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Este estudio actualiza la diversidad, distribución y fenología de mosquitos y flebotomos en Extremadura (España), así como con la detección en los mismos de ADN de Dirofilaria y Leishmania, respectivamente. Un total de 1495 mosquitos de 16 especies y 1095 flebotomos de 5 especies fueron capturados durante 2012-2013, utilizando trampas CDC en 13 de 20 comarcas. Se encontraron cinco nuevos registros de mosquitos para esta región a saber: An. maculipennis s.s., Orthopodomyia pulcripalpis, Aedes (Ochlerotatus) punctor, Cx. europaeus, y Cx. modestus; mientras que para flebotomos, Phlebotomus ariasi, P. papatasi, and P. sergenti constituyen el primer informe en Badajoz. Además, este estudio demuestra mediante estudios morfológicos y filogenéticos comparativos, que Cx. univittatus, vector del virus del Nilo Occidental, está presente en la península ibérica. Además, en un único ejemplar de Culex pipiens, fue detectado ADN de Dirofilaria immitis tanto en las secciones de cabeza-tórax y abdomen, así como de ADN de filáridos de especie desconocida en ocho especímenes adicionales de Cx. pipiens, revelando, por primera vez en España, la ocurrencia probable de infección madura de D. immitis en este mosquito, así como también de estos nematodos no caracterizados, apoyando su papel potencial como vector de estos parásitos. Por otra parte, la caracterización de las secuencias de ADN de Leishmania obtenidas en flebotomos, reveló una estrecha relación con L. tarentolae y L. infantum en S. minuta y en P. perniciosus, respectivamente. El estudio de preferencias hemáticas mostró una amplia gama de hospedadores, lo que indica un comportamiento oportunista de los flebotomos., This study updates the diversity, distribution, and seasonal trends of mosquitoes and sand flies in Extremadura (Spain), with detection of Dirofilaria and Leishmania DNA, respectively. A total of 1,495 mosquitoes of 16 species and 1095 sand flies of 5 species were captured during 2012-2013 using CDC miniature light-traps in 13 of 20 counties. Five new records of mosquitoes were found for this región: An. maculipennis s.s., Orthopodomyia pulcripalpis, Aedes (Ochlerotatus) punctor, Cx. europaeus, and Cx. modestus; while for sand flies, Phlebotomus ariasi, P. papatasi, and P. sergenti constitute the first report of sand flies species in Badajoz, the most southern province of Extremadura region. In addition, this survey demonstrates, by morphological and molecular phylogenetic comparison, that the West Nile virus vector Cx. univittatus is present in the Iberian Peninsula. Forthermore, in a single Culex pipiens specimen D. immitis DNA was detected both in the head-thorax and abdomen sections, and Filarioid nematode DNA was also evidenced in eight additional Cx. pipiens specimens, revealing, for the first time in Spain, the occurrence of likely mature infection of D. immitis in Cx. pipiens, as well as with other yet uncharacterized nematodes, supporting its role as a potential vector of these filarids. On the other hand, characterization of obtained Leishmania DNA sequences in sand flies by phylogenetic analyses revealed close relatedness with Leishmania tarentolae in S. minuta and L. infantum in P. perniciosus. Haematic preferences showed a wide range of hosts, namely: swine, humans, sheep, chickens, horses, donkeys and turkeys.

Recientemente se ha determinado el asentamiento de la mosca invasora Protophormia terraenovae en condiciones naturales en Extremadura. Por tanto, quisimos determinar si esta especie es capaz de provocar miasis traumáticas en la ganadería ovina en las condiciones climáticas de la región. Para ello, se realizó una infestación experimental con distintas dosis de L1 de esta especie (obtenidas del asticot comercializado en tiendas de pesca) en ovejas merinas, para comprobar la patogenicidad de las mismas en el desarrollo de la posible miasis. Los resultados obtenidos determinaron que, en las condiciones experimentales de este proyecto, las L1 de P. terraenovae no provocaron una miasis traumática propiamente dicha, ya que no se produjeron alteraciones patológicas reseñables ni a nivel sistémico ni a nivel local. Más bien, aquellas larvas que fueron capaces de desarrollarse en la zona de la lesión provocaron una reducción significativa del número de bacterias y del tejido necrótico en esta localización, con lo que estos resultados incitan a pensar en el uso de estas larvas para la larvoterapia animal, previa esterilización y control de cría de las mismas. Recently the establishment of the invasive fly Protophormia terraenovae in natural conditions in Extremadura has been confirmed. Therefore, we wanted to determine if this species is able to provoke traumatic myiasis in the ovine hut in the climatic conditions of the region. For this, an experimental infestation with different doses of L1 of this species (obtained from the asticot marketed in fishing shops) was carried out in merino sheep, to verify their pathogenic influence in the development of the possible myiasis. The results determined that, in the experimental conditions of this project, the L1 of P. terraenovae did not provoke a traumatic myiasis, since pathological alterations were observed neither at the systemic level nor at the local one. Rather than that, those larvae that were able to develop in the lesion area, caused a significant reduction in the number of bacteria and necrotic tissue at this location, so these results suggest the use of these larvae for maggot debridement therapy, prior sterilization and breeding control of the same ones.

This work was funded by Consejería de Economía e Infraestructura of the Junta de Extremadura and the European Regional Development Fund, a Way to Make Europe, through the research project (grant numbers IB20089 and IB16135), and PR (19_ECO_0070) project Aves y Enfermedades Infecciosas Emergentes: Impacto de las Especies Ex´oticas y Migratorias en la transmisi´on de Malaria aviar y el virus del Nilo Occidental - AvEIEs from Ayudas Fundaci´on BBVA a Equipos de Investigaci´on Científica 2019. CISA (INIA-CSIC) was funded by INIA projects number ERTA2015– 00002-CO2–01 and AEI PID2020–116768RR-C21. MF was supported by a Juan de la Cierva 2017 Formaci´on contract (FJCI- 2017–34394) from the Ministry of Science, Innovation and Universities, and she is currently funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie (grant agreement No 844285, ‘EpiEcoMod’). JM was supported by a postdoctoral grant from the Juan de la Cierva Subprogram (FJCI- 2017–34109), with the financial sponsorship of the MICINN. FG-C is supported by Ayudas a la Formaci´on Investigador Predoctoral 2018 (PD18056) from the Extremadura Government. PA-S is supported by an FPI-SGIT predoctoral contract from INIA/CISA., We explore the presence of zoonotic flaviviruses (West Nile virus (WNV) and Usutu virus (USUV)) neutralizing antibodies in rarely studied passerine bird species. We report, for the first time in Europe, WNV-specific antibodies in red avadavat and cetti’s warbler, and USUV in yellow-crowned bishop. The evidence of WNV and USUV circulating in resident and migratory species has implications for both animal and public health. Future outbreaks in avian reservoir hosts may occur and passerines should be considered as priority target species in flavivirus surveillance programmes., Consejería de Economía e Infraestructura of the Junta de Extremadura and the European Regional Development Fund IB20089 IB16135 PR (19_ECO_0070), INIA projects number ERTA2015– 00002-CO2–01 AEI PID2020–116768RR-C21, Juan de la Cierva 2017 Formaci´on contract (FJCI- 2017–34394), European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie (grant agreement No 844285, ‘EpiEcoMod’), Juan de la Cierva Subprogram (FJCI- 2017–34109), Ayudas a la Formaci´on Investigador Predoctoral 2018 (PD18056), FPI-SGIT predoctoral contract from INIA/CISA

13 Pág. Centro de Investigación en Sanidad Animal (CISA), West Nile virus (WNV) is a mosquito-borne emerging virus in Europe with capacity to cause neurological complications such as encephalitis or meningoencephalitis in humans, birds or equids. In Spain, WNV is actively circulating in mosquitoes, birds and horses in different regions, but never has been deeply studied in Extremadura. Therefore, the aim of this study was to evaluate the seroprevalence of WNV in equids of those areas and to analyse the risk factors associated with exposure to the virus. A total of 199 out of 725 equids presented antibodies against WNV by competition ELISA (27.45%), while 22 were doubtful (3.03%). Anti-WNV IgM antibodies were detected in 16 equids (2.21%), and 3 animals were doubtful (0.41%). All ELISA-reactive positive/doubtful sera (N = 226) were further tested by micro-virus neutralization test (VNT), and a total of 143 horses were confirmed as positive for WNV, obtaining a seroprevalence of 19.72% in equids of western Spain. In addition, specific antibodies against USUV were confirmed in 11 equids. In 24 equids, a specific flavivirus species (detected by ELISA test) could not be determined. The generalized linear mixed-effects models showed that the significant risk factors associated with individual WNV infection in equids were the age (adults) and hair coat colour (light), whereas in USUV infections, it was the breed (pure). Data demonstrated that WNV and USUV are circulating in regions of western Spain. Given the high WNV seroprevalence found in equids from the studied areas, it is important to improve the surveillance programmes of public health to detect undiagnosed human cases and to establish a vaccination programme in equid herds in these regions., This research was supported financially by grant IB16135 funded by the Regional Ministry of Economy and Infrastructure, Extremadura Government and the European Regional Development Fund ‘A way to make Europe’ and grant E-RTA2015-00002-C02-00 funded by INIA. FG is supported by Ayudas a la Formación Investigador Predoctoral 2018 Formation contract (PD18056) from the Extremadura Government. PAS is funded by a FPI predoctoral fellowship from INIA. MF is currently funded by a Juan de la Cierva 2017 Formación contract (FJCI-2017-34394) from the Ministry of Science, Innovation and Universities.

10 Pág. Centro de Investigación en Sanidad Animal (CISA), West Nile virus (WNV) is an emerging flavivirus transmitted generally by mosquitoes of Culex genus. It is maintained in an enzootic life cycle where birds act as reservoir hosts. Humans and horses are also susceptible to infection, and occasionally, they suffer from neurological complications. However, they do not transmit the virus to other vectors, behaving as dead-end hosts. Sporadic WNV outbreaks observed in horses and wild birds from Extremadura (western Spain) during 2016 and 2017 seasons prompted to carry out this survey in wild birds, focused on specimens coming from two wildlife rehabilitation centres. Between October 2017 and December 2019, samples from 391 wild birds, belonging to 56 different species were collected and analysed in search of evidence of WNV infection. The analysis of serum samples for WNV-specific antibodies by ELISA, whose specificity was subsequently confirmed by virus-neutralisation test (VNT) showed positive results in 18.23 % birds belonging to 18 different species. Pelecaniformes (33.33 %), Accipitriformes (25.77 %) and Strigiformes (22.92 %) orders had the higher seroprevalences. Remarkably, WNV-specific antibodies were found in a black stork for the first time in Europe. Analysis by real time RT-PCR in symptomatic birds confirmed the presence of WNV lineage 1 RNA in griffon vulture and little owls. Specificity analysis of ELISA positive and doubtful sera was performed by differential VNT titration against WNV and two other cross-reacting avian flaviviruses found in Spain: Usutu virus (USUV) and Bagaza virus (BAGV). Only four samples showed USUV-specific antibodies (1.04 %) corresponding to three species: Eurasian eagle-owl, griffon vulture and great bustard (first detection in Europe) whereas no samples were found reactive to BAGV. Differential VNT yielded undetermined flavivirus result in 16 samples (4.17 %). This is the first study carried out on wild birds from Extremadura (western Spain). It highlights the widespread circulation of WNV in the region and its co-circulation with USUV., Research funded by Consejería de Economía e Infraestructura of the Junta de Extremadura and the European Regional Development Fund, a Way to Make Europe, through the research project (grant numbers IB16135 and GR18148) and the National Institute for Agricultural and food Research and Technology (INIA-CISA) (grant number E-RTA2015-00002-C02-00)

17 Pág., Phytoestrogens (PE) are compounds found in plants such as soy (isoflavones), flax seeds and cereals (lignans) and pomegranates (ellagitannins). PE have shown estrogenic/antiestrogenic, antioxidant, anti-inflammatory, antineoplastic and apoptotic activities. The human studies are showing promising although inconsistent results about the beneficial effects of PE on ameliorating the menopausal symptoms or reducing the risk of certain cancers, cardiovascular disease or diabetes. The effects of PE on the organism are mediated by the intestinal microbiota, which transforms them into bioactive PE such as genistein, equol, enterolignans and certain urolithins. In this work, we review the most recent findings about the bacteria able to metabolize PE, together with the latest studies on the effects of PE on health. In addition, we describe the possible factors hindering the demonstration of the beneficial effect of PE on health, evincing the importance of measuring the actual circulating PE in order to encompass the variability of PE metabolism due to the intestinal microbiota. With this in mind, we also explore an approach to ensure the access to bioactive PE., This work was supported by projects RM2012-00004-00-00 and RTA2017-00002-00-00 from the Ministry of Science, Innovation and Universities (Spain)