Leishmania Infection in Wild Lagomorphs and Domestic Dogs in North-East Spain.

Leishmania infantum is a zoonotic protozoan parasite distributed worldwide that is transmitted by phlebotomine sandflies. Dogs are the main reservoir for human infections. However, in recent years, the capacity of lagomorphs to contribute to Leishmania transmission has been confirmed. The present study aimed to assess Leishmania spp. exposure and infection in lagomorphs and sympatric domestic dogs in NE Spain. Sera from European hares, European rabbits, and rural dogs were tested for antibodies against L. infantum using an in-house indirect ELISA. PCR analysis targeting Leishmania spp. was performed in spleens from L. europaeus. Antibodies against Leishmania spp. were detected in all the species analyzed. Total sample prevalence was significantly higher in O. cuniculus (27.9%) than in L. europaeus (2.0%). Results of the PCR were all negative. The present study expands knowledge about Leishmania infections in free-ranging lagomorphs in the Iberian Peninsula, suggesting a more important role of O. cuniculus in the study area. Given the strong correlation between lagomorph densities and human leishmaniasis outbreaks in Spain, the high rabbit and human densities in NE Spain, and the high Leishmania spp. seroprevalence in rabbits, it becomes imperative to establish surveillance programs for lagomorphs in this region.

Adipocytes in synovial fluid cytology: An approach for diagnosing synovial lipomatosis.

A 2-year-old neutered male bullmastiff dog was presented with chronic left hind limb lameness. Physical examination revealed left stifle effusion and medial buttress without cranial tibial thrust. Radiographs showed joint effusion and new bone formation at the patella apex. Magnetic resonance imaging showed increased synovial fluid, widening of the joint space, abnormal infrapatellar fat body and thinning of the cranial cruciate ligament. Synoviocentesis and cytologic evaluation of synovial fluid revealed marked mononuclear inflammation with abundant fatty tissue, suggesting synovial lipomatosis in conjunction with the imaging findings. The disease was confirmed histologically after sampling the lesion during arthrotomy. Synovial lipomatosis, characterized by extensive synovial adipose tissue proliferation of the synovial membrane, is a rare "tumor-like" disorder that usually affects the stifle. Although the etiology remains unclear, joint trauma, inflammation, instability, and lipid abnormalities have been proposed as causes. Inflammatory factors may promote synoviocyte and adipocyte hyperplasia that perpetuate the process. Surgical removal may be suggested to eliminate triggers and prevent future recurrences. The report provides the first cytological description of adipocytes in synovial fluid associated with the diagnosis of synovial lipomatosis in dogs. This case report underscores the potential effectiveness of cytologic analysis of synovial fluid smears, in combination with magnetic resonance imaging (MRI), for diagnosing this condition and reducing complications associated with arthrotomy for sampling purposes. Additionally, the case highlights that synovial lipomatosis should be considered as a potential differential diagnosis for synovial masses in dogs. Further cases are needed to validate these observations in veterinary medicine.

Recombination between non-structural and structural genes as a mechanism of selection in lagoviruses: The evolutionary dead-end of an RHDV2 isolated from European hare.

The genus Lagovirus, belonging to the family Caliciviridae, emerged around the 1980s. It includes highly pathogenic species, rabbit hemorrhagic disease virus (RHDV/GI.1) and European brown hare syndrome virus (EBHSV/GII.1), which cause fatal hepatitis, and nonpathogenic viruses with enteric tropism, rabbit calicivirus (RCV/GI.3,4) and hare calicivirus (HaCV/GII.2). Lagoviruses have evolved along two independent genetic lineages: GI (RHDV and RCV) in rabbits and GII (EBHSV and HaCV) in hares. To be emphasized is that genomes of lagoviruses, like other caliciviruses, are highly conserved at RdRp-VP60 junctions, favoring intergenotypic recombination events at this point. The recombination between an RCV (genotype GI.3), donor of non-structural (NS) genes, and an unknown virus, donor of structural (S) genes, likely led to the emergence of a new lagovirus in the European rabbit, called RHDV type 2 (GI.2), identified in Europe in 2010. New RHDV2 intergenotypic recombinants isolated in rabbits in Europe and Australia originated from similar events between RHDV2 (GI.2) and RHDV (GI.1) or RCV (GI.3,4). RHDV2 (GI.2) rapidly spread worldwide, replacing RHDV and showing several lagomorph species as secondary hosts. The recombination events in RHDV2 viruses have led to a number of viruses with very different combinations of NS and S genes. Recombinant RHDV2 with NS genes from hare lineage (GII) was recently identified in the European hare. This study investigated the first RHDV2 (GI.2) identified in Italy in European hare (RHDV2_Bg12), demonstrating that it was a new virus that originated from the recombination between RHDV2, as an S-gene donor and a hare lagovirus, not yet identified but presumably nonpathogenic, as an NS gene donor. When rabbits were inoculated with RHDV2_Bg12, neither deaths nor seroconversions were recorded, demonstrating that RHDV2_Bg12 cannot infect the rabbit. Furthermore, despite intensive and continuous field surveillance, RHDV2_Bg12 has never again been identified in either hares or rabbits in Italy or elsewhere. This result showed that the host specificity of lagoviruses can depend not only on S genes, as expected until today, but potentially also on some species-specific NS gene sequences. Therefore, because RHDV2 (GI.2) infects several lagomorphs, which in turn probably harbor several specific nonpathogenic lagoviruses, the possibility of new speciation, especially in those other than rabbits, is real. RHDV2 Bg_12 demonstrated this, although the attempt apparently failed.

A new HaCV-EBHSV recombinant lagovirus circulating in European brown hares (Lepus europaeus) from Catalonia, Spain.

In 2020/2021, several European brown hare syndrome virus (EBHSV) outbreaks were recorded in European hares (Lepus europaeus) from Catalonia, Spain. Recombination analysis combined with phylogenetic reconstruction and estimation of genetic distances of the complete coding sequences revealed that 5 strains were recombinants. The recombination breakpoint is located within the non-structural protein 2C-like RNA helicase (nucleotide position ~ 1889). For the genomic fragment upstream of the breakpoint, a non-pathogenic EBHSV-related strain (hare calicivirus, HaCV; GII.2) was the most closely related sequence; for the rest of the genome, the most similar strains were the European brown hare syndrome virus (EBHSV) strains recovered from the same 2020/2021 outbreaks, suggesting a recent origin. While the functional impact of the atypical recombination breakpoint remains undetermined, the novel recombinant strain was detected in different European brown hare populations from Catalonia, located 20-100 km apart, and seems to have caused a fatal disease both in juvenile and adult animals, confirming its viability and ability to spread and establish infection. This is the first report of a recombination event involving HaCV and EBHSV and, despite the recombination with a non-pathogenic strain, it appears to be associated with mortality in European brown hares, which warrants close monitoring.

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.

First detection of herpesvirus and hemosporidians in the endangered Pyrenean Capercaillie (Tetrao urogallus aquitanicus).

Pathogens affect wild bird populations worldwide, contributing to their decline. Considering the scarce health data regarding the endangered Pyrenean Capercaillie (Tetrao urogallus aquitanicus), we molecularly surveyed selected pathogens (Newcastle disease virus, Avian influenza virus, Chlamydia psittaci, avian pathogenic Escherichia coli, Campylobacter jejuni, and Salmonella spp.) in 30 Pyrenean Capercaillie feces collected in the field (Catalonia, northeastern Spain). Additionally, swab and tissue samples from eight wild Pyrenean Capercaillies of Catalonia and Andorra were molecularly tested for herpesvirus and hemosporidians (Plasmodium spp., Haemoproteus spp., and Leucocytozoon spp.). All fecal samples were negative for the pathogens tested. Nevertheless, we detected a novel herpesvirus in 50% (4/8) of the Pyrenean Capercaillies, and hemosporidian DNA in 62.5% (5/8) of the tissue samples (i.e., Haemoproteus sp. [4 of 8] and/or Leucocytozoon sp. [3 of 8]). To our knowledge, this is the first detection of herpesvirus and hemosporidians infections in Pyrenean Capercaillies. The putative novel herpesvirus belongs to the genus Iltovirus. The presence of hemosporidian parasites in this mountain bird species is of concern, and could be related to the marked increase in the average temperature in the Pyrenees as a consequence of climate change. Our findings are fundamental to improve the conservation plans for the endangered Pyrenean Capercaillie population.

Diseases of Iberian ibex (Capra pyrenaica).

Iberian ibex (Capra pyrenaica) is an ecologically and economically relevant medium-sized emblematic mountain ungulate. Diseases participate in the population dynamics of the species as a regulating agent, but can also threaten the conservation and viability of vulnerable population units. Moreover, Iberian ibex can also be a carrier or even a reservoir of pathogens shared with domestic animals and/or humans, being therefore a concern for livestock and public health. The objective of this review is to compile the currently available knowledge on (1) diseases of Iberian ibex, presented according to their relevance on the health and demography of free-ranging populations; (2) diseases subjected to heath surveillance plans; (3) other diseases reported in the species; and (4) diseases with particular relevance in captive Iberian ibex populations. The systematic review of all the information on diseases affecting the species unveils unpublished reports, scientific communications in meetings, and scientific articles, allowing the first comprehensive compilation of Iberian ibex diseases. This review identifies the gaps in knowledge regarding pathogenesis, immune response, diagnostic methods, treatment, and management of diseases in Iberian ibex, providing a base for future research. Moreover, this challenges wildlife and livestock disease and wildlife population managers to assess the priorities and policies currently implemented in Iberian ibex health surveillance and monitoring and disease management.

Tissue-specific assessment of oxidative status: Wild boar as a case study.

In recent decades, there has been a fast-growing interest in using biomarkers of oxidative stress (BOS) in conservation programs of many vertebrate species. Biomarkers of oxidative stress can be measured in different biological samples (e.g., body fluids and tissues). However, since comparisons of the same battery of BOS among tissues of the same individual are scarce in the literature, the chosen target tissues regularly rely on arbitrary decisions. Our research aimed to determine if the oxidative status of free-ranging wild boar (Sus scrofa) naturally infected with Mycobacterium spp (etiological agent of tuberculosis, TB), varies depending on the sample where it was quantified. We compared antioxidant p-nitrophenyl esterase activity (EA), glutathione peroxidase (GPX) concentrations, and total oxidative status (TOS) in serum, lung, spleen, kidney, and muscle of 63 wild boar hunter-harvested in central Spain. Biomarkers of oxidative stress in serum had higher concentrations than in other tissues. The poor agreement between serum and other tissues highlights the importance of running complete BOS assessments in the same fluid or tissue. Further, low concentrations of BOS in tissues of TB-affected individuals were observed, and significant differences between healthy and sick boar were only detected in the serum of individuals developing mild TB and in the muscle of individuals with mild or severe disease status. However, all organs from wild boars affected with mild TB were not in oxidative imbalance compared to healthy control animals, suggesting that wild boars may cope well with TB. Our data indicate that serum and other tissues can be used as BOS in field conservation programs to monitor wildlife population health. Still, context-specific validations are needed to determine the most appropriate samples to use.

Absence of anti-Besnoitia spp. specific antibodies in European wild lagomorphs from the Iberian Peninsula.

Besnoitia besnoiti is an apicomplexan parasite whose life cycle is not completely understood. It is assumed that this parasite might have an indirect life cycle with a carnivore as a definitive host able to shed oocysts after the ingestion of mature cysts in tissues of an infected intermediate host. Cattle and wild cervids on the Iberian Peninsula can act as intermediate hosts of B. besnoiti, and exposure to the parasite has been demonstrated in equids. In this study, we aimed to assess the presence of members of the genera Besnoitia in wild lagomorphs from the Iberian Peninsula and the potential role of these host species in the life cycle of B. besnoiti, as all the animals were sampled from 19 regions of the Iberian Peninsula where cases of bovine besnoitiosis have been previously detected. Serum samples (Oryctolagus cuniculus: n = 552; Lepus europaeus: n = 122) were first analysed by ELISA and subsequently confirmed by Western blot (WB). Specific antibodies against B. besnoiti were not found in any sampled animal by WB. In addition, lung samples from a subset of wild rabbits (n = 16) were tested by PCR and Besnoitia spp. DNA was not detected. These results suggest that Besnoitia spp. are unlikely to circulate in wild lagomorphs in the Iberian Peninsula. Thus, lagomorphs are not expected to play a key role in the biological cycle of B. besnoiti. Further studies are necessary to assess whether different micromammal species, such as rodents, can serve as natural reservoirs of Besnoitia spp. in other European regions.

Assessing mammal trapping standards in wild boar drop-net capture.

Applying contemporary trapping standards when managing wildlife should no longer be an option, but a duty. Increasing wild boar populations originate a growing number of conflicts and hunting is the only cost-effective management option in most cases. However, new scenarios where hunting is unfeasible emerge and trapping necessities cope with lacking regulatory frameworks and technical guidelines. In this research, we evaluated drop nets, a capture method not considered by the international trapping standards, to capture Eurasian wild boar (Sus scrofa), a wildlife species not included in the list of mammal species under the scope of the Agreement on International Humane Trapping Standards (AIHTS). Less than 20% of the captured wild boars presented moderate or severe injuries attributable to the capture method, hence fulfilling the acceptance thresholds of the outdated AIHTS. Based on the new standards thresholds of acceptance, the humaneness of drop-nets in our study ranged 66-78%, under the 85% required. The capture success and selectivity were 100%, as ensured by operator-driven triggering, which should be considered the main strengths of this method, together with the minimization of animal suffering owing the short duration of the stressful situation. Additionally, in spite of the socially adverse environment, with people contrary to wild boar removal, no disturbances against the capture system or operations occurred. This is the first assessment of a drop-net capture method according to internationally accepted mammal trapping standards, with unconclusive results. However, there is a need for adapted procedures and thresholds of acceptance aimed at not-mechanical traps in general, and specifically at drop-nets. Compared to other live-capture methods, drop-nets minimize the duration of the stressful situation -at the expense of a strong adrenergic acute response-, maximize the probabilities of capturing entire sounders of prosocial species, which may be also considered as more humane, and has the ability to coordinate higher values of capture success, absolute selectivity and adaptability to difficult environments.

Generalized tuberculosis due to Mycobacterium caprae in a red fox phylogenetically related to livestock breakdowns.

BACKGROUND: Tuberculosis (TB) due to Mycobacterium caprae is endemic in goat herds and free-ranging wild boars in Spain, causing infections in other livestock or wild animals to a lesser extent. TB infection in foxes is infrequently reported and they are usually considered spillover hosts of TB. CASE PRESENTATION: A blind, depressed and severely emaciated red fox (Vulpes vulpes) was admitted to a rehabilitation center. After clinical examination it was humanely sacrificed. At necropsy, generalized TB lesions were observed that were subsequently confirmed by histopathology along with a co-infection with canine distemper virus. M. caprae was isolated from mycobacterial culture and spoligotype SB0415 was identified. Whole genome sequencing (WGS) of the isolated M. caprae was carried out and single nucleotide polymorphisms (SNP) were compared with other sequences of M. caprae isolated from livestock and wildlife of the same area throughout the last decade. CONCLUSIONS: This is the first reported case of TB due to M. caprae in a fox in the Iberian Peninsula. WGS and SNP analysis, together with spatial-temporal investigations, associated this case with recent M. caprae outbreaks in cattle and goat herds of the area. The results indicated transmission of M. caprae between livestock and the fox, suggesting that this species may occasionally play a role in the epidemiology of animal TB.

Coinfection of Chytrid Fungi in Urodeles during an Outbreak of Chytridiomycosis in Spain.

Chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), has had an unprecedented impact on amphibian biodiversity. Although Bd is globally widespread, Bsal is currently spreading, increasing the probability that these pathogens will co-occur in individual amphibians. Interactions among coinfecting parasites can have significant outcomes on disease dynamics and impact and, therefore, may have important consequences for amphibian conservation. We analyzed the patterns of Bd-Bsal coinfections in two species of free-ranging urodeles during an outbreak of chytridiomycosis in Spain. Our goals were to assess 1) the probability of co-occurrence of both chytrid species and 2) the correlation of pathogen loads in coinfected hosts. We detected coinfections in 81.58% of Triturus marmoratus (n=38) and in 18.75% of Ichthyosaura alpestris (n=16). Histopathologic lesions of chytridiomycosis were observed only in T. marmoratus. Our results demonstrate a positive relationship between Bd and Bsal loads in T. marmoratus, whereas the co-occurrence analysis showed a random association among pathogens in both urodele species. Overall, we show that Bd-Bsal coinfections intensify pathogen load in T. marmoratus and could, therefore, increase disease severity and have important consequences for the conservation of some amphibian species.

First report of interspecific transmission of sarcoptic mange from Iberian ibex to wild boar.

BACKGROUND: Sarcoptic mange is a globally distributed parasitic disease caused by the burrowing mite Sarcoptes scabiei. This mite has a certain degree of host specificity, although interspecific transmission can occur among phylogenetically related species or through prey-predator mediated exposure. In 2018, a wild boar (Sus scrofa) with lesions compatible with sarcoptic mange was hunted in Ports de Tortosa i Beseit Natural Park (PTB, north-eastern Spain), where an active epizootic outbreak of sarcoptic mange is affecting Iberian ibexes (Capra pyrenaica) since 2014. METHODS: A complete necropsy, skin scrapings and skin digestions with hydroxide potassium were performed to confirm the diagnosis. Routine histopathological analysis, toluidine blue staining and immunohistochemistry were used to characterize the lesions and the inflammatory infiltrate. Finally, 10 specific S. scabiei microsatellites were molecularly genotyped through polymerase chain reactions in mites obtained from the affected wild boar. For phylogenetic comparison, mites obtained from sympatric Iberian ibexes and allopatric wild boars and Iberian ibexes from southern Spain were analysed. RESULTS: Sarcoptes scabiei was visually and molecularly identified in the infested wild boar from PTB, causing skin lesions with dermal inflammatory infiltrate rich in T and B cells, which indicate an adaptive immune response. Three S. scabiei genetic clusters were identified: one included mites from southern Iberian ibexes, another included mites from southern wild boars, and a third one distinctively grouped the wild boar from PTB with the sympatric ibexes. CONCLUSIONS: To the authors' knowledge, this is the first reported case of sarcoptic mange in wild boar in Spain and the first documented case of S. scabiei cross-transmission from a wild ruminant host to a wild boar. The wild boar presented an ordinary scabies type reaction, which is typical of the self-limiting infestations reported in other cases of interspecific transmission.

Spillover event of recombinant Lagovirus europaeus/GI.2 into the Iberian hare (Lepus granatensis) in Spain.

Viruses that affect lagomorphs have decades of reported history of spillover events. One of these viruses is the causative agent of the so-called rabbit or 'lagomorph' haemorrhagic disease (e.g. Lagovirus europaeus/GI.1 and L. europaeus/GI.2). In particular, L. europaeus/GI.2 has shown a great capacity to recombine with existing lagoviruses. In fact, it has replaced the former GI.1 genotype in the wild, and recently, an increase on spillover events has been detected among several lagomorph species including European and North American species of hares. In this study, we report for the first time the infection of a wild Iberian hare with GI.2 (RHDV2/b), potential shedding and associated histopathological alterations. We identify the recombinant GI.4P-GI.2 as causative of the infection and discuss plausible causes regarding the origin of the spillover event and its potential consequences for the Iberian hare wild populations, which is an endemic species of the Iberian Peninsula as well as an important game and prey species for many predators, including endangered species.

Protective Effect of Intestinal Helminthiasis Against Tuberculosis Progression Is Abrogated by Intermittent Food Deprivation.

Background: Tuberculosis (TB) is still a major challenge for humankind. Because regions with the highest incidence also have a high prevalence of helminthiasis and nutritional scarcity, we wanted to understand the impact of these on TB progression. Methods: We have developed an experimental murine model for active TB in C3HeB/FeJ, coinfected with Trichuris muris and Heligmosomoides polygyrus nematodes, and exposed to an environmental mycobacterium (M. manresensis) and intermittent fasting. Cause-effect relationships among these factors were explored with Partial Least Squares Path modelling (PLSPM). Results: Previous parasitization had a major anti-inflammatory effect and reduced systemic levels of ADA, haptoglobin, local pulmonary levels of IL-1ß, IL-6, TNF-α, CXCL-1, CXCL-5 and IL-10. Oral administration of heat-killed M. manresensis resulted in a similar outcome. Both interventions diminished pulmonary pathology and bacillary load, but intermittent food deprivation reduced this protective effect increasing stress and inflammation. The PLSPM revealed nematodes might have protective effects against TB progression. Conclusions: Significantly higher cortisol levels in food-deprivation groups showed it is a stressful condition, which might explain its deleterious effect. This highlights the impact of food security on TB eradication policies and the need to prioritize food supply over deworming activities.

Characterization of New Molecular Markers of Three Botflies Parasitizing Cervid Hosts.

Specific identification of oestrid larvae is usually problematic not only when using morphobiometric features, but also when applying molecular criteria, since very few molecular markers have been described for this group of flies. New molecular markers for oestrid are needed for more reliable species identification, diagnostic purposes, and epidemiological surveys; moreover, they can help in phylogenetic reconstruction. Here, we report the characterization of COI, 28S rDNA, ITS1, and ITS2 in Cephenemyia stimulator from roe deer and in Cephenemyia auribarbis and Pharyngomyia picta from red deer. The COI and 28S rDNA are very uniform in length, while the ITSs sequences are highly variable at both intraspecific and interspecific levels. The described ITSs sequences were longer than those described for other dipteran species by the presence of simple repeats and tandem repeat sequences. In C. auribarbis both ITS1 and ITS2 appeared as two variants, one short and the other long. In general, the analyzed markers present low intraspecific genetic variation and high interspecific variation. ITSs showed the greatest amount of intraspecific and interspecific variation. Phylogenetic analysis demonstrated that the characterized sequences differentiate the species and genera of Oestridae.

First description of sarcoptic mange in an Iberian hare (Lepus granatensis).

The Iberian hare (Lepus granatensis) is a popular small game species in the Iberian Peninsula, and it has never been reported to be affected by sarcoptic mange. An adult female Iberian hare with overt skin lesions on forelimbs and ventral thorax, suggestive of sarcoptic mange, was culled in Quart de les Valls municipality in the Valencian Community, Spain, in 2019. Skin scrapings were digested in 10% KOH solutions to confirm the presence of mites. Ten Sarcoptes microsatellite markers were used to characterize the genetic structure of mites obtained from the hare, and from sympatric and allopatric wild rabbits (Oryctolagus cuniculus) and red foxes (Vulpes vulpes). A total of 56 alleles were counted across the 10 microsatellite loci. Six private alleles were found at four loci (Sarms 33, 38, 41, 45). The multivariate analysis characterized three main clusters, corresponding to mites collected on foxes originating from Catalonia, foxes from Valencia and the hare plus wild rabbits. To our knowledge, this is the first reported case of sarcoptic mange in the Iberian hare. The origin was molecularly traced back to contacts with endemically infected wild rabbits. We encourage further investigations on cross-transmissibility of S. scabiei between wild rabbits and the diverse representatives of Lepus spp.

Endemic occurrence of Fasciola hepatica in an alpine ecosystem, Pyrenees, Northeastern Spain.

Fasciola hepatica is a liver parasite of ruminants whose distribution is determined by its intermediate host, the freshwater snail Galba truncatula. In Europe, F. hepatica is mostly associated with lowlands. Infection from sympatric domestic reservoirs is rarely reported in wild mountain ungulates. This study explores F. hepatica in a multi-host system in a European alpine area. Serum samples (n = 1,209) from Pyrenean chamois (Rupicapra p. pyrenaica), European mouflon (Ovis aries musimon), domestic sheep (Ovis aries) and domestic cattle (Bos taurus) were collected in the National Game Reserve of Freser-Setcases (NGRFS) in Catalonia, Northeastern Spain, from 2008 to 2019, and tested for antibodies against F. hepatica. During the same period, the livers of 214 chamois hunted in the NGRFS were inspected for F. hepatica and associated pathological changes. Finally, 907 freshwater snails were collected in summer 2016 between 1559 and 2,224 metres above sea level (asl) in the NGRFS, and F. hepatica DNA sought by PCR. Antibodies against F. hepatica were detected in all four species, with a higher prevalence in cattle and sheep than in chamois. Fasciola hepatica and hepatic lesions were concurrently observed in 13/214 of the chamois livers inspected (6.1%, CI95 2.9%-9.3%). Fasciola hepatica DNA was detected in one out of the 907 snails (0.1%, Cl95 0.1% - 0.3%; Ct value 33.3) and collected at 2054 m asl. Fasciola hepatica was consistently detected in a high mountain multi-host system, suggesting that its life cycle is completed and that it occurs endemically at the highest elevation reported in Europe. Transhumant livestock are the likely source in this alpine ecosystem, which according to rare occurrence of F. hepatica DNA in G. truncatula is still a suboptimal habitat for F. hepatica life cycle. Studying parasites at their highest distribution range can be useful to monitor climate change in seasonal mountain environments.

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.

Genetic characterization of Carnivore Parvoviruses in Spanish wildlife reveals domestic dog and cat-related sequences.

The impact of carnivore parvovirus infection on wild populations is not yet understood; disease signs are mainly developed in pups and assessing the health of litters in wild carnivores has big limitations. This study aims to shed light on the virus dynamics among wild carnivores thanks to the analysis of 213 samples collected between 1994 and 2013 in wild ecosystems from Spain. We determined the presence of carnivore parvovirus DNA by real-time PCR and sequenced the vp2 gen from 22 positive samples to characterize the strains and to perform phylogenetic analysis. The presence of carnivore parvovirus DNA was confirmed in 18% of the samples, with a higher prevalence detected in wolves (Canis lupus signatus, 70%). Fourteen sequences belonging to nine wolves, three Eurasian badgers (Meles meles), a common genet (Genetta genetta) and a European wildcat (Felis silvestris) were classified as canine parvovirus 2c (CPV-2c); five sequences from three wolves, a red fox (Vulpes vulpes) and a stone marten (Martes foina) as CPV-2b; and three sequences from a badger, a genet and a stone marten as feline parvovirus (FPV). This was the first report of a wildcat infected with a canine strain. Sequences described in this study were identical or very close related to others previously found in domestic carnivores from distant countries, suggesting that cross-species transmission takes place and that the parvovirus epidemiology in Spain, as elsewhere, could be influenced by global factors.