HEALTH ASSESSMENT OF CAPTIVE BEARDED VULTURES (GYPAETUS BARBATUS) IN CATALONIA (SPAIN).

Bearded vulture (Gypaetus barbatus) populations are declining worldwide primarily due to anthropogenic factors. A captive breeding program has been established in Spain, a country with one of the largest free-living populations in Europe, to further enhance the conservation efforts of this emblematic species. However, captive vulture populations can be exposed to different risks through food, such as drug residues and antimicrobial-resistant (AMR) bacteria. Health surveillance of species involved in captive breeding programs is important to face introduction of healthy animals in situ and to obtain baseline clinical data. The objective of this study was to assess the general health status of bearded vultures held in captivity in Catalonia (northeastern Spain) by carrying out hematologic, biochemical, toxicologic, and bacteriologic analyses. A total of 16 bearded vultures were sampled; the data obtained from one vulture, with a chronic tibiotarsal fracture, were excluded from the statistical analysis. Hematologic and biochemical parameters of the bearded vultures were mostly within the range of standard values as stated in previous studies. Basal feather and serum corticosterone levels were analyzed and described for the first time in this species. A total of 15 Escherichia coli isolates were obtained that were resistant to fluoroquinolones (80%), tetracycline (60%), trimethoprim and ampicillin (40%), sulfamethoxazole (33%), and colistin (20%), with 40% of them being multidrug resistant. Three of 15 isolates were carriers of the mcr-1 gene. Only the injured bird previously treated with enrofloxacin was positive for fluoroquinolone residues. Periodic monitoring for the presence of AMR bacteria would be recommended in captive breeding programs as a preventive action to establish future therapies.

Hotspot of Crimean-Congo Hemorrhagic Fever Virus Seropositivity in Wildlife, Northeastern Spain.

We conducted a serosurvey for Crimean-Congo hemorrhagic fever virus antibodies in various wildlife species in Catalonia, northeastern Spain. We detected high seroprevalence in southern Catalonia, close to the Ebro Delta wetland, a key stopover for birds migrating from Africa. Our findings could indicate that competent virus vectors are present in the region.

African swine fever virus infection in Classical swine fever subclinically infected wild boars.

BACKGROUND: Recently moderate-virulence classical swine fever virus (CSFV) strains have been proven capable of generating postnatal persistent infection (PI), defined by the maintenance of viremia and the inability to generate CSFV-specific immune responses in animals. These animals also showed a type I interferon blockade in the absence of clinical signs. In this study, we assessed the infection generated in 7-week-old CSFV PI wild boars after infection with the African swine fever virus (ASFV). The wild boars were divided in two groups and were infected with ASFV. Group A comprised boars who were CSFV PI in a subclinical form and Group B comprised pestivirus-free wild boars. Some relevant parameters related to CSFV replication and the immune response of CSFV PI animals were studied. Additionally, serum soluble factors such as IFN-α, TNF-α, IL-6, IL-10, IFN-γ and sCD163 were analysed before and after ASFV infection to assess their role in disease progression. RESULTS: After ASFV infection, only the CSFV PI wild boars showed progressive acute haemorrhagic disease; however, the survival rates following ASFV infection was similar in both experimental groups. Notwithstanding, the CSFV RNA load of CSFV PI animals remained unaltered over the study; likewise, the ASFV DNA load detected after infection was similar between groups. Interestingly, systemic type I FN-α and IL-10 levels in sera were almost undetectable in CSFV PI animals, yet detectable in Group B, while detectable levels of IFN-γ were found in both groups. Finally, the flow cytometry analysis showed an increase in myelomonocytic cells (CD172a+) and a decrease in CD4+ T cells in the PBMCs from CSFV PI animals after ASFV infection. CONCLUSIONS: Our results showed that the immune response plays a role in the progression of disease in CSFV subclinically infected wild boars after ASFV infection, and the immune response comprised the systemic type I interferon blockade. ASFV does not produce any interference with CSFV replication, or vice versa. ASFV infection could be a trigger factor for the disease progression in CSFV PI animals, as their survival after ASFV was similar to that of the pestivirus-free ASFV-infected group. This fact suggests a high resistance in CSFV PI animals even against a virus like ASFV; this may mean that there are relevant implications for CSF control in endemic countries. The diagnosis of ASFV and CSFV co-infection in endemic countries cannot be ruled out and need to be studied in greater depth.

Absence of antibodies specific to Besnoitia spp. in European sheep and goats from areas in Spain where bovine besnoitiosis is endemic.

Besnoitia besnoiti and B. caprae, which infect bovids (cattle and antelopes) and goats, respectively, are responsible for besnoitiosis, a chronic and debilitating disease. Bovine besnoitiosis is considered to be a reemerging disease in Central and Western Europe. In addition, infection by Besnoitia spp. has been reported in reindeer from Sweden and Finland. Recently, the parasite was also detected in roe deer and red deer from Spain, where an interconnection between the domestic and sylvatic cycles of B. besnoiti has been presumed. In contrast, caprine besnoitiosis seems to be enzootic to Kenya and Iran. The presence of Besnoitia spp. in small domestic ruminants has never been explored in Europe, and the role that these species might play in the epidemiology of bovine besnoitiosis, as intermediate hosts or reservoirs of B. besnoiti, remains unknown. Herein, the first serosurvey conducted in European sheep and goats from areas in Spain where bovine besnoitiosis is endemic is described. Convenience sampling was conducted of 1943 sheep and 342 goats close to cattle from the Pyrenees and Central Spain that were infected with endemic Besnoitia spp. Serum samples were first analyzed by ELISA and then by confirmatory Western blot. Specific antibodies were not found in any sampled animal. Thus, sheep are unlikely to play a role in the epidemiology of bovine besnoitiosis, at least in the sampled areas. A larger serosurvey is necessary to determine whether goats might be a putative reservoir. To confirm the results of this study, sheep and goats should be further studied in other European countries and regions where their numbers are high and where bovine besnoitiosis is spreading.

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.

First report of Sarcocystis halieti (Apicomplexa) in bearded vulture (Gypaetus barbatus).

At least three Sarcocystis species (S. falcatula, S. halieti and S. wobeseri-like) have been detected infecting raptorial birds. By histopathology and PCR-sequencing of the ITS1 marker, S. halieti was detected in a bearded vulture (Gypaetus barbatus) and a black kite (Milvus migrans) from the Catalonia region in North Spain. The 241 bp-long sequences obtained from the Sarcocystis organisms detected in both raptors showed 97.5-99.6% and 97.9-100% similarity with those of previously identified S. halieti; also, the phylogenetic trees generated placed the identified sequences together with other sequences of S. halieti available in GenBank. In sum, the description of the bearded vulture as a new intermediate host for S. halieti adds new insights on the complex epidemiology of the genus involving avian hosts.

Mycoplasma conjunctivae in domestic small ruminants from high mountain habitats in Northern Spain.

BACKGROUND: Infectious keratoconjunctivitis (IKC) is a clinical condition affecting eyes of domestic and wild Caprinae worldwide, and Mycoplasma conjunctivae is considered the primary causative agent of IKC in sheep, goats and wild Caprinae. Domestic ruminants from high mountain habitats share grazing areas with wild mountain ungulates, such as chamois (Rupicapra spp.), Alpine ibex (Capra ibex) and European mouflon (Ovis aries musimon), and domestic sheep seem to act as M. conjunctivae reservoir. In this study, the presence of M. conjunctivae in domestic sheep and goats from the two main mountain ranges of Northern Spain, the Pyrenees and the Cantabrian Mountains, has been investigated. RESULTS: Eye swabs were obtained from 439 domestic small ruminants selected from flocks that seasonally graze in alpine meadows during three consecutive years (2011-2012-2013). Seventy-nine out of the 378 domestic sheep (20.9%) tested positive to a M. conjunctivae specific real time-PCR (rt-PCR) in at least one eye, whereas all the 61 sampled domestic goats were negative. Statistically significant higher prevalence and higher proportion of infected flocks (P < 0.001) was observed in the Pyrenees (25.7%; 12 flocks out of 13), where M. conjunctivae is widespread and probably endemic in domestic sheep, than in the Cantabrian Mountains (7.8%; one flock out of six). Twenty-five sheep (three from the Pyrenees and 22 from the Cantabrian Mountains) which showed clinical signs consistent with infectious keratoconjunctivitis (IKC) were negative by rt-PCR. In contrast, 62 out of the 71 (87.3%) M. conjunctivae-positive sheep from the Pyrenees and the eight positive sheep from the Cantabrian Mountains were asymptomatic. CONCLUSIONS: This study provides rt-PCR-based evidences of M. conjunctivae maintenance in domestic sheep, as well as a relationship between prevalence in domestic sheep and previously reported M. conjunctivae and IKC in wild ruminants. Domestic goats do not seem to play an important role in the epidemiology of M. conjunctivae in alpine habitats from Northern Spain.

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.

New Potential Role of European Mouflon (Ovis aries musimon) in the Epidemiology of Border Disease in the Pyrenees.

Since 2001, high-mortality outbreaks of border disease (BD) have negatively affected populations of Pyrenean chamois (Rupicapra pyrenaica pyrenaica). Studies in the affected areas determined that sympatric wild ruminants did not seem to have an epidemiologic role in the circulation of border disease virus (BDV). However, the recent increase in European mouflon (Ovis aries musimon) densities might enhance the risk of pathogen transmission among chamois and mouflons. We conducted a serologic and virologic investigation of BDV in European mouflon from the Spanish Pyrenees, with the aim of determining potential changes in the role of this species in BDV epidemiology. From 2018 to 2022, we detected antibodies against BDV in 31/185 (16.7%) animals but did not detect BDV RNA in any spleen sample (0/65). These results indicate that BDV infection is occurring in these mouflon populations to a greater extent than previously described, which could shift the current understanding of BD epidemiology in the Pyrenees and cause an unpredictable effect on both chamois and mouflon populations. Further studies on the molecular identification of BDV in mouflon and chamois are required to better understand the contribution of mouflon in the epidemiology of BD.

The fecal bacterial microbiome of the Kuhl's pipistrelle bat (Pipistrellus kuhlii) reflects landscape anthropogenic pressure.

BACKGROUND: Anthropogenic disturbance has the potential to negatively affect wildlife health by altering food availability and diet composition, increasing the exposure to agrochemicals, and intensifying the contact with humans, domestic animals, and their pathogens. However, the impact of these factors on the fecal microbiome composition of wildlife hosts and its link to host health modulation remains barely explored. Here we investigated the composition of the fecal bacterial microbiome of the insectivorous bat Kuhl's pipistrelle (Pipistrellus kuhlii) dwelling in four environmental contexts with different levels of anthropogenic pressure. We analyzed their microbiome composition, structure and diversity through full-length 16S rRNA metabarcoding using the nanopore long-read sequencer MinION™. We hypothesized that the bacterial community structure of fecal samples would vary across the different scenarios, showing a decreased diversity and richness in samples from disturbed ecosystems. RESULTS: The fecal microbiomes of 31 bats from 4 scenarios were sequenced. A total of 4,829,302 reads were obtained with a taxonomic assignment percentage of 99.9% at genus level. Most abundant genera across all scenarios were Enterococcus, Escherichia/Shigella, Bacillus and Enterobacter. Alpha diversity varied significantly between the four scenarios (p < 0.05), showing the lowest Shannon index in bats from urban and intensive agriculture landscapes, while the highest alpha diversity value was found in near pristine landscapes. Beta diversity obtained by Bray-Curtis distance showed weak statistical differentiation of bacterial taxonomic profiles among scenarios. Furthermore, core community analysis showed that 1,293 genera were shared among localities. Differential abundance analyses showed that the highest differentially abundant taxa were found in near pristine landscapes, with the exception of the family Alcaligenaceae, which was also overrepresented in urban and intensive agriculture landscapes. CONCLUSIONS: This study suggests that near pristine and undisturbed landscapes could promote a more resilient gut microbiome in wild populations of P. kuhlii. These results highlight the potential of the fecal microbiome as a non-invasive bioindicator to assess insectivorous bats' health and as a key element of landscape conservation strategies.

Foodborne Pathogens at the Livestock-Wildlife-Human Interface in Rural Western Uganda.

Foodborne pathogens are an important cause of morbidity and mortality worldwide. To assess the presence of Salmonella, Campylobacter and Arcobacter spp. in livestock, wildlife, and humans from different regions across western Uganda, 479 faecal samples were tested by PCR. Salmonella and Campylobacter spp. were more frequently detected in livestock (5.1% and 23.5%, respectively) compared to wildlife (1.9% and 16.8%, respectively). Wildlife from remote areas showed lower Salmonella and Campylobacter spp. occurrence than in areas where interactions with livestock are common, suggesting that spill-over may exist from livestock or humans. Further studies are needed to better understand the transmission dynamics of these pathogens at the wildlife-livestock-human interface in western Uganda.

Eurasian griffon vultures carry widespread antimicrobial resistant Salmonella and Campylobacter of public health concern.

The global emergence of antimicrobial-resistant (AMR) strains of Salmonella and Campylobacter is a serious public health concern. Both bacteria are leading causes of human gastrointestinal foodborne infections and the two most reported zoonoses in the European Union. By feeding on livestock carcasses, especially from intensive farming, as well as on landfill sites, obligate avian scavengers can become infected with zoonotic pathogens and AMR strains, and can be considered large-scale sentinels of the environmental burden. In this study, we assessed the occurrence and AMR of Salmonella spp. and Campylobacter spp. in 218 Eurasian griffon vultures (Gyps fulvus) captured in north-eastern Spain. We isolated Salmonella from 8.1 % of individuals and Campylobacter lari from 4.7 %. Among the 10 different Salmonella serovars found, monophasic S. Typhimurium was the most frequent. Genotyping analysis revealed same strains of monophasic S. Typhimurium shared by gulls, livestock and humans. Isolates from both bacterial species presented AMR to important antimicrobials (tetracyclines, fluoroquinolones and ß-lactams). In conclusion, this study shows that Eurasian griffon vultures in north-eastern Spain are carriers of widespread AMR zoonotic Salmonella and Campylobacter. More comprehensive analyses are still needed to understand the potential risk of spill-over from those wild birds to humans.

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.

Experimental infection of pigs with Border disease virus isolated from Pyrenean chamois (Rupicapra pyrenaica).

Between 2001 and 2007, several outbreaks of disease associated with Border disease virus (BDV) infection were reported in the central Pyrenees (northeast Spain) and were associated with a major reduction in chamois (Rupicapra pyrenaica) populations. At the same time, wild boars (Sus scrofa) from the same area were found to be seropositive to this pestivirus, without showing clinical signs. The present study examines the susceptibility of domestic swine and the course of the infection with a BDV strain isolated from naturally infected chamois. Twenty pigs were inoculated with 1 x 10(7) TCID(50) (50% tissue culture infective dose) by oronasal route, and 16 control pigs received Eagles sterile Minimal Essential Medium. Serologic (enzyme-linked immunosorbent assay and virus neutralization test) and reverse transcription polymerase chain reaction assays were performed on serum samples obtained at 0, 3, 7, 10, 14, 21, and 31 days postinoculation (dpi). All infected pigs were viremic from 3 to 14 dpi. After 14 dpi, all infected animals developed an antibody response against the homologous virus. Clinical signs or histologic lesions were not observed in inoculated pigs. The present work demonstrates the susceptibility of domestic swine to a BDV strain of chamois origin.

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.

NSAIDs detected in Iberian avian scavengers and carrion after diclofenac registration for veterinary use in Spain.

Despite the now well recognised impact of diclofenac on vultures across the Indian subcontinent, this non-steroidal anti-inflammatory drug (NSAID) was registered in 2013 for livestock treatment in Spain, Europe's main vulture stronghold. We assessed the risk of exposure to diclofenac and nine other NSAIDs in avian scavengers in the Iberian Peninsula (Spain and Portugal) after the onset of diclofenac commercialization. We sampled 228 livestock carcasses from vulture feeding sites, primarily pig (n = 156) and sheep (n = 45). We also sampled tissues of 389 avian scavenger carcasses (306 Eurasian griffon vultures, 15 cinereous vultures, 11 Egyptian vultures, 12 bearded vultures and 45 other facultative scavengers). Samples were analysed by liquid chromatography with mass spectrometry (LCMS). Seven livestock carcasses (3.07%) contained NSAID residues: flunixin (1.75%), ketoprofen, diclofenac and meloxicam (0.44% each). NSAID residues were only detected in sheep (4.44%) and pig (3.21%) carcasses. Fourteen dead avian scavengers (3.60%) had NSAID residues in kidney and liver, specifically flunixin (1.03%) and meloxicam (2.57%). Flunixin was associated with visceral gout and/or kidney damage in three (0.98%) dead Eurasian griffons. To date, diclofenac poisoning has not been observed in Spain and Portugal, however, flunixin would appear to pose an immediate and clear risk. This work supports the need for well managed carrion disposal, alongside appropriate risk labelling on veterinary NSAIDs and other pharmaceuticals potentially toxic to avian scavengers.

Peste des Petits Ruminants at the Wildlife-Livestock Interface in the Northern Albertine Rift and Nile Basin, East Africa.

In the recent past, peste des petits ruminants (PPR) emerged in East Africa causing outbreaks in small livestock across different countries, with evidences of spillover to wildlife. In order to understand better PPR at the wildlife-livestock interface, we investigated patterns of peste des petits ruminants virus (PPRV) exposure, disease outbreaks, and viral sequences in the northern Albertine Rift. PPRV antibodies indicated a widespread exposure in apparently healthy wildlife from South Sudan (2013) and Uganda (2015, 2017). African buffaloes and Uganda kobs <1-year-old from Queen Elizabeth National Park (2015) had antibodies against PPRV N-antigen and local serosurvey captured a subsequent spread of PPRV in livestock. Outbreaks with PPR-like syndrome in sheep and goats were recorded around the Greater Virunga Landscape in Kasese (2016), Kisoro and Kabale (2017) from western Uganda, and in North Kivu (2017) from eastern Democratic Republic of the Congo (DRC). This landscape would not be considered typical for PPR persistence as it is a mixed forest-savannah ecosystem with mostly sedentary livestock. PPRV sequences from DRC (2017) were identical to strains from Burundi (2018) and confirmed a transboundary spread of PPRV. Our results indicate an epidemiological linkage between epizootic cycles in livestock and exposure in wildlife, denoting the importance of PPR surveillance on wild artiodactyls for both conservation and eradication programs.

Border disease virus among chamois, Spain.

Approximately 3,000 Pyrenean chamois (Rupicapra pyrenaica pyrenaica) died in northeastern Spain during 2005-2007. Border disease virus infection was identified by reverse transcription-PCR and sequencing analysis. These results implicate this virus as the primary cause of death, similar to findings in the previous epizootic in 2001.