Introduction
Wild mammals, including large predators such as the brown bear (Ursus arctos), play an important role in ecosystems and provide multiple public benefits, e.g., ecosystem services like seed dispersal and cycling organic matter, or economic benefits through nature tourism for bear-watching (Tattoni et al., Reference Tattoni, Galaverni, Pollutri, Preatoni, Martinoli and Araña2023). However, an increase in various zoonotic diseases over the past decades has highlighted the role of wild mammals in disseminating hazardous pathogens, which can affect domesticated animals and humans. This has brought into the limelight the One Health concept that emphasizes the interconnectedness of all life and states that as we share the environment, the health of people is highly dependent on the health of animals. One of the major tasks related to the One Health concept is the need for detailed knowledge of zoonotic parasites spread by different wild animal species and populations (e.g., Thompson, Reference Thompson2013; Jenkins et al., Reference Jenkins, Simon, Bachand and Stephen2015; Goulet et al., Reference Goulet, de Garine-Wichatitsky, Chardonnet, de Klerk, Kock, Muset, Suu-Ire and Caron2024).
The brown bear is the largest mammalian predator in mainland Europe and despite being a protected species in the European Union, its populations in several countries remain small and endangered, notably in southern and central areas. In eastern and northern Europe, the populations are generally larger; however, habitat loss and hunting pressure remain major risk factors and constant monitoring of population status is therefore needed. In Estonia, the brown bear population size is, according to the official data, 900–950 individuals (Veeroja et al., Reference Veeroja, Männil, Jõgisalu and Kübarsepp2023). Of reported observations, 40–50% of females with cubs-of-the-year have been recorded in the core area of the northeastern part of Estonia. Despite the fact that about half of the country is covered by forest, only 12% of the forested area is strictly protected. According to Tammeleht et al. (Reference Tammeleht, Kull and Pärna2020), areas suitable for denning are relatively rare in these protected areas and are most commonly found in economically managed forests. Intensive forest management for timber production can disturb hibernating bears and result in the death of cubs, as well as decrease the habitat quality. Another potential threat is hunting. The current hunting pressure (nearly 10% of the presumed population size) is officially not regarded as a major threat, as a general upward trend in the population size has been reported for the last two decades (Veeroja et al., Reference Veeroja, Männil, Jõgisalu and Kübarsepp2023). The history of brown bears in the territory of Estonia has been relatively long. Studies based on mitochondrial DNA (mtDNA) have suggested that after the Last Ice Age, migration waves from two refuge areas (Carpathian and Altai-Sayan) contributed most to the colonization of the current territory of Estonia (Saarma et al., Reference Saarma, Ho, Pybus, Kaljuste, Tumanov, Kojola, Vorobiev, Markov, Saveljev, Valdmann, Lyapunova, Abramov, Männil, Korsten, Vulla, Pazetnov, Pazetnov, Putchkovskiy and Rõkov2007; Korsten et al., Reference Korsten, Ho, Davison, Pähn, Vulla, Roht, Tumanov, Kojola, Andersone-Lilley, Ozolins, Pilot, Mertzanis, Giannakopoulos, Vorobiev, Markov, Saveljev, Lyapunova, Abramov, Männil, Valdmann and Saarma2009; Davison et al., Reference Davison, Ho, Bray, Korsten, Tammeleht, Hindrikson, Østbye, Østbye, Lauritzen, Austin, Cooper and Saarma2011; Keis et al., Reference Keis, Remm, Ho, Davison, Tammeleht, Tumanov, Saveljev, Männil, Kojola, Abramov, Margus and Saarma2013; Anijalg et al., Reference Anijalg, Ho, Davison, Keis, Tammeleht, Bobowik, Tumanov, Saveljev, Lyapunova, Vorobiev, Markov, Kryukov, Kojola, Swenson, Hagen, Eiken, Paule and Saarma2018). Due to human activities, primarily the high hunting pressure, but also habitat destruction, the brown bear population went through a severe genetic bottleneck from 1890 to 1940. The number of bears was reduced to near extinction in the 1920s, with only 20–30 individuals remaining (Kaal, Reference Kaal1980). Due to protective measures, the population started to recover in the following decades, and it has been proposed that the core areas in northeastern (‘Alutaguse’) and central Estonia (‘Vahe-Eesti’), with their high forest coverage and low human density, were the primary refuge areas for bears that supported the population recovery. Despite the increase in population size, the re-inhabiting process has been slow, especially in the southern and western parts of Estonia (Kaal, Reference Kaal1980; Valdmann et al., Reference Valdmann, Saarma and Karis2001; Anijalg et al., Reference Anijalg, Remm, Tammeleht, Keis, Valdmann and Saarma2020). Moreover, the gene flow between the bear population of Estonia and those in neighbouring countries seems rather limited (Tammeleht et al., Reference Tammeleht, Remm, Korsten, Davison, Tumanov, Saveljev, Männil, Kojola and Saarma2010). Thus, considering the severe demographic bottleneck and limited gene flow, we expect that the parasite fauna of brown bears should largely depend on local environmental conditions, food items, and the parasite fauna of other local mammals who share the territories with brown bears. As the majority of parasites are acquired from the environment through feeding, the data on food habits is especially important for understanding the circulation of parasites. Studies on brown bear food habits in Estonia have revealed a highly diverse diet, including 72 plant, 31 animal, and 1 fungal taxa (Vulla et al., Reference Vulla, Hobson, Korsten, Leht, Martin, Lind, Männil, Valdmann and Saarma2009; Keis et al., Reference Keis, Tammeleht, Valdmann and Saarma2019).
Before the current study, brown bear parasites have not been investigated in Estonia. However, parasitological studies of several other wild mammal species have been carried out in the country, namely for the red fox (Vulpes vulpes), raccoon dog (Nyctereutes procyonoides), golden jackal (Canis aureus), grey wolf (Canis lupus), and Eurasian lynx (Lynx lynx). The common denominator for these species is a very high helminth prevalence, ranging from 91% for the jackal (Tull et al., Reference Tull, Valdmann, Tammeleht, Kaasiku, Rannap and Saarma2022) and 98% for the raccoon dog (Laurimaa et al., Reference Laurimaa, Süld, Davidson, Moks, Valdmann and Saarma2016a), up to 100% for the lynx (Valdmann et al., Reference Valdmann, Moks and Talvik2004), wolf (Moks et al., Reference Moks, Jõgisalu, Saarma, Talvik, Järvis and Valdmann2006), and fox (Laurimaa et al., Reference Laurimaa, Moks, Soe, Valdmann and Saarma2016b). In Europe, studies on the brown bear helminth fauna have been carried out in Poland (Borecka et al., Reference Borecka, Gawor and Zięba2013), Croatia (Aghazadeh et al., Reference Aghazadeh, Elson-Riggins, Reljić, De, Huber, Majnarić and Hermosilla2015); Slovakia (Orosova et al., 2016), Bosnia and Herzegovina (Omeragić et al., Reference Omeragić, Kapo, Škapur, Softić, Goletić, Šaljić and Goletić2024), Romania (Borka-Vitális et al., Reference Borka-Vitális, Domokos, Földvári and Majoros2017), Spain (Costa et al., Reference Costa, Hartasánchez, Santos and Camarão2022; Cano et al., Reference Cano, Penteriani, Vega, del Mar Delgado, González-Bernardo, Bombieri, Zarzo-Arias, Sánchez-Andrade Fernández and Paz-Silva2024; Remesar et al., Reference Remesar, Busto, Diaz, Rivas, Lopez-Bao, Ballesteros and Garcia-Dios2024), and central Italy (Paoletti et al., Reference Paoletti, Iorio, Traversa, Francesco, Gentile, Angelucci, Amicucci, Bartolini, Marangi and Cesare2017). However, these reports cover only a small fraction of the brown bear distribution area in Europe. Studies on the seasonal variation of helminth parasites are even more scarce, with only two studies addressing the topic, namely for bears of Slovakia (Orosová et al., Reference Orosová, Goldová, Ciberej and Štrkolcová2016) and the Cantabrian Mountains of Spain (Cano et al., Reference Cano, Penteriani, Vega, del Mar Delgado, González-Bernardo, Bombieri, Zarzo-Arias, Sánchez-Andrade Fernández and Paz-Silva2024). According to the published studies, the parasite prevalence in brown bear scats ranges from 16% to 79%, and various parasites are known to infect the brown bear, including zoonotic taxa of protozoa (Cryptosporidium spp., Coccidia sp., Sarcocystis sp., Eimeria sp., Giardia spp.), nematodes (Ancylostoma sp., Uncinaria sp., Baylisascaris transfuga, Crenosoma sp., Spirurida sp., Trichinella sp., Trichuris sp.), cestodes (Taenia sp., Dicrocoelium dendriticum) and trematodes. As a long-living mammal with a highly diverse omnivorous diet, it can be assumed that the brown bear plays an important role in spreading zoonotic parasites also in Estonia.
Separate research has been done on the bear-specific nematode B. transfuga in Slovakia, Croatia, and Poland (Finnegan, Reference Finnegan2009; Ambrogi, Reference Ambrogi2011; Gawor et al., Reference Gawor, Gawor, Gromadka, Zwijacz-Kozica and Zieba2017; Štrkolcová et al., Reference Štrkolcová, Goldová, Šnábel, Špakulová, Orosová, Halán and Mojžišová2018; Molnár et al., Reference Molnár, Königová, Major, Vasilková, Tomková and Várady2020). These studies have found that the prevalence of B. transfuga can range from 10% to 91%, but it needs to be taken into consideration that B. transfuga likely lays eggs periodically, like B. procyonis, which can sometimes result in underestimates of the parasite’s prevalence (Aghazadeh et al., Reference Aghazadeh, Elson-Riggins, Reljić, De, Huber, Majnarić and Hermosilla2015). Since B. transfuga can infect and cause health problems for >100 species, including mammals, birds, and humans (Kazacos, Reference Kazacos2016), and can be highly prevalent in bear scats, it is important to pay special attention to this nematode.
As there is a lack of knowledge of brown bear parasites in northern and eastern European countries, including Estonia, and only two of the existing publications have examined the seasonal dynamics of brown bear parasite infections, with this study, we aim to fill these gaps by analysing brown bear parasite fauna and its seasonal dynamics in Estonia. Considering the relatively high parasite prevalence in the other mammalian predators investigated in Estonia, we hypothesize that the parasite prevalence of brown bears in Estonia is among the highest in Europe. We also hypothesize that the prevalence is lowest in spring, after the hibernation period, and highest in late summer-early autumn, after the period of hyperphagia.
Materials and methods
Sampling
Sampling was conducted in the brown bear core area in northeastern Estonia (~ 4,000 km2) and in the south-eastern region (~600 km2) (Figure 1). In total, 198 scats were collected over a one-year period (April 2022–April 2023) from the forest and various forest roads. Scats were placed into numbered plastic bags, and additional information was recorded, such as the date and GPS location. Samples were frozen and kept at −80°C for at least two weeks to inactivate zoonotic endoparasites, as several life-threatening parasites, including Echinococcus multilocularis and E. granulosus s.l., are endemic in Estonia (Moks et al., Reference Moks, Jõgisalu, Saarma, Talvik, Järvis and Valdmann2006, Reference Moks, Jõgisalu, Valdmann and Saarma2008; Laurimaa et al., Reference Laurimaa, Davison, Süld, Plumer, Oja, Moks, Keis, Hindrikson, Kinkar, Laurimäe, Abner, Remm, Anijalg and Saarma2015a, Reference Laurimaa, Davison, Plumer, Süld, Oja, Moks, Keis, Hindrikson, Kinkar, Laurimäe, Abner, Remm, Anijalg and Saarma2015b).
Figure 1. Locations of brown bear scats in Estonia (n = 148), collected from April 2022 to April 2023.
Sample preparation for flotation and genetic analysis
The frozen scats were thawed at room temperature, after which 10 grams of material was weighed and homogenised for further processing. The sample was divided between two plastic cups, with 4 grams of homogenised scat placed into each cup. A total of 40 mL of distilled water was added and the mixture was stirred with a plastic stick every 5 minutes for a total of 30 minutes. The mixture was drained through a 0.5 mm stainless steel sieve into a 50 mL conical tube. The walls of the cups were rinsed with distilled water and poured through the sieve into the tubes, bringing the total volume in both tubes to 50 mL. The tubes were centrifuged at 1550 g (rcf) for 5 minutes at room temperature, and the supernatant was discarded. One of the sediments was used for DNA extraction to determine the host via genetic analysis, and the other for flotation to identify the endoparasite taxa based on the morphology of eggs/oocysts.
Genetic host identification
All samples were subjected to genetic analysis to select only the brown bear samples for further analysis. Briefly, the genomic DNA was extracted, and part of the mtDNA control region was PCR-amplified and sequenced to determine the host species.
DNA was extracted from the scat with the NucleoSpin DNA Stool Kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s protocol with minor changes:
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• Before extraction, the samples were heated at 98°C for 7 minutes (to break the shells of the parasite eggs more efficiently; although not required for host identification, this step was performed to enable genetic parasite identification, if needed).
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• To increase the efficiency of precipitation, incubation was carried out at 2°C for 10 minutes.
A 361 bp fragment of the mtDNA control region was PCR-amplified using the primers Canis7F-CCCTATGTACGTCGTGCATTA (a newly designed primer) and Canis3R-TGTGTGATCATGGGCTGATT (Plumer et al., Reference Plumer, Talvi, Männil and Saarma2018). The reaction mix, a total of 20 μL, contained 1 μL of purified DNA, 1 μL of primers (final concentration of 0.25 μM), 4 μL of 5× HOT FIREPol MultiPlex Mix (Solis BioDyne, Tartu, Estonia), and 14 μL of milliQ water. The cycling conditions for the PCR were: the primary denaturation of DNA was done for 12 minutes at 95°C, followed by 10 cycles in the touch-down regime: 20 s 95°C, 30 s 55°C (lowering the temperature by 0.5°C for each cycle), 45 s at 72°C; this was followed by 35 cycles: 20 s 98°C, 30 s 50°C, 45 s 72°C, and finally 2 minutes at 72°C. The resultant PCR products were purified at 37°C for 30 minutes with the enzymes FastAP and ExoI, using 1 unit of each (Thermo Fisher Scientific, USA), and the enzymes were then inactivated at 80°C for 15 minutes.
The purified PCR products were sent to the core sequencing facility at the Institute of Genomics, University of Tartu, for sequencing with the primer Canis 7F. The resultant DNA sequences were used to identify the host species, using a homology search with the Nucleotide BLAST (National Library of Medicine, USA).
Further analyses were performed only with the samples that were genetically confirmed as belonging to the brown bear (n = 148).
Morphological identification of parasites
A modified McMaster’s method (Roepstorff and Nansen, Reference Roepstorff and Nansen1998) was used to detect and identify endoparasite eggs/oocysts according to their morphology from the sediment. Flotation analysis was performed using a concentrated salt-sugar water mixture (400 g of NaCl and 500 g of white sugar dissolved in 1 L of water) with a specific gravity of 1.28 g/cm3. The solution was added to the sediment until 5 mL of general capacity (in the 50 mL Falcon tube). The mixture was stirred by hand or vortexed until homogeneous, and left to settle in a rack for 30 seconds, after which 2–3 mL of the top layer was collected with a pipette and transferred into two McMaster chambers. The sample was left to settle for 2 minutes, after which a microscopic analysis was conducted using the microscope Leica DM3000 LED with 100–200× magnification. Helminth eggs and protozoan oocysts were identified and counted, with a maximum of 100 per sample (due to the labour intensive nature of manual counting, and higher counts would not provide further significant data), and identified based on morphology and size (Schaul, Reference Schaul2006; Bowman, Reference Bowman2014; Borka-Vitalis et al., 2017; De Silva et al., Reference De Silva, Rajakaruna, Mohotti, Rajapakse and Perera2022).
Genetic verification of Baylisascaris transfuga
The eggs of B. transfuga are morphologically similar to those of other species in the genus and can be difficult to distinguish. Standard diagnostic techniques, such as the flotation combined with microscopical analysis, can be used to identify the presence of Baylisascaris-type ascarid eggs. However, it cannot be used for definitive species identification. Although other members of the genus Baylisascaris have never been recorded in Estonia, to verify the presence of B. transfuga, we conducted a genetic analysis for eight randomly chosen samples where morphological examination suggested B. transfuga. New primers Bay1F-TGGAGGTTGAGTAGTAATTGAGAGC and Bay1R-CCCTAACTCTACTTTACTACAACTTACTC were designed to amplify and sequence a 310 bp fragment of the mtDNA 12S ribosomal RNA gene, corresponding to positions 711-1020 of the reference sequence NC_015924.1 in the GenBank database (Xie et al., Reference Xie, Zhang, Wang, Lan, Li, Chen, Fu, Nie, Yan, Gu, Wang, Peng and Yang2011). The PCR and sequencing protocols were identical to those used for the genetic host identification, except the annealing temperature, which followed a touchdown regime from 60°C to 50°C.
Data analysis
The data analyses were conducted to identify patterns in the seasonality (three different seasons) of parasite infections and compare the prevalence (frequency of occurrence, FO) of different parasites, as well as the infection intensity of B. transfuga. To analyse the seasonal variation of infection with helminths and protozoa the year was divided into three different seasons based on bear feeding ecology (Vulla et al., Reference Vulla, Hobson, Korsten, Leht, Martin, Lind, Männil, Valdmann and Saarma2009): samples collected from April to the last week of May were categorized as spring samples, samples collected from the last week of May to July were categorized as summer samples, and samples collected from August to November were categorized as autumn samples. The identified parasites were categorized as protozoa, nematodes, trematodes, and cestodes. The programs MS Excel and R Studio (version 2021.09.1+372, The R Foundation, 2024) were used to analyse the statistically significant associations between parasite prevalence and season, using the Chi-square and Fisher’s Exact tests for preliminary assessment. Considering that many samples did not contain certain parasite eggs or oocysts, we used Firth’s bias-reduced logistic regression model (logistf) for more detailed data analysis.
Results
Of the collected scats, 148 were confirmed by genetic analysis to belong to brown bears, and among these, 75% contained parasite eggs/oocysts (Table 1). Protozoa (Eimeria, Giardia, Coccidia) were found in 16% and helminths in 63% of samples. Of the latter, 7% were cestodes (Taenia, Diphyllobothrium), 60% nematodes (Strongylidae, Ascarididae, Capillaria, B. transfuga), and 4% trematodes. The most frequent of all parasites was the nematode B. transfuga, recorded in 51% of all analysed samples, followed by coccidia with 12% prevalence (Table 2).
Table 1. Prevalence (frequency of occurrence, %) of different parasite groups found in brown bear scats of Estonia (n = 148), collected from April 2022 to April 2023
Table 2. Identified parasite taxa in brown bear scats (n = 148) of Estonia and their prevalence (%)
B. transfuga, Baylisascaris transfuga.
During the genetic verification of B. transfuga, eight samples were successfully analysed, resulting in 295 bp sequences of mtDNA 12S rRNA gene. Based on the nucleotide BLAST search (https://blast.ncbi.nlm.nih.gov), all eight samples were verified to contain B. transfuga. Two haplotypes were found. Sequences of six samples corresponded to haplotype Bt-Est1, which was 100% identical across the 295 bp reference sequence NC_015924.1 from the GenBank database. Two sequences were designated as haplotype Bt-Est2, having a single nucleotide difference at position 846 (A compared to G in the reference). Both haplotypes were deposited in the GenBank database (accession codes: PV834818 for haplotype Bt-Est1, and PV834819 for Bt-Est2).
In spring, the most frequent were protozoa, present in 41% of all spring samples, whereas among summer and autumn samples, the most prevalent were nematodes, with 54% and 77%, respectively (Table 3). Statistically significant (p < 0.05) associations were identified between seasons and the prevalence of protozoa (χ2 = 22.496, df = 2, p < 0.001), roundworms (χ2 = 32.374, df = 2, p < 0.001), and tapeworms (Fisher’s test p-value = 0.01), with protozoa infections being most common in spring, tapeworm in summer and roundworms in autumn (Figure 2). The most common endoparasite, B. transfuga, exhibited significant seasonal variation (χ2 = 47.461, df = 2, p < 0.001), with the frequency of occurrence being highest in autumn (present in 72%) and lowest in spring (3%), and the same trend was also revealed for the infection intensity (Figure 3).
Table 3. Seasonal variation in the prevalence (%) of different parasite taxa found in brown bear scats of Estonia (n = 148)
B. transfuga, Baylisascaris transfuga.
Table 4. Prevalence of different parasite taxa in summer and autumn, compared to their occurrence in spring (according to the Firth’s bias-reduced logistic regression model)
Figure 2. Variation of parasite prevalence (frequency of occurrence, %) in brown bear scats in different seasons: spring (n = 34), summer (n = 28), and autumn (n = 86).
Figure 3. Seasonal variation in the infection intensity (egg quantity) of the bear nematode Baylisascaris transfuga in brown bear scats (eggs were counted up to 100; scats with zero eggs are not included). The box (interquartile range) shows the range of values that 50% of the samples have. The dark horizontal line in the box represents the mean value, the whiskers show values of 1.5 times the interquartile range, and the dots outside the whiskers are extreme values of egg counts.
Compared to spring, protozoa are 0.33× less likely to be present in scats collected in summer and 0.1× less likely to be present in scats collected in autumn (p = 0.05 and p < 0.01, respectively; Table 3, Table 4), whereas nematode eggs are 4.2× more likely to be present in scats collected in summer and 12× more likely in scats collected in autumn (p < 0.01). Cestode and trematode infections’ association with seasons was not statistically significant (p-values 0.1 and 0.6, respectively).
Discussion
Brown bears can spread and act as a reservoir host for many zoonotic pathogens (Sheikh et al., Reference Sheikh, Tak, Fazili, Bhat and Wani2018; Salvo and Chomel, Reference Salvo and Chomel2020; Costa et al., Reference Costa, Hartasánchez, Santos and Camarão2022). With the exception of Eimeria sp., all parasite taxa identified in this research are either zoonotic or potentially zoonotic (Lindsay and Todd Jr, Reference Lindsay, Todd and Kreier1993; Acha and Szyfres, Reference Acha and Szyfres2003; Scholz et al., Reference Scholz, Garcia, Kuchta and Wicht2009; Kazacos, Reference Kazacos2016; Deplazes et al., Reference Deplazes, Eichenberger and Grimm2019; Otranto and Deplazes, Reference Otranto and Deplazes2019; Mathison et al., Reference Mathison, Mehta and Couturier2021). The latter category includes B. transfuga, the most frequent of all helminth parasites found in this study. Even though the zoonotic potential of B. transfuga has not been confirmed yet, all nine species of Baylisascaris have been considered to be able to cause visceral, ocular, and/or neural larva migrans in more than a hundred species, including birds, a wide range of mammals, and humans (Bauer, Reference Bauer2013; Kazacos, Reference Kazacos2016). Since B. transfuga was found to cause fatal neurological diseases in a colony of Japanese macaques (Macaca fuscata fuscata) (Sato et al., Reference Sato, Une, Kawakami, Saito, Kamiya, Akao and Furuoka2005), it is likely that the nematode is able to cause serious diseases in other primates, including humans. In this work, we did not find any representative of Uncinaria spp. Apparently, these parasites are rare in bears of Estonia, although Uncinaria stenocephala has been recorded in other mammalian carnivores in the country, such as in the fox, jackal, raccoon dog, wolf, and American mink (Neovison vison) (Moks et al., Reference Moks, Jõgisalu, Saarma, Talvik, Järvis and Valdmann2006; Laurimaa et al., Reference Laurimaa, Süld, Davidson, Moks, Valdmann and Saarma2016a, b; Tull et al., Reference Tull, Valdmann, Tammeleht, Kaasiku, Rannap and Saarma2022). The Ascarididae eggs most likely originated from Toxocara sp., as according to Holland (Reference Holland2023), Toxocara sp. infects a wide range of wild animals and has been shown to infect at least 19 species of wild canids, including the red fox, wolf, golden jackal, and raccoon dogs. Considering the overlap in food objects between brown bears and the listed canids, it is likely that the brown bears can also be infected with Toxocara sp. On the other hand, the eggs in their scats may have originated from ingesting prey that contained Toxocara sp. eggs.
Analysing seasonal differences in parasite infection provides important data for brown bear parasite dynamics. In general terms, the seasonal dynamics of the brown bear parasite infections in Estonia follow the same trend as for bears in Spain (Cano et al., Reference Cano, Penteriani, Vega, del Mar Delgado, González-Bernardo, Bombieri, Zarzo-Arias, Sánchez-Andrade Fernández and Paz-Silva2024) and Slovakia (Orosová et al., Reference Orosová, Goldová, Ciberej and Štrkolcová2016), namely that endoparasite eggs and oocysts had the lowest prevalence in scats collected in spring and the highest prevalence in scats collected in the autumn. Bears in Estonia have two periods that are probably the most significant in terms of parasite infection: hyperphagia in late summer/autumn and hibernation in winter. In Estonia, brown bears enter into hyperphagia in late summer when berries such as blueberry and raspberry start to mature (later cowberry and cranberry) and hibernate usually from November–December to March–April. The period of hibernation varies to some extent, depending on environmental and individual factors, for example, the weather temperature, food availability, animal’s age, sex, presence of cubs, etc. (Evans et al., Reference Evans, Singh, Friebe, Arnemo, Laske, Fröbert, Swenson and Blanc2016; Pigeon et al., Reference Pigeon, Stenhouse and Côté2016). When during hyperphagia bears eat as much as possible to accumulate fat reserves for hibernation, then during hibernation, bears do not eat or drink, and significant changes take place in their body temperature and metabolism. Both can impact the life of parasites inhabiting the bear’s gastrointestinal system (Finnegan, Reference Finnegan2009). Our analysis revealed statistically significant seasonal differences in the prevalence of roundworms and protozoa: roundworms had the highest prevalence in autumn and the lowest in spring, whereas the opposite was found for protozoa. The most likely explanation is that during the hyperphagia in autumn, the roundworms, especially the B. transfuga with its large body size (up to 24 cm; Sapp et al., Reference Sapp, Gupta, Martin, Murray, Neidringhaus, Pfaff and Yabsley2017), have plenty of nutrients available and thrive. On the other hand, it has been speculated by Finnegan (Reference Finnegan2009) that during hibernation, due to the reduced availability of nutrients, majority of nematodes may starve to death, while the conditions can improve for protozoa. In spring, when brown bears begin to acquire roundworm infections once again, the infection with protozoa diminishes. This opinion is supported by research on the parasite fauna of sledge dogs in Poland, where, compared to dogs that also had a roundworm infection, protozoa (Cryptosporidium spp. and Giardia spp.) infections were 2–3 times more frequent in dogs who were not infected with roundworms (Bajer et al., Reference Bajer, Bednarska and Rodo2011). They also found a negative correlation between the dogs’ age and infections with nematodes and protozoa: older dogs were less likely to have a roundworm infection, whereas protozoa followed the opposite trend.
Why are roundworms and protozoa reciprocally affected? One possibility is that the excreted metabolites of nematodes, such as ammonia and excretory-secretory proteins (ESPs), can be toxic to protozoa. The ESPs include various proteases, nucleotidases, esterases, glycases, dismutases, and others connected to infectivity, immune evasion, and pathogenicity (Graeff-Texeira et al., Reference Graeff-Teixeira, Morassutti and Kazacos2016; Mehrdana and Buchmann, Reference Mehrdana and Buchmann2017).
In comparison to bear populations in other European countries, the current study revealed that the endoparasite prevalence in Estonia (75%) is among the highest in Europe. The closest infection values have been recorded in Slovakia (76%; Orosová et al., Reference Orosová, Goldová, Ciberej and Štrkolcová2016) and Spain (71% and 79%; Costa et al., Reference Costa, Hartasánchez, Santos and Camarão2022; Remesar et al., Reference Remesar, Busto, Diaz, Rivas, Lopez-Bao, Ballesteros and Garcia-Dios2024). However, the majority of other studies did not analyse or detect any protozoa. When excluding the protozoa infections from this research, the endoparasite prevalence is 63%, which is still at the higher end in Europe, where the prevalence of helminths ranges between 0% and 64%. The most likely explanation for the high prevalence is the relatively high environmental presence of helminths in Estonia. This is supported, as mentioned above, by very high helminth prevalence (91–100%) among other mammalian predators in Estonia. In addition, the relatively large number of feeding grounds for the wild boar (~3000; Environmental Agency of Estonia) serves as a place of gathering for many mammalian species and acts as a hotspot for parasite circulation (Süld et al., Reference Süld, Valdmann, Laurimaa, Soe, Davison and Saarma2014; Oja et al., Reference Oja, Velström, Moks, Jokelainen and Lassen2017). The use of feeding grounds is part of a traditional hunting strategy also for bears and other game species.
From the One Health perspective, it is important to note that the majority of the recorded parasite taxa in brown bears of Estonia are zoonotic or potentially so. Among others, the finding of an acanthocephalan also poses a concern, as human infections have been on the rise globally in recent years. One reason for this could be that medical professionals have not been familiar enough with this taxon, which has resulted in underestimates in the past (Mathison et al., Reference Mathison, Mehta and Couturier2021). Monitoring parasites of animals is therefore important, as it can provide data on the repertoire of parasites present at the animal-human interface and can direct attention to lesser-known parasites that have not yet been detected (due to misidentification?) in humans. There is also another aspect to consider. Brown bears can serve as a long-time reservoir for zoonotic parasites and keep them circulating in the ecosystem. As an omnivore, the brown bear can acquire parasites from different sources (e.g., animals, plants, soil, water), which means they can harbour and spread a diverse community of parasites. Due to the relatively large size of bear scats compared to those of many other mammals, they can be considered an efficient means to spread parasites, as the larger amount of scat protects parasite eggs and oocysts from abiotic factors, such as sunlight (UV radiation) and temperature. Although brown bears tend to avoid humans, they often come close to human settlements by visiting beehives and orchards, where the parasites in bear scats can spread to domestic animals and humans. This highlights the need for regular monitoring of zoonotic parasites spread by the brown bear and other wild mammals.
Acknowledgements
We would like to thank Teivi Laurimäe for her assistance and an anonymous reviewer for helpful suggestions.
Financial support
This work was supported by the Estonian Research Council (grants PRG1209 and TK215).
Competing interests
The authors declare no conflicts of interest.
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