European brown hare syndrome virus in free-ranging European brown hares from Argentina

From 1998 to 2000, serum samples of 80 shot European brown hares (Lepus europaeus) from Argentina were examined for antibodies against European brown hare syndrome virus (EBHSV) and 80 spleen samples were tested for EBHSV-antigen by enzyme linked immunosorbent assay (ELISA). Nine hares were positive for EBHSV-antigen. Antibodies against EBHSV were detected in only one individual. Based on negative staining electron microscopy of spleen homogenates, we observed calicivirus in one of five EBHSV-antigen positive hares. However, EBHS has not been reported to cause abnormal mortality in these hares. This is the first report of antibodies to EBHSV, EBHSV-antigen, and electron microscopy findings in free-ranging European brown hares from South America.     

The occurrence of mountain hare mitochondrial DNA in wild brown hares

If interspecific hybrids are fertile and backcross to either parental species, transmission of mitochondrial DNA over the species barrier can occur. To investigate if such transmission has occurred between the brown hare Lepus europeus Pall and the mountain hare L. timidus L. in Scandinavia, an analysis of genetic variation in mitochondrial DNA from 36 hares, collected from 15 localities, was performed. Sequence divergence of mtDNA between species was estimated at 8 ± 1% (SD). Intraspecific mtDNA sequence divergence varied between 0.09 and 0.38% in brown hares and 0.10 and 1.44% in mountain hares. In six out of 18 brown hares examined, two different haplotypes of mountain hare origin were detected, demonstrating a transmission of mtDNA haplotypes from mountain hares to brown hares. The results indicate that interspecific hybridization between the two species occurs in wild populations.     

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.     

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.     

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.     

Natural toxoplasma gondii infections in European brown hares and mountain hares in Finland: proportional mortality rate, antibody prevalence, and genetic characterization

In material examined postmortem in Finland from May 2006 to April 2009, acute generalized toxoplasmosis was the immunohistochemically confirmed cause of death in 14 (8.1%) of 173 European brown hares (Lepus europaeus) and four (2.7%) of 148 mountain hares (Lepus timidus). Sera from 116 of the European brown hares and 99 of the mountain hares were screened with a commercial direct agglutination test for Toxoplasma gondii-specific IgG antibodies at a dilution of 1:40. All sera from cases of fatal toxoplasmosis had high titers of antibodies reactive to T. gondii. In contrast, none of 107 European brown hares and four (4%) of 96 mountain hares that died of other causes were antibody-positive. The proportional mortality rates and the T. gondii antibody prevalences among noncases differed significantly between the two host species (P<0.05). Direct genetic characterization of the causative agent was performed on DNA extracted from formalin-fixed, paraffin-embedded tissue of the hares with fatal toxoplasmosis. Based on the results with six microsatellite markers (B18, TUB2, TgM-A, W35, B17, and M33; all six in 15 cases and four in three cases), all the cases were caused by T. gondii genotype II; the size of the PCR product at the seventh marker (M48) varied (213-229 base pairs). The presence of T. gondii genotype II, which is endemic in Europe, is now confirmed in Finnish wildlife: Natural infections with T. gondii parasites belonging to this widespread genotype caused fatal generalized toxoplasmosis in the two species of wild hares.     

European brown hare syndrome virus: relationship to rabbit hemorrhagic disease virus and other caliciviruses.

Monoclonal antibodies directed against the capsid protein of rabbit hemorrhagic disease virus (RHDV) were used to identify field cases of European brown hare syndrome (EBHS) and to distinguish between RHDV and the virus responsible for EBHS. Western blot (immunoblot) analysis of liver extract of an EBHS virus (EBHSV)-infected hare revealed a single major capsid protein species of approximately 60 kDa that shared epitopes with the capsid protein of RHDV. RNA isolated from the liver of an EBHSV-infected hare contained two viral RNA species of 7.5 and 2.2 kb that comigrated with the genomic and subgenomic RNAs of RHDV and were recognized by labeled RHDV cDNA in Northern (RNA) hybridizations. The nucleotide sequence of the 3' 2.8 kb of the EBHSV genome was determined from four overlapping cDNA clones. Sequence analysis revealed an open reading frame that contains part of the putative RNA polymerase gene and the complete capsid protein gene. This particular genome organization is shared by RHDV but not by other known caliciviruses. The deduced amino acid sequence of the capsid protein of EBHSV was compared with the capsid protein sequences of RDDV and other caliciviruses. The amino acid sequence comparisons revealed that EBHSV is closely related to RHDV and distantly related to other caliciviruses. On the basis of their genome organization, it is suggested that caliciviruses be divided into three groups.     

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.     

The ubiquitous mountain hare mitochondria: multiple introgressive hybridization in hares, genus Lepus

Climatic oscillations during the glaciations forced dramatic changes in species distributions, such that some presently temperate regions were alternately occupied by temperate and arctic species. These species could have met and hybridized during climatic transitions. This phenomenon happened for three hare species present in Iberia (Lepus granatensis, Lepus europaeus and Lepus castroviejoi), which display high frequencies of mitochondrial DNA (mtDNA) from Lepus timidus, an arctic/boreal species presently extinct in Iberia. Here, we extend our previous geographical survey to determine whether the distribution of this mtDNA lineage extends beyond the northern half of the Iberian Peninsula, where it is found at high frequencies. We also review the taxonomy, distribution and molecular phylogeny of the genus Lepus. The phylogenetic inference reveals the presence of L. timidus-like mtDNA in several other hare species in Asia and North America, suggesting that the mitochondrial introgression observed in Iberia might be generalized. Comparison with the available nuclear gene phylogenies suggests that introgression could have happened repeatedly, possibly during different climatic transitions. We discuss demographic and adaptive scenarios that could account for the repetition in time and space of this spectacular phenomenon and suggest ways to improve our understanding of its determinants and consequences. Such high levels of introgressive hybridization should discourage attempts to revise hare taxonomy based solely on mtDNA.     

Scattered woody vegetation promotes European brown hare population

European brown hare populations have declined during the last decades. Agricultural intensification has been identified as a relevant driver of this process and agri-environment schemes have been implemented to foster biodiversity in agricultural landscapes. Because species-specific outcomes of measures strongly depend on tailored design of the policy framework and the local management, while changing climate may pose additional challenges, policy and management need science-based information of which landscape composition should be promoted to achieve set biodiversity goals.Here, we used direct observations of European brown hares over 20 years for evaluating the effects of landscape composition and weather conditions on European brown hare density. For the first time, our analysis compared the estimates of machine learning (gradient boosting machine) and linear mixed models in terms of importance of a wide range of explanatory variables for European brown hare densities and effect trends.Scattered woody vegetation, as represented by the two variables transitional woodland-shrub and small woody features, was on top rankings among the predictors and greater proportions of these elements were accompanied by sharp increases of European brown hare density. Also warmer winter temperature had a positive effect.We conclude that promoting scattered woody vegetation in agricultural landscapes is a powerful tool for improving European brown hare habitat quality. Particularly with the increasing dynamic in agriculture due to climate change, incentives and regulations that create a long-lasting heterogeneity in the landscape composition through near-natural elements can support the population of this popular mammal.     

Brown hares on the edge: Genetic population structure of the Danish brown hare

Denmark lies on the edge of the distributional range of the brown hareLepus europaeus Pallas, 1778, where population differentiation is most likely to occur. A total of 369 brown hares from eight geographically distinct Danish European brown hare populations were used to study the genetic population structure. In all, 480bp of the mitochondrial D-loop were sequenced in both directions. Observed genetic diversity (π) was relatively low (π=0.41%) while haplotype diversity (h=0.808) and the number of unique haplotypes (19) were similar to levels found in other European brown hare populations. The observed population structure was pronounced (pairwise conventionalF _ST and ϕ _st ranged between 6.9–57% and 5–69.8%, respectively). There was no correlation between the geographic and the genetic distance. Population structure was influenced by genetic drift, anthropogenic effects (eg translocation and escapes from hare-farms) and by post-glacial recolonization from southern refuges or refuges north east of the Black Sea. Analysis of historical population expansion/fluctuation events indicated that the populations have experienced different demographic events in the recent past. Relatively high sequence divergence between some populations might be explained by multiple recolonization events after the last Pleistocene glaciations or by stocking effects. Colonization from southern refuges was supported by the observation that haplotype 2 in the Danish brown hare was identical to the central European ancestral haplotype c07.     

Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males

This study aimed to establish artificial insemination (AI) protocols to predictably initiate pregnancy during the breeding season in the European brown hare (EBH) (Lepus europaeus PALLAS, 1778). Semen was collected from seven captive and eight free-ranging males by means of electroejaculation. Semen from the free-ranging males was cryopreserved using directional freezing. Total motility/integrity of fresh and frozen-thawed semen was 91.6%/87.7% and 46.9%/53.8%, respectively. Ovulation was induced in ultrasonographically preselected females using a gonadotropin-releasing hormone analogue. Each female was inseminated with 1 mL fresh (Group A, n = 16) or frozen-thawed semen (Group B, n = 9) at a concentration of 100 × 10⁶ spermatozoa/mL. The use of ultrasonography (10 to 22 MHz) confirmed the intracervical semen deposit, the success of artificial ovulation induction (formation of postovulatory corpus luteum), and permitted the monitoring of individual pregnancies. Although sperm motility/integrity was significantly different between groups, no significant difference was detected in conception rates (A, 87.50%; B, 77.78%). Because of embryonic resorption, there was a slight difference in fertility rate between groups (A, 62.5%; B, 77.78%). Overall, AI in captive EBH using fresh and frozen-thawed semen achieved successful fertility rates. Long-term cryopreserved semen was used to bring new genetic material from the wild into a genetically limited captive population without extensive animal transport. Therefore, AI has the potential to enhance breeding programs for EBH especially when cryopreserved semen from wild donors is used.     

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.     

Unpreferred plants affect patch choice and spatial distribution of European brown hares

Many herbivore species prefer to forage on patches of intermediate biomass. Plant quality and forage efficiency are predicted to decrease with increasing plant standing crop which explains the lower preference of the herbivore. However, often is ignored that on the long-term, plant species composition is predicted to change with increasing plant standing crop. The amount of low-quality, unpreferred food plants increases with increasing plant standing crop. In the present study the effects of unpreferred plants on patch choice and distribution of European brown hare in a salt-marsh system were studied. In one experiment, unpreferred plants were removed from plots. In the second experiment, plots were planted with different densities of an unpreferred artificial plant. Removal of unpreferred plants increased hare-grazing pressure more than fivefold compared to unmanipulated plots. Planting of unpreferred plants reduced hare-grazing pressure, with a significant reduction of grazing already occurring at low unpreferred plant density. Spatial distribution of hares within this salt-marsh system was related to spatial arrangement of unpreferred plants. Hare-grazing intensity decreased strongly with increasing abundance of unpreferred plants despite a high abundance of principal food plants. The results of this study indicate that plant species replacement is an important factor determining patch choice and spatial distribution of hares next to changing plant quality. Increasing abundance of unpreferred plant species can strengthen the decreasing patch quality with increasing standing crop and can decrease grazing intensity when preferred food plants are still abundantly present.     

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.