First report of Eimeria myoxi in the garden dormouse (Eliomys quercinus Linnaeus, 1766) from Doñana Natural Area (Andalusia, SW Spain).

This work reports for the first time the presence and molecular characterization of Eimeria myoxi in the garden dormouse (Eliomys quercinus) from the Doñana Natural Area (Andalusia, SW Spain). Fresh faecal samples were collected from a total of 28 garden dormice, which were caught following current guidelines for the ethical use of animals in research, and processing by a standard flotation technique with saturated saline solution. Then, wet drops were examined microscopically, and the number of oocysts was semi-quantified. Eimeria oocysts were observed in 16 of the 28 (57.1%) faecal samples, showing most of them a very low number of oocysts (≤1 oocyst per microscopic field × 400). The unsporulated oocysts visualized in 16 faecal samples were subspherical and of length 19.2 ± 1.2 µm and width 17.4 ± 1.1 µm, being morphologically compatible with E. myoxi. This finding was supported by molecular analysis of the small subunit ribosomal RNA (SSU-rRNA) gene, identifying the same species in 22 of the 28 (78.6%) dormice, including 15 samples in which oocyst size was compatible with E. myoxi. Moreover, the subsequent analyses of the apicoplast open reading frame 470 (ORF470) and the mitochondrial cytochrome c oxidase subunit I (COI) genes confirmed the molecular identification of the isolates as E. myoxi. The phylogeny analyses were consistent with previous phylogenetic studies and support the existence of three lineages of rodent-infecting Eimeria species.

Long-term determinants of the seroprevalence of the bluetongue virus in deer species in southern Spain.

Bluetongue is a vector-borne disease affecting domestic and wild ruminants, with a major socioeconomic impact. Endemic circulation of the bluetongue virus serotype 4 (BTV-4) and BTV-1 have occurred in Spain since 2004 and 2007, respectively. However, epidemiological studies have seldom been approached from a long-term perspective in wild ruminants. A total of 881 deer (red deer and fallow deer) were necropsied from 2005 to 2018 as part of the DNP health-monitoring program. Serum samples were tested for antibodies against BTV with the aims of assessing the temporal trend and to evaluate the modulating factors: individual, populational, environmental, and stochastic. Red deer displayed statistically significant higher seroprevalences of BTV (SBT; 78.6 ± 3.8%) than fallow deer (53.1 ± 4.7%). The detection of BTV-1 and BTV-4 by the serum neutralization test in calves suggested the circulation of both serotypes over the study period. For red deer, wet years together with high densities could provide suitable conditions for vector borne BTV transmission. Moreover, proximity to high suitability habitat for Culicoides, permanent pasturelands, was associated with higher SBT. The differences in the ecology and behaviour of deer species influencing the exposure to the vectors could determine the differences found in the SBT patterns. This study evidences the role that deer species may play in the maintenance of BTV, however, elucidating the epidemiological role of host in different contexts as well as the consequences of climate change on the competent vector populations and its potential effect on the dynamics of BTV infection in hosts communities deserve further research.

Long-Term Determinants of the Seroprevalence of Toxoplasma gondii in a Wild Ungulate Community.

Toxoplasma gondii is an obligate intracellular protozoan which infects warm-blooded vertebrates, including humans, worldwide. In the present study, the epidemiology of T. gondii was studied in the wild ungulate host community (wild boar, red deer, and fallow deer) of Doñana National Park (DNP, south-western Spain) for 13 years (2005-2018). We assessed several variables which potentially operate in the medium and long-term (environmental features, population, and stochastic factors). Overall, the wild ungulate host community of DNP had high seroprevalence values of T. gondii (STG; % ± confidence interval (CI) 95%; wild boar (Sus scrofa) 39 ± 3.3, n = 698; red deer (Cervus elaphus) 30.7 ± 4.4, n = 423; fallow deer (Dama dama) 29.7 ± 4.2, n = 452). The complex interplay of hosts and ecological/epidemiological niches, together with the optimal climatic conditions for the survival of oocysts that converge in this area may favor the spread of the parasite in its host community. The temporal evolution of STG oscillated considerably, mostly in deer species. The relationships shown by statistical models indicated that several factors determined species patterns. Concomitance of effects among species, indicated that relevant drivers of risk operated at the community level. Our focus, addressing factors operating at broad temporal scale, allows showing their impacts on the epidemiology of T. gondii and its trends. This approach is key to understanding the epidemiology and ecology to T. gondii infection in wild host communities in a context where the decline in seroprevalence leads to loss of immunity in humans.

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.

Spatio-temporal trends in the frequency of interspecific interactions between domestic and wild ungulates from Mediterranean Spain.

Controlling infections shared by wildlife and livestock requires the understanding and quantification of interspecific interactions between the species involved. This is particularly important in extensive multi-host systems, in which controlled domestic animals interact with uncontrolled, abundant and expanding wild species, such as wild ungulates. We have, therefore, quantified the interspecific interactions between wild boar (Sus scrofa) and free-ranging cattle in Mediterranean Spain, along with their spatio-temporal variability. GPS-GSM-collars were used to monitor 12 cows and 14 wild boar in the Doñana National Park between 2011 and 2013. Interactions were defined as encounters between cattle and wild boar within a spatio-temporal window of 52 m and 1 hour. On average, each wild boar interacted with one cow 1.5 ± (SE) 0.5 times per day, while each cow interacted with one wild boar 1.3 ± 0.4 times per day. The frequency of interaction was significantly higher during crepuscular hours owing to the overlap of both species' activity, and also during spring and autumn, probably owing to a higher individual aggregation around shared resources. Finally, the frequency of interaction was higher near the most significant shared resources (e.g. water points) but was lower in areas with dense vegetation. The results presented here show the usefulness of GPS monitoring as regards quantifying interactions and helping to clarify the process of pathogen transmission at the wildlife-livestock interface in Mediterranean Spain, along with the main spatio-temporal risk factors. In a changing scenario in which European populations of wild ungulates are increasing, more efficient measures with which to control interactions are required to meet the demands of farmers and managers. Our results, therefore, provide directional hypotheses that could be used to design disease control programmes.

Long-term patterns in Iberian hare population dynamics in a protected area (Doñana National Park) in the southwestern Iberian Peninsula: Effects of weather conditions and plant cover.

The Iberian hare (Lepus granatensis) is a widely distributed endemic species in the Iberian Peninsula. To improve our knowledge of its population dynamics, the relative abundance and population trends of the Iberian hare were studied in the autumns of 1995-2012 in a protected area (Doñana National Park) by spotlighting in 2 different habitats: marshland and ecotones. The average relative abundance was 0.38 hare/km (SD = 0.63) in the marshland and 3.6 hares/km (SD = 4.09) in ecotones. The Iberian hare population exhibited local interannual fluctuations and a negative population trend during the study period (1995-2012). The results suggest that its populations are in decline. The flooding of parts of the marshland in June, July and October favor hare abundance in the ecotone. Hare abundance in the marshland increases as the flooded surface area increases in October. These effects are more pronounced if the rains are early (October) and partially flood the marsh. By contrast, when marsh grasses and graminoids are very high and thick (as measured using the aerial herbaceous biomass [biomass marshland] as a proxy), the abundance of hares decreases dramatically as does the area of the marsh that is flooded (in November).

Spatiotemporal interactions between wild boar and cattle: implications for cross-species disease transmission.

Controlling infectious diseases at the wildlife/livestock interface is often difficult because the ecological processes driving transmission between wildlife reservoirs and sympatric livestock populations are poorly understood. Thus, assessing how animals use their environment and how this affects interspecific interactions is an important factor in determining the local risk for disease transmission and maintenance. We used data from concurrently monitored GPS-collared domestic cattle and wild boar (Sus scrofa) to assess spatiotemporal interactions and associated implications for bovine tuberculosis (TB) transmission in a complex ecological and epidemiological system, Doñana National Park (DNP, South Spain). We found that fine-scale spatial overlap of cattle and wild boar was seasonally high in some habitats. In general, spatial interactions between the two species were highest in the marsh-shrub ecotone and at permanent water sources, whereas shrub-woodlands and seasonal grass-marshlands were areas with lower predicted relative interactions. Wild boar and cattle generally used different resources during winter and spring in DNP. Conversely, limited differences in resource selection during summer and autumn, when food and water availability were limiting, resulted in negligible spatial segregation and thus probably high encounter rates. The spatial gradient in potential overlap between the two species across DNP corresponded well with the spatial variation in the observed incidence of TB in cattle and prevalence of TB in wild boar. We suggest that the marsh-shrub ecotone and permanent water sources act as important points of TB transmission in our system, particularly during summer and autumn. Targeted management actions are suggested to reduce potential interactions between cattle and wild boar in order to prevent disease transmission and design effective control strategies.

Independent lineage of lymphocytic choriomeningitis virus in wood mice (Apodemus sylvaticus), Spain.

To clarify the presence of lymphocytic choriomeningitis virus (LCMV) in Spain, we examined blood and tissue specimens from 866 small mammals. LCMV RNA was detected in 3 of 694 wood mice (Apodemus sylvaticus). Phylogenetic analyses suggest that the strains constitute a new evolutionary lineage. LCMV antibodies were detected in 4 of 10 rodent species tested.

Small-scale indirect effects determine the outcome of a tripartite plant-disperser-granivore interaction.

The microhabitat in which plants grow affects the outcome of their interactions with animals, particularly non-specialist consumers. Nevertheless, as most research on this topic has dealt with either mutualists or antagonists, little is known about the indirect effects of plant microhabitats on the outcome of tripartite interactions involving plants and both mutualists (e.g. seed dispersers) and antagonists (e.g. granivores). During three consecutive years, we analysed small-scale variations in the interaction of a perennial myrmecochore, Helleborus foetidus, with its seed dispersers and consumers as a function of the intensity of plant cover. Most seeds were released during the day and were rapidly removed by ants. Nevertheless, the proportion of ant-removed seeds was higher for plants located in open microhabitats than for plants surrounded by dense vegetation and rocky cover. Ant sampling revealed that seed removers were equally abundant, irrespective of the level of cover. By contrast, a few tiny ant species that feed on the reward without transporting the seeds were more abundant in highly covered microhabitats, irrespective of hellebore diaspore availability. These "cheaters" decrease the chance of removal by removers and increase the probability of seeds remaining on the ground until night, when granivore mice Apodemus sylvaticus become active. Mice also preferred foraging in covered microhabitats, where they consumed a larger proportion of seeds. Therefore, the density of cover indirectly increased seed predation risk by attracting more seed predators and cheater ants that contribute to increase seed availability for seed predators. Our results emphasize the importance of considering the indirect effects of plant microhabitat on their dispersal success. They highlight the indirect effect of cheaters that are likely to interfere in mutualisms and may lead to their collapse unless external factors such as spatio-temporal heterogeneity in seed availability constrain their effect.

Synchrony between fruit maturation and effective dispersers' foraging activity increases seed protection against seed predators.

The evolution of pollination and seed dispersal mutualisms is conditioned by the spatial and temporal co-occurrence of animals and plants. In the present study we explore the timing of seed release of a myrmecochorous plant (Helleborus foetidus) and ant activity in two populations in southern Spain during 2 consecutive years. The results indicate that fruit dehiscence and seed shedding occur mostly in the morning and correspond to the period of maximum foraging activity of the most effective ant dispersers. By contrast, ant species that do not transport seeds and/or that do not abound near the plants are active either before or after H. foetidus diaspores are released. Experimental analysis of diet preference for three kinds of food shows that effective ant dispersers are mostly scavengers that readily feed on insect corpses and sugars. Artificial seed depots suggest that seeds deposited on the ground out of the natural daily time window of diaspore releasing are not removed by ants and suffer strong predation by nocturnal rodents Apodemus sylvaticus. Nevertheless, important inter-annual variations in rodent populations cast doubts on their real importance as selection agents. We argue that traits allowing synchrony between seed presentation and effective partners may constitute a crucial pre-adaptation for the evolution of plant-animal mutualisms involving numerous animal partners.