Study of Canine Distemper Virus Presence in Catalonia's Wild Carnivores through H Gene Amplification and Sequencing.

Canine distemper virus (CDV) is recognised worldwide as an important pathogen in both domestic and wild carnivores. Few data are available on its impact and spread on the wildlife/wildlife-domestic animal-environment interface. This study, aimed at developing a conservation-oriented control strategy, analysed 89 sick or deceased animals from 2019 to 2023 at the Wildlife Rehabilitation Centre in Torreferrussa. RT-PCR and sequencing of the partial H gene were used to detect and analyse CDV in tissues. The total positive percentage was 20.22% (18/89), comprising 13 red foxes (44.8%), 4 European badgers (28.6%), and 1 American mink (4.5%), while 24 Eurasian otters tested negative. Phylogenetic analysis indicated that all of the CDV strains belong to the European lineage. Geographically distant individuals and different species shared the same viral strain, suggesting a strong capacity of CDV for interspecies and long-distance transmission. This calls for further research, particularly focusing on potential impacts of CDV on endangered carnivores.

Morbidity and Prognostic Factors Associated with Wild Hedgehogs Admitted to a Wildlife Rehabilitation Center in Catalonia (NE Spain) from 1995 to 2020.

Wildlife rehabilitation centers (WRC) play a crucial role in the collection of data and the monitoring of hedgehog populations. The main objective of this study was to identify the morbidity and prognostic factors associated with the mortality of wild hedgehogs admitted at a WRC in Catalonia. A total number of 3397 hedgehogs admitted from 1995 to 2020 were studied. The principal cause of admission was orphaned/young category (41%) followed by misplacement (19%), natural disease (17%), and trauma (14%). The best outcomes for release were for misplacement (93.6%), orphaned/young (72.3%), and other causes (77.6%), and the lowest proportion of released animals were found for natural disease (41.4%) and trauma (44.7%) categories. The most common macroscopic findings were the respiratory and digestive lesions. Internal parasites were also prevalent in 61% of the animals but with no association with a higher mortality. In the multivariate analyses, the prognostic indicators related with the mortality outcome were the presence of systemic (OR = 3.6, CI 95%: 2.8-4.6) and neurological (OR = 4.3, CI 95%: 2.9-6.4) signs. Morbidity and prognostic factors in wildlife rehabilitation are essential for providing effective care, making informed decisions, optimizing resources, and improving rehabilitation success rates.

Genomic insights into the Montseny brook newt (Calotriton arnoldi), a Critically Endangered glacial relict.

The Montseny brook newt (Calotriton arnoldi), considered the most endangered amphibian in Europe, is a relict salamandrid species endemic to a small massif located in northeastern Spain. Although conservation efforts should always be guided by genomic studies, those are yet scarce among urodeles, hampered by the extreme sizes of their genomes. Here, we present the third available genome assembly for the order Caudata, and the first genomic study of the species and its sister taxon, the Pyrenean brook newt (Calotriton asper), combining whole-genome and ddRADseq data. Our results reveal significant demographic oscillations which accurately mirrored Europe's climatic history. Although severe bottlenecks have led to depauperate genomic diversity and long runs of homozygosity along a gigantic genome, inbreeding might have been avoided by assortative mating strategies. Other life history traits, however, seem to have been less advantageous, and the lack of land dispersal has driven to exceptional levels of population fragmentation.

Lack of Detection of Toxoplasma gondii in Pipistrellus spp. Bats from Densely Cat-Populated Areas of NE Spain.

Toxoplasma gondii infection in healthy animals is often asymptomatic. However, some species with little history of contact with the parasite, such as marsupials and New World primates, present high mortality rates after infection. Despite its potential conservation concern, T. gondii infection in insectivorous bats has received little attention, and its impact on bat populations' health is unknown. To assess the putative role of insectivorous bats in the cycle of T. gondii, samples of three species of bats (Pipistrellus pipistrellus, P. pygmaeus and P. kuhlii) collected between 2019 and 2021 in NE Spain were tested for the presence of the parasite using a qPCR. All tissues resulted negative (0.0% prevalence with 95% CI: [0.0-2.6]) for the presence of T. gondii. Unlike previous studies on insectivorous bats from Europe, Asia and America, the present study suggests that Pipistrellus spp. bats do not play a significant role in the epidemiology of T. gondii in NE Spain. Further studies are encouraged to elucidate both the epidemiology of T. gondii and its potential impact on the health of microchiropteran species in Europe.

West Nile Virus Lineage 2 Spreads Westwards in Europe and Overwinters in North-Eastern Spain (2017-2020).

West Nile virus lineage 2 (WNV-L2) emerged in Europe in 2004; since then, it has spread across the continent, causing outbreaks in humans and animals. During 2017 and 2020, WNV-L2 was detected and isolated from four northern goshawks in two provinces of Catalonia (north-eastern Spain). In order to characterise the first Spanish WNV-L2 isolates and elucidate the potential overwintering of the virus in this Mediterranean region, complete genome sequencing, phylogenetic analyses, and a study of phenotypic characterisation were performed. Our results showed that these Spanish isolates belonged to the central-southern WNV-L2 clade. In more detail, they were related to the Lombardy cluster that emerged in Italy in 2013 and has been able to spread westwards, causing outbreaks in France (2018) and Spain (2017 and 2020). Phenotypic characterisation performed in vitro showed that these isolates presented characteristics corresponding to strains of moderate to high virulence. All these findings evidence that these WNV-L2 strains have been able to circulate and overwinter in the region, and are pathogenic, at least in northern goshawks, which seem to be very susceptible to WNV infection and may be good indicators of WNV-L2 circulation. Due to the increasing number of human and animal cases in Europe in the last years, this zoonotic flavivirus should be kept under extensive surveillance, following a One-Health approach.

Widespread Circulation of Flaviviruses in Horses and Birds in Northeastern Spain (Catalonia) between 2010 and 2019.

The surveillance for West Nile virus (WNV) in Catalonia (northeastern Spain) has consistently detected flaviviruses not identified as WNV. With the aim of characterizing the flaviviruses circulating in Catalonia, serum samples from birds and horses collected between 2010 and 2019 and positive by panflavivirus competition ELISA (cELISA) were analyzed by microneutralization test (MNT) against different flaviviruses. A third of the samples tested were inconclusive by MNT, highlighting the limitations of current diagnostic techniques. Our results evidenced the widespread circulation of flaviviruses, in particular WNV, but also Usutu virus (USUV), and suggest that chicken and horses could serve as sentinels for both viruses. In several regions, WNV and USUV overlapped, but no significant geographical aggregation was observed. Bagaza virus (BAGV) was not detected in birds, while positivity to tick-borne encephalitis virus (TBEV) was sporadically detected in horses although no endemic foci were observed. So far, no human infections by WNV, USUV, or TBEV have been reported in Catalonia. However, these zoonotic flaviviruses need to be kept under surveillance, ideally within a One Health framework.

Extended-Spectrum ß-Lactam Resistant Klebsiella pneumoniae and Escherichia coli in Wild European Hedgehogs (Erinaceus europeus) Living in Populated Areas.

Wildlife has been suggested to be a good sentinel of environmental health because of its close interaction with human populations, domestic animals, and natural ecosystems. The alarming emergence of antimicrobial resistance (AMR) in human and veterinary medicine has activated/triggered the awareness of monitoring the levels of AMR pollution in wildlife. European hedgehogs (Erinaceus europaeus) are common wild species habiting urban areas in Europe. However, there are few studies conducted in hedgehogs as reservoirs of AMR bacteria or genes. The aim of this study was to assess the occurrence and distribution of ESBL, AmpC, and carbapenem-resistant enterobacteria and AMR genes in wild European hedgehogs in Catalonia, a densely populated region of NE Spain. A total of 115 hedgehogs admitted at the Wildlife Rehabilitation Center of Torreferrussa were studied. To our knowledge, this is the first description of ß-lactam resistant enterobacteria in wild hedgehogs. Interestingly, 36.8% (42/114) of the animals were detected as carriers of ß-lactamase/carbapenemase resistance genes. Klebsiella spp. (59.6%), and specifically K. pneumoniae (84.6%), were the bacteria with the highest proportion of resistance genes, followed by E. coli (34.6%) and C. freundii (5.8%). The most frequently detected genetic variants were blaCTX-M-15 (19.3%), blaSHV-28 (10.5%), blaCMY-1 (9.7%), blaCMY-2 (8.8%), and blaOXA-48 (1.7%). In addition, 52% (27/52) of the isolates presented a multidrug resistance (MDR) phenotype and 31% had an extended drug resistance (XDR) profile. No clustering of animals with AMR genes within the study region was shown in the spatial analysis, nor differences in the proportion of positive animals among regions, were detected. The results of this study suggest that wild European hedgehogs could be good sentinels of AMR environmental pollution, especially in areas with a high human population density, because they either inhabit and/or feed in an anthropogenic environment. In conclusion, it is crucial to raise awareness of the strong interconnection between habitats and compartments, and therefore this implies that AMR issues must be tackled under the One Health approach.

Tracking Toxoplasma gondii in freshwater ecosystems: interaction with the invasive American mink (Neovison vison) in Spain.

Water-borne transmission may play an important role in the epidemiology of Toxoplasma gondii. Mammals closely related to freshwater ecosystems, such as the American mink (Neovison vison), are potentially valuable sentinels for T. gondii. To assess the importance of freshwater ecosystems in T. gondii epidemiology, sera of 678 American minks collected during the 2010 to 2015 Spanish national eradication campaigns were tested for the presence of T. gondii antibodies using the modified agglutination test (MAT, cut-off 1:25). A high prevalence of samples, 78.8% (CI95%: 75.5-81.8), were seropositive. In addition, a specific real-time PCR was performed in 120 brain samples and the parasite DNA was detected in 9.2% (CI95%: 5.2-15.7). Significant differences in seroprevalence were detected among bioregions, with the highest levels detected in coastal areas, and by age. The higher seroprevalence observed in older animals (80.0% adults versus 68.7% juveniles) confirms the importance of the horizontal transmission. These results indicate a widespread presence of T. gondii oocysts in freshwater ecosystems from Spain and further support the importance of water-borne transmission in the epidemiology of T. gondii.

First cases of European bat lyssavirus type 1 in Iberian serotine bats: Implications for the molecular epidemiology of bat rabies in Europe.

Previous studies have shown that EBLV-1 strains exclusively hosted by Eptesicus isabellinus bats in the Iberian Peninsula cluster in a specific monophyletic group that is related to the EBLV-1b lineage found in the rest of Europe. More recently, enhanced passive surveillance has allowed the detection of the first EBLV-1 strains associated to Eptesicus serotinus south of the Pyrenees. The aim of this study is the reconstruction of the EBLV-1 phylogeny and phylodynamics in the Iberian Peninsula in the context of the European continent. We have sequenced 23 EBLV-1 strains detected on nine E. serotinus and 14 E. isabellinus. Phylogenetic analyses were performed on the first 400-bp-5' fragment of the Nucleoprotein (N) gene together with other 162 sequences from Europe. Besides, fragments of the variable region of the phosphoprotein (P) gene and the glycoprotein-polymerase (G-L) intergenic region were studied on Spanish samples. Phylogenies show that two of the new EBLV-1a strains from Iberian E. serotinus clustered together with French strains from the North of the Pyrenees, suggesting a recent expansion southwards of this subtype. The remaining seven Iberian strains from E. serotinus grouped, instead, within the cluster linked, so far, to E. isabellinus, indicating that spatial distribution prevails over species specificity in explaining rabies distribution and supporting interspecific transmission. The structure found within the Iberian Peninsula for EBLV-1b is in concordance with that described previously for E. isabellinus. Finally, we have found that the current EBLV-1 European strains could have emerged only 175 years ago according to our evolutionary dynamics analyses.

Getting off to a good start? Genetic evaluation of the ex situ conservation project of the Critically Endangered Montseny brook newt (Calotriton arnoldi).

Ex situ management strategies play an important role in the conservation of threatened species when the wild survival of the species cannot be ensured. Molecular markers have become an outstanding tool for the evaluation and management of captive breeding programs. Two main genetic objectives should be prioritized when planning breeding programs: the maintenance of maximum neutral genetic diversity, and to obtain "self-sustaining" captive populations. In this study, we use 24 microsatellite loci to analyze and evaluate the genetic representativity of the initial phases of the captive breeding program of the Montseny brook newt, Calotriton arnoldi, an Iberian endemic listed as Critically Endangered. The results show that the initial captive stock has 74-78% of the alleles present in the wild populations, and captures roughly 93-95% of their total genetic diversity as observed in a previous study on wild newts, although it does not reach the desired 97.5%. Moreover, the percentage of unrelatedness among individuals does not exceed 95%. Therefore, we conclude that the genetic diversity of the captive stock should be improved by incorporating genetic material from unrelated wild newts. In recognition of the previously described significant genetic and morphological differentiation between eastern and western wild populations of C. arnoldi, we suggest maintaining two distinct breeding lines, and we do not recommend outbreeding between these lines. Our comparisons of genetic diversity estimates between real and distinct sample-sized simulated populations corroborated that a minimum of 20 individuals are needed for each captive population, in order to match the level of genetic diversity present in the wild populations. Thus, the current initial stock should be reinforced by adding wild specimens. The captive stock and subsequent cohorts should be monitored in order to preserve genetic variation. In order to avoid genetic adaptation to captivity, occasionally incorporating previously genotyped individuals from the wild into the captive populations is recommended.

Multidrug-resistant Salmonella enterica Serovar Typhimurium Monophasic Variant 4,12:i:- Isolated from Asymptomatic Wildlife in a Catalonian Wildlife Rehabilitation Center, Spain.

Wildlife can act as long-term asymptomatic reservoirs for zoonotic bacteria, such as Salmonella. The prevalence and antimicrobial-susceptibility profiles of Salmonella spp. were assessed in 263 cases in wildlife from 22 animal orders from a wildlife rehabilitation center in Catalonia (NE Spain), September 2013-May 2014. Eleven of 263 tested animals were positive for Salmonella spp., representing an overall prevalence of 4.2%. Prevalences by taxonomic categories were 2% in mammals, 4.7% in birds, and 4.5% in reptiles. By species, one each of European hedgehog (Erinaceus europeus; from a sample of n = 26), Eurasian Eagle Owl (Bubo bubo; n = 2), Barn Owl (Tyto alba; n = 3), Tawny Owl (Strix aluco; n = 20), Egyptian Vulture (Neophron percnopterus; n = 1), Griffon Vulture (Gyps fulvus; n = 1), and Hoopoe (Upupa epops; n = 2), and two each Common Kestrels (Falco tinnunculus; n = 16) and pond sliders (Trachemys scripta; n = 25) were positive for Salmonella. By serotyping, seven of eleven isolates were classified as S. enterica subsp. enterica serovar Typhimurium, and five of seven belonged to the monophasic variant 4,12:i:-. All the monophasic variants were isolated from birds (4/5 in raptors) and showed a multidrug-resistance (MDR) profile to at least ampicillin, streptomycin, sulfonamide, and tetracycline (R-type ASSuT), and up to 12 antibiotics. The large proportion of S. Typhimurium monophasic MDR strains detected in wildlife never treated with antibiotics, especially in raptors, adds more complexity to the epidemiologic control of one of the most frequent serovars involved in human and livestock infection.

Interspecific and geographical differences in anticoagulant rodenticide residues of predatory wildlife from the Mediterranean region of Spain.

We studied the prevalence of anticoagulant rodenticides (ARs) in the liver of 344 individuals representing 11 species of predatory wildlife that were found dead in the Mediterranean region of Spain (Catalonia and Majorca Island). Six different ARs (brodifacoum, bromadiolone, difenacoum, flocoumafen, difethialone, warfarin) were found in the liver of 216 (62.8%) animals and >1 AR co-occurred in 119 individuals (34.6%). The occurrence of ARs was positively correlated with the human population density. Catalonia and Majorca showed similar prevalence of AR detection (64.4 and 60.4%, respectively), but a higher prevalence was found in the resident population of Eurasian scops owl (Otus scops) from Majorca (57.7%) compared to the migratory population from Catalonia (14.3%). Birds of prey had lower levels of bromadiolone than hedgehogs, whereas no difference was found for other ARs. The risk of SGAR poisoning in wild predators in NE Spain is believed to be elevated, because 23.3% of the individuals exhibited hepatic concentration of ARs exceeding 200 ng/g.

A comparative study of corneal sensitivity in birds of prey.

OBJECTIVE: To determine and compare the corneal sensitivity in healthy wild diurnal and nocturnal birds of prey (BP) indigenous to Catalonia (Spain), and to establish if age is a determining factor in corneal sensitivity in those species. METHODS: Ophthalmic examination was performed in 105 BP. Only birds with no ocular abnormalities were included in the study (n = 81): 21 diurnal BP (Falco tinnunculus: 16 fledglings, 5 adults) and 60 nocturnal BP (20 Athene noctua [9 fledglings, 11 adults], 20 Strix aluco [15 fledglings, 5 adults], and 20 Otus scops [6 fledglings and 14 adults]). Corneal touch threshold (CTT) was determined for each eye in five different corneal regions. Five attempts to cause a blink reflex were made in each region, and when three or more reflexes were positive, the pressure was deemed the CTT. Statistical analysis was performed using a Student's t-test for independent data or an anova model. The results between species and age groups were compared using the Generalized Estimated Equations model. RESULTS: There were no significant differences between any of the corneal regions (P = 0.25), or between the right (CTT = 4.9 ± 1.7 cm) and left (CTT = 4.8 ± 1.7 cm) eye in any of the species (P = 0.692). No difference was found between diurnal and nocturnal species (P = 0.913). Considering all the species, a significant difference was found between the mean CTT of fledglings (5.4 ± 1.2 cm) and adults (4.1 ± 2 cm), P < 0.001. A significant difference was found between fledglings and adults of A. noctua (P < 0.001) and S. aluco (P = 0.002). CONCLUSIONS: There is no significant difference in CTT between the different corneal regions in all the species studied. Corneal sensitivity is similar between diurnal and nocturnal birds of prey. Age is a determining factor in the CTT of A. noctua and S. aluco, with fledglings having a significantly higher CTT.

Seropositivity and risk factors associated with Toxoplasma gondii infection in wild birds from Spain.

Toxoplasma gondii is a zoonotic intracellular protozoan parasite of worldwide distribution that infects many species of warm-blooded animals, including birds. To date, there is scant information about the seropositivity of T. gondii and the risk factors associated with T. gondii infection in wild bird populations. In the present study, T. gondii infection was evaluated on sera obtained from 1079 wild birds belonging to 56 species (including Falconiformes (n=610), Strigiformes (n=260), Ciconiiformes (n=156), Gruiformes (n=21), and other orders (n=32), from different areas of Spain. Antibodies to T. gondii (modified agglutination test, MAT titer ≥1:25) were found in 282 (26.1%, IC(95%:)23.5-28.7) of the 1079 birds. This study constitute the first extensive survey in wild birds species in Spain and reports for the first time T. gondii antibodies in the griffon vulture (Gyps fulvus), short-toed snake-eagle (Circaetus gallicus), Bonelli's eagle (Aquila fasciata), golden eagle (Aquila chrysaetos), bearded vulture (Gypaetus barbatus), osprey (Pandion haliaetus), Montagu's harrier (Circus pygargus), Western marsh-harrier (Circus aeruginosus), peregrine falcon (Falco peregrinus), long-eared owl (Asio otus), common scops owl (Otus scops), Eurasian spoonbill (Platalea leucorodia), white stork (Ciconia ciconia), grey heron (Ardea cinerea), common moorhen (Gallinula chloropus); in the International Union for Conservation of Nature (IUCN) "vulnerable" Spanish imperial eagle (Aquila adalberti), lesser kestrel (Falco naumanni) and great bustard (Otis tarda); and in the IUCN "near threatened" red kite (Milvus milvus). The highest seropositivity by species was observed in the Eurasian eagle owl (Bubo bubo) (68.1%, 98 of 144). The main risk factors associated with T. gondii seropositivity in wild birds were age and diet, with the highest exposure in older animals and in carnivorous wild birds. The results showed that T. gondii infection is widespread and can be at a high level in many wild birds in Spain, most likely related to their feeding behaviour.