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Article

Assessing Parasite Prevalence and Health Status of the Eurasian Tree Sparrow (Passer montanus) in Green Urban Areas of a Southern European City

by
Aida Vega
1,*,
Michael J. Yabsley
2,3,4,
Sonia M. Hernández
2,3,4,
Kayla B. Garrett
2,3,
Jose I. Aguirre
1 and
Eva Banda
1
1
Biodiversity Monitoring Group, Department of Biodiversity, Ecology and Evolution, Facultad de Ciencias Biológicas, Universidad Complutense de Madrid, 28040 Madrid, Spain
2
Southeastern Cooperative Wildlife Disease Study, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA
3
Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602, USA
4
Center for Ecology of Infectious Diseases, University of Georgia, Athens, GA 30602, USA
*
Author to whom correspondence should be addressed.
Birds 2025, 6(3), 43; https://doi.org/10.3390/birds6030043 (registering DOI)
Submission received: 13 June 2025 / Revised: 9 August 2025 / Accepted: 11 August 2025 / Published: 16 August 2025
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Simple Summary

Urban environments generate new types of ecosystems whose ecological characteristics vary with local planning strategies. In Europe, the expansion of urban areas is a key conservation goal. However, the impact on wildlife health remains a relatively understudied area. The present study assesses the prevalence and parasite burden of Eurasian Tree Sparrows in five urban green areas in Madrid, Spain, over a period of four years. These green areas differed in their green infrastructure, providing a useful model to assess how green area design influences urban biodiversity. We examined parasite presence along with measures of body condition and immune status. Blood parasites were detected in 29% of individuals, and gastrointestinal parasites were found in 4% of birds. Notably, blood parasite prevalence was significantly higher in green areas characterized by the presence of stagnant, untreated water and muddy margins, which may be optimal breeding sites for vectors. Our findings highlight the role of water management in urban green areas as a key factor influencing parasite transmission. Incorporating parasite risk into green area planning can improve wildlife conservation and reduce potential health risks to humans in urban areas.

Abstract

Urban landscapes have given rise to novel ecosystems (e.g., green areas), which differ in design and ecological quality depending on local planning strategies. Europe has the goal to increase conservation through increasing greenspace; however, urban wildlife health impacts, particularly on birds, are poorly studied. This study investigates associations between haemosporidians and intestinal coccidia in the Eurasian Tree Sparrow (Passer montanus), as well as their body condition and immunological status, from five urban green areas in Madrid, Spain, from 2019 to 2022. These green areas differ in green infrastructure, and because these birds are adapted to urban environments, they are a good model to evaluate how green area infrastructure may affect the birds’ health. We detected a 29% prevalence of haemosporidians (Haemoproteus being the most common, followed by Leucocytozoon and Plasmodium) and a 4% prevalence of intestinal coccidia. We found that haemosporidian prevalence was significantly higher in green areas with untreated stagnant water surrounded by muddy areas, ideal conditions for vector reproduction. Therefore, effective management strategies, especially related to water treatment, are essential for protecting urban wildlife and human health. This study provides valuable information for researchers and urban wildlife managers to incorporate appropriate management strategies into urban green area planning to preserve urban biodiversity and protect public health.

1. Introduction

Urban green areas play a critical role in shaping biodiversity and ecosystem functions in cities. In Europe, strategies aimed at promoting biodiversity within urban areas increasingly focus on enhancing green infrastructure, including urban green areas, street trees, and water bodies. In Spain, local planning frameworks aim to integrate biodiversity goals into urban design [1]. These strategies promote the appropriate management planning (including freshwater) and protect and restore biodiversity and well-functioning ecosystems, which are key to enhancing resilience and preventing the emergence and spread of future disease outbreaks [2]. Understanding how specific features of urban green spaces influence wildlife is therefore a key challenge in modern urban ecology.
The growth of cities is transforming natural environments into urbanized areas. This transformation has led to the development of novel ecosystems, such as gardens and urban green areas [3]. As the focus on these areas and their associated wildlife increases, new challenges arise in balancing human needs with those of wildlife [4].
The European Commission defines green infrastructure as “a strategically planned network of natural and semi-natural areas with other environmental features designed and managed to deliver a wide range of ecosystem functions. It incorporates green spaces (or blue if aquatic ecosystems are concerned) and other physical features in terrestrial (including coastal) and marine areas.” [5]. (Semi)-natural areas mainly consist of green areas such as parks and trees along streets, blue areas including ponds and rivers, and gray-green-blue areas such as green walls or roofs [6].
Today, the promotion of green infrastructure, healthy ecosystems, and nature-based solutions is usually integrated systematically into urban planning, including public spaces, the design of buildings, and their surroundings [7]. In fact, urban green areas not only act as a recreational area and a refuge for biodiversity but also play a crucial role in the ecology of infectious diseases.
The ecology of infectious diseases and their transmission dynamics are closely linked to the abiotic and biotic factors contained in the green infrastructure [8]. Although green infrastructures can act as a barrier to the amplification and spread of some diseases in humans and animals, they also have the potential to act as a channel that facilitates disease transmission. The management structure and composition of the green infrastructure, including the connectivity between green spaces and water bodies, can influence the abundance and distribution of vectors and hosts [9]. Studies have shown that these areas can facilitate the spread of heteroxenous parasites [10,11]. Water in urban green areas can act as concentration points for animals such as birds, enhancing the opportunities for vector-host interactions. It has been noted that the presence of water resources favors breeding sites for vectors of parasites [12,13].
The Eurasian Tree Sparrow Passer montanus Linnaeus, 1758, is common in European urban green areas as it has high adaptability to these environments [14] and is thus classified as an urban exploiter [15]. Long-term studies have shown that this species tends to persist and increase in urbanized landscapes, unlike other closely related sparrows that have been declining with increasing urban intensity [16]. In Spain, it is considered a species of Least Concern, and globally it is listed as Least Concern by the IUCN Red List [17]. The Eurasian Tree Sparrow is a cavity-nesting species, often using natural cavities as well as artificial nest boxes when available. In our study, all green areas except two had nest boxes for long-term bird monitoring and biodiversity enhancement programs. The species is also highly social and often nests in loose groups or forms flocks outside the breeding season [18]. This may increase opportunities for parasite transmission through close contact or shared vector exposure. Due to its non-migratory nature, this species shares its habitat with a variety of vectors and parasites throughout the year. Therefore, the Eurasian Tree Sparrow is an ideal model species for understanding how urban green infrastructure could, in the long term, impact its health.
Several studies have shown that the ecology of parasites is influenced by host and environmental factors [19,20]. Infection prevalence often differs between ecological bird groups that occupy distinct habitats, likely due to differences in exposure to vectors [21]. Seasonal changes are also relevant, as haemosporidian infections prevalence typically occurs in spring and summer, coinciding with increased vector activity [22]. Furthermore, infection dynamics could be affected by the host’s age and sex: juveniles frequently exhibit higher parasite loads and are more prone to acute infections, while adults are more likely to sustain low-intensity infections [23].
Parasitic infections can exert costs on the host, affecting body condition and immune status, which are used to assess overall health. While experimental studies have supported that prevalence and parasite load can significantly reduce body condition [24], other research has suggested that there may not be a direct relationship between body condition and the likelihood of parasitic infection [25]. Moreno-Rueda [26] reported that only individuals in good condition are able to cope with high parasite loads, while those with poorer body conditions may experience parasite-induced mortality.
Immune status reflects the host’s ability to protect itself from parasitic infections, and it plays a crucial role in these dynamics. Leukocytes, or white blood cells (WBC), are important indicators of a portion of the immune system [27]. A compromised immune system could increase the susceptibility to parasites, which may lead to an increase in energy demands that could disrupt other physiological processes [28]. Thus, the body condition and immune status of the Eurasian Tree Sparrow not only provide information on their health but also act as an indicator of the potential quality of the green infrastructure. The species could turn into a sentinel to monitor vector-host interactions and pathogen dynamics, providing valuable insights into how urban planning affects wildlife health and public health.
Madrid is a big city (606 square kilometers) with diverse urban green infrastructure due to differential management, maintenance of gardens, and water management according to the urban planning and the Biodiversity Promotion and Management Plan [29]. This study had two main objectives: (1) to assess the prevalence and burden of blood and gastrointestinal parasites in Eurasian Tree Sparrow across five green areas in Madrid, and (2) to evaluate their relationship with host body condition and immune status. We hypothesized that the parasite prevalence and burden would be higher in green areas with stagnant, untreated water and muddy margins, as these conditions, often associated with urban green space management, are known to provide optimal breeding habitats for the vectors of haemosporidian parasites. We also expected that individuals from more heavily parasitized areas would exhibit lower body condition scores and higher white blood cell count (WBC), a crude indicator of the immune system function of the individual birds.

2. Materials and Methods

2.1. Study Sites

Madrid is the capital of Spain with approximately 3.3 million inhabitants and a population density of around 5400 inhabitants per square kilometer [30]. The city is located in the central Iberian Peninsula (40° N, 3° W), within the Mediterranean continental climatic zone, characterized by hot, dry summers and cold winters, with annual precipitation of 436 mm and an average annual temperature of 14.6 °C. During the study period (2019–2022), weather conditions were broadly consistent with long-term climatic averages [31]. In terms of vegetation, Madrid lies within the Mesomediterranean bioclimatic belt, where typical flora includes holm oak, Quercus rotundifolia Lam., Mediterranean shrubs, and riparian species in wetter zones. As part of its urban planning and ecological strategy, Madrid maintains over 33% green and blue coverage, including historical green areas, peri-urban forests, and artificial water bodies, with more than 250,000 street trees and 150 fountains and ponds [29].
Data were collected at five constant effort ringing stations (CESs) between August 2019 and March 2022 (32-month period). All CESs are located in urban green areas in Madrid city, except for one located in a peri-urban site (Figure 1). The five sites are Finca El Garzo, Encinar de San Pedro, Parque del Oeste, Parque Juan Carlos I, and Madrid Río.
Finca El Garzo is a 400-hectare peri-urban non-managed natural area located in Las Rozas de Madrid, 10 km from the city center of Madrid. It is located in the protected area of Cuenca Alta del Manzanares Regional Park. It was listed as a Site of Community Interest (SCI) of the Red Natura 2000 and declared a Biosphere Reserve by UNESCO [32]. The site is bordered by mixed-use areas, including residential developments. Light industrial facilities and transportation infrastructure. In this area, there are dense meadows that stay green all year round since the soil is kept moist by the presence of a small creek that does not have a paved shore. This creek is fed by the train repair factory wastewater treatment plant, which flows with a designed and authorized discharge of 200 m3/day, continuous throughout the year. The creek is surrounded by deciduous species such as ash Fraxinus angustifolia Vahl, silver poplar Populus alba L., and blackberry Rubus ulmifolius Schott. There are also holm oaks in the surrounding area. Even though the area has restricted access and low recreational use, occasional hikers and locals visit. The Eurasian Tree Sparrow is a common resident at his site and breeds in natural cavities found in older trees, as well as in artificial nest boxes installed.
Encinar de San Pedro is a semi-natural area with low management located in Casa de Campo, in the western area of the city of Madrid. It is an 80-hectare area with restricted access, whose priority action is the conservation and regeneration of the Mediterranean scrub [33]. The green area is surrounded by extensive woodland, the Madrid Zoo to the south, and urban neighborhoods to the east, creating a buffer between natural and urban areas. The vegetation is dominated by holm oak, with a shrub border of yellow broom Retama sphaerocarpa (L.) Boiss and gum rockrose Cistus ladanifer L. The area has three untreated ponds with unpaved shores where species related to riparian forests can be found. The ponds are also used as bathing sites for wild pigs, Sus scrofa L., 1758, whose activities result in the formation of mud around the pond edges. Public access is limited, and recreational use is minimal due to the conservation aim of the area. The Eurasian Tree Sparrow is observed all year round, particularly near pond edges. The site includes artificial nest boxes.
Parque del Oeste is located in the NW area of the city of Madrid and covers an area of 98 hectares. The green area was created in 1893, and it is one of the five historical green areas in the city [34]. The green area is surrounded by dense urban development, including residential buildings, major roads, and the nearby university district. Despite its urban setting, it retains a mixture of formal gardens and semi-natural areas. In one of the peripheral areas of the green area, there is a birding area, created in 1992, where the CES is located. It has a feeding area and autochthonous plants such as holly Ilex aquifolium L., dog rose Rosa canina L., and laurustinus Viburnum tinus L. [35]. It also includes an artificial paved pond with nontreated water, where domestic animals bathe, and as a result, mud is accumulated along the pond’s perimeter. Only the birding area is subject to specific management; the rest of the green area is maintained as a conventional urban green space with ornamental gardening. The Eurasian Tree Sparrow is commonly present in less disturbed corners and near feeding areas and ponds. The green area and the birding zone include nest boxes.
Parque Juan Carlos I is an urban green area that covers an area of 220 hectares, 21 of which are occupied by olive trees Olea europaea L., as a project to recover the old Olivar La Hinajosa [36]. Water is one of the fundamental elements of the green area, and consists of a river, a canal, and a biologically treated, paved, and reclaimed water lake of 30,000 m2 with thousands of common carp Cyprinus carpio L. for mosquito control. The CES is an adjunct to the lake and has restricted access. The area is dominated by species associated with humid forests, including ivy Hedera helix L., primrose jasmine Jasminum mesnyi Hance, silver poplar, and ash trees. Management intensity varies across the green area. While some areas, such as the olive grove, receive minimal intervention and maintain a more natural character, other zones are subject to intensive ornamental gardening and landscape maintenance. Recreational use of the green area is high, with walking paths, bike lanes, playgrounds, and open lawns. The Eurasian Tree Sparrow is frequently observed throughout the green area, especially near dense vegetation and water features. No artificial nest boxes are present.
Madrid Río is a 429-hectare linear urban artificial green area with high management. It was designed in 2003, coinciding with the undertaking of the original highway ring around downtown. This green area aimed to create a green corridor and regenerate the fluvial ecosystem of the Manzanares River. It stretches from El Pardo in the NW to Getafe in the NE, linking gardens and urban green areas [37]. The green area is surrounded by a mix of densely built residential neighborhoods (such as Arganzuela, Legazpi, and Usera), historical landmarks (e.g., the Royal Palace and Matadero Madrid), and major transportation infrastructures, including the M-30 ring and several bridges and tunnels. Its urban layout makes it a transit-adjacent green corridor, offering continuous access along the riverfront. The green area includes 47 species of trees, 38 species of shrubs, and 21 hectares of pastures. Access to the Manzanares River basin is forbidden to people and domestic animals. The only exception is an urban beach, where there are some water fountains with cement pavement. In addition to the river, 13 ornamental fountains are located along Madrid Río. The CES is near one of these fountains with stagnant treated water and cement pavement. The CES is located in a pasture area where holm oak, strawberry tree Arbutus unedo L., and common box Buxus sempervirens L. predominate as autochthonous species, and boxwood Buxus colchica Pojark., and the genus Albizzia Benth., as allochthonous species.

Description of the Habitat

To characterize the habitat, a 1 × 1 km2 digital satellite image was plotted around each CES on a grid divided into 100 cells. Each cell was assigned a score for four major land cover types: vegetation cover, building density, percent pavement, and water percentage as described by [38] (Table 1). Additionally, we considered whether the water was treated or untreated and if the water was flowing or stagnant. These land cover types capture important differences in the green infrastructure at each site.

2.2. Birds Sampling

Eurasian Tree Sparrows were captured at ringing stations using mist nets. Each station followed a protocol based on the CES scheme [39], which maintains a constant ringing effort, with the same number and placement of the nets every 10 days. Mist nets are set from dawn for five hours, which is the peak capture period [40].
Birds were individually marked with aluminum rings with a unique alphanumeric code [41]. Upon capture, they were weighed and measured (wing cord and tarsus-metatarsus length). Adults were sexed based on morphological characteristics such as brood patch or cloacal protuberance since there is no sexual dimorphism in plumage for this species, and age classes were assigned (first year and adult) according to Demongin, Moss [42] and Blasco-Zumeta, Heinze [43]. Body condition was assessed as an indicator of overall health for each individual and was calculated as the standardized residuals of a major axis regression (RMA) of tarsus length on body mass [44]. This yielded a unitless index, where positive values indicate better-than-average condition for a given body size.
Blood samples were obtained from each individual (n = 108) via jugular vein using a 30-gauge needle. The number of individuals sampled at each site was as follows: Finca El Garzo (n = 8), Encinar de San Pedro (n = 21), Parque del Oeste (n = 37), Juan Carlos I (n = 15), and Madrid Rio (n = 27). Two drops of blood were used to make two thin blood smears, and the rest was preserved in 96% ethanol for DNA amplification [45]. One set of fecal samples (n= 66) was preserved in a 2% potassium dichromate (K2Cr2O7) solution to identify sporulated oocysts [46]. Other fecal samples (n = 47) were immediately transferred to 1.5 mL Eppendorf tubes with 70% ethanol in an amount sufficient to cover the fecal material. All fecal samples corresponded to individuals from whom blood was also collected. Only 21 individuals provided blood samples along with both types of fecal samples.

2.3. Laboratory Work

2.3.1. Blood Parasite Detection and Leukocyte Profile

Blood smears were fixed with absolute ethanol, and later they were stained with a Kit for Rapid Staining in Hematology (Rapid Panopticon) (CE-IVD) for clinical diagnosis (Panreac AppliChem, ITW Reagents, Barcelona, Spain) [47].
Blood smears were examined at 1000× magnification with oil immersion using a MOTIC Model BA210LED light microscope (Motic, Kowloon, Hong Kong, China) to calculate a differential blood count of leucocytes and to determine the parasitemia for blood parasites. Parasitemias were calculated after examination of approximately 50,000 erythrocytes. This reduces the possibility of underestimating the prevalence and provides a more accurate parasitemia. Parasites were identified according to Valkiūnas [48] and Valkiūnas, Iezhova [49] keys.
To calculate a differential white blood cell count, the total number of white blood cells (WBC) per 10,000 red blood cells was also recorded, and each leukocyte was classified as heterophil, lymphocyte, basophil, monocyte, or eosinophil. The percentage of each white cell type was calculated and multiplied by the total WBC count to obtain the absolute count [50]. The ratio of heterophils to lymphocytes (H/L ratio) was calculated by dividing the relative heterophil counts by the relative lymphocyte counts. The H/L ratio is often associated with environmental stress [51,52].
Blood smear examination and parasite counts were conducted by two observers (A.V. and E.B.). Prior to analysis, we performed a repeatability test (r = 0.93) by reviewing a subset of smears to establish and agree on identification and counting criteria, ensuring consistency across all observations.

2.3.2. DNA Extraction and Nested PCR Analysis

Approximately 50 μL of ethanol-fixed blood (equivalent to ~5 μL of whole blood) was allowed to dry in a plastic snap-top tube with 10 μL of dPBS to ensure blood did not fully dry out but that ethanol was fully evaporated. Genomic DNA was extracted from ~5 μL of whole blood samples using a DNeasy® Blood & Tissue Kit Protocol for Animal Blood (Hilden, Germany) according to the manufacturer’s protocol for nucleated erythrocytes.
For the detection of Haemoproteus, Leucocytozoon, and Plasmodium species, a nested PCR protocol for targeting the cytochrome b (cyt-b) gene of the parasite mitochondrial genome (mtDNA) was performed as described [53,54]. The first round of PCR primers targeted all three genera using primers HaemNFI [5′-CATATATTAAGAGAAITATGGAG-3′] and HaemNR3 [5′-ATAGAAAGATAAGAAATACCATTC-3′]. Two sets of second-round PCRs were run targeting Haemoproteus and Plasmodium using primers HAEMF [5′-ATGGTGCTTTCGATATATGCATG-3′] and HAEMR2 [5′-GCATTATCTGGATGTGATAATGGT-3′] and Leucocytozoon species using primers HaemFL [5′-ATGGTGTTTTAGATACTTACATT-3′] and HaemR2L [5′-CATTATCTGGATGAGATAATGGIGC-3′].
Amplicons were gel purified with the Qiagen QIAquick® Gel Extraction Kit (QIAGEN, Hilden, Germany) and bidirectionally sequenced at Genewiz (South Plainfield, NJ, USA). Sequences were assembled in Geneious 2023.2.1 (Biomatters Limited, Auckland, New Zealand [http://www.geneious.com/]) and compared to related sequences using the BLASTN algorithm in GenBank in the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 19 July 2024)).
Nested PCR has been used in conjunction with direct microscopic visualization to exploit the strengths of each method. While nested PCR is highly sensitive for detecting parasites, particularly low parasitemia, it cannot quantify parasite load [55]. In addition, PCR may underestimate the prevalence in co-infection because it tends to detect only the most abundant parasite. Furthermore, locally circulating haemosporidian variants may differ at primer binding regions, reducing PCR amplification.

2.3.3. Coprological Examinations

The fecal samples in 2% K2Cr2O7 solution were kept at room temperature for at least 7 days to allow oocysts to sporulate. A saturated saline solution of sodium chloride (NaCl) with a specific gravity (SG) of 1.2 was used for the flotation technique. Samples were allowed to float undisturbed for 5 min before analysis with a McMaster chamber [56].
A combined sedimentation-flotation method was followed for fecal samples in 70% ethanol. The samples were filtered and centrifuged. The supernatant was decanted, and the sediment was resuspended with 1 mL of saturated saline solution (NaCl) or zinc sulfate heptahydrate flotation solution (ZnSO4·7H2O), both with an SG of 1.2. Five smears with five drops of the floated sediment were analyzed microscopically. The rest of the sample was then evaluated in a McMaster chamber [57].

2.4. Data Analysis

Generalized linear models (GLMs) were conducted using a binomial family distribution to analyze the influence of the month, body condition, and green area on blood parasite prevalence (0 absence, 1 presence, combined results of blood smear and PCR analysis). Prevalence data from PCR and microscopy were pooled, with parasite load estimates derived only from microscopy. The combination of these two methods appears to be the most effective approach for determining true infection. In addition, GLMs using a Gaussian family distribution were conducted to analyze the blood parasite load (number of parasitized cells per individual) and the variation in body condition and immunological condition (percentage of heterophils, lymphocytes, basophils, monocytes, and eosinophils) using parasite prevalence and parasite load, month, body condition, and green area as independent variables.
The Akaike Information Criterion (AIC), along with model weights and delta values, was used for model selection among the multiple ones constructed. The ‘stepAIC’ function (package: MASS, version: 7.3-60) was used for a backward approach variable selection, systematically removing non-significant variables. Statistical significance was assessed using a p-value threshold of 0.05. All statistical analyses were conducted in R Version 4.3.3 [58].

3. Results

Of the 108 blood smears analyzed, 29% (n = 31) were positive for blood parasites in the genera Haemoproteus Kruse, 1890, Leucocytozoon Berestneff, 1904, and/or Plasmodium Marchiafava et Celli, 1895 (Figure 2). Haemoproteus was the most prevalent haemosporidian parasite, whereas Plasmodium was the least common (Table 2). Since Plasmodium and Leucocytozoon were only detected in low prevalences, the analysis of parasite load models focused on the genus Haemoproteus. Two individuals had co-infections: one sample was co-infected with Haemoproteus and Plasmodium, and the other with Haemoproteus and Leucocytozoon. No additional blood parasite taxa were detected in the samples analyzed.
In addition, marked variability in parasite prevalence was observed between the different sampling sites (Figure 3). For example, the overall parasite presence in the Madrid Río green area showed fewer infections and parasite diversity compared to the Parque del Oeste and Encinar de San Pedro green areas. Haemoproteus was the dominant parasite in the Parque del Oeste green area.
Sequence analysis revealed that the haemosporidian lineages detected in this study included Haemoproteus passeris (Kruse, 1890) [59] (n = 8) and Plasmodium relictum (Grassi and Feletti, 1891) [60] (n = 2). Molecular characterization of blood parasites is a complementary technique to species-level parasite detection, as it facilitates the differentiation of morphologically similar species (Table 2).
In total, 66 samples preserved in 2% K2Cr2O7 solution and 42 fecal samples preserved in 70% ethanol were examined for intestinal parasites. No gastrointestinal helminths were detected in 70% ethanol, but a coccidian prevalence of 4% (n = 3) was detected in 2% K2Cr2O7 solution. The sporulating oocyst is spherical (25 × 25 μm) with a smooth, colorless, or pale yellowish wall. Both sporocysts are lemon-shaped, and the Stieda body can be seen in the polar region as a hyaline mass. Each sporocyst contains four elongated sporozoites (Figure 4). Due to its morphological features, the coccidian species is compatible with the genus Isospora Schneider, 1881 [61].
Binomial GLMs were performed to evaluate the blood parasite prevalence of the three genera. The best model for prevalence included only the variable green area. Model estimates indicated that individuals with blood parasites were primarily located in Parque del Oeste and Encinar de San Pedro compared to the other sites (Table 3; Figure 5A).
Gaussian GLMs were used to assess the Haemoproteus load. The best model included only the green area. Encinar de San Pedro showed a significant trend, and Parque del Oeste was significant (Table 4; Figure 5B). The model indicates that these two green areas have the highest parasite load.
Gaussian GLMs were performed to evaluate the factors influencing body condition. The best model included Haemoproteus parasitemia, green area, and month as predictors (Figure 6). Individuals who presented a better body condition had a higher Haemoproteus parasitemia. During the summer months, between May and August, individuals exhibited lower body condition.
The GLM analysis of the leukocyte differential data demonstrated significant differences in the lymphocyte and eosinophil percentage, but no differences were found for the rest of the immune cells or the H/L ratio. The best GLM for lymphocytes revealed that green area and body condition significantly influenced lymphocyte percentage. There was a significant negative association between lymphocyte percentage and body condition (Table 5; Figure 7). The GLM analysis for eosinophil percentage showed a significant effect of green area and a negative association with Haemoproteus infection (Table 5; Figure 8).

4. Discussion

In this study, we investigated the prevalence and parasitic load of blood and gastrointestinal parasites in the Eurasian Tree Sparrow, as well as the effects on the body condition and immune status of individuals across multiple urban green areas in Madrid city and surroundings, characterized by different types of water management and green infrastructure. The main results revealed that the Haemoproteus prevalence and parasitemia were different among the five green areas studied (Figure 3, Table 3 and Table 4). Additionally, following our predictions, there were measurable effects in heavily parasitized areas on body condition and immune status.
The potential for movement of Eurasian Tree Sparrow between green areas was considered as a limitation of this study. Although dispersal cannot be entirely excluded, available data suggest that regular inter-park movements are unlikely. Radiotracking studies indicate that this species is largely sedentary, with average home ranges between 0.8 and 11.5 hectares and daily movement distances of approximately 600 m [62,63]. In our long-term ringing program operating 10 Constant Effort Sites across Madrid, the recapture rate of marked individuals between parks is less than 0.001% [64], supporting this inference. Given that the green areas studied range from 21 to 220 hectares and are separated by several kilometers of urban matrix (Figure 1). Thus, each site was treated as an ecological unit in our analyses. While this approach does not explicitly model green area-specific variables, these characteristics were described qualitatively (Table 1) and guided the interpretation of observed differences in prevalence and burden of parasite and host condition.
As predicted, the blood parasite prevalence was significantly higher in green areas with stagnant untreated water surrounded by muddy areas, suggesting that the urban green infrastructure and the type of water management promote biotic and abiotic factors that favor vectors and parasite transmission. In this study, each green area was analyzed as an ecological unit, treating its integrated green infrastructure features (e.g., water type and shoreline structure) as part of the broader site context. These aimed to identify ecological patterns across sites with different combinations of percent habitat characteristics, rather than isolate the statistical contribution of each individual variable. Although these features were not included as covariates in our statistical models, they were recorded and are described in detail (Table 1) to support the ecological interpretation of our findings.
Previous research [65,66] indicated that haemosporidian parasites are typically transmitted by specific dipteran vectors: Haemoproteus by Culicoides biting midges, Plasmodium by Culex mosquitoes, and Leucocytozoon by black flies, Simuliidae [67,68,69]. These vectors often breed in habitats with stagnant, organic-rich water and muddy margins, conditions that enhance oviposition and larval development that could increase parasite transmission risk [70].
In our study, the green areas with the highest Haemoproteus prevalence shared these features: stagnant, untreated water sources and muddy shorelines with limited maintenance. In contrast, green areas with treated and flowing water and regular cleaning showed lower parasitemia levels, likely due to reduced suitability for vector development [71]. Furthermore, natural, or unpaved, shorelines that retain soft soil and organic debris may facilitate vector reproduction, while engineered or paved edges limit these opportunities [72]. These findings emphasize the role of local habitat in influencing vector-borne parasite transmission, reinforcing the value of microhabitat-level assessment over broad urban-rural comparisons [73,74]
The higher detection rate of haemosporidians by microscopy compared to nested PCR in Table 2 is counterintuitive, as molecular methods are generally more sensitive [55]. One potential explanation for this phenomenon is that parasitemia was estimated after screening 50,000 erythrocytes per individual—approximately five times the conventional 10,000 erythrocytes. This may greatly reduce false negatives at low intensities and could outperform PCR under very low burdens [75]. In accordance with these findings, nested PCR did not identify additional positives beyond those evident microscopically.
In addition to blood parasites, gastrointestinal parasites were also examined, though their detection was limited. Isospora was detected only rarely in our study. The low detection rate may be influenced by the timing of our sampling, as Isospora oocysts have been demonstrated to be excreted by passerines in the late afternoon or evening [76]. However, no studies have specifically examined the excretion patterns of Isospora in Eurasian Tree Sparrows. Moreover, the probability of Isospora transmission in urban areas is associated with factors such as elevated population density and constrained spatial dispersion of individuals, which can result in increased fecal contamination [77]. The implementation of management practices in urban areas, such as regular maintenance and removal of fecal matter, could limit the transmission potential.
Recent European and local policies emphasize biodiversity-friendly urban planning. The EU Nature Restoration Law (Regulation EU 2023/411) calls for integrating multifunctional green infrastructure and rewilding approaches to enhance urban ecosystems and their functions [78]. In Madrid, the Biodiversity Promotion and Management Plan [29] emphasizes the use of more natural designs for urban green areas and water features and their role in ecological connectivity, flood mitigation, and social well-being [79].
While these strategies are grounded in ecological principles, our findings suggest that insufficient maintenance of certain green areas may inadvertently create favorable conditions for parasite vectors and reservoirs and may lead to potential consequences for urban wildlife and public health [80]. Therefore, maintenance efforts should be accompanied by targeted monitoring to avoid compromising animal, human, and ecosystem health.
We found a positive relationship between Haemoproteus parasitemia and body condition (Figure 6A). Although this seems counterintuitive, this could be explained by differences in mortality rates between individuals [81]. Birds with better body condition may be better able to cope with physiological stress and higher parasite loads [82]. In addition, the use of mist nets as a capture method for wild birds could influence the results of the study, as it selects for individuals that can withstand the demands of high parasitemia, while weaker individuals would not be captured, as they may experience morbidity or mortality. Lastly, Haemoproteus, Plasmodium, and Leucocytozoon spp. in their natural hosts generally do not pose a health threat [48].
Site-specific differences in Haemoproteus parasitemia and body condition by site and month could be due to several factors. Different green areas have variable food availability [83] and microclimate differences in roosting sites that may increase the vulnerability of birds to major stressors such as predation pressure or noise and light pollution [84], and variations in green infrastructure. Additionally, the Eurasian Tree Sparrow exhibits biparental care, and the breeding season extends from April or May to July [85]. During the breeding season, activities such as nest building, incubation, and chick feeding require considerable energy from the parents. The increased energy demands and stress on birds during this period contribute to a seasonal decline in the energy reserves of individuals, which can lead to poorer body condition [86]. Similar patterns have been reported in Pied Flycatchers nesting near forest water bodies, where individuals exhibited higher haemosporidian infection rates and reduced body condition compared to those nesting farther from water sources [87]. These findings suggest that seemingly favorable habitats could act as ecological traps when they also support high densities of vectors.
As expected, we observed variations in WBC counts and differentials related to body condition and parasite prevalence. Individuals with lower body conditions tended to have higher lymphocyte percentages (Figure 7A, Table 5). Irizarry-Rovira [88] and Samani [89] reported that elevated lymphocytes indicate that individuals with poorer body conditions are likely to be experiencing acute processes such as acute inflammation caused by stressors like parasites. Similarly, eosinophil counts were higher in infected individuals (Figure 8A, Table 5), consistent with the role of these cells in defending against parasitic infections. Eosinophils contain granules rich in peroxidase, lysozyme, and arginine, which are key components of parasite suppression [90].

5. Conclusions

Our results highlighted differences in parasite prevalence between five green areas in Madrid city and surroundings, Spain, with a higher prevalence in areas with stagnant and untreated water surrounded by muddy areas. These findings suggest that some urban green spaces have favorable habitats for parasite transmission. Still, instead of recommending interventions like water treatment, which could unintentionally produce environmental impacts, we propose to design urban green spaces that support structurally diverse ecosystems. Nevertheless, it has to be considered that some areas in Madrid have already implemented Bacillus thuringiensis israelensis (Bti) for the management of black fly control.
The Eurasian Tree Sparrow could serve as an indicator species, and its parasite burden can provide insights into the overall quality and biodiversity of urban green spaces. Additionally, our results showed a correlation between Haemoproteus parasitemia, body condition, and immune status. This result implies that individuals with better body condition may endure higher parasite loads. In contrast, those individuals with poorer body conditions tend to exhibit higher lymphocyte percentages that could be linked to higher stress or infection. Additionally, parasite presence was associated with a higher eosinophil percentage that reflects the role of the cells in combating parasitic infections.
Finally, the rare detection rate of Isospora suggests that urban management practices may help limit its prevalence. Further studies on excretion patterns in Eurasian Tree Sparrows are needed to clarify the influence of sampling timing.
To ensure effective wildlife health, it is necessary to establish a network of CES across urban green areas. Such a network would provide valuable data on parasitic infections in wildlife through continuous and systematic monitoring, which would facilitate early detection of emerging zoonotic diseases.

Author Contributions

Conceptualization, E.B. and J.I.A.; methodology, A.V., E.B. and J.I.A.; formal analysis, A.V., E.B. and J.I.A.; investigation, A.V., E.B., J.I.A., M.J.Y., S.M.H. and K.B.G.; data curation, A.V., E.B., J.I.A., M.J.Y., S.M.H. and K.B.G.; writing—original draft preparation, A.V.; writing—review and editing, E.B., J.I.A., M.J.Y., S.M.H. and K.B.G.; supervision, E.B. and J.I.A.; project administration, E.B. and J.I.A.; funding acquisition, E.B., J.I.A., M.J.Y., S.M.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by ENARA E.A. and the Biodiversity Monitoring Group from the Universidad Complutense de Madrid, through the Ayuntamiento de Madrid (grant numbers EXP. 300/2022/00034 and EXP. DT07C003P1528813). Aida Vega was supported by the Funding Program of Complutense University of Madrid and Santander Bank (grant number CT25/24).

Institutional Review Board Statement

This study was performed with all the necessary permits issued by Consejería del Medio Ambiente, Vivienda y Agricultura, Área de Conservación de Flora y Fauna, Comunidad de Madrid, and biological sampling licenses required according to the Spanish law (Ref: 10/073306.9/23, PROEX 172.8/23). Birds were released in the same area of capture immediately after ringing and sampling without any apparent changes in behavior or health.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We wish to thank Beatriz Martínez Miranzo and Jorge Belinchón Aznar for their invaluable assistance with fieldwork and sample collection.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Map of the community of Madrid in central Spain showing the sites of the five constant effort ringing stations (CESs).
Figure 1. Map of the community of Madrid in central Spain showing the sites of the five constant effort ringing stations (CESs).
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Figure 2. Morphological characteristics of three genera of blood parasites from Eurasian Tree Sparrow (Passer montanus). Almost fully grown gametocyte of Haemoproteus (a); gametocyte of Leucocytozoon (b); and mature erythrocytic meront of Plasmodium (c). Simple triangular arrowheads, host cell nuclei. Simple arrows, parasite nuclei. Scale bar = 10 μm.
Figure 2. Morphological characteristics of three genera of blood parasites from Eurasian Tree Sparrow (Passer montanus). Almost fully grown gametocyte of Haemoproteus (a); gametocyte of Leucocytozoon (b); and mature erythrocytic meront of Plasmodium (c). Simple triangular arrowheads, host cell nuclei. Simple arrows, parasite nuclei. Scale bar = 10 μm.
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Figure 3. Geographical graph with latitude and longitude as the axes showing the green areas sampled. Below the name of each site, the total number of samples collected and the percentage and total number of samples positive for infection are recorded. The colors of the pie chart show a different category according to the proportion and number of positive samples for each genus of blood parasite and uninfected Eurasian Tree Sparrow (Passer montanus) individuals. Orange: percentage of samples showing no infection; Purple: percentage of samples positive for Haemoproteus; Blue: percentage of samples positive for Leucocytozoon; Pink: percentage of samples positive for Plasmodium.
Figure 3. Geographical graph with latitude and longitude as the axes showing the green areas sampled. Below the name of each site, the total number of samples collected and the percentage and total number of samples positive for infection are recorded. The colors of the pie chart show a different category according to the proportion and number of positive samples for each genus of blood parasite and uninfected Eurasian Tree Sparrow (Passer montanus) individuals. Orange: percentage of samples showing no infection; Purple: percentage of samples positive for Haemoproteus; Blue: percentage of samples positive for Leucocytozoon; Pink: percentage of samples positive for Plasmodium.
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Figure 4. Sporulated oocyst of Isospora spp. with two oblong-shaped sporocysts and four elongated sporozoites in each sporocyst. W: oocyst wall; SP: sporocyst; SB: Stieda body; SE: Sporozoite. Scale bar = 10 μm.
Figure 4. Sporulated oocyst of Isospora spp. with two oblong-shaped sporocysts and four elongated sporozoites in each sporocyst. W: oocyst wall; SP: sporocyst; SB: Stieda body; SE: Sporozoite. Scale bar = 10 μm.
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Figure 5. Predicted values of blood parasite prevalence (A) and Haemoproteus spp. load (B) in different green areas according to the fitted model. The dots represent the predicted values for each green area, while the vertical lines indicate the confidence intervals.
Figure 5. Predicted values of blood parasite prevalence (A) and Haemoproteus spp. load (B) in different green areas according to the fitted model. The dots represent the predicted values for each green area, while the vertical lines indicate the confidence intervals.
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Figure 6. Positive relation between Haemoproteus parasitemia (per 50,000 erythrocytes) and body condition (A); body condition related to the green area (B); and body condition during the months (C). Body condition refers to the standardized residuals from major axis regression of body mass on tarsus length. Positive values indicate better-than-average condition for a given body size.
Figure 6. Positive relation between Haemoproteus parasitemia (per 50,000 erythrocytes) and body condition (A); body condition related to the green area (B); and body condition during the months (C). Body condition refers to the standardized residuals from major axis regression of body mass on tarsus length. Positive values indicate better-than-average condition for a given body size.
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Figure 7. Negative relationship between lymphocyte percentage and body condition (A); lymphocyte percentage in different green areas of Madrid (B).
Figure 7. Negative relationship between lymphocyte percentage and body condition (A); lymphocyte percentage in different green areas of Madrid (B).
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Figure 8. Percentage of eosinophils without and with Haemoproteus spp. (A); eosinophil percentage in different green areas of Madrid (B).
Figure 8. Percentage of eosinophils without and with Haemoproteus spp. (A); eosinophil percentage in different green areas of Madrid (B).
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Table 1. Habitat description scores by land cover type. Numbers are expressed as percentages.
Table 1. Habitat description scores by land cover type. Numbers are expressed as percentages.
Green Area% Vegetation% Buildings% Pavement% WaterType of Water
Finca El Garzo94.52.42.30.8Untreated; Flowing;
Unpaved shore
Encinar de San Pedro94.40.35.30.1Untreated; Stagnant;
Unpaved shore
Parque del Oeste61.410.327.90.4Untreated; Stagnant;
Paved shore
Parque Juan Carlos I55.415.723.45.5Treated; Stagnant;
Paved shore
Madrid Río23.437.733.35.7Treated; Flowing;
Paved shore
Table 2. Comparison of the prevalence of blood parasites in Eurasian Tree Sparrows (Passer montanus) detected by microscopic techniques, nested PCR, and both techniques combined.
Table 2. Comparison of the prevalence of blood parasites in Eurasian Tree Sparrows (Passer montanus) detected by microscopic techniques, nested PCR, and both techniques combined.
TechniqueGenusNo. PositivePrevalence (%; N = 108)
Microscopic techniqueHaemoproteus2220%
Leucocytozoon76%
Plasmodium22%
Nested PCR techniqueHaemoproteus87%
Leucocytozoon00%
Plasmodium22%
Both techniques combinedHaemoproteus2220%
Leucocytozoon76%
Plasmodium22%
Table 3. Results for the selection of the best binomial GLMs and detailed results of the best model for all genera of blood parasite prevalence. Significant p-values are highlighted in bold (p ≤ 0.05).
Table 3. Results for the selection of the best binomial GLMs and detailed results of the best model for all genera of blood parasite prevalence. Significant p-values are highlighted in bold (p ≤ 0.05).
Independent VariableEstimateStd. Errorz-Valuep-Value
Finca El Garzo0.29240.47440.6160.5377
Encinar de San Pedro0.95020.42032.2820.0225
Parque del Oeste−1.34220.3307−4.059<0.001
Parque Juan Carlos I0.6490.48781.3310.1833
Madrid Río−1.29690.8673−1.4950.1348
Table 4. Results for the selection of the best Gaussian GLMs for Haemoproteus parasitemia and detailed results for each location. Significant p-values are highlighted in bold (p ≤ 0.05), and marginally significant trends (p ≤ 0.1) are highlighted in italic.
Table 4. Results for the selection of the best Gaussian GLMs for Haemoproteus parasitemia and detailed results for each location. Significant p-values are highlighted in bold (p ≤ 0.05), and marginally significant trends (p ≤ 0.1) are highlighted in italic.
Independent VariableEstimateStd. Errorz-Valuep-Value
Finca El Garzo−30.9330.3−1.0210.3096
Encinar de San Pedro32.918.011.8270.0706
Parque del Oeste80.6332.982.4450.0162
Parque Juan Carlos I15.7227.520.5710.569
Madrid Río−32.937.32−0.8810.3801
Table 5. Results of the best model for lymphocyte and eosinophil percentage with their respective predictors. Significant p-values are shown in bold (p ≤ 0.05).
Table 5. Results of the best model for lymphocyte and eosinophil percentage with their respective predictors. Significant p-values are shown in bold (p ≤ 0.05).
Independent VariableEstimateStd. Errorz-Valuep-Value
LymphocyteFinca El Garzo−4.9622.7123−1.8290.0703
Encinar de San Pedro−0.29422.4848−0.1180.906
Parque del Oeste76.08991.610947.234<0.001
Parque Juan Carlos I4.50432.95061.5270.13
Madrid Río−7.34133.467−2.1170.0367
Body condition−2.38771.0501−2.2740.0251
Eosinophil
Finca El Garzo2.72790.93462.1910.004
Encinar de San Pedro−2.02050.8789−2.2990.0235
Parque del Oeste3.18950.69754.573<0.001
Parque Juan Carlos I0.18771.01880.1840.8541
Madrid Río0.04531.16710.0390.969
Haemoproteus prevalence−2.12150.6329−3.3520.001
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Vega, A.; Yabsley, M.J.; Hernández, S.M.; Garrett, K.B.; Aguirre, J.I.; Banda, E. Assessing Parasite Prevalence and Health Status of the Eurasian Tree Sparrow (Passer montanus) in Green Urban Areas of a Southern European City. Birds 2025, 6, 43. https://doi.org/10.3390/birds6030043

AMA Style

Vega A, Yabsley MJ, Hernández SM, Garrett KB, Aguirre JI, Banda E. Assessing Parasite Prevalence and Health Status of the Eurasian Tree Sparrow (Passer montanus) in Green Urban Areas of a Southern European City. Birds. 2025; 6(3):43. https://doi.org/10.3390/birds6030043

Chicago/Turabian Style

Vega, Aida, Michael J. Yabsley, Sonia M. Hernández, Kayla B. Garrett, Jose I. Aguirre, and Eva Banda. 2025. "Assessing Parasite Prevalence and Health Status of the Eurasian Tree Sparrow (Passer montanus) in Green Urban Areas of a Southern European City" Birds 6, no. 3: 43. https://doi.org/10.3390/birds6030043

APA Style

Vega, A., Yabsley, M. J., Hernández, S. M., Garrett, K. B., Aguirre, J. I., & Banda, E. (2025). Assessing Parasite Prevalence and Health Status of the Eurasian Tree Sparrow (Passer montanus) in Green Urban Areas of a Southern European City. Birds, 6(3), 43. https://doi.org/10.3390/birds6030043

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