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[Introduction]: Wild birds are considered reservoirs of poultry pathogens although transmission routes have not been conclusively established. Here we use camera trapping to study wild bird communities on commercial layer and red-legged partridge farms over a one-year timeframe. We also analyze direct and indirect interactions of other bird species with the house sparrow (Passer domesticus), a potential bridge host., [Methods]: We conducted camera trapping events between January 2018 and October 2019, in two caged layer farms, one free-range layer farm, and two red-legged partridge farms in South-Central Spain., [Results and discussion]: We observed wild bird visits on all types of farms, with the significantly highest occurrence on red-legged partridge farms where food and water are more easily accessible, followed by commercial caged layer farms, and free-range chicken farms. The house sparrow (Passer domesticus) followed by spotless starlings (Sturnus unicolor) was the most encountered species on all farms, with the highest frequency in caged layer farms. On partridge farms, the house sparrow accounted for 58% of the wild bird detections, while on the free-range chicken farm, it made up 11% of the detections. Notably, the breeding season, when food and water are scarce in Mediterranean climates, saw the highest number of wild bird visits to the farms. Our findings confirm that the house sparrow, is in direct and indirect contact with layers and red-legged partridges and other wild birds independent of the type of farm. Contacts between house sparrows and other bird species were most frequent during the breeding season followed by the spring migration period. The species most frequently involved in interactions with the house sparrow belonged to the order Passeriformes. The study provides a comparative description of the composition and seasonal variations of bird communities in different types of layer/ poultry farms in Southern Spain i.e. a Mediterranean climate. It confirms the effectiveness of biosecurity measures that restrict access to feed and water. Additionally, it underscores the importance of synanthropic species, particularly the house sparrow, as potential bridge vector of avian pathogens.

Bagaza virus (BAGV) is a mosquito-borne flavivirus of the Ntaya serocomplex that emerged in 2010 in Spain in an outbreak in gamebirds, primarily red-legged partridges. It reappeared in 2019 and again in 2021 when it emerged also in Portugal. An experimental infection confirmed the high susceptibility of red-legged partridges to the virus and direct transmission to uninfected contacts. Here we study pathogenesis and viral antigen distribution in experimentally infected partridges in comparison to what was found in field cases. Sixteen five-month-old red-legged partridges were inoculated with BAGV (strain Spain RLP-Hcc1/2010). Four additional partridges were kept as contacts in the same cage, and ten sham-inoculated partridges as controls. Two partridges were euthanized at 4, 7 and 10 days post-infection (dpi) and another three birds died on days 6, 7 and 10 dpi, respectively. Tissues (brain, lung, pancreas, liver, kidney, spleen, heart, bursa of Fabricius, large and small intestines) were fixed in 10% neutral buffered formalin and processed for histopathology and immunohistochemistry. Slides were scored for lesion severity and number and type of viral antigen positive cells per 0.2mm2. Partridges euthanized at 4 dpi only had congestion and inflammatory infiltrates in spleen and heart, while from 7dpi on, the main findings were congestion and endothelial hypertrophy in addition to mononuclear inflammatory infiltrates in the spleen, kidney, liver and heart. Perivascular cuffs of inflammatory cells, endothelial cell swelling, and neuronal necrosis and gliosis were observed in the brain, especially of birds euthanized/dead on 10 dpi. Hemosiderosis, one of the hallmarks of BAGV infection in free-living red-legged partridges, was only observed sporadically. BAGV antigen was found on 4 dpi in the caeca and spleen. On 7 and 10 dpi, BAGV antigen were mostly identified in the spleen, bursa, heart, and kidney. No BAGV antigen was detected in the brain except for two Purkinje ce

Spain houses approximately 75% of the world’s population of griffon vultures (Gyps fulvus). While vultures are generally resistant to infectious disease agents, during the 2022 breeding season highly pathogenic avian influenza virus H5N1 affected griffon and bearded (Gypaetus barbatus) vultures. We report epidemiologic information gathered in the context of a program on movement ecology of Spanish griffon vultures that could help to understand risks in future breeding seasons. A total of 91 nestling and adult griffon vultures were ringed and radiotagged in 2021 (n = 50) and 2022 (n=41) and vascular feather and blood samples were taken to study exposure to Flaviviruses. Tissue including feather pulp or bone marrow from decomposed or previously frozen (-20ºC) carcasses (n=11) from northern and southern Spain were subjected to Real Time RTPCRs for influenza A matrix and H5 AIV. Samples from positive birds were sent to the Central veterinary laboratory for confirmation. We confirmed highly pathogenic H5N1 AIV in feather pulp and bone of deceased, highly decomposed, individuals. Feathers of all other vultures except a nestling that died after tagging were negative by Real time RTPCR. None of the vultures sampled in 2021 had antibodies against H5 AIV, while three ELISA positive nestlings from 2022 had high titres of antibodies against H5N1 by haemgglutination inhibition test, showing that at least two nestlings had survived the infection and fledged. Retrospective analysis of stored tissues from adult griffon vultures thought to have died from poisoning confirmed HPAIV H5N1 in at least one individual. Our results suggest that the current H5N1 AIV is highly pathogenic for griffon vultures but that not all individuals with systemic infections succumb. Further investigations should determine whether such survivors could become persistently infected and spread the disease. Our results suggest that the current H5N1 AIV is highly pathogenic for griffon vultures but that not all

Landfills have played a significant role in the recovery of white storks (Ciconia ciconia) populations across various European countries. While there is ample information about the populational-level effects, there is a lack of knowledge regarding the individual effects of using this food resource for feeding nestlings. This study aims to assess the nutritional status and oxidative stress balance of nestlings with varying degrees of exposure to landfill-provided food This study aims to assess the nutritional status and oxidative stress balance of nestlings with different use of landfill-provided food. Nestlings fed with food foraged by breeding pairs from landfills exhibited better nutritional status compared to individuals located farther from landfills. This can be attributed to a higher ingestion rate, resulting in increased plasmatic values of cholesterol, triglycerides, and HDL in plasma. However, the oxidative stress balance varied across different years, with individuals raised in 2014 showing higher values of Vitamin E and lower values of LDH compared to those raised in 2013. Furthermore, the impact of landfills on certain oxidative stress parameters also depended on the year of study., In 2013, the Total Antioxidant Capacity (TAC) of plasma showed a positive correlation with the distance to landfills, while the concentration of Malondialdehyde (MDA), an indicator of lipid peroxidation, exhibited a negative correlation. These findings suggest that the use of landfills as a food resource has a consistently positive effect on the nutritional status of white stork nestling. However, the relationship with oxidative stress is highly dependent on the climatic conditions of each year, emphasizing the importance of considering these factors when evaluating the use of landfills as a food resource.

[ES] Los antibióticos han mejorado la esperanza de vida, pero su uso excesivo ha aumentado la resistencia bacteriana. La resistencia, causada por mutaciones genéticas o transferencia horizontal, no implica virulencia, pero ambas están relacionadas. En las depuradoras, los vertederos y las explotaciones ganaderas existe el riesgo de contaminación por bacterias resistentes a los antibióticos. Como las cigüeñas blancas se han adaptado a alimentarse en los vertederos, al ingerir bacterias resistentes a los antibióticos procedentes de los vertederos, podrían convertirse en reservorios y vectores de bacterias resistentes/resistencias. Para investigar el papel de las cigüeñas blancas en la dispersión de genes de resistencia y virulencia, estudiamos comparativamente el agua de los humedales y los excrementos de cigüeña blanca recogidos en humedales y vertederos. Nuestro estudio se centra en cinco localidades de las provincias de Ciudad Real y Toledo. Se analizaron trece muestras de agua y 61 “pooles” compuestos por 299 muestras fecales de cigüeña blanca. En todas las muestras se detectaron genes de resistencia y virulencia mediante PCR en tiempo real. La prevalencia de genes de resistencia fue similar en las heces y el agua, excepto para los genes qnrS y mcr-1 que solo se encontraron en las heces. Los genes que proporcionan resistencia a los carbapenémicos (blaKPC y blaNDM) no se detectaron. La diversidad de genes de virulencia fue mayor en las heces que en el agua del humedal. En concreto, el gen de virulencia bfpA sólo se detectó en heces y con una frecuencia significativamente mayor en vertederos que en humedales (p=0,005), el gen de virulencia stx1 sólo estuvo presente en excrementos de cigüeña recogidos en el Parque Nacional de Las Tablas de Daimiel durante la migración primaveral y el gen est (ST) se encontró con una frecuencia significativamente mayor en excrementos de cigüeña recogidos en el vertedero de Alcázar de San Juan y en el embalse de Cazalegas durante la mi, [EN] Antibiotics have improved life expectancy, but their overuse has increased bacterial resistance. Resistance, caused by genetic mutations or horizontal transfer, does not imply virulence, but the two are related. In sewage treatment plants, landfills and livestock farms, there is a risk of contamination by antibiotic-resistant bacteria. As white storks have adapted to feed in landfills, by ingesting antibiotic-resistant bacteria from landfills, they could become reservoirs and vectors of resistant/resistant bacteria. To investigate the role of white storks in the dispersal of resistance and virulence genes, we comparatively studied wetland water and white stork droppings collected from wetlands and landfills. Our study focuses on five localities in the provinces of Ciudad Real and Toledo, in Spain. Thirteen water samples and 61 pools composed of 299 white stork faecal samples were analysed. Resistance and virulence genes were detected in all samples by real-time PCR. The prevalence of resistance genes was similar in faeces and water, except for the qnrS and mcr-1 genes which were only found in faeces. Genes providing resistance to carbapenems (blaKPC and blaNDM) were not detected. The diversity of virulence genes was higher in faeces than in wetland water. In particular, the virulence gene bfpA was only detected in faeces and with a significantly higher frequency in landfill than in wetlands (p=0.005), the virulence gene stx1 was only present in stork droppings collected in Las Tablas de Daimiel National Park during spring migration and the est (ST) gene was found with a significantly higher frequency in stork droppings collected at the Alcázar de San Juan landfill and Cazalegas reservoir during postnuptial migration and wintering period. The similar prevalence of certain resistance genes in stork droppings and water suggests a possible gene exchange between faeces and water, while differences in the prevalence of virulence genes indicate an association of resis

Migratory storks could be vectors of transmission of bacteria of public health concern mediated by the colonization, persistence and excretion of such bacteria. This study aims to determine genera/species diversity, prevalence, and co-colonization indices of bacteria obtained from tracheal (T) and nasal (N) samples from storks in relation to exposure to point sources through foraging. One-hundred and thirty-six samples from 87 nestlings of colonies of parent white storks with different foraging habits (natural habitat and landfills) were obtained (84 T-samples and 52 N-samples) and processed. Morphologically distinct colonies (up to 12/sample) were randomly selected and identified by MALDI-TOF-MS. About 87.2% of the total 806 isolates recovered were identified: 398 from T-samples (56.6%) and 305 from N-samples (43.4%). Among identified isolates, 17 genera and 46 species of Gram-positive and Gram-negative bacteria were detected, Staphylococcus (58.0%) and Enterococcus (20.5%) being the most prevalent genera. S. sciuri was the most prevalent species from T (36.7%) and N (34.4%) cavities of total isolates, followed by E. faecalis (11.1% each from T and N), and S. aureus [T (6.5%), N (13.4%)]. Of N-samples, E. faecium was significantly associated with nestlings of parent storks foraging in landfills (p = 0.018). S. sciuri (p = 0.0034) and M. caseolyticus (p = 0.032) from T-samples were significantly higher among nestlings of parent storks foraging in natural habitats. More than 80% of bacterial species in the T and N cavities showed 1–10% co-colonization indices with one another, but few had ≥ 40% indices. S. sciuri and E. faecalis were the most frequent species identified in the stork nestlings. Moreover, they were highly colonized by other diverse and potentially pathogenic bacteria. Thus, storks could be sentinels of point sources and vehicles of bacterial transmission across the “One Health” ecosystems.

Table S1. Prevalence pattern and association of S. aureus in white stork nestlings with the foraging habits of their parents., Table S2. Primers and conditions of the PCR performed in this study.

The molecular ecology of Staphylococcus aureus in migratory birds (such as white storks) is necessary to understand their relevance in the “One Health” ecosystems. This study determined the nasotracheal carriage rates of S. aureus from white storks in Southern Spain and genetically characterized the within-host diversity. A collection of 67 S. aureus strains, previously obtained from 87 white stork nestlings (52 nasal and 85 tracheal samples) fed by their parents with food foraged in natural and landfill habitats, were tested for their antimicrobial resistance (AMR) phenotypes. Moreover, the AMR genotypes, immune evasion cluster (IEC), virulence genes and the detection of CC398 lineage were studied by PCR. The spa types and multilocus-sequencing-typing (MLST) were also determined by PCR and sequencing. Staphylococcus aureus carriage was found in 31% of storks (36.5%/11.9% in nasal/tracheal samples). All isolates were methicillin-susceptible (MSSA) and 8.8% of them were also susceptible to all tested antibiotics. The AMR phenotype/percentage/genes detected were as follows: penicillin/79.1%/blaZ; erythromycin-clindamycin-inducible/19.1%/ermA, ermT; tetracycline/11.9%/tetK; clindamycin/4.5%/lnuA and ciprofloxacin/4.5%. Twenty-one different spa types, including 2 new ones (t7778-ST15-CC15 and t18009-ST26-CC25), were detected and ascribed to 11 clonal complexes (CCs). MSSA-CC398 (8.2%), MSSA-CC15 (7.1%) and MSSA-ST291 (5.9%) were the most prevalent lineages in storks. Moreover, tst-positive (MSSA-CC22-t223 and MSSA-CC30-t1654), eta-positive (MSSA-CC9-t209) and etb-positive strains (MSSA-CC45-t015) were detected in four storks. The 18.5% of storks harboured distinct MSSA strains (with different lineages and/or AMR genes). Nestlings of storks foraging in landfills (10 CCs) had more diverse S. aureus strains than those of parents foraging in natural habitats (3 CCs). Low level of AMR was demonstrated among S. aureus strains. The predominance of MSSA-CC398 (an emergent clade

[EN] The house sparrow (Passer domesticus) is an ubiquitous species, distributed worldwide in rural, urban and humanized environments. In Europe, sparrow populations have suffered an alarming decline in recent decades, especially in urbanized areas. In the present study we analyzed, the effects of urbanization on body condition, pathogen prevalence and changes in the serum proteome. For this, we captured sparrows in four locations with two different degrees of urbanization. Biometric data, blood samples and oral and cloacal swabs were collected and stored at -80° C until further analysis. Rural sparrows were in significantly better physical condition than urban sparrows and there were no significant differences between sexes. After testing for haemoparasites, avian influenza virus (AIV), West Nile virus (WNV), other flaviviruses and serum proteomics, a significantly higher prevalence of haemoparasites was found in rural sparrows, 70.16% (87/124), compared to urban sparrows 50.00% (27/54). No AIV or WNV genetic material was detected in any sparrow, although one urban sparrow (0.58%) had antibodies to AIV. Serum proteomics revealed that in comparison to the normalized mean, rural sparrows had elevated values, of certain proteins involved in the metabolism, in contrast to proteins ofthe immune system and the coagulation system, which were found to be elevated in urban sparrows. The results obtained confirm that urban sparrows have a lower body condition and an activation of the immune system, above the normalized values, and rural sparrows have a higher prevalence of avian malaria. Furthermore, these results confirm that sparrows are at least occasionally exposed to AIVs and may be involved in virus transmission and maintenance., [ES] El gorrión común (Passer domesticus), es ubicuo, distribuido a nivel mundial en zonas rurales y ambientes urbanos y humanizados. En Europa en las últimas décadas, las poblaciones de gorriones han sufrido un alarmante descenso, especialmente en áreas urbanizadas. En el presente estudio se analizaron los efectos de la urbanización, en la condición corporal y la prevalencia de patógenos, en relación con los cambios en el proteoma sérico. Para llevar a cabo este estudio, se capturaron gorriones en cuatro localizaciones con dos diferentes grados de urbanización y se recolectaron datos biométricos, muestras de sangre e hisopos orales y cloacales, conservadas a -80º C hasta su análisis. Los gorriones rurales estaban en significativamente mejor condición física que los urbanos y no había diferencias significativas entre sexos. Tras analizar la presencia de hemoparásitos, virus de influenza aviar (VIA), virus del Nilo Occidental (VNO), otros flavivirus y la proteómica sérica, se encontraron prevalencias de hemoparásitos significativamente más altas en gorriones rurales 70,16% (87/124) que en gorriones urbanos 50,00% (27/54). En ningún gorrión se detectó material genético de VIA, o VNO, aunque un gorrión de procedencia urbana (0,58%), presentaba anticuerpos frente a VIA. La proteómica sérica reveló que los gorriones rurales presentaban valores elevados, frenteal valor normalizado, de ciertas proteínas implicadas en el metabolismo, a diferencia de las proteínas del sistema inmune y del sistema de coagulación, que se encontraron elevadas en gorriones urbanos. Los resultados obtenidos confirman que los gorriones de ambientes urbanizados presentan una menor condición corporal y una activación del sistema inmune, por encima de los valores normalizados, y los gorriones rurales presentan una mayor prevalencia a malaria aviar. Además, estos resultados confirman que los gorriones están al menos ocasionalmente expuestos a los VIA y podrán participar en la transmisión y mantenimien

[Background] Connections between habitats are key to a full understanding of anthropic impacts on ecosystems. Freshwater habitats are especially biodiverse, yet depend on exchange with terrestrial habitats. White storks (Ciconia ciconia) are widespread opportunists that often forage in landfills and then visit wetlands, among other habitats. It is well known that white storks ingest contaminants at landfills (such as plastics and antibiotic resistant bacteria), which can be then deposited in other habitats through their faeces and regurgitated pellets., [Methods] We characterized the role of white storks in habitat connectivity by analyzing GPS data from populations breeding in Germany and wintering from Spain to Morocco. We overlaid GPS tracks on a land-use surface to construct a spatially-explicit network in which nodes were sites, and links were direct flights. We then calculated centrality metrics, identified spatial modules, and quantified overall connections between habitat types. For regional networks in southern Spain and northern Morocco, we built Exponential Random Graph Models (ERGMs) to explain network topologies as a response to node habitat., [Results] For Spain and Morocco combined, we built a directed spatial network with 114 nodes and 370 valued links. Landfills were the habitat type most connected to others, as measured by direct flights. The relevance of landfills was confirmed in both ERGMs, with significant positive effects of this habitat as a source of flights. In the ERGM for southern Spain, we found significant positive effects of rice fields and salines (solar saltworks) as sinks for flights. By contrast, in the ERGM for northern Morocco, we found a significant positive effect of marshes as a sink for flights., [Conclusions] These results illustrate how white storks connect landfills with terrestrial and aquatic habitats, some of which are managed for food production. We identified specific interconnected habitat patches across Spain and Morocco that could be used for further studies on biovectoring of pollutants, pathogens and other propagules.

This study determined the carriage rates and antimicrobial resistance (AMR) genes of enterococci from nasotracheal samples of three healthy animal species and in-contact humans. Nasal samples were collected from 27 dog-owning households (34 dogs, 41 humans) and 4 pig-farms (40 pigs, 10 pig-farmers), and they were processed for enterococci recovery (MALDI-TOF-MS identification). Also, a collection of 144 enterococci previously recovered of tracheal/nasal samples from 87 white stork nestlings were characterized. The AMR phenotypes were determined in all enterococci and AMR genes were studied by PCR/sequencing. MultiLocus-Sequence-Typing was performed for selected isolates. About 72.5% and 60% of the pigs and pig-farmers, and 29.4% and 4.9%, of healthy dogs and owners were enterococci nasal carriers, respectively. In storks, 43.5% of tracheal and 69.2% of nasal samples had enterococci carriages. Enterococci carrying multidrug-resistance phenotype was identified in 72.5%/40.0%/50.0%/23.5%/1.1% of pigs/pig-farmers/dogs/dogs' owners/storks, respectively. Of special relevance was the detection of linezolid-resistant enterococci (LRE) in (a) 33.3% of pigs (E. faecalis-carrying optrA and/or cfrD of ST59, ST330 or ST474 lineages; E. casseliflavus-carrying optrA and cfrD); (b) 10% of pig farmers (E. faecalis-ST330-carrying optrA); (c) 2.9% of dogs (E. faecalis-ST585-carrying optrA); and (d) 1.7% of storks (E. faecium-ST1736-carrying poxtA). The fexA gene was found in all optrA-positive E. faecalis and E. casseliflavus isolates, while fexB was detected in the poxtA-positive E. faecium isolate. The enterococci diversity and AMR rates from the four hosts reflect differences in antimicrobial selection pressure. The detection of LRE carrying acquired and transferable genes in all the hosts emphasizes the need to monitor LRE using a One-Health approach.

A collection of 259 staphylococci of 13 different species [212 coagulase-negative (CoNS) and 47 coagulase-positive (CoPS)] recovered from nasotracheal samples of 87 healthy nestling white storks was tested by the spot-on-lawn method for antimicrobial-activity (AA) against 14 indicator bacteria. Moreover, extracts of AP isolates were obtained [cell-free-supernatants (CFS) both crude and concentrated and butanol extracts] and tested against the 14 indicator bacteria. The microbiota modulation capacity of AP isolates was tested considering: (a) intra-sample AA, against all Gram-positive bacteria recovered in the same stork nasotracheal sample; (b) inter-sample AA against a selection of representative Gram-positive bacteria of the nasotracheal microbiota of all the storks (30 isolates of 29 different species and nine genera). In addition, enzymatic susceptibility test was carried out in selected AP isolates and bacteriocin encoding genes was studied by PCR/sequencing. In this respect, nine isolates (3.5%; seven CoNS and two CoPS) showed AA against at least one indicator bacteria and were considered antimicrobial-producing (AP) isolates. The AP isolates showed AA only for Gram-positive bacteria. Three of these AP isolates (S. hominis X3764, S. sciuri X4000, and S. chromogenes X4620) revealed AA on all extract conditions; other four AP isolates only showed activity in extracts after concentration; the remaining two AP isolates did not show AA in any of extract conditions. As for the microbiota modulation evaluation, three of the nine AP-isolates revealed intra-sample AA. It is to highlight the potent inter-sample AA of the X3764 isolate inhibiting 73% of the 29 representative Gram-positive species of the nasotracheal stork microbiota population. On the other hand, enzymatic analysis carried out in the two highest AP isolates (X3764 and X4000) verified the proteinaceous nature of the antimicrobial compound and PCR analysis revealed the presence of lantibiotic-like encoding

A bearded vulture (Gypaetus barbatus) found dead in northern Spain presented external lesions consistent with electrocution as the cause of death. During forensic examination, macroscopic lesions suggested potential comorbidity, so samples were collected for molecular and toxicological analyses. Gastric content and liver were analysed for toxic substances, and pentobarbital (a common pharmaceutical used for euthanasia in domestic animals) was detected at a concentration of 37.3 and 0.05 µg/g, respectively. Other toxicological, viral and endoparasite analyses (avian malaria, avian influenza and flaviviruses) were negative. Thus, although the cause of death was electrocution, pentobarbital intoxication likely impaired the equilibrium and reflexes of the individual, possibly causing the bird to contact energized wires that it would not have otherwise. These results underline the importance of comprehensive analysis of forensic cases of wildlife deaths and reveal barbiturate poisoning as an additional threat for the conservation of the bearded vulture in Europe.

West Nile virus (WNV) is the most widespread flavivirus in the world with a wide vertebrate host range. Its geographic expansion and activity continue to increase with important human and equine outbreaks and local bird mortality. In a previous experiment, we demonstrated the susceptibility of 7-week-old red-legged partridges (Alectoris rufa) to Mediterranean WNV isolates Morocco/2003 and Spain/2007, which varied in virulence for this gallinaceous species. Here we study the pathogenesis of the infection with these two strains to explain the different course of infection and mortality. Day six post-inoculation was critical in the course of infection, with the highest viral load in tissues, the most widespread virus antigen, and more severe lesions. The most affected organs were the heart, liver, and spleen. Comparing infections with Morocco/2003 and Spain/2007, differences were observed in the viral load, virus antigen distribution, and lesion nature and severity. A more acute and marked inflammatory reaction (characterized by participation of microglia and CD3+ T cells) as well as neuronal necrosis in the brain were observed in partridges infected with Morocco/2003 as compared to those infected with Spain/2007. This suggests a higher neurovirulence of Morocco/2003, probably related to one or more specific molecular determinants of virulence different from Spain/2007.

Flaviviruses such as West Nile (WNV), Usutu (USUV) and Bagaza (BAGV) virus and avian malaria parasites are vector borne pathogens that circulate naturally between avian and mosquito hosts. WNV and USUV and potentially also BAGV constitute zoonoses. Temporal and spatial cocirculation and coinfection with Plasmodium spp., and West Nile virus has been documented in birds and mosquito vectors, and fatally USUV-infected passerines coinfected with Plasmodium spp. had more severe lesions. Also, WNV, USUV and BAGV have been found to cocirculate. Yet little is known about the interaction of BAGV and malaria parasites during consecutive or coinfections of avian hosts. Here we report mortality of free-living red-legged partridges in a hunting estate in Southern Spain that were coinfected with BAGV and Plasmodium spp. The outbreak occurred in the area where BAGV first emerged in Europe in 2010 and where cocirculation of BAGV, USUV and WNV was confirmed in 2011 and 2013. Partridges were found dead in early October 2019. Birds had mottled locally pale pectoral muscles, enlarged, congestive greenish-black tinged livers and enlarged kidneys. Microscopically congestion and predominantly mononuclear inflammatory infiltrates were evident and Plasmodium phanerozoites were present in the liver, spleen, kidneys, muscle and skin. Molecular testing and sequencing detected Plasmodium spp. and BAGV in different tissues of the partridges, and immunohistochemistry confirmed the presence and colocalization of both pathogens in the liver and spleen. Due to the importance of the red-legged partridge in the ecosystem of the Iberian Peninsula and as driver of regional economy such mortalities are of concern. Such outbreaks may reflect climate change related shifts in host, vector and pathogen ecology and interactions that could emerge similarly for other pathogens.

Bagaza virus emerged in Spain in 2010 and was not reported in other countries in Europe until 2021, when the virus was detected by molecular methods in a corn bunting and several red-legged partridges in Portugal. Sequencing revealed high similarity between the 2021 strains from Portugal and the 2010 strains from Spain.