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Hepatitis B virus (HBV) and Hepatitis E virus (HEV) are zoonotic pathogens posing significant health concerns in rural Amazonia, a region marked by high endemicity, poverty, and limited healthcare access. However, the epidemiology of HBV and HEV in this ecosystem remains underexplored. This study examines the circulation of HBV and HEV at the human–wildlife interface and identifies risk factors within an isolated Amazonian indigenous community reliant on hunting for subsistence. Antibodies against HBV core antigens (HBcAbs) were found in three wildlife species: Cuniculus paca (0.8%), Tayassu pecari (1.6%), and Mazama americana (4.1%), marking the first record of HBV antibodies in free-ranging wildlife in the Amazon. However, further research is necessary to identify circulating strains and their relation to human HBV. HBcAbs were also detected in 9.1% of human samples, confirming exposure to HBV in the region. HEV IgG antibodies were present in 17.1% of humans and were associated with higher age. All wildlife and domestic animal samples tested negative for HEV, but transmission through consumption of wild animals and contaminated water needs further investigation. The identified risk factors highlight the urgent need for measures to promote safer food handling, improved sanitation, hygiene, and practices related to contact with wild animals.

Serosurvey results for Crimean-Congo hemorrhagic fever virus antibodies in dromedary camels in Algeria indicate that the pathogen is circulating endemically in desertic areas, despite the hostile environment. Thus, dromedaries are suitable sentinels for detecting human risk for Crimean-Congo hemorrhagic fever in desertic areas.

Identifying the role that host species play in pathogen transmission and maintenance is crucial for disease control, but it is a difficult task, in particular for vector-borne and multi-host pathogens, and especially when wildlife species are involved. This is the case for a Crimean-Congo hemorrhagic fever virus (CCHFV) hotspot in north-eastern Spain, where Iberian ibex (Capra pyrenaica) and wild boar (Sus scrofa) are involved, but their roles in disease transmission are unclear. In this context, we studied the dynamics of CCHFV transmission in these two species during the collapse of an Iberian ibex population due to a sarcoptic mange outbreak. We carried out a repeated cross-sectional study measuring the trends of CCHFV seroprevalence in Iberian ibex and wild boar and their abundances. In addition, we identified the tick species present in this area on the vegetation and on wild boars, and evaluated relevant meteorological factors. Results show that while the trends in CCHFV seroprevalence in Iberian Ibex and density of wild boars remained constant (p = 1.0 and p = 0.8, respectively), both the trends in Iberian ibex census and CCHFV seroprevalence in wild boars decreased significantly (p = 0.003 and p = 0.0001, respectively), and were correlated (Spearman's rank, 0.02

Toxoplasma gondii is a generalist zoonotic parasite that involves a wide range of warm-blooded animals as intermediate hosts and felines as definitive hosts. Recent studies have proved significant positive associations between human population density and T. gondii seroprevalence in wildlife. However, there is limited data regarding T. gondii wildlife in urban areas, where the highest human density occurs. The present study aimed to analyse the T. gondii exposure in urban hedgehogs from the Metropolitan Area of Barcelona, NE Spain. One hundred eighteen hedgehogs were analysed for the presence of antibodies (modified agglutination test; n = 55) and parasite DNA (qPCR; heart = 34; brain = 60). Antibodies were detected in 69.09% of hedgehogs. T. gondii DNA was not detected in any of the analysed samples. The present study reports a high T. gondii seroprevalence in urban hedgehogs in areas surrounding Barcelona, the most densely human-populated area of NE Spain, reinforcing the association between human population density and environmental T. gondii oocysts. The lack of detection by molecular techniques warrants more studies. In the last few decades, the distribution and abundance of European hedgehogs have declined, including their urban populations. Further research is needed to investigate the impact of T. gondii on hedgehog populations.

Toxoplasma gondii is a ubiquitous zoonotic protozoan parasite that infects a wide variety range of warm-blooded animals. This study describes the epidemiological scenario of T. gondii in an indigenous community that relies on subsistence hunting in a well-conserved and isolated area of the Peruvian Amazon. The high seropositivity against T. gondii in humans (83.3% IgG and 6.1% IgM), wild mammals (30.45%, 17 species), peri-domestic rodents (10.0% Rattus sp.), and domestic animals (94.1% dogs and 100% cats) indicates the existence of a sylvatic cycle in the community under study. Individual age was found to be positively associated with IgG detection against T. gondii but not with IgM. It is estimated that each family consumed 5.67 infected animals per year with terrestrial species having higher infective rates than arboreal species. The main risk factors included improper handling and cooking of wild meat, poor hygiene practices, and feeding uncooked offal to domestic animals. This scenario results in a continuous process of infection and reinfection within the indigenous community with cats, dogs, and peri-domestic animals becoming infected through the ingestion of infected raw viscera. Our results emphasize the need to promote safe food handling practices and disposal of waste materials from hunted animals in such communities.

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.

Abstract 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

We conducted a retrospective serosurvey for antibodies against Crimean-Congo hemorrhagic fever virus in wild ungulates along the eastern Mediterranean Coast of Spain. The virus has been endemic in this region since 2010 but is mainly restricted to geographic clusters with extremely high seropositivity associated with high density of bovids.

The use of passive integrated transponder (PIT) tags in urodeles has become popular for individual marking in population and disease ecology studies. However, mark loss or mark-induced mortality can introduce biases and decrease precision in parameter estimates, leading to ineffective population management strategies. In this study we aimed to 1) analyze the existing literature on the use of PIT tags in urodeles; 2) determine whether species characteristics and PIT tagging methods influenced PIT tag rejection across studies; and 3) experimentally assess the adequacy of a subcutaneous PIT tagging method without anesthesia in three European urodele species. We systematically and quantitatively reviewed a database of literature related to the use of PIT tags in urodeles, classified and examined urodele species details, study design, PIT tagging methods, and outcomes across studies. Among the 51 peer-reviewed papers that fit our criteria, the most striking finding was the lack of reporting and standardization of the PIT tagging procedures. The majority of studies presented incomplete information on factors that could strongly influence the probability of PIT tag rejection as well as impact individual welfare (i.e. PIT tag size, its anatomical placement in the animal, anesthesia use, sterility or skin closure methods). We could not identify significant predictors of PIT tag loss, suggesting that the effectivity of PIT tags may be highly specific to the species and method used. Our PIT tagging method proved reliable in Salamandra salamandra and Pleurodeles waltl, whereas it did not seem a suitable technique for Calotriton asper (PIT tag loss was 0% and 66.6%, respectively, and significantly different among species). Overall, we recommend a greater emphasis on reporting implantation methods, ensuring animal welfare and performing species and protocol specific laboratory trials before using PIT tags in urodeles in the field. Critically analyzing PIT tagging methods as well as testing their use in different species is essential to ensure the validity of future research studies and conservation strategies in urodeles.

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.

Abstract Background Livestock play an important role as reservoir of enteric pathogens and antimicrobial resistance (AMR), a health and economic concern worldwide. However, little is known regarding the transmission and maintenance of these pathogens at the wildlife-livestock interface. In this study, we assessed the occurrence, genetic diversity and AMR of Campylobacter spp. and Salmonella spp. shed by sympatric free-ranging livestock and a wild herbivore in an alpine ecosystem. Results Campylobacter spp. was isolated from 23.3 % of cattle and 7.7 % of sheep but was not isolated from horses nor Pyrenean chamois (Rupicapra pyrenaica). Campylobacter jejuni was the most frequent species. A high genetic diversity and certain host specificity of C. jejuni isolates was observed. The main AMR detected in Campylobacter isolates was to nalidixic acid (88.2 %), ciprofloxacin (82.4 %) and tetracycline (82.4 %); only 11.7 % of the isolates were pan-susceptible and 17.6 % were multi-resistant. Salmonella ser. Newport was isolated only from one Pyrenean chamois and was pan-susceptible. Conclusions Results show that free-ranging cattle and sheep are spreaders of Campylobacter as well as their AMR strains in the alpine environment. Therefore, contaminated alpine pastures or streams may constitute a source for the dissemination of AMR enteropathogens. However, apparently, alpine wild ungulates such as Pyrenean chamois play a negligible role in the epidemiology of zoonotic enteropathogens and AMR, and are not potential bioindicators of the burden of alpine environments.

Urban wild boars (Sus scrofa) from Barcelona, Spain, harbor great diversity of Streptococcus suis strains, including strains with the cps2 gene and with the same molecular profile as local human cases. The increasing trend of potential effective contacts for S. suis transmission is of public health concern.

Antimicrobial resistance (AMR) has spread worldwide due to the inappropriate use of antimicrobial drugs in human and veterinary medicine, becoming a public health problem. However, little is known about its occurrence and maintenance in wild animals, and very few studies have been carried out in ecosystems subjected to low human pressure. In our study, nasal and lung swabs were collected from hunted Pyrenean chamois (Rupicapra pyrenaica), and nasal swabs from sympatric domestic sheep were also collected. The swabs were cultured in agar plates to obtain bacterial isolates from the Pasteurellaceae family. The presence of AMR was assessed in a total of 28 Pasteurellaceae isolates from 45 Pyrenean chamois, and 9 isolates from sympatric domestic sheep found in the National Hunting Reserve of Freser-Setcases (Northeastern Pyrenees, Spain). The isolates belonged to one of the following three species: Pasteurella multocida, Mannheimia haemolytica and Bibersteinia trehalosi. Some P. multocida and M. haemolytica isolates tested positive for AMR. The statistical analysis revealed no differences between the AMR levels from chamois and domestic sheep isolates. However, one P. multocida of chamois origin presented resistance to cephalosporins and fluoroquinolones, which are antibiotics of critical importance for human health. Further studies are required to elucidate potential routes of dissemination of AMR genes in natural environments and assess any significant persistence in wildlife to design risk mitigation actions.

Abstract 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.

Infections by Chlamydiae are associated with ocular disease in humans and animals. In this study, the presence and diversity of Chlamydia spp. was assessed in diseased and healthy eyes of domestic sheep and wild ruminants that share mountain habitats in northern Spain. The presence of Chlamydia spp. was tested by real-time PCR in 1786 conjunctival swabs collected from both eyes of 893 animals from mountain habitats in northern Spain, and chlamydial species were identified in the positive samples by ArrayTube microarray methods. Chlamydial DNA was detected in 0.6% (CI95% 0.2–1.3) of the Pyrenean chamois (Rupicapra pyrenaica) and 1.4% (CI95% Ovis aries) sampled, with Chlamydia pecorum the only chlamydial species identified. No association of C. pecorum with ocular disease or co-infection with Mycoplasma conjunctivae was found. Further studies on the pathogenesis of infectious keratoconjunctivitis are needed to better understand the ecology of C. pecorum and its possible role as a ruminant pathogen at the wildlife–livestock interface.

The presence of respiratory viruses and pestiviruses in sheep has been widely demonstrated, and their ability to cause injury and predispose to respiratory processes have been proven experimentally. A longitudinal observational study was performed to determine the seroprevalence of bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1 (BHV-1) and pestiviruses in 120 lambs at the beginning and the end of the fattening period. During this time, the animals were clinically monitored, their growth was recorded, and post-mortem examinations were performed in order to identify the presence of pneumonic lesions in the animals. Seroconversion to all viruses tested except BHV-1 was detected at the end of the period. Initially, BPIV-3 antibodies were the most frequently found, while the most common seroconversion through the analysed period occurred to BRSV. Only 10.8% of the lambs showed no detectable levels of antibodies against any of the tested viruses at the end of the survey. In addition, no statistical differences were found in the presentation of respiratory clinical signs, pneumonic lesions nor in the production performance between lambs that seroconverted and those which did not, except in the case of pestiviruses. The seroconversion to pestiviruses was associated with a reduction in the final weight of the lambs.

Porcine circovirus 3 (PCV-3) is the third member of the family Circoviridae, genus Circovirus, able to infect swine. A high prevalence of viral DNA has been recorded in wild boars. Recently, PCV-3 DNA was identified in Italian wild ruminants. Based on these previous results, this study assessed the frequency of PCV-3 DNA detection in free-ranging ruminants and Lagomorpha species in Spain. In addition, the genetic characterization of the PCV-3 PCR-positive samples was performed. A total of 801 serum samples, including red deer (Cervus elaphus, [CE]; n = 108), roe deer (Capreolus capreolus, [CC]; n = 87), Pyrenean chamois (Rupicapra pyrenaica, [RP]; n = 133), Iberian ibex (Capra pyrenaica, [CP]; n = 92), mouflon (Ovis aries, [OA]; n = 91), fallow deer (Dama dama, [DD]; n = 104), European rabbit (Oryctolagus cuniculus, [OC]; n = 101), and European hare (Lepus europaeus, [LE]; n = 85) from Catalonia (northeast Spain) were tested by conventional polymerase chain reaction (PCR) and, when positive, sequenced. Overall, PCV-3 DNA was found in three out of 801 analyzed sera (0.37%) corresponding to one red deer (1/108, 0.9%), one mouflon (1/91, 1.1%), and one fallow deer (1/104, 0.96%). None of the samples collected from Lagomorpha species resulted PCR positive. The partial genome sequences detected in positive samples displayed high identity with some PCV-3 sequences detected in wild boars and domestic pigs (99.7% and 100%, respectively). In conclusion, the present study indicated that free-ranging ruminant and Lagomorpha species are not relevant in the epidemiology of PCV-3 in Spain.

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

The increase in human population and domestic pets, such as cats, are generating important consequences in terms of habitat loss and pathogen pollution of coastal ecosystems with potential to generate negative impacts in marine biodiversity. Toxoplasma gondii is the etiological agent of zoonotic disease toxoplasmosis, and is associated with cat abundance and anthropogenic disturbance. The presence of T. gondii oocysts in the ocean has negatively affected the health status of the threatened Southern sea otter (Enhydra lutris nereis) populations. The present study analyzed seroprevalence and presence of T. gondii DNA in American mink (Neovison vison), Southern river otters (Lontra provocax) and domestic cats (Felis silvestris catus) in four different areas in Southern Chile comprising studies in rivers and lakes in Andean foothills and mountains, marine habitat and island coastal ecosystems. Mean seroprevalence of T. gondii in the study was 64% of 151 total animals sampled: 59% of 73 American mink, 77% of 13 Southern river otters, 68% of 65 domestic cats and in two of two kodkods (Leopardus guigna). Toxoplasma gondii DNA was detected in tissues from one American mink and one Southern river otter. The present study confirms the widespread distribution of T. gondii in Southern Chile, and shows a high exposure of semiaquatic mustelids and domestic cats to the parasite. Cats and anthropogenic disturbance have a role in the maintenance of T. gondii infection in ecosystems of southern Chile.

Abstract Background Pyrenean chamois (Rupicapra pyrenaica pyrenaica) is a nearly monomorphic mountain ungulate with an unbiased sex-specific overwinter adult survival. Few differences in gastrointestinal parasitism have been reported by coprology as yet. This study aims to assess diversity, prevalence, intensity of infection and aggregation of gastrointestinal nematodes in male and female adult chamois. We expect no differences in the parasite infection rates between sexes. Findings Gastrointestinal tracts of 28 harvested Pyrenean chamois in the Catalan Pyrenees (autumn 2012 and 2013) were necropsied and sexual differences in the diversity and structure of parasite community, prevalence, intensity of infection, and richness were investigated. We found 25 helminth species belonging to 13 different genera. Conclusions Contrary to our expectations, male chamois showed different parasite communities, higher prevalence, intensity of infection and richness than females. Such sexual differences were clear irrespective of age of individuals. Hence, male chamois must cope with a more diverse and abundant parasite community than females, without apparent biological cost. Further research will be required to confirm this hypothesis.

Functional roles of domestic and wild host populations in infectious keratoconjunctivitis (IKC) epidemiology have been extensively discussed claiming a domestic reservoir for the more susceptible wild hosts, however, based on limited data. With the aim to better assess IKC epidemiology in complex host-pathogen alpine systems, the long-term infectious dynamics and molecular epidemiology of Mycoplasma conjunctivae was investigated in all host populations from six study areas in the Pyrenees and one in the Cantabrian Mountains (Northern Spain). Detection of M. conjunctivae was performed by qPCR on 3600 eye swabs collected during seven years from hunted wild ungulates and sympatric domestic sheep (n = 1800 animals), and cluster analyses of the strains were performed including previous reported local strains. Mycoplasma conjunctivae was consistently detected in three Pyrenean chamois (Rupicapra p. pyrenaica) populations, as well as in sheep flocks (17.0% of sheep) and occasionally in mouflon (Ovis aries musimon) from the Pyrenees (22.2% in one year/area); statistically associated with ocular clinical signs only in chamois. Chamois populations showed different infection dynamics with low but steady prevalence (4.9%) and significant yearly fluctuations (0.0%- 40.0%). Persistence of specific M. conjunctivae strain clusters in wild host populations is demonstrated for six and nine years. Cross-species transmission between chamois and sheep and chamois and mouflon were also sporadically evidenced. Overall, independent M. conjunctivae sylvatic and domestic cycles occurred at the wildlife-livestock interface in the alpine ecosystems from the Pyrenees with sheep and chamois as the key host species for each cycle, and mouflon as a spill-over host. Host population characteristics and M. conjunctivae strains resulted in different epidemiological scenarios in chamois, ranging from the fading out of the mycoplasma to the epidemic and endemic long-term persistence. These findings highlight the capacity of M. conjunctivae to establish diverse interactions and persist in host populations, also with different transmission conditions.

Two groups with three wild boars each were used: Group A (animals 1 to 3) served as the control, and Group B (animals 4 to 6) was postnatally persistently infected with the Cat01 strain of CSFV (primary virus). The animals, six weeks old and clinically healthy, were inoculated with the virulent strain Margarita (secondary virus). For exclusive detection of the Margarita strain, a specific qRT-PCR assay was designed, which proved not to have cross-reactivity with the Cat01 strain. The wild boars persistently infected with CSFV were protected from superinfection by the virulent CSFV Margarita strain, as evidenced by the absence of clinical signs and the absence of Margarita RNA detection in serum, swabs and tissue samples. Additionally, in PBMCs, a well-known target for CSFV viral replication, only the primary infecting virus RNA (Cat01 strain) could be detected, even after the isolation in ST cells, demonstrating SIE at the tissue level in vivo. Furthermore, the data analysis of the Margarita qRT-PCR, by means of calculated ΔCt values, supported that PBMCs from persistently infected animals were substantially protected from superinfection after in vitro inoculation with the Margarita virus strain, while this virus was able to infect naive PBMCs efficiently. In parallel, IFN-α values were undetectable in the sera from animals in Group B after inoculation with the CSFV Margarita strain. Furthermore, these animals were unable to elicit adaptive humoral (no E2-specific or neutralising antibodies) or cellular immune responses (in terms of IFN-γ-producing cells) after inoculation with the second virus. Finally, a sequence analysis could not detect CSFV Margarita RNA in the samples tested from Group B. Our results suggested that the SIE phenomenon might be involved in the evolution and phylogeny of the virus, as well as in CSFV control by vaccination. To the best of our knowledge, this study was one of the first showing efficient suppression of superinfection in animals, especially in the absence of IFN-α, which might be associated with the lack of innate immune mechanisms.

Understanding the spread of Toxoplasma gondii (T. gondii) in wild birds, particularly in those with opportunistic feeding behavior, is of interest for elucidating the epidemiological involvement of these birds in the maintenance and dissemination of the parasite. Overall, from 2009 to 2011, we collected sera from 525 seagull chicks (Yellow-legged gull (Larus michahellis) and Audouin's gull (L. audouinii)) from 6 breeding colonies in Spain and tested them using the modified agglutination test (MAT) for the presence of antibodies against T. gondii. Chick age was estimated from bill length. Main food source of seagull chicks was evaluated using stable isotope analyses from growing scapular feathers. Overall T. gondii seroprevalence was 21.0% (IC95% 17.5-24.4). A generalized linear mixed-effects model indicated that year (2009) and food source (freshwater) were risk factors associated to the individual risk of infection by T. gondii, while age (days) was close to significance. Freshwater food origin was related to the highest seroprevalence levels, followed by marine origin, supporting freshwater and sewages as important routes of dispersion of T. gondii. Year differences could indicate fluctuating rates of exposure of seagull chicks to T. gondii. Age ranged from 4 to 30 days and seropositivity tended to increase with age (P = 0.07), supporting that seropositivity is related to T. gondii infection rather than to maternal transfer of antibodies, which in gulls is known to sharply decrease with chick age. This study is the first to report T. gondii antibodies in Yellow-legged and Audouin's gulls, thereby extending the range of intermediate hosts for this parasite and underscoring the complexity of its epidemiology.

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

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.

Since 2001 several outbreaks of a new disease associated with Border disease virus (BDV) infection have caused important declines in Pyrenean chamois (Rupicapra pyrenaica) populations in the Pyrenees. The goal of this study was to analyze the post-outbreak BDV epidemiology in the first two areas affected by disease with the aim to establish if the infection has become endemic. We also investigated if BDV infected wild and domestic ruminants sharing habitat with chamois. Unexpectedly, we found different epidemiological scenarios in each population. Since the disease outbreaks, some chamois populations recuperated quickly, while others did not recover as expected. In chamois from the first areas, prevalence was high (73.47%) and constant throughout the whole study period and did not differ between chamois born before and after the BDV outbreak; in all, BDV was detected by RT-PCR in six chamois. In the other areas, prevalence was lower (52.79%) and decreased during the study period; as well, prevalence was significantly lower in chamois born after the disease outbreak. No BDV were detected in this population. A comparative virus neutralisation test performed with four BDV strains and one Bovine viral diarrhoea virus (BVDV) strain showed that all the chamois had BDV-specific antibodies. Pestivirus antibodies were detected in all the rest of analyzed species, with low prevalence values in wild ruminants and moderate values in domestic ruminants. No viruses were detected in these species. These results confirm the hypothesis that outbreaks of BDV infection only affect the Pyrenean chamois, although other wild ruminants can occasionally be infected. In conclusion, two different scenarios have appeared since the first border disease outbreaks in Pyrenean chamois: on the one hand frequent BDV circulation with possible negative impact on population dynamics in some areas and on the other, lack of virus circulation and quick recovery of the chamois population.

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.

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), considered a zoonotic agent of wildlife origin, can infect various animal species, including wildlife in free-range and captive environments. Detecting susceptible species and potential reservoirs is crucial for preventing the transmission, spread, genetic evolution, and further emergence of viral variants that are major threats to global health. This study aimed to detect exposure or acute infection by SARS-CoV-2 in 420 animals from 40 different wildlife species, including terrestrial and aquatic mammals, from different regions of Spain during the 2020–2023 coronavirus disease 19 (COVID-19) pandemic. In total, 8/137 animals were positive for SARS-CoV-2 antibodies against the receptor binding domain and/or viral nucleoprotein according to independent ELISAs. However, only one ELISA-positive sample of a captive bottlenose dolphin (Tursiops truncatus) tested positive for SARS-CoV-2 neutralizing antibodies with a low titre (SNT50 38.15) according to a virus neutralization test. Cetaceans are expected to have a high risk of infection with SARS-CoV-2 according to early predictive studies due to the similarity of their angiotensin converting enzyme 2 cell receptor to that of humans. Moreover, of 283 animals analysed for SARS-CoV-2 RNA using RT-qPCR, none tested positive. Our results reinforce the importance of considering cetaceans at risk for SARS-CoV-2 infection and support taking preventive biosecurity measures when interacting with them, especially in the presence of individuals with suspected or confirmed COVID-19. Although most animals in this study tested negative for acute infection or viral exposure, ongoing surveillance of wildlife species and potentially susceptible animals is important to prevent future spillover events and detect potential novel reservoirs.