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Abstract 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.

La Organización Mundial de la Salud ha estimado que alrededor de dos millones de niños muerenanualmente de enfermedades diarreicas en países en vías de desarrollo. Entre los agentes etiológicos clásicos queproducen un cuadro diarreico importante se encuentra Escherichia coli enteropatogénica (EPEC). Se conoce que elprincipal reservorio natural es el ser humano, especialmente niños con diarrea y niños o adultos que son portadoresasintomáticos, pero también se han aislado EPEC de diversos serotipos de animales salvajes. Debido a la reconocidaimportancia que tiene EPEC a nivel epidemiológico, en este estudio se utilizan polluelos de cigüeña para evaluar cómoinfluyen diferentes dietas (basadas en vertederos o en zonas naturales) en el papel potencial de las cigüeñas blancas comoreservorios de EPEC

Flaviviruses 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 co-circulation and co-infection 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 co-circulate. Yet little is known about the interaction of BAGV and malaria parasites during consecutive or co-infections of avian hosts. Here we report mortality of free-living red-legged partridges in a hunting estate in Southern Spain due to coinfection with BAGV and Plasmodium spp. The outbreak occurred in the area where BAGV first emerged in Europe in 2010 and where co-circulation 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 co-localization of both pathogens in the liver and spleen. Although Plasmodium spp. are known to be highly prevalent in red-legged partridges, this is the first account of mortality caused by co-infection with BAGV and Plasmodium sp. 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. Also, they may reflect climate change related changes in host, vector and pathogen ecology and interactions that could emerge similarly in other pathogens.

This article belongs to the Special Issue Viral Shedding and Transmission in Zoonotic Diseases., The mosquito-borne flaviviruses USUV and WNV are known to co-circulate in large parts of Europe. Both are a public health concern, and USUV has been the cause of epizootics in both wild and domestic birds, and neurological cases in humans in Europe. Here, we explore the susceptibility of magpies to experimental USUV infection, and how previous exposure to USUV would affect infection with WNV. None of the magpies exposed to USUV showed clinical signs, viremia, or detectable neutralizing antibodies. After challenge with a neurovirulent WNV strain, neither viremia, viral titer of WNV in vascular feathers, nor neutralizing antibody titers of previously USUV-exposed magpies differed significantly with respect to magpies that had not previously been exposed to USUV. However, 75% (6/8) of the USUV-exposed birds survived, while only 22.2% (2/9) of those not previously exposed to USUV survived. WNV antigen labeling by immunohistochemistry in tissues was less evident and more restricted in magpies exposed to USUV prior to challenge with WNV. Our data indicate that previous exposure to USUV partially protects magpies against a lethal challenge with WNV, while it does not prevent viremia and direct transmission, although the mechanism is unclear. These results are relevant for flavivirus ecology and contention., This work was supported by grants RTA2015-009 and E-RTA2017-003-C02 from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA).

Most methicillin resistant Staphylococcus aureus (MRSA) isolates harboring mecC gene belong to clonal complex CC130. This lineage has traditionally been regarded as animal-associated as it lacks the human specific immune evasion cluster (IEC), and has been recovered from a broad range of animal hosts. Nevertheless, sporadic mecC-MRSA human infections have been reported, with evidence of zoonotic transmission in some cases. The objective of this study was to investigate the whole-genome sequences of 18 S. aureus CC130 isolates [13 methicillin-resistant (mecC-MRSA) and five methicillin-susceptible (MSSA)] from different sequences types, obtained from a variety of host species and origins (human, livestock, wild birds and mammals, and water), and from different geographic locations, in order to identify characteristic markers and genomic features. Antibiotic resistance genes found among MRSA-CC130 were those associated with the SSCmecXI element. Most MRSA-CC130 strains carried a similar virulence gene profile. Additionally, six MRSA-CC130 possessed scn-sak and one MSSA-ST130 had lukMF’. The MSSA-ST700 strains were most divergent in their resistance and virulence genes. The pan-genome analysis showed that 29 genes were present solely in MRSA-CC130 (associated with SCCmecXI) and 21 among MSSA-CC130 isolates (associated with phages). The SCCmecXI, PBP3, GdpP, and AcrB were identical at the amino acid level in all strains, but some differences were found in PBP1, PBP2, PBP4, and YjbH proteins. An examination of the host markers showed that the 3’ region of the bacteriophage φ3 was nearly identical to the reference sequence. Truncated hlb gene was also found in scn-negative strains (two of them carrying sak-type gene). The dtlB gene of wild rabbit isolates included novel mutations. The vwbp gene was found in the three MSSA-ST700 strains from small ruminants and in one MSSA-ST130 from a red deer; these strains also carried a scn-type gene, different from the human and equine variants. Finally, a phylogenetic analysis showed that the three MSSA-ST700 strains and the two MSSA-ST130 strains cluster separately from the remaining MRSA-CC130 strains with the etD2 gene as marker for the main lineage. The presence of the human IEC cluster in some mecC-MRSA-CC130 strains suggests that these isolates may have had a human origin., The work performed in the University of La Rioja was financed by projects SAF2016-76571-R and PID2019-106158RB-I00 of the Agencia Estatal de Investigación (AEI) of Spain and the Fondo Europeo de Desarrollo Regional (FEDER) of EU. Work in Cambridge was supported by a UK-China AMR Partnership Initiative (MR/P007201/1) held by the Department of Veterinary Medicine, University of Cambridge.

This article belongs to the Special Issue West Nile Virus Infection., 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., This study has been supported by the project PAC08-0296-7771 (JCCM), and from INIA-MARM funds (INIA CC08-020). Elena Sotelo was a fellow from INIA. Elisa Perez Ramirez was a fellow of the National Research Council (CSIC) and Ana Valeria Gutierrez was a fellow of the JCCM.

The use of landfills as foraging areas by white storks (Ciconia ciconia) is a recent well-known behaviour. While several studies have highlighted positive effects at a populational level others suggest that the presence of pollutants, pathogens and the lower presence of antioxidants in the food could pose a health risk for individuals. The objective of this study was to evaluate potential effects of the use of landfills as a food resource on the physiology and health of white stork nestlings, by a multidisciplinary approach based on the analysis of nutritional status, body condition, blood parameters, oxidative stress balance and the presence of pathogens. Results showed better body condition in individuals associated with landfills compared to the ones feeding on natural resources, as well as better nutritional status, as indicated by higher levels of albumin, cholesterol, and triglycerides in plasma. As many pollutants have a pro-oxidant effect, we evaluated oxidative stress balance, with no differences in the indicators of damage except for methaemoglobin (metHb), significantly higher in nestlings associated with landfill-origin food. Regarding antioxidants, GSH was higher in nestlings associated with landfills, which may suggest a hormetic response induced potentially by the presence of pollutants in waste. Nestlings fed food from landfills also had a higher presence of Escherichia coli with a multiresistant phenotype to antibiotics. In conclusion, our results show that nestlings fed with a higher proportion of food from landfills present a better nutritional status and body condition than those fed with a higher proportion of natural diet, being the only indicators of negative effects of the use of this food resource the higher percentage of metHb in the peripheral blood and the presence of antibiotic-resistant E. coli., This work was partly supported by grant RTA2011-00111_C03-02 financed by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). We thank Pablo Camarero for his help during laboratory analysis with the HPLC. J.P.P was funded by a grant from the Complutense University of Madrid (CT45/15-CT46/15). MMH is currently funded by Junta de Comunidades de Castilla-La Mancha and the European Regional Development Fund (SBPLY/17/180501/000514). This paper is a contribution of J.I.A to project CGL2017-85637-P of the Spanish Ministry of Economy and Competitiveness.

Telomere length (TL) and shortening is increasingly shown to predict variation in survival and lifespan, raising the question of what causes variation in these traits. Oxidative stress is well known to accelerate telomere attrition in vitro, but its importance in vivo is largely hypothetical. We tested this hypothesis experimentally by supplementing white stork (Ciconia ciconia) chicks with antioxidants. Individuals received either a control treatment, or a supply of tocopherol (vitamin E) and selenium, which both have antioxidant properties. The antioxidant treatment increased the concentration of tocopherol for up to two weeks after treatment but did not affect growth. Using the telomere restriction fragment technique, we evaluated erythrocyte TL and its dynamics. Telomeres shortened significantly over the 21 days between the baseline and final sample, independent of sex, mass, size and hatching order. The antioxidant treatment significantly mitigated shortening rate of average TL (−31% in shorter telomeres; percentiles 10th, 20th and 30th). Thus, our results support the hypothesis that oxidative stress shortens telomeres in vivo., Diego San Mauro for his financial support for the telomere assays (Project CGL2017-85637-P financed by the Spanish Ministry of Economy and Competitiveness). Fieldwork for this study and oxidative stress assays were partly supported by project RTA2011-00111-C03-02 financed by the Spanish National Institute for Agricultural Research INIA. J.P.-P. was funded by a grant from the Complutense University of Madrid (CT45/15-CT46/15).

The objective of this study was to determine the prevalence and diversity of coagulase-negative staphylococci (CoNS) species from wild birds in Spain, as well as to analyze the antimicrobial resistance phenotype/genotype and the virulence gene content. During 2015&ndash, 2016, tracheal samples of 242 wild birds were collected in different regions of Spain for staphylococci recovery. The species identification was performed using MALDI-TOF. The antimicrobial resistance phenotype and genotype was investigated by the disk diffusion method and by PCR, respectively. The presence of the virulence genes lukF/S-PV, tst, eta, etb, etd and scn was investigated by PCR. Moreover, CoNS carrying the mecA gene were subjected to SCCmec typing. Of the tested animals, 60% were CoNS-carriers, and 173 CoNS isolates were recovered from the 146 positive animals, which belonged to 11 species, with predominance of S. sciuri (n = 118) and S. lentus (n = 25). A total of 34% of CoNS isolates showed a multidrug resistance phenotype, and 42 mecA-positive methicillin-resistant CoNS (MRCoNS) were detected. The isolates showed resistance to the following antimicrobials (percentage of resistant isolates/antimicrobial resistance genes detected): penicillin (49/ blaZ, mecA), cefoxitin (24/ mecA), erythromycin and/or clindamycin (92/ erm(B), erm(C), erm(43), msr(A), mph(C), lnu(A), lsa(B), vga(A) and sal(A)), gentamicin and/or tobramycin (5/ aac(6&prime, )-Ie-aph(2&Prime, )-Ia, ant(4&prime, )-Ia), streptomycin (12/str), tetracycline (17/ tet(K), tet(L), tet(M)), ciprofloxacin (4), chloramphenicol (1/ fexA), fusidic acid (86/ fusB, fusD) and trimethoprim&ndash, sulfamethoxazole (1/ dfrK). None of the isolates harbored the lukF/S-PV, eta, etb, etd and scn genes, but two S. sciuri isolates (1%) carried the tst gene. Wild birds are frequently colonized by CoNS species, especially S. sciuri. We identified scavenging on intensively produced livestock and feeding on landfills as risk factors for CoNS carriage. High proportions of MRCoNS and multidrug resistant CoNS were detected, which coupled with the presence of important virulence genes is of concern.

The poultry red mite (PRM), Dermanyssus gallinae, is a hematophagous ectoparasite considered as the major pest in the egg-laying industry. Its pesticide-based control is only partially successful and requires the development of new control interventions such as vaccines. In this study, we follow a vaccinology approach to identify PRM candidate protective antigens. Based on proteomic data from fed and unfed nymph and adult mites, we selected a novel PRM protein, calumenin (Deg-CALU), which is tested as a vaccine candidate on an on-hen trial. Rhipicephalus microplus Subolesin (Rhm-SUB) was chosen as a positive control. Deg-CALU and Rhm-SUB reduced the mite oviposition by 35 and 44%, respectively. These results support Deg-CALU and Rhm-SUB as candidate protective antigens for the PRM control., This work was partially supported by the European Cooperation in Science and Technology (COST), Improving current understanding and research for sustainable control of the poultry red mite Dermanyssus gallinae – COREMI project (Action FA1404). JFLB was supported by Ministerio de Ciencia, Innovación y Universidades (Spain), Doctorado Industrial contract (DI-14-06917) and Sabiotec SA. MV was supported by the University of Castilla- La Mancha (Spain).

The mosquito-borne West Nile virus (WNV) is a highly neurovirulent Flavivirus currently representing an emergent zoonotic concern. WNV cycles in nature between mosquito vectors and birds that act as amplifier hosts and play an essential role in virus ecology, being, thus, WNV a threat to many species. Availability of an efficient avian vaccine would benefit certain avian populations, both birds grown for hunting and restocking activities, as well as endangered species in captive breeding projects, wildlife reservations, and recreation installations, and would be useful to prevent and contain outbreaks. Avian vaccination would be also of interest to limit WNV spillover to humans or horses from susceptible bird species that live in urbanized landscapes, like magpies. Herein, we have addressed the efficacy of a single dose of a WNV recombinant subviral particle (RSP) vaccine in susceptible magpie (Pica pica). The protective capacity of the RSP-based vaccine was demonstrated upon challenge of magpies with 5 × 103 plaque forming units of a neurovirulent WNV strain. A significant improvement in survival rates of immunized birds was recorded when compared to vehicle-inoculated animals (71.4 vs. 22.2%, respectively). Viremia, which is directly related to the capacity of a host to be competent for virus transmission, was reduced in vaccinated animals, as was the presence of infectious virus in feather follicles. Bird-to-bird transmission was recorded in three of six unchallenged (contact) magpies housed with non-vaccinated WNV-infected birds, but not in contact animals housed with vaccinated WNV-infected magpies. These results demonstrate the protective efficacy of the RSP-based vaccine in susceptible birds against WNV infection and its value in controlling the spread of the virus., This work was supported by grants RTA2015-009 and E-RTA20017-003-C02 from INIA, and P2013/ABI-2906 and P2018/BAA-4370 from the Comunidad Autónoma de Madrid.

Esta monografía constituye la quinta publicación técnica del programa Migra de SEO/BirdLife (www. migraciondeaves.org)., [ES]: Esta publicación trata sobre la ecología del movimiento de la cigüeña blanca (Ciconia ciconia) a lo largo de todo el ciclo anual: temporadas de cría y de invernada y ambas migraciones entre estas zonas. Se trata de la quinta monografía del programa Migra (www.migraciondeaves.org) desarrollado por SEO/BirdLife desde 2011, cuyo objetivo es conocer los movimientos y la migración de las aves presentes en España, y que para el estudio de esta especie ha contado con la colaboración de la organización suiza Storch Schweiz, la Estación Biológica de Doñana (EBD-CSIC), el Instituto MaxPlanck de Ornitología en Radolfzell (Alemania) y el Grupo SaBio del IREC (CSIC-UCLM-JCCM). La cigüeña blanca es un ave de gran tamaño, muy planeadora y que depende de las corrientes térmicas para el vuelo. Hasta hace relativamente poco tiempo, era considerada un migrador transahariano obligado que solo se observaba en territorio ibérico durante la época de reproducción. Sin embargo, en las últimas décadas está cambiando su comportamiento migratorio, y en la actualidad se cuenta con un importante número de aves que invernan en España, tanto aves nativas invernantes como ejemplares de origen centroeuropeo. Durante la migración transahariana la península Ibérica representa un área de paso obligado para las cigüeñas blancas de Europa Occidental y centroeuropeas, que viajan hasta el área de Gibraltar para cruzar el Estrecho entre finales de agosto y septiembre, más tarde que las aves ibéricas. Una vez en África continúan la migración atravesando el desierto del Sahara hasta llegar al Sahel, donde se encuentra la principal área de invernada de la población occidental. En España, la población reproductora se distribuye fundamentalmente en la mitad occidental de la Península y en el valle del Ebro. Además, cría en Galicia y en localidades aisladas de la fachada cantábrica, pero falta en grandes áreas de la costa mediterránea y este de la meseta. El último censo nacional corresponde a 2004, cuando el tamaño de la población reproductora española se cifró en 33.217 parejas reproductoras y 31.229 ejemplares invernando., [EN]: The present publication describes the movement ecology of white storks (Ciconia ciconia) across the complete annual cycle: breeding and wintering seasons, and both migratory periods between wintering and breeding grounds. It is the fifth monograph published in the context of the Migra program (www.migraciondeaves.org/en/) run by SEO/BirdLife since 2011 in order to describe the movements and migratory routes of the avian species present in Spain. For this particular species SEO/BirdLife has received collaboration from the Swiss non-governmental organization Storch Schweiz, the Doñana Biological Research Station (EBD-CSIC), the Max-Planck Institute of Ornithology in Radolfzell (Germany), and the Health and Biotechnology Research Group of the Spanish Institute of Game and Wildlife Research (IREC, CSIC-UCLM-JCCM). The white stork is a large conspicuous soaring bird that depends on thermals and air currents for its flights. Until recently it was considered an obligatory trans-Saharan migrant that was only present in the Iberian Peninsula during the breeding season. However the white storks’ migratory behaviour has been changing throughout the past decades and at present a large number of birds winter in Spain, both native resident storks and birds from Central Europe that spend the winter in Spanish territory. During trans-Saharan migration the Iberian Peninsula constitutes an obligatory part of the route of white storks of Western and Central Europe that travel to Gibraltar and cross the strait between the end of August and September, later than storks from the Peninsula. Once in Africa these birds travel onwards crossing the Sahara to reach the Sahel zone, as sub-Saharan Africa is the primary wintering area of the western white stork population. In Spain the breeding population of white storks occupies predominantly the western half of the Iberian Peninsula and the Ebro river valley. In addition, they breed in Galicia and in the Cantabria part of the Atlantic coast, but are absent in large parts of the Mediterranean and the Eastern plateau. The most recent national census from 2004 noted 33,217 breeding pairs and 33,229 wintering individuals., Los datos recogidos en esta monografía se obtuvieron gracias a la financiación obtenida en el marco de los siguientes proyectos: • El Gobierno de La Rioja financió el marcado con emisor satélite GPS de un adulto de cigüeña blanca en La Rioja. • El Ayuntamiento de Vitoria-Gasteiz en el entorno de la “Green Capital”, el Gobierno Vasco y la Diputación Foral de Álava hicieron posible el trabajo de captura y marcaje de un adulto de cigüeña blanca en Álava. • Once de las cigüeñas adultas fueron marcadas con transmisor satélite en el marco de un proyecto para la determinación del papel de la cigüeña blanca en la epidemiología de los virus de influenza aviar de alta y baja patogenicidad, financiado por el Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (proyecto RTA2011-00088-C03-02), y desarrollado por el Grupo SaBio del IREC (CSIC-UCLM-JCCM). • El trabajo en el área de Doñana fue financiado por el Ministerio de Economía y Competitividad y la Comunidad Europea (proyecto CGL2012-32544 concedido a J. Blas), el Ministerio de Agricultura, Alimentación y Medio Ambiente (Organismo Autónomo Parques Nacionales, subvención 511/2012 a J. Blas), la Universidad de Saskatchewan (beca Isabel María López Martínez Memorial concedida a F.T-M), y el Consejo Superior de Investigaciones Científicas CSIC (contrato Ramón y Cajal 2010-07120 y proyecto I-LINK0564 a J. Blas). Estos proyectos permitieron financiar y reforzar las colaboraciones científicas entre la Estación Biológica de Doñana y el Instituto Max-Planck de Ornitología en Radolfzell (Alemania) para el estudio de la ecología de las cigüeñas en el sur de España. • La organización suiza Storch Schweiz ha financiado el marcaje de numerosas cigüeñas blancas en España y Centroeuropa dentro del proyecto conjunto “SOS Cigüeña-Migración en cambio” entre esta entidad y SEO/BirdLife. Los trabajos de captura, marcaje y seguimiento de las aves, y las labores de edición y maquetación de esta publicación se han llevado a cabo en el marco de este programa, cuyo principal financiador es la Fundación Iberdrola España.

More than 70% of new human pathogens are zoonotic and many originate from the wildlife reservoir. Wildlife rehabilitation centres (WRC) are an easily accessible source for sample and data collection for preventive surveillance, but data collected this way may be biased. We use white storks (Ciconia ciconia) as a model to compare pathogen prevalence obtained in the field and WRC. We address factors that may affect disease prevalence data like origin, the age group and the “diseased” state of WRC admissions. In this study we compared prevalence of Escherichia coli and Salmonella spp. in the digestive tract; antibodies against West Nile virus, avian influenza and Newcastle disease virus, and antimicrobial resistance patterns of E. coli between nestling and adult wild storks established in different habitats (n = 90) and storks admitted to two different WRC (n = 30) in the same region. When age groups and colonies of origin were disregarded, the mean enterobacteria (E. coli, Salmonella) and viral antibody prevalence of the wild population (n = 90) were similar to prevalence observed in the individuals admitted to WRC (n = 30). However, in fledgling juvenile storks admitted to WRC, the prevalence of Salmonella spp. (13.3%), E. coli showing resistance to cefotaxime (37.9%) and against two antimicrobials at once (41.4%) were more similar to the prevalence in stork nestlings from landfill-associated colonies (7.9%, 37.1% and 48.6%, respectively for prevalence of Salmonella spp. and E. coli displaying, cefotaxime resistance and resistance against two antimicrobials), and significantly higher than in colonies located in natural habitats (0%; 10.5% and 15.8%, respectively). Thus, pathogen surveillance in individuals from an abundant species admitted to WRC is useful to monitor overall mean prevalence, but for certain pathogens may not be sufficient to detect differences between local populations. In addition, the ecology of the tested species and the specific temporal, spatial and age group distribution of WRC admissions have to be taken into account., This work was funded by the projects: RTA2011-00111-C03-02 and ERTA2013-0013 funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, INIA. María Cruz Camacho is a fellow of the Junta de Comunidades de Castilla – La Mancha (JCCM).

These results were presented as oral exposition at the 27th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Vienna, April 2017., The aim of this study was to determine the carriage rate of coagulase-positive staphylococci (CoPS) in wild birds and to characterize recovered isolates. Tracheal samples from 324 wild birds, obtained in different Spanish regions during 2015–2016, were screened for CoPS carriage. The antimicrobial resistance profile and the virulence gene content were investigated. Molecular typing was performed by spa, agr, MLST, SCCmec, and S. delphini group classification. CoPS were recovered from 26 samples of wild birds (8.3%), and 27 isolates were further characterized. Two CoPS species were detected: S. aureus (n = 15; eight cinereous vultures and seven magpies) and S. delphini (n = 12; 11 cinereous vultures and one red kite). Thirteen S. aureus were methicillin-resistant (MRSA) and the remaining two strains were methicillin-susceptible (MSSA). Twelve MRSA were mecC-positive, typed as t843-ST1583/ST1945/ST1581/ST1571 (n = 11) and t1535-ST1945 (n = 1) (all of clonal-complex CC130); they were susceptible to the non-β-lactams tested. The remaining MRSA strain carried the mecA gene, was typed as t011-ST398-CC398-agrI-SCCmec-V, and showed a multiresistance phenotype. MSSA isolates were ascribed to lineages ST97-CC97 and ST425-CC425. All S. aureus lacked the studied virulence genes (lukS/F-PV, tst, eta, etb, and etd), and the IEC type E (with scn and sak genes) was detected in four mecC-positive and one MSSA isolates. S. delphini strains were methicillin-susceptible but showed resistance to at least one of the antimicrobials tested, with high penicillin (75%, with blaZ gene) and tetracycline [58%, with tet(K)± tet(L)] resistance rates. All S. delphini isolates presented the virulence genes lukS-I, siet, and se-int, and four carried the clindamycin-resistance lnu(A) gene., This work was supported by project SAF2016-76571-R from the Ministerio de Economía y Competitividad (MINECO) of Spain and FEDER. Laura Ruiz-Ripa has a pre-doctoral fellowship from the Universidad de La Rioja (Spain).

Wild birds often harbor infectious microorganisms. Some of these infectious microorganisms may present a risk to domestic animals and humans through spillover events. Detections of certain microorganisms have been shown to increase host susceptibility to infections by other microorganisms, leading to coinfections and altered host-to-host transmission patterns. However, little is known about the frequency of coinfections and its impact on wild bird populations. In order to verify whether avian influenza virus (AIV) natural infection in wild waterbirds was related to the excretion of other microorganisms, 73 AIV-positive samples (feces and cloacal swabs) were coupled with 73 AIV-negative samples of the same sampling characteristics and tested by real-time PCR specific for the following microorganisms: West Nile virus, avian avulavirus 1,Las aves silvestres frecuentemente albergan microorganismos infecciosos. Algunos suponen un riesgo por su posible transmisión a animales domésticos o representar un problema de salud pública si son zoonóticos. Se ha relacionado la detección de algunos microorganismos microbianos con una mayor susceptibilidad del hospedador a la infección por otros, llevando a una coinfección y a una alteración de los patrones de transmisión entre hospedadores. Sin embargo, poco se sabe sobre la frecuencia y el impacto de estas coinfecciones en la epidemiologia de las enfermedades en las aves silvestres. Con el ánimo de determinar si una infección natural con el virus de la influenza aviar (VIA) en aves acuáticas se relaciona con la excreción de otros microorganismos, se seleccionaron 73 muestras positivas a VIA y un número igual de negativas de similares características y se sometieron a análisis por PRC a tiempo real para la detección de los siguientes agentes: virus del Nilo occidental, avulavirus aviar de tipo 1

Preliminary results were presented at the 35th meeting of the Wildlife Fauna Ecopathology Study Group (Groupe d’Étude sur l’Ecopathologie de la Faune Sauvage de Montagne/Grupo de Estudios de Ecopatología de Fauna Salvaje de Montaña, GEEFSM), Cofrentes Muela de Cortes, Valencia, Spain, 1st–4th June 2017., In the period from October 2016 to February 2017, the urinary tracts of 390 wild boar (Sus scrofa) from four areas of south central Spain (102 from Doñana National Park; 150 from Sierra Morena and the Toledo Mountains; 84 from Sierra Nevada; 54 from Sierra de Cazorla, Segura y Las Villas Natural Park) were examined for the presence of adult specimens of Stephanurus dentatus (Nematoda: Strongyloidea). This parasite was only detected in the wild boar population of Doñana National Park, with high prevalence (76.5 ± 4.2%; 78/102), mean intensity (43.2 ± 4.4) and mean abundance (33.1 ± 3.8). Juvenile wild boar had significantly lower prevalence and abundance than subadult and adult wild boar. The intensity of infestation was significantly higher in male than in female wild boar. The detection of a focus of S. dentatus infestation in the wild boar population in Doñana National Park will provide further opportunities for understanding the epidemiology of this parasite., This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO; grant number AGL2016-76358-R). Pelayo Acevedo and Samantha Moratal were supported by the MINECO and the Universidad de Castilla-La Mancha (UCLM) through a ‘Ramón y Cajal’ contract (RYC-2012-11970) and a predoctoral fellowship (FPU16/02036), respectively.

Infectious and non-infectious diseases affect raptors around the globe. This chapter aims to describe and illustrate the most important disease entities in raptors. While anthropogenic accidental and malicious trauma and emaciation are the most common causes of death across species, malicious or inadvertent poisoning of vultures and other raptors and a variety of viral (e.g., avian pox, West Nile virus, and Newcastle’s disease) and protozoal diseases regularly cause disease and death in free ranging raptors. Prey is a common source of both toxins and infectious pathogens in these top predators. Furthermore, endoparasites and ectoparasites are common in free ranging raptors. Aspergillosis, gout, and staphylococcal pododermatitis are common in raptors in captivity; protozoal diseases of nonendemic raptors (e.g., malaria) and nutritional diseases including obesity and atherosclerosis are also frequent in the captive settings. Bacterial, congenital, and neoplastic diseases are of a sporadic nature.

Objectives: The objective of this study was to analyse the prevalence of tracheal carriage of Staphylococcus aureus/MRSA in storks and to study the resistance and virulence genes in the obtained isolates. Methods: Tracheal samples from 92 stork nestlings of two landfill-associated and two natural-habitat colonies were inoculated in specific media for S. aureus and MRSA recovery. Antimicrobial susceptibility was tested, and the presence of resistance, virulence and immune evasion cluster (IEC) genes was analysed by PCR. S. aureus isolates were characterized by spa and agr typing. Staphylococcal cassette chromosome (SCC) mec type was determined for mecC-positive isolates, and MLST was performed for 17 selected S. aureus isolates. Results: S. aureus isolateswere identified in 32/92 samples (34.8%), and 38 isolateswere recovered. The prevalence of S. aureus was higher in nestlings from landfills (24/43, 55.8%) than in those fromnatural habitats (8/49, 16.3%). Three birds from landfill-associated colonies carried MRSA, two with mecA-positive strains [clonal complex (CC) 5-spa-t002 and CC398-spa-t011] and one with a mecC-positive strain [sequence type (ST) 3061-CC130-spat843- agr-III-SCCmecXI). None of the MRSA isolates presented IEC genes. Thirty-five MSSA isolates, which showed 18 different spa types (ascribed to CC5, CC7, CC22, CC30, CC45, CC59, CC133 and CC398), were obtained. The agr types detected were I (63%), II (29%) and III (8%). Resistance and virulence genes identified in MSSA were blaZ (n=25), erm(T) (n=9), erm(A) (n=1), tet(M) (n=2), fexA (n=3), str (n=2), tst (n=2), eta (n=1) and cna (n=15). The IEC types B, C, D and G were found in MSSA isolates, and two new STs were identified (ST3060 and ST3061). Conclusions: White storks are frequently tracheal carriers of S. aureus, including ST398 isolates. MRSA isolates of lineages CC398-mecA and CC130-mecC were detected in storks from landfill-associated colonies exposed to human residues. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

Aquatic wild birds have been intensively studied to better understand their role in avian influenza virus (AIV) maintenance and spread. To date, AIV surveillance has primarily focused on natural aquatic environments where different bird species aggregate and viral survival is enhanced. However, artificial habitats such as landfills are attracting substantial numbers of wild birds, AIV reservoir species included. The use of landfills as a predictable food source has significantly influenced population size, migratory traits, and feeding behavior of white storks (Ciconia ciconia) and black-headed gulls (Chroicocephalus ridibundus) among others. Considering the proximity of landfills to urban settlements and frequently poultry-farms, targeted monitoring of AIV in bird species that forage at landfills but are known to also frequent urban and agricultural habitats could be a useful means for monitoring of AIV, especially during periods of bird aggregation. During the wintering season 2014-2015, the prevalence of AIV in five avian species at two landfills in South-Central Spain was explored by rRT-PCR and species related temporal variation in AIV prevalence determined. We collected and tested 1,186 fresh fecal samples from white storks (N = 689), cattle egrets (Bubulcus ibis, N = 116) and mixed flocks of gulls (N = 381) as well as cloacal and oral swabs from five birds found dead. Seven samples contained AIV, five from gulls and one each from a stork and a cattle egret. Overall, AIV prevalence was 0.60%. No significant temporal variation was observed in AIV prevalence. Prevalence differed significantly among the sampled taxonomic groups, being highest in gulls (1.31%). H16N3 subtype was detected from a cattle egret and H11N9 subtype from a white stork, whereas gulls harbored both subtypes in addition to H11N3 subtype. H16 subtype detection in a cattle egret evidences its host range may not be restricted to gulls. Our results indicate that wild birds foraging at landfills may carry different LPAIV subtypes., This work was funded by the RTA2011-00111-C03 grant, financed by the National Institute for Agricultural Science and Technology (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, INIA) of Spain. OT was recipient of an INIA predoctoral grant.

The poultry red mite (PRM), Dermanyssus gallinae, is a hematophagous ectoparasite of birds with worldwide distribution that causes economic losses in the egg-production sector of the poultry industry. Traditional control methods, mainly based on acaricides, have been only partially successful, and new vaccine-based interventions are required for the control of PRM. Vaccination with insect Akirin (AKR) and its homolog in ticks, Subolesin (SUB), have shown protective efficacy for the control of ectoparasite infestations and pathogen infection/transmission. The aim of this study was the identification of the akr gene from D. gallinae (Deg-akr), the production of the recombinant Deg-AKR protein, and evaluation of its efficacy as a vaccine candidate for the control of PRM. The anti-Deg-AKR serum IgY antibodies in hen sera and egg yolk were higher in vaccinated than control animals throughout the experiment. The results demonstrated the efficacy of the vaccination with Deg-AKR for the control of PRM by reducing mite oviposition by 42% following feeding on vaccinated hens. A negative correlation between the levels of serum anti-Deg-AKR IgY and mite oviposition was obtained. These results support Deg-AKR as a candidate protective antigen for the control of PRM population growth., This work was partially supported by COST Action FA 1404 (COREMI) (https://www.coremi.eu/home.html). JFLB was supported by Doctorado Industrial contract DI-14-06917 from Ministerio de Ciencia, Innovación y Universidades (Spain) and Sabiotec S.A. MV was supported by the Research Plan of the University of Castilla-La Mancha (Spain).

The objectives were to evaluate the presence of vancomycin-resistant enterococci with acquired (VRE-a) and intrinsic (VRE-i) resistance mechanisms in fecal samples from different wild animals, and analyze their phenotypes and genotypes of antimicrobial resistance. A total of 348 cloacal/rectal samples from red-legged partridges (127), white storks (81), red kites (59), and wild boars (81) (June 2014/February 2015) were inoculated in Slanetz-Bartley agar supplemented with vancomycin (4 μg/mL). We investigated the susceptibility to 12 antimicrobials and the presence of 19 antimicrobial resistance and five virulence genes. In addition, we performed multilocus sequence typing, detection of IS16 and studied Tn1546 structure. One VRE-a isolate was identified in one wild boar. This isolate was identified as Enterococcus faecium, harbored vanA gene included into Tn1546 (truncated with IS1542/IS1216), and belonged to the new ST993. This isolate contained the erm(A), erm(B), tet(M), dfrG, and dfrK genes. Neither element IS16 nor the studied virulence genes were detected. Ninety-six VRE-i isolates were identified (89 Enterococcus gallinarum and seven Enterococcus casseliflavus), with the following prevalence: red kites (71.2 %), white storks (46.9 %), red-legged partridges (7.9 %), and wild boars (4.9 %). Most E. gallinarum isolates showed resistance to tetracycline (66.3 %) and/or erythromycin (46.1 %). High-level resistance to aminoglycosides was present among our VRE-i isolates: kanamycin (22.9 %), streptomycin (11.5 %), and gentamicin (9.4 %). In general, VRE-i isolates of red kites showed higher rates of resistance for non-glycopeptide agents than those of other animal species. The dissemination of acquired resistance mechanisms in natural environments could have implications in the global spread of resistance with public health implications., This work was supported by Project SAF2012-35474 from the Ministerio de Economía y Competitividad (MINECO) of Spain and the Fondo Europeo de Desarrollo Regional (FEDER), the project POIC-2014-001-P of the regional government of Castilla–La Mancha, and by the project RTA2011-00111-C03-02 from the National Institute for Research in Agricultural and Alimentary Technology (INIA), by CDTI (Centro para el Desarrollo Tecnológico Industrial, MINECO). Carmen Lozano has a contract associated with Project SAF2012-35474.

This chapter intends to identify research priorities and current trends in wildlife diseases. This is an ever-growing field where veterinary science has important overlaps and interactions with many related scientific fields such as molecular biology, wildlife ecology, human medicine and animal science, among others. This emerging research field is in the core of the One Health idea., This is a contribution to EU-H2020 grant COMPARE (377/14) and to MINECO Spain Plan Nacional de I + D grant AGL2014-56305 and FEDER.

As a response to the need for improved and cost-efficient West Nile virus (WNV) and other flavivirus surveillance tools, we tested 887 juvenile free-living red deer, 742 free-living juvenile wild boar, and 327 farmed deer, to detect temporal variability in exposure to these viruses. Thirty of 742 wild boar samples (4%; 95% CI 2.8,5.7) yielded a positive ELISA result. Antibody-positive individuals had been sampled between 2003 and 2011 in localities from central and southern Spain. No wild boar from the northern half of Spain (n=120) tested positive. Regarding juvenile wild red deer, only two out of 887 samples yielded a positive ELISA result (0.2%; 95% CI 0.1,0.8). These two samples came from the same site and sampling year. The likelihood of detecting contact with WNV or cross-reacting flaviviruses was 18 times higher among juvenile wild boar than among juvenile red deer. ELISA positivity among farmed deer increased 10-fold after local flavivirus outbreaks recorded in the summer and autumn of 2010. This survey demonstrated the potential usefulness of juvenile wild ungulates, particularly wild boar, as suitable flavivirus sentinels in southwestern Europe, and that systematic serum banking of samples from hunter-harvested wildlife or from individual farmed ungulates provides valuable material for retrospective epidemiological surveys and future disease monitoring., This study was supported by POII09-0141-8176 (Castilla-La Mancha) and by JCCM PPIC10-0226-0243. The authors also acknowledge the support of the Ministerio de Medio Ambiente, Rural y Marino (MARM).

The Iberian Peninsula is located along the East Atlantic and Black Sea/Mediterranean flyways and is the third ranking European country as wintering quarter for wild migrating birds after Turkey and Rumania. For these reasons, Spanish wetlands are of importance in AIV surveillance, and of great interest for the study of the epidemiology of LPAIV under Mediterranean climate conditions. Nevertheless, information on prevalence of LPAIV viruses in Spain is still scarce and is restricted to two serological surveys carried out in the south of the country during 1990 and 1994 and one virological study performed recently in North East Spain. In the present study we analysed the prevalence of AIV circulating in wild birds in continental wetlands in central Spain and determined temporal, spatial and species variation. Real time RTPCR was performed on 1435 faecal samples and cloacal swabs from 54 species. An overall AIV prevalence of 2.6% was detected with a peak during November and December, when thousands of migrating wild birds arrive to Spain for wintering. Highest prevalence rates were detected in Phoenicopteriformes and Anseriformes. AIV prevalence obtained from cloacal swabs and fresh faeces did not vary significantly, which supports faecal sampling as an appropriate method for large scale LPAIV surveillance programs. Viral culture was achieved in samples obtained from two Mallards and a White stork, in which subtypes H7N9 and H11N9, respectively, were identified. Our results reflect a similar scenario in AIV epidemiology in small continental wetlands as compared to large coastal humid areas in Europe and underline the importance of including species such as flamingos and storks in surveillance programs, since their role in AIV ecology in these areas could be more important than previously considered., We are grateful to all the personnel of the Wildlife Rehabilitation Centres in Castilla-La Mancha for their collaboration and to all the people that helped in the field work. We also thank JCCM for support, Tomás Velasco for sharing his knowledge of wetlands and waterbirds in Central Spain and Vega Álvarez (NEIKER) for technical support. We wish to thank the “Spanish National Reference Laboratory” for official confirmation of isolates. This work is a contribution to grant INIA RTA2005-111-00, grant JCCM PA298-05 and grant MEC/INIA FAU2006-019-C03. E. Pérez-Ramírez currently has an I3p fellowship from the Spanish Science Council (CSIC).

Wild birds often harbor infectious microorganisms. Some of these infectious microorganisms may present a risk to domestic animals and humans through spillover events. Detections of certain microorganisms have been shown to increase host susceptibility to infections by other microorganisms, leading to coinfections and altered host-to-host transmission patterns. However, little is known about the frequency of coinfections and its impact on wild bird populations. In order to verify whether avian influenza virus (AIV) natural infection in wild waterbirds was related to the excretion of other microorganisms, 73 AIV-positive samples (feces and cloacal swabs) were coupled with 73 AIV-negative samples of the same sampling characteristics and tested by real-time PCR specific for the following microorganisms: West Nile virus, avian avulavirus 1, Salmonella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, Mycobacterium avium subspecies, Mycobacterium tuberculosis complex, and Mycobacterium spp. Concurrent detections were found in 47.9% (35/73) of the AIV-positive samples and in 23.3% (17/73) of the AIV-negative samples (P = 0.003). Mycobacterium spp. and Salmonella spp. were found to be significantly more prevalent among the AIV-positive samples than among the AIV-negative samples (42.9% vs. 22.8%; P = 0.024 and 15.2% vs. 0.0%; P = 0.0015, respectively). Prevalence of concurrent detections differed significantly among sampling years (P = 0.001), host families (P = 0.002), host species (P = 0.003), AIV subtypes (P = 0.003), and type of sample (P = 0.009). Multiple concurrent detections (more than one of the tested microorganisms excluding AIV) were found in 9.6% (7/73) of all the AIV-positive samples, accounting for 20% (7/35) of the concurrent detection cases. In contrast, in AIV-negative samples we never detected more than one of the selected microorganisms. These results show that AIV detection was associated with the detection of the monitored microorganisms. Further studies of a larger field sample set or under experimental conditions are necessary to infer causality in these trends.