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. 2026 May 7;22:376. doi: 10.1186/s12917-026-05466-z

Identification of Ctenocephalides felis felis and the detection and characterization of associated Wolbachia endosymbiont in Pakistan

Nadia Nadia 1, Muhammad Numan 1, Ahmad H Khan 2,3, Sudais Rahman 1, Sidra Batoool 1, Zhihua Sun 4, Asma F Alotaibi 5, Mashal M Almutairi 6, Abid Ali 1,✉, Alanoud T Aljasham 5,✉
PMCID: PMC13322021  PMID: 42098841

Abstract

Fleas are holometabolous, blood-feeding ectoparasites capable of transmitting diverse pathogens of significant veterinary and public health concerns. Their occurrence and abundance within a given habitat depend on environmental factors and the availability of suitable hosts. In Pakistan, research on flea fauna and their associated pathogens has been neglected. To date, no reports have documented the molecular characterization of Ctenocephalides felis felis and their associated bacteria in Pakistan. In the present study, three hundred and eighty morphologically identified C. felis felis specimens-comprising 184 collected from free roaming (stray) dogs (n = 69) and 196 from cats (n = 86), were subjected to DNA extraction followed by amplification of the cytochrome oxidase subunit 1 gene (cox1) and citrate synthase gene (gltA) for flea identity confirmation and screening for the presence of associated bacteria, respectively. Amplicons of appropriate base pair sizes were sequenced and submitted to BLASTn and subsequently subjected to phylogenetic analyses. The obtained cox1 sequence from the morphologically identified C. felis felis in this study showed 100% identity and phylogenetically clustered with C. felis felis sequences from India, Australia, Thailand, China, and Laos in GenBank. Similarly, gltA sequences showed 100% identity with the Wolbachia endosymbiont of C. felis reported from the United Kingdom. This study provides the first genetic characterization of C. felis felis infesting dogs and cats, and their associated Wolbachia endosymbiont in Khyber Pakhtunkhwa (KP), Pakistan. These findings provide baseline molecular data and highlight the need for systematic surveillance and management measures to mitigate any potential veterinary and public health threats.

Keywords: Ctenocephalides felis felis, cox1, Wolbachia, Siphonaptera, Phylogeny, Pakistan

Introduction

Fleas (Insecta: Siphonaptera) are obligatory hematophagous ectoparasites of veterinary and public health significance. They are small (2–10 mm), laterally flattened, wingless, and highly specialized for a blood-feeding lifestyle, exhibiting distinct morphological and physiological adaptations, including laterally flattened bodies, piercing-sucking mouthparts, and enlarged hind legs adapted for jumping and efficient host acquisition [37, 38]. The importance of fleas is largely related to their ability to transmit infectious agents during blood meals [66]. Globally, there are 16 recognized families within the order Siphonaptera, representing over 2,500 species in 240 genera, with the highest diversity in temperate regions [9, 47]. Most flea species of medical and veterinary relevance belong to the Pulicidae and Ceratophyllidae [ 25].

Within the Pulicidae, the cat flea Ctenocephalides felis (Bouché, 1835), and the dog flea Ctenocephalides canis (Curtis, 1826) comprise most flea species infesting companion animals [6, 32, 51]. Among these, C. felis exhibits substantial genetic diversity and cosmopolitan distribution originating from the Ethiopian region [33, 45]. Based on molecular approaches, the genus Ctenocephalides is classified as having three recognized subspecies of C. felis: the cosmopolitan C. felis felis and two Ethiopian region subspecies: C. felis damarensis and C. felis strongylus [33]. Several factors contribute to its global distribution, including its broad resilience to habitat conditions within human dwellings, tolerance to climatic fluctuations, and larvae can exploit organic debris and other flea-associated resources [14, 28].

In addition to cats and dogs, C. felis felis has been reported from a wide range of wild hosts, including opossums and rodents [36], as well as raccoons [49], and is frequently encountered in human dwellings [7, 17, 41]. Accurate identification of flea species is essential, as different species are associated with distinct epidemiological risks [39]. While traditional morphological methods remain fundamental, the use of molecular markers has significantly enhanced species confirmation and phylogenetic resolution [62, 67]. The molecular marker cytochrome oxidase subunit 1 gene (cox1) has been widely used for the identification, detection of intraspecies variations, and phylogenetic relationships of various arthropods, particularly ticks and Ctenocephalides spp. [54, 56].

The significance of C. felis felis is attributed to its parasitism and transmission of zoonotic pathogens, including Rickettsia spp., Bartonella spp. [9], and Wolbachia spp. [11, 40, 43, 50]. Wolbachia endosymbionts are estimated to infect approximately 40–66% arthropod species and many filarial nematodes [27, 53, 63]. Although the veterinary and public health significance of fleas has been studied extensively in other climes [30, 34, 61], such studies in Pakistan remain limited. To date, only a few flea species have been morphologically reported from Pakistan, including Pulex irritans, C. felis, C. canis [46, 55] Neopsylla sp., Ischnopsyllus intermedius, Nosopsyllus sp., I. octactenus, Xenopsylla astia, X. cheopis, Synosternus pallidus, and S. cleopatrae [23].

The climate and geography of Pakistan offer suitable conditions for the establishment of many flea species, yet there is no genetic data available to describe these species. Despite their importance, molecular insights into Wolbachia endosymbionts in fleas in Pakistan remain scarce. To our knowledge, no molecular studies have addressed the detection of Wolbachia bacteria in C. felis felis from the Oriental region, including Pakistan, to date. The present study aimed to undertake the morphological identification and molecular characterization of fleas infesting dogs and cats in Pakistan and to investigate the occurrence of associated bacteria.

Material and methods

Study area and flea collections

During September–November 2024 and February-August 2025, fleas were collected from free roaming (stray) dogs and cats across four selected districts of Khyber Pakhtunkhwa province of Pakistan, including Nowshera (34.005152º N, 71.948503º E), Charsadda (34.159023º N, 71.754861º E), Mardan (34.169412º N, 72.082828º E), and Swabi (34.129097º N, 72.465734º E). No samples were found in December 2024 and January 2025 due to winter seasons, as flea activities are significantly reduced during these months. The geographical coordinates of each sampling site were determined using a Global Positioning System (GPS), and the map was created with ArcGIS (v. 10.3.1) (ESRI, Redlands, CA, USA) (Fig. 1). These selected districts were chosen based on their high animal density, accessibility, and representation of different ecological conditions within the province. All samples were collected opportunistically in public areas, and no animals were examined in veterinary clinics or under clinical conditions. Animals were physically examined under field conditions, and fleas were collected by hand and using a flea comb and forceps. Flea collection was carried out only once per animal host. The collected flea samples were preserved in sterile vials containing 70% ethanol and properly labeled with host type, host age, date, season, and location of collection. The preserved fleas were used for morphological and molecular analyses.

Fig. 1.

Fig. 1

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Map of Pakistan highlighting the selected sampling sites

Morphological identification

The collected flea specimens were examined under a stereomicroscope (Luxeo 6Z, LABOMED, United States) and identified, where possible to the species and subspecies level with the aid of standard taxonomic keys [10, 31, 32, 35, 48].

DNA extraction and polymerase chain reaction

The collected flea specimens were processed for DNA extraction (five specimens per host from each study site; Table 1). Before DNA extraction, specimens were removed from ethanol and air-dried or incubated at 37 °C for 20–30 min to eliminate the residual ethanol. Subsequently, the fleas were dissected using sterile scalpels and scissors. Genomic DNA was extracted using the phenol–chloroform method [52] and quantified using a NanoDrop spectrophotometer (OPTIZEN, Daejeon, South Korea).

Table 1.

Prevalence and distribution patterns of fleas and Wolbachia endosymbiont’s gltA PCR results

Location Host No. infested/examined host (%) No. of collected fleas (♂/♀) No. subjected to molecular analyses gltA-positive
Nowshera Dog 18/30 (60) 28 (12/16) 5 (3F, 2 M) 4 (2F, 2 M)
Cat 6/12 (50) 48 (20/28) 5 (5F, 0 M) 4 (4F)
Mardan Dog 11/25 (44) 49 (27/22) 5 (5F, 0 M) 3 (3F)
Cat 43/48 (89.6) 56 (18/38) 5 (3F, 2 M) 2 (2F)
Swabi Dog 17/28 (60.7) 40 (17/23) 5 (4F, 1 M) 3 (2F, 1 M)
Cat 25/31 (80.6) 43 (19/24) 5 (0F, 5 M) 2 (2 M)
Charsadda Dog 23/34 (67.6) 67 (20/47) 5 (3F, 2 M) 2 (2F)
Cat 12/15 (80) 49 (15/34) 5 (5F, 0 M) 2 (2F)
Total dogs 69/117 (59) 184 (76/108) 20 (15F, 5 M) 12 (9F, 3 M)
Total cats 86/106 (81.1) 196 (72/124) 20 (13F, 7 M) 10 (8F, 2 M)
Overall 155/223 (51.6) 380 (148/232) 40 (28F, 12 M) 22 (17F, 5 M)
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A conventional PCR was employed for the partial amplification of the cox1 gene using the primers HCO2198: TAA A C TTC AGG GTG ACC AAA AAA TCA and LCO1490: GGT CAA CAA ATC ATA AAG ATA TTG G [18]. In addition, PCR amplification of the citrate synthase (gltA) fragment was carried out using primer BhCS781p: GGG GAC CAG CTC ATG GTG G and BhCS1137n: AAT GCA AAA A G AAC AGT AAA CA [44]. Each PCR reaction mixture was performed in a final volume of 20 µl, including 1 µL of each primer (forward and reverse: 10 µM), 3 µL of genomic DNA (100 ng/µL), 3 µL nuclease-free water, and 12 µL of DreamTaq Green Master Mix (2X) (Thermo Scientific, Waltham, MA, USA).

The conditions for thermocycling included an initial denaturation at 95 °C for 3 min, then 39 cycles including denaturation at 95 °C for 15 s, annealing (30 s) at 48 °C for cox1 or at 56 °C for gltA, extension at 72 °C for 30 s, and a final extension at 72 °C for 7 min. Each PCR experiment included a negative control (no template DNA) to monitor contamination and a positive control (previously confirmed Rhipicephalus microplus DNA sample) to verify amplification success. Amplicons of expected size were determined by comparison with a DNA ladder. The amplification was analyzed by electrophoresis on a 2% agarose gel, stained with ethidium bromide, and visualized using a Gel Documentation System (BioDoc-It™ Imaging Systems, Upland, CA, USA). The amplified DNA was purified using the GeneClean® II kit from Qbiogene (Illkirch-Graffenstaden, France).

Sequencing and phylogenetic analyses

All the PCR-positive amplicons for both the cox1 and gltA fragments were sequenced bi-directionally using the Sanger sequencing approach at a commercial facility (Macrogen, Inc., Seoul, South Korea). The obtained high-quality sequences were trimmed and assembled using SeqMan v. 5 (DNASTAR, Madison, WI, USA) to remove the poor-reading sequence region. Multiple identical sequences for both genes were considered as a single sequence. Subsequently, the sequences were subject to the Basic Local Alignment Search Tool (BLASTn) in the National Center for Biotechnology Information (NCBI) [2] to determine their similarities to sequences in the GenBank. The obtained sequences and the GenBank-retrieved sequences, along with an outgroup, were aligned in BioEdit V. 7.0.5 (Raleigh, NC, USA) [24], using the ClustalW multiple alignment tool [60]. Phylogenetic analyses were conducted using the Maximum Likelihood (ML) method in MEGA-11 software. The best-fit nucleotide substitution Kimura 2-parameter model [58] was selected based on model testing. The robustness of the tree topology was assessed using 1,000 bootstrap replicates.

Statistical analyses

The dataset of recorded observations was assembled and structured in Microsoft Excel 2023 spreadsheets, followed by descriptive statistics and graphical analyses.

Results

A total of 450 fleas were collected from the 155 examined animals. From this number, 380 were identified as C. felis felis (Table 1; Fig. 2a), and the other 70 specimens remained unidentified due to morphological damage and incomplete diagnostic features, which prevented accurate taxonomic identification. The morphological identification of C. felis felis was based on the following characteristics: head length was generally approximately twice its height and pointed or angled frons (Fig. 2d); both the pronotal and genal comb were present on the head with both having eight or nine horizontally aligned spines (Fig. 2d); the first spine of the genal comb was slightly smaller in length than the second spine (Fig. 2d) and two bristles were found in the lateral metanomal area (Fig. 2e); tibia of all 6 legs had 5 notches with one small stout bristle in the interval between apical and postmedian long bristles on dorsal margin of the hind tibia (Fig. 2f-g); in female, the abdomen was aligned with the head having smooth aedeagal location (Fig. 2c); while it was triangular in male (Fig. 2b).

Fig. 2.

Fig. 2

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Morphological features of the identified C. felis felis: (a), lateral view showing the general anatomical features, including the eyes (i), ocular bristle (ii), pronotal comb (iii), genal comb (iv), mouthparts (maxillary and labial palps (v, vi), mesopleuron (vii) legs (coxa) (viii), trochanter (ix), femur (x), tibia (xi) and tarsus (xii) and abdominal tergites (xiii) and sternites (xiv), and the pygidium (xv). Male (b), Female (c), and main morphological features: Presence of both pronotal and genal comb (d), first genal spine slightly shorter than the second (d) Two bristles in the lateral metanotal area (e), tibia having 5 notches (f), One small stout bristle on hind tibia (g)

Prevalence and distribution of fleas

Of the 155 infested animals, 73/155 (46.45%) male and 82/155 (53.55%) female hosts were infested with fleas. Infestation rates were 60% (n = 18) in dogs and 50% (n = 6) in cats in Nowshera, 44% (n = 11) in dogs and 89.6% (n = 43) in cats in Mardan, 60.7% (n = 17) and 80.6% (n = 25) in dogs and cats in Swabi and 67.6% (n = 23) in dogs and 80% (n = 12) in cats in the Charsadda district (Table 1).

Overall, the infestation rates of C. felis felis were relatively similar for both male and female hosts. However, male dogs exhibited the highest infestation rate in Charsadda and the lowest in Mardan. Similarly, female cats had the highest infestation as compared to female dogs and male cats. Age-wise infestation showed that older hosts had the highest overall infestation rate compared to younger hosts (Fig. 3a-b).

Fig. 3.

Fig. 3

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Factors associated with C. felis felis infestation in dogs and cats in the study areas. (a) shows infestation rates of host (%) by age group, (b) infestation rates of host (%) by sex, (c) frequency rate of flea infestation (%) by sex, (d) seasonal variation in flea infestation (%), and (e) frequency of flea infestation (%) by body area

Infestation rates were higher during the summer than in winter. Additionally, the number of infested sites on the hosts were higher in the head and around the genital area (40.52%) than at other sites such as the neck and ventral sides (30.52%) and the legs and tail region (28.94%). Cats were preferred hosts for flea infestation (51.57%) than dogs (48.42%). In the four localities, female C. felis felis were found more dominant than males, collected from two hosts (Fig. 3c-e).

Analyses of flea’s cox1 and Wolbachia endosymbiont’s gltA sequences

BLAST results of the obtained cox1 sequence showed 100% identity with C. felis felis from Thailand, India, Laos, Australia, and China. Phylogenetic analyses based on 30 sequences retrieved from the NCBI database and sequences obtained in the current study, clustered within a single well-supported clade. This clade was clearly separated from other Ctenocephalides species, including C. orientis (KP229381.1 and KY800499.1) and C. canis (KY865412.1 and MN173766.1) (Fig. 4).

Fig. 4.

Fig. 4

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Maximum likelihood (ML) phylogenetic tree based on the cox1 sequence of C. felis felis. Bootstrap values were calculated using 1,000 replicates. The sequence generated in this study was highlighted by a black circle, bold, and underlined. The cox1 sequence of Leptopsylla segnis was used as an outgroup

Wolbachia endosymbionts were detected in the fleas collected from all four selected locations with an overall prevalence of 55% (22/40) (Table 1). BLASTn result showed that the gltA nucleotide sequences obtained in this study had 100% identity with the Wolbachia endosymbiont of the C. felis isolate wCfeJ from the UK reported by [19]. The phylogenetic analyses indicated that the obtained Wolbachia sequence clustered within Supergroup B, supported by high bootstrap values, and showed close similarity to the previously reported sequences (Fig. 5).

Fig. 5.

Fig. 5

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Maximum likelihood phylogenetic tree based on the gltA sequence of Wolbachia endosymbiont. Bootstrap values were calculated using 1,000 replicates. The sequence generated in this study was highlighted by a black circle, bold, and underlined. The gltA sequence of Anaplasma marginale was used as an outgroup

Discussion

To the best of our knowledge, studies using molecular methods to characterize C. felis felis and its associated Wolbachia species in fleas infesting stray dogs and cats in Pakistan, along with their veterinary and public health importance, have not received adequate attention. Therefore, this study was designed to address this critical knowledge gap. The prevalence of C. felis felis observed in this study provides insight into its distribution patterns. This finding is consistent with earlier studies from Pakistan, where C. felis felis has been reported as the most common flea species infesting dogs and cats, likely due to suitable environmental conditions [3, 42]. Similar prevalence patterns have also been reported in other tropical and subtropical regions, such as India and Iran, where environmental conditions like high temperature and humidity favor flea survival and reproduction [5, 26, 57]. In contrast, lower prevalence in parts of Europe has been associated with improved animal management and routine use of ectoparasiticides [4, 6, 12, 21]. These differences highlight the influence of environmental and management factors on flea infestation patterns.

Ctenocephalides felis felis has a broad host range, which contributes to its persistence in diverse habitats. In this study, higher infestation rates were observed during the warm season, consistent with previous reports indicating that flea populations increase under favorable environmental conditions such as higher temperature and humidity [15]. The infestation rate in the study area was higher in cats than in dogs, which aligns with earlier findings by [1]. This high infestation rate in cats could be due to the role of felines as the primary host for this species. Additionally, the overall prevalence of infestation was higher in younger dogs and cats than in older animals, a pattern reported in previous studies [16, 65]. Our results are also comparable with those of [13] who stated that highest infestation of ectoparasites occur in young animals due to their poor grooming, compared to older animals.

Molecular characterization based on the cox1 gene confirmed the identity of C. felis felis with sequences clustering in the phylogenetic tree with those reported from multiple geographic regions, including Thailand, India, Laos, Australia, and China. This clustering further supports the genetic conservation of C. felis felis across different regions. Furthermore, phylogenetic analyses showed that C. felis felis forms a sister clade with the subspecies C. felis strongylus and C. felis damarensis, indicating limited evolutionary divergence among C. felis subspecies. In contrast, C. orientis formed a distinct clade, supporting previous findings that it is genetically separate from C. felis and more closely related to C. canis [20, 31].

In this study, Wolbachia endosymbionts were detected in 55% of C. felis felis specimens collected from dogs and cats, indicating a moderately prevalent association of Wolbachia with the C. felis felis species. However, this prevalence is lower than that reported by Tay [59], but higher than that reported by [22] in C. felis and other flea species. Such variations may occur due to the differences in host populations and preferences, geographic location, and detection methods. Although Wolbachia was broadly characterized in arthropods by [29] its presence in fleas was not confirmed at that time. The first conclusive molecular detection of Wolbachia pipientis in fleas, including C. felis felis, was reported by [50]. In the present study, the obtained sequences showed high identity and phylogenetically clustered with flea-derived Wolbachia strains, rather than Wolbachia from other arthropod hosts. These results are consistent with patterns reported in previous studies, where Wolbachia lineages are often structured according to their arthropod hosts [64].

It is important to note that the gltA primers used in this study were originally designed for the detection of Bartonella spp.; however, they successfully amplified Wolbachia sequences, a finding consistent with previous reports of cross-amplification due to conserved regions within the gltA gene among Alphaproteobacteria [8]. While this approach enabled detection, it represents a limitation, as it does not allow precise characterization of Wolbachia at the species or strain level. Thus, the results obtained should be interpreted with caution. Future studies should employ Wolbachia-specific markers such as wsp, ftsZ, and 16S rRNA genes to improve specificity and provide deeper insight into genetic diversity.

These findings have important implications for veterinary and public health, as C. felis felis is a known vector of zoonotic pathogens such as Rickettsia and Bartonella species. Also, the presence of Wolbachia in C. felis felis broadens our understanding of its distribution across different host taxa and highlights its potential relevance for veterinary and public health because this endosymbiont is known in arthropods to influence host biology, including reproduction and interaction with pathogens. Understanding the presence of Wolbachia in fleas could inform the development of alternative vector control strategies that could complement conventional measures, such as regular surveillance, treatment of companion animals, and environmental management.

Conclusion

This study provides the first molecular evidence of C. felis felis from stray dogs and cats, and its associated Wolbachia endosymbiont in Pakistan. Although no zoonotic pathogens were directly investigated in this study, the presence of C. felis felis highlights its potential role as a vector. These findings emphasize the need for further studies on the diversity of fleas infesting domestic and stray animals, as well as the prevalence and role of associated Wolbachia endosymbionts and other pathogens across the country.

Acknowledgements

The authors acknowledge the financial support provided by the Higher Education Commission (HEC), Pakistan, and the Pakistan Science Foundation (PSF). The authors thank the Ongoing Research Funding Program (ORF-2026-1440), King Saud University, Riyadh, Saudi Arabia.

Abbreviations

BLAST

Basic local alignment search tool

cox1

Cytochrome c oxidase Subunit I

gltA

Citrate synthase

KP

Khyber Pakhtunkhwa

NCBI

National Center for Biotechnology Information

ML

Maximum likelihood

PCR

Polymerase chain reaction

Authors’ contributions

A.Ali designed the study. A.Ali, M.M.A, A.T.A acquired the budget. N.N., M.N., Z.S., A.F.A., S.R., M.M.A., Z.S., A.F.A., S.B., A.T.A., A.H.K. and A.Ali collected the samples, performed the experimental work, carried out the sequences analyses and designed the location map. All the authors carried out a critical review and authorized the final manuscript.

Funding

The authors thank the Ongoing Research Funding Program (ORF-2026–1440), King Saud University, Riyadh, Saudi Arabia.

Data availability

The datasets generated and/or analyzed during the current study are available in the GenBank repository (NCBI) under accession numbers PX901761 (https://www.ncbi.nlm.nih.gov/nuccore/PX901761) and PX906551 (https://www.ncbi.nlm.nih.gov/nuccore/PX906551.1).

Declarations

Ethics approval and consent to participate

The current study was approved by the Advanced Study and Research Board (Dir/A&R/AWKUM/2025/1709) of Abdul Wali Khan University, Mardan, Pakistan. As the animals included in this study were free roaming (stray), no owner consent was applicable. Flea collection was conducted in public areas with minimal handling of animals. All procedures were carried out in accordance with institutional guidelines and were performed carefully to avoid harm, stress, or discomfort to the animals.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Abid Ali, Email: uop_ali@yahoo.com.

Alanoud T. Aljasham, Email: Aaljasham@ksu.edu.sa

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated and/or analyzed during the current study are available in the GenBank repository (NCBI) under accession numbers PX901761 (https://www.ncbi.nlm.nih.gov/nuccore/PX901761) and PX906551 (https://www.ncbi.nlm.nih.gov/nuccore/PX906551.1).

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