Research Article

Molecular Characterization of Paramphistomum cervi (Trematoda: Digenea: Paramphistomatidae) from Uzbekistan

Maxfuza S. Taylakova1, Asadullo S. Daminov1, Komoliddin X. Urokov1, Latofat A. Xujanova1, Oybek O. Amirov2*, Ruziboy Q. Shapaotov2

1Samarkand State University of Veterinary Medicine, Animal Husbandry and Biotechnology, Ulugbek St 94, Samarkand, Uzbekistan; 2Institute of Zoology of the Academy of Sciences of the Republic of Uzbekistan, 232B Bagishamol Street, Tashkent, Uzbekistan.

Received | August 30, 2025; Accepted | September 23, 2025; Published | January 24, 2026

*Correspondence | Oybek O. Amirov, Institute of Zoology of the Academy of Sciences of the Republic of Uzbekistan, 232b Bagishamol Street, Tashkent, Uzbekistan; Email: [email protected]

Citation | Taylakova MS, Daminov AS, Urokov KX, Xujanova LA, Amirov OO, Shapaotov RQ (2026). Molecular characterization of Paramphistomum cervi (Trematoda: Digenea: Paramphistomatidae) from Uzbekistan. Adv. Anim. Vet. Sci., 14(2):319-327.

DOI | https://dx.doi.org/10.17582/journal.aavs/2026/14.2.319.327

ISSN (Online) | 2307-8316

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

The genus Paramphistomum (Fischoeder, 1901) currently includes several species, among them P. cervi (Zeder, 1790), P. liorchis (Fischoeder, 1901), P. gracile (Fischoeder, 1901), P. epiclitum (Fischoeder, 1904), P. gotoi (Fukui, 1922), P. ichikawai (Fukui, 1922), P. leydeni (Näsmark, 1937), and P. hiberniae (Willmott, 1950). One of the most widely studied representatives, Paramphistomum cervi (Zeder, 1790) Fischoeder, 1901, was initially recorded by Daubenton in 1754 in the digestive tract of cattle and later confirmed by Zeder in 1790 in deer (Kennedy et al., 1985). In subsequent decades, some researchers, relying on limited morphological descriptions, proposed different names for the same species, including Fasciola cervi (Schrank, 1790), Fasciola elaphi (Sinha, 1950), and Monostoma conicum (Zeder, 1790).

P. cervi (Trematoda: Digenea: Paramphistomatidae) is a parasitic fluke whose adult form colonizes the rumen of ruminants, whereas the immature stages are usually found in the duodenum. This parasite has been documented in domestic livestock such as cattle, sheep, and goats, as well as in several wild ungulates (Adane and Demelash, 2020). Taxonomic classification within Digenea is still under revision, with modern molecular data suggesting reductions of certain orders and regrouping at the superfamily level. Nevertheless, families remain the most stable taxonomic units. Currently, the order Echinostomida (subclass Digenea) encompasses the superfamily Paramphistomoidea (Olson et al., 2003), which comprises the families Paramphistomidae and Gastrothylacidae, both of which harbor the majority of paramphistome species found in ruminants.

This parasite has a wide geographical range and has been reported from many parts of the world (Rangel-Ruiz et al., 2003; Ayaz et al., 2013). While infections with adult flukes are generally mild, infestations in young animals may result in severe gastroenteritis (Ayaz et al., 2013; Horak, 1971; Olsen, 1974). Immature paramphistomosis is still considered a neglected parasitic disease (Hajipour et al., 2021), frequently overlooked in diagnosis (Phiri et al., 2007). Nonetheless, it can lead to substantial losses due to mortality, reduced growth, and diminished milk production (Chaudhry et al., 2017). Even in areas where prevalence is reported to be low, such as the Pakong and Pasean regions, outbreaks in calves can result in high mortality and significant tissue damage, thereby reducing the economic value of livestock (Aryani et al., 2022; Hassan et al., 2011; Zhang and Hewitt, 1997).

Research on P. cervi to date has mostly focused on its morphology, life cycle, and epidemiology (Rangel-Ruiz et al., 2003; Ayaz et al., 2013; Wang et al., 2006), while molecular-level studies are comparatively scarce. More recently, however, both the complete mitochondrial genome and ITS2 rDNA region of P. cervi have been reported (Yan et al., 2013; Bazsalovicsova et al., 2010).

In eukaryotes, nuclear rDNA occurs as tandem repeats, each repeat containing three coding regions (18S, 5.8S and 28S rRNA genes), two internal transcribed spacers (ITS1 and ITS2), and an intergenic spacer (IGS) between transcriptional units (Long and Dawid, 1980). Because these rDNA regions evolve at different rates, they represent valuable genetic markers for phylogenetic and taxonomic analyses. Among them, the ITS sequences are particularly informative for parasite identification (Dai et al., 2012; Orosova et al., 2010; Yamada et al., 2011; Wang et al., 2012). The IGS region, which often includes repetitive elements, shows significant variability within and between parasite species (Zhao et al., 2011).

The present study aims to characterize Paramphistomum cervi (Zeder, 1790) from small and large ruminants using molecular analysis of the rDNA ITS2 region.

Materials and Methods

Collection of helminthological samples

In the present study, parasitic specimens were recovered from the digestive tracts of both large ruminants (cattle) is correct and small ruminants (sheep). Helminthological samples were collected from privately owned farms across four districts of the Samarkand region: Ishtikhon (sheep, n = 2), Payariq (sheep, n = 3), Pastdargom (cattle, n = 1), and Akdarya (sheep, n = 2). All specimens were preserved in a 70% ethanol solution for subsequent morphological and molecular analyses (Figure 1).

 

Morphological and morphometric analysis

Species identification of Paramphistomum cervi (genus Paramphistomum) was carried out using Meiji ML5000 and Nexcope NSZ818 light microscopes. Diagnostic features such as the overall body shape, structure of anterior and posterior suckers, reproductive organs and distribution of tegumental papillae were examined to confirm species identity (Choudhary et al., 2015).

DNA extraction

Genomic DNA was isolated from specimens preserved in 70% ethanol using the DNeasy Blood and Tissue Kit (Qiagen, https://www.qiagen.com). DNA concentration and purity were assessed with a Thermo Fisher Scientific spectrophotometer (China). All DNA extracts were kept at –20 °C until further PCR amplification.

PCR amplification

Molecular identification was performed by targeting the rDNA ITS1–5.8S–ITS2 region, a widely applied marker for helminth taxonomy (Subbotin et al., 2001). PCR reactions (20 μl total volume) consisted of 16.1 μl nuclease-free water, 2 μl 10× PCR buffer, 0.4 μl dNTPs, 2 μl of each primer [forward primer TW81 (5`-GTTTCCGTAGGTGAACCTGC-3`) and reverse primer AB28 (5`-ATATGCTTAAGTTCAGCGGGT-3`)], and 0.4 μl Taq DNA polymerase. The amplification protocol consisted of an

 

Table 1: Information on genetic samples obtained from the GenBank database.

No.	Species name	Accession number	Geographic origin	Host species
1	Paramphistomum cervi	KX274233	Croatia	Cervus elaphus
2	Paramphistomum cervi	KJ459936	China	Sheep
3	Paramphistomum cervi	KJ459935	China	Sheep
4	Paramphistomum cervi	OK216191	Egypt	Rumen
5	Paramphistomum cervi	OK216190	Egypt	Rumen
6	Paramphistomum cervi	OK216189	Egypt	Rumen
7	Paramphistomum cervi	HM026462	Slovakia	Cervus elaphus
8	Calicophoron clavula	MK416145	Egypt	–
9	Calicophoron phillerouxi	PP854140	South Africa	African buffalo
10	Calicophoron clavula	KX668944	USA	Kenyan ruminants
11	Calicophoron clavula	OQ842708	Germany	Cattle
12	Calicophoron microbothrium	MN912249	Egypt	–
13	Calicophoron phillerouxi	KX668977	USA	Kenyan ruminants
14	Calicophoron phillerouxi	KX668965	USA	Kenyan ruminants
15	Calicophoron phillerouxi	KX668964	USA	Kenyan ruminants
16	Calicophoron microbothrium	KX668921	USA	Kenyan ruminants
17	Calicophoron microbothrium	KX668919	USA	Kenyan ruminants
18	Calicophoron microbothrium	KX668918	USA	Kenyan ruminants
19	Calicophoron microbothrium	KP639631	Zimbabwe	Cattle
20	Carmyerius gregarius	OQ842678	Germany	Sheep

 

initial denaturation step at 98 °C for 30 s, followed by 40 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 10 min (Kuchboev et al., 2020).

Gel electrophoresis, purification, and sequencing

PCR products were visualized on a 1% agarose gel under 100 V. Target DNA bands were excised and purified using reagents supplied by “Sileks M” (Moscow, Russia), following the manufacturer’s instructions. Sequencing was performed using the ABI PRISM® BigDye™ Terminator v3.1 Cycle Sequencing Kit, with services provided by GATC Biotech AG. Sequence data were subsequently processed and aligned using BioEdit*, ClustalX2*, DNASTAR™* and PAUP* software packages (Larkin et al., 2007).

Phylogenetic tree construction

To investigate the molecular phylogenetic relationships of Paramphistomum cervi, nucleotide sequences of the ITS2 region were analyzed. These sequences were compared with homologous genetic data of other species retrieved from the GenBank database (Table 1).

Nucleotide sequences of the ITS2 region were used for phylogenetic inference. A sequence from a different genus was incorporated as an outgroup. Multiple sequence alignment was first conducted with the MAFFT algorithm (Katoh et al., 2002), and subsequently, ambiguous regions and evident misalignments were manually refined using BioEdit* software (version 7.0.5.2; Hall, 1999). Gaps and missing data were retained and treated as missing characters rather than being removed, as this approach helps preserve phylogenetic signal and provides a more reliable representation of evolutionary relationships. Phylogenetic tree reconstruction was performed in IQ-TREE 2 (Minh et al., 2020) under the Maximum Likelihood (ML) framework. The most appropriate nucleotide substitution model was identified automatically with the Model Finder module. Node support was assessed with 1000 bootstrap replicates (Felsenstein, 1985). The resulting tree was visualized and graphically customized using the Interactive Tree of Life (iTOL) web tool, which provides a clear and flexible representation of phylogenetic relationships.

Results and Discussion

Morphological and morphometric analysis

The sentence is very long. Consider breaking it up for clarity: Helminthological studies revealed that P. cervi, isolated from the digestive systems of cattle and sheep, had a pale reddish coloration and a pear-shaped body. An oral sucker was located at the anterior end, and a ventral sucker (acetabulum) was positioned at the posterior end. The total body length (2.8 mm to 8.3 mm) indicates notable intraspecific variability among the parasites. The mean body length was 6.4 mm, suggesting these specimens belong to a relatively large group of organisms. The standard deviation of 1.07 mm reflects a moderate degree of variation (Table 2, Figure 2).

 

Table 2: Morphometric characteristics of Paramphistomum cervi (n = 10).

Morphological features	Measurements (mm), n=10
Body length	2.8 – 8.3, (6.4±1.07)
Body width	1.2 – 3.6, (2.7±0.9)
Diameter of oral sucker	0.18 – 0.32, (0.23±0.3)
Diameter of ventral sucker	0.45 – 0.80, (0.61±0.4)
Egg size (length × width)	0.11–0.16 × 0.7–0.9

 

Body width also demonstrated a certain level of variability. The average width was 2.7 mm, which appears proportionate to body length. A relatively high standard deviation (±0.9 mm) indicates noticeable morphological differences in this parameter as well. The diameter of the oral sucker was relatively small, with an average of 0.23 mm and a very low standard deviation (±0.03 mm), suggesting that this structure is morphologically stable and shows minimal variation among individuals.

The ventral sucker (acetabulum) was significantly larger than the oral sucker, likely to facilitate firm attachment within the host’s tissue. Its average diameter was 0.61 mm, with a low standard deviation (±0.04 mm), indicating structural consistency. Egg dimensions fall within the micrometric range, which requires specialized measurement techniques. The differences between length and width were minimal, and overall, the egg size is within the typical range reported for trematodes. Therefore, egg morphology may serve as a useful criterion for species identification of P. cervi.

 

The oral sucker is funnel-shaped, widening towards the posterior. The intestines are broad, interconnected in a serpentine manner, and their terminal portions are positioned more dorsally than laterally. The genital pore is located anterior to the intestinal bifurcation. The acetabulum (posterior sucker) is situated near the posterior end of the body (subterminal position), measuring approximately one-fourth to one-fifth of the body length.

 

Molecular-genetic analysis

Following sequencing and chromatogram analysis, a 286-base-pair fragment of the rDNA ITS2 region was successfully amplified and identified from P. cervi (genus Paramphistomum, Fischoeder, 1901). For comparative analysis, reference sequences were obtained from the international GenBank database (https://blast.ncbi.nlm.nih.gov), including Paramphistomum cervi (accession number: HM026462) and Calicophoron clavula (genus Calicophoron, Näsmark, 1937; accession number: OQ842699) (Figure 3).

This section is very repetitive and hard to follow. It should be significantly condensed and presented in a more synthetic way. For example: Nucleotide variations were observed among the P. cervi samples from different regions (Payariq, Pastdargom, Ishtikhon, Akdarya) and the reference sequence (HM026462). Key variable positions included sites 64, 126, 168, 186, 230, 246, and 254. For instance, at position 126, samples from Payariq (P. cervi_Pay) had a thymine (T), whereas samples from other regions and the reference sequence had a cytosine (C). Greater sequence divergence was observed between all P. cervi samples and the congeneric species Calicophoron clavula (OQ842699), with consistent differences at positions 186 (A in P. cervi vs. G in C. clavula), 246 (C vs. T), and 254 (T vs. C).

In P. cervi_Pas, at position 64, G-guanine was found, while in P. cervi_Ish, T-thymine was present. At position 168, P. cervi_Pas contained T-thymine, while P. cervi_Ish contained C-cytosine. At position 64, P. cervi_Pas contained G-guanine, while P. cervi_Oqd contained T-thymine. At position 64, P. cervi_Pas contained G-guanine, while C. clavula OQ842699 contained T-thymine. At positions 168 and 254, P. cervi_Pas contained T-thymine, whereas C. clavula OQ842699 contained C-cytosine. At position 186, P. cervi_Pas contained A-adenine, while C. clavula OQ842699 contained G-guanine. At position 246, P. cervi_Pas contained C-cytosine, while C. clavula OQ842699 contained T-thymine.

In P. cervi_Ish, at position 186 A-adenine was present, while in C. clavula, OQ842699 contained a G-guanine; at position 246, P. cervi_Ish contained a C-cytosine, while C. clavula OQ842699 contained T-thymine; at position 254, P. cervi_Ish contained T-thymine, while C. clavula OQ842699 contained C-cytosine. Similarly, in P. cervi_Oqd, at position 186 A-adenine was present, while in C. clavula, OQ842699 contained a G-guanine; at position 246, P. cervi_Oqd contained a C-cytosine, while C. clavula OQ842699 contained T-thymine; and at position 254, P. cervi_Oqd contained T-thymine, while C. clavula OQ842699 contained C-cytosine.

The observed substitutions within the ITS2 region are located in a non-coding segment of rDNA. Although these mutations are not expected to alter protein structure, they may still influence secondary DNA structure or ribosomal RNA processing. In this study, recurrent substitutions at positions 126 and 230 consistently differentiated P. cervi from Calicophoron species and also showed variability among local P. cervi populations. Such patterns suggest that these mutations, while synonymous in nature, may hold phylogenetic and taxonomic value for distinguishing closely related species and geographic isolates.

Phylogenetic analysis

The Paramphistomum cluster (marked with a blue line) includes only P. cervi samples. The genetic similarity among these species is very high, and the main node is strongly supported with a bootstrap value of 94. Within the group, samples obtained from different geographical regions (Ishtikhon (Sheep), Payariq (Sheep), Pastdargom (Cattle), Akdarya (Sheep)), as well as sequences retrieved from GenBank, are present, and they are positioned almost identically in the phylogenetic tree (Figure 4).

 

C. gregarious (OQ842678) was used as an outgroup to root the tree and provide phylogenetic orientation. The molecular distance scale (0.005) represents the number of substitutions per site. Bootstrap support values (shown at the nodes) indicate the statistical reliability of branching, where values above 70% are generally considered strong support.

The Calicophoron cluster (separated by a red line) is divided into two sub-branches. The first branch consists of C. clavula and C. phillerouxi species, with the main node showing a bootstrap value of 35. The second branch unites C. microbothrium samples, with a bootstrap value of 43 at its main node. The overall node for the entire Calicophoron group has a bootstrap value of 42, indicating a moderate level of confidence in interspecific differentiation. As an outgroup, the sample Carmyerius gregarius (OQ842678) was used, and it was clearly separated from all other species. The molecular distance scale represents 0.005 units. The results demonstrate that species belonging to the genera Paramphistomum and Calicophoron are well distinguished phylogenetically. The high bootstrap values for P. cervi samples confirm their genetic integrity, whereas the internal sub-branches within the Calicophoron group are separated with relatively low confidence.

Discussion

Paramphistomiasis is highly prevalent in ruminants, and morphological studies along with genetic approaches are used in species discrimination. The morphological analysis of the current specimens revealed that they belong to the superfamily Paramphistomidea by comparison with other trematodes (Jones et al., 2002).

During this study, the morphological and morphometric characteristics of Paramphistomum cervi were analyzed and compared with data from existing literature to clarify the diagnostic features of the species. The parasite’s light reddish coloration, pear-shaped body structure, and the presence of two suckers one funnel-shaped oral sucker located anteriorly and a posterior acetabulum represent classical morphological traits of this species.

The observed body length (ranging from 2.8 to 8.3 mm, with an average of 6.4 mm) and width (1.2 to 3.6 mm, average 2.7 mm) fall within the range reported by many authors and indicate the species’ morphometric variability. Notably, the subterminal position of the acetabulum, located near the posterior end and occupying approximately one-fourth to one-fifth of the body, serves as a key diagnostic feature that distinguishes this species from other representatives of the Paramphistomum genus.

The wide, serpentine-shaped intestinal branches and the posteriorly expanding funnel-like oral opening reflect adaptations in the digestive system of the species. Additionally, the genital pore located just posterior to the intestinal bifurcation is also an important morphological trait for species identification.

Furthermore, the identification of P. cervi in the digestive systems of sheep and cattle indicates its widespread distribution among ruminants and highlights its ecological adaptability. These morphological analyses play a crucial role in the accurate identification, differential diagnosis, and development of effective treatment and prevention strategies for this parasitic species.

The results of the phylogenetic analysis based on ITS rRNA gene sequences demonstrated clear phylogenetic differentiation between representatives of the genera Paramphistomum and Calicophoron. In the constructed phylogenetic tree, all P. cervi samples formed a monophyletic cluster, and the high bootstrap value (94) at the main node confirmed their genetic coherence and phylogenetic stability. This cluster included four autochthonous samples obtained from the study areas (P. cervi_Pay, P. cervi_Ish, P. cervi_Oqd – isolated from sheep; P. cervi_Pas – isolated from cattle) as well as sequences available in the GenBank database, originating from Croatia (Cervus elaphus), China (sheep), Egypt (rumen sample), and Slovakia (Cervus elaphus).

In Uzbekistan, molecular investigations of the local fauna have been expanding in recent years, encompassing studies on insects (Kimyonazarov et al., 2024; Kadirov et al., 2024), nematodes (Aliyev et al., 2024; Mirzaev et al., 2024; Turgunov et al., 2024), and fish (Quvatov et al., 2023; Ubaydullayev et al., 2025).

Earlier research has shown that the ITS2 region of rDNA tends to be more conserved than ITS1 (Luton et al., 1992), potentially due to the presence of varying types and quantities of repeat sequences. Both long and short repeats, which can cause size differences, have been documented in various helminth groups such as trematodes (Warberg et al., 2005), cestodes (Bowles et al., 1995), and nematodes (Subbotin et al., 2011). However, in the current study, no length variation was observed among the ITS2 sequences of any P. cervi specimens.

The minimal genetic distance observed between local and international samples suggests that P. cervi, despite its wide host range (including sheep, cattle, and wild ungulates), exhibits a high level of genetic conservation. This suggests that its evolutionary history may reflect its ability to parasitize a broad spectrum of hosts with limited host-specific adaptations. Moreover, the monophyletic structure of the cluster provides strong evidence of the intraspecific phylogenetic homogeneity of P. cervi.

In contrast, the Calicophoron group was divided into two subclusters: The first comprising C. clavula and C. phillerouxi, and the second consisting of C. microbothrium samples. The bootstrap values for these subclusters were relatively low (35 and 43), indicating weaker genetic differentiation among these species compared to P. cervi. According to the data table, these species are geographically widespread, found in Egypt, the USA (including Kenyan domestic animals), Germany (cattle), South Africa (African buffalo), and Zimbabwe (cattle). The wide distribution range, particularly in species such as C. microbothrium-suggests a broad ecological amplitude and a high degree of host plasticity or low host specificity.

The outgroup Carmyerius gregarius (Germany, sheep) was clearly separated from the main clusters and placed at the root of the tree, demonstrating the phylogenetic adequacy of the chosen outgroup. The low value of the molecular distance scale (0.005) indicates relatively low nucleotide divergence among samples, while still confirming clear phylogenetic separation at the genus level.

While recent studies have highlighted the scarcity of molecular investigations on paramphistomid flukes (Mitchell et al., 2020), they have generally focused on broad species-level identification and global distribution without providing a critical assessment of existing phylogenies. In this context, the present work introduces a novel contribution by presenting the first molecular-genetic characterization of Paramphistomum cervi from Uzbekistan, including isolates from both sheep and cattle. Unlike previous studies that primarily emphasized the universal applicability of ITS2 sequencing, our research provides a detailed comparative analysis of nucleotide polymorphisms across local isolates and reference sequences. The identification of consistent diagnostic substitutions at specific positions (64, 126, 168, 230, 246, and 254) not only confirms the high conservatism of P. cervi but also establishes reliable markers for distinguishing between closely related taxa (Paramphistomum vs. Calicophoron). Furthermore, by integrating newly generated sequences with international GenBank data, we critically evaluated the robustness of genus-level clades, demonstrating strong phylogenetic stability in P. cervi and revealing relatively weaker resolution within Calicophoron. This dual approach local molecular characterization combined with a critical evaluation of existing phylogenies-extends beyond earlier reports and provides a regional reference dataset that can be applied to future phylogeographic and epidemiological studies in Central Asia.

Overall, the findings indicate that P. cervi, despite its wide geographical distribution and broad host range, remains genetically stable and conservative, whereas the genus Calicophoron exhibits comparatively higher levels of internal genetic diversity. These results provide important insights into host adaptation strategies, dispersal dynamics, and phylogenetic evolution of these parasites.

Conclusion

The present study provides a comprehensive morphological, morphometric, and molecular characterization of Paramphistomum cervi, a trematode parasite isolated from the digestive systems of cattle and sheep in the Samarkand region. Morphologically, P. cervi exhibits a light reddish, pear-shaped body with distinct anatomical features including anterior and posterior suckers, a funnel-shaped oral opening, serpentine intestinal branches, and clearly positioned genital pore and acetabulum. These features are consistent with previously described characteristics of the species.

Morphometric analysis revealed that body length ranged from 2.8 to 8.3 mm (mean ± SD: 6.4 ± 1.07 mm) and body width from 1.2 to 3.6 mm (2.7 ± 0.9 mm), indicating moderate intraspecific variation. The oral sucker was relatively small (0.23 ± 0.03 mm), while the ventral sucker (acetabulum) was significantly larger (0.61 ± 0.04 mm), suggesting functional adaptation for host attachment. Egg dimensions were within typical micrometric ranges reported for trematodes, showing minimal variation and offering diagnostic value for species identification.

Molecular and bioinformatic analysis targeting the ITS2 region of rDNA confirmed the genetic identity of the species. Despite minor nucleotide variations indicating potential inter-population differences, phylogenetic reconstruction revealed that all P. cervi samples formed a strongly supported clade (bootstrap = 94), highlighting a high degree of genetic homogeneity. Furthermore, the analysis distinctly separated the genera Paramphistomum and Calicophoron, underscoring their evolutionary divergence.

Collectively, these findings confirm the taxonomic status of P. cervi and emphasize the importance of integrating morphological and molecular approaches for accurate parasite identification. The results hold practical relevance for parasitological diagnostics, epidemiological surveillance, and the development of targeted control and prevention strategies against paramphistomosis in ruminants.

Acknowledgements

This study was conducted as part of the 2025–2029 research program of the Institute of Zoology, Academy of Sciences of the Republic of Uzbekistan, under the project “1.2. Development of a digital information system for the fauna of Bukhara and Navoi regions”, funded by the state budget.

Novelty Statement

This study provides the first molecular characterization of P. cervi from Uzbekistan, thereby filling a significant gap in the parasitological database of Central Asia. These results not only expand the regional knowledge of trematode biodiversity but also contribute novel reference data for future epidemiological and phylogenetic research.

Author’s Contribution

Field sampling, morphological identification, and statistical analyses were carried out by MT, AD, KU, and LX. Molecular experiments and sequencing were performed by RS. Data interpretation and manuscript preparation were undertaken by OA. All authors reviewed and approved the final version of the manuscript.

Generative AI and AI-assisted technology statement

The authors declare that no generative AI was used to conceive, analyze, or interpret the research data.

Conflict of interest

The authors have declared no conflict interests related to this work.

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