Factors influencing wild chimpanzee (Pan troglodytes verus) relative abundance in an agriculture-swamp matrix outside protected areas.

Human population growth and anthropogenic activities are exacerbating pressures on biodiversity globally. Land conversion is aggravating habitat fragmentation and non-human primates are increasingly compelled to live in forest-agricultural mosaics. In Sierra Leone, more than half of the wild chimpanzee population (Pan troglodytes verus) occurs outside protected areas and competes for resources with farmers. Our study area, in the Moyamba district in south-western Sierra Leone, is practically devoid of forest and is dominated by cultivated and fallow fields, swamps and mangroves. In this region, traditional slash-and-burn agriculture modifies annually the landscape, sparing swamps and mangroves and semi-domesticated oil palms (Elaeis guineensis). This study aimed to explore ecological and anthropogenic factors influencing chimpanzee relative abundance across this highly degraded and human-impacted landscape. Between 2015 and 2016, we deployed 24 camera traps systematically across 27 1.25x1.25 km grid cells. Cameras were operational over a period of 8 months. We used binomial iCAR models to examine to what extent anthropogenic (roads, settlements, abandoned settlements and human presence) and habitat variables (swamps, farmland and mangroves) shape chimpanzee relative abundance. The best model explained 43.16% of the variation with distance to roads and swamps emerging as the best predictors of chimpanzee relative abundance. Our results suggest that chimpanzees avoid roads and prefer to maintain proximity to swamps. There was no significant effect of settlements, abandoned settlements, mangroves or human presence. It appears that chimpanzees do not avoid areas frequented by people; although, our findings suggest temporal avoidance between the two species. We highlight the importance of studying chimpanzee populations living in anthropogenic habitats like agricultural-swamp matrixes to better understand factors influencing their distribution and inform conservation planning outside protected areas.

Porcine circovirus 3 is highly prevalent in serum and tissues and may persistently infect wild boar (Sus scrofa scrofa).

Porcine circovirus 3 (PCV-3) prevalence has been minimally investigated in wild boar; dynamics of infection and viral tissue distribution are currently unknown. In this study, serum samples from 518 wild boar (from years 2004 to 2018) were used to study frequency of infection. Also, serum samples from 19 boar captured and recaptured at least two times for a period of time from 1 month to 1 year were collected to determine PCV-3 infection dynamics. Finally, to elucidate PCV-3 DNA organic distribution, sera, different tissues and faeces were obtained from 35 additional wild boar. PCV-3 DNA was extracted and amplified with a conventional PCR. For the PCV-3 PCR-positive sera from the longitudinally sampled and different tissue types, a quantitative PCR was performed. Genome sequence was obtained from a number of PCV-3 PCR-positive samples from different years, different time-points of infection and tissues. Obtained results confirmed the susceptibility of wild boar to the virus, showing high frequency of PCV-3 detection (221 out of 518, 42.66%) and demonstrating circulation at least since 2004. Compiled data indicate the possibility of long-term infections, since 5 out of 10 PCV-3 PCR-positive boars longitudinally sampled showed positivity in samplings separated for more than 5 months. All tested tissue types' harboured PCV-3 genome, with the highest percentage of PCR positivity in submandibular lymph node, tonsil, lung, liver, spleen and kidney. The amount of DNA in all tested PCV-3 PCR-positive samples was moderate to low. All partial and complete PCV-3 sequences obtained from wild boar displayed high nucleotide identity, higher than 98%. In conclusion, this study further confirms that wild boar is susceptible to PCV-3 infection, showing high frequency of detection in this animal species. Furthermore, PCV-3 can be found in different tissues of wild boar and is apparently able to cause persistent infection.

Plasma biochemistry RIs and age effect in European Strigiformes.

BACKGROUND: Blood biochemistry and hematology are essential in the laboratory diagnosis of disease. In Strigiformes, little information regarding reference values and influence of different preanalytic factors is available, while age is known to have an effect on some biochemistry analytes, especially in early life characterized by a rapid growth rate and increase in body mass. OBJECTIVE: The objective of this study was to determine baseline data for 29 blood biochemistry variables in 5 species of Iberian Strigiformes assigned to different age classes. METHODS: Healthy nocturnal birds living in wildlife health centers of Catalonia, Northeastern Spain were assigned to different age classes (chicks, juveniles, adults where possible and available) and their blood was collected and analyzed for different standard biochemistry variables. Species included Tawny owls, Little owls, Long-eared owls, Scop owls, and Barn owls. RESULTS: A total of 276 clinically healthy animals were sampled, including between 179 chicks, 52 juveniles, and 45 adults. The RIs of cholesterol, albumin, and osmolality were the only variables that did not show interspecific variability. Common trends between age class groups were observed in 4 species. Phosphorus and calcium concentrations and ALP activities decreased with increasing age, while concentrations of sodium and γ-globulins tended to increase in the Strigidae family. Differences were concentrated in chicks and no differences were observed between juveniles and adults in any variable. CONCLUSIONS: Our results demonstrate the importance of defining biochemical RIs specific for certain age classes.

Hematologic reference intervals and age effect in European Strigiformes.

BACKGROUND: The clinical importance of hematologic testing in avian veterinary medicine is reflected in the increasing number of studies for the establishment of hematologic RIs of Strigiformes and other species. Age is an important physiologic factor in birds and the effect on hematology variable should be understood. OBJECTIVES: The objective of this study was to determine baseline data of hematologic variables in 5 species of Iberian Strigiformes in different age classes. METHODS: Nocturnal birds of prey were sampled at Wildlife Health Centers. Packed cell volume was determined by the microhematocrit centrifugation method, and RBC and WBC counts were determined using the direct hemocytometer count method with Natt and Herrick solution. Hemoglobin concentration was measured spectrophotometrically. The MCV, MCHC, and MHC were calculated using the standard formulas. The differential WBC count was performed by the routine microscopic evaluation of 200 cells on a blood smear manually stained with Wright stain. Thrombocyte blood count estimate was obtained from the blood film. RESULTS: No differences were observed between juveniles and adults for any variable evaluated in Tawny owl, Little owl, Scops owl, Long-eared owl, and Barn owl. In addition, PCV, RBC, and HGB of chicks were statistically significantly lower than in juveniles and adults, and total WBC was significantly higher in Tawny owl, Little owl, Scops owl, and Long-eared owl. CONCLUSIONS: Our findings provide evidence that laboratory data from chicks of Strigiformes are different compared to juveniles and adults; therefore, separate RIs were defined.

Determination of fluoroquinolone antibiotic residues in the plasma of Eurasian griffon vultures (Gyps fulvus) in Spain.

Due to the possible toxicological impact, the accumulation of pharmaceuticals in wildlife as a consequence of human practices is of growing concern. The consumption of carrion at feeding stations - the so-called 'vulture restaurants' - with no management of the veterinary drugs it contains may expose scavengers to pharmaceuticals. To demonstrate this, we analyzed plasma from Eurasian griffon vultures (Gyps fulvus) originating from two different areas of Spain for antibiotics such as enrofloxacin and ciprofloxacin, its primary metabolite. Quinolone residues were detected in about 65% (n=106) of birds, of which 15.1% (16/106) had quantifiable amounts of enrofloxacin (0.049±0.102µg/mL) and 5.7% (6/106) of ciprofloxacin (0.009±0.007µg/mL). The differences in exposure between the two sampled areas are attributable to different types of carrion management: the vultures that fed in areas with a high density of dead livestock (supplied directly to feeding stations) were more prone to exposure than those that sought food in areas where carcass availability is more unpredictable. Our findings are evidence that vultures have access to medicated livestock and that there are quantifiable levels of livestock antibiotics in vulture plasma. However, the vultures analyzed in this study had maximum antibiotic concentrations of only 0.4µg/mL, much less than the concentrations used in the clinical treatment of scavengers and a level that is probably too small to cause intoxication.

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.

Streptococcus pharyngis sp. nov., a novel streptococcal species isolated from the respiratory tract of wild rabbits.

Four isolates of an unknown Gram-stain-positive, catalase-negative coccus-shaped organism, isolated from the pharynx of four wild rabbits, were characterized by phenotypic and molecular genetic methods. The micro-organisms were tentatively assigned to the genus Streptococcus based on cellular morphological and biochemical criteria, although the organisms did not appear to correspond to any species with a validly published name. Comparative 16S rRNA gene sequencing confirmed their identification as members of the genus Streptococcus, being most closely related phylogenetically to Streptococcus porcorum 682-03(T) (96.9% 16S rRNA gene sequence similarity). Analysis of rpoB and sodA gene sequences showed divergence values between the novel species and S. porcorum 682-03(T) (the closest phylogenetic relative determined from 16S rRNA gene sequences) of 18.1 and 23.9%, respectively. The novel bacterial isolate could be distinguished from the type strain of S. porcorum by several biochemical characteristics, such as the production of glycyl-tryptophan arylamidase and α-chymotrypsin, and the non-acidification of different sugars. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be assigned to a novel species of the genus Streptococcus, and named Streptococcus pharyngis sp. nov. The type strain is DICM10-00796B(T) ( = CECT 8754(T) = CCUG 66496(T)).

Hematologic and biochemical reference intervals for Wild Boar (Sus scrofa) captured by cage trap.

BACKGROUND: Establishing reference intervals (RI) for hematologic and biochemical variables in wild animals presents great challenges because capture stress or anesthesia during sampling can affect blood variables. OBJECTIVES: The aims of this study were to establish RI for hematologic and blood biochemistry variables for Wild Boar (Sus scrofa) caught using cage traps, and provide information on the studied variables for different age groups. METHODS: Blood samples were obtained from 89 Wild Boars captured by cage trap between 2005 and 2013 in northeastern Spain. Piglets were handled without anesthesia, while juvenile and adult animals were anesthetized using a combination of tiletamine and zolazepam. Blood samples were collected from the anterior vena cava and were placed into plain and EDTA tubes. Thirteen hematologic and 21 biochemical variables were determined. Reference intervals for piglets and juvenile and adult groups were determined, and differences between these groups were statistically analyzed. RESULTS: Adults had higher HGB, PCV, MCH, MCHC, neutrophil count, and total protein, albumin, creatinine, and chloride concentrations than juveniles; in contrast, juveniles had higher values for lymphocyte count, cholesterol concentration, and ALP activity. CONCLUSIONS: Reference intervals determined in this study provide a baseline for interpreting hematologic and biochemical results in Wild Boar at different age stages, and contribute to optimization of the management of this species.

Uses and limitations of faecal egg count for assessing worm burden in wild boars.

The most widely used technique to assess helminth infection in both domestic and wild mammals is the faecal egg count (FEC). Most efforts to test the reliability of FEC as a proxy for parasite load are in small ruminant studies and limited work has evaluated the use of FEC in pigs. The aim of this study was to explore whether FEC is a reliable indicator of helminth load, and to evaluate the effects of sample storage on FEC accuracy in 59 wild boars. Though FEC was useful for assessing most helminth infections (e.g., Metastrongylus spp., Ascaris suum, Trichuris suis), stomach nematodes were often missed. The accuracy of FEC decreased over time, and thus it is recommended that samples be processed within 5 days of collection.

Estimation of cultivable bacterial diversity in the cloacae and pharynx in Eurasian griffon vultures (Gyps fulvus).

In this work, we describe the biodiversity of cloacal and pharynx culture-based bacteria (commensal and pathogenic), in 75 Eurasian griffon vultures (Gyps fulvus) from two geographic areas. We address the question of whether the cultivable microbiota of vultures is organised into assemblages occurring by chance. In addition, we assess bacterial diversity in both anatomic regions and geographic areas. Bacterial diversity was represented by 26 Gram-negative and 20 Gram-positive genera. The most common genera were Escherichia, Enterococcus, Staphylococcus, Clostridium and Lactococcus. Escherichia coli and Enterococcus faecalis were the most common species in cloacal and pharyngeal samples. Staphylococcus and Erysipelothrix were isolated from the pharynx and Salmonella and Corynebacterium from the cloacae, and no Campylobacter was isolated from the cloacal swabs. Ten cloacal swabs were positive for Salmonella, of which five isolates were Salmonella enterica serotype 4,(5),12:i:-, one isolate was S. enterica serotype Derby, three isolates were S. enterica serotype 61:k:1,5,7 and one isolate was S. enterica serotype Infantis. The null modelling approach revealed that the commensal bacteria of vultures are not structured in assemblages. On the other hand, differences in bacterial genus and species richness between cloacal and pharyngeal samples or between geographic areas were clear, with the pharynx in vultures from both geographic areas being richer. The results of this study indicate also that vultures can serve as a reservoir of certain pathogenic zoonotic bacteria. The dissemination of these zoonotic pathogens in wildlife could be prevented by periodic sanitary surveys.

Assessment of the exposure to heavy metals in Griffon vultures (Gyps fulvus) from the Iberian Peninsula.

Griffon vultures (Gyps fulvus), by virtue of their position at the top of the food chain and as obligate scavengers, are at risk of accumulating and concentrating heavy metals in their tissues and may be more predisposed to their toxic effects. The aim of this study is to investigate heavy metal concentrations in Griffon vultures in Portugal and Catalonia, Spain and to determine if heavy metal concentrations in the blood of weak and/or injured Griffon vultures admitted to wildlife rehabilitation centres (WRC) reflect contamination profiles in the local, free-living and outwardly healthy population. Whole-blood samples taken from 121 Griffon vultures caught in the wild or admitted to WRC in Portugal and Catalonia, Spain were examined for cadmium (Cd), mercury (Hg) and lead (Pb) by inductively coupled plasma mass spectrometry (ICP-MS). Cd and Hg were not detected in most samples (98.3% and 95%, respectively), while Pb was detected in all birds in concentrations ranging between 4.97 and 300.23 µg/dl. Birds admitted to WRC had significantly lower Pb concentrations (24.15 ± 15.07 and 25.98 ± 18.04 µg/dl in Portugal and Catalonia, Spain, respectively) than animals caught in the wild (29.67 ± 13.19 and 42.22 ± 50.08 µg/dl in Portugal and Catalonia, Spain, respectively) (p<0.05). This may be explained by the fact that malnutrition was the main cause of admission of Griffon vultures to WRC, as ingestion has been described as the most significant pathway for Pb exposure in raptors. Therefore Griffon vultures admitted to WRC do not seem to be representative of the local, free-flying populations, so it remains necessary to continue catching when one intends to monitor Pb exposure in this species. The population of vultures captured in Catalonia, Spain showed the highest mean blood Pb concentration, perhaps due to the municipal rubbish dump located near the feeding station, with rubbish providing a significant fraction of their trophic needs. The ingestion of game meat with bullet fragments in carcasses or with Pb shots embedded in their flesh could also be the cause of the high blood Pb concentrations found in some vultures. The potential risk of Pb exposure in Griffon vulture populations must be given consideration, since most individuals evaluated had Pb concentrations between 20 and 100µg/dl, which is considered to be subclinical exposure to Pb, and which is above the threshold level at which Pb can affect antioxidant system in this species (15 µg/dl).

Management of a caseous lymphadenitis outbreak in a new Iberian ibex (Capra pyrenaica) stock reservoir.

BACKGROUND: In 2010, an Iberian ibex (Capra pyrenaica hispanica) stock reservoir was established for conservation purposes in north-eastern Spain. Eighteen ibexes were captured in the wild and housed in a 17 hectare enclosure. Once in captivity, a caseous lymphadenitis (CLA) outbreak occurred and ibex handlings were carried out at six-month intervals between 2010 and 2013 to perform health examinations and sampling. Treatment with a bacterin-based autovaccine and penicillin G benzatine was added during the third and subsequent handlings, when infection by Corynebacterium pseudotuberculosis was confirmed. Changes in lesion score, serum anti-C. pseudotuberculosis antibodies and haematological parameters were analyzed to assess captivity effects, disease emergence and treatment efficacy. Serum acute phase proteins (APP) Haptoglobin (Hp), Amyloid A (SAA) and Acid Soluble Glycoprotein (ASG) concentrations were also determined to evaluate their usefulness as indicators of clinical status. Once in captivity, 12 out of 14 ibexes (85.7%) seroconverted, preceding the emergence of clinical signs; moreover, TP, WBC, eosinophil and platelet cell counts increased while monocyte and basophil cell counts decreased. After treatment, casualties and fistulas disappeared and both packed cell volume (PCV) and haemoglobin concentration significantly increased. Hp, SAA and ASG values were under the limit of detection or showed no significant differences. CONCLUSIONS: A role for captivity in contagion rate is suggested by the increase in antibody levels against C. pseudotuberculosis and the emergence of clinical signs. Although boosted by captivity, this is the first report of an outbreak of caseous lymphadenitis displaying high morbidity and mortality in wild ungulates. Treatment consisting of both vaccination and antibiotic therapy seemed to prevent mortality and alleviate disease severity, but was not reflected in the humoural response. Haematology and APP were not useful indicators in our study, perhaps due to the sampling frequency. Presumably endemic and irrelevant in the wild, this common disease of domestic small ruminants is complicating conservation efforts for the Iberian ibex in north-eastern Spain.

Carriage of Staphylococcus aureus by free-living wild animals in Spain.

The presence of methicillin-susceptible Staphylococcus aureus (MSSA) was analyzed in different free-living wild animals to assess the genetic diversity and predominant genotypes on each animal species. Samples were taken from the skin and/or nares, and isolates were characterized by spa typing, multilocus sequence typing (MLST) and antimicrobial susceptibility testing. The proportion of MSSA carriers were 5.00, 22.93, 19.78, and 17.67% in Eurasian griffon vulture, Iberian ibex, red deer, and wild boar, respectively (P = 0.057). A higher proportion of isolates (P = 0.000) were recovered from nasal samples (78.51%) than skin samples (21.49%), but the 9.26% of red deer and 18.25% of wild boar would have been undetected if only nasal samples had been tested. Sixty-three different spa types were identified, including 25 new spa types. The most common were t528 (43.59%) in Iberian ibex, t548 and t11212 (15.79% and 14.04%) in red deer, and t3750 (36.11%) in wild boar. By MLST, 27 STs were detected, of which 12 had not been described previously. The most frequent were ST581 for Iberian ibex (48.72%), ST425 for red deer (29.82%), and ST2328 for wild boar (42.36%). Isolates from Eurasian griffon vulture belong to ST133. Host specificity has been observed for the most frequent spa types and STs (P = 0.000). The highest resistance percentage was found against benzylpenicillin (average, 22.2%), although most of the S. aureus isolates were susceptible to all antimicrobial tested. Basically, MSSA isolates were different from those MRSA isolates previously detected in the same animal species.

Food-borne zoonotic pathogens and antimicrobial resistance of indicator bacteria in urban wild boars in Barcelona, Spain.

Wildlife is increasingly abundant in urban environments, but little is known about the zoonotic pathogens carried by these populations. Urban wild boars are of particular concern because this species is well-known as a pathogen reservoir, and thus, we studied selected zoonotic pathogens in urban wild boars in Barcelona, Spain (n=41). Salmonella enterica was found in 5.00% (95% CI 0.61-16.91) and Campylobacter coli in 4.88% (95% CI 0.6-16.53) of the animals. E. coli O157:H7 and C. jejuni were not found. Other thermophilic Campylobacter were moderately prevalent (19.51%, 95% CI 8.82-34.87). Additionally, we screened for antimicrobial resistance in indicator bacteria: resistance was most frequent in Enterococcus faecium (95% of the isolates were resistant to at least one antimicrobial agent), followed by Enterococcus faecalis (50%) and Escherichia coli (10%). For the first time resistance to linezolid in bacteria carried by wildlife is reported. These findings pose a concern for public health, and thus, further research is needed on wildlife in urban environments.

Methicillin resistant Staphylococcus aureus (MRSA) carriage in different free-living wild animal species in Spain.

Methicillin-resistant Staphylococcus aureus (MRSA) is a life-threatening pathogen in humans and its presence in animals is a public health concern. The aim of this study was to measure the prevalence of MRSA in free-living wild animals. Samples from red deer (n=273), Iberian ibex (n=212), Eurasian Griffon vulture (n=40) and wild boar (n=817) taken from different areas in Spain between June 2008 and November 2011 were analyzed. Characterization of the isolates was performed by spa typing, multi-locus sequence typing (MLST) and antimicrobial susceptibility testing. A low prevalence of MRSA was found with 13 isolates obtained from 12 animals (0.89%; 95% CI: 0.46-1.56). All MRSA sequence types belonged to ST398 (t011 and t1451) and ST1 (t127). Genotypes and antimicrobial susceptibility patterns (tetracycline resistance in ST398 and clindamycin-erythromycin-tetracycline resistance in ST1) suggest that the MRSA found probably originated in livestock (ST398) or humans (ST1). This is the first report of MRSA carriers in free-living wild animals in Europe. Although our data showed that MRSA prevalence is currently low, free-living wild animals might act as reservoir and represent a potential risk for human health.

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.

Effect of acepromazine and haloperidol in male Iberian Ibex (Capra pyrenaica) captured by box-trap.

Short-acting neuroleptic drugs are used to prevent adverse effects of stress in wildlife. We compared the effect of acepromazine and haloperidol in Iberian ibex (Capra pyrenaica) captured with box-traps. We captured 23 male Iberian ibex at the National Game Reserve of Ports de Tortosa i Beseit, northeastern Spain, March 2003-June 2005. Seven animals received 0.1 mg/kg of acepromazine maleate, eight received 0.33 mg/kg of haloperidol and eight animals acted as controls. Clinical, hematologic, and serum biochemical parameters were analyzed. Both treatments decreased rectal temperature, white blood cells, lymphocytes, and concentrations of creatinine, creatine kinase, and lactate dehydrogenase. Acepromazine also decreased red blood cells, packed cell volume, hemoglobin concentration, neutrophils, and concentrations of glucose and cholesterol. Haloperidol also decreased heart rate and concentrations of urea and potassium. Our results demonstrate the suitability of using acepromazine and haloperidol in capture operations to reduce stress and prevent its adverse effects.

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.

Border disease virus shedding and detection in naturally infected Pyrenean chamois (Rupicapra pyrenaica).

Pyrenean chamois (Rupicapra pyrenaica) populations of the central and eastern Pyrenees have been affected by severe outbreaks associated with Border disease virus (BDV) since 2001. Eight Pyrenean chamois (7 males and 1 female) from 1 to 8 years of age with clinical signs consistent with BDV infection were studied. At necropsy, whole blood, tissue samples (skin, brain, prescapular lymph node, thyroid gland, lung, liver, spleen, kidney, small intestine, bone marrow, and testicle), urine, and nasal, oral, and rectal swabs were obtained. The fetus from a pregnant female was also studied. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the virus in all samples, and virus isolation was performed. Sera and tissue samples were positive to RT-PCR, and the virus was isolated from all chamois. The nasal, oral, and rectal swabs and urine samples were RT-PCR positive in 100%, 85.71%, 71.43%, and 100% of chamois, respectively, confirming the excretion of the virus via these 4 routes. In addition, sera were tested for BDV antibodies using enzyme-linked immunosorbent assay and seroneutralization techniques, with negative results. Sequence analysis of the 5' untranslated region in 7 of the chamois confirmed that the virus is grouped into the BDV-4 genotype, the same BDV previously described in Pyrenean chamois. To the authors' knowledge, this is the first study of naturally infected Pyrenean chamois, providing evidence that infected animals shed BDV through nasal, oral, fecal, and urinary excretion routes.