Molecular detection and COX1-based phylogenetic placement of Dipylidium caninum caninum in stray dogs from Iğdır Province, eastern Türkiye

Introduction

Dipylidium caninum (Cestoda: Dipylidiidae) is a globally distributed tapeworm that inhabits the small intestine of domestic dogs and cats and may occasionally infect humans, particularly children (Bowman et al., 2002; Jiang et al., 2017). Given the documented genetic heterogeneity and unresolved taxonomy within the Dipylidium caninum group, the lineage detected in the present study is hereafter referred to as Dipylidium caninum within the D. caninum species complex, unless otherwise specified. Transmission occurs through an indirect life cycle in which cysticercoids develop in flea intermediate hosts, mainly Ctenocephalides felis and Ctenocephalides canis, and definitive hosts become infected by ingesting infected fleas (Bowman et al., 2002).

Although D. caninum is among the most frequently reported cestodes in companion animals, human dipylidiasis has traditionally been considered rare. However, confirmed human cases reported in Asia and Eastern Europe indicate that zoonotic transmission continues in regions with high flea exposure (Ramana et al., 2011; Jiang et al., 2017; Bronstein et al., 2020). A recent global synthesis further supports the ongoing public health relevance of this parasite (Rousseau et al., 2022).

Recent molecular studies have challenged the long-standing view of D. caninum as a genetically uniform species. Substantial mitochondrial divergence has been documented, and at least two host-associated genotypes, adapted to dogs and cats, respectively, have been proposed (Labuschagne et al., 2018; Beugnet et al., 2018). In addition, morphologically similar taxa such as D. carracidoi may have been historically misidentified as D. caninum, highlighting unresolved taxonomic complexity within Dipylidiidae (Millán & Casanova, 2009; Enitez-Bolivar et al., 2022).

In Türkiye, numerous coprological surveys have reported D. caninum in both owned and shepherd dog populations across multiple provinces, with prevalence varying by geography, diagnostic approach and dog population structure (Yaman et al., 2006; Orhun & Ayaz, 2006; Kozan et al., 2007; Yıldırım et al., 2007; Öter et al., 2011; Işık et al., 2014; Nas & Biçek, 2018; Karakuş & Denizhan, 2019; Ceylan et al., 2024). These findings are consistent with earlier studies dating back to the late 1970s, indicating long-term endemic transmission in both rural and urban settings (Güralp et al., 1977; Taşan, 1983; Tınar et al., 1989; Doğanay & Öge, 1993; Şahin et al., 1993; Aydenizöz, 1997; Ayçiçek et al., 1998).

Despite this extensive parasitological background, molecular data on Dipylidium caninum in dogs from Türkiye remain scarce. Although recent work has demonstrated the presence of D. caninum DNA in flea vectors (Özdemir et al., 2025), molecular prevalence and sequence-based characterization studies in canine hosts are still limited, particularly at the regional level. In eastern Türkiye, previous studies have mainly focused on the molecular epidemiology of zoonotic and parasitic agents in animals and environmental samples, highlighting their prevalence and potential public health significance (Ertaş et al., 2022; Ertaş & Ayan, 2022; Kılınç et al., 2023; Koca et al., 2023; Akkuş & Ertaş Oğuz, 2024; Ertaş Oğuz et al., 2025). However, molecular information on Dipylidiidae cestodes in shepherd dog populations from this region remains limited.

Therefore, the present study aimed to investigate the molecular presence of Dipylidiidae cestodes in shepherd dogs from Iğdır Province, eastern Türkiye, using a COX1-based PCR approach and to characterize the detected lineage through partial COX1 gene sequencing. The resulting data provide new molecular evidence from an under-studied region and contribute to a better understanding of Dipylidium diversity and transmission dynamics in shepherd dog populations.

Materials and Methods

Literature data extraction

To place the findings from Iğdır in a broader epidemiological context, published prevalence data for Dipylidium caninum infections in dogs were compiled from the literature. For each country, mean prevalence values were calculated across available studies irrespective of diagnostic method, and these country-level estimates were subsequently grouped to generate continent-level summaries. Although this approach does not constitute a formal meta-analysis, it provides a comparative framework for interpreting regional variation and situating the Iğdır data within global transmission patterns. A descriptive overview of the global prevalence of D. caninum in dogs is presented in Supplementary Figure S1. Data handling and visualization were performed using R statistical software (R Core Team, 2025).

Supplementary Fig. S1.

Results

Out of 100 screened fecal samples, eight (8 %) yielded a PCR product of the expected size for the COX1 gene fragment. All positive samples produced identical sequences across the analyzed region, indicating the presence of a single mitochondrial haplotype among the infected dogs.

The obtained 396 bp partial mitochondrial COX1 sequence of Dipylidium caninum isolate Igdir_Dog1 from a shepherd dog in Iğdır Province, Türkiye, was deposited in GenBank under accession number PZ299077.1.

BLASTn analysis showed that the sequence matched D. caninum entries deposited in GenBank, including partial COX1 sequences and complete mitochondrial genome records. The highest identity was observed with a dog-derived sequence from Ghana (PX765914.1; 92.12 % identity, 93 % query cover), followed by bush dog-derived Colombian sequences (OR251824.1 and OR251823.1), a canine complete mitogenome from the USA (OK523384.1), and a dog-derived Croatian sequence (PZ165824.1), each showing 91.92 % identity and 100 % query cover. Turkish dog-derived sequences from Elazığ (PP842203.1 and PP842202.1) showed 91.27 % identity with 95 % query cover. Cat-associated sequences were also included in the comparison and showed lower identity values, ranging approximately from 88.71 % to 89.80 %. The full BLASTn comparison is provided in Supplementary Table 1.

Discussion

The present study provides molecular evidence of Dipylidium caninum in dogs from Iğdır Province, eastern Türkiye, and represents one of the first COX1-based molecular characterizations of Dipylidiidae in this region. Although D. caninum has long been reported in Türkiye using conventional parasitological methods, molecular data remain limited. Two unpublished GenBank records from Elazığ (PP842202.1 and PP842203.1; Aslan Celik et al., GenBank direct submissions) and the molecular detection of D. caninum DNA in fleas from Tokat indicate that molecular evidence from Türkiye is gradually increasing (Özdemir et al., 2025). In this context, the Iğdır sequence reported here adds new COX1 data from eastern Türkiye and helps expand the limited molecular data-set available for Türkiye.

In the phylogenetic tree, the analyzed D. caninum sequences were arranged into three broad groups, but the separation between dog- and cat-associated lineages was unclear (Fig. 1). The first group mainly contained cat-associated and mixed-host sequences from South Africa, Mexico, the USA, Italy, Egypt, Croatia, Pakistan and Ethiopia (Labuschagne et al., 2018; Jesudoss Chelladurai et al., 2023; Camacho-Giles et al., 2024; Citterio et al., 2021; Satour et al., 2026; Beck & Šikić, 2026; Alvi et al., 2022; Dumendiak et al., 2024). This group also included some non-cat hosts, suggesting that host origin alone may not fully explain the observed phylogenetic pattern. Dogs dominated the second group and other canine-derived sequences from Ghana, Colombia, the USA, Croatia, China and Türkiye (Xie et al., 2019; Uribe et al., 2023; Jesudoss Chelladurai et al., 2023; Addy et al., 2026; Beck & Šikić, 2026; Aslan Çelik et al., 2024). The Iğdır isolate PZ299077.1 was positioned within this broad canine-derived group, together with the previously submitted Turkish dog sequences from Elazığ and several dog or wild-canid sequences from other regions. This placement indicates that the Iğdır sequence is not isolated from the global D. caninum dataset, but falls within a group largely represented by canine hosts. The third group included mainly Iraqi and Pakistani sequences obtained from both dog and cat hosts, forming a mixed regional cluster (Al-Ardi, 2022; Alvi et al., 2022). This pattern may reflect regional sequence similarity rather than a strict host-associated structure. Overall, the phylogenetic pattern supports the identification of the Iğdır isolate D. caninum and shows that it contributes new eastern Turkish COX1 data to the limited molecular records available from Türkiye. However, because several branches showed low to moderate support values and the analyzed COX1 fragment was relatively short, the observed clustering should be interpreted cautiously rather than as definitive evidence of host-specific lineages.

Fig. 1.

Previous molecular and genomic studies have shown that D. caninum contains largely host-associated canine and feline lineages, with marked genetic differentiation between these groups (Labuschagne et al., 2018; Rousseau et al., 2022; Jesudoss Chelladurai et al., 2023). However, host association is not strict, and dogs may carry parasites related to different genotype groups. Recent data from Ghana also reported both canine and feline–canine-associated genotypes, although most dog-derived isolates were assigned to the canine genotype (Addy et al., 2026). Therefore, the placement of PZ299077.1 near Ghanaian dog-derived sequences and other canine-host records supports its affinity with the canine-derived group. Considering the known mitochondrial diversity within the D. caninum complex, the relatively low COX1 identity values observed here do not preclude this placement. However, because the analysis was based on a partial COX1 fragment, the result should be interpreted as molecular placement within the canine-derived group rather than as a final genotype-level conclusion.

Earlier parasitological surveys conducted in different provinces of Türkiye have repeatedly reported D. caninum in dogs using copromicroscopic examination or necropsy-based approaches, with prevalence values varying widely depending on geographic region, dog population structure, and diagnostic methodology (Güralp et al., 1977; Taşan, 1983; Tınar et al., 1989; Doğanay & Öge, 1993; Aydenizöz, 1997; Ayçiçek et al., 1998; Yaman et al., 2006; Kozan et al., 2007; Yıldırım et al., 2007; Öter et al., 2011; Işık et al., 2014; Nas & Biçek, 2018; Karakuş & Denizhan, 2019; Ceylan et al., 2024). However, most of these investigations lacked molecular confirmation, leaving uncertainties regarding species identity and the extent of genetic diversity within Dipylidiidae. The national overview presented in Figure 2 supports this pattern. It shows that D. caninum has been reported from both the western and eastern regions of Türkiye, using both dog-based surveys and flea-based molecular detection. The 5 % molecular prevalence detected in Iğdır therefore adds PCR-based evidence from eastern Türkiye and supports the need for further molecular surveillance. Additionally, the Tokat flea-based PCR record was considered as supporting evidence for transmission rather than as directly comparable dog prevalence data (Özdemir et al., 2025).

Fig. 2.

In conclusion, this study provides a new COX1-based molecular record of D. caninum in dogs from Iğdır Province, eastern Türkiye. It adds new mitochondrial data to the limited molecular dataset currently available from Türkiye. The analyzed sequence showed affinity with canine-derived D. caninum sequences in the phylogenetic analysis and contributes additional information to the growing body of evidence for genetic diversity within the D. caninum complex. In line with recent studies, the present findings also highlight the need for broader sampling and the inclusion of additional mitochondrial and nuclear markers from underrepresented regions to understand better the evolutionary structure and global diversity of this parasite. From a One Health perspective, continued molecular surveillance of dogs, together with monitoring of flea intermediate hosts, may contribute to an improved understanding of the transmission dynamics and zoonotic risk associated with canine dipylidiasis.