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.     

Evolutionary relationships among hares from North Africa (Lepus sp. or Lepus spp.), cape hares (L. capensis) from South Africa, and brown hares (L. europaeus), as inferred from mtDNA PCR-RFLP and allozyme data

Systematics and taxonomy of hares of the genus Lepus (Lagomorpha) are under contentious debate, and phylogenetic relationships among many taxa are not well understood. Here we study genetic differentiation and evolutionary relationships among North African hares, currently considered subspecies of Lepus capensis , cape hares ( L. capensis ) from the Cape province in South Africa, and brown hares ( L. europeaus ) from Europe and Anatolia, using maternally (mtDNA) and biparentally (allozymes) inherited markers. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of a c. 1.8 kb long segment of the mitochondrial control region using eight hexanucleotide-recognizing restriction endonucleases yielded 28 haplotypes, and horizontal starch gel electrophoresis of proteins encoded by 25 structural gene loci revealed 52 alleles at 18 polymorphic loci. Diverse phylogenetic analyses (neighbor joining dendrogram, median joining network, multidimensional scaling of pairwise distances, AMOVA, F -statistics, hierarchical F -statistics) of genetic variants revealed marked substructuring of mtDNA into three phylogeographic groups, namely an African, a central European, and an Anatolian, but a somewhat less pronounced overall differentiation of the nuclear genome, despite a relatively high number of population-specific (private) alleles. However, all our results are not incongruent with Petter's (1959: Mammalia 23 , 41; 1961: Z. f. Säugetierkunde 26 , 30; 1972 : Société Des Sciences Naturelles et Physiques du Maroc 52 , 122) hypothesis that North African hares generally belong to L. capensis and that brown hares should be included in this species as well.     

Mitochondrial Cytochrome b Gene Sequence Diversity in the Korean Hare, Lepus coreanus Thomas (Mammalia, Lagomorpha)

Partial sequences of the mitochondrial cytochrome b gene of the Korean hare (Lepus coreanus) were analyzed to determine the degree of genetic diversity. Nine haplotypes were observed, and the maximum Tamura-Nei nucleotide distance among them was 2.8%, indicating that genetic diversity of L. coreanus is moderate. In order to clarify the Korean hare's taxonomic status and relationship with the Manchurian hare (L. mandshuricus) and the Chinese hare (L. sinensis), these nine haplotypes of the Korean hare were compared with 13 haplotypes from five other species of eastern Asian Lepus including L. mandshuricus and L. sinensis. The Korean hare was distinct in its cytochrome b gene, and it is confirmed that L. coreanus is a valid species, as noted by Jones and Johnson (1965, Univ. Kansas Publ. (Mus. Nat. Hist.) 16:357). Further analyses of mtDNA cytochrome b gene with additional specimens of L. coreanus from North Korea and other species of Lepus from eastern Asia are needed to clarify the taxonomic status of the divergent mtDNA clades of L. mandshuricus and L. sinensis.     

Behaviour, population changes and dispersal of mountain hares (Lepus timidus) in Scotland

Mountain hares in north-east Scotland spent the day in forms in long heather and moved downhill in the evening to feed in hill pastures. Here they grazed intensively, often in groups of 4–6 individuals. In June and July adult females, then pregnant or lactating, grazed in daylight on the pastures. Leverets spent less time grazing, and more in play and exploration. They spent the day in cover near the feeding areas, and if disturbed during grazing crouched or went into cover, while adults fled. During 1982 and 1983 males predominated in groups of grazing hares, but in 1984 both sexes were equally represented. These changes in sex ratio were reflected in the increased proportion of females trapped for marking between February and late July during 1982–86. There was a dominance order related to weight among male hares and dominant hares approached more females. There was no firm evidence of mate-guarding. Males approached females regardless of their oestrous state and were usually rebuffed with varying degrees of intensity including striking and chasing. There were no interactions between females. Adults of both sexes chased leverets for short distances but leverets joined groups of feeding adults. Neither leverets nor first-winter hares showed evidence of dispersal. Mountain hares avoided sheep and cattle and there were fewer hares after the arrival of sheep in May.     

Winter locomotor activity patterns of European hares (Lepus europaeus)

In this study, activity patterns of the European hare (Lepus europaeus) were analyzed during winter using global positioning system (GPS) collars on 24 hares in two study areas located in central Italy. We programmed the collars to collect 12 location points per day, for a duration of three months. Results show two distinct phases of activities related to the day–night cycle. The daytime phase is characterized by inactivity at the form while the second phase is characterized by movements. Males were more active than females, showing a constant locomotor activity during the whole night. Females showed two peaks of activity during the night with a reduction in the middle of this time period. The comparison between females of the two study areas showed difference in interfix distance in particular around sunset and sunrise. In fact the minimum daily movement between the two areas shows that foraging sites of area B are more distant than those of area A. The recent possibility to apply GPS collars on small–medium mammals provides a powerful instrument to study the behavioral ecology of the European hare, and consequently promote an effective population management strategy for the species conservation.     

Mitochondrial HVI sequence variation in Anatolian hares (Lepus europaeus Pallas, 1778)

Variability and phylogenetic relationships of sequences of the hypervariable domain I (HVI) of the mitochondrial DNA was studied in 46 brown hares (Lepus europaeus) from Anatolia, to test the hypotheses that (i) hares from several islands off the Anatolian coast and from Cyprus are phylogenetically close to mainland Anatolian hares, (ii) Anatolian hare sequence variability is higher than that of typical European brown hares, and to iii) infer possible Anatolian source populations of hares from some islands in the eastern Mediterranean. Neighbor joining and Maximum Parsimony analyses revealed reciprocal monophyly for sequences from Anatolia, the considered eastern Mediterranean islands off the Anatolian coast, Cyprus, and those sequences published earlier form NE Greece that were supposed to originate from earlier immigration via the late-Pleistocene/early-Holocene land bridge that connected SE Europe and W Anatolia (Kasapidis et al., 2005. Mol. Phylogenet. Evol. 34, 55–66). A high sequence idiosyncrasy was found among the Anatolian samples. Almost all approaches to compare variability between Anatolian and the downloaded European sequence data indicated higher sequence diversity in Anatolia, in accordance with earlier findings for allozyme loci. Network and principal coordinate analyses of the Anatolian sequences and those from the islands off the Anatolian coast as well as the Anatolian-type NE Greek sequences suggested high mitochondrial gene exchange among local populations in Anatolia with little effect of possible geographic barriers, and did not provide clues for tracing possible origins of island populations.     

Excretion of corticosteroids in urine and faeces of hares (Lepus europaeus)

Increased production of glucocorticoids by the adrenal cortex is found in mammals under stress. As cortisol itself is absent in the faeces, an enzyme immunoassay (11-oxoaetiocholanolone) measuring 11,17-dioxoandrostanes has already been established to measure faecal cortisol metabolites in ruminants for non-invasive monitoring of adrenocortical activity. The aim of this study was to establish route and delay of excretion of glucocorticoids in hares and to determine whether a cortisol-, corticosterone- or this new enzyme immunoassay is best suited to detect faecal glucocorticoid metabolites. In the first experiment radioactive-labelled glucocorticoids (¹⁴C-cortisol and ³H-corticosterone) were administered intravenously to two groups of three hares in metabolic cages. All voided urine and faecal samples were collected for 4 days. Metabolites of both steroids were found predominantly in the urine (91 ± 4%). Peak concentrations were observed in the first urinary sample following infusion (13 ± 6 h) and in the faeces with a delay of about 1 day (23 ± 7 h). Most of the radioactivity was not extractable with diethylether, indicating that the metabolites excreted in urine and faeces are mainly conjugated or polar unconjugated ones. This was confirmed by reverse-phase high-performance liquid chromatography separations of the metabolites, which also revealed marked differences concerning the metabolism of the two glucocorticoids injected. Compared with the cortisol and the corticosterone enzyme immunoassay, only the group-specific enzyme immunoassay for 11,17-dioxoandrostanes detected high quantities of immunoreactive metabolites. In a second experiment hares (n=20) were stressed by rousing them three times (5 min, 10 min and another 5 min) with a 20-min break in-between. Faecal samples were collected 2 days before until 4 days after stress and analysed using the 11-oxoaetiocholanolone enzyme immunoassay. After stress significantly (P < 0.001) increased 11,17-dioxoandrostane concentrations were found. Based on these results, measuring 11,17-dioxoandrostanes in faeces enables non-invasive monitoring of disturbances in hares and thus provides a tool for field investigations elucidating the role of stress in hare populations. Accepted: 24 November 1999     

Annual production of young in European hares (Lepus europaeus) in the Netherlands

In the Netherlands, hares have a period of sexual inactivity in autumn. Even though a reactivation of spermatogenesis was found from the second half of November onwards, first conceptions did not occur until the last week of December. Throughout January, the proportion of pregnant females increased to 80% in February in healthy adults, but only to 8% in diseased ones. Due to the spread in the start of the breeding season caused by variations in health and age, no synchronization in birth was found. Consequently, in order to be able to determine the annual production of young, the gradual change in pregnancy rate and mean litter size over the year had to be taken into account. Taking mean monthly values for healthy adult females, an average annual production of nearly 11 young was found. Compared with other European countries, annual production in the Netherlands is relatively high which is probably due to a comparatively long breeding season.     

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.     

Colour change of Mountain hares (Lepus timidus scoticus) in north-east Scotland

A two-year field study of colour change in a population of Mountain hares showed that the rate of colour change was significantly faster in a warm spring than in a cold one. The same result was found in individually marked wild hares, indicating that the response is not due to differences in age or sex ratios. It is postulated that the white coat is for camouflage, and the duration for which it is worn is correlated with temperature because of the coat's thickness. Moulting is probably timed by daylength, and the rate of moult affected by temperature.     

Speciation and paraphyly in western mediterranean hares (Lepus castroviejoi,L. europaeus,L. granatensis,andL. capensis) revealed by mitochondrial DNA phylogeny

Mitochondrial DNA (mtDNA) variation among specimens of the northwestern African hare (Lepus capensis schlumbergeri) and three European hares sampled in Spain (L. castroviejoi andL. granatensis, which are endemic to the Iberian Peninsula, andL. europaeus) was analyzed using seven restriction endonucleases. Fourteen haplotypes were found among the 34 animals examined. Restriction site maps were constructed and the phylogeny of the haplotypes was inferred. mtDNA ofL. capensis was the most divergent, which is consistent with its allopatric African distribution and with an African origin of European hares. We estimated that mtDNA in hares diverges at a rate of 1.5–1.8% per MY assuming that the European and African populations separated 5–6 MYBP. Maximum intraspecies nucleotide divergences were 1.3% inL. capensis, 2.7% inL. castroviejoi, and 2.3% inL. granatensis but 13.0% inL. europaeus. The latter species contained two main mtDNA lineages, one on the branch leading toL. castroviejoi and the other on that leading toL. granatensis. The separation of these two lineages from theL. castroviejoi orL. granatensis lineages appears to be much older than the first paleontological record ofL. europaeus in the Iberian peninsula. This suggests that the apparent polyphyly ofL. europaeus is due not to secondary introgression, but to the retention of ancestral polymorphism inL. europaeus. The results suggest thatL. europaeus either has evolved as a very large population for a long time or has been fractionated. Such a pattern of persistence of very divergent lineages has also been reported in other species of highly mobile terrestrial mammals. As far as mtDNA is concerned,L. europaeus appears to be the common phylogenetic trunk which has diversified during dispersion over the European continent and from whichL. castroviejoi andL. granatensis speciated separately in southwest Europe.     

Molecular phylogeny of North American Branchiobdellida (Annelida: Clitellata)

Branchiobdellidans, or crayfish worms, are ectosymbiotic clitellate annelids associated primarily with freshwater crayfishes. The main objectives of our study were to infer a molecular phylogeny for the North American Branchiobdellida, examine its congruence with morphology-based hypotheses of relationships at the subfamily and genus level, and use our dataset to assess consistency of GenBank-archived branchiobdellidan sequences. We used nucleotide sequence data from two mtDNA genes (COI and 16S rDNA) and three nuclear genes (28S rDNA, 18S rDNA, and ITS1) to estimate phylogenetic relationships among 47 described and one undescribed species of Branchiobdellida. We recovered a monophyletic branchiobdellidan clade with generally short branch lengths, suggesting that a large portion of the taxon has likely undergone a recent and rapid radiation in North America. Results from our phylogenetic analyses indicate that current taxonomic groupings are largely unsupported by the molecular data. All four subfamilies are either paraphyletic or polyphyletic, and only three of seven sampled non-monotypic genera were monophyletic. We found a high rate (49%) of inconsistency in GenBank-archived sequences, over 70% of which can be attributed to field- or laboratory-based error.     

Body composition and reproduction in mountain hares (Lepus timidus scoticus) in north-east Scotland

Mountain hares in north-east Scotland are pregnant from Mid-February to mid-July, although breeding is at a peak from April to June when all adult females are either pregnant or lactating or both. The onset of reproduction coincides with the highest body fat composition and the end of the breeding season coincides with the greatest depletion of body fat.     

Reproduction of Mountain hares (Lepus timidus) in relation to density and physical condition

Population estimates of Mountain hares were done on three islands off the west coast of Sweden. Three different relative measures of hare reproduction were negatively related to density. Most of this relationship was accounted for through a positive correlation with body condition. However, there was no significant density trend in litter size or number of litters. The start of the reproductive season each year was related to mean air temperature during spring, so when spring was early so was conception.     

Diet selection by European hares (Lepus europaeus) in the alpine zone of the Snowy Mountains, Australia

European hares (Lepus europaeus) are grazers and open grassland specialists that are replaced in mountain areas of their natural range in the northern hemisphere by browsing/intermediate feeding mountain hares (Lepus timidus), but in their introduced range in the southern hemisphere, occupy the alpine zone. We used micro-histological identification of plant fragments and germination of seeds in faecal pellets of L. europaeus from the Snowy Mountains, Australia, to determine diet. We asked whether diet shifted and/or diet breadth expanded in response to seasonally reduced food availability, particularly during winter. If so, did the constraints of food availability in the alpine zone lead to the diet mirroring that of L. timidus in its native alpine habitat. The diet of L. europaeus was dominated by grasses, herbs and shrubs. The main diet items in summer were grasses (70 %) and herbs (28 %). Grasses declined in the diet between summer and autumn when herbs increased to co-dominance, with a further change after establishment of the winter snowpack to a greater preponderance of shrubs (43 % compared with a maximum of 3 % in snow-free months). L. europaeus selected a wider range of plants in winter (59 species compared with 39 in summer) and diet was significantly more variable in winter than in autumn or summer (and in autumn than summer). We concluded that the persistence of L. europaeus in alpine areas of the southern hemisphere is testament to their ability to expand their dietary breadth to occupy mountain climatic zones normally occupied by L. timidus.