Survival and divergence in a small group: The extraordinary genomic history of the endangered Apennine brown bear stragglers.

About 100 km east of Rome, in the central Apennine Mountains, a critically endangered population of ∼50 brown bears live in complete isolation. Mating outside this population is prevented by several 100 km of bear-free territories. We exploited this natural experiment to better understand the gene and genomic consequences of surviving at extremely small population size. We found that brown bear populations in Europe lost connectivity since Neolithic times, when farming communities expanded and forest burning was used for land clearance. In central Italy, this resulted in a 40-fold population decline. The overall genomic impact of this decline included the complete loss of variation in the mitochondrial genome and along long stretches of the nuclear genome. Several private and deleterious amino acid changes were fixed by random drift; predicted effects include energy deficit, muscle weakness, anomalies in cranial and skeletal development, and reduced aggressiveness. Despite this extreme loss of diversity, Apennine bear genomes show nonrandom peaks of high variation, possibly maintained by balancing selection, at genomic regions significantly enriched for genes associated with immune and olfactory systems. Challenging the paradigm of increased extinction risk in small populations, we suggest that random fixation of deleterious alleles (i) can be an important driver of divergence in isolation, (ii) can be tolerated when balancing selection prevents random loss of variation at important genes, and (iii) is followed by or results directly in favorable behavioral changes.

IMMOBILIZING THE VULNERABLE APENNINE CHAMOIS (RUPICAPRA PYRENAICA ORNATA) WITH A LOW-DOSE XYLAZINE-KETAMINE COMBINATION, REVERSED WITH IDAZOXAN OR ATIPAMEZOLE.

Little information is available on chemical capture of the vulnerable subspecies within the genus Rupicapra. Low-dosage combinations of xylazine and ketamine were tested for immobilization of captive and free-ranging Apennine chamois, Rupicapra pyrenaica ornata (85 and 66 immobilizations, respectively) in a retrospective analysis. Of the six dosage groups, all of them providing an acceptable level of immobilization, the optimal trade-off between safety and efficacy was found following administration of a mean dosage of 0.24±0.03 mg/kg xylazine and 1.07±0.15 mg/kg ketamine, resulting in 7.50±3.31 min induction time, deep sedation with no or limited reaction to handling in 96% of the chamois, minimal deviation of physiologic parameters from previously reported physiologic values for anesthetized or physically restrained chamois, and no mortality. Intravenous injection of idazoxan (0.05±0.01 mg/kg) or atipamezole (0.38±0.37 mg/kg) resulted in faster reversal than intravenous injection of tolazoline (1.05±0.15 mg/kg) in 1.3 vs. 4.1 min. When free-ranging chamois were darted with similar xylazine and ketamine dosages, induction time was 8.49±5.48 min, 88% of the animals were deeply sedated, and a single animal died from respiratory arrest (1.5% mortality). Intramuscular atipamezole provided smoother reversal than intravenous idazoxan. The results of this study suggest that xylazine/ketamine combinations, at remarkably lower dosage than previously published in Caprinae, may be safely and effectively used in chemical capture protocols of Apennine chamois, to facilitate conservation-oriented relocation and research.

Molecular Detection and Phylogenetic Analysis of Canine Distemper Virus in Marsican Brown Bear (Ursus arctos marsicanus).

In this paper, we report the first molecular detection of the canine distemper virus in the Marsican brown bear (Ursus arctos marsicanus). Three subadults and one adult were live-trapped and checked for the main viral pathogens responsible for infectious diseases in this species. The four bears were found to be negative for all investigated viruses except for one, which resulted in a positive outcome for CDV by means of RT-PCR targeting fragments of viral N and H genes. The sequence analysis revealed the specificity of amplicons for the Europe Wildlife lineage of CDV, the same viral strain recovered from three foxes and two unvaccinated dogs coming from the same territories where the positive bear was captured. These results confirm the receptivity of Marsican brown bear for CDV, apparently without any pathological consequences for the positive animal, and suggest the presence in the studied area of a unique wild host-adapted lineage of the virus, able to spread in domestic animals, too. In this respect, continuous and specifically targeted surveillance systems are necessary in order to highlight any changes in the epidemiology of the infection in the territories where the Marsican brown bear lives, along with a more effective vaccination program for domestic dogs co-existing with this endangered species.

Pelodera strongyloides in the critically endangered Apennine brown bear (Ursus arctos marsicanus).

Skin biopsies from 20 Apennine brown bears (Ursus arctos marsicanus), 17 of which displaying skin lesions, were investigated by histopathology. Different degrees of dermatitis characterized by folliculitis and furunculosis accompanied by epidermal hyperplasia and epidermal and follicular hyperkeratosis were detected. In the most severe lesions, the superimposition of traumatic wounds, probably self-induced by scratching, was observed. In 8 out of 17 (47.0%) affected bears, cross- and longitudinally-sectioned nematode larvae were present within the lumen of hair follicles, whose localization and morphological characteristics were consistent with Pelodera strongyloides. P. strongyloides is a free-living saprophytic nematode whose third-stage larvae can invade the skin causing pruritic dermatitis in several mammalian species. This is the first report of Pelodera infection in the brown bear. Although capable of causing primary dermatitis, the finding of Pelodera is not sufficient to conclude that it is the cause of the lesions observed in bears. Nevertheless, the high prevalence of the infection is indicative of a diffuse phenomenon that requires further specific investigations given the interest and conservational relevance of this relict bear population.

Management Models Applied to the Human-Wolf Conflict in Agro-Forestry-Pastoral Territories of Two Italian Protected Areas and One Spanish Game Area.

Our work shows that, despite the persistence of persecutory actions, conservation activity has proved successful for the return of numerous wild mammals to different habitats, including the wolf. The human-wolf conflict is still described in all countries where the wolf is present. This is evidenced by the high number of damages on livestock, and the corpses of wolves found both in protected areas and in those where hunting is permitted. The diagnosis of road accidents, together with poisoning and poaching, are major causes of mortality. Although hunting records the highest percentage of kills in Spain, the demographic stability reported by the censuses suggests that this activity does not have a consistent influence on the Iberian wolf population's survival. In Italy, where wolf hunting is prohibited, wolf populations are to be increasing. In some Italian situations, wolf attacks on horses seem to cause unwanted damage to foals, but they represent a very precious source of information about the habits of carnivores. A simple management plan would be sufficient to help the coexistence between the productive parts and the ecosystem services ensured by the presence of the wolf. The presence of hybrids is a negative factor.

Helminth infections in faecal samples of Apennine wolf (Canis lupus italicus) and Marsican brown bear (Ursus arctos marsicanus) in two protected national parks of central Italy

This article reports the results of a copromicroscopic and molecular investigation carried out on faecal samples of wolves (n=37) and brown bears (n=80) collected in two protected national parks of central Italy (Abruzzo Region). Twenty-three (62.2%) samples from wolves were positive for parasite eggs. Eight (34.78%) samples scored positive for single infections, i.e. E. aerophilus (21.74%), Ancylostoma/Uncinaria (4.34%), Trichuris vulpis (4.34%), T. canis (4.34%). Polyspecific infections were found in 15 samples (65.21%), these being the most frequent association: E. aerophilus and Ancylostoma/Uncinaria. Thirty-seven (46.25%) out of the 80 faecal samples from bears were positive for parasite eggs. Fourteen (37.83%) samples were positive for B. transfuga, and six (16.21%) of them also contained Ancylostoma/Uncinaria, one (2.7%) E. aerophilus and one (2.7%) both E. aerophilus and Ancylostoma/Uncinaria. Of the other samples, 19 (51.35%) were positive for Ancylostoma/Uncinaria, two (5.4%) for E. aerophilus and two (5.4%) for both. Molecular analysis found the roundworm and capillariid eggs found in wolves and bear samples to be Toxocara canis, Baylisascaris transfuga and Eucoleus aerophilus (syn. Capillaria aerophila). Considering the high prevalence of zoonotic intestinal helminths detected in this study, it is important to improve the knowledge and awareness of the general public and park operators regarding the potential health risk associated with infections in wildlife.

Serologic evidence for selected infectious diseases in Marsican brown bears (Ursus arctos marsicanus) in Italy (2004-09).

We tested 30 serum samples collected during 2004-09 from 22 free-ranging Marsican brown bears (Ursus arctos marsicanus) in the National Park of Abruzzo, Lazio, and Molise, Italy, for antibodies against canine distemper virus (CDV), canine adenovirus type 2 (CAV-2), canine parvovirus type 2 (CPV-2), Brucella spp., and eight Leptospira interrogans sensu lato serovars. Antibody to CDV was detected in 11 samples (37%); only two bears (10%) had detectable CAV-2 and Brucella spp. antibodies; three bears were positive for L. interrogans serovar Bratislava; and one sample had antibody against L. interrogans serovar Copenhageni. All samples were positive for CPV-2 antibody. The CPV-2 antibody titers varied from 1∶640 to 1∶10,240, suggesting that transmission was still active. Fifty percent of bears were positive for antibody to two or more pathogens. Our results highlight the need to consider infectious diseases as a potential risk for Marsican brown bear conservation.

Arctic lineage-canine distemper virus as a cause of death in Apennine wolves (Canis lupus) in Italy.

Canine distemper virus (CDV) infection is a primary threat affecting a wide number of carnivore species, including wild animals. In January 2013, two carcasses of Apennine wolves (Canis lupus) were collected in Ortona dei Marsi (L'Aquila province, Italy) by the local Veterinary Services. CDV was immediately identified either by RT-PCR or immunohistochemistry in lung and central nervous tissue samples. At the same time, severe clinical signs consistent with CDV infection were identified and taped (Videos S1-S3) from three wolves rescued in the areas surrounding the National Parks of the Abruzzi region by the Veterinary Services. The samples collected from these symptomatic animals also turned out CDV positive by RT-PCR. So far, 30 carcasses of wolves were screened and CDV was detected in 20 of them. The sequencing of the haemagglutinin gene and subsequent phylogenetic analysis demonstrated that the identified virus belonged to the CDV Arctic lineage. Strains belonging to this lineage are known to circulate in Italy and in Eastern Europe amongst domestic dogs. To the best of our knowledge this is the first report of CDV Arctic lineage epidemics in the wild population in Europe.