Next Article in Journal
Taurine Supplementation Enhances the Resistance of Litopenaeus vannamei Postlarvae to Low-Salinity Stress
Previous Article in Journal
Harnessing the Power of Microbiota: How Do Key Lactobacillus Species Aid in Clearing High-Risk Human Papilloma Virus Infection and Promoting the Regression of Cervical Dysplasia?
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Biology
Volume 14
Issue 8
10.3390/biology14081083
biology-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Parasitic Fauna of Lepus europaeus and Lepus timidus in Kazakhstan: Parasitological Profile and Molecular Identification

by
Vladimir Kiyan
1,2,*,
Ainura Smagulova
1,
Nurassyl Manapov
1,
Karina Jazina
1,
Rabiga Uakhit
1,
Aitbay Bulashev
3,
Lyudmila Lider
3 and
Sergey Leontyev
1
1
Laboratory of Biodiversity and Genetic Resources, National Center for Biotechnology, Astana 010000, Kazakhstan
2
Scientific Center for Biological Research, Astana 010000, Kazakhstan
3
Department of Veterinary Medicine, S. Seifullin Kazakh Agrotechnical Research University, Astana 010000, Kazakhstan
*
Author to whom correspondence should be addressed.
Biology 2025, 14(8), 1083; https://doi.org/10.3390/biology14081083
Submission received: 17 July 2025 / Revised: 12 August 2025 / Accepted: 18 August 2025 / Published: 19 August 2025
Browse Figures
Versions Notes

Simple Summary

Hares are important wild animals in Kazakhstan, both for the environment and for local hunting traditions. Like all animals, hares can carry parasites that may affect their health and the health of other animals and people. However, little was known about the species and spread of these parasites in hares living in Kazakhstan. In this study, hares from two regions were collected and examined to determine the types and prevalence of internal parasites. Modern genetic methods were used to identify the parasites more accurately and to explore their relationships to similar species found in other regions. Several species of helminths and protozoan parasites were detected, some of which are known to cause disease. The rates of infection varied among different parasites but were generally low to moderate. This is the first detailed study of hare parasites in Kazakhstan that combines traditional and genetic approaches. The results help improve our knowledge about wildlife health in the region and will assist in managing hare populations and protecting other animals. Understanding these parasite infections also supports efforts to maintain biodiversity and reduce risks to domestic animals and people.

Abstract

Hares (Lepus europaeus and Lepus timidus) play important ecological and economic roles in Kazakhstan; however, data on their parasitic fauna are scarce. This study aimed to assess the diversity, prevalence, and genetic characteristics of endoparasites in wild hare populations from central and northern Kazakhstan. From November 2022 to February 2025, 107 hares (L. europaeus, n = 46; L. timidus, n = 61) were collected in the Akmola and Karaganda regions. Parasitological and coprological analyses were conducted. Genetic and phylogenetic analyses were performed using partial sequences of the mitochondrial cox1 and nad1 genes and a partial 28S rDNA region to assess the genetic diversity and evolutionary relationships of selected parasite genera. Seven parasite taxa were identified: Mosgovoyia pectinata (6.5% in L. europaeus, 11.5% in L. timidus), Cysticercus pisiformis (4.3% in Le, 3.3% in Lt), Coenurus serialis (3.3%, only in Lt), Passalurus ambiguus (6.5% in Le, 4.9% in Lt), Nematodirus leporis (36.6% in Le, 13.4% in Lt), Eimeria spp. (9.1% in Le, 3.8% in Lt), and Trichuris leporis (1.9%, only in Lt). Genetic methods confirmed species identity and revealed phylogenetic relationships. This is the first genetically confirmed survey of endoparasites in hares in Kazakhstan. The results contribute valuable data to regional parasitology and support the need for continued wildlife health monitoring.

1. Introduction

The European hare (Lepus europaeus) and the mountain hare (Lepus timidus) are well-known lagomorph species widely distributed across Europe, inhabiting a broad range of environments from agricultural landscapes to mountainous regions [1]. The mountain hare primarily inhabits forested and shrubby areas, adjacent steppe zones, the shores of reed-covered lakes and swamps, and, in exceptional cases, entirely treeless landscapes. The proportion of individuals found in these primary biotopes ranges from 51% to 63% of the total mountain hare population, depending on the year. The European hare predominantly occupies steppe, meadow, and agricultural lands as its main biotopes, although it has also been observed within forested and shrubby areas. These primary biotopes account for 80.0% to 84% of the total winter–spring population of the European hare [2].
In recent decades, significant population declines have been reported in many areas, attributed to various environmental pressures including habitat degradation, predation, and infectious diseases. Among these, parasitic infections have been identified as a critical factor influencing hare health and survival [3,4,5].
Numerous studies have documented a diverse array of helminths affecting hares, particularly gastrointestinal parasites such as Trichostrongylus retortaeformis, Graphidium strigosum, Eimeria spp., as well as cestodes including Mosgovoyia pectinata and Taenia pisiformis [6,7,8]. These infections can lead to severe health issues, reducing host fitness and potentially destabilizing local populations [9,10].
Beyond their direct impact on hare health and mortality, some parasites and pathogens associated with hares are of zoonotic concern, posing potential risks to human health [11,12,13]. Advances in molecular identification techniques have substantially enhanced our understanding of parasite diversity, transmission dynamics, and epidemiology, emphasizing the importance of detailed, species-specific studies [14].
Research conducted in various European countries including Italy, Germany, and Scotland has revealed considerable variation in parasite prevalence, species composition, and infection intensity. These differences underscore the role of geographic and ecological factors in shaping helminth communities [15,16,17]. For instance, high prevalence rates of gastrointestinal nematodes such as T. retortaeformis and G. strigosum are frequently reported, with significant associations noted between parasite burden and the health status of hares [18,19].
In European hares, the main parasitic pathogens identified include Trichuris leporis, Passalurus ambiguus, T. retortaeformis, M. pectinata, Strongyloides papillosus, Protostrongylus pulmonalis, Andrya spp., Dicrocoelium dendriticum, Cysticercus spp., Cittotenia spp., Nematodirus aspinosus, and Eimeria spp. [3,4,5,6,7,17,20,21]. In mountain hares, the pathogens detected include M. pectinata, P. ambiguus, G. strigosum, T. retortaeformis, P. pulmonalis, D. dendriticum, N. aspinosus, and Eimeria spp. [8,15,20,22,23,24].
Given the ecological and economic importance of hares, particularly in the context of game management and biodiversity conservation, continued helminthological research is essential. The present study aims to contribute to this growing body of knowledge by investigating the diversity, prevalence, and health impacts of parasitic infections in Lepus europaeus and Lepus timidus populations in Kazakhstan. The findings are intended to support the development of more effective wildlife health monitoring and management strategies in the region.

2. Materials and Methods

2.1. Sample Collection and Analysis

Sample collection was carried out between November 2022 and February 2025. During this period, a total of 107 hares Lepus europaeus (n = 46) and Lepus timidus (n = 61) were obtained from the Akmola and Karaganda regions of Kazakhstan.
Akmola Region is situated in the temperate zone, within the West Siberian climatic region. It features a sharply continental, arid climate with hot summers and cold winters, and significant daily and annual temperature fluctuations. Annual temperatures typically range from −19 °C to +26 °C, rarely falling below −29 °C or exceeding +32 °C. The landscape consists of a dissected small-hill terrain with predominant chernozem soils and includes forest-steppe and steppe zones.
Karaganda Region is located within the elevated Kazakh Uplands (Saryarka) and is characterized by a sharply continental, dry climate with long, hot summers and cold, snowy winters. Annual temperatures range from −20 °C to +26 °C. The region features a diverse relief including hills, valleys, dry riverbeds, endorheic, and lake basins. Its territory encompasses steppes, deserts, and mountainous areas, with mountain spurs present in the eastern part.
All animals were legally hunted by private amateur hunters in accordance with established national regulations and under officially issued hunting quotas. The species of hares belonging to the genus Lepus were identified based on morphological characteristics in accordance with identification guides [25].

2.2. Parasitological Methods

All samples were delivered to the Parasitology Laboratory of the Faculty of Veterinary Medicine, S. Seifullin Kazakh Agrotechnical Research University for helminthological dissection. The intestinal organs and muscle tissues of each animal were examined for the presence of helminths according to the method described by Skrjabin [26]. For the parasitological analysis, each animal was first examined for endoparasites with a special focus on the gastrointestinal tract. Therefore, each organ was examined externally and sectioned to search for intraparenchymatous and intraluminal parasites. Larvae of Cysticercus spp., adult worms of M. pectinata, and pinworms were isolated, washed in physiological saline, identified based on morphological characteristics [8,27,28], and preserved in 70% ethanol for further analysis.

2.3. Coprological Investigations

Fecal samples from each hare were collected directly from the rectum and large intestine and subjected to qualitative copromicroscopic analysis using two flotation solutions of low (50% ZnCl2, specific gravity 1300) and high-density Schizer’s solution with a solution density of 1.2–1.25 (454 g sugar per 1 L of water) [29,30].

2.4. DNA Extraction Method

Genomic DNA was extracted by homogenizing adult parasites or cysticercus in an Eppendorf centrifuge tube (Aptaca, Canelli, AT, Italy), using the standard phenol–chloroform method with proteinase K, followed by ethanol precipitation [31]. The concentration and purity of the extracted DNA were assessed by measuring absorbance at 260 and 280 nm using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The DNA was dissolved in double-distilled water (ddH2O) and stored at −20 °C until further use.

2.5. PCR and Sequencing

Polymerase chain reaction (PCR) was employed to assess the genetic diversity of different parasite species. Specific primers (Table 1) were used for parasite species identification and gene fragment amplification. Primers JB11F/JB12R were used to amplify a fragment of the NADH dehydrogenase subunit 1 (nad1) gene from T. pisiformis and T. serialis; primers COX-F/COX-R were used to amplify a fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene from M. pectinata; and primers C1F/D2R were used to amplify a fragment of the 28S ribosomal RNA gene from P. ambiguus. PCR reactions were performed in a 25 μL mixture containing 2× DreamTaq PCR Master Mix (Thermo Fisher Scientific, Carlsbad, CA, USA), nuclease-free water, 10 pmol of each primer, and 20 ng of template genomic DNA. Specific PCR conditions for amplifying marker regions are detailed in Table 1. Amplified products were separated by electrophoresis on a 1.5% agarose gel prepared with 1× TBE buffer, stained with ethidium bromide (8 ng/μL), and visualized under UV light.
All PCR-positive products were purified using the Quick PCR Purification Kit (QIAGEN, Germantown, MD, USA) according to the manufacturer’s instructions, then subjected to sequencing and genotyping. Sequencing was performed on a 3730xl DNA Analyzer 96-Capillary Array (Thermo Fisher Scientific, Applied Biosystems, Foster City, CA, USA). The genetic sequences obtained in this study have been deposited in GenBank under the following accession numbers: T. pisiformis (PV871991, PV871992, PV871993), T. serialis (PV871989, PV871990), M. pectinata (PV855722, PV855723), P. ambiguus (PV855725, PV855726, PV855727). The resulting nucleotide sequences were manually edited and compared with reference sequences from the GenBank database using the BLAST algorithm (https://www.ncbi.nlm.nih.gov/ (accessed on 9 May 2025)).

2.6. Phylogenetic Analysis

The obtained sequences were manually edited, and sequence similarity searches were performed using the BLAST algorithm (https://blast.ncbi.nlm.nih.gov (accessed on 9 May 2025)) to compare them with reference sequences from GenBank. Nucleotide sequences of various partial genes were aligned using the MUSCLE multiple sequence alignment program. A maximum-likelihood phylogenetic tree was constructed based on the Tamura–Nei model using MEGA version 11 software [32].

2.7. Statistical Analysis

To assess the risk of infection with parasites of mountain (Lepus timidus) and European (Lepus europaeus) hares, the odds of further infection (chances) were calculated separately for each helminth species. For all parasites in Table 2, the Fisher metric test was used, since small frequencies (<5) were observed. Values p > 0.05 indicate the absence of statistically significant infection signals between calendar hares. The frequency of detected parasites was statistically compared using the chi-square test, with a 95% confidence interval. The p-value was calculated, with statistical significance established at p < 0.05.

3. Results

3.1. Parasitological Research

During the study, 107 hares (L. europaeus and L. timidus) were examined. Initial inspection of the abdominal cavity and muscle tissue enabled the identification of the causative agents of cysticercosis. In 2 out of 46 European hares, similar clinical manifestations in the form of grape-like conglomerates characteristic of Cysticercus pisiformis (the larval stage of T. pisiformis) were detected in the abdominal organs (Figure 1A). The prevalence of C. pisiformis in European hares was 4.3% (Table 2). In 2 out of 61 mountain hares, grape-like conglomerates were also found in the abdominal organs. These structures were morphologically similar to those observed in European hares and are characteristic of C. pisiformis (Figure 1A). In another two mountain hares, translucent coenuri containing clusters of protoscoleces characteristic of Coenurus serialis (the larval stage of T. serialis) were detected in the muscle tissue (Figure 1B) [27]. The prevalence of C. pisiformis and C. serialis in mountain hares was 3.3% each (Table 2).
Examination of the intestinal contents of the hare revealed two types of intestinal parasites: cestodes and small nematodes, specifically pinworms. Macro- and microscopic analyses of the recovered cestodes confirmed morphological characteristics typical of M. pectinata. The adult form of the cestode was characterized by a segmented body with an average length of 14.5 ± 4.3 cm (n = 23) (Figure 2A), with an anterior end bearing four suckers used for attachment to the host’s intestinal wall (Figure 2B).
The body consists of numerous segments (proglottids), which gradually increase in size toward the posterior end. Genital openings are located in the middle third of the lateral margin of each segment. Mature proglottids contain eggs (Figure 2C). The eggs are spherical or oval in shape, measuring approximately 30–40 µm in diameter, and possess a thick shell that protects them from external environmental factors (Figure 2D) [8,18,33,34]. The prevalence of the cestode M. pectinata was 11.5% in mountain hares and 6.5% in European hares (Table 2).
Nematodes, represented by pinworms of the species Passalurus ambiguus, were primarily found parasitizing the large intestine of hares. Adult females measured 7–9 mm (n = 95) in length, while males ranged from 4–6 mm (n = 71). The body was thin and whitish in appearance (Figure 3A). The anterior portion of the worm bore an esophagus, bulb, and papillae, used for attachment to the intestinal mucosa (Figure 3B). The posterior part is narrowed, and males possess a sexual spicule (Figure 3C). The eggs were oval-shaped, measuring 30–40 µm, and had a well-defined shell (Figure 3D) [18,28]. The prevalence of the nematode P. ambiguus was 4.9% in mountain hares and 6.5% in European hares (Table 2).
The highest odds value was observed for the mountain hare when infected with M. pectinata and amounted to 0.1296, which indicates a relatively high probability of infection compared to other helminths in this species. The similar value for the European hare was 0.0698, which indicates a lower probability of infection with M. pectinata in this species. Infections with T. pisiformis and P. ambiguus were detected in both hare species, with similar odds of infection in the mountain hare (odds = 0.0339) and slightly higher values for the European hare (0.0455 and 0.0750, respectively), which may indicate similar levels of susceptibility to these helminths. For T. serialis, the odds of infection were 0.0339 in mountain hares, while this helminth was not detected at all in European hares.
A study was also conducted on the prevalence of fecal-transmitted parasitic infections in hares, identified by the presence of helminth eggs and oocysts in fecal samples. A total of 63 hare fecal samples were examined. The results confirmed the presence of four pathogens: Nematodirus leporis, Trichuris leporis, and Eimeria spp. The results of the copromicroscopic analyses are presented in Table 3, and images of helminth eggs and coccidian oocysts are shown in Figure 4.
Nematodirus leporis infection is approximately 2.7 times more common in European hares than in mountain hares (Table 3). The wide confidence interval and high p-value mean that there is no statistically significant difference. This is due to the extremely small number of infected individuals in both groups. Trichuris leporis was excluded from the calculation due to the absence of infection cases in one of the hare species, which makes the statistical analysis of OR incorrect and mathematically unstable with such small samples. Although the OR values indicate an increased risk of infection in European hares, all p-values > 0.05 and the confidence intervals include 1. This means that the differences are not statistically significant and can be explained by random variation, especially given the small sample size, especially for Lepus europaeus (only 11 individuals).

3.2. Molecular Genetics Identification

Molecular genetics and phylogenetic analyses were conducted using partial sequences of the mitochondrial cox1 and nad1 genes, as well as a partial 28S rDNA region, to assess the genetic diversity and evolutionary relationships of the isolated parasites.
Molecular identification of Taenia spp. was carried out using primers specific to partial regions of the mitochondrial nad1 gene, producing an amplicon of approximately 500 bp. The resulting sequences of T. pisiformis and T. serialis were submitted to GenBank and subsequently included in the phylogenetic analysis (Figure 5). A phylogenetic tree was constructed to determine the evolutionary relationships among Taenia spp. involved in cysticercosis, based on the Maximum Likelihood (ML) method using the Tamura–Nei model [35]. The analysis included 16 nucleotide sequences with a total alignment length of 893 positions. Bootstrap analysis with 1000 replicates was performed to assess the robustness of the inferred clades [36]. All evolutionary analyses were conducted in MEGA11 [32]. The Mesocestoides sp. (PP972730) was used as an outgroup for rooting the tree.
To genetically characterize M. pectinata, partial fragments of the mitochondrial cox1 gene were amplified using specific primers, generating a PCR product of around 650 bp. The generated sequences were deposited in GenBank and used for phylogenetic reconstruction (Figure 6). A phylogenetic tree was constructed using the Maximum Likelihood method and based on the Tamura–Nei model to evaluate the evolutionary relationships of M. pectinata with related cestodes. The analysis involved nine nucleotide sequences and a total alignment length of 13,490 positions. Phylogenetic reliability was assessed with 1000 bootstrap replicates. The Diphyllobothrium latum (AB302389) was used as an outgroup for rooting the tree.
The identification of Passalurus spp. was based on amplification of partial 28S rDNA gene regions, resulting in a ~781 bp fragment. The sequence obtained for P. ambiguus was submitted to GenBank and incorporated into the phylogenetic analysis (Figure 7).

4. Discussion

The mountain hare (L. timidus) and European hare (L. europaeus) are the most widespread hare species in Kazakhstan and are considered valuable game animals, commonly targeted in recreational and sport hunting. Their population sizes are not regulated and can fluctuate significantly from year to year, depending on various factors, including disease outbreaks. To date, no comprehensive studies on the parasite fauna of hares in Northern and Central Kazakhstan have been conducted; existing data are limited to brief reports based primarily on coprological analyses.
This study represents the first in-depth investigation of endoparasites in these two-hare species in Kazakhstan. A total of seven parasite genera were identified, including M. pectinata, T. serialis, T. pisiformis, P. ambiguus, N. leporis, Eimeria spp., and T. leporis. The research was geographically limited to two regions, and the sample size was not proportional to the total hare population in each area. Specimens, including hare carcasses, infected organs, and fecal samples, were obtained from private recreational hunters who voluntarily contributed to the study.
Mosgovoyia pectinata. The cestode M. pectinata (Spasskii, 1951) is a common parasite of hares and belongs to the family Anoplocephalidae, order Cyclophyllidea [37]. Its life cycle involves two hosts: oribatid mites as intermediate hosts and hares as definitive hosts [38,39]. According to various sources, species such as Trichocalumna curva, Scheloribates spp. [40], and Ceratoppia bipilis [41] may serve as intermediate hosts for M. pectinata. However, the complete life cycle of M. pectinata, the specific identity of its intermediate hosts, and the overall species diversity of oribatid mites in Kazakhstan remain poorly studied and represent important directions for future research. In our study, the overall prevalence of M. pectinata among the two examined hare species was 6.5% in European hares (L. europaeus) and 11.5% in mountain hares (L. timidus). The pathogen was detected in both the Akmola and Karaganda regions, with no significant correlation between location and infection rate, indicating a widespread distribution of M. pectinata across the northern and central parts of Kazakhstan. The two isolates obtained in this study (PV855722 and PV855723), marked with red circles, clustered together with high bootstrap support (89–95%) alongside reference sequences of M. pectinata, forming a distinct and well-supported monophyletic clade and share a similar genetic sequence with those found in Finland (AY568211) and Denmark (OQ421548, ON754342). This confirms the accurate molecular identification of the isolates as M. pectinata. The M. pectinata clade was clearly separated from other cestode taxa, including Moniezia expansa and T. hydatigena, which formed their own distinct branches with strong bootstrap support (100%), demonstrating phylogenetic divergence at the genus and species levels. These results provide strong molecular evidence for the classification of the newly sequenced isolates within the M. pectinata lineage.
These results are comparable to data reported from Norway and Ireland, where 12% and 14% of mountain hares were infected with M. pectinata, respectively [39,42]. However, the observed prevalence was considerably lower than that reported in other regions. For instance, in Finland, the average infection rate was 13% in brown hares and 30% in mountain hares [20]. In Russia, the prevalence of M. pectinata in brown hares varied across different regions, including the European northeast, the Kirov and Astrakhan regions, and Yakutia, where it reached up to 32%, depending on the study area [43,44,45,46]. In mountain hares, infection has also been recorded in Ukraine and Crimea, with prevalence reaching up to 58% [47,48,49]. In northwestern Iran, M. pectinata was found in 17% of wild European hares (L. europaeus) [33]. Additionally, reports of M. pectinata infection in hares exist from Kyrgyzstan [50], Azerbaijan [51], Armenia [52], Turkmenistan [53], and several other countries. According to the phylogenetic analysis, the isolates from this study of M. pectinata share a similar genetic sequence with those found in Finland (AY568211) and Denmark (OQ421548, ON754342).
Taenia spp. The conducted studies revealed the presence of an intermediate stage of cysticercosis in hares, caused by two species of cestodes: T. serialis and T. pisiformis. The prevalence of C. pisiformis (the larval stage of T. pisiformis) in European hares and mountain hares was 4.3% and 3.3%, respectively. This was recorded in two regions of Kazakhstan—Akmola and Karaganda. C. serialis (the larval stage of T. serialis) was detected only in mountain hares from the Akmola region, with a prevalence of 3.3%. The three T. pisiformis isolates obtained in this study (PV871992, PV871993, and PV871991) clustered closely with reference sequences JX677976.1 and JN870127.1, forming a strongly supported monophyletic clade (bootstrap = 89%). This grouping confirms the species identity of the field isolates and suggests low intraspecific divergence within T. pisiformis. The clade also includes MZ287427.1, further supporting genetic homogeneity among global T. pisiformis strains. These findings align with previously published data by Jia et al. (2010) [54], which confirmed the monophyly of T. pisiformis based on mitochondrial markers. Similarly, two additional isolates (PV871989 and PV871990) grouped within the T. serialis clade, along with reference sequences AM503337.1, AM503336.1, and DQ401137.1. Although the bootstrap support for this subgroup was moderate (54%), the overall topology reflects expected species-level relationships. Other closely related Taenia species, such as T. ovis, T. asiatica, T. multiceps, and T. krabbei, formed distinct branches, confirming the genetic divergence within the genus.
T. pisiformis (Bloch, 1780) is a cestode with a global distribution. In its life cycle, canids serve as the definitive hosts (occasionally felids), while lagomorphs (hares and rabbits) and rodents serve as intermediate hosts [55]. The intermediate stage, C. pisiformis, has been detected in hares in various regions worldwide. The prevalence of C. pisiformis in hares has been reported as follows: 10.38% in Poland [6], 14.8% in Italy [56], 8% in Canada [57], 70% in Mexico [58], 22.7% in Belarus [59], 17.53% in Ukraine [60], 15.1% in Russia [61], and 13.33% in Iran [62]. No data regarding T. pisiformis-induced cysticercosis in hares has been found in open sources for Kazakhstan, indicating that the findings presented here are the first such report and warrant further large-scale studies across the country. The identity of the definitive host of T. pisiformis in Kazakhstan remains uncertain, as our previous investigations of helminths in wild carnivores did not reveal the presence of this species among the identified cestodes [63,64]. However, there is evidence that T. pisiformis circulates among domestic dogs throughout most regions of Kazakhstan [65,66], highlighting the need for further analysis, including molecular identification methods.
T. serialis is a rare and poorly studied parasite belonging to the Taeniidae family. The adult form parasitizes the small intestine of definitive hosts (carnivores, primarily canines), while the larval stage, C. serialis, develops in the subcutaneous and intermuscular connective tissues of intermediate hosts such as rabbits, hares, and other rodents [55,67]. Coenurosis caused by T. serialis has been reported in animals worldwide, particularly in Europe, Africa, the Middle East, Australia, and the Americas [68,69,70,71]. It has also been recorded in hares in Russia [46], Ukraine (Crimea) [49], Kyrgyzstan [50], and China [72]. However, no scientific data exist regarding the distribution of T. serialis in Kazakhstan. Given the current gaps in knowledge regarding the taxonomy and life cycle of T. serialis especially concerning its intermediate and definitive hosts [73] and the documented cases of human infection [74], the detection of this parasite in Kazakhstan is of particular importance. These findings highlight the need for comprehensive research into its occurrence and epidemiology within the region.
Passalurus ambiguous. P. ambiguus has a direct life cycle that does not involve an intermediate host. Eggs are deposited around the anus, and ingestion typically occurs during cecotrophy [75]. In this study, nematodes of the species P. ambiguus (pinworms) were identified in both mountain hares and brown hares across the entire study area, with a prevalence of 4.9% and 6.5%, respectively. Comparable prevalence rates have been reported in Finland (3%) [76], Germany (3%) [77], Iran (6.66%) [62], and Armenia (8.6%) [78]. The prevalence rates observed in our study were lower than those reported in Italy (ranging from 9.3% to 12.9%) [5,7], Egypt (45%) [28], and the United Kingdom (30%) [79]. P. ambiguus has also been documented in Ukraine (Crimea) [49], Russia [80], and Uzbekistan [81].
The tree (Figure 7) shows a well-supported monophyletic cluster comprising three isolates from this study (PV855725, PV855726, and PV855727), marked with red circles, which grouped closely with a reference sequence of P. ambiguus (KY990018.1), forming a monophyletic group supported by a bootstrap value of 91%. This confirms the identity of the field isolates and supports the intraspecific genetic homogeneity within the P. ambiguus clade. This finding is consistent with those reported by Hussein et al. (2022) and Abdel-Gaber et al. (2019) [16,82], who also observed the monophyly of P. ambiguus in phylogenetic trees based on ribosomal markers. The P. ambiguus clade was clearly separated from other Oxyurid nematodes, including Thelandros filiformis, Parapharyngodon micipsae, Trichostrongylus retortaeformis, Strongyloides papillosus, and Heteroxynema cucullatum, formed distinct branches, confirming the phylogenetic divergence from the Passalurus clade. Bootstrap values above 70% (e.g., 91%, 99%) indicate reliable topologies, while lower values (e.g., 37%, 47%) suggest moderate to low support for some internal nodes. The phylogenetic inference was based on 1000 bootstrap replicates.
P. ambiguus is considered the most common intestinal nematode in rabbits and hares. Although typically not highly pathogenic, outbreaks with high mortality have been reported in juvenile animals [8]. Young rabbits may suffer from heavy infections, characterized by perianal irritation, and large worm burdens can contribute to enteritis complex development [83]. Our findings expand the current understanding of the geographic distribution of P. ambiguus and its prevalence among wild hare populations in Kazakhstan. To the best of our knowledge, this is the first genetically confirmed report of the occurrence of P. ambiguus in central and northern regions of Kazakhstan. Therefore, systematic monitoring and control by national veterinary services are essential to prevent mass mortality in both domestic and farmed lagomorph populations.
Fecal-transmitted parasitic infections. An attempt was made to assess the main types of helminth infections present in the feces of hares collected within the study area. Coprological analysis allowed the identification of three genera: Nematodirus leporis, Eimeria spp., and Trichuris leporis (Figure 4, Table 3). In the mountain hare, Nematodirus leporis was the most common parasite with a prevalence of 13.4% (7/52) and odds = 0.1556. In the European hare, the same parasite was found with an even higher prevalence of 36.6% (4/11) and odds = 0.5714. In both cases, the p-values calculated using the binomial test with H0: p = 0 were <0.000001, indicating a statistically significant presence of this parasite in the populations of both hare species. For Eimeria spp., the prevalence in mountain hares was 3.8% (2/52), and in European hares it was 9.1% (1/11). However, in both cases, the p-value = 1.0000, indicating the absence of a statistically significant difference and inconclusive evidence for the presence of the parasite in the population. Trichuris leporis was detected in only one mountain hare (1/52, 1.9%) and was not recorded in European hares, which prevented statistical comparison. The identified pathogens are considered common and significant for lagomorphs, and their presence has been reported in various countries, including European nations [3,4,5,6,7,18], Iran [62], Ukraine [47,49], Russia [45,46], Azerbaijan [51], and several Central Asian countries [50,53,81]. The main limiting factor in conducting genetic identification of these parasites in the present study was the low intensity of infection observed in the hare samples. However, given the veterinary relevance of the detected species, especially Eimeria spp., the question of their species diversity and distribution among hare populations in Kazakhstan remains highly relevant and warrants further molecular investigation.
The mountain hare (L. timidus) and the European hare (L. europaeus) are closely related species, and it is expected that they share a similar set of helminth parasites. Although they inhabit the same general territory, they occupy different ecological niches. The mountain hare tends to prefer areas with dense, tall vegetation and the presence of woody and shrubby growth, while the European hare primarily inhabits open flatlands, often near outskirts of populated areas. Their diets also differ: twigs dominate the mountain hare’s diet, whereas grasses are the main component of the European hare’s diet.
These ecological differences may influence not only their exposure to parasites but also the structure of the food web in which they participate. For instance, the mountain hare is a key prey species for the lynx, which inhabits forested regions, while the European hare is more commonly hunted by wolves and foxes in steppe habitats. Consequently, the composition of definitive hosts (e.g., wild carnivores and stray dogs) involved in the life cycles of many helminths may vary between habitats.
A similar pattern is observed with intermediate hosts, particularly invertebrates such as insects and oribatid mites, whose abundance and distribution are influenced by climatic conditions. For example, oribatid mites inhabit soil, decaying plant material, tree bark, and the organic layers of forests—conditions more typical of habitats occupied by mountain hares. This may explain the higher prevalence of M. pectinata observed in L. timidus in our study. Thus, even when the two hare species are exposed to the same spectrum of potential parasites, differences in habitat, diet, and host interactions can result in variation in infection prevalence, intensity, and transmission dynamics.

5. Conclusions

The findings provide important baseline data on the prevalence and diversity of parasites in L. europaeus and L. timidus, as well as their geographic distribution and potential risk factors for parasitic infections in hare populations and other wildlife species. These results underscore the need for continued monitoring of parasitic infections in hares by national veterinary authorities, targeted outreach to inform recreational hunters about infection risks and transmission routes, and the development of management strategies for regulating hare populations in the affected regions.

Author Contributions

Conceptualization, V.K. and A.B.; methodology, V.K., L.L. and R.U.; software, N.M. and R.U.; validation, A.S., N.M. and K.J.; formal analysis, V.K. and A.B.; investigation, R.U., A.S. and S.L.; resources, V.K. and S.L.; data curation, V.K. and L.L.; writing—original draft preparation, V.K., A.S. and R.U.; writing—review and editing, V.K. and R.U.; visualization, A.S. and N.M.; supervision, V.K.; project administration, V.K.; funding acquisition, V.K. All authors have read and agreed to the published version of the manuscript.

Funding

The research was carried out with financial support from the Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan within the framework of project AP19576421 for 2023–2025.

Institutional Review Board Statement

The animal study protocol was approved by the Animal Ethics Committee of the National Center for Biotechnology (approval based on protocol extracts No. 10 dated 10 November 2022) for studies involving animals. All procedures were performed in compliance with ethical guidelines for animal research, in line with the principles of the World Medical Association’s Declaration of Helsinki (http://ec.europa.eu/environment/chemicals/lab_animals/legal_en.htm (accessed on 5 November 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data are contained within this article.

Acknowledgments

The authors would like to express their sincere gratitude to all hunters who participated in this study and provided biological material for analysis.

Conflicts of Interest

The authors declare that they have no conflicts of interest to disclose.

Abbreviations

The following abbreviations are used in this manuscript:
Nnumber
bpbase pairs
CACalifornia
CIconfidence interval
cox1cytochrome c oxidase subunit 1
DNAdeoxyribonucleic acid
FForward
MDMaryland
MLMaximum Likelihood
nad1NADH dehydrogenase subunit 1
OROdds Ratio
RRevers
SDstandard deviation
TBETris-borate-EDTA buffer
USAUnited States of America
UVultraviolet

References

  1. Smith, R.K.; Vaughan Jennings, N.; Harris, S. A quantitative analysis of the abundance and demography of European hares Lepus europaeus in relation to habitat type, intensity of agriculture and climate. Mammal Rev. 2005, 35, 1–24. [Google Scholar] [CrossRef]
  2. Vilkov, V.S.; Pashkov, S.V. The state of the populations of white hare (Lepus timidus) and hare (L. europaeus) in the forest-steppe of Kazakhstan. Ecosystems 2019, 20, 149–162. (In Russian) [Google Scholar]
  3. Dubinský, P.; Vasilková, Z.; Hurníková, Z.; Miterpáková, M.; Slamečka, J.; Jurčík, R. Parasitic infections of the European brown hare (Lepus europaeus Pallas, 1778) in south-western Slovakia. Helminthologia 2010, 47, 219–225. [Google Scholar] [CrossRef]
  4. Kornaś, S.; Wierzbowska, I.A.; Wajdzik, M.; Kowal, J.; Basiaga, M.; Paweł Nosal, P. Endoparasites of European Brown Hare (Lepus Europaeus) from Southern Poland Based on Necropsy. Ann. Anim. Sci. 2014, 14, 297–306. [Google Scholar] [CrossRef]
  5. Sergi, V.; Romeo, G.; Serafini, M.; Torretta, E.; Macchioni, F. Endoparasites of the European Hare (Lepus Europaeus) (Pallas, 1778) in Central Italy. Helminthologia 2018, 55, 127–133. [Google Scholar] [CrossRef] [PubMed]
  6. Samorek-Pieróg, M.; Karamon, J.; Brzana, A.; Bilska-Zając, E.; Zdybel, J.; Cencek, T. Molecular Confirmation of Massive Taenia pisiformis Cysticercosis in One Rabbit in Poland. Pathogens 2021, 10, 1029. [Google Scholar] [CrossRef] [PubMed]
  7. Brustenga, L.; Franciosini, M.P.; Diaferia, M.; Rigamonti, G.; Musa, L.; Russomanno, B.L.; Veronesi, F. Parasitological Survey in European Brown Hare (Lepus europaeus Pallas, 1778) Breeding Facilities in Southern Italy. Pathogens 2023, 12, 208. [Google Scholar] [CrossRef]
  8. Magnussen, E.; Stensvold, C.R.; Berg, R.; Jokelainen, P.; Haukisalmi, V. Identification of the tapeworm Mosgovoyia pectinata (Anoplocephalidae) in Faroese mountain hares (Lepus timidus). Int. J. Parasitol. Parasites Wildl. 2023, 21, 17–21. [Google Scholar] [CrossRef]
  9. Wells, K.; Gibson, D.I.; Clark, N.J.; Ribas, A.; Morand, S.; McCallum, H.I. Global spread of helminth parasites at the human-domestic animal-wildlife interface. Glob. Change Biol. 2018, 24, 3254–3265. [Google Scholar] [CrossRef]
  10. Macchioni, F.; Romeo, G.; Trocchi, V.; Usai, F.; Cecchi, F.; Monni, G.; Stancampiano, L. Intestinal helminths of the endemic Italian hare, Lepus corsicanus (De Winton, 1898), in Sicily (Italy). Hystrix Ital. J. Mammal. 2022, 33, 98–100. [Google Scholar] [CrossRef]
  11. Treml, F.; Pikula, J.; Bandouchova, H.; Horakova, J. European brown hare as a potential source of zoonotic agents. Vet. Med. 2007, 52, 451–456. [Google Scholar] [CrossRef]
  12. Ebani, V.V.; Poli, A.; Rocchigiani, G.; Bertelloni, F.; Nardoni, S.; Papini, R.A.; Mancianti, F. Serological survey on some pathogens in wild brown hares (Lepus europaeus) in Central Italy. Asian Pac. J. Trop. Med. 2016, 9, 465–469. [Google Scholar] [CrossRef]
  13. Molina, R.; Jiménez, M.I.; Cruz, I.; Iriso, A.; Martín-Martín, I.; Sevillano, O.; Melero, S.; Bernal, J. The hare (Lepus granatensis) as potential sylvatic reservoir of Leishmania infantum in Spain. Vet. Parasitol. 2012, 190, 268–271. [Google Scholar] [CrossRef]
  14. Yan, X.; He, S.; Liu, Y.; Han, B.; Zhang, N.; Deng, H.; Wang, Y.; Liu, M. Molecular identification and phylogenetic analysis of gastrointestinal nematodes in different populations of Kazakh sheep. Exp. Parasitol. 2023, 254, 108625. [Google Scholar] [CrossRef] [PubMed]
  15. Newey, S.; Shaw, D.J.; Kirby, A.; Montieth, P.; Hudson, P.J.; Thirgood, S.J. Prevalence, intensity and aggregation of intestinal parasites in mountain hares and their potential impact on population dynamics. Int. J. Parasitol. 2005, 35, 367–373. [Google Scholar] [CrossRef] [PubMed]
  16. Hussein, N.M.; Rabie, S.A.H.; Abuelwafa, W.A.; El Din, M.M.M. Morphometry, molecular identification and histopathology of Passalurus ambiguus Rudolphi, 1819 in domestic rabbits (Oryctolagus cuniculus) in Qena, Upper Egypt. J. Parasit. Dis. 2022, 46, 511–525. [Google Scholar] [CrossRef] [PubMed]
  17. Faehndrich, M.; Woelfing, B.; Klink, J.C.; Roller, M.; Baumgärtner, W.; Wohlsein, P.; Raue, K.; Strube, C.; Ewers, C.; Prenger-Berninghoff, E.; et al. Pathomorphological Findings and Infectious Diseases in Selecte European Brown Hare (Lepus europaeus Pallas, 1778) Populations from Schleswig-Holstein, Germany. Pathogens 2023, 12, 1317. [Google Scholar] [CrossRef]
  18. Hughes, K. Endoparasites of rabbits and hares. J. Vet. Diagn. Investig. 2024, 36, 599–616. [Google Scholar] [CrossRef]
  19. Eberhardt, A.T.; Robles, M.D.R.; Monje, L.D.; Beldomenico, P.M.; Callejón, R. A new Trichuris species (Nematoda: Trichuridae) from capybaras: Morphological-molecular characterization and phylogenetic relationships. Acta Trop. 2019, 190, 244–252. [Google Scholar] [CrossRef]
  20. Ryzhikov, K.M.; Gubanov, N.M.; Fedorov, K.P. Biology of Mosgovoyia pectinata, hare cestodes. Sci. Notes V. I. Lenin Mosc. State Pedagog. Inst. 1956, 96, 147–150. (In Russian) [Google Scholar]
  21. Fadeyev, V.A. European hare—Lepus europaeus Pall., 1778. In Mammalia of Kazakhstan; Sludskiy, A.A., Strautman, E.A., Eds.; Science of the Kazakh SSR: Alma-Ata, Kazakhstan, 1980; Volume 2, pp. 79–112. (In Russian) [Google Scholar]
  22. Hulbert, I.A.; Boag, B. The potential role of habitat on intestinal helminths of mountain hares, Lepus timidus. J. Helminthol. 2001, 75, 345–349. [Google Scholar] [CrossRef]
  23. Boag, B.; Iason, G. The occurrence and abundance of helminth parasites of the mountain hare Lepus timidus (L.) and the wild rabbit Oryctolagus cuniculus (L.) in Aberdeenshire, Scotland. J. Helminthol. 1986, 60, 92–98. [Google Scholar] [CrossRef] [PubMed]
  24. Kapitonov, V.I.; Utinov, S.R. Mountain hare—Lepus timidus Linnaeus, 1758. In Mammalia of Kazakhstan; Sludskiy, A.A., Strautman, E.A., Eds.; Science of the Kazakh SSR: Alma-Ata, Kazakhstan, 1980; Volume 2, pp. 9–51. (In Russian) [Google Scholar]
  25. Bobrinsky, N.A.; Kuznetsov, B.A.; Kuzyakin, A.P. Identifier of Mammals of the USSR; Prosveshchenie: Moscow, Russia, 1965; pp. 244–245. (In Russian) [Google Scholar]
  26. Skrjabin, K.I. Method of Complete Helminthological Dissection of Vertebrates, Including Humans; Publishing House of 1st Moscow State University: Moscow, Russia, 1928; pp. 40–48. (In Russian) [Google Scholar]
  27. Deplazes, P.; Eckert, J.; Mathis, A.; Von Samson-Himmelstjerna, G.; Zahner, H. Parasitology in Veterinary Medicine; Wageningen Academic Publishers: Wageningen, The Netherlands, 2016; pp. 224–239. [Google Scholar]
  28. De Bellocq, J.G.; Ferté, H.; Depaquit, J.; Justine, J.L.; Tillier, A.; Durette-Desset, M.C. Phylogeny of the Trichostrongylina (Nematoda) inferred from 28S rDNA sequences. Mol. Phylogenet. Evol. 2001, 19, 430–442. [Google Scholar] [CrossRef] [PubMed]
  29. Dryden, M.W.; Payne, P.A.; Smith, V. Comparison of common fecal flotation techniques for the recovery of parasite eggs and oocysts. Vet. Ther. 2005, 6, 15–18. [Google Scholar] [PubMed]
  30. Zajac, A.M.; Conboy, G.A.; Little, S.E.; Reichard, M.V. Veterinary Clinical Parasitology, 9th ed.; Wiley & Sons: Hoboken, NJ, USA, 2021; pp. 4–8. [Google Scholar]
  31. Cunningham, S.W.; Tessler, M.; Johnson-Rosemond, J.; Whittaker, I.S.; Brugler, M.R. Environmental DNA Isolation, Validation, and Preservation Methods. Methods Mol. Biol. 2024, 2744, 171–180. [Google Scholar] [CrossRef]
  32. Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef]
  33. Baran, A.I.; Alvandi, G.; Firouzamandi, M.; Norouzi, R.; Katiraee, F. Study of morphology and phylogeny of Mosgovoyia pectinata (Cestoda; Anoplocephalidae) from European wild hares (Lepus europaeus) in Northwestern Iran. J. Anim. Environ. 2021, 13, 13–20. [Google Scholar] [CrossRef]
  34. Kalmykov, A.P.; Vlasenkov, S.A.; Tulendeev, R.N. The first registration of the cestode Mosgovoyia pectinata (Goeze, 1782) Spassky, 1951 at the European hare (Lepus Europaeus Pall. 1821) in the Volga delta. In Biodiversity, Rational Use of Biological Resources and Biotechnology. Proceedings of the International Scientific and Practical Online Conference, Astrakhan State University, Astrakhan, Russia; Publishing house “Astrakhan University”: Astrakhan, Russia, 2021; pp. 12–15. (In Russian) [Google Scholar]
  35. Tamura, K.; Nei, M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 1993, 10, 512–526. [Google Scholar] [CrossRef]
  36. Felsenstein, J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 1985, 39, 783–791. [Google Scholar] [CrossRef]
  37. Khalil, L.F.; Jones, A.; Bray, R.A. Order Cyclophyllidea (diagnosis and key to families). In Keys to the Cestode Parasites of Vertebrates; Khalil, L.F., Jones, A., Bray, R.A., Eds.; CAB International: Wallingford, UK, 1994; pp. 305–307. [Google Scholar]
  38. Stunkard, H.W. Studies on the life history of the Anoplocephaline Cestodes of Hares and Rabbits. J. Parasit. Urbana. 1941, XXVII, 299–325. [Google Scholar] [CrossRef]
  39. Ball, S.; Kelly, T.; Butler, F. Endoparasites of the endemic Irish hare Lepus timidus hibernicus. J. Wildl. Dis. 2020, 19, 337–341. [Google Scholar] [CrossRef]
  40. Lin, Y.; Lingxian, H. Studies on the life cycle of Ctenotaenia citelli (Kirshenblat) and Mosgovoyia pectinata (Goeze). Acta Zool. Sin. 1986, 32, 144–151. [Google Scholar]
  41. Berg, C. Endoparasitism in Wild Norwegian Hares (Lepus timidus). A National Survey and a More Detailed Study of Two Isolated Subpopulations. Bachelor’s Thesis, Veterinary College of Norway, Oslo, Norway, 1980; p. 71. [Google Scholar]
  42. Soveri, T.; Valtonen, M. Endoparasites of hares (Lepus timidus L. and L. europaeus Pallas) in Finland. J. Wildl. Dis. 1983, 19, 337–341. [Google Scholar] [CrossRef] [PubMed]
  43. Yushkov, V.F. Fauna of the European North-East of Russia; Nauka: St. Petersburg, Russia, 1995; pp. 80–85. (In Russian) [Google Scholar]
  44. Maslennikova, O.V.; Limendova, S.A. Monitoring of helminths of some mammal species in the Kirov region. In Ecology of the native land: Problems and solutions. Proceedings of the XVII All-Russian Scientific and Practical Conference with International Participation, Vyatka State University, Kirov, Russia, 26–27 April 2022; Vyatka State University Publishing House: Kirov, Russia, 2022; pp. 317–320. (In Russian) [Google Scholar]
  45. Sedalishchev, V.T.; Anufriev, A.I. Ecological and physiological characteristics of the mountain hare Lepus timidus (Leporidae) in Central Yakutia. Bull. North-East. Sci. Cent. Far East. Branch Russ. Acad. Sci. 2013, 1, 69–74. (In Russian) [Google Scholar]
  46. Siben, A.N.; Nikonova, A.A. Helminthofauna of the white hare (Lepus timidus) in Russia. Literature review. Agro-Ind. Complex Innov. Technol. 2022, 4, 31–38. (In Russian) [Google Scholar] [CrossRef]
  47. Vysotskaya, S.O. Helminths of small mammals of the Transcarpathian region (Eastern Carpathians, Ukraine). Parasitology 1997, 31, 346–355. (In Russian) [Google Scholar]
  48. Cadenatsii, A.N. Helminth fauna of mammals of Crimea and experience of recovery of domestic animals from main helminthiases. Work. GELAN 1957, 1, 109. (In Russian) [Google Scholar]
  49. Stryukov, A.A.; Leonov, S.V.; Sorgin, D.A. Endoparasites of the brown hare (Lepus europaeus) in the Crimea: An annotated checklist and invasion indicators. Ecosystems 2022, 29, 58–69. (In Russian) [Google Scholar]
  50. Khidaibergenov, A.D.j. Current status of trematodes and cestodes wild mammals of the Kyrgyzstan. Sci. New Technol. 2015, 2, 113–115. (In Russian) [Google Scholar]
  51. Fataliev, G.G. Helminth fauna of the brown hare of Azerbaijan and the ways of its formation. Bull. Zaporizhzhskiy Natl. Univ. Collect. Sci. Work. Biol. Sci. 2013, 3, 53–59. [Google Scholar]
  52. Movsesyan, S.O.; Chubaryan, F.A.; Nikogosyan, M.A. Cestodes of the Fauna of the Southern Lesser Caucasus; Nauka, Institute of Parasitology RAS: Moscow, Russia, 2006; 331p. (In Russian) [Google Scholar]
  53. Kontrimavicius, V.L. Helminth fauna of hares of the USSR and the experience of its ecological-geographical analysis. Work. GELAN 1959, 9, 133–144. [Google Scholar]
  54. Jia, W.Z.; Yan, H.B.; Guo, A.J.; Zhu, X.Q.; Wang, Y.C.; Shi, W.G.; Chen, H.T.; Zhan, F.; Zhang, S.H.; Fu, B.Q.; et al. Complete mitochondrial genomes of Taenia multiceps, T. hydatigena and T. pisiformis: Additional molecular markers for a tapeworm genus of human and animal health significance. BMC Genom. 2010, 11, 447. [Google Scholar] [CrossRef]
  55. Loos-Frank, B. An up-date of Verster’s (1969) “Taxonomic revision of the genus Taenia Linnaeus” (Cestoda) in table format. Syst. Parasitol. 2000, 45, 155–183. [Google Scholar] [CrossRef]
  56. Stancampiano, L.; Ravagnan, S.; Capelli, G.; Militerno, G. Cysticercosis by Taenia pisiformis in Brown Hare (Lepus europaeus) in Northern Italy: Epidemiologic and pathologic features. Int. J. Parasitol. Parasites Wild 2019, 9, 139–143. [Google Scholar] [CrossRef] [PubMed]
  57. Keith, L.B.; Cary, J.R.; Yuill, T.M.; Keith, I.M. Prevalence of helminths in a cyclic snowshoe hare population. J. Wild Dis. 1985, 21, 233–254. [Google Scholar] [CrossRef] [PubMed]
  58. Dominguez-Roldan, R.; Perez-Martinez, M.; Rosetti, M.F.; Arias-Hernandez, D.; Bernal-Fernandez, G.; Flores-Perez, F.I.; Hallal Calleros, C. High frequency of Taenia pisiformis metacestodes and high sex-associated susceptibility to cysticercosis in naturally infected wild rabbits. Parasitol. Res. 2018, 117, 2201–2206. [Google Scholar] [CrossRef] [PubMed]
  59. Dubina, I.N.; Subbotin, A.M. Epizootology of Taenia pisiformis and its larval stage Cysticercus pisiformis. Vet. Sci. Abstr. J. 2000, 1, 71–74. (In Russian) [Google Scholar]
  60. Mykhailiutenko, S.; Kruchynenko, O.; Kuzmenko, L.; Klymenko, O.; Dmytrenko, N. Morpho-anatomical traits of Cysticercus pisiformis in European rabbits (Oryctolagus cuniculus). Bulg. J. Vet. Med. 2024, 27, 593–601. [Google Scholar] [CrossRef]
  61. Krivko, M.S.; Tambiev, T.S.; Krivko, A.S.; Kazhanova, M.D.; Lipkovich, A.D.; Tazayan, A.N.; Gak, Y.M.; Ivanchenko, Y.V. The incidence of Cysticercosis in brown hares in Rostov oblast. J. Agric. Environ. 2024, 5, 45. [Google Scholar] [CrossRef]
  62. Garedaghi, Y.; Firouzivand, Y.; Luca, I. Prevalence of Endoparasites and their Zoonotic Significance in Wild Rabbits of Ahar City, Iran. Am. J. Anim. Vet. Sci. 2022, 17, 31–34. [Google Scholar] [CrossRef]
  63. Zhaxylykov, A.; Uakhit, R.; Kiyan, V. Study of prevalence and morphological features of cestodes in foxes (Vulpes vulpes) in the territory of Kazakhstan. J. Biol. Res. 2024, 1, 31–38. [Google Scholar] [CrossRef]
  64. Uakhit, R.; Smagulova, A.; Lider, L.; Leontyev, S.; Kiyan, V. Epizootiological monitoring of wolf helminths in Northern and Central Kazakhstan. Vet. World 2024, 17, 1648–1654. [Google Scholar] [CrossRef] [PubMed]
  65. Akhmetbekov, N.A. Zonal distribution of helminthiasis in Akmola and Karaganda regions. Sci. Bull. S. Seifullin Kazakh Agrotech. Univ. 2001, 4, 55–61. [Google Scholar]
  66. Zhaksylykova, A.A.; Abdybekova, F.M.; Sayakova, Z.Z.; Kenesary, S.A.; Kydyrkhanova, E.A. Epizootic situation of echinococcosis of animals (dogs) in the territory of the Republic of Kazakhstan. Endless Light Sci. 2024, 30, 5–10. [Google Scholar]
  67. Hoberg, E.P. Taenia tapeworms: Their biology, evolution and socioeconomic significance. Microbes Infect. 2002, 4, 859–866. [Google Scholar] [CrossRef]
  68. Eckert, J. Interactions between cestodes and their vertebrate hosts. In Parasite-Host Associations: Coexistence or Conflict? Toft, C.A., Aeschlimann, A., Bolis, L., Eds.; Oxford University Press: Oxford, UK, 1991; pp. 201–227. [Google Scholar] [CrossRef]
  69. Morandi, B.; Bazzucchi, A.; Gambini, S.; Crotti, S.; Cruciani, D.; Morandi, F.; Napoleoni, M.; Piseddu, T.; Di Donato, A.; Gavaudan, S. A novel intermediate host for Taenia serialis (Gervais, 1847): The European roe deer (Capreolus capreolus L. 1758) from the Monti Sibillini National Park (MSNP), Italy. Int. J. Parasitol. Parasites Wildl. 2021, 17, 110–113. [Google Scholar] [CrossRef]
  70. Hough, I. Subcutaneous larval Taenia serialis in a ring-tailed possum (Pseudocheirus peregrinus). Aust. Vet. J. 2000, 78, 468. [Google Scholar] [CrossRef]
  71. Segovia, J.M.; Torres, J.; Miquel, J.; Llaneza, L.; Feliu, C. Helminths in the wolf, Canis lupus, from north-western Spain. J. Helminthol. 2001, 75, 183–192. [Google Scholar] [CrossRef]
  72. Zhang, X.Y.; Jian, Y.N.; Ma, L.Q.; Li, X.P.; Karanis, P. A Case of Coenurosis in a Wild Rabbit (Lepus sinensis) Caused by Taenia serialis Metacestode in Qinghai Tibetan Plateau Area, China. Korean J. Parasitol. 2018, 56, 195–198. [Google Scholar] [CrossRef]
  73. Schneider-Crease, I.; Griffin, R.H.; Gomery, M.A.; Dorny, P.; Noh, J.C.; Handali, S.; Chastain, H.M.; Wilkins, P.P.; Nunn, C.L.; Snyder-Mackler, N.; et al. Identifying wildlife reservoirs of neglected taeniid tapeworms: Non-invasive diagnosis of endemic Taenia serialis infection in a wild primate population. PLoS Neglected Trop. Dis. 2017, 11, e0005709. [Google Scholar] [CrossRef]
  74. Tappe, D.; Berkholz, J.; Mahlke, U.; Lobeck, H.; Nagel, T.; Haeupler, A.; Muntau, B.; Racz, P.; Poppert, S. Molecular Identification of Zoonotic Tissue-Invasive Tapeworm Larvae Other than Taenia solium in Suspected Human Cysticercosis Cases. J. Clin. Microbiol. 2016, 54, 172–174. [Google Scholar] [CrossRef]
  75. Rinaldi, L.; Russo, T.; Schioppi, M.; Pennacchio, S.; Cringoli, G. Passalurus ambiguus: New insights into copromicroscopic diagnosis and circadian rhythm of egg excretion. Parasitol. Res. 2007, 101, 557–561. [Google Scholar] [CrossRef]
  76. Mäkitaipale, J.; Karvinen, I.; Virtala, A.K.; Näreaho, A. Prevalence of intestinal parasites and risk factor analysis for Eimeria infections in Finnish pet rabbits. Vet. Parasitol. Reg. Stud. Rep. 2017, 9, 34–40. [Google Scholar] [CrossRef]
  77. Raue, K.; Heuer, L.; Böhm, C.; Wolken, S.; Epe, C.; Strube, C. 10-year parasitological examination results (2003 to 2012) of faecal samples from horses, ruminants, pigs, dogs, cats, rabbits and hedgehogs. Parasitol. Res. 2017, 116, 3315–3330. [Google Scholar] [CrossRef] [PubMed]
  78. Grigoryan, G.A. On the study of the fauna of parasitic worms of hares (Lepus europaeus cyrensis Sat., 1905) of Armenia. Zool. Collect. 1950, 7, 111–120. (In Russian) [Google Scholar]
  79. Allan, J.C.; Craig, P.S.; Sherington, J.; Rogan, M.T.; Storey, D.M.; Heath, S.; Iball, K. Helminth parasites of the wild rabbit Oryctolagus cuniculus near Malham Tarn, Yorkshire, UK. J. Helminthol. 1999, 73, 289–294. [Google Scholar] [CrossRef] [PubMed]
  80. Grishina, E.A. Study of markers of blood lymphocytes apoptosis in animals at helminthiases. Russ. J. Parasitol. 2017, 40, 162–167. (In Russian) [Google Scholar]
  81. Kabilov, T.K.; Saidullaev, S.S.; Dadaev, S.; Kabilova, S.h.T. On the study of helminths of rare and endangered species of wild mammals of the fauna of Uzbekistan. In Proceedings of the Scientific Conference Problems of Protection and Sustainable Use of Biodiversity of the Animal World of Kazakhstan, Almaty, Kazakhstan, 6–8 April 1999; p. 67. [Google Scholar]
  82. Abdel-Gaber, R.; Ataya, F.; Fouad, D.; Daoud, M.; Alzuhairy, S. Prevalence, Morphological and Molecular Phylogenetic Analyses of the Rabbit Pinworm, Passalurus ambiguus Rudolphi 1819, in the Domestic Rabbits Oryctolagus cuniculus. Acta Parasitol. 2019, 64, 316–330. [Google Scholar] [CrossRef]
  83. Harcourt-Brown, F.M. Digestive system disease. In BSAVA Manual of Rabbit Medicine; Meredith, A., Lord, B., Eds.; British Small Animal Veterinary Association: Gloucester, UK, 2014; pp. 168–190. [Google Scholar]
Biology 14 01083 g001
Figure 1. Macroscopic appearance of larval cestodes in hares. (A) Cysticercus pisiformis in the abdominal cavity of a European hare. (B) Coenurus serialis in the muscle tissue of a mountain hare.
Figure 1. Macroscopic appearance of larval cestodes in hares. (A) Cysticercus pisiformis in the abdominal cavity of a European hare. (B) Coenurus serialis in the muscle tissue of a mountain hare.
Biology 14 01083 g001
Biology 14 01083 g002
Figure 2. Main morphological features of the cestode Mosgovoyia pectinata: (A) general body structure; (B) anterior end (scolex) with suckers; (C) proglottids with visible genital openings and eggs; (D) cestode eggs.
Figure 2. Main morphological features of the cestode Mosgovoyia pectinata: (A) general body structure; (B) anterior end (scolex) with suckers; (C) proglottids with visible genital openings and eggs; (D) cestode eggs.
Biology 14 01083 g002
Biology 14 01083 g003
Figure 3. Morphological features of the pinworm Passalurus ambiguus found in the large intestine of hares: (A) general appearance of an pinworms; (B) anterior end showing the esophagus, bulb, and papillae; (C) posterior end with spicule; (D) characteristic oval-shaped egg with a distinct shell.
Figure 3. Morphological features of the pinworm Passalurus ambiguus found in the large intestine of hares: (A) general appearance of an pinworms; (B) anterior end showing the esophagus, bulb, and papillae; (C) posterior end with spicule; (D) characteristic oval-shaped egg with a distinct shell.
Biology 14 01083 g003
Biology 14 01083 g004
Figure 4. Eggs of helminths and coccidia detected in the survey: (A,B) Nematodirus leporis; (C) Eimeria spp.; (D) Trichuris leporis.
Figure 4. Eggs of helminths and coccidia detected in the survey: (A,B) Nematodirus leporis; (C) Eimeria spp.; (D) Trichuris leporis.
Biology 14 01083 g004
Biology 14 01083 g005
Figure 5. Phylogenetic tree constructed using the Maximum Likelihood method based on the Tamura–Nei model for cysticercosis species. (Red circles indicate isolates from this study).
Figure 5. Phylogenetic tree constructed using the Maximum Likelihood method based on the Tamura–Nei model for cysticercosis species. (Red circles indicate isolates from this study).
Biology 14 01083 g005
Biology 14 01083 g006
Figure 6. Phylogenetic tree constructed using the Maximum Likelihood method based on the Tamura–Nei model for Mosgovoyia pectinata species. (Red circles indicate isolates from this study).
Figure 6. Phylogenetic tree constructed using the Maximum Likelihood method based on the Tamura–Nei model for Mosgovoyia pectinata species. (Red circles indicate isolates from this study).
Biology 14 01083 g006
Biology 14 01083 g007
Figure 7. Phylogenetic tree constructed using the Maximum Likelihood method based on the Tamura–Nei model for Passalurus ambiguus species. (Red circles indicate isolates from this study).
Figure 7. Phylogenetic tree constructed using the Maximum Likelihood method based on the Tamura–Nei model for Passalurus ambiguus species. (Red circles indicate isolates from this study).
Biology 14 01083 g007
Table 1. Characteristics of primers used and PCR conditions.
Table 1. Characteristics of primers used and PCR conditions.
No.TargetPrimerSequencePCR ParametersReference
1nad1JB11F
JB12R		5′-ACCACTAACTAATTCACTTTC-3′
5′-AGATTCGTAAGGGGCCTAATA-3′		initial denaturation 95 °C 3 min[3]
35 cycles
denaturation at 95 °C for 60 s
annealing at 50 °C for 60 s
extension at 72 °C for 60 s
final extension at 72 °C 5 min
2cox1COX-F
COX-R		5′-GATGTTTTCTTTACATTTATCTGGTG-3′
5′-GCCACCACAAATCAAGTATC-3′		initial denaturation 95 °C 3 min[8]
35 cycles
denaturation at 95 °C for 30 s
annealing at 53 °C for 60 s
extension at 72 °C for 60 s
final extension at 72 °C 7 min
328SC1F
D2R		5′-ACCCGCTGAATTTAAGCAT-3′
5′-TCCGTGTTTCAAGACGG-3′		initial denaturation 95 °C 2 min[16,28]
35 cycles
denaturation at 95 °C for 15 s
annealing at 50 °C for 20 s
extension at 72 °C for 30 s
final extension at 72 °C 1 min
Table 2. Prevalence and intensity of helminths found in the mountain and European hares.
Table 2. Prevalence and intensity of helminths found in the mountain and European hares.
HostSpecies IdentifiedN Infected/N Examined% Prevalence (95% CI)N Helminth FoundRange of IntensityMean (SD) IntensityOddsp-Value
>0.05
Mountain hare
(Lepus timidus)		Mosgovoyia pectinata7/6111.5 (4.7–22.2)553–157.9 (4.5)0.12960.5100
Taenia serialis2/613.3 (0.4–11.3)7030–4035.0 (7.1)0.03390.5052
Taenia pisiformis2/613.3 (0.4–11.3)14834–11474.0 (56.57)0.03391.0000
Passalurus ambiguus3/614.9 (10.3–13.7)1031–8734.3 (49.7)0.05171.0000
European hare
(Lepus europaeus)		Mosgovoyia pectinata3/466.5 (1.4–18.0)342–2811.3 (13.1)0.06980.5100
Taenia pisiformis2/464.3 (0.5–14.9)10746–6153.5 (10.6)0.04551.0000
Passalurus ambiguus3/466.5 (1.4–18.0)1415–9547.0 (45.6)0.06981.0000
95% CI: 95% confidence interval; SD: standard deviation.
Table 3. Study of helminths found in the feces samples of the mountain and European hares.
Table 3. Study of helminths found in the feces samples of the mountain and European hares.
HostSpecies IdentifiedN Infected/N Examined% Prevalence (95% CI)Odds Ratio (95% CI)p-Value
>0.05
Mountain hare
(Lepus timidus)		Nematodirus leporis7/5213.4 (5.6–25.8)0.1556 (6.68–25.27)0.0000
Eimeria spp.2/523.8 (0.4–13.2)0.40 (0.03–4.85)1.0000
Trichuris leporis1/521.9 (0.5–10.3)--
European hare
(Lepus europaeus)		Nematodirus leporis4/1136.6 (10.1–69.2)0.5714 (15.17–64.62)0.0000
Eimeria spp.1/119.1 (0.2–41.3)2.50 (0.21–30.29)1.0000
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Kiyan, V.; Smagulova, A.; Manapov, N.; Jazina, K.; Uakhit, R.; Bulashev, A.; Lider, L.; Leontyev, S. Parasitic Fauna of Lepus europaeus and Lepus timidus in Kazakhstan: Parasitological Profile and Molecular Identification. Biology 2025, 14, 1083. https://doi.org/10.3390/biology14081083

AMA Style

Kiyan V, Smagulova A, Manapov N, Jazina K, Uakhit R, Bulashev A, Lider L, Leontyev S. Parasitic Fauna of Lepus europaeus and Lepus timidus in Kazakhstan: Parasitological Profile and Molecular Identification. Biology. 2025; 14(8):1083. https://doi.org/10.3390/biology14081083

Chicago/Turabian Style

Kiyan, Vladimir, Ainura Smagulova, Nurassyl Manapov, Karina Jazina, Rabiga Uakhit, Aitbay Bulashev, Lyudmila Lider, and Sergey Leontyev. 2025. "Parasitic Fauna of Lepus europaeus and Lepus timidus in Kazakhstan: Parasitological Profile and Molecular Identification" Biology 14, no. 8: 1083. https://doi.org/10.3390/biology14081083

APA Style

Kiyan, V., Smagulova, A., Manapov, N., Jazina, K., Uakhit, R., Bulashev, A., Lider, L., & Leontyev, S. (2025). Parasitic Fauna of Lepus europaeus and Lepus timidus in Kazakhstan: Parasitological Profile and Molecular Identification. Biology, 14(8), 1083. https://doi.org/10.3390/biology14081083

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop