Molecular identification of a new pestivirus associated with increased mortality in the Pyrenean Chamois (Rupicapra pyrenaica pyrenaica) in Spain

Pestivirus infection was identified in 16 of 17 chamois during an outbreak of a previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in northeastern Spain in 2001-02. By analysis of the 5' noncoding regions of the virus, we assigned it to the border disease virus cluster with pairwise similarity values ranging from 82.1% to 88.1%. It will be important to investigate the association of this pestivirus with disease in Pyrenean chamois.     

Two Different Epidemiological Scenarios of Border Disease in the Populations of Pyrenean chamois (Rupicapra p. pyrenaica) after the First Disease Outbreaks

Since 2001 several outbreaks of a new disease associated with Border disease virus (BDV) infection have caused important declines in Pyrenean chamois (Rupicapra pyrenaica) populations in the Pyrenees. The goal of this study was to analyze the post-outbreak BDV epidemiology in the first two areas affected by disease with the aim to establish if the infection has become endemic. We also investigated if BDV infected wild and domestic ruminants sharing habitat with chamois. Unexpectedly, we found different epidemiological scenarios in each population. Since the disease outbreaks, some chamois populations recuperated quickly, while others did not recover as expected. In chamois from the first areas, prevalence was high (73.47%) and constant throughout the whole study period and did not differ between chamois born before and after the BDV outbreak; in all, BDV was detected by RT-PCR in six chamois. In the other areas, prevalence was lower (52.79%) and decreased during the study period; as well, prevalence was significantly lower in chamois born after the disease outbreak. No BDV were detected in this population. A comparative virus neutralisation test performed with four BDV strains and one Bovine viral diarrhoea virus (BVDV) strain showed that all the chamois had BDV-specific antibodies. Pestivirus antibodies were detected in all the rest of analyzed species, with low prevalence values in wild ruminants and moderate values in domestic ruminants. No viruses were detected in these species. These results confirm the hypothesis that outbreaks of BDV infection only affect the Pyrenean chamois, although other wild ruminants can occasionally be infected. In conclusion, two different scenarios have appeared since the first border disease outbreaks in Pyrenean chamois: on the one hand frequent BDV circulation with possible negative impact on population dynamics in some areas and on the other, lack of virus circulation and quick recovery of the chamois population.     

Prevalence of antibodies against selected agents shared between Cantabrian chamois (Rupicapra pyrenaica parva) and domestic goats

Southern chamois (Rupicapra pyrenaica) share the habitat with domestic ungulates, and may, therefore, play a role in the epidemiology of shared agents. The objective of this study was to determine the seroprevalence for Brucella spp., Mycobacterium avium ssp. paratuberculosis (MAP), pestivirus, and Sarcoptes scabiei in Cantabrian chamois (Rupicapra pyrenaica parva) and compare these data with those of sympatric domestic goats (Capra hircus). From 2005 to 2008, blood samples were obtained from 236 adult Cantabrian chamois in two different populations, the western one and the eastern one. Seroprevalence for Brucella spp. and pestivirus was assessed using commercial ELISA kits, whereas specifically designed ELISA tests were used for MAP and S. scabiei. No antibodies against Brucella spp. were detected. Conversely, antibodies against MAP, pestivirus (chamois 3.8%; goat 2.3%), and S. scabiei (chamois 11.9%; goat 12.8%) were detected in both species. Seroprevalence for MAP was significantly higher for domestic goats (26%) than for chamois (9.7%). In chamois, seroprevalence for pestivirus was higher in the west (6.5%) than in the east (range 0–1.8%), whereas seroprevalence for S. scabiei followed the opposite trend (west 4.6%; east 16.7–21.4%). We suggest that certain diseases could circulate between Cantabrian chamois and domestic goat populations, and domestic livestock may suppose a threat for the health status of sympatric Cantabrian chamois.     

Survey of Pestivirus infection in wild and domestic ungulates from south-western Italian Alps

The transmission of pestiviruses between domestic and wild ruminants has not been documented in communal alpine pastures shared between wildlife and livestock. The aim of this study was to investigate the role of domestic and wild ungulates species from Varaita Valley (SW Italian Alps) in the epidemiology of Pestivirus infections. Sera from free-ranging alpine chamois (Rupicapra rupicapra) and roe deer (Capreolus capreolus) were collected from 1994 to 2009 and 2001 to 2009, respectively. Also, sera from cattle and sheep sampled in 2009 were studied. Sera were tested for the presence of antibodies against pestivirus with an ELISA assay. Sera from positive animals were subsequently tested with a comparative virus neutralisation test using the BVDV-NADL and BDV-137/4 strains. Sera were tested for the presence of pestiviral antigen and the presence of viral RNA with a commercial ELISA assay and RT-PCR. Antibodies against Pestivirus were detected in 132 out of 312 (42%) chamois, in 30 out of 175 (17%) cattle and 6 out of 24 (25%) sheep. No antibodies were found in roe deer. No Pestivirus antigen or RNA was detected in any of the samples. Results indicate circulation of pestiviruses among the studied chamois, cattle and sheep populations. However the role of wild ungulates in the dynamics of Pestivirus infection is still unknown and monitoring the presence of these viruses in wild ungulates would be of importance, especially in the chamois population, where pestiviruses seem to circulate extensively.     

Vigilance behavior of Pyrenean chamois Rupicapra pyrenaica pyrenaica: Effect of sex and position in the herd

The Pyrenean chamois Rupicapra pyrenaica pyrenaica is a mountain-dwelling ungulate with an extensive presence in open areas. Optimal group size results from the trade off between advantages (a reduction in the risk of predation) and disadvantages (competition between members of the herd) of group living. In addition, advantages and disadvantages of group living may vary depending on the position of each individual within the herd. Our objective was to study the effect of central vs. Peripheral position in the herd on feeding and vigilance behavior in male and female Pyrenean chamois and to ascertain if a group size effect existed. We used focal animal sampling and recorded social interactions when a focal animal was involved. With males, vigilance rate was higher in the central part of the group than at the periphery, probably due to a higher density of animals in the central part of the herd and a higher probability of being disturbed by conspecifics. With females, vigilance rate did not differ according to position in the herd. Females spent more time feeding than males, and males showed a higher frequency of the vigilance behavior than females. We did not observe a clear relationship between group size and vigilance behavior. The differences in vigilance behavior might be due to social interactions.     

Predicting seasonal and spatial variations in diet quality of Pyrenean chamois (Rupicapra pyrenaica pyrenaica) using near infrared reflectance spectroscopy

Monitoring nutritional status of wildlife is key to understanding the population response to changes in food availability. Several direct and indirect methods have been proposed for such purposes, but faecal nitrogen (FN) is by far the most commonly used indirect non-invasive indicator of free-ranging ruminant diet quality. In this work, two methods were used to estimate the FN content in 291 pellet samples of Pyrenean chamois (Rupicapra pyrenaica pyrenaica) collected during a 2-year period (May 2009 to May 2011) in two chamois populations from the Catalan Pyrenees, Spain: the Dumas LECO analyzer and near-infrared reflectance spectroscopy (NIRS). In order to optimize further FN estimations we performed three different NIRS calibrations: (1) general, across year, season and populations; (2) population-specific; and (3) season-specific, i.e., different periods of vegetation growth. FN ranged from 1.66 % to 2.85 % (dry matter basis) and was higher in the main vegetation growing season than in winter. Concerning NIRS calibration, our study confirmed that FN of Pyrenean chamois can be accurately determined by NIRS, since the general calibration was accurate enough to predict FN. It is concluded that NIRS represents a non-destructive, emission-free and low-cost analytical technique which may reduce the use of conventional laborious methods for estimating FN in long-term wildlife monitoring.     

A panel of polymorphic microsatellites in the threatened Apennine chamois (Rupicapra pyrenaica ornata)

The Apennine chamois (Rupicapra pyrenaica ornata) is a highly endangered mountain ungulate. About 800 individuals still survive in central Apennines, Italy. Current conservation projects involve genetic identification of single animals, paternity tests, and assessment of relatedness. A suite of 60 microsatellite primer pairs from domestic bovids and cervids revealed polymorphisms for 10 loci, providing evidence for a low level of genetic variability, which might be a consequence of severe population bottlenecks and prolonged permanence at low numbers.     

Diversity and evolution of the Mhc-DRB1 gene in the two endemic Iberian subspecies of Pyrenean chamois, Rupicapra pyrenaica

Major histocompatibility complex class II locus DRB variation was investigated by single-strand conformation polymorphism analysis and sequence analysis in the two subspecies of Pyrenean chamois (Rupicapra pyrenaica) endemic to the Iberian Peninsula. Low levels of genetic variation were detected in both subspecies, with seven different alleles in R. p. pyrenaica and only three in the R. p. parva. After applying the rarefaction method to cope with the differences in sample size, the low allele number of parva was highlighted. The low allelic repertoire of the R. p. parva subspecies is most likely the result of bottlenecks caused by hunting pressure and recent parasitic infections by sarcoptic mange. A phylogenetic analysis of both Pyrenean chamois and DRB alleles from 10 different caprinid species revealed that the chamois alleles form two monophyletic groups. In comparison with other Caprinae DRB sequences, the Rupicapra alleles displayed a species-specific clustering that reflects a large temporal divergence of the chamois from other caprinids, as well as a possible difference in the selective environment for these species.     

Semen evaluation in the chamois of Abruzzi (Rupicapra pyrenaica ornata)

The chamois of Abruzzi (Rupicapra pyrenaica ornata) has been classified as endangered by the World Union for Conservation. The objective of this study was to analyze seasonal differences in the characteristics of various male reproductive organs and in semen quality. The study was conduced during 2004 in the reserve of Lama dei Peligni (Italy) on three chamois males aged between 2 and 5 years. Males were captured during March-May months and October-December months. Various testicular and scrotal measurements were taken and semen was collected using an electroejaculator. Sperm motility pattern was evaluated using computer assisted sperm analyzer, membrane integrity using differential staining and morphology with phase contrast microscopy. Testicular size, sperm motility membrane integrity and the percent of morphological normal spermatozoa were greater during October-December. The authors concluded that sperm characteristics are strongly influenced by season and that semen collected in this period (breeding season) has suitable quality for manipulation and long-term preservation.     

Increased hormonal stress response of Apennine chamois induced by interspecific interactions and anthropogenic disturbance

Responses of animals to environmental changes and their interactions with other species play an important role in conservation. Sharing a common habitat may lead to interspecific competition for resources, but field assessment of these biological events is not always easily accomplished. By using a non-invasive method, we evaluated the physiological stress responses of Apennine chamois (Rupicapra pyrenaica ornata) to the presence of cattle, sheep and goat, red deer (Cervus elaphus), people (hikers), and predators to identify which factors may affect this endangered species. During September 2012, November 2012, and July 2013, a total of 318 faecal samples were collected in representative sites and analysed for faecal cortisol metabolites (FCM). FCM concentration was analysed through linear mixed-effect models. A significant increase in FCM values in Apennine chamois sharing their habitat with domestic animals was recorded during all study periods. On the contrary, stress responses to red deer and people were limited in time and emerged only during summer months, when hikers are more frequent and red deer extend their altitudinal range reaching chamois’ habitat. The observed effects of domestic animals, red deer, and hikers should be considered in future Apennine chamois management plans, which should include the regulation of pastured domestic livestock, anthropogenic disturbances, and possible interferences with other wild species within parks.     

Complete Genome Sequence of a Novel Pestivirus from Sheep

We report here the complete genome sequence of pestivirus strain Aydin/04-TR, which is the prototype of a group of similar viruses currently present in sheep and goats in Turkey. Sequence data from this virus showed that it clusters separately from the established and previously proposed tentative pestivirus species.     

Pestivirus infection in small ruminants: virological and histopathological findings.

Pestivirus infection was detected in several flocks of sheep and goats located in the south of Italy by means of serological, virological and histopathological investigations. From four animals, two lambs and two kids, showing enteric symptoms which died during the first week of life, four pestivirus strains were isolated and typed as BVDV-like (three isolates) and "tipic" BDV strains (one isolate). The histopathological lesions consisted of areas of hypomyelination in the brain, and a slight depletion of thymic medullary lymphocytes associated with an increase in reticular cells.     

Giraffe strain of pestivirus: its taxonomic status based on the 5'-untranslated region

The 5'-untranslated region (5'-UTR) of the 'Giraffe' strain of pestivirus was sequenced for comparison with those of other pestiviruses from cattle, sheep, goats, and swine. A phylogenetic tree constructed with these strains suggested that the 'Giraffe' strain was allocated to a new taxon. This observation was also confirmed by a newly proposed method based on palindromic nucleotide substitutions (PNS) at the three variable regions in the 5'-UTR. Other reported pestivirus strains isolated from deer were assigned as bovine viral disease virus (BVDV)-1 according to the PNS as well as phylogenetic analysis, suggesting that BVDV-1 strains can cross-infect deer as well as cattle, sheep, goats, and swine, and that wild deer may serve as a reservoir of BVDV-1. We also identified the genovar of a deer isolate, SH9/11, as BVDV-1c by the PNS method.     

Social Interactions in Isard Kids Rupicapra pyrenaica pyrenaica (Artiodactyla: Bovidae): Possible Influence on Development of Social Bonding

Assuming that social experiences at an early age may be a determinant factor in the process of formation of social network and attachments, we studied the social interactions of isard, or Pyrenean chamois, kids (Rupicapra pyrenaica pyrenaica) during their first year, to understand “how” sociality arises in isards rather than “why”. Interactions with the mother were affiliative, and approaches of kid to mother or vice versa were generally followed by another “positive” interaction. Interactions between kids were also frequent and playful. Interactions with both mother and peers provided positive reinforcement which may strengthen bonds. However, early interactions of kids with yearlings and other adults provided negative experience, no attachment thus developing from them. Social affinities of adults as well as the social and spatial organization of groups may be understood when ontogenetical history and immediate causality are highlighted.     

Dung avoidance as a possible mechanism in competition between wild and domestic ungulates: two experiments with chamois <Emphasis Type="Italic">Rupicapra rupicapra</Emphasis>

Wild ungulates often adjust spatial behaviour where they coexist with livestock. In European mountains, chamois Rupicapra rupicapra or R. pyrenaica commonly avoid alpine pastures used by domestic sheep. Mechanisms leading to competitive losing out of the wild species are not well understood, but mostly, resource competition is inferred and sometimes demonstrated. We hypothesised that chamois need to minimise the risk of intestinal parasite uptake and therefore would avoid pastures contaminated with sheep dung. We tested this in two experiments by contaminating feeding patches of wild and captive chamois with (parasite-free) sheep faeces. Wild chamois did not avoid grazing sites or plots on which sheep dung had been placed at a (low) density representing the commonly encountered situation in the Swiss Alps. Captive chamois strongly reduced browsing time on small trees given for food when the surroundings of the trees were sprayed with a watery faecal solution. We concluded that the odour signalled a potential high risk of parasite infection to the captive chamois, whereas the density of sheep dung on the grazing site of wild chamois was not high enough to be perceived as a risk. The need to minimise endoparasite uptake from faeces may therefore play a role in driving spatial behaviour of wild ungulates and could result in competitive imbalance between wild and domestic ungulates.     

Feeding habits,forage selection,and diet overlap in Alpine chamois (<Emphasis Type="Italic">Rupicapra rupicapra</Emphasis> L.) and domestic sheep

Fecal microhistological analyses have been used to assess the summer diet of chamois (Rupicapra rupicapra L., 1758) and domestic sheep and to evaluate diet overlap. The aim of our work was to investigate interspecific interactions and to assess the effects of sheep grazing on the summer feeding habits of chamois. A high dietary overlap (Pianka’s index = 0.93–0.99) was found, despite differences in the use of some plant groups. Sheep presence strongly affected chamois feeding habits: a reduction of highly digestible forbs was observed in the chamois diet during August, when both species grazed in the same range. As a consequence of sheep grazing, chamois may have been forced to reduce niche breadth and to change their food habits, increasing percentages of monocotyledons in the diet and feeding mainly on Cyperaceae. The diet overlap between sheep and chamois suggests that resource-mediated interactions as well as direct competition must be taken into account when evaluating chamois and sheep compatibility on Alpine meadows.     

Clinical infection of Cantabrian chamois (Rupicapra pyrenaica parva) by louping ill virus: new concern for mountain ungulate conservation?

Louping ill virus (LIV) was recently involved in an outbreak of encephalitis in domestic goats from Asturias region, northwestern Spain. Since livestock and wildlife in Asturias are frequently in close contact, we designed a retrospective survey for LIV antibody prevalence in wild ungulates by testing sera from 51 red deer (Cervus elaphus), 19 Cantabrian chamois (Rupicapra pyrenaica parva) and 8 roe deer (Capreolus capreolus) by the haemagglutination inhibition (HI) test. Only two Cantabrian chamois out of the 78 tested (2.6?±?3.5 %) gave positive results. Seroprevalence in chamois was 10.5?±?13.8 %. One of these chamois was found dead after falling down a cliff and the other one was found alive but with neurological signs. Histological examination of brain samples revealed that both animals showed severe inflammatory lesions compatible with a viral encephalitis caused by LIV, but LIV antigen was not detectable by specific immunohistochemistry. Real time RT-PCR was performed on formalin-fixed paraffin embedded sections of brain but was unable to confirm the presence of LIV RNA due to poor sample quality. By testing one of two HI positive sera from chamois by virus neutralization test and plaque reduction neutralization test against West Nile virus, Bagaza virus, Usutu virus, LIV and tick-borne encephalitis virus, we confirmed the presence of high antibody titres (1:10240) against LIV in the absence of antibodies to another Flavivirus. This work describes the first association between LIV and clinical encephalitis in chamois, which suggests that special attention should be paid to the impact on chamois conservation and management in Asturias, and perhaps in other European regions.     

Late Pleistocene and Holocene chamois in Italy

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Effect of domestic sheep on chamois activity,distribution and abundance on sub-alpine pastures

Resource competition and disease transmission may occur when domestic and wild ungulates live sympatricly. We investigated if the release of sheep (Ovis aries) onto alpine pasture in Switzerland affected chamois (Rupicapra rupicapra) activity budgets, local population size and spatial distribution. We also evaluated the risk of transmission of Mycoplasma conjunctivae (causing a contagious eye disease) from sheep to chamois by examining if the two species had close contact with one another. We carried out the study in an alpine valley containing two adjacent areas: one containing sheep (Fochsenflue) and one where sheep were excluded (Spitzflue). We found no difference between the activity budgets of the chamois at the two sites. At the Fochsenflue, chamois and sheep mainly used separate areas. However, after approximately 1 month, sheep started to move twice per day, into the main area of the chamois. The percentage time feeding, spatial distribution and numbers of chamois did not change in response. Sheep were responsible for all encounters in which the two species came closer than 50 m to each other. The encounters were brief, body contact never occurred, they were not concentrated at saltlicks and chamois mainly ended them. The results suggest that the presence of sheep had little effect on the chamois. However, competition between the two species could still be occurring over a longer time scale. Finally, we found that the risk of inter-specific transmission of IKC through direct body contact is likely to be low, but the risk through indirect means (flies or aerosols) remains.