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.     

Epizootiologic and ecologic investigations of European brown hares (Lepus europaeus) in selected populations from Schleswig-Holstein, Germany

From 1997-99 European brown hare (Lepus europaeus) population densities were estimated by spotlight surveys within different areas in Schleswig-Holstein, Germany. These areas showed a wide variation in local hare population densities. In addition, red fox (Vulpes vulpes) densities were estimated in 1997 by surveys of fox dens and litters. Sera of 321 hares (shot between 1998-2000) from four study areas were examined for antibodies against European brown hare syndrome virus (EBHSV) by enzyme linked immunosorbent assay (ELISA), Yersinia spp. (n = 299) and Francisella tularensis (n = 299) by western blotting, Brucella spp. by Rose Bengal test, and Toxoplasma gondii by Sabin-Feldman test (n = 318). Tissue samples comprising lung, liver, spleen, kidney, heart, and adrenal glands were collected for histopathology. Liver (n = 201) and spleen (n = 201) samples were processed for the detection of T. gondii-antigen in tissue sections and 321 liver and spleen samples were investigated for EBHSV-antigen by ELISA. Furthermore, 116 hares were examined macro- and microscopically for lungworms. Significant negative correlations between hare and fox densities were found in spring and autumn 1997. Antibodies against EBHSV were detected in 92 of 321 (29%), against Yersinia spp. in 163 of 299 (55%), and against T. gondii in 147 of 318 (46%) hares. We evaluated the potential influence of origin and hunting season on exposure rates of hares using logistic regression analysis. A strong association between hare densities and exposure rates was observed for various agents. One hundred and eight of 201 (57%) hares were positive for T. gondii-antigen. All sera were negative for antibodies against Brucella spp. and F. tularensis and all lung samples were negative for lungworms. In conclusion, variation in red fox densities may have an impact on the hare populations examined and the infectious diseases we studied seem to play a subordinate role in the dynamics of European brown hare populations from Schleswig-Holstein.     

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.     

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.     

Population density,roads and altitude influences on spatial distribution of hares positive to EBHSV

Understanding of the ecology of infected animals facilitates disease risk assessment and is also crucial for wildlife conservation. Relatively little is known about the spatial distribution of infected wild mammals in relation to environmental factors. In neighboring Mediterranean ecosystems 250 European brown hares (Lepus europaeus) were collected and examined with RT-PCR to detect European Brown Hare Syndrome Virus (EBHSV). Multivariate statistics and Geographical Information System (GIS) analysis were applied to estimate spatial patterns of biotic and abiotic factors and human activities as determinants of EBHSV positivity. Hare population abundance was estimated using faeces counts and belt drive censuses. The study showed that EBHSV infected hares had widespread distribution even in isolated areas. However, EBHSV infection prevalence was higher in areas with higher hare abundance, closer to paved road networks and at lower altitudes. The risk map revealed the potential distribution of EBHSV-infected hares. This study shows that host abundance and landscape influence the ecology of the disease, a finding that should be taken into account in future studies. The management of harvest and restocking of hares is also discussed for population conservation.     

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.     

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.     

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 Relationships Among Tunisian Hares (Lepus sp.), South African Cape Hares (L. capensis), and European Brown Hares (L. europaeus)

Tunisian hares (n = 45), currently assigned to Lepus capensis, were assayed for allelic variation at 40 allozyme loci, and allele frequencies at 32 loci were directly compared with earlier data of South African cape hares (L. capensis, n = 9) and European brown hares (L. europaeus, n = 244) to reveal genetic relationships among them. European mountain hares (L. timidus, n = 200) were used for outgroup comparison. In the Tunisian hares 27.5% of the loci were polymorphic with 2–4 alleles. Among all alleles at polymorphic loci, 15.1% occurred exclusively in Tunisian hares, 5.7% exclusively in cape hares, and 7.5% exclusively in brown hares at low frequencies. Not a single locus showed alternately fixed alleles between the samples of the L. capensis/L. europaeus complex. Levels of absolute and relative genetic differentiation among the samples of the L. capensis/ L. europaeus complex were low, relative to pairwise comparisons involving mountain hares. Diverse cluster analyses and multidimensional scaling of various pairwise genetic distance matrices concordantly grouped Tunisian hares with brown hares, and South African cape hares clustered only slightly farther apart, whereas mountain hares were distinctly separate. These results suggest regionally distinct phylogenetic units within an overall cohesive gene pool in the L. capensis/ L. europaeus complex, supporting Petter's view that all North African hares belong to L. capensis except for one local population of savanna hares, and that cape hares and brown hares are conspecific.     

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.     

Genetic diversity within Anatolian brown hares (Lepus europaeus Pallas, 1778) and differentiation among Anatolian and European populations

Genetic variability of Anatolian hares and relationships between Anatolian and European populations were assessed by a multilocus allozyme approach to infer evolutionary relationships between hares from Asia Minor and Europe. Of the 48 loci assayed, 19 (39.6%) were polymorphic with two to four alleles in the Anatolian hares. Among all Anatolian alleles, 14 were so far not found in the compared 717 brown hares from Europe. Overall, genetic diversity was highest in Anatolian hares, intermediate in brown hares from the southern and southeastern Balkans and lowest in central European populations. The rich genetic diversity in Anatolian hares might be a consequence of Anatolias biogeographic position with the chance of multiple gene flow from neighbouring regions, and the likelihood of long-term presence of hares during the last ice age, when large parts of more northern latitudes did not provide suitable habitats.However, among 28 loci used for the comparison between European and Anatolian populations, most common alleles of European brown hares were also common in Anatolian populations and no alternately fixed alleles were found for Anatolian and European populations. This together with only little or moderately varying allele frequencies produced low genetic divergence between Anatolian and European populations. Genetic differentiation among Anatolian populations was also low. Even between the two forms with different coat colour (brownish and yellowish) in Anatolian hares, there was little genetic differentiation. Altogether, all Anatolian hares studied presently are closely related to European brown hare populations, and only some distantly spaced population pairs revealed increased genetic divergence.

Zusammenfassung

Genetische Diversität anatolischer Feldhasen (Lepus europaeus Pallas, 1778) und Differenzierung zwischen anatolischen und europäischen PopulationenZur Beurteilung der phylogenetischen Beziehungen zwischen anatolischen Hasen und europäischen Feldhasenpopulationen wurde die allelische Variabilität anatolischer Hasen mittels horizontaler Stärkegelelektrophorese erfaßt und gemeinsam mit unmittelbar vergleichbaren Daten griechischer, bulgarischer und österreichischer Populationen aus früheren Studien populationsgenetischen Analysen unterzogen. Neunzehn der 48 untersuchten Loci der anatolischen Hasen zeigten allelische Variabilität. Unter den anatolischen Allelen kamen 14 bisher in den europäischen Polulationen nicht vor. Insgesamt zeigten anatolische Hasen die höchste und österreichische Populationen die niedrigste genetische Diversität; die jeweiligen Werte der griechischen und bulgarischen Populationen lagen dazwischen. Dies entspricht unserer Hypothese hoher genetischer Diversität in Anatolien, auf Grund der biogeografischen Position und der klimatischen bzw. Lebensraumbedingungen während des Pleistozäns, die, im Gegensatz zu Mitteleuropa, kontinuierliche Hasenpopulation in Anatolien wahrscheinlich erscheinen lassen. Kontinuierliche Populationen und Genflüsse aus verschiedenen Nachbarregionen könnten bei langfristig relative ungestörten Populationen zur Anreicherung genetischer Varianten in Anatolien geführt haben, während mitteleuropäische Feldhasenpopulationen im Zuge ihrer postglazialen Einwanderung aus Refugial-gebieten an genetischer Vielfalt eingebüßt haben. Allerdings waren die häufigen Allele der anatolischen Hasen ebenfalls häufig bei den europäischen Feldhasen vertreten; somit ergab sich insgesamt nur eine geringe genetische Differenzierung zwischen anatolischen und europäischen Feldhasen. Die zwei in Anatolien gefundenen Fellfärbungstypen (brauner vs. gelber Grundton) zeigten ebenfalls keine besondere genetische Differenzierung.     

High mtDNA haplotype diversity among introduced Swedish brown haresLepus europaeus

The brown hareLepus europaeus Pallas, 1778 occurs naturally in central Eurasia, but has been introduced to parts of northern Europe, South- and North America, Australia and New Zealand. Brown hares were introduced to Sweden from central Europe for hunting purposes during the 19th century. We investigated how the human--mediated brown hare colonisation of Sweden is reflected in the amount of genetic variation present by assessing variation and composition of mitochondrial DNA (mtDNA) lineages among Swedish brown hares. MtDNA from a total of 40 brown hare specimens from 15 localities were analysed for Restriction Fragment Length Polymorphisms. The haplotype diversity is surprisingly high (0.893 ± 0.002) when compared to the mtDNA diversity among brown hares on the European continent as well as to other mammalian species. Admixture of haplotypes from different source populations combined with a reduced effect of random genetic drift and a relaxed selection pressure due to rapid population growth after introduction are mechanisms that are likely to account for the observed high mtDNA haplotype diversity.     

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.     

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.     

Granulomatous peritonitis in a European brown bear caused by Baylisascaris transfuga

We report a case of granulomatous peritonitis due to Baylisascaris transfuga in a young male European brown bear (Ursus arctus). At necropsy, there were extensive abdominal adhesions and extensive granulomatous tissue on the peritoneum and liver capsule. In the gastrointestinal tract, there were 58 nematodes that were identified as Baylisascaris transfuga using light and scanning electron microscopy.     

Mitogenetic structure of brown bears (Ursus arctos L.) in northeastern Europe and a new time frame for the formation of European brown bear lineages

We estimated the phylogenetic relationships of brown bear maternal haplotypes from countries of northeastern Europe (Estonia, Finland and European Russia), using sequences of mitochondrial DNA (mtDNA) control region of 231 bears. Twenty-five mtDNA haplotypes were identified. The brown bear population in northeastern Europe can be divided into three haplogroups: one with bears from all three countries, one with bears from Finland and Russia, and the third composed almost exclusively of bears from European Russia. Four haplotypes from Finland and European Russia matched exactly with haplotypes from Slovakia, suggesting the significance of the current territory of Slovakia in ancient demographic processes of brown bears. Based on the results of this study and those from the recent literature, we hypothesize that the West Carpathian Mountains have served either as one of the northernmost refuge areas or as an important movement corridor for brown bears of the Eastern lineage towards northern Europe during or after the last ice age. Bayesian analyses were performed to investigate the temporal framework of brown bear lineages in Europe. The molecular clock was calibrated using Beringian brown bear sequences derived from radiocarbon-dated ancient samples, and the estimated mutation rate was 29.8% (13.3%-47.6%) per million years. The whole European population and Western and Eastern lineages formed about 175,000, 70,000 and 25,000 years before present, respectively. Our approach to estimating the time frame of brown bear evolution demonstrates the importance of using an appropriate mutation rate, and this has implications for other studies of Pleistocene populations.     

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.