Warm winters are associated to more intense West Nile virus circulation in southern Spain.

West Nile virus (WNV) is the most widely distributed mosquito-borne flavivirus in the world. This flavivirus can infect humans causing in some cases a fatal neurological disease and birds are the main reservoir hosts. WNV is endemic in Spain, and human cases have been reported since 2004. Although different studies analyse how climatic conditions can affect the dynamics of WNV infection, very few use long-term datasets. Between 2003 and 2020 a total of 2,724 serum samples from 1,707 common coots (Fulica atra) were analysed for the presence of WNV-specific antibodies. Mean (SD) annual seroprevalence was 24.67% (0.28) but showed high year-to-year variations ranging from 5.06% (0.17) to 68.89% (0.29). Significant positive correlations (p < 0.01) were observed between seroprevalence and maximum winter temperature and mean spring temperature. The unprecedented WNV outbreak in humans in the south of Spain in 2020 was preceded by a prolonged period of escalating WNV local circulation. Given current global and local climatic trends, WNV circulation is expected to increase in the next decades. This underscores the necessity of implementing One Health approaches to reduce the risk of future WNV outbreaks in humans. Our results suggest that higher winter and spring temperatures may be used as an early warning signal of more intense WNV circulation among wildlife in Spain, and consequently highlight the need of more intense vector control and surveillance in human inhabited areas.

Landscape and mosquito community impact the avian Plasmodium infection in Culex pipiens.

Avian malaria parasites provide an important model for studying host-pathogen interactions, yet understanding their dynamics in vectors under natural conditions is limited. We investigated the effect of vector abundance, species richness and diversity, and habitat characteristics on avian Plasmodium prevalence and lineage richness in Culex pipiens across 45 urban, natural, and rural localities in southern Spain. Analyzing 16,574 mosquitoes grouped in 768 mosquito pools, 32.7% exhibited parasite presence. 13 different Plasmodium lineages were identified, with the lineage SYAT05 being the most commonly found. Parasite prevalence positively correlated with the distance to saltmarshes and rivers, but negatively with the distance to total water source. Parasite lineage diversity was higher in natural than in rural areas and positively correlated with mosquito species richness. These results emphasize the complex dynamics of avian Plasmodium in the wild, with habitat characteristics and vector community driving the parasite transmission by mosquito vectors.

The prohibition of recreational hunting of wild ungulates in Spanish National Parks: Challenges and opportunities.

A new regulation has led to the prohibition of recreational hunting on estates located within Spanish National Parks (NPs). Before the ban, eleven NPs in Spain had already reported negative ecological consequences associated with high densities of wild ungulates. The new situation that has occurred after the ban signifies that policies with which to control populations of wild ungulates in NPs, most of which do not have a sufficient natural capacity to regulate populations, depend exclusively on the parks' authorities. The banning of recreational hunting implies a series of social, ecological, economic and logistic challenges. The control of wild ungulate populations in NPs requires: i) the legal basis for culling; ii) social acceptance as regards removing animals and the extractive procedures employed in NPs; iii) the long-term monitoring of wild ungulates and the damages that they cause, and iv) sufficient financial and human resources. A more integrated management and policy plan is, therefore, required, which should be supported by two pillars: i) the sustainability of natural resources and the conservation of functional environments, and ii) providing society with explanations regarding the need to manage wild ungulates. In order to bridge the potential gap between these key pillars, it is important to involve stakeholders in the decision-making processes concerning wild ungulate management. The forthcoming changes in Spanish NPs provide a promising opportunity to make a substantial improvement to wild ungulate management in these protected areas. This management approach could, moreover, serve as an example and be transferred to other protected spaces.

Long-term serological surveillance for West Nile and Usutu virus in horses in south-West Spain.

West Nile virus (WNV) is a re-emerging zoonotic pathogen with increasing incidence in Europe, producing a recent outbreak in 2020 in Spain with 77 human cases and eight fatalities. However, the factors explaining the observed changes in the incidence of WNV in Europe are not completely understood. Longitudinal monitoring of WNV in wild animals across Europe is a useful approach to understand the eco-epidemiology of WNV in the wild and the risk of spillover into humans. However, such studies are very scarce up to now. Here, we analysed the occurrence of WNV and Usutu virus (USUV) antibodies in 2102 samples collected between 2005 and 2020 from a population of feral horses in Doñana National Park. The prevalence of WNV antibodies varied between years, with a mean seroprevalence of 8.1% (range 0%-25%) and seasonally. Climate conditions including mean minimum annual temperatures and mean rainy days per year were positively correlated with WNV seroprevalence, while the annual rainfall was negatively. We also detected the highest incidence of seroconversions in 2020 coinciding with the human outbreak in southern Spain. Usutu virus-specific antibodies were detected in the horse population since 2011. The WNV outbreak in humans was preceded by a long period of increasing circulation of WNV among horses with a very high exposure in the year of the outbreak. These results highlight the utility of One Health approaches to better understand the transmission dynamics of zoonotics pathogens.

The local skin cellular immune response determines the clinical outcome of sarcoptic mange in Iberian ibex (Capra pyrenaica).

Introduction: Sarcoptic mange, caused by Sarcoptes scabiei, is a disease with implications for wildlife conservation and management. Its severity depends on the host's local skin immune response, which is largely unknown in Iberian ibex (Capra pyrenaica), a mountain ungulate dramatically affected by mange. In this species, the clinical outcome of sarcoptic mange varies among individuals, and the local immune response could be key to controlling the infestation. This study aims to characterize the local cellular immune response and its relationship with the clinical outcome. Methods: Fourteen Iberian ibexes were experimentally infested with S. scabiei and six more served as controls. Clinical signs were monitored, and skin biopsies were collected from the withers at 26, 46, and 103 days post-infection (dpi). The presence and distribution of macrophages (including M1 and M2 phenotypes), T lymphocytes, B lymphocytes, plasma cells, and interleukine 10 were quantitatively evaluated using immunohistochemical techniques. Results: An inflammatory infiltrate that decreased significantly from 26 to 103 dpi was observed in all the infested ibexes. The predominant inflammatory cell population in the skin of the mangy ibexes was formed by macrophages (mainly the M2 phenotype) followed by T lymphocytes, with lower numbers of B lymphocytes and plasma cells. Three clinical courses were identified: total recovery, partial recovery, and terminal stage. The inflammatory infiltrates were less pronounced in the fully recovered ibexes than in those that progressed to the terminal stage throughout the study. Discussion: The results suggest an exacerbated but effective Th1-type cellular immune response controlling mange in Iberian ibex. Furthermore, the local immune response appears to determine the variability of the clinical responses to S. scabiei infestation in this species. This first report on the progression of local skin immune cells is relevant not only for individuals but also for population management and conservation.

Genomic Analysis of West Nile Virus Lineage 1 Detected in Mosquitoes during the 2020-2021 Outbreaks in Andalusia, Spain.

Emerging infectious diseases are one of the most important global health challenges because of their impact on human and animal health. The vector-borne West Nile virus (WNV) is transmitted between birds by mosquitos, but it can also infect humans and horses causing disease. The local circulation of WNV in Spain has been known for decades, and since 2010, there have been regular outbreaks in horses, although only six cases were reported in humans until 2019. In 2020, Spain experienced a major outbreak with 77 human cases, which was followed by 6 additional cases in 2021, most of them in the Andalusian region (southern Spain). This study aimed to characterize the genomes of the WNV circulating in wild-trapped mosquitoes during 2020 and 2021 in Andalusia. We sequenced the WNV consensus genome from two mosquito pools and carried out the phylogenetic analyses. We also compared the obtained genomes with those sequenced from human samples obtained during the outbreak and the genomes obtained previously in Spain from birds (2007 and 2017), mosquitoes (2008) and horses (2010) to better understand the eco-epidemiology of WNV in Spain. As expected, the WNV genomes recovered from mosquito pools in 2020 were closely related to those recovered from humans of the same outbreak. In addition, the strain of WNV circulating in 2021 was highly related to the WNV strain that caused the 2020 outbreak, suggesting that WNV is overwintering in the area. Consequently, future outbreaks of the same strain may occur in in the future.

Co-exposure to pathogens in wild ungulates from Doñana National Park, South Spain.

Multiple infections or co-exposure to pathogens should be considered systematically in wildlife to better understand the ecology and evolution of host-pathogen relationships, so as to better determine the potential use of multiple pathogens as indicators to guide health management. We describe the pattern of co-exposure to several pathogens (i.e. simultaneous positive diagnosis to pathogens in an individual considering Mycobacterium tuberculosis complex lesions, and the presence of antibodies against Toxoplasma gondii, bluetongue virus, and hepatitis E virus) and assessed their main drivers in the wild ungulate community from Doñana National Park (red deer, fallow deer, and wild boar) for a 13-years longitudinal study. The lower-than-expected frequency of co-exposure registered in all species was consistent with non-mutually exclusive hypotheses (e.g. antagonism or disease-related mortality), which requires further investigation. The habitat generalist species (red deer and wild boar) were exposed to a greater diversity of pathogens (frequency of co-exposure around 50%) and/or risk factors than fallow deer (25.0% ± CI95% 4.9). Positive relationships between pathogens were evidenced, which may be explained by common risk factors favouring exposure. The specific combination of pathogens in individuals was mainly driven by different groups of factors (individual, environmental, stochastic, and populational), as well as its interaction, defining a complex eco-epidemiological landscape. To deepen into the main determinants and consequences of co-infections in a complex assemblage of wild hosts, and at the interface with humans and livestock, there also is needed to expand the range of pathogens and compare diverse assemblages of hosts under different environmental and management circumstances.

Oxidative status in relation to blood parasite infections in house sparrows living along an urbanization gradient.

Living organisms are exposed to a wide range of substances - internal and external - which act like reactive oxygen species (ROS). Oxidative damage accurs when the balance between ROS and antioxidant defenses is altered. Urbanization and parasite infection are both important sources of ROS with different harmful effects on wildlife health, but the potential synergies between both factors are poorly known. Here, we analyse the oxidative stress of wild juvenile male house sparrows (Passer domesticus) along an urbanization gradient in relation to the infection status by three common blood parasites (Plasmodium, Haemoproteus and Leucocytozoon) and bird body condition. We analysed samples from 688 birds captured at 45 localities from southern Spain grouped into triplets including an urban, a rural and a natural habitat, with 15 localities per habitat type. We measured i) thiobarbituric acid reactive substances (TBARS) levels as indicator of the oxidative damage to lipids, and the activity of three antioxidant enzymes ii) glutathione peroxidase (GPx), iii) superoxide dismutase (SOD) and iv) glutathione reductase (GR) as indicators of bird's antioxidant capacity. Birds infected with Haemoproteus and urban birds showed significantly and marginally higher levels of TBARS than uninfected and rural birds, respectively. The relationship between TBARS and body condition is different regarding the infection status (significative) and habitat (marginally significant) being negative for Haemoproteus infected and urban birds but positive for uninfected and non-urban birds. The antioxidant activity was significantly lower in Plasmodium infected birds but marginally higher in Leucocytozoon infected birds than in uninfected ones. Individuals with higher body condition had higher GPx and SOD activity in relation to a lower GR activity. Overall, these results suggest that blood parasites infections and urbanization affect the oxidative status of wild birds and highlight the role of bird's body condition on the regulation of the oxidative stress status.

A One Health view of the West Nile virus outbreak in Andalusia (Spain) in 2020.

Reports of West Nile virus (WNV) associated disease in humans were scarce in Spain until summer 2020, when 77 cases were reported, eight fatal. Most cases occurred next to the Guadalquivir River in the Sevillian villages of Puebla del Río and Coria del Río. Detection of WNV disease in humans was preceded by a large increase in the abundance of Culex perexiguus in the neighbourhood of the villages where most human cases occurred. The first WNV infected mosquitoes were captured approximately one month before the detection of the first human cases. Overall, 33 positive pools of Cx. perexiguus and one pool of Culex pipiens were found. Serology of wild birds confirmed WNV circulation inside the affected villages, that transmission to humans also occurred in urban settings and suggests that virus circulation was geographically more widespread than disease cases in humans or horses may indicate. A high prevalence of antibodies was detected in blackbirds (Turdus merula) suggesting that this species played an important role in the amplification of WNV in urban areas. Culex perexiguus was the main vector of WNV among birds in natural and agricultural areas, while its role in urban areas needs to be investigated in more detail. Culex pipiens may have played some role as bridge vector of WNV between birds and humans once the enzootic transmission cycle driven by Cx. perexiguus occurred inside the villages. Surveillance of virus in mosquitoes has the potential to detect WNV well in advance of the first human cases.

Population genetic structure of the elephant tick Amblyomma tholloni from different elephant populations in Kenya.

Understanding factors that shape tick population genetic structure is important as they may be exploited in crafting strategies for vector control. Amblyomma tholloni, or "elephant tick" is a three-host tick whose adults preferentially parasitize African elephants. The aim of this study was to determine the influence of fragmentation of the host populations on the genetic structure of this tick species from different ecosystems in Kenya, using the second internal transcribed spacer (ITS-2) and mitochondrial cytochrome oxidase 1 (CO1) loci. Population genetic analysis of ticks collected from four elephant populations using ITS-2 and CO1 loci revealed high gene diversity. Gene diversity at the ITS-2 locus was 0.91 and the nucleotide diversity was, 0.01. ITS-2 gene diversity was highest in Laikipia-Samburu ecosystem (0.947) and lowest in Tsavo (0.80). The CO1 locus also had high gene diversity, 0.790, and low nucleotide diversity, 0.006, and like ITS-2, gene diversity was higher in Laikipia-Samburu ecosystem (1.00) and lower in Tsavo (0.286). There was a modest statistically significant genetic differentiation among the four tick populations based on ITS-2 (FST = 0.104, P < 0.001; ΦST = 0.105, P < 0.001), and a 10% of molecular variance attributed to genetic variation between populations. There was also statistically significant differentiation among tick populations using haplotype frequencies for CO1 locus (FST = 0.167, P < 0.001) accounting for 17% of genetic variance among populations, but not modelled genetic distances (ΦST = 0.029, P = 0.095) suggesting very recent genetic differentiation. In addition, populations of A. tholloni in Kenya had a significantly negative Tajima D and Fu & Li's F* and D* at the CO1 locus suggesting recent positive selection. The extensive acaricide use in livestock, which host the larval stage, could be driving purifying selection and genetic hitchhiking of the CO1 locus. However, tests sensitive to demography such as Fu's FS, Ramos-Onsins & Rozas's R2 and raggedness index r were statistically significant at the ITS-2 locus suggesting ancient demographic expansion. Elephant population fragmentation appears to shape the genetic structure of A. tholloni, while agro-ecological factors could influence the genetic diversity of ticks.

Differences in fatty acids composition between Plasmodium infected and uninfected house sparrows along an urbanization gradient.

Anthropogenic activities such as intensification of agriculture, animal husbandry and expansion of cities can negatively impact wildlife through its influence on the availability of high-quality food resources and pathogen transmission. The house sparrow (Passer domesticus), an urban exploiter, is undergoing a population decline. Nutritional constrains and infectious diseases has been highlighted as potential causes. Fatty acids (FAs) play an important role in modulating certain immune responses needed to combat parasite infections. FAs are highly influenced by dietary availability and have been shown to vary between urban and rural birds. Habitat anthropization also affects avian malaria epidemiology but little attention has been given to the relationship between blood parasite infection, host FAs composition and anthropization. Here, we analysed 165 juvenile birds either infected by Plasmodium or uninfected, captured at 15 localities grouped in triplets containing urban, rural and natural habitats. The total level of FAs was higher in birds from urban than from rural habitats, suggesting a greater availability of fat-rich foods sources. Furthermore, Plasmodium infected birds had higher relative levels of ω-3 polyunsaturated fatty acids (PUFAs) but lower of ω-6 PUFAs than uninfected birds. In concordance, the ω-6/ω-3 ratio was also lower in infected than in uninfected birds, but only from natural habitats, likely driven by the slightly higher ω-3 PUFAs in infected birds from natural habitats. Birds from anthropized environments may metabolize the ω-3 PUFAs to promote anti-inflammatory responses against stressors, which would result in lower ω-3 affecting their response against Plasmodium. Alternatively, lower ω-6 PUFAs may influence birds susceptibility to infection due to a weaker pro-inflammatory response. These descriptive results do not allow us to identify the causality of these associations but highlight the need to further investigate the relevance of FAs for birds to fight infectious diseases in habitats with different degree of urbanization.

First record of Pharyngomyia picta (Diptera: Oestridae) parasitizing Cervus elaphus in Sierra Nevada National Park.

The necropsy made to two adult individuals of red deer shot in Sierra Nevada National Park revealed infestation by second- and third-instar larvae of the oestrid Pharyngomyia picta (Meigen 1824). The animals were shot in Dehesa de las Hoyas, about 2000 m above sea level. This is the first record of this parasite in Sierra Nevada National Park. Furthermore, the occurrence of adult P. picta at this altitude could indicate significant changes of the climatic conditions, now matching better the environmental requirements of this bot fly. Its monitoring may, therefore, be of great help for detecting global change signs.

A field test of the dilution effect hypothesis in four avian multi-host pathogens.

The Dilution Effect Hypothesis (DEH) argues that greater biodiversity lowers the risk of disease and reduces the rates of pathogen transmission since more diverse communities harbour fewer competent hosts for any given pathogen, thereby reducing host exposure to the pathogen. DEH is expected to operate most intensely in vector-borne pathogens and when species-rich communities are not associated with increased host density. Overall, dilution will occur if greater species diversity leads to a lower contact rate between infected vectors and susceptible hosts, and between infected hosts and susceptible vectors. Field-based tests simultaneously analysing the prevalence of several multi-host pathogens in relation to host and vector diversity are required to validate DEH. We tested the relationship between the prevalence in house sparrows (Passer domesticus) of four vector-borne pathogens-three avian haemosporidians (including the avian malaria parasite Plasmodium and the malaria-like parasites Haemoproteus and Leucocytozoon) and West Nile virus (WNV)-and vertebrate diversity. Birds were sampled at 45 localities in SW Spain for which extensive data on vector (mosquitoes) and vertebrate communities exist. Vertebrate censuses were conducted to quantify avian and mammal density, species richness and evenness. Contrary to the predictions of DEH, WNV seroprevalence and haemosporidian prevalence were not negatively associated with either vertebrate species richness or evenness. Indeed, the opposite pattern was found, with positive relationships between avian species richness and WNV seroprevalence, and Leucocytozoon prevalence being detected. When vector (mosquito) richness and evenness were incorporated into the models, all the previous associations between WNV prevalence and the vertebrate community variables remained unchanged. No significant association was found for Plasmodium prevalence and vertebrate community variables in any of the models tested. Despite the studied system having several characteristics that should favour the dilution effect (i.e., vector-borne pathogens, an area where vector and host densities are unrelated, and where host richness is not associated with an increase in host density), none of the relationships between host species diversity and species richness, and pathogen prevalence supported DEH and, in fact, amplification was found for three of the four pathogens tested. Consequently, the range of pathogens and communities studied needs to be broadened if we are to understand the ecological factors that favour dilution and how often these conditions occur in nature.

Urbanization effects on temporal variations of avian haemosporidian infections.

Urbanization is an important human-driven process that leads to biodiversity loss and alters the interactions between organisms, including disease transmission. Although urbanization affects both host and vector communities, the effects on vector-borne pathogens are still poorly understood. Here, we monitored variation in prevalence and richness of three common blood parasites in birds (Plasmodium, Haemoproteus and Leucocytozoon) from localities with different land uses (urban, rural and natural) during two consecutive years (2013 and 2014). Overall, 1400 juvenile house sparrows (Passer domesticus) from 15 localities in southern Spain were included in this study. Haemoproteus and Leucocytozoon prevalence was higher in 2013 than in 2014, particularly in urban and natural habitats for the case of Leucocytozoon. Prevalence was correlated between years for Haemoproteus and Leucocytozoon, independently of the habitat. Additionally, rural habitats harboured significantly higher Haemoproteus lineage richness compared to urban and natural habitats during 2014. Leucocytozoon lineage richness was negatively correlated between years in rural habitats but positively correlated in urban and natural habitats in comparison. Parasite lineages found in birds were homogeneously distributed along habitats and years and the common lineages prevalence were not influenced by them. Our results highlight different patterns of infection depending on the parasite genera probably related to the composition and density of vector communities. The specific reproductive environmental requirements of the different groups of vectors involved in the transmission may be affected by climatic conditions and landscape features.

Understanding host utilization by mosquitoes: determinants, challenges and future directions.

Mosquito host utilization is a key factor in the transmission of vector-borne pathogens given that it greatly influences host-vector contact rates. Blood-feeding patterns of mosquitoes are not random, as some mosquitoes feed on particular species and/or individuals more than expected by chance. Mosquitoes use a number of cues including visual, olfactory, acoustic, and thermal stimuli emitted by vertebrate hosts to locate and identify their blood meal sources. Thus, differences in the quality/intensity of the released cues may drive host selection by mosquitoes at both inter- and intra-specific levels. Such patterns of host selection by mosquitoes in space and time can be structured by factors related to mosquitoes (e.g. innate host preference, behavioural plasticity), to hosts (e.g. emission of host-seeking cues, host availability) or to both (e.g. pathogen infection). In this study, we review current evidence, from phenomena to mechanisms, of how these factors influence host utilization by mosquitoes. We also review the methodologies commonly used in this research field and identify the major challenges for future studies. To bridge the knowledge gaps, we propose improvements to strengthen traditional approaches and the use of a functional trait-based approach to infer mosquito host utilization in natural communities.

The role of different Culex mosquito species in the transmission of West Nile virus and avian malaria parasites in Mediterranean areas.

Vector-borne diseases, especially those transmitted by mosquitoes, have severe impacts on public health and economy. West Nile virus (WNV) and avian malaria parasites of the genus Plasmodium are mosquito-borne pathogens that may produce severe disease and illness in humans and birds, respectively, and circulate in an endemic form in southern Europe. Here, we used field-collected data to identify the impact of Culex pipiens, Cx. perexiguus and Cx. modestus, on the circulation of both WNV and Plasmodium in Andalusia (SW Spain) using mathematical modelling of the basic reproduction number (R0 ). Models were calibrated with field-collected data on WNV seroprevalence and Plasmodium infection in wild house sparrows, presence of WNV and Plasmodium in mosquito pools, and mosquito blood-feeding patterns. This approach allowed us to determine the contribution of each vector species to pathogen amplification. Overall, 0.7% and 29.6% of house sparrows were positive to WNV antibodies and Plasmodium infection, respectively. In addition, the prevalence of Plasmodium was higher in Cx. pipiens (2.0%), followed by Cx. perexiguus (1.8%) and Cx. modestus (0.7%). Three pools of Cx. perexiguus were positive to WVN. Models identified Cx. perexiguus as the most important species contributing to the amplification of WNV in southern Spain. For Plasmodium models, R0 values were higher when Cx. pipiens was present in the population, either alone or in combination with the other mosquito species. These results suggest that the transmission of these vector-borne pathogens depends on different Culex species, and consequently, their transmission niches will present different spatial and temporal patterns. For WNV, targeted surveillance and control of Cx. perexiguus populations appear as the most effective measure to reduce WNV amplification. Also, preventing Culex populations near human settlements, or reducing the abundance of these species, are potential strategies to reduce WNV spillover into human populations in southern Spain.

Mosquitoes in an Urban Zoo: Identification of Blood Meals, Flight Distances of Engorged Females, and Avian Malaria Infections.

Zoological gardens are home to a large number of vertebrate species and as such are suitable sites for both mosquito breeding and maintenance. They are excellent places for entomological studies of mosquito phenology, diversity, and blood-feeding patterns, as well as for xenomonitoring. During 2016, we sampled mosquitoes in Barcelona Zoo and used molecular methods to determine their blood-feeding patterns and the prevalence and diversity of avian malaria parasites. We also estimated the flight distance of engorged mosquitoes in the area. Overall, 1,384 adult Culex pipiens s.l., Culiseta longiareolata, and Aedes albopictus were captured. Birds dominated the diet of Cx. pipiens s.l. (n = 87) and Cs. longiareolata (n = 6), while humans were the only blood-meal source of Ae. albopictus (n = 3). Mosquitoes had a mean flight distance of 95.67 m after feeding on blood (range 38.71-168.51 m). Blood parasites were detected in the abdomen of 13 engorged Cx. pipiens s.l., eight of which had fed on magpies. Four Plasmodium lineages and a single lineage of the malaria-like parasite Haemoproteus were identified. These results suggest that Cx. pipiens s.l. is involved in the local transmission of avian Plasmodium, which potentially affects the circulation of parasites between and within wildlife and enclosed animals. Vigilance regarding possible mosquito breeding sites in this zoo is thus recommended.

Long-Term Determinants of Tuberculosis in the Ungulate Host Community of Doñana National Park.

Animal tuberculosis (TB) is endemic in wild boar (Sus scrofa), red deer (Cervus elaphus), fallow deer (Dama dama) and cattle in south and central Spain. In order to clarify the processes that operate in the medium and long-term, we studied TB at the wildlife-livestock interface in Doñana National Park for 14 years (2006-2018) in relation to host density, stochastic factors (rainfall) and environmental features (e.g., aggregation points such as waterholes). Wild boar showed the highest prevalence of TB (76.7%), followed by red deer (42.5%), fallow deer (14.4%) and cattle (10.7%). We found evidence of relevant epidemiological processes which operate over the long-term and interact with host and community ecology. Interestingly, the effect of high wild boar population density on increased TB rates was mediated by sows, which could determine high incidence in young individuals already in maternal groups. Rainfall significantly determined a higher risk of TB in male red deer, probably mediated by sex-related differences in life history traits that determined more susceptibility and/or exposure in comparison to females. The positive association between the prevalence of TB in fallow deer and cattle may indicate significant interspecies transmission (in either direction) and/or similar exposure to risk factors mediated by ecological overlapping of grazing species. The identification of long-term drivers of TB provided evidence that its control in extensive pastoral systems can only be achieved by targeting all relevant hosts and integrating measures related to all the factors involved, such as: population abundance and the aggregation of wild and domestic ungulates, environmental exposure to mycobacteria, cattle testing and culling campaigns and adjustments of appropriate densities.

Mosquitoes are attracted by the odour of Plasmodium-infected birds.

Parasites can manipulate their hosts to increase their transmission success. Avian malaria parasites (Plasmodium) are thought to alter the cues such as host odour, used by host-seeking mosquitoes. Bird odour is affected by secretions from the uropygial gland and may play a role in modulating vector-host interactions. We tested the hypothesis that mosquitoes are more attracted to the uropygial secretions and/or whole-body odour (headspace) of Plasmodium-infected house sparrows (Passer domesticus) than to those of uninfected birds. We tested the attraction of nulliparous (e.g. uninfected mosquitoes without previous access to blood) Culex pipiens females towards these stimuli in a dual-choice olfactometer. We used Gas Chromatography-Mass Spectrometry (GC-MS) analyses to assess whether Plasmodium infection is associated with differences in the chemical composition of uropygial secretions. Mosquitoes were more attracted to the odours of infected than uninfected birds, regardless of sex. However, the significant interaction between infection status and the stimuli (uropygial secretion or headspace) showed that mosquitoes were more attracted to the headspace of infected birds; no differences were found in the case of uropygial secretions. The compounds in the volatile lipophilic fraction of the uropygial secretion did not differ between infected and uninfected birds. These results support the host manipulation hypothesis since avian Plasmodium parasites may be capable of altering their host's body odour, thereby making infected individuals more attractive to mosquitoes.