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Lachryphagous males of Phortica variegata (Fallén, 1823) are gaining increasing attention in Europe, as they act as vectors of the nematode Thelazia callipaeda Railliet & Henry, 1910, causal agent of thelaziosis, an emergent zoonotic disease. Currently, there are no effective control strategies against the vector, and surveillance and monitoring rely on time-consuming and nonselective sampling methods. Our aim was to improve the knowledge about the population dynamics and the chemical ecology of the species. A total of 5,726 P. variegata flies (96.4% males and 3.6% females, mostly gravid) were collected in field experiments during June–September of 2020 in an oak forest in northern Spain. Our results indicate that 1) by means of sweep netting a significantly higher number of captures were found both around the collector´s body and in the air than at ground level; 2) a positive relationship was detected between the abundance of Phortica flies and temperature, with two significant peaks of abundance at 24 and 33°C; 3) the blend of red wine and cider vinegar was the most attractive bait; 4) yellow traps captured fewer flies compared to black and transparent traps; and 5) a significant reduction toward vinegar and wine was detected in presence of the phenolic monoterpenoid carvacrol. In addition, all the males (n = 690) analyzed by both molecular detection and dissection resulted negative for the presence of T. callipaeda larvae. Overall, these findings provide a better understanding of the vector in terms of monitoring and management strategies., Special thanks are given to the manager of the Izki Golf Club for providing the funding to complete part of the study. M.F. is currently funded by European Union Horizon 2020 Marie Sklodowska-Curie Actions (844285; ‘EpiEcoMod’). We also thank the anonymous reviewers for their many insightful comments and suggestions which helped us to significantly improve the manuscript. This study is part of a work awarded in 2021 with the "III Premio Nacional Felix Pérez y Pérez" of the Illustrious Official College of Veterinarians of Burgos, Spain.

Tabanids (Diptera: Tabanidae) are large haematophagous flies that cause both direct (by biting nuisance) and indirect (primarily by mechanical transmission of diseases) damage to host species. Research studies on this family have received little attention in some parts of Europe. Our aims were to characterise the species richness, abundance, and peak of activity of tabanid fly species in a region of Northern Spain. Home-made canopy traps, sweep nets, and Malaise traps were employed for the collection of tabanids across four cattle farms, two equestrian centres, and two golf courses during a 3-month period in the summer of 2020. A total of 300 specimens of 27 tabanid species belonging to eight genera were identified. The most prevalent species were Haematopota pluvialis (23.3%), Tabanus eggeri (20.0%), and Tabanus bromius (8.0%). The former species was recorded biting humans and therefore should be considered of relevance to public health. Tabanids were more diverse and abundant in scrubland and grazing pastures [relative abundance (RA) = > 10%; species richness (S) = 8–12; Shannon-Index (H´) = 1.5 − 2.1] compared to crop landscapes (RA =

Animal sanctuaries are important organisations that promote animal welfare and health as well as social awareness. Following the appearance of several donkeys with severe and distinctive skin lesions in a sanctuary from western Spain in 2021, a multicomponent study was performed to rule out the possible causes. The lesions were mainly concentrated on the extremities and, to a lesser extent, on the face and/or chest. The use of cotton leggings on their extremities as an external barrier to prevent them from bites and treatments with antiparasitic, antiseptic washes and dermatitis lotion showed to be effective measures to improve the donkey lesions. Skin scraping was negative for any relevant causative agents. Histopathological examination of the lesions showed an inflammatory infiltrate at the superficial dermal level, compatible with diffuse chronic dermatitis. A field entomological study was conducted from May to October 2021 to ascertain if any Diptera was responsible for these lesions. Considerable numbers of the stable fly Stomoxys calcitrans were recorded in both sticky traps and decomposing straw bedding. Several species of hematophagous Diptera were also recorded in lower numbers in other traps placed on the farm facilities. According to the entomological data, the location of the bites and clinical signs, it was concluded that S. calcitrans was the main cause of the skin lesions in the donkeys. To our best knowledge, this is the first clinical case of donkeys affected by the stable fly in Spain. For differential diagnosis, a brief discussion of the evidence caused by other biting Diptera groups is provided.

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.

An important element of vector control and surveillance of mosquito-borne diseases is updated information on vector species distribution. The aim of this study was to collect available information about mosquito species reported in Extremadura between 1920 and 2020 and create a catalog that would combine both published data and our recent field identifications. An exhaustive list is hereby presented, including species status and detailed distribution maps at a municipal level as well as their importance for public health. A total of 33 species, classified into five genera: Anopheles (five species), Aedes (14), Culex (nine), Culiseta (four), and Orthopodomyia (one) has been recorded, including 31 autochthonous, one invasive, Aedes (Stegomyia) albopictus, and one disappeared since 1953, Aedes (Stegomyia) aegypti. For the first time in Extremadura, we report the presence of important vectors such as Aedes (Aedimorphus) vexans vexans and Culex (Culex) perexiguus, and the new record of six species in the province of Badajoz, namely: Aedes (Dahliana) echinus, Aedes (Fredwardsius) vittatus, Aedes (Ochlerotatus) berlandi, Aedes (Ochlerotatus) pulcritarsis, Culex (Culex) mimeticus, and Culiseta (Culiseta) subochrea. Nineteen of these species are potential vectors of medical and veterinary relevance.

The large diversity of new tick-borne phleboviruses, and the negative impacts of the virulent viruses on human/animal health have led to a growing interest in their analysis. In this report, new insights are brought out into the diversity of putative phleboviruses circulating in Portugal (both the continental territory and the islands of Sao Miguel, in the Azores, and Madeira), as well as in the Spanish western regions of Extremadura and Castilla and Leon. Phlebovirus sequences were frequently detected (L-segment) from both questing and feeding ticks, but especially in Rhipicephalus sanguineus sensu lato (s.l.) specimens. These sequences were detected in adult ticks, as well as nymphs and eggs, supporting the hypothesis of viral maintenance by vertical transmission. Though multiple genetic groups could be identified in phylogenetic trees (AnLuc, KarMa, RiPar virus 1, and Spanish group 1 and 2), all the sequences from Portugal and Spain shared common ancestry with other viral sequence obtained from samples collected over a large geographic coverage. Spatiotemporal analysis placed Middle-East as the geographic origin of the most recent common ancestor (MRCA) of all phleboviruses analysed in the present study. More recent viral transitions might include migrations from Spain to continental Portugal, and from there to the Portuguese Islands. Our findings suggest that the time of the MRCA of phleboviruses was dated around 225 years ago [95% HPD: 124–387 year before the last sampling date].

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)

The detection of three Gongylonema sp. infective larvae in two specimens of the dung beetle Geotrupes mutator (Marsham, 1802) from western Spain is reported here for the first time in Europe. Scanning electron microscopy confirmed that the analyzed specimens belong to the genus Gongylonema, but it was not possible to determine the species identity by the lack of morphological information in the literature and because many of the phenotypic characteristics had not yet fully developed at this juvenile stage. Nevertheless, a phylogenetic analysis using amplified cox1 nucleotide sequences has revealed that the studied larvae could be clearly discriminated (< 89% identity) from all the other Gongylonema cox1 sequences available in public genetic databases. While our results are limited by the scarcity of genetic information available for this genus, the possibility that the analyzed specimens might correspond to a new species should not be ruled out, and more studies are needed. The results provided in this report indicate that G. mutator is involved in the transmission cycle of Gongylonema sp. to vertebrates in Europe., The Authors also appreciate the technical and human support provided by the facility of analysis and characterization of solids and surfaces of SAIUEx (financed by UEX, Junta de Extremadura, MICINN, FEDER, and FSE).

Phlebotomus (Transphlebotomus) mascittii Grassi, 1908 (Diptera: Psychodidae) has been found in several European countries. In Spain, sporadic records were reported in the early ’80s in Catalonia (Northeast Spain), and it was never detected again. Recent entomological surveys carried out between 2004 and 2020 revealed the presence of several specimens of P. mascittii in Spain. The species identification was confirmed by both morphological and molecular analyses. The analyzed specimens belonged to the haplotype (COI_2) defined by one polymorphic site compared to other European specimens. Phlebotomus mascittii was found in low population densities in rural areas associated with livestock farms and in an urban cemetery during the summer season. This study provides the first records of this species in various localities along the Cantabrian cornice (Northern Spain) and represents its westernmost observation in the Palearctic region. The implications of the finding of this uncommon species are discussed at different levels, with emphasis on its suspected role in the transmission of leishmaniosis.

Spirocerca lupi infection in dogs (Canis domesticus) is associated with esophageal lesions that may evolve to a neoplastic stage in the form of esophageal sarcoma. In the red fox (Vulpes vulpes) infected with the closely related Spirocerca vulpis, similar lesions may occur in the stomach, but neoplastic forms have not been reported. We characterize Spirocerca vulpis-induced lesions in the fox, using pathology and immunohistochemical (IHC) techniques. Seventy-one out of 163 Spirocerca vulpis-positive red foxes were selected and subjected to histopathological study. Lesions were classified as patchy or diffuse. Ten patchy and 10 diffuse lesion samples were studied using three IHC markers (CD68, CD3, and CD79α for macrophages, T lymphocytes, and B lymphocytes, respectively) and H&E stain for neutrophils and eosinophils. Intensity of necrosis, hemorrhages, and the presence of collagen was also analyzed. Of the S. vulpis-positive red foxes, 96.9% had S. vulpis nodules localized in the gastric area (wall and/or omentum), and 3.1% had nodules in the small intestine. All the samples had a moderate to severe lymphoplasmacytic infiltrate. Mild eosinophil infiltration was observed in both types of lesions, while neutrophil infiltration was significatively higher in the patchy than in the diffuse lesions. Fibrosis with mature collagen fibers was also predominant in the patchy lesions along with the presence of T lymphocytes and macrophages. Both the patchy and diffuse patterns had very few B lymphocytes. These findings suggest that the diffuse form is an earlier stage of the lesion, which eventually evolves into patchy forms. Neoplastic forms were not seen. Although more studies are necessary, this study describes the lesions, characterizes the inflammatory infiltrates, and establishes a possible evolution of the different pathological forms of S. vulpis infection in the red fox.

Changes in environmental conditions, whether related or not to human activities, are continuously modifying the geographic distribution of vectors, which in turn affects the dynamics and distribution of vector-borne infectious diseases. Determining the main ecological drivers of vector distribution and how predicted changes in these drivers may alter their future distributions is therefore of major importance. However, the drivers of vector populations are largely specific to each vector species and region. Here, we identify the most important human-activity-related and bioclimatic predictors affecting the current distribution and habitat suitability of the mosquito Culex pipiens and potential future changes in its distribution in Spain. We determined the niche of occurrence (NOO) of the species, which considers only those areas lying within the range of suitable environmental conditions using presence data. Although almost ubiquitous, the distribution of Cx. pipiens is mostly explained by elevation and the degree of urbanization but also, to a lesser extent, by mean temperatures during the wettest season and temperature seasonality. The combination of these predictors highlights the existence of a heterogeneous pattern of habitat suitability, with most suitable areas located in the southern and northeastern coastal areas of Spain, and unsuitable areas located at higher altitude and in colder regions. Future climatic predictions indicate a net decrease in distribution of up to 29.55%, probably due to warming and greater temperature oscillations. Despite these predicted changes in vector distribution, their effects on the incidence of infectious diseases are, however, difficult to forecast since different processes such as local adaptation to temperature, vector-pathogen interactions, and human-derived changes in landscape may play important roles in shaping the future dynamics of pathogen transmission. info:eu-repo/semantics/acceptedVersion

Leishmaniosis caused by Leishmania infantum is present in Mediterranean countries, with high prevalence in areas of the center and south of Spain. However, in some regions such as Extremadura (in southwest of Spain), data has not been updated since 1997. The aim of this work was (i) to provide information about the distribution of phlebotomine sand fly species in western of Spain (Extremadura region), (ii) to determine risk factors for the presence of sand fly vectors and (iii) to detect Leishmania DNA and identify blood meal sources in wild caught females. During 2012–2013, sand flies were surveyed using CDC miniature light-traps in 13 of 20 counties in Extremadura. Specimens were identified morphologically and females were used for molecular detection of Leishmania DNA by kDNA, ITS-1 and cyt-B. In addition, blood meals origins were analyzed by a PCR based in vertebrate cyt b gene. A total of 1083 sand flies of both gender were captured and identified. Five species were collected, Phlebotomus perniciosus (60.76%), Sergentomyia minuta (29.92%), P. ariasi (7.11%), P. papatasi (1.48%) and P. sergenti (0.74%). The last three species constitute the first report in Badajoz, the most southern province of Extremadura region. Leishmania DNA was detected in three out of 435 females (one P. pernicious and two S. minuta). Characterization of obtained DNA sequences 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, rabbits, horses, donkeys and turkeys. The simultaneous presence of P. perniciosus and P. ariasi vectors, the analysis of blood meals, together with the detection of L. infantum and in S. minuta of L. tarentolae, confirms the ideal conditions for the transmission of this parasitosis in the western of Spain. These results improve the epidemiological knowledge of leishmaniosis and its vectors in this part of Spain, highlighting the need for ongoing entomological and parasitological surveillance.

The main aim of this study was to not only establish the prevalence of the recently described Spirocerca vulpis parasite in the wild-life cycle of carnivores in western Spain but to also elaborate a model to explain the risk of infestation based on 16 topo-climatic and habitat variables. During the period from June 2016 to November 2017, 1644 carcasses of red foxes (Vulpes vulpes) and another 105 wild mammals, legally hunted or killed in car accidents, were analyzed. Parasitic nodules of Spirocerca were found in 6% of the foxes, and the molecular analyses established a homology of our samples with the species S. vulpis. There were no differences in the occurrence of the infestation between sexes, but there were differences in terms of age, such that infestation was proportionally more frequent among young individuals. In terms of temporality, a higher percentage of positive cases was observed during the late-autumn and winter months, especially between December and February. This study provides new data on the factors that predispose S. vulpis infection in the red fox. Model results indicate that a spatial pattern exists in the occurrence and prevalence of this species in the studied area (higher probabilities to the west), and that this pattern seems to mainly be associated with topo-climatic variables., This research did notreceive any specific grant from funding agencies of the public, commercial, or not-for-profit sectors.

Estudio financiado por la Junta de Extremadura y el Fondo Euro-peo FEDER “Una manera de hacer Europa”, mediante el proyectocon ref. IB16135.

A study was performed to evaluate the implication of Protophormia terraenovae larvae as a surgical therapy for wounded skin. Three groups of sheep (n = 25) were considered based on larval doses. Groups 1 and 2 were artificially infested with low and high concentrations of L1 stage P. terraenovae, respectively, and group 0 served as a control. Skin biopsies were taken at 4 and 14 d postinfestation (D.P.If). A histopathological study was carried out to evaluate the lesions with a score, numbers of eosinophils and mast cells, and an immunohistochemical analysis of CD3, CD79α, and CD68 as T lymphocytes, B lymphosytes, and macrophages, respectively. The results indicated that higher larval doses led to faster regeneration by 14 D.P.If. Furthermore, the higher larval doses showed a high number of the CD68 marker and eosinophils and a low number of CD3 and CD79α markers and mast cells. In addition, the number of mast cells, T lymphocytes, and macrophage markers increased when the lesion progressed; however, a low number of immunolabeled CD79α cells and eosinophils were observed. The results indicate a possible positive effect of larvae in the healing of certain wounds.

Dirofilaria immitis is one of the most frequently detected mosquito-transmitted zoonotic filarioid nematode in mammals in Europe, being canine dirofilariosis a major animal health problem, endemic in the Mediterranean area. This study, focused on Southwest Spain, in order to bring new insights into (i) the epidemiology of Dirofilaria spp., (ii) the species of Culicid vectors possibly involved in their transmission and (iii) the genetic variability of those potential vectors. A total of 881 adult female mosquitoes from 11 different species, were captured during 2012-2013, and detection of filarioid DNA was attempted by PCR using specific primers (ITS-2 and COI), followed by DNA sequencing. In a single Culex pipiens specimen D. immitis DNA was detected both in the head-thorax and abdomen sections. Filarioid nematode DNA was also detected in eight additional Cx. pipiens specimens also in both the thorax and the abdomen, but analysis of sequence data did not allow unambiguous assignment of any of the obtained sequences to a previously defined species. All Cx. pipiens with filarioid DNA were individually analysed by CQ11 to discriminate between pipiens, molestus, and hybrid forms. Besides, rDNA ITS-2 sequence analysis revealed the presence of haplotype H1 and H2 of Cx. pipiens. To our knowledge this study revealed, 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.

Phlebotomine sand flies (Diptera, Psychodidae) are vectors of multiple Leishmania species, among which Leishmania infantum stands out as a being frequently pathogenic to humans and dogs in Mediterranean countries. In this study, Sergentomyia minuta sand flies were collected using CDC miniature light traps in different 431 biotopes from Southwest Spain. A total of 114 females were tested for the presence of Leishmania DNA by targeting ITS-1 and cyt-B sequences by PCR. Leishmania DNA was detected in one S. minuta. Characterization of the obtained DNA sequences by phylogenetic analyses revealed close relatedness with Leishmania tarentolae Wenyon, 1921 as well as with both human and canine pathogenic strains of Asian origin (China), previously described as Leishmania sp. To our knowledge, this is the first report of phlebotomine sand flies naturally infected with L. tarentolae-like in Spain. The possible infection of sand flies with novel Leishmania species should be taken into consideration in epidemiological studies of vector species in areas where leishmaniosis is endemic.

Background Culex univittatus and Culex perexiguus mosquitoes (Diptera: Culicidae) are competent arbovirus vectors, but with unclear morphological differentiation. In Europe, and in the Iberian Peninsula in particular, the presence of either or both species is controversial. However, in order to conduct adequate surveillance for arboviruses in this region, it is crucial to clarify whether Cx. univittatus is present or not, as well as to critically assess existing differentiation tools. This study aimed to clarify this situation, by morphological and molecular phylogenetic comparison of Iberian specimens deemed as Cx. univittatus, with others of South African origin, i.e. from the type-locality region. Methods Thus, morphological characteristics useful to distinguish both species, such as midfemur pale line, hindfemur R ratio, seta g R1 ratio, seta f shape, length of ventral arm of phalosome and number of setae on IX tergal abdominal segment, were observed. A phylogenetic analysis based on cox1 mtDNA, of which there were no sequences from Cx. univittatus yet available in the GenBank database, was performed. Results This analysis showed that Iberian and South African specimens are morphologically similar, except for the length of the ventral arm of the phalosome, which was higher in the Iberian specimens. Although the Iberian specimens could not be accurately identified using BOLD Systems, phylogenetic analysis still grouped these closer to South African Cx. univittatus, than to Cx. perexiguus from Turkey and Pakistan, despite the observed segregation of both taxa as two individual monophyletic clusters with shared common ancestry. Conclusions This survey demonstrates that the West Nile virus vector Cx. univittatus is present in the Iberian Peninsula. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1877-7) contains supplementary material, which is available to authorized users.

This study updates the diversity, distribution, and seasonal trends of mosquitoes in a western region of Spain, assesses ecological determinants of Culex pipiens s.l., and determines form composition of Cx. pipiens s.s. populations. A total of 1,495 mosquitoes of 16 species was collected during 2012–2013, of which Cx. pipiens s.l. and Cx. theileri were the most abundant. Five new records for An. maculipennis s.s., Orthopodomyia pulcripalpis, Aedes (Ochlerotatus) punctor, Cx. europaeus, and Cx. modestus were found for this region. Cx. pipiens density varied across weather and habitat patterns, correlating positively with high temperatures and with a preference for urbanized areas and rural areas within a proximity of ovine farms. Moreover, molecular identification by CQ11FL was performed in 467 Cx. pipiens s.s., detecting both pipiens (66%) and molestus (8.4%) forms coexisting in different habitats (urban, peri-urban and rural) aboveground with a high degree of hybridization (25.7%). The abundance of Cx. pipiens in urban areas and farms, with the presence of hybrids, may increase their capacity to act as bridge vectors for the transmission of arboviral infections. These data will be helpful for further implementation of entomological programs focused on risk assessment for arboviruses or other mosquito-borne pathogens.