Spatial and Temporal Phylogeny of Border Disease Virus in Pyrenean Chamois (Rupicapra p. pyrenaica).

Border disease virus (BDV) affects a wide range of ruminants worldwide, mainly domestic sheep and goat. Since 2001 several outbreaks of disease associated to BDV infection have been described in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in Spain, France and Andorra. In order to reconstruct the most probable places of origin and pathways of dispersion of BDV among Pyrenean chamois, a phylogenetic analysis of 95 BDV 5'untranslated sequences has been performed on chamois and domestic ungulates, including novel sequences and retrieved from public databases, using a Bayesian Markov Chain Monte Carlo method. Discrete and continuous space phylogeography have been applied on chamois sequences dataset, using centroid positions and latitude and longitude coordinates of the animals, respectively. The estimated mean evolutionary rate of BDV sequences was 2.9×10-3 subs/site/year (95% HPD: 1.5-4.6×10-3). All the Pyrenean chamois isolates clustered in a unique highly significant clade, that originated from BDV-4a ovine clade. The introduction from sheep (dated back to the early 90s) generated a founder effect on the chamois population and the most probable place of origin of Pyrenean chamois BDV was estimated at coordinates 42.42 N and 1.9 E. The pathways of virus dispersion showed two main routes: the first started on the early 90s of the past century with a westward direction and the second arise in Central Pyrenees. The virus spread westward for more than 125 km and southward for about 50km and the estimated epidemic diffusion rate was about 13.1 km/year (95% HPD 5.2-21.4 km/year). The strong spatial structure, with strains from a single locality segregating together in homogeneous groups, and the significant pathways of viral dispersion among the areas, allowed to reconstruct both events of infection in a single area and of migrations, occurring between neighboring areas.

The two sides of border disease in Pyrenean chamois (Rupicapra pyrenaica): silent persistence and population collapse.

In 2001, border disease virus (BDV) was identified as the cause of a previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica) in Spain. Since then, the disease has caused a dramatic decrease, and in some cases collapse, of chamois populations and has expanded to nearly the entire distribution area in the Pyrenees. Chamois BDV was characterized as BDV-4 genotype and experimental studies confirmed that it was the primary agent of the disease. The infection has become endemic in the Central and Eastern Pyrenees. However, while most Pyrenean chamois populations have been severely affected by the disease, others have not, despite the circulation of BDV in apparently healthy individuals, suggesting the existence of different viral strategies for persisting in the host population. Changes in the interplay of pathogen, host and environmental factors may lead to the formation of different disease patterns. A key factor influencing disease emergence may be pathogen invasiveness through viral mutation. Host factors, such as behavior, immunity at the population level and genetic variability, may also have driven different epidemiological scenarios. Climatic and other ecological factors may have favored secondary infections, such as pneumonia, that under particular circumstances have been major contributing factors in the high mortality observed in some areas.

Border Disease Virus: An Exceptional Driver of Chamois Populations Among Other Threats.

Though it is accepted that emerging infectious diseases are a threat to planet biodiversity, little information exists about their role as drivers of species extinction. Populations are also affected by natural catastrophes and other pathogens, making it difficult to estimate the particular impact of emerging infectious diseases. Border disease virus genogroup 4 (BDV-4) caused a previously unreported decrease in populations of Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in Spain. Using a population viability analysis, we compared probabilities of extinction of a virtual chamois population affected by winter conditions, density dependence, keratoconjunctivitis, sarcoptic mange, and BD outbreaks. BD-affected populations showed double risk of becoming extinct in 50 years, confirming the exceptional ability of this virus to drive chamois populations.

Mineral levels in Pyrenean chamois (Rupicapra pyrenaica).

The effects of mineral deficiencies are often sub-clinical, and the importance of mineral status is often underestimated in wildlife populations. To our knowledge, this is the first study that gives reference intervals of hepatic minerals for Pyrenean chamois (Rupicapra pyrenaica). We determined macro and trace mineral concentrations in liver samples from 100 animals (44 healthy and 56 sick) collected in the Catalan Pyrenees (NE Spain) from 1995 to 2008. After wet digestion, we determined Na, K, Ca, P, Mg, S, and Fe concentrations by inductively coupled plasma optical emission spectrometry (ICP-OES) and Cu, Zn, and Mo concentrations by inductively coupled plasma mass spectrometry (ICP-MS). We observed low hepatic concentrations of Cu in a considerable percentage of chamois, without evidences that these low concentrations increased their susceptibility to infectious diseases. The group of sick chamois had very similar percentage of animals (10/56) with low concentration of Cu (<20 ppm DW) than the group of healthy chamois (9/44). On the other hand, we observed that infectious diseases increased significantly the hepatic concentrations of Na, Ca, Mg, Fe and Zn, very likely, as a consequence of processes associated with the acute phase inflammatory response. The obtained values of liver mineral levels and their sources of variation, such as sex, age and disease, mostly fall within the range of those described for other ruminants, but possible deficiencies and differences between individuals and populations require further study.

Effect of perphenazine enanthate in Pyrenean chamois (Rupicapra pyrenaica).

Perphenazine enanthate was used to allow adaptation to captivity in 11 Pyrenean chamois (Rupicapra pyrenaica). At the time of capture, all animals received 0.10 mg/kg of acepromazine maleate and 2.5 mg/kg of perphenazine enanthate intramuscularly. The effect was evaluated by means of three behaviors: alertness, defecation, and flight distance. The tranquilization and lack of fear of humans of all animals were determined and the usefulness of this long-acting tranquilizer for chamois adaptation to captivity was confirmed.

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.

Surveillance of border disease in wild ungulates and an outbreak in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in Andorra.

The Principality of Andorra is surrounded by areas in which Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations were severely affected by infection with border disease virus (BDV) which caused disease outbreaks between 2001 and 2009. Nevertheless, the Andorran chamois populations were not affected during this period. In light of the severe impact of BDV on several of the neighboring Pyrenean chamois populations, we monitored local Andorran populations in an effort to detect pestivirus antibodies and BDV in wild ungulates. In addition, an episode of mortality between 2009 and 2010 in chamois was investigated. We analyzed samples (spleen or serum) from 175 Pyrenean chamois, 284 European mouflon (Ovis orientalis musimon), 13 roe deer (Capreolus capreolus capreolus), and five wild boars (Sus scrofa castilianus). With the exception of three dead chamois found between 2009 and 2010, all samples came from healthy animals hunted during the hunting season. A commercial blocking enzyme-linked immunosorbent assay (ELISA) was used to test sera for antibodies against pestivirus. Positive sera were tested with a comparative virus neutralization test (VNT) using three BDV strains and a bovine viral diarrhea virus strain. Reverse-transcription-polymerase chain reaction (RT-PCR) was performed on all sera and spleen homogenates. Antibodies against pestivirus were detected by ELISA in four of the 69 chamois (5%; 95% CI= 1.29-13.11). The VNT confirmed three of these chamois were infected with a BDV. Viral RNA was detected by RT-PCR in three chamois-one apparently healthy animal hunted in 2009 and two dead animals. Viral sequences showed that the three chamois were infected with a BDV-4, the same genotype that was involved in previous episodes of mortality in the Pyrenees. Although Pyrenean chamois from Andorra had had little contact with the pestiviruses until 2009, in this year BDV was associated with a severe disease outbreak.

Evaluation of the efficacy of commercial vaccines against bluetongue virus serotypes 1 and 8 in experimentally infected red deer (Cervus elaphus).

Red deer (Cervus elaphus) is a widespread and abundant species susceptible to bluetongue virus (BTV) infection. Inclusion of red deer vaccination among BTV control measures should be considered. Four out of twelve BTV antibody negative deer were vaccinated against serotype 1 (BTV-1), and four against serotype 8 (BTV-8). The remaining four deer acted as unvaccinated controls. Forty-two days after vaccination (dpv), all deer were inoculated with a low cell passage of the corresponding BTV strains. Serological and virological responses were analyzed from vaccination until 28 days after inoculation (dpi). The vaccinated deer reached statistically significant (P<0.05) higher specific antibody levels than the non vaccinated deer from 34 (BTV-8) and 42 (BTV-1) dpv, maintaining stable neutralizing antibodies until 28 dpi. The non vaccinated deer remained seronegative until challenge, showing neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of the non vaccinated deer from 2 to 28 dpi, whereas no BTV RNA was found in the vaccinated deer. BTV was isolated from the blood of non vaccinated deer from 7 to 28 dpi (BTV-1) and from 9 to 11 dpi (BTV-8). BTV RNA could be identified by RT-PCR at 28 dpi in spleen and lymph nodes, but BTV could not be isolated from these samples. BT-compatible clinical signs were inapparent and no gross lesions were found at necropsy. The results obtained in the present study confirm that monovalent BTV-1 and BTV-8 vaccines are safe and effective to prevent BTV infection in red deer. This finding indicates that vaccination programs on farmed or translocated red deer could be a useful tool to control BTV.

Experimental infection with chamois border disease virus causes long-lasting viraemia and disease in Pyrenean chamois (Rupicapra pyrenaica).

Since 2001, severe outbreaks of disease associated with border disease virus (BDV) infection have been reported in Pyrenean chamois. The disease is characterized by variable degrees of cachexia, alopecia and neurological manifestations prior to death. The aim of this study was to investigate this disease under experimental conditions. To assess viral virulence, humoral immune response, dissemination and probable routes of transmission, seven chamois (five seronegative and two seropositive for BDV) were inoculated with a BDV isolated from a naturally infected chamois. A group of three chamois were maintained as uninfected controls. The five seronegative chamois became viraemic from day 2 post-inoculation (p.i.) until their death (three animals) or the end of the experiment (on day 34 p.i.) and developed neutralizing antibodies from day 18 p.i. until the end of the study. Continuous shedding of the virus was detected by RT-PCR in oral, nasal and rectal swabs in viraemic chamois from day 5 p.i. Despite none of the viraemic chamois showing obvious neurological signs, all of them had a non-suppurative meningoencephalitis as seen in naturally infected chamois. The two inoculated BDV-seropositive chamois did not become viraemic. This study confirms that BDV is the primary agent of the disease that has been affecting chamois populations in recent years in the Pyrenees and that previously acquired humoral immunity is protective.

Trichinella sp. in red foxes (Vulpes vulpes) from Catalonia, NE Spain.

European legislation allows the official recognition of Trichinella-free pig holdings, provided Trichinella sp. infection is absent from humans and prevalence of Trichinella sp. infection in red foxes (Vulpes vulpes) is below 0.1% in the area, region or country. Tibialis anterior muscle samples from 1,319 red foxes captured in Catalonia (NE Spain) between 1998 and 2007 were analyzed for Trichinella sp. using the digestion method. Four foxes resulted positive (one in 1999, one in 2002 and two in 2006), accounting for a low prevalence (0.3%). However, this prevalence was concentrated in mountain or rural areas with a low sample size, reaching high local prevalences. The two positive samples in 2006 were characterized as Trichinella britovi, and a sylvatic cycle of trichinellosis seems to occur, at least in the rural insufficiently sampled regions of Catalonia. Overall, the results obtained do not currently allow the establishment of Trichinella-free pig holdings in the study area, but further research is needed to better know the prevalence and cycle of Trichinella sp. in Catalonia.

Retrospective study of pestivirus infection in Pyrenean chamois (Rupicapra pyrenaica) and other ungulates in the Pyrenees (NE Spain).

In 2001 a new Pestivirus (Family Flaviviridae) was associated with an outbreak of a previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica) in the Pyrenees (NE Spain). Molecular characterization assigned this virus to the Border Disease Virus (BDV) cluster, BDV-4 genotype. A retrospective study was performed in archived sera and spleen of 74 Pyrenean chamois and in archived sera of 28 mouflon (Ovis ammon), 56 red deer (Cervus elaphus), 43 roe deer (Capreolus capreolus) and 29 fallow deer (Dama dama) from the Pyrenees between the years 1990 and 2000. Thirty six of 74 (48.6%) sera of Pyrenean chamois, one of mouflon and one of red deer were positive by an ELISA antibody test. Comparative virus neutralization tests were performed on 26 seropositive chamois, one mouflon and one red deer, using five pestivirus strains. An ELISA antigen test was performed on 37 seronegative chamois and yielded positive results in one chamois and inconclusive result in two. RT-PCR and virus isolation performed on spleen samples from these three animals gave positive results in the positive and one inconclusive animal. Sequence analysis in the 5' unstranslated region revealed that they were grouped into the BDV-4 genotype. Virological and serological data of the present study indicate that BDV infection has been present in the chamois population since at least 1990, 11 years before the first outbreak of disease. Therefore, the emergence of the disease in 2001 is apparently due to other factors rather than the introduction of a new virus in the chamois population.