred the dense, forested habitats, which seemed to provide food resources as well as shelter from predators throughout the year.