Biodiversity and prevalence of gastrointestinal helminths in roe deer (Capreolus capreolus L.) in relation to wind farm presence in central Poland

Biodiversity and prevalence of gastrointestinal helminths in roe deer (Capreolus capreolus L.) in relation to wind farm presence in central Poland | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biodiversity and prevalence of gastrointestinal helminths in roe deer (Capreolus capreolus L.) in relation to wind farm presence in central Poland Anna Maria Pyziel, Kateryna Slivinska, Rusłan Sałamatin, Joanna Werszko, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9245327/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Background The roe deer ( Capreolus capreolus ) is a widespread cervid in Europe and an important reservoir of gastrointestinal helminths of veterinary relevance. Although environmental and host-related drivers of parasite communities are well studied, the effects of anthropogenic infrastructure, such as wind farms, on host–parasite interactions remain poorly understood. This study aimed to characterize gastrointestinal helminth diversity in roe deer from central Poland and evaluate the impact of wind farm presence on nematode infections. Methods A total of 102 roe deer were examined from three regions in central Poland between 2022 and 2024. Animals were categorized based on occurrence within or outside wind farm areas (39 and 63 individuals, respectively). Helminths were collected from the abomasum, small intestine, large intestine, and cecum using standard parasitological methods. For molecular identification, nematodes morphologically assigned to the genera Oesophagostomum and Chabertia were analyzed based on the ITS2 gene region, whereas the cox1 gene was used for cestodes of the genus Moniezia . The effects of wind farm presence on parasite abundance were analyzed using generalized linear models with a negative binomial distribution. Results A diverse helminth community was identified, comprising 7 species in the abomasum, 7 in the small intestine, and 4 species each in the large intestine and cecum. Haemonchus contortus dominated the abomasum; Chabertia ovina was the most prevalent species in the small and large intestine; and Trichuri s sp. was the most prevalent parasite in the cecum. Tapeworms of the genus Moniezia were found in the small intestine of 4 animals. Molecular analyses confirmed Oe. venulosum and C. ovina , and revealed genetically distinct Moniezia sp. Apart from Trichuris sp. infection, roe deer from wind farm areas exhibited higher helminth abundance and greater species richness than those from control areas, with infection levels approximately twice as high. Conclusions The increased parasite burden in wind farm areas suggests that anthropogenic disturbance may influence host–parasite dynamics, potentially via stress-related mechanisms. These findings highlight the importance of incorporating wildlife health considerations into renewable energy impact assessments. wind farms impact gastrointestinal parasites roe deer (Capreolus capreolus) central Poland Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background The European roe deer ( Capreolus capreolus ) is one of the most abundant wild ungulates in Europe, including Poland, where its population has been steadily increasing in recent decades (1, 2). As a widely distributed species inhabiting agricultural and forest mosaics, roe deer play an important ecological role. At the same time, they act as reservoirs and transmitters of numerous parasites, including gastrointestinal helminths of both veterinary and ecological significance (3, 4). Gastrointestinal nematodes (GINs) of ruminants belong to six families: Trichostrongylidae Leiper, 1912 (genera: Ostertagia, Spiculopteragia, Mazamastrongylus, Trichostrongylus, Haemonchus, Ashworthius ), Molineidae Skrjabin and Schultz, 1937 (genus Nematodirus ), Capillaridae Neveu-Lemaire, 1936 (genus Aonchotheca ), Trichuridae Baird, 1853 (genus Trichuris ), Ancylostomatidae Lane, 1907 (genus Bunostomum ), and Chabertidae Popova, 1952 (genera Chabertia and Oesophagostomum ) (5, 6). These nematodes occupy different regions of the host digestive tract: representatives of the Trichostrongylidae are mainly located in the stomach, whereas members of the Molineidae, Capillaridae, and Ancylostomatidae are typically found in the small intestine. In contrast, nematodes of the families Trichuridae and Chabertidae inhabit the large intestine. Recent large-scale syntheses indicate that cervids in Europe harbour a wide range of generalist nematodes with broad host spectra, including species commonly associated with livestock, such as Haemonchus contortus, Ostertagia ostertagi, Trichostrongylus vitrinus, T. axei, T. capricola, Oesophagostomum venulosum , and Chabertia ovina , among others (7). Notably, it has been demonstrated that some abomasal nematodes of cervids can be transmitted to domestic ruminants, including anthelmintic-resistant strains of H. contortus (8). This underscores the epidemiological importance of parasite exchange between wild ruminants and livestock (9). Consequently, such transmission can negatively affect the profitability of animal production, particularly in the case of infections with blood-feeding helminths, such as H. contortus and A. sidemi , which may cause haemorrhagic anaemia in the host (10, 11). A combination of environmental, ecological, and host-related factors shapes helminth communities in roe deer. Among these, habitat type has been identified as a key determinant of abomasal nematode community composition, with distinct parasite assemblages associated with forest and agricultural environments (12). More broadly, anthropogenic changes are increasingly recognized as major drivers of host-parasite dynamics (13). While their ecological impacts on wildlife behaviour and habitat use have been widely documented (14), their effects on wildlife health remain less well understood. In this context, recent studies suggest that wind turbines may act as chronic stressors for terrestrial mammals, potentially affecting their physiology and immune responses (15, 16). Despite growing interest in wildlife parasitology, the potential influence of wind farm infrastructure on gastrointestinal parasite communities in wild ungulates has not yet been directly examined. This gap in knowledge highlights the need for studies addressing how renewable energy development may influence ecosystem health. Therefore, this study aimed to: characterize the diversity and prevalence of gastrointestinal helminths in roe deer from central Poland; assess spatial variation in parasite communities across different regions and sections of the digestive tract; and evaluate the potential impact of wind farm presence on helminth species composition and prevalence. Methods Study Area European roe deer samples were collected in Central Poland, within the Mazowieckie and Łódzkie voivodeships, across three sites: Iłża (51°10'7"N, 21°11'0"E), Rawa Mazowiecka (51°48'22"N, 20°4'30"E), and Węgrów (52°23'3"N, 21°50'0"E) (Fig. 1). These areas, part of the Central European Plain, are characterized by predominantly flat terrain with an average elevation of 173 m a.s.l. (17). Shaped by glacial processes, the landscape comprises diverse landforms and soils-from fertile loams to sandy substrates-affecting vegetation structure and microhabitat availability for intermediate parasite hosts. Major rivers, including the Vistula in Mazowieckie, create moist riparian zones that favor the survival and development of helminth eggs and larvae. The temperate climate, with cold winters, warm summers, and peak precipitation during the growing season (18), further influences the seasonal dynamics of gastrointestinal parasites, resulting in a heterogeneous environment conducive to parasite persistence and transmission in wild ruminants. All three sites contain wind farms within agricultural landscapes that partially overlap the hunting grounds where roe deer were sampled. Based on their position relative to the turbines, deer were categorized as occurring inside or outside the wind farm. The wind farm area was defined as the space between turbines forming the cluster, plus a 700 m buffer zone, a distance previously used to assess wind turbine impacts on terrestrial mammals, including roe deer (19, 20). Examined animals Hunting management was actively practiced in the study areas, with licensed hunters harvesting a predetermined number of game animals, including roe deer, each year. Between 2022 and 2024, samples were collected in collaboration with local hunting clubs under formal agreements. For each individual, metadata including sex, age, date, and location of acquisition were recorded. Hunters provided the entire digestive tract post-mortem, which was then transported to the laboratory for parasitological analysis. No animals were killed specifically for this study; all samples were obtained as by-products of routine hunting activities, in compliance with Polish hunting regulations and management plans (21). The sampling strategy aimed to ensure that animals originated from comparable environmental conditions-agricultural landscapes of Central Poland-and experienced similar hunting pressure. In total, 102 roe deer were sampled: 39 from wind farm areas and 63 from outside these areas. Regionally, this included 48 individuals from Iłża (18 on wind farms, 30 outside), 34 from Rawa Mazowiecka (12 on wind farms, 22 outside), and 20 from Węgrów (9 on wind farms, 11 outside) (Fig. 1). Sections of the digestive tract - including the abomasum, small intestine, large intestine, and cecum - were collected from each animal for analysis. In some instances, specific sections could not be collected due to damage (Table 1). Collection of helminths In each case, the contents of the abomasum, small intestine, large intestine, and cecum were mixed separately with tap water at a ratio of 1:10, decanted several times, and washed through a sieve (mesh size: 100–150 µm). The resulting sediment was then examined for the presence of parasites using a Delta Optical SZ-450 T stereomicroscope (Delta Optical, Mińska Mazowiecki, Poland). Collected parasites were placed in tubes containing 70% ethanol for subsequent morphological and molecular analyses. Morphological examination The anterior and posterior parts of the nematode specimens were excised and transferred into a drop of lactophenol on a microscope slide to render the cuticle transparent and to allow visualization of the oral cavity and spicules of males (6). The middle body parts of the examined specimens were individually transferred into Eppendorf tubes containing 70% ethanol and appropriately labelled. Species identification of the nematodes was performed using a LAB40 light microscope (OPTA-TECH, Warsaw, Poland) at magnifications of 100×-400× (22, 23). Microphotographs were captured using a digital camera, and measurements were obtained with OPTA View-15 software (OPTA-TECH). Due to the poor preservation of the cestode specimens, morphometric analyses could not be conducted; therefore, molecular methods were applied instead. DNA extraction, amplification, and sequencing Nematodes Genomic DNA was individually extracted from 50 intestinal nematodes to enable molecular species identification (25 individuals of Oesophagostomum sp. and 25 of Chabertia sp.) using a NucleoSpin Tissue DNA extraction kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s protocol. Partial regions of the internal transcribed spacer 2 (ITS2) and the large subunit (LSU) ribosomal RNA gene were amplified by polymerase chain reaction (PCR) using two oligonucleotide primers: forward NC1 (5′-ACG TCT GGT TCA GGG TTG TT-3′) and reverse NC2 (5′-TTA GTT TCT TTT CCT CCG CT-3′) (24). PCR amplification was performed in a total reaction volume of 50 µL, containing 20 µL of molecular biology grade water (Sigma-Aldrich, St. Louis, MO, USA), 25 µL of AccuStart II PCR ToughMix (2× concentration) (Quantabio, Beverly, MA, USA), 1 µL of GelTrack Loading Dye (50× concentration) (Quantabio), 1 µL of each primer (20 mM), and 2 µL of template DNA. Negative (molecular biology grade water) control was included in each run. Thermal cycling conditions followed those described previously (25). The resulting sequences were assembled into contigs using CodonCode Aligner version 8.0 (CodonCode Corporation, Centerville, MA, USA). The obtained nucleotide sequences were compared with those available in the NCBI database using the Basic Local Alignment Search Tool (BLAST) and subsequently submitted to GenBank. Cestodes The tissue samples of two tapeworms recovered from the small intestine of roe deer originating from the Węgów and Iłża regions were rinsed with phosphate-buffered saline (PBS) to remove residual ethanol. Genomic DNA was extracted using the Genomic Mini Kit (A&A Biotechnology, Gdańsk, Poland) in accordance with the manufacturer’s instructions. PCR amplification was performed using primers JB3 (5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB4.5 (5′-TAAAGAAAGAACATAATGAAA-ATG-3′) (26) to obtain a 396 bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Each PCR reaction was carried out in a total volume of 25 µl, containing 18 µl of deionized water, 1.5 µl of 25 µM MgCl₂ solution, 0.25 µl of Allegro Taq DNA polymerase (5 U/µl) (Novazym, Poznań, Poland), 0.25 µl of dNTP mix (10 mM), 2.5 µl of 10× Taq DNA polymerase buffer (containing 25 mM MgCl₂), 0.25 µl of each primer (20 pmol/µl), and 4 µl of template DNA. In the negative control, nuclease-free water was used instead of template DNA. PCR conditions followed the protocol described previously (26). Amplification products were visualized on 1% agarose gels stained with SimplySafe™ (EURx, Gdańsk, Poland). The gels were documented using a ChemiDoc system and MP Lab software (Image Lab, Bio-Rad, Hercules, USA). The PCR products were purified using the Agarose-Out DNA Purification Kit (EURx, Gdańsk, Poland) and subsequently sequenced by Genomed (Warsaw, Poland). The resulting nucleotide sequences were assembled using CLC Main Workbench version 6.9.1 and analyzed with NCBI BLAST (27). Finally, the sequences were submitted to GenBank. Phylogenetic reconstruction of cestodes The 62 nucleotide sequences showing the highest similarity in the NCBI BLAST analysis were selected for phylogenetic tree reconstruction using Bayesian inference implemented in MrBayes version 3.2.7 (28). The sequence of Echinococcus canadensis (GenBank: LC184604) was used as an outgroup. Statistical analysis of the impact of selected factors on the number of nematodes We used a generalized linear model with a negative binomial distribution and a log link function to evaluate the potential impact of selected factors on the number of nematodes in different sections of the roe deer digestive tract. Negative binomial models are suitable for handling overdispersion in count data (29). Separate models were constructed for each section (abomasum, small intestine, large intestine, and cecum) to account for differences in total abundance and species composition between sections. In each model, the dependent variable was the total number of nematodes in a given section of the digestive tract. Four explanatory variables were included: sex and age of the animals, study area, and the presence of roe deer within wind farm areas. The inclusion of the latter variable was motivated by the potential impact of wind farm infrastructure on roe deer stress levels and health (15, 30). The effects of the selected factors on nematode abundance were assessed using a model selection approach. Specifically, all possible model variants (i.e., all combinations of explanatory variables), including the null model, were fitted to identify the best-supported model (31). Models were ranked according to the Akaike Information Criterion (AIC), with the model showing the lowest AIC value considered the best-fitting. Only the results of the highest-ranked models are presented. All statistical analyses were performed using IBM SPSS Statistics 29.0 (Armonk, New York). Results Abomasal nematodes A total of seven nematode species were identified in the abomasum of roe deer in central Poland (Table 2; Fig. 2). The most dominant species was the blood-sucking H. contortus , which was commonly found in roe deer across all examined regions. The prevalence of H. contortus ranged from 35.4% in the Iłża region to 47.4% in the Węgrów region. Moreover, this species exhibited the highest infection intensity in all regions, with a maximum of 2,558 individuals recorded in a single host from the Węgrów region. Notably, the composition of abomasal nematode communities differed between regions. In the Rawa Mazowiecka region, only monoinfections with H. contortus were observed, whereas mixed infections comprising four and five species were recorded in the Węgrów and Iłża regions, respectively. The highest species richness was found in roe deer from the Iłża region, where the presence of the alien, blood-sucking species A. sidemi was recorded exclusively. Additionally, Spiculopteragia boehmi was identified in animals from the Iłża region, together with Ostertagia leptospicularis and its minor morph, O. kolchida ; however, these parasites were detected in only a single individual. Interestingly, O. leptospicularis was also found in a roe deer from the Węgrów region, where Mazamastrongylus dagestanica and Trichostrongylus capricola were also recorded. Small intestine nematodes Together, seven species of nematodes were identified in the small intestine of the examined roe deer (Table 3; Fig. 3). All species were detected in animals from the Iłża region, whereas lower species richness was observed in the two remaining areas. Molecular analyses indicated that Oesophagostomum sp. corresponded to Oe. venulosum , while Chabertia sp. was identified as Ch. ovina (GenBank: PV920951 and PV920952, respectively). All obtained partial nucleotide sequences of the ITS2 and LSU regions for each species were homologous both to one another and to reference isolates of Oe. venulosum and Ch. ovina available in GenBank. The length of the obtained sequences ranged from 240 to 300 bp. Nevertheless, nematodes such as Aonchotheca sp., Ch. ovina , Bunostomum trigonocephalum , and Oe. venulosum were common parasites in animals from all studied locations. The most dominant species across all regions was Ch. ovina , with prevalence ranging from 15.2% to 31.6% in the Iłża and Węgrów regions, respectively. Moreover, the highest infection intensity was also recorded for Ch. ovina , regardless of region, reaching a maximum of 66 individuals in a single host from the Węgrów region. Additionally, single infections of Nematodirus europaeus , H. contortus , and B. phlebotomum were detected exclusively in roe deer from the Iłża region. Small intestine cestodes Fragmental strobiles of tapeworms were found in the contents of the small intestine of four roe deer, including two animals from the Węgrów and the Iłża regions (prevalence: 10.5% and 4.3%, respectively). Their general prevalence was 4.1%, as tapeworms were found in 4 of 98 examined small intestines. Two partial sequences of the cox1 gene, each of a length of 396 bp, were obtained from 2 cestodes, one from the Węgrów region, and the second from Iłża (GenBank: PX511568, PX511569, respectively). The obtained sequences differ at the nucleotide level but encode the same amino acid sequence. The obtained isolates showed a percentage identity ranging from 87.6% to 93.5% with Moniezia species strains from Denmark, China, India, Senegal, Ethiopia, Turkey, Vietnam, and Taiwan, derived from both wild and domestic ruminants, including European bison, sheep, buffalo, cows, and goats. More detailed data are presented in Fig. 5. Due to the high level of variation in the examined fragment of the cox1 gene, the species-level determination of the tapeworms was currently unaccomplishable. Therefore, they were classified as Moniezia sp. Large intestine nematodes A total of four nematode species were identified in the large intestine of roe deer (Table 4; Fig. 3). Nematodes such as Ch. ovina , Oe. venulosum , and Trichuris sp. were commonly observed regardless of the animals’ geographic origin. However, Ch. ovina was the dominant species across all locations, with prevalence ranging from 63.2% in the Węgrów region to 70.8% in the Rawa Mazowiecka region. The highest intensity of Ch. ovina infection was recorded in a single individual from the Iłża region, in which 117 specimens were found. The fourth species, B. trigonocephalum , was detected exclusively in one roe deer from the Rawa Mazowiecka region. Cecum nematodes Together, four nematode species were identified in the cecum of the roe deer (Table 5; Fig. 3). Trichuris sp. and Oe. venulosum were present in animals inhabiting all three regions. Moreover, Trichuris sp. was the most prevalent cecal parasite, with prevalence ranging from 30% in the Węgrów region to 55.3% in the Iłża region. The maximum intensity of Trichuris sp. infection was 46 individuals recorded in a single roe deer from the Iłża region. Other nematodes detected in this part of the intestine included Ch. ovina , found in animals from the Iłża and Rawa Mazowiecka regions, and H. contortus , the least prevalent species, recorded in a single animal from the Iłża region. Prevalence of parasites in animals living on wind farms and outside wind farm areas Regarding abomasum and small intestine nematodes, the prevalence of each species was higher in animals inhabiting areas within the wind farms. This pattern did not apply to worms of the genus Trichuris , which were diagnosed in the large intestine and cecum. Specifically, Trichuris sp. was the only nematode whose prevalence was higher in animals from areas outside the wind farms compared to those inside. Moreover, nematode species richness was consistently higher in hosts from areas within the wind farms. In particular, species composition was enriched by M. dagestanica , O. kolchida , S. boehmi , and T. capricola among abomasum nematodes (Table 6); B. phlebotomum and H. contortus among small intestine nematodes (Table 7); and B. trigonocephalum and H. contortus among large intestine and cecum nematodes, respectively (Table 8). Tapeworms of Moniezia sp. were also more prevalent in animals inhabiting wind farm areas compared to roe deer from areas outside the wind farms (Table 7). Impact of selected factors on the number of nematodes in sections of the digestive tract For all sections of the digestive tract of roe deer, unique models that significantly explained the variability in nematode counts were obtained. However, not all best-fitting models for individual digestive tract sections included all variables as components. The presence of the wind farm significantly explained the variability in nematode counts in three of the four digestive tract sections: the abomasum, small intestine, and cecum (Table S1). In the case of the large intestine, this variable was excluded during model selection. In all cases where the wind farm was a significant explanatory variable, nematode counts were higher in the wind farm areas compared to the control areas (Fig. 4). The marginal mean in wind farm areas was approximately twice as high as in control areas. However, the standard error for individuals residing in wind farms was consistently greater (Fig. 4). The study area was an important variable in all best-fitting models for individual sections of the digestive tract, although not all comparisons between study areas were statistically significant (Table S1). Nevertheless, the study area's effect was unclear, as indicated by the varying estimates across study areas. Sex and age of animals were also important variables in the models, with sex appearing in all models and age in only two of four (exclusively in models for abomasum and small intestine). Regarding the sex of animals, females always showed lower nematode numbers than males (Table S1). Discussion The high diversity of GINs recorded in this study confirms that roe deer harbour complex parasite communities typical of European cervids. All GIN parasites identified here have previously been reported in this host (6, 32, 33, 34), although earlier data from Poland indicate an even greater species richness, including O. antipini , O. lyrata , S. asymmetrica , S. mathevossiani , T. askivalli , and T. axei (6, 12, 32). Moreover, additional species have been reported in roe deer from other European countries and Russia, such as Cooperia oncophora , Marshallagia marshalli , N. europeus , N. filicollis , O. occidentalis , O. ostertagi , and T. colubriformis (34, 35, 36, 37). Together, these findings highlight both the richness and geographical variability of helminth communities in this host. In contrast to nematodes, knowledge of cestode diversity in roe deer remains limited. To date, only two tapeworm species, M. expansa and M. benedeni , have been reported in Poland based on morphological identification (33, 38). The detection of Moniezia sp. with low genetic similarity to reference sequences in the present study supports earlier suggestions that insufficient molecular data hinder accurate species identification in wild ruminants (39). This finding points to potential cryptic diversity within the genus and suggests the presence of a third, previously unrecognized Moniezia species in Poland, emphasizing the need for integrative taxonomic approaches combining molecular and morphological methods. Similarly, the inability to identify Aonchotheca and Trichuris to species level in this study is consistent with previous reports of T. globulosa and A. bovis (syn. Capillaria bovis ) in roe deer in Poland (33). Particular attention should be paid to the occurrence of hematophagous nematodes of the subfamily Haemonchinae. Both A. sidemi and H. contortus were recorded by us, in agreement with some studies (12), but contrasting with others that report the dominance of only one of these species (6, 32, 33, 35, 37). The dominance of H. contortus observed here supports growing evidence that this species is becoming increasingly important in wildlife and reflects ongoing parasite exchange between wild and domestic ruminants, particularly in areas where grazing habitats overlap (4). An additional noteworthy observation was the presence of a single H. contortus individual in the caecum, which represents an atypical localization. This may be explained by post-mortem displacement or abnormal migration within the gastrointestinal tract; however, aberrant localization cannot be excluded, especially under conditions of high infection intensity (184 individuals were found in the abomasum of the infected roe deer) or host physiological stress, such as that potentially associated with wind farm environments. The parasite assemblage identified in this study includes numerous species with well-documented pathogenic effects. Hematophagous nematodes such as H. contortus and A. sidemi can cause severe anemia and reduced fitness (40), while abomasal species, including O. kolchida , O. leptospicularis , M. dagestanica , and S. boehmi , may induce gastric disturbances and weight loss (41). Intestinal nematodes such as Ch. ovina , Oe. venulosum , N. europaeus , and Trichuris sp. are associated with enteritis and secondary infections (42). In addition, hookworms ( B. phlebotomum , B. trigonocephalum ) contribute to anemia, whereas less pathogenic taxa, including Aonchotheca sp. and Moniezia sp., may still affect host condition under heavy infection. Importantly, mixed infections are likely to have synergistic effects, further exacerbating negative impacts on host health (40, 42). One of the most significant findings of this study is the clear effect of wind farm presence on parasite infections. Roe deer inhabiting wind farm areas exhibited higher nematode abundance and species richness, suggesting that anthropogenic infrastructure can influence host-parasite interactions. This, to our knowledge, represents the first evidence linking wind farms to gastrointestinal helminth dynamics in terrestrial mammals. Previous research in such environments has focused mainly on behavioral and ecological responses (44, 45, 46, 47), with some studies indicating that cumulative stressors associated with wind farms may negatively affect fitness and survival (48, 49). Supporting this interpretation, anthropogenic disturbance has been shown to induce physiological stress responses, including elevated glucocorticoid levels (15, 16), as well as reduced body condition (19, 50, 51), and developmental abnormalities (20). Since chronic stress can impair immune function (52), it is plausible that such mechanisms contribute to increased susceptibility to parasitic infections. Although direct evidence linking these processes to disease dynamics has been limited (14), the present study suggests that they may result in measurable differences in parasite burden. Together, these findings underline the complex interplay between environmental factors, host condition, and parasite ecology. Conclusions This study confirms that a complex interplay of environmental, host-related, and anthropogenic factors shapes gastrointestinal helminth communities in roe deer. Importantly, it provides novel evidence that wind farm infrastructure may influence parasite infections in wild ruminants, likely through stress-mediated mechanisms. Abbreviations a.s.l. – above sea level A. – Ashworthius B. – Bunostomum Ch. – Chabertia H. – Haemonchus M. – Mazamastrongylus N. – Nematodirus O. – Ostertagia Oe. – Oesophagostomum S. – Spiculopteragia T. - Trichostrongylus Declarations Ethics approval and consent to participate Studies on animal tissues obtained post-mortem do not require the approval of the Ethics Committee. All the samples were taken exclusively from roe deer legally hunted during the hunting seasons 2023 and 2024 according to Polish hunting law (Act of the Polish Parliament dated 13 October 1995, item 713, the Hunting Law). Consent for publication Not applicable. Availability of data and materials The nucleotide sequences obtained in the study are available in the GenBank database under the following numbers: PV920951 www.ncbi.nlm.nih.gov/nuccore/pv920951, PV920952 www.ncbi.nlm.nih/nuccore/pv920952, PX511568 www.ncbi.nlm.nih.gov/nuccore/px511568, PX511569 www.ncbi.nlm.nih.gov/nuccore/px511569. Competing interests The authors have no conflict of interest. Funding The study was partially financed by the National Science Centre, Poland (Grant number: 2021/41/B/NZ9/04442), and by the European Union (MSCA4Ukraine project https://cordis.europa.eu/project/id/101101923). Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the MSCA4Ukraine Consortium as a whole nor any individual member institutions of the MSCA4Ukraine Consortium can be held responsible for them. 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Effects of wind turbines on area use and behaviour of semi-domestic reindeer in enclosures. Rangifer. 2004;24:55-66. Walter WD, Leslie DM, Jenks JA. Response of Rocky Mountain Elk ( Cervus elaphus ) to wind-power development. Am Midl Nat. 2006; 156:363-375. Skarin A, Nellemann C, Rönnegård L, Sandström P, Lundqvist H. Wind farm construction impacts reindeer migration and movement corridors. Landsc Ecol. 2015; 30:1527-1540. Kim S, Dhakal T, Cho KH, Kim T, Woo S, Kim J, Lee D, Jang G. Occupancy of roe deer, water deer, and wild boar in wind farm‐integrated forest ecosystems: a case study in Korea. Ecosphere. 2025;16:e70258. Taylor KL, Beck JL, Huzurbazar SV. Factors influencing winter mortality risk for pronghorn exposed to wind energy development. Rangel Ecol Manag. 2016; 69:108-116. Milligan MC, Johnston AN, Beck JL, Smith KT, Taylor KL, Hall E, Knox L, Cufaude T, Wallace C, Chong G, Kauffman MJ. Variable effects of wind energy development on seasonal habitat selection of pronghorn. Ecosphere. 2021;12:e03850. Agnew RC, Smith VJ, Fowkes RC. Wind turbines cause chronic stress in badgers ( Meles meles ) in Great Britain. J Wildl Dis. 2016;52:459-467. Karwowska M, Mikołajczak J, Dolatowski ZJ, Borowski S. The effect of varying distances from the wind turbine on meat quality of growing-finishing pigs. Ann Anim Sci. 2015;15:1043-1054. Sapolsky RM, Romero LM, Munck AU. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory and preparative actions. Endocr Rev. 2000;21:55-89. Tables Tables 1 to 8 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Table2.docx Table3.docx Table4.docx Table5.docx Table6.docx Table7.docx Table8.docx TableS1.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 27 Apr, 2026 Reviews received at journal 23 Apr, 2026 Reviews received at journal 09 Apr, 2026 Reviewers agreed at journal 05 Apr, 2026 Reviewers agreed at journal 03 Apr, 2026 Reviewers agreed at journal 02 Apr, 2026 Reviewers invited by journal 02 Apr, 2026 Editor assigned by journal 02 Apr, 2026 Editor invited by journal 01 Apr, 2026 Submission checks completed at journal 31 Mar, 2026 First submitted to journal 31 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9245327","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":617777768,"identity":"dafe3295-dd3b-46aa-8e16-2b07e734c1a2","order_by":0,"name":"Anna Maria Pyziel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYDADNgbmxgcMDAkMDOzEa2FsNgBrYSbeHsY2CaK08M/uPfiBccdhez72xrZq3pw0BnNCWiTunEuWYDxzmJmN52Dbbd5tOQyWzYSccyPHQIKx7TAbm0QiSEsFg8FhAjrkb+QY/wBq4WGTf9hWTJQWgxs5ZiBbJNiAJDPIYQS1GAK1WCSeSTdg40lslpy7LY2HoF/kgA678XGHtb18++GDH95uS5YzZ28goAcEEhsQJvMYEKEBGIkNdQgOcVpGwSgYBaNgJAEATT08rm2vGa0AAAAASUVORK5CYII=","orcid":"","institution":"Nicolaus Copernicus University in Toruń","correspondingAuthor":true,"prefix":"","firstName":"Anna","middleName":"Maria","lastName":"Pyziel","suffix":""},{"id":617777769,"identity":"b7d5d7c0-99c2-46d7-9a39-cab6a7f75a25","order_by":1,"name":"Kateryna Slivinska","email":"","orcid":"","institution":"Museum and Institute of Zoology of the Polish Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kateryna","middleName":"","lastName":"Slivinska","suffix":""},{"id":617777770,"identity":"cd403fb2-ecbb-458d-ab37-7f3cf2ef65cf","order_by":2,"name":"Rusłan Sałamatin","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Rusłan","middleName":"","lastName":"Sałamatin","suffix":""},{"id":617777771,"identity":"0bf23a9b-934d-49c1-9e15-38d5e289d287","order_by":3,"name":"Joanna Werszko","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Joanna","middleName":"","lastName":"Werszko","suffix":""},{"id":617777772,"identity":"32414bf5-8ae5-4878-ab77-e35c130d8db8","order_by":4,"name":"Marta Kloch","email":"","orcid":"","institution":"Warsaw University of Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Kloch","suffix":""},{"id":617777775,"identity":"8efeeb66-a764-4627-9b2a-59fd4e54960f","order_by":5,"name":"Marcin Świątek","email":"","orcid":"","institution":"Warsaw University of Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Marcin","middleName":"","lastName":"Świątek","suffix":""},{"id":617777776,"identity":"1c9eac0f-785c-46b1-b5f1-42eb74a1e9f1","order_by":6,"name":"Olena Zhytova","email":"","orcid":"","institution":"Polissia National University","correspondingAuthor":false,"prefix":"","firstName":"Olena","middleName":"","lastName":"Zhytova","suffix":""},{"id":617777779,"identity":"f5f308bf-77d9-4a88-83af-d5a455e8e290","order_by":7,"name":"Daniel Klich","email":"","orcid":"","institution":"Warsaw University of Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Klich","suffix":""}],"badges":[],"createdAt":"2026-03-27 13:39:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9245327/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9245327/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106724094,"identity":"34e235a2-9522-48a7-be3c-81d7ea7e54d7","added_by":"auto","created_at":"2026-04-12 18:25:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":784270,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of study areas in Poland with the number of obtained roe deer samples.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/0865fca746091d8afd2f72f2.png"},{"id":106724163,"identity":"faec580b-e8c9-439c-a798-a31012c76978","added_by":"auto","created_at":"2026-04-12 18:26:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1117806,"visible":true,"origin":"","legend":"\u003cp\u003eMale distal body parts of abomasal nematodes found in roe deer. (2A) \u003cem\u003eAshworthius sidemi\u003c/em\u003e (×100) Bar = 100 μm; (2B) \u003cem\u003eAshworthius sidemi\u003c/em\u003e (×100) Bar = 100 μm; (2C) \u003cem\u003eHaemonchus contortus\u003c/em\u003e (×100) Bar = 100 μm; (2D) \u003cem\u003eMazamastrongylus dagestanica\u003c/em\u003e (×200) Bar = 100 μm; (2E) \u003cem\u003eOstartagia kolchida\u003c/em\u003e (×200) Bar = 100 μm; (2F) \u003cem\u003eOstertagia leptospicularis\u003c/em\u003e (×200) Bar = 100 μm; (2G) \u003cem\u003eSpiculopteragia boehmi\u003c/em\u003e (×200) Bar = 100 μm; (2H) \u003cem\u003eTrichostrongylus capricola\u003c/em\u003e (×200) Bar = 100 μm\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/755254b762cd62f602143b65.png"},{"id":106444473,"identity":"b71dc17f-56f3-4ce8-af2e-734fef308ce3","added_by":"auto","created_at":"2026-04-08 15:17:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2140612,"visible":true,"origin":"","legend":"\u003cp\u003eNematodes found in the intestine of roe deer. (3A) \u003cem\u003eAonchotheca\u003c/em\u003e sp. (×40) Bar = 130 μm; (3B) \u003cem\u003eBunostomum phlebotomum\u003c/em\u003e, anterior end (×200) Bar = 100 μm; (3C) \u003cem\u003eBunostomum phlebotomum\u003c/em\u003e, male posterior end (×100) Bar = 100 μm; (3D) \u003cem\u003eBunostomum phlebotomum\u003c/em\u003e, female posterior end (×100) Bar = 100 μm; (3E) \u003cem\u003eBunostomum trigonocephalum\u003c/em\u003e, anterior end (×200) Bar = 100 μm; (2F) \u003cem\u003eBunostomum trigonocephalum\u003c/em\u003e, male posterior end (×100) Bar = 100 μm; (2G) \u003cem\u003eBunostomum trigonocephalum\u003c/em\u003e, female posterior end (×40) Bar = 130 μm; (2H) \u003cem\u003eChabertia ovina\u003c/em\u003e, anterior end (×100) Bar = 100 μm; (2I) \u003cem\u003eChabertia ovina\u003c/em\u003e, male posterior end (×40) Bar = 130 μm; (2J) \u003cem\u003eChabertia ovina\u003c/em\u003e, female posterior end (×100) Bar = 100 μm; (2K) Haemonchus contortus (×100) Bar = 100 μm; (2L) \u003cem\u003eNematodirus europeus\u003c/em\u003e(×100) Bar = 100 μm; (2M) \u003cem\u003eOesophagostomum venulosum\u003c/em\u003e, anterior end (×100) Bar = 100 μm; (2N) \u003cem\u003eOesophagostomum venulosum\u003c/em\u003e, male posterior end (×40) Bar = 130 μm; (2O) \u003cem\u003eOesophagostomum venulosum\u003c/em\u003e, female posterior end (×100) Bar = 100 μm; (2P) \u003cem\u003eTrichuris\u003c/em\u003e sp. Bar = 20 mm.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/b59273822054e55708d5641c.png"},{"id":106724167,"identity":"fecd8bfe-d8d1-4668-ab93-71780fded0b0","added_by":"auto","created_at":"2026-04-12 18:26:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":174041,"visible":true,"origin":"","legend":"\u003cp\u003eMean numbers of nematodes in selected parts of the digestive tract in roe deer dwelling on wind farm areas and control areas. Marginal means calculated in generalized linear models with negative binomial distribution and log link function, based on the highest-ranked model composition. The figure also includes pairwise comparison of the means with the Least Significant Difference test in the same highest-ranked models.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/5e6ef87daa6d6615764d702e.png"},{"id":106444475,"identity":"e95f170b-c20d-46a1-9504-9ea362e84159","added_by":"auto","created_at":"2026-04-08 15:17:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":252051,"visible":true,"origin":"","legend":"\u003cp\u003eBayesian inference tree based on the 370‑bp fragment of 62 distinct sequences of the cox1 gene for cestodes of family Anoplocephalidae (outgroup not shown). The number of sequences from each species is shown in parentheses. The Bayesian posterior probabilities are shown adjacent to branch nodes\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/8bf333910822ba3dc366a3be.jpg"},{"id":107704823,"identity":"577a9664-c973-4d29-b880-a59a3b9fbe1b","added_by":"auto","created_at":"2026-04-24 08:59:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4428853,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/bff7f272-0c6d-4670-be45-a1340c0e46bf.pdf"},{"id":106444469,"identity":"2848c555-98b1-4378-9077-534fb2cfed23","added_by":"auto","created_at":"2026-04-08 15:17:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16157,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/eb559427ce66aa80dc425136.docx"},{"id":106724218,"identity":"fd9a3a02-e1f1-4c85-b028-2faed1a888b2","added_by":"auto","created_at":"2026-04-12 18:26:46","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18950,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/051b36b93218386bd9aec4e7.docx"},{"id":106724742,"identity":"a603c37d-a694-4d98-bdeb-a259b141e3aa","added_by":"auto","created_at":"2026-04-12 18:29:27","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18604,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/16cde4697343494787d62b82.docx"},{"id":106444474,"identity":"8c595b30-e9c3-41f3-aee3-8b366036887f","added_by":"auto","created_at":"2026-04-08 15:17:18","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17914,"visible":true,"origin":"","legend":"","description":"","filename":"Table4.docx","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/7a8ef66a132c62c391dc67e0.docx"},{"id":106444477,"identity":"f3db6370-3641-4a96-8471-dc6fb2a36f37","added_by":"auto","created_at":"2026-04-08 15:17:18","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":17374,"visible":true,"origin":"","legend":"","description":"","filename":"Table5.docx","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/f9fd47657549eb28faae2466.docx"},{"id":106724069,"identity":"353f95e9-db76-4761-94d6-487f490f27bc","added_by":"auto","created_at":"2026-04-12 18:25:20","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":17515,"visible":true,"origin":"","legend":"","description":"","filename":"Table6.docx","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/1ed27c5bf044b4e01d1d2ac5.docx"},{"id":106724207,"identity":"1244ca2e-4aa9-4fc5-8e0e-016a838012ba","added_by":"auto","created_at":"2026-04-12 18:26:41","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":17276,"visible":true,"origin":"","legend":"","description":"","filename":"Table7.docx","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/6a0177f4e484171c56eefab1.docx"},{"id":106444481,"identity":"a14a1c45-ab0e-4e40-b333-3930a86f8b7b","added_by":"auto","created_at":"2026-04-08 15:17:19","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":17109,"visible":true,"origin":"","legend":"","description":"","filename":"Table8.docx","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/f62742822b14ac2f7a39bf1b.docx"},{"id":106444480,"identity":"be17bfa0-e879-4185-9688-b24f4953a3ea","added_by":"auto","created_at":"2026-04-08 15:17:19","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":21580,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9245327/v1/622b15037451bf9bbfe3d1ef.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biodiversity and prevalence of gastrointestinal helminths in roe deer (Capreolus capreolus L.) in relation to wind farm presence in central Poland","fulltext":[{"header":"Background","content":"\u003cp\u003eThe European roe deer (\u003cem\u003eCapreolus capreolus\u003c/em\u003e) is one of the most abundant wild ungulates in Europe, including Poland, where its population has been steadily increasing in recent decades (1, 2). As a widely distributed species inhabiting agricultural and forest mosaics, roe deer play an important ecological role. At the same time, they act as reservoirs and transmitters of numerous parasites, including gastrointestinal helminths of both veterinary and ecological significance (3, 4).\u003c/p\u003e \u003cp\u003eGastrointestinal nematodes (GINs) of ruminants belong to six families: Trichostrongylidae Leiper, 1912 (genera: \u003cem\u003eOstertagia, Spiculopteragia, Mazamastrongylus, Trichostrongylus, Haemonchus, Ashworthius\u003c/em\u003e), Molineidae Skrjabin and Schultz, 1937 (genus \u003cem\u003eNematodirus\u003c/em\u003e), Capillaridae Neveu-Lemaire, 1936 (genus \u003cem\u003eAonchotheca\u003c/em\u003e), Trichuridae Baird, 1853 (genus \u003cem\u003eTrichuris\u003c/em\u003e), Ancylostomatidae Lane, 1907 (genus \u003cem\u003eBunostomum\u003c/em\u003e), and Chabertidae Popova, 1952 (genera \u003cem\u003eChabertia\u003c/em\u003e and \u003cem\u003eOesophagostomum\u003c/em\u003e) (5, 6). These nematodes occupy different regions of the host digestive tract: representatives of the Trichostrongylidae are mainly located in the stomach, whereas members of the Molineidae, Capillaridae, and Ancylostomatidae are typically found in the small intestine. In contrast, nematodes of the families Trichuridae and Chabertidae inhabit the large intestine.\u003c/p\u003e \u003cp\u003eRecent large-scale syntheses indicate that cervids in Europe harbour a wide range of generalist nematodes with broad host spectra, including species commonly associated with livestock, such as \u003cem\u003eHaemonchus contortus, Ostertagia ostertagi, Trichostrongylus vitrinus, T. axei, T. capricola, Oesophagostomum venulosum\u003c/em\u003e, and \u003cem\u003eChabertia ovina\u003c/em\u003e, among others (7). Notably, it has been demonstrated that some abomasal nematodes of cervids can be transmitted to domestic ruminants, including anthelmintic-resistant strains of \u003cem\u003eH. contortus\u003c/em\u003e (8). This underscores the epidemiological importance of parasite exchange between wild ruminants and livestock (9). Consequently, such transmission can negatively affect the profitability of animal production, particularly in the case of infections with blood-feeding helminths, such as \u003cem\u003eH. contortus\u003c/em\u003e and \u003cem\u003eA. sidemi\u003c/em\u003e, which may cause haemorrhagic anaemia in the host (10, 11).\u003c/p\u003e \u003cp\u003eA combination of environmental, ecological, and host-related factors shapes helminth communities in roe deer. Among these, habitat type has been identified as a key determinant of abomasal nematode community composition, with distinct parasite assemblages associated with forest and agricultural environments (12). More broadly, anthropogenic changes are increasingly recognized as major drivers of host-parasite dynamics (13). While their ecological impacts on wildlife behaviour and habitat use have been widely documented (14), their effects on wildlife health remain less well understood.\u003c/p\u003e \u003cp\u003eIn this context, recent studies suggest that wind turbines may act as chronic stressors for terrestrial mammals, potentially affecting their physiology and immune responses (15, 16). Despite growing interest in wildlife parasitology, the potential influence of wind farm infrastructure on gastrointestinal parasite communities in wild ungulates has not yet been directly examined. This gap in knowledge highlights the need for studies addressing how renewable energy development may influence ecosystem health.\u003c/p\u003e \u003cp\u003eTherefore, this study aimed to: characterize the diversity and prevalence of gastrointestinal helminths in roe deer from central Poland; assess spatial variation in parasite communities across different regions and sections of the digestive tract; and evaluate the potential impact of wind farm presence on helminth species composition and prevalence.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eStudy Area\u003c/h2\u003e\n \u003cp\u003eEuropean roe deer samples were collected in Central Poland, within the Mazowieckie and Łódzkie voivodeships, across three sites: Iłża (51°10'7\"N, 21°11'0\"E), Rawa Mazowiecka (51°48'22\"N, 20°4'30\"E), and Węgrów (52°23'3\"N, 21°50'0\"E) (Fig. 1). These areas, part of the Central European Plain, are characterized by predominantly flat terrain with an average elevation of 173 m a.s.l. (17). Shaped by glacial processes, the landscape comprises diverse landforms and soils-from fertile loams to sandy substrates-affecting vegetation structure and microhabitat availability for intermediate parasite hosts. Major rivers, including the Vistula in Mazowieckie, create moist riparian zones that favor the survival and development of helminth eggs and larvae.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eThe temperate climate, with cold winters, warm summers, and peak precipitation during the growing season (18), further influences the seasonal dynamics of gastrointestinal parasites, resulting in a heterogeneous environment conducive to parasite persistence and transmission in wild ruminants.\u003c/p\u003e\n \u003cp\u003eAll three sites contain wind farms within agricultural landscapes that partially overlap the hunting grounds where roe deer were sampled. Based on their position relative to the turbines, deer were categorized as occurring inside or outside the wind farm. The wind farm area was defined as the space between turbines forming the cluster, plus a 700 m buffer zone, a distance previously used to assess wind turbine impacts on terrestrial mammals, including roe deer\u003c/p\u003e\n \u003cp\u003e(19, 20).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eExamined animals\u003c/h3\u003e\n\u003cp\u003eHunting management was actively practiced in the study areas, with licensed hunters harvesting a predetermined number of game animals, including roe deer, each year. Between 2022 and 2024, samples were collected in collaboration with local hunting clubs under formal agreements. For each individual, metadata including sex, age, date, and location of acquisition were recorded. Hunters provided the entire digestive tract post-mortem, which was then transported to the laboratory for parasitological analysis. No animals were killed specifically for this study; all samples were obtained as by-products of routine hunting activities, in compliance with Polish hunting regulations and management plans (21).\u003c/p\u003e\n\u003cp\u003eThe sampling strategy aimed to ensure that animals originated from comparable environmental conditions-agricultural landscapes of Central Poland-and experienced similar hunting pressure. In total, 102 roe deer were sampled: 39 from wind farm areas and 63 from outside these areas. Regionally, this included 48 individuals from Iłża (18 on wind farms, 30 outside), 34 from Rawa Mazowiecka (12 on wind farms, 22 outside), and 20 from Węgrów (9 on wind farms, 11 outside) (Fig. 1).\u003c/p\u003e\n\u003cp\u003eSections of the digestive tract - including the abomasum, small intestine, large intestine, and cecum - were collected from each animal for analysis. In some instances, specific sections could not be collected due to damage (Table 1).\u003c/p\u003e\n\u003ch3\u003eCollection of helminths\u003c/h3\u003e\n\u003cp\u003eIn each case, the contents of the abomasum, small intestine, large intestine, and cecum were mixed separately with tap water at a ratio of 1:10, decanted several times, and washed through a sieve (mesh size: 100–150 µm). The resulting sediment was then examined for the presence of parasites using a Delta Optical SZ-450 T stereomicroscope (Delta Optical, Mińska Mazowiecki, Poland). Collected parasites were placed in tubes containing 70% ethanol for subsequent morphological and molecular analyses.\u003c/p\u003e\n\u003ch3\u003eMorphological examination\u003c/h3\u003e\n\u003cp\u003eThe anterior and posterior parts of the nematode specimens were excised and transferred into a drop of lactophenol on a microscope slide to render the cuticle transparent and to allow visualization of the oral cavity and spicules of males (6). The middle body parts of the examined specimens were individually transferred into Eppendorf tubes containing 70% ethanol and appropriately labelled.\u003c/p\u003e\n\u003cp\u003eSpecies identification of the nematodes was performed using a LAB40 light microscope (OPTA-TECH, Warsaw, Poland) at magnifications of 100×-400× (22, 23). Microphotographs were captured using a digital camera, and measurements were obtained with OPTA View-15 software (OPTA-TECH).\u003c/p\u003e\n\u003cp\u003eDue to the poor preservation of the cestode specimens, morphometric analyses could not be conducted; therefore, molecular methods were applied instead.\u003c/p\u003e\n\u003ch3\u003eDNA extraction, amplification, and sequencing\u003c/h3\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eNematodes\u003c/h2\u003e\n \u003cp\u003eGenomic DNA was individually extracted from 50 intestinal nematodes to enable molecular species identification (25 individuals of \u003cem\u003eOesophagostomum\u003c/em\u003e sp. and 25 of \u003cem\u003eChabertia\u003c/em\u003e sp.) using a NucleoSpin Tissue DNA extraction kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s protocol.\u003c/p\u003e\n \u003cp\u003ePartial regions of the internal transcribed spacer 2 (ITS2) and the large subunit (LSU) ribosomal RNA gene were amplified by polymerase chain reaction (PCR) using two oligonucleotide primers: forward NC1 (5′-ACG TCT GGT TCA GGG TTG TT-3′) and reverse NC2 (5′-TTA GTT TCT TTT CCT CCG CT-3′) (24).\u003c/p\u003e\n \u003cp\u003ePCR amplification was performed in a total reaction volume of 50 µL, containing 20 µL of molecular biology grade water (Sigma-Aldrich, St. Louis, MO, USA), 25 µL of AccuStart II PCR ToughMix (2× concentration) (Quantabio, Beverly, MA, USA), 1 µL of GelTrack Loading Dye (50× concentration) (Quantabio), 1 µL of each primer (20 mM), and 2 µL of template DNA. Negative (molecular biology grade water) control was included in each run.\u003c/p\u003e\n \u003cp\u003eThermal cycling conditions followed those described previously (25). The resulting sequences were assembled into contigs using CodonCode Aligner version 8.0 (CodonCode Corporation, Centerville, MA, USA). The obtained nucleotide sequences were compared with those available in the NCBI database using the Basic Local Alignment Search Tool (BLAST) and subsequently submitted to GenBank.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eCestodes\u003c/h3\u003e\n\u003cp\u003eThe tissue samples of two tapeworms recovered from the small intestine of roe deer originating from the Węgów and Iłża regions were rinsed with phosphate-buffered saline (PBS) to remove residual ethanol. Genomic DNA was extracted using the Genomic Mini Kit (A\u0026amp;A Biotechnology, Gdańsk, Poland) in accordance with the manufacturer’s instructions.\u003c/p\u003e\n\u003cp\u003ePCR amplification was performed using primers JB3 (5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB4.5 (5′-TAAAGAAAGAACATAATGAAA-ATG-3′) (26) to obtain a 396 bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene.\u003c/p\u003e\n\u003cp\u003eEach PCR reaction was carried out in a total volume of 25 µl, containing 18 µl of deionized water, 1.5 µl of 25 µM MgCl₂ solution, 0.25 µl of Allegro Taq DNA polymerase (5 U/µl) (Novazym, Poznań, Poland), 0.25 µl of dNTP mix (10 mM), 2.5 µl of 10× Taq DNA polymerase buffer (containing 25 mM MgCl₂), 0.25 µl of each primer (20 pmol/µl), and 4 µl of template DNA. In the negative control, nuclease-free water was used instead of template DNA.\u003c/p\u003e\n\u003cp\u003ePCR conditions followed the protocol described previously (26). Amplification products were visualized on 1% agarose gels stained with SimplySafe™ (EURx, Gdańsk, Poland). The gels were documented using a ChemiDoc system and MP Lab software (Image Lab, Bio-Rad, Hercules, USA).\u003c/p\u003e\n\u003cp\u003eThe PCR products were purified using the Agarose-Out DNA Purification Kit (EURx, Gdańsk, Poland) and subsequently sequenced by Genomed (Warsaw, Poland). The resulting nucleotide sequences were assembled using CLC Main Workbench version 6.9.1 and analyzed with NCBI BLAST (27). Finally, the sequences were submitted to GenBank.\u003c/p\u003e\n\u003ch3\u003ePhylogenetic reconstruction of cestodes\u003c/h3\u003e\n\u003cp\u003eThe 62 nucleotide sequences showing the highest similarity in the NCBI BLAST analysis were selected for phylogenetic tree reconstruction using Bayesian inference implemented in MrBayes version 3.2.7 (28). The sequence of \u003cem\u003eEchinococcus canadensis\u003c/em\u003e (GenBank: LC184604) was used as an outgroup.\u003c/p\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eStatistical analysis of the impact of selected factors on the number of nematodes\u003c/h2\u003e\n \u003cp\u003eWe used a generalized linear model with a negative binomial distribution and a log link function to evaluate the potential impact of selected factors on the number of nematodes in different sections of the roe deer digestive tract. Negative binomial models are suitable for handling overdispersion in count data (29). Separate models were constructed for each section (abomasum, small intestine, large intestine, and cecum) to account for differences in total abundance and species composition between sections.\u003c/p\u003e\n \u003cp\u003eIn each model, the dependent variable was the total number of nematodes in a given section of the digestive tract. Four explanatory variables were included: sex and age of the animals, study area, and the presence of roe deer within wind farm areas. The inclusion of the latter variable was motivated by the potential impact of wind farm infrastructure on roe deer stress levels and health (15, 30).\u003c/p\u003e\n \u003cp\u003eThe effects of the selected factors on nematode abundance were assessed using a model selection approach. Specifically, all possible model variants (i.e., all combinations of explanatory variables), including the null model, were fitted to identify the best-supported model (31). Models were ranked according to the Akaike Information Criterion (AIC), with the model showing the lowest AIC value considered the best-fitting. Only the results of the highest-ranked models are presented.\u003c/p\u003e\n \u003cp\u003eAll statistical analyses were performed using IBM SPSS Statistics 29.0 (Armonk, New York).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eAbomasal nematodes\u003c/h2\u003e\n \u003cp\u003eA total of seven nematode species were identified in the abomasum of roe deer in central Poland (Table 2; Fig. 2). The most dominant species was the blood-sucking \u003cem\u003eH. contortus\u003c/em\u003e, which was commonly found in roe deer across all examined regions. The prevalence of \u003cem\u003eH. contortus\u003c/em\u003e ranged from 35.4% in the Iłża region to 47.4% in the Węgrów region. Moreover, this species exhibited the highest infection intensity in all regions, with a maximum of 2,558 individuals recorded in a single host from the Węgrów region.\u003c/p\u003e\n \u003cp\u003eNotably, the composition of abomasal nematode communities differed between regions. In the Rawa Mazowiecka region, only monoinfections with \u003cem\u003eH. contortus\u003c/em\u003e were observed, whereas mixed infections comprising four and five species were recorded in the Węgrów and Iłża regions, respectively. The highest species richness was found in roe deer from the Iłża region, where the presence of the alien, blood-sucking species \u003cem\u003eA. sidemi\u003c/em\u003e was recorded exclusively.\u003c/p\u003e\n \u003cp\u003eAdditionally, \u003cem\u003eSpiculopteragia boehmi\u003c/em\u003e was identified in animals from the Iłża region, together with \u003cem\u003eOstertagia leptospicularis\u003c/em\u003e and its minor morph, \u003cem\u003eO. kolchida\u003c/em\u003e; however, these parasites were detected in only a single individual. Interestingly, \u003cem\u003eO. leptospicularis\u003c/em\u003e was also found in a roe deer from the Węgrów region, where \u003cem\u003eMazamastrongylus dagestanica\u003c/em\u003e and \u003cem\u003eTrichostrongylus capricola\u003c/em\u003e were also recorded.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eSmall intestine nematodes\u003c/h2\u003e\n \u003cp\u003eTogether, seven species of nematodes were identified in the small intestine of the examined roe deer (Table 3; Fig. 3). All species were detected in animals from the Iłża region, whereas lower species richness was observed in the two remaining areas. Molecular analyses indicated that \u003cem\u003eOesophagostomum\u003c/em\u003e sp. corresponded to \u003cem\u003eOe. venulosum\u003c/em\u003e, while \u003cem\u003eChabertia\u003c/em\u003e sp. was identified as \u003cem\u003eCh. ovina\u003c/em\u003e (GenBank: PV920951 and PV920952, respectively). All obtained partial nucleotide sequences of the ITS2 and LSU regions for each species were homologous both to one another and to reference isolates of \u003cem\u003eOe. venulosum\u003c/em\u003e and \u003cem\u003eCh. ovina\u003c/em\u003e available in GenBank. The length of the obtained sequences ranged from 240 to 300 bp.\u003c/p\u003e\n \u003cp\u003eNevertheless, nematodes such as \u003cem\u003eAonchotheca\u003c/em\u003e sp., \u003cem\u003eCh. ovina\u003c/em\u003e, \u003cem\u003eBunostomum trigonocephalum\u003c/em\u003e, and \u003cem\u003eOe. venulosum\u003c/em\u003e were common parasites in animals from all studied locations. The most dominant species across all regions was \u003cem\u003eCh. ovina\u003c/em\u003e, with prevalence ranging from 15.2% to 31.6% in the Iłża and Węgrów regions, respectively. Moreover, the highest infection intensity was also recorded for \u003cem\u003eCh. ovina\u003c/em\u003e, regardless of region, reaching a maximum of 66 individuals in a single host from the Węgrów region. Additionally, single infections of \u003cem\u003eNematodirus europaeus\u003c/em\u003e, \u003cem\u003eH. contortus\u003c/em\u003e, and \u003cem\u003eB. phlebotomum\u003c/em\u003e were detected exclusively in roe deer from the Iłża region.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eSmall intestine cestodes\u003c/h2\u003e\n \u003cp\u003eFragmental strobiles of tapeworms were found in the contents of the small intestine of four roe deer, including two animals from the Węgrów and the Iłża regions (prevalence: 10.5% and 4.3%, respectively). Their general prevalence was 4.1%, as tapeworms were found in 4 of 98 examined small intestines.\u003c/p\u003e\n \u003cp\u003eTwo partial sequences of the cox1 gene, each of a length of 396 bp, were obtained from 2 cestodes, one from the Węgrów region, and the second from Iłża (GenBank: PX511568, PX511569, respectively). The obtained sequences differ at the nucleotide level but encode the same amino acid sequence. The obtained isolates showed a percentage identity ranging from 87.6% to 93.5% with \u003cem\u003eMoniezia\u003c/em\u003e species strains from Denmark, China, India, Senegal, Ethiopia, Turkey, Vietnam, and Taiwan, derived from both wild and domestic ruminants, including European bison, sheep, buffalo, cows, and goats. More detailed data are presented in Fig. 5. Due to the high level of variation in the examined fragment of the cox1 gene, the species-level determination of the tapeworms was currently unaccomplishable. Therefore, they were classified as \u003cem\u003eMoniezia\u003c/em\u003e sp.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003eLarge intestine nematodes\u003c/h2\u003e\n \u003cp\u003eA total of four nematode species were identified in the large intestine of roe deer (Table 4; Fig. 3). Nematodes such as \u003cem\u003eCh. ovina\u003c/em\u003e, \u003cem\u003eOe. venulosum\u003c/em\u003e, and \u003cem\u003eTrichuris\u003c/em\u003e sp. were commonly observed regardless of the animals’ geographic origin. However, \u003cem\u003eCh. ovina\u003c/em\u003e was the dominant species across all locations, with prevalence ranging from 63.2% in the Węgrów region to 70.8% in the Rawa Mazowiecka region. The highest intensity of \u003cem\u003eCh. ovina\u003c/em\u003e infection was recorded in a single individual from the Iłża region, in which 117 specimens were found. The fourth species, \u003cem\u003eB. trigonocephalum\u003c/em\u003e, was detected exclusively in one roe deer from the Rawa Mazowiecka region.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003eCecum nematodes\u003c/h2\u003e\n \u003cp\u003eTogether, four nematode species were identified in the cecum of the roe deer (Table 5; Fig. 3). \u003cem\u003eTrichuris\u003c/em\u003e sp. and \u003cem\u003eOe. venulosum\u003c/em\u003e were present in animals inhabiting all three regions. Moreover, \u003cem\u003eTrichuris\u003c/em\u003e sp. was the most prevalent cecal parasite, with prevalence ranging from 30% in the Węgrów region to 55.3% in the Iłża region. The maximum intensity of \u003cem\u003eTrichuris\u003c/em\u003e sp. infection was 46 individuals recorded in a single roe deer from the Iłża region. Other nematodes detected in this part of the intestine included \u003cem\u003eCh. ovina\u003c/em\u003e, found in animals from the Iłża and Rawa Mazowiecka regions, and \u003cem\u003eH. contortus\u003c/em\u003e, the least prevalent species, recorded in a single animal from the Iłża region.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003ePrevalence of parasites in animals living on wind farms and outside wind farm areas\u003c/h2\u003e\n \u003cp\u003eRegarding abomasum and small intestine nematodes, the prevalence of each species was higher in animals inhabiting areas within the wind farms. This pattern did not apply to worms of the genus \u003cem\u003eTrichuris\u003c/em\u003e, which were diagnosed in the large intestine and cecum. Specifically, \u003cem\u003eTrichuris\u003c/em\u003e sp. was the only nematode whose prevalence was higher in animals from areas outside the wind farms compared to those inside. Moreover, nematode species richness was consistently higher in hosts from areas within the wind farms. In particular, species composition was enriched by \u003cem\u003eM. dagestanica\u003c/em\u003e, \u003cem\u003eO. kolchida\u003c/em\u003e, \u003cem\u003eS. boehmi\u003c/em\u003e, and \u003cem\u003eT. capricola\u003c/em\u003e among abomasum nematodes (Table 6); \u003cem\u003eB. phlebotomum\u003c/em\u003e and \u003cem\u003eH. contortus\u003c/em\u003e among small intestine nematodes (Table 7); and \u003cem\u003eB. trigonocephalum\u003c/em\u003e and \u003cem\u003eH. contortus\u003c/em\u003e among large intestine and cecum nematodes, respectively (Table 8). Tapeworms of \u003cem\u003eMoniezia\u003c/em\u003e sp. were also more prevalent in animals inhabiting wind farm areas compared to roe deer from areas outside the wind farms (Table 7).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003eImpact of selected factors on the number of nematodes in sections of the digestive tract\u003c/h2\u003e\n \u003cp\u003eFor all sections of the digestive tract of roe deer, unique models that significantly explained the variability in nematode counts were obtained. However, not all best-fitting models for individual digestive tract sections included all variables as components. The presence of the wind farm significantly explained the variability in nematode counts in three of the four digestive tract sections: the abomasum, small intestine, and cecum (Table S1). In the case of the large intestine, this variable was excluded during model selection. In all cases where the wind farm was a significant explanatory variable, nematode counts were higher in the wind farm areas compared to the control areas (Fig. 4). The marginal mean in wind farm areas was approximately twice as high as in control areas. However, the standard error for individuals residing in wind farms was consistently greater (Fig. 4).\u003c/p\u003e\n \u003cp\u003eThe study area was an important variable in all best-fitting models for individual sections of the digestive tract, although not all comparisons between study areas were statistically significant (Table S1). Nevertheless, the study area's effect was unclear, as indicated by the varying estimates across study areas.\u003c/p\u003e\n \u003cp\u003eSex and age of animals were also important variables in the models, with sex appearing in all models and age in only two of four (exclusively in models for abomasum and small intestine). Regarding the sex of animals, females always showed lower nematode numbers than males (Table S1).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe high diversity of GINs recorded in this study confirms that roe deer harbour complex parasite communities typical of European cervids. All GIN parasites identified here have previously been reported in this host (6, 32, 33, 34), although earlier data from Poland indicate an even greater species richness, including \u003cem\u003eO. antipini\u003c/em\u003e, \u003cem\u003eO. lyrata\u003c/em\u003e, \u003cem\u003eS. asymmetrica\u003c/em\u003e, \u003cem\u003eS. mathevossiani\u003c/em\u003e, \u003cem\u003eT. askivalli\u003c/em\u003e, and \u003cem\u003eT. axei\u003c/em\u003e (6, 12, 32). Moreover, additional species have been reported in roe deer from other European countries and Russia, such as \u003cem\u003eCooperia oncophora\u003c/em\u003e, \u003cem\u003eMarshallagia marshalli\u003c/em\u003e, \u003cem\u003eN. europeus\u003c/em\u003e, \u003cem\u003eN. filicollis\u003c/em\u003e, \u003cem\u003eO. occidentalis\u003c/em\u003e, \u003cem\u003eO. ostertagi\u003c/em\u003e, and \u003cem\u003eT. colubriformis\u003c/em\u003e (34, 35, 36, 37). Together, these findings highlight both the richness and geographical variability of helminth communities in this host.\u003c/p\u003e \u003cp\u003eIn contrast to nematodes, knowledge of cestode diversity in roe deer remains limited. To date, only two tapeworm species, \u003cem\u003eM. expansa\u003c/em\u003e and \u003cem\u003eM. benedeni\u003c/em\u003e, have been reported in Poland based on morphological identification (33, 38). The detection of \u003cem\u003eMoniezia\u003c/em\u003e sp. with low genetic similarity to reference sequences in the present study supports earlier suggestions that insufficient molecular data hinder accurate species identification in wild ruminants (39). This finding points to potential cryptic diversity within the genus and suggests the presence of a third, previously unrecognized \u003cem\u003eMoniezia\u003c/em\u003e species in Poland, emphasizing the need for integrative taxonomic approaches combining molecular and morphological methods. Similarly, the inability to identify \u003cem\u003eAonchotheca\u003c/em\u003e and \u003cem\u003eTrichuris\u003c/em\u003e to species level in this study is consistent with previous reports of \u003cem\u003eT. globulosa\u003c/em\u003e and \u003cem\u003eA. bovis\u003c/em\u003e (syn. \u003cem\u003eCapillaria bovis\u003c/em\u003e) in roe deer in Poland (33).\u003c/p\u003e \u003cp\u003eParticular attention should be paid to the occurrence of hematophagous nematodes of the subfamily Haemonchinae. Both \u003cem\u003eA. sidemi\u003c/em\u003e and \u003cem\u003eH. contortus\u003c/em\u003e were recorded by us, in agreement with some studies (12), but contrasting with others that report the dominance of only one of these species (6, 32, 33, 35, 37). The dominance of \u003cem\u003eH. contortus\u003c/em\u003e observed here supports growing evidence that this species is becoming increasingly important in wildlife and reflects ongoing parasite exchange between wild and domestic ruminants, particularly in areas where grazing habitats overlap (4). An additional noteworthy observation was the presence of a single \u003cem\u003eH. contortus\u003c/em\u003e individual in the caecum, which represents an atypical localization. This may be explained by post-mortem displacement or abnormal migration within the gastrointestinal tract; however, aberrant localization cannot be excluded, especially under conditions of high infection intensity (184 individuals were found in the abomasum of the infected roe deer) or host physiological stress, such as that potentially associated with wind farm environments.\u003c/p\u003e \u003cp\u003eThe parasite assemblage identified in this study includes numerous species with well-documented pathogenic effects. Hematophagous nematodes such as \u003cem\u003eH. contortus\u003c/em\u003e and \u003cem\u003eA. sidemi\u003c/em\u003e can cause severe anemia and reduced fitness (40), while abomasal species, including \u003cem\u003eO. kolchida\u003c/em\u003e, \u003cem\u003eO. leptospicularis\u003c/em\u003e, \u003cem\u003eM. dagestanica\u003c/em\u003e, and \u003cem\u003eS. boehmi\u003c/em\u003e, may induce gastric disturbances and weight loss (41). Intestinal nematodes such as \u003cem\u003eCh. ovina\u003c/em\u003e, \u003cem\u003eOe. venulosum\u003c/em\u003e, \u003cem\u003eN. europaeus\u003c/em\u003e, and \u003cem\u003eTrichuris\u003c/em\u003e sp. are associated with enteritis and secondary infections (42). In addition, hookworms (\u003cem\u003eB. phlebotomum\u003c/em\u003e, \u003cem\u003eB. trigonocephalum\u003c/em\u003e) contribute to anemia, whereas less pathogenic taxa, including \u003cem\u003eAonchotheca\u003c/em\u003e sp. and \u003cem\u003eMoniezia\u003c/em\u003e sp., may still affect host condition under heavy infection. Importantly, mixed infections are likely to have synergistic effects, further exacerbating negative impacts on host health (40, 42).\u003c/p\u003e \u003cp\u003eOne of the most significant findings of this study is the clear effect of wind farm presence on parasite infections. Roe deer inhabiting wind farm areas exhibited higher nematode abundance and species richness, suggesting that anthropogenic infrastructure can influence host-parasite interactions. This, to our knowledge, represents the first evidence linking wind farms to gastrointestinal helminth dynamics in terrestrial mammals. Previous research in such environments has focused mainly on behavioral and ecological responses (44, 45, 46, 47), with some studies indicating that cumulative stressors associated with wind farms may negatively affect fitness and survival (48, 49). Supporting this interpretation, anthropogenic disturbance has been shown to induce physiological stress responses, including elevated glucocorticoid levels (15, 16), as well as reduced body condition (19, 50, 51), and developmental abnormalities (20). Since chronic stress can impair immune function (52), it is plausible that such mechanisms contribute to increased susceptibility to parasitic infections. Although direct evidence linking these processes to disease dynamics has been limited (14), the present study suggests that they may result in measurable differences in parasite burden.\u003c/p\u003e \u003cp\u003eTogether, these findings underline the complex interplay between environmental factors, host condition, and parasite ecology.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study confirms that a complex interplay of environmental, host-related, and anthropogenic factors shapes gastrointestinal helminth communities in roe deer. Importantly, it provides novel evidence that wind farm infrastructure may influence parasite infections in wild ruminants, likely through stress-mediated mechanisms.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ea.s.l. \u0026ndash; above sea level\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA.\u0026nbsp;\u003c/em\u003e\u003cem\u003e\u0026ndash; Ashworthius\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eB. \u0026nbsp;\u0026ndash; Bunostomum\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCh. \u0026ndash; Chabertia\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eH. \u0026ndash; Haemonchus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. \u0026ndash; Mazamastrongylus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eN. \u0026ndash; Nematodirus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eO. \u0026ndash; Ostertagia\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOe. \u0026ndash; Oesophagostomum\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. \u0026ndash; Spiculopteragia\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eT. - Trichostrongylus\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudies on animal tissues obtained post-mortem do not require the approval of the Ethics Committee. All the samples were taken exclusively from roe deer legally hunted during the hunting seasons 2023 and 2024 according to Polish hunting law (Act of the Polish Parliament dated 13 October 1995, item 713, the Hunting Law).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe nucleotide sequences obtained in the study are available in the GenBank database under the following numbers: PV920951 www.ncbi.nlm.nih.gov/nuccore/pv920951, PV920952 www.ncbi.nlm.nih/nuccore/pv920952, PX511568 www.ncbi.nlm.nih.gov/nuccore/px511568, PX511569 www.ncbi.nlm.nih.gov/nuccore/px511569. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was partially financed by the National Science Centre, Poland (Grant number: 2021/41/B/NZ9/04442), and by the European Union (MSCA4Ukraine project https://cordis.europa.eu/project/id/101101923). Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the MSCA4Ukraine Consortium as a whole nor any individual member institutions of the MSCA4Ukraine Consortium can be held responsible for them.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAMP\u0026nbsp;\u003c/strong\u003econceptualization,morphological and molecular analyses of nematodes; photographic documentation; writing – original draft, review and editing, supervision;\u003cstrong\u003e\u0026nbsp;KS\u0026nbsp;\u003c/strong\u003efunding acquisition, methodology of collecting the worms, writing – original draft, review and editing;\u003cstrong\u003e\u0026nbsp;RS\u0026nbsp;\u003c/strong\u003emolecular analyses of cestodes, writing – review and editing;\u003cstrong\u003e\u0026nbsp;JW\u0026nbsp;\u003c/strong\u003emolecular analyses of cestodes, writing – original draft, review and editing;\u003cstrong\u003e\u0026nbsp;MK\u0026nbsp;\u003c/strong\u003ecollecting ofsamples;\u003cstrong\u003e\u0026nbsp;MŚ\u0026nbsp;\u003c/strong\u003ecollecting ofsamples;\u003cstrong\u003e\u0026nbsp;OZ\u0026nbsp;\u003c/strong\u003emethodology of collecting the worms;\u0026nbsp;\u003cstrong\u003eDK\u0026nbsp;\u003c/strong\u003efunding acquisition, conceptualization, statistical analysis, writing – original draft, review and editing, supervision.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eApollonio M, Andresen R, Putman R. 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Landsc Ecol. 2015; 30:1527-1540.\u003c/li\u003e\n\u003cli\u003eKim S, Dhakal T, Cho KH, Kim T, Woo S, Kim J, Lee D, Jang G. Occupancy of roe deer, water deer, and wild boar in wind farm‐integrated forest ecosystems: a case study in Korea. Ecosphere. 2025;16:e70258. \u003c/li\u003e\n\u003cli\u003eTaylor KL, Beck JL, Huzurbazar SV. Factors influencing winter mortality risk for pronghorn exposed to wind energy development. Rangel Ecol Manag. 2016; 69:108-116.\u003c/li\u003e\n\u003cli\u003eMilligan MC, Johnston AN, Beck JL, Smith KT, Taylor KL, Hall E, Knox L, Cufaude T, Wallace C, Chong G, Kauffman MJ. Variable effects of wind energy development on seasonal habitat selection of pronghorn. Ecosphere. 2021;12:e03850.\u003c/li\u003e\n\u003cli\u003eAgnew RC, Smith VJ, Fowkes RC. Wind turbines cause chronic stress in badgers (\u003cem\u003eMeles meles\u003c/em\u003e) in Great Britain. J Wildl Dis. 2016;52:459-467.\u003c/li\u003e\n\u003cli\u003eKarwowska M, Mikołajczak J, Dolatowski ZJ, Borowski S. The effect of varying distances from the wind turbine on meat quality of growing-finishing pigs. Ann Anim Sci. 2015;15:1043-1054. \u003c/li\u003e\n\u003cli\u003eSapolsky RM, Romero LM, Munck AU. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory and preparative actions. Endocr Rev. 2000;21:55-89.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 8 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"wind farms impact, gastrointestinal parasites, roe deer (Capreolus capreolus), central Poland","lastPublishedDoi":"10.21203/rs.3.rs-9245327/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9245327/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe roe deer (\u003cem\u003eCapreolus capreolus\u003c/em\u003e) is a widespread cervid in Europe and an important reservoir of gastrointestinal helminths of veterinary relevance. Although environmental and host-related drivers of parasite communities are well studied, the effects of anthropogenic infrastructure, such as wind farms, on host\u0026ndash;parasite interactions remain poorly understood. This study aimed to characterize gastrointestinal helminth diversity in roe deer from central Poland and evaluate the impact of wind farm presence on nematode infections.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 102 roe deer were examined from three regions in central Poland between 2022 and 2024. Animals were categorized based on occurrence within or outside wind farm areas (39 and 63 individuals, respectively). Helminths were collected from the abomasum, small intestine, large intestine, and cecum using standard parasitological methods. For molecular identification, nematodes morphologically assigned to the genera \u003cem\u003eOesophagostomum\u003c/em\u003e and \u003cem\u003eChabertia\u003c/em\u003e were analyzed based on the ITS2 gene region, whereas the cox1 gene was used for cestodes of the genus \u003cem\u003eMoniezia\u003c/em\u003e. The effects of wind farm presence on parasite abundance were analyzed using generalized linear models with a negative binomial distribution.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA diverse helminth community was identified, comprising 7 species in the abomasum, 7 in the small intestine, and 4 species each in the large intestine and cecum. \u003cem\u003eHaemonchus contortus\u003c/em\u003e dominated the abomasum; \u003cem\u003eChabertia ovina\u003c/em\u003e was the most prevalent species in the small and large intestine; and \u003cem\u003eTrichuri\u003c/em\u003es sp. was the most prevalent parasite in the cecum. Tapeworms of the genus \u003cem\u003eMoniezia\u003c/em\u003e were found in the small intestine of 4 animals. Molecular analyses confirmed \u003cem\u003eOe. venulosum\u003c/em\u003e and \u003cem\u003eC. ovina\u003c/em\u003e, and revealed genetically distinct \u003cem\u003eMoniezia\u003c/em\u003e sp. Apart from \u003cem\u003eTrichuris\u003c/em\u003e sp. infection, roe deer from wind farm areas exhibited higher helminth abundance and greater species richness than those from control areas, with infection levels approximately twice as high.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe increased parasite burden in wind farm areas suggests that anthropogenic disturbance may influence host\u0026ndash;parasite dynamics, potentially via stress-related mechanisms. These findings highlight the importance of incorporating wildlife health considerations into renewable energy impact assessments.\u003c/p\u003e","manuscriptTitle":"Biodiversity and prevalence of gastrointestinal helminths in roe deer (Capreolus capreolus L.) in relation to wind farm presence in central Poland","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-08 15:17:10","doi":"10.21203/rs.3.rs-9245327/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-27T05:27:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-23T21:11:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T07:33:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"242968536310189189307797900913774407156","date":"2026-04-05T11:48:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"270930775885749047684564872079511207768","date":"2026-04-03T09:36:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"137822382836047604703329675688592886250","date":"2026-04-02T08:49:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-02T07:13:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-02T04:26:14+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-01T14:53:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-31T09:19:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2026-03-31T08:50:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ce2f6391-3188-4ccd-92fc-d9335beda7ec","owner":[],"postedDate":"April 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T05:39:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-08 15:17:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9245327","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9245327","identity":"rs-9245327","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}