Batrachochytrium dendrobatidis infection of amphibians in the Doñana National Park, Spain

Amphibian chytridiomycosis, caused by infection with the non-hyphal, zoosporic chytrid fungus Batrachochytrium dendrobatidis (Bd), is an emerging infectious disease recognised as a cause of recent amphibian population declines and extinctions worldwide. The Do?ana National Park (DNP) is located in southwestern Spain, a country with widespread Bd infection. This protected area has a great diversity of aquatic habitats that constitute important breeding habitats for 11 native amphibian species. We sampled 625 amphibians in December 2007 and February to March 2008, months that correspond to the early and intermediate breeding seasons for amphibians, respectively. We found 7 of 9 sampled species to be infected with Bd and found differences in prevalence between sampling periods. Although some amphibians tested had higher intensities of infection than others, all animals sampled were apparently healthy and, so far, there has been no evidence of either unusually high rates of mortality or amphibian population declines in the DNP.     

Formation and growth of a heronry in a managed wetland in Doñana,southwest Spain

Capsule A newly created wetland has been beneficial for the breeding of five heron species and for the settlement and expansion of Glossy Ibises in the region.

Aim To determine whether created wetland would lead to the establishment of a heron and ibis breeding colony, and if so, to determine its effects on the distribution of these species in the wider region.

Methods Number of pairs breeding at the new wetland and in the region were estimated annually from 1996 to 2008. We investigated whether the growth of the new colony was explained by redistribution of breeding pairs within the region.

Results Purple Herons, Squacco Herons and Glossy Ibises colonised the new wetland in 1996. Black‐crowned Night Herons settled in 1998, while Cattle and Little Egrets were the last species to establish in the mixed colony in 2001. The population increase was particularly marked for Glossy Ibises (which had bred in Doñana only sporadically through the 20th century). Our findings suggest that simple redistribution of pairs does not explain the formation and growth of the new colony. Since the first year of establishment numbers of Glossy Ibises and Purple Herons have shown a significant growth trend at both the new colony and in Doñana.

Conclusion The creation and management of a new wetland has been successful for the conservation of heron and egret species and promoted the expansion of the Glossy Ibis population across the western Mediterranean region.     

Primary moult of continental Black-tailed Godwits Limosa limosa limosa in the Doñana wetlands,Spain

Capsule: Most Continental Black-tailed Godwits Limosa limosa limosa using the Doñana wetlands during post-breeding migration appear to begin moult before they arrive and suspend moult before they migrate onwards to West Africa.

Aims: We aim to describe the primary moult strategies and patterns in the Continental Black-tailed Godwits using the Doñana wetlands, a major passage and wintering area for waterbirds in southern Spain.

Methods: Individual godwits were captured, marked and their primary moult was scored in Doñana during the non-breeding season (June–March) in 2011 and 2012. Data from resightings of colour-marked godwits and birds equipped with satellite transmitters were used to estimate stopover duration during post-breeding migration (June–September) to determine if godwits move to West Africa before completing their primary moult.

Results: Average primary moult duration was estimated to be 84 days?±?9 se, during 29 June–21 September and did not differ between sexes. Only 2% of individuals were observed with suspended moult. We estimated stopover duration in Doñana to be 13 days?±?2 se before migrating to West Africa.

Conclusions: Most godwits stage for about two weeks in the Doñana wetlands during southward migration, moult their primaries and appear to suspend moult before crossing the Sahara. Others may complete their primary moult in Doñana, or elsewhere in Europe and overwinter in Doñana where increasing numbers of godwits have been detected in recent years. A few individuals may finish the moult in Doñana and migrate to West Africa late in the post-breeding season.     

Acidobacteria in freshwater ponds at Doñana National Park, Spain

The Acidobacteria show a widespread distribution in natural ecosystems. In this study, we analyzed the presence of Acidobacteria in freshwater ponds at Do?ana National Park (southwestern Spain). Nucleic acid sequence analysis, quantitative, real-time RT-PCR, and fluorescence in situ hybridization (FISH) were carried out. Acidobacteria in these aquatic environments were investigated using their 16S and 23S rDNA sequences and acidobacterial specific primer pairs through phylogenetic approaches. The presence of up to five subdivisions of Acidobacteria was detected during this study. The analyzed ponds exhibited distinctive patterns of acidobacterial clades. In order to detect their role in ecosystem functions, metabolically active Acidobacteria were detected based upon rRNA analyses. Quantitative, real-time RT-PCR showed a low percentage of metabolically active Acidobacteria at suboxic zones within the water column covered by surface Fe-rich films. Oxygen-saturated areas showed around 4% of total bacterial RNA belonging to Acidobacteria both in the water column and the sediment surface. The morphology of the most abundant Acidobacteria was revealed by FISH as cocci generally in pairs or chains. Enrichment cultures were also obtained and indicated a putative metabolism based on aerobic and heterotrophic characteristics likely taking advantage of the abundant organic matter present at the investigated sites. These results represent a significant contribution toward understanding the distribution and ecological role of the phylum Acidobacteria in natural ecosystems, specifically at Do?ana National Park freshwater ponds.     

Influence of grazing on the decomposition of Pinus pinea L. needles in a silvopastoral system in Doñana, Spain

Aims

The aim of this study was to determine whether goat grazing in the understory of a pine forest at Doñana Natural Park could accelerate the decomposition of the pine needles accumulated on the soil surface and, if so, through which mechanisms. Specifically, the roles of trampling (mechanical fragmentation) and nutrient enrichment through defecation (fertilization) were evaluated in terms of their effect on pine needle decomposition rates.

Methods

An experiment was conducted featuring the following 4 treatments: 1) intact needles (control), 2) trampled needles, 3) intact needles fertilized with liquid manure, and 4) trampled needles fertilized with liquid manure. Litter decomposition was determined as a function of mass loss over time, using the litter-bag method. Bags were recovered 4, 8, 16, 24 and 36 months after burial in soil, dried and weighed. Needle length, leaf mass per area and C and N concentration were also measured in the buried litter-bags.

Results

Four months after burial, mass loss was greater in the trampled (23–27 %) than non-trampled (14–16 %) treatments. However, from 8 months onwards, decomposition rates in the fertilized treatments were significantly higher than those in the non-fertilized treatments (between 5 % and 15 % less mass loss). Meanwhile, fertilized treatments presented higher N content (2.1 %) than the non-fertilized ones (1.2 %), with a significantly lower C:N ratio also found in the in the fertilized treatment.

Conclusions

Trampling and fertilization during grazing accelerates litter decomposition and thus promotes the incorporation of N into the system. Acceleration of decomposition reduces the accumulation of pine needles on the soil surface, reducing the risk of fire.     

Crustacean and rotifer composition of temporary ponds in the Doñana National Park (SW Spain) during floods

The zooplankton of 18 temporary ponds in the Doñana National Park (SW Spain) was studied during floods in February and May 1997. A total of 37 rotifer taxa and 34 crustaceans species were identified (17 cladocerans, 2 ostracans, 4 diaptomids, 7 cyclopodids, 1 harpacticoid, 1 anostracan, 1 notostracan and 1 conchostracan). Zooplankton samples were collected separately from the littoral and the open-water of 12 different ponds. Commonly distributed zooplankton species (frequency of appearance 50%) were not segregated to either the littoral or the open-water according to a chi-square test (P>0.005). The study ponds were divided into seasonal, intermediate and ephemeral ponds according to the length of their hydroperiod. The total numbers of both crustacean and rotifer taxa were highest in the intermediate-hydroperiod ponds (26 and 32 taxa, respectively). The total number of zooplankton taxa collected in both February and May was not significantly correlated to the hydroperiod of the temporary ponds of Doñana during the study period (r=0.165, P=0.526). 21 rotifer taxa and 20 crustacean species were found in the ephemeral ponds; the number of restricted species was also relatively high (3 rotifers and 4 crustaceans). Therefore, the ephemeral ponds held a relatively rich community during floods compared to other temporary ponds of Doñana despite their small size and short wet phase.     

Epidemiology of Ancylostoma spp. in the endangered Iberian lynx (Lynx pardinus) in the Doñana National Park, south-west Spain

The epidemiology of Ancylostoma spp. was studied in the endangered Iberian lynx (Lynx pardinus) in the Do?ana National Park, south-west Spain. Faecal samples were collected throughout a complete annual cycle (August 1997 to September 1998). The overall egg prevalence of Ancylostoma spp. was 57.8%. The pattern of abundance of Ancylostoma spp. eggs in faeces was overdispersed. Juvenile lynx demonstrated a statistically higher prevalence and abundance of Ancylostoma spp. than in adults. These levels of egg output (maximum 21195 epg), as previously reported in free ranging large felid cubs, could be close to disease involvement. The potential pathogenicity of hookworms and the influence of individual and ecological factors on hookworm transmission in the Iberian lynx from the Do?ana National Park population are discussed.     

Species Richness and Distribution of Copepods and Cladocerans and their Relation to Hydroperiod and Other Environmental Variables in Doñana, South-west Spain

Twenty-five sites located in five wetland zones within Doñana, south-west Spain were studied for copepod and cladoceran species richness and composition in relation to habitat characteristics from January to March 2004. The γ-diversity of copepods and cladocerans combined varied between wetland zones, which differed significantly in conductivity, surface area and abundance of vegetation. However, there were no significant differences between zones in local species richness of either copepods or cladocerans or the two combined. Species richness was significantly higher in sites with intermediate hydroperiods (duration between 3 and 5 months) than in sites with shorter or longer hydroperiods. CCA analyses performed separately for copepods and cladocerans extracted conductivity, surface area, and vegetation cover as the main factors structuring species composition of both groups. Ours is one of the first studies of zooplankton in the natural marshes of the Doñana World Heritage Site, and we recorded five species new to the Doñana area.     

Rotifers in ephemeral ponds of Doñana National Park

32 ephemeral dune ponds were studied at the end of January 1990. 34 rotifer species were identified. The distribution of only a few species was related to salinity, suspended matter, chlorophyll-a and total water protein concentrations. However, most of the species seem to be better related to the colonization possibilities than to the environmental characteristics of the ponds.     

Variability of the sediment phosphate composition of a temporary pond (Doñana National Park,SW Spain)

This study reports on the spatial and temporal variability of the phosphate composition in the sediment of a temporary pond over a period of 3 years using the EDTA-method for P-fractionation. Sediment samples were collected at three different sites (open-water, littoral and flood plain) to compare the effect of the length of the wet/dry phase on the sediment phosphate composition, with special emphasis on the potential bioavailability of the P-fractions.Fine sediments (<0.1 mm) were rich in organic matter (9–25%) and contained high mean concentrations of Tot-P (182–655 mg kg^–1 d.w.), especially in the flood plain sediment. The sediment P composition was dominated by P-organic fractions at all sites (64–94%). The average C/N ratios were 8.8, 6.0 and 5.9 for sediments of the flood plain, littoral and open-water sites, respectively. The flood plain sediment was significantly poorer in iron-bound P (FeOOHP), but richer in the P-organic fractions extracted by EDTA than the sediment of the open-water site (P<0.01). The percentage of organic matter increased significantly in the sediment of the open-water site at the end of each dry season (P<0.05), while it decreased in the sediment of the flood plain site (P<0.01). In all sediments, the fraction of Fe(OOH)P decreased at the end of each dry season and some of these changes were significant (P<0.05). The decrease in the fraction of Fe(OOH)P was not related to changes in the sediment redox potential. Although the flood plain site was dry longer than the open-water site during the study period, the differences between the sediment composition of both sites were probably due to the effect of plant growth on the dry sediments of the flood plain site rather than to a direct effect of desiccation.     

A quantitative hydrogeomorphic approach to the classification of temporary wetlands in the Doñana National Park (SW Spain)

A quantitative hydrogeomorphic approach was applied to an extensive survey of temporary wetlands in the Doñana National Park (SW Spain) in search for quantitative thresholds for wetland classification. Twenty freshwater ponds on the aeolian sand mantle and 46 sites on silty-clay substrate, located in the southern marshland, were surveyed during the heaviest rainy period so far recorded (October 1995–September 1997). On average, temporary ponds showed higher water depth, longer flooding period, lower conductivity (<0.5 mS cm^?1), lower pH (6.7), lower phosphate concentration (0.4 μM) and a more balanced proportion of Ca²⁺/Na⁺ than temporary marshes. During floods, marshland sites exhibited higher water transparency, pH (9.5), alkalinity (3.5 meq l^?1), conductivity (8.2 mS cm^?1), phosphate concentration (0.7 μM), Na⁺ and Ca²⁺ concentrations (97.2 and 3.5 meq l^?1, respectively) than ponds. Study sites were significantly segregated (ANOSIM test: R = 0.88, P < 0.01, n = 92) in relation to water depth and conductivity. A conductivity of 1.6 mS cm^?1 is proposed as a threshold between marshland sites and ponds during floods. Marshland sites were further segregated into two groups (ANOSIM test: R = 0.777, P < 0.01, n = 23) according to the Na⁺/Ca²⁺ ratio (in meq l^?1) at a threshold value of 25. An ordination by PCA showed that five variables grouped 81.4% of the total variance in two axes. The first PCA axis (60.7% of variance) separated temporary wetlands into ponds and marshland sites according to variables related to substrate and hydrology (Na⁺/Ca²⁺ ratio, conductivity, water depth and flooding period). Other variables (e.g., water transparency, alkalinity, pH, submersed macrophyte biomass, phosphate, nitrate and planktonic chlorophyll concentrations) did not produce a significant segregation between marshland and pond sites during floods. Further discrimination within each wetland type was thus not achieved.     

Regulation and Spatiotemporal Patterns of Extracellular Enzyme Activities in a Coastal,Sandy Aquifer System (Doñana,SW Spain)

A seasonal study of extracellular enzyme activities (EEA) was conducted in the coastal, sandy aquifer system located in the greater fluvial–littoral ecosystem of Doñana (SW, Spain). Glucosidase, leucine aminopeptidase, alkaline phosphatase, and phenol oxidase activities were determined over a 2-year period in 30 piezometers spread in an area of approximately 100 km². Taking into account all enzymes, piezometers, and seasons, EEA ranged over several orders of magnitude, from 1.01?×?10^?5?±?2.92?×?10^?6 to 1.37?±?0.13 nmol (methylumbelliferyl, amido-4-methylcoumarin, or dihydroxyphenylalanine) mL^?1 h^?1. The quality, much more than the quantity, of organic matter and nutrients seemed to be the major variables that controlled the spatiotemporal patterns showed by EEA. EEA patterns obtained in this study agree with several functional models of microbial communities, such as optimal resource allocation and nutrient co-limitation. This study probably represents the first one in which these functional models have been tested in subsurface systems. Results obtained in this study seem to suggest that microbial communities inhabiting groundwaters in Doñana are not dead or compromised cells. By contrast, these communities play relevant roles in carbon and nutrient cycling, continue with the decomposition process that begins in the sediments of the shallow lakes located in the area, provide remineralized carbon and nutrients to producers of these surface aquatic systems, and close energy and matter cycles. This study proposes that groundwater systems should be considered dynamic systems, comparable in functional complexity to surface systems.     

Ongoing anthropogenic eutrophication of the catchment area threatens the Doñana World Heritage Site (South-west Spain)

Eutrophication is a major cause of wetland degradation worldwide. In recent decades, reductions in nutrient inputs have led to improvements in water quality in many rivers and lakes in central and northern Europe, but long-term trends are less clear in southern Europe. We conducted the first comprehensive study of water quality in Doñana (SW Spain), one of the most important wetland complexes in Europe and the Mediterranean region. The core area of Doñana is a large shallow, seasonal marsh (UNESCO World Heritage Site—WHS) that floods during rainy, cool winter months, then dries out during the summer. The marsh is fed by three main streams whose catchments are within a Biosphere Reserve but are impacted by greenhouses (for cultivating fruit, irrigated with groundwater), poorly treated urban wastewaters and tourism. From 2013 to 2016, we monitored nutrient (Total P, Total N, PO₄^?3, NH₄⁺, NO₃^?, NO₂^?) and phytoplankton chlorophyll-a (chla) concentrations in surface waters of the Doñana marsh and the three main streams. We quantified changes in greenhouse cover since 1995 using satellite images. Nutrient concentrations in streams were consistently higher than in the marsh, particularly in the Partido and Rocina streams that regularly reached concentrations equivalent to a “bad physico-chemical status” under the EU Water Framework Directive (WFD), and whose catchments suffered a fivefold expansion of greenhouses from 1995 to 2016. The Partido was the most polluted stream, and the most affected by wastewater effluents, and had particularly high concentrations of NH₄⁺ (Geometric Mean?=?0.3 mg L^?1) and NO₂^? (GM?=?0.52 mg L^?1) across seasons. Patterns in chla concentrations were less consistent, but streams (GM?=?6.78 µg L^?1) generally had higher concentrations than the marsh (GM?=?4.27 µg L^?1). Nutrient concentrations in spot samples within the marsh largely depended on a combination of evaporation (as revealed by higher stable isotope δ²H values in the water column) and spatial processes (concentrations increase close to stream entry points, where conductivity is lower). Anthropogenic nutrient pollution in entry streams is a serious problem in Doñana, with extensive stretches too toxic for fish. Reinforcement of policies aimed at reducing nutrient inputs to Doñana are urgently required to meet the biodiversity conservation and environmental objectives for the WHS and WFD, respectively. Paradoxically, the marsh is currently relied upon to purify the water entering from streams.

     

Long-term vegetation changes on the stabilized dunes of Doñana National Park (SW Spain)

Changes in the vegetation of stabilized sand dunes of Doñana National Park in the last four centuries have been analysed, combining ecological and historical sources of information.Quantitative vegetation data have been obtained from 5 inventories of a 15 km long transect, consisting of 34 points where nearby woody plants have been recorded. Inventories were taken in 1636, 1647, 1652, 1682 and 1859. The analysis of these data, combined with climatic and management information on the area, reveals that until 1636 a mediterranean forest dominated byQuercus suber, existed; it was used as a game preserve. From 1636 to 1682 vegetation underwent profound changes with a marked drop in trees and mature scrub. The vegetation was replaced by a pioneer scrub species. This rapid change was brought about by the introduction of cattle and pigs, timber and cork exploitation, and the use of prescribed fire. The climatic crisis that arose circa 1700 led to erosion of the sandy soils, which made the vegetation change irreversibly. Livestock pressure hampered natural forest regeneration until 1737 whenPinus pinea plantations started. From then onwards little change in vegetation is known to have occurred.It is suggested that the main steps of change in Doñana vegetation history may have occurred in many other areas of the Mediterranean Basin.     

Recolonisation potential of zooplankton propagule banks in natural and agriculturally modified sections of a semiarid temporary stream (Doñana,Southwest Spain)

The viable propagule banks of a temporary stream were studied from sections with different agricultural history. Hatching of zooplankton (copepods, rotifers and cladocerans) was recorded in the laboratory under controlled temperature and light conditions from an agriculturally modified area with average hydroperiods of about a week per year and two semi-natural reference areas with average hydroperiods of more than 3 weeks per year. We found significant differences in both taxon richness and abundance of zooplankton hatching between areas, which were lower in the agriculturally modified section, compared to the reference sections. Another factor likely to have influenced hatching in our experiment was conductivity, which differed between the two reference sections and might have affected hatching at high conductivities. For restoration purposes, hydrological reconnection of stream segments is important to facilitate dispersal from the high diversity upstream segments to the depleted sites downstream.     

Recovery of zooplankton diversity in a restored Mediterranean temporary marsh in Doñana National Park (SW Spain)

Over 30 years after drainage for agriculture, a 2700 ha temporary marshland was recently restored in Doñana National Park. We describe the recovery of zooplankton communities (copepods, cladocerans and rotifers) in 47 new temporary ponds excavated as part of the restoration project during the first two hydroperiods (April 2006 and 2007), and compare them to those of eight reference sites in the surrounding marshland. Major changes in the species composition and abundance occurred in new ponds between years. While rotifers and cyclopoid copepods dominated in terms of number of individuals in 2006, calanoid copepods and cladocerans were the most abundant groups in 2007. Rotifer species richness was significantly lower in 2007, but there was an increase in Simpson and β-diversity in 2007 owing largely to a dramatic decline in the abundance of Hexarthra cf. fennica (rare in reference sites) from 93% of all rotifer individuals in new ponds in 2006 to only 32% in 2007. In contrast, species richness of copepods and cladocerans was significantly higher in new ponds in 2007, but there were no changes in Simpson diversity. β-Diversity of cladocerans was also significantly higher in 2007. In 2006, the species richness of cladocera and copepods was significantly lower in new ponds than in reference sites, but by 2007 there were no differences in richness or Simpson diversity. Overall, 7 copepod, 13 cladoceran and 26 rotifer taxa were recorded in new ponds, including 80% of taxa recorded in reference sites. These results indicate that zooplankton communities can be rapidly restored in Mediterranean temporary wetlands, at least when large source populations in the surrounding area reduce dispersal limitation. They also illustrate the importance of comparing different metrics of richness and diversity in studies of zooplankton restoration.     

The influence of ammonia on the growth and photosynthesis of Ruppia drepanensis Tineo from Doñana National Park (SW Spain)

In a laboratory experiment, Ruppia drepanensis Tineo seedlings from a brackish marsh in Southern Spain were grown at 20 and 30 °C, at three different nitrogen levels. These levels were obtained by the addition of a slow release fertilizer (23% NH₄NO₃ by weight) to a sediment mixture of sand and clay (3:1). Several morphometric parameters were recorded during the first five weeks of the experiment, and photosynthesis and respiration were measured after 7 weeks of growth. Results showed a significant reduction of growth and development with increasing nitrogen and temperature levels. Dark respiration increased strongly at high nitrogen levels. At the same time, net photosynthesis at 250 and 500 µE m^-2 s^-1, Pm, Km and LCP were not affected by either factor. We attribute these phenomena to ammonia toxicity, since relatively high total ammonia (NH₃ + NH_f4 ^p+) levels were found in the interstitial water.     

Utility of proteomics to assess pollutant response of clams from the Doñana bank of Guadalquivir Estuary (SW Spain)

The utility of proteomics to assess pollutant response of Scrobicularia plana clams from three sites of Guadalquivir Estuary at the southern end of the National Park of Do?ana (SW Spain) has been studied. Protein expression profiles were analyzed by 2-DE in soluble fractions of S. plana gills. Nearly 2000 well-resolved spots were detected in silver-stained gels, with focused areas in the 4-6.5 pH range. Different protein expression signatures were found at each site, with the highest number of more intense spots in animals with the highest metal content. Nineteen more intense protein spots were analyzed out by nanospray-ion trap tandem mass spectrometry, de novo sequencing and a bioinformatics search for their possible identification. While sequence tags of 16 more intense protein spots were obtained, including several proteins induced by pollutant exposure of model organisms, only 2 proteins were unambiguously identified: hypoxanthine-guanine phosphoribosyltransferase (HPRT) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Both enzymes were significantly higher in animals with the highest metal contents. Thus, we propose these two proteins, HPRT and G3PDH, as novel pollution biomarkers.     

Microbial Activities in a Coastal,Sandy Aquifer System (Doñana Natural Protected Area,SW Spain)

We conducted a seasonal research of the activities of microbial communities in a coastal, sandy aquifer system located in the Doñana Natural Protected Area (SW, Spain). Groundwaters from 30 piezometers were sampled over a two-year period. The proportion of active microbial biomass ranged from 0.02 to 6.36% of the total microbial biomass, while the active microbial biomass ranged from 7.42 10 ^?3 2.20 10 ^?4 to 17.30 3.71 ngC mL ^?1 . Bacterial carbon production, measured through the incorporation of [ ³ H]leucine into cellular proteins, showed a mean value of 0.18 0.72 ngC mL ^?1 h ^?1 in all wells and all seasons. Bacterial growth rates ranged from 0.03 to 87.26 days. These activities exhibited spatiotemporal patterns. Temperature and the presence of nutrients and organic matter appear to be important factors controlling these patterns. However, hydrogeological flows, both local and regional, seemed to constitute the most important factor determining these spatiotemporal patterns, probably because the distribution of nutrients in aquifer systems is mainly controlled by these hydrogeological flows. The well-known hydrological flows connecting surface waters and groundwaters in Doñana support the assumption that both water compartments form a unique entity (called hydroecosystem), which functions as a whole. Consequently, not only microbial processes in surface waters can influence ecological processes in groundwaters, the characteristics of surface waters can also be affected by groundwater chemical processes, among others, mediated by the activities of microbial communities.     

Antibodies to Mycobacterium bovis in wild carnivores from Doñana National Park (Spain)

We conducted a retrospective serologic survey for antibodies against the MPB70 protein of Mycobacterium bovis in wild carnivores from Do?ana National Park (southwestern Spain). Serum samples from 118 red foxes (Vulpes vulpes), 39 Iberian lynx (Lynx pardinus), 31 Eurasian badgers (Meles meles), five Egyptian mongoose (Herpestes ichneumon), four European genet (Genetta genetta), and one Eurasian otter (Lutra lutra) were analyzed using an indirect competitive enzyme-linked immunoassay. Antibodies against the MPB70 protein of M. bovis were detected in seven badgers, five foxes, and one lynx. The frequency of positive animals was significantly higher in badger (23%) than in lynx (3%) and fox (4%). Antibodies were not detected in other species. Annual antibody frequency peaked at 38% in badgers and 11% for red fox. These species may contribute to persistence of bovine tuberculosis in Do?ana.