1 Introduction:

Many ectoparasites infect poultry, causing inconvenience, anxiety, and the transmission of pathogens. Fleas are among the ectoparasites that infect chickens. Echidnophaga gallinacea (Siphonaptera: Pulicidae) (Westwood) is known as the sticktight flea. It is common among the flocks of poultry, particularly around the eyes, combs, wattles, and on bare areas. It is an occasional pest of cats, dogs and has also been recorded in horses and humans [1]. In young birds, large numbers of these fleas can cause anemia, exsanguination, and subsequent death, which leads to economic loss for farmers [2]. In Mexico, these species of flea have been recorded in raptors [3]. Restlessness, irritability, weakness, anemia, loss of appetite, eyelid ulceration, corneal opacities, and blindness are the symptoms of flea infestation in birds [4]. Ehlers et al. [5] and Diakou et al. [6] examined flea samples using PCR, and discovered a rickettsia similar to Rickettsia felis. Furthermore, Kumsa et al. [7] found that the chicken flea also transmits Bartonella henselae. At night, adult female sticktights that are attached to the host lay eggs, which fall to the ground under the bird. After four days, the eggs hatch, and white larvae emerge in the soil. The larvae feed on the organic matter present on the floor of the barn, molt several times, turn into pupae inside a cocoon, and develop into adult insects. This process generally takes a week to a month time depending on the temperature, and has a one-month latency period in the absence of the host. When an adult flea emerges, it looks for a host to feed on it in order to complete its life cycle [8].

Numerous studies have investigated the prevalence of fleas in birds and mammals. In Iran, 52.8% of chickens were reported to be infected with ectoparasites, and 8% were infected with fleas [9]. In India, 25.54% infection rate of chicken fleas has been reported [4]. In addition, one species of flea (E. gallinacea) has been recorded infecting chickens in California, with an infection rate of 20% [10]. Genetic identification of E. gallinacea has been performed in many countries, including Egypt, Australia, and Cameroon [11,12,13]. However, we were unable to trace any research on this parasite in Saudi Arabia. In the year 2019, we studied the prevalence of external parasite infections in the chickens of Al-Baha region of the Kingdom of Saudi Arabia. Among these parasites, the fleas were morphologically classified as galenic, infecting 59.38% of all the chickens examined [14]. Because of the high rates of infection and damage caused by this parasite in chickens and other farm animals, it is necessary to determine the rates of infection among chickens. We also studied its morphological and genetic characteristics and its genetic relationship with other insects. This information is crucial in identifying this most widespread species and to select the best methods to control and limit their spread.

2 Materials and methods

2.1 Samples

A total of 102 adult chickens (20 males and 82 females) from three traditional farms in the Al-Baha region were examined during the August 2022. The samples were collected manually using tweezers, placed directly in ethyl alcohol (70%), and stored in a refrigerator (− 20 °C).

Study area

The study was conducted in the Al-Baha region, which is located in the southwest of the Kingdom of Saudi Arabia (20° 1′ 0″ North, 41° 28′ 0″ East).

2.2 Morphological structures

The flea samples were examined using a stereomicroscope and identified morphologically based on the previous research [11, 15, 16].

2.3 Molecular structures

2.3.1 Step (1): DNA extraction

Each E. gallinacea sample was processed to obtain its total genomic DNA (gDNA) using a mini-prep DNA isolation kit (GeneAll® ExgeneTM Clinic SV DNA Isolation Kit; GeneAll Research Institute, Seoul, Korea) with suitable modifications. The samples were homogenized, and then, each sample was digested for 16 h at 56 °C on a heat block in 200 µL of lysis buffer (CL) and 20 mL of proteinase K. Subsequent procedures were carried out following the manufacturer's instructions. Finally, the entire DNA sample was eluted using 30 mL of elution solution (Tris buffer, pH 8.5, warmed to 70 °C).

2.3.2 Step (2): cytochrome oxidase subunit 1 gene amplification

The genomic DNA from each E. gallinacea specimen was sequenced and polymerase chain reaction (PCR) was performed. Conventional PCR was used to amplify a mitochondrial cytochrome oxidase subunit 1 (COI) fragment with a specific amplicon size (710 bp), which confirmed that the specimens belonged to the E. gallinacea species [17]. LCO1490 (5′-GTCAACAAATCATAAAGATATTGG-3′) and HC02198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) were utilized for the PCR reaction. The reaction mixture for PCR amplification (total reaction volume, 50 mL) consisted of 20 mL of BioMix Red and 2x (Bioline, BIO-25006), 5 mL of DNA template, and 2 mL of each primer (0.4 M final concentration). Nuclease-free water was used as a negative control. The PCR process included 40 cycles, starting with a denaturation step.

2.3.3 Step (3): analysis of phylogenetic tree

Reference sequences were selected by performing a search with the Basic Local Alignment Search Tool (NCBI, Bethesda, MD, USA) to find the closest nucleotide sequence matches [18] available in the NCBI GenBank database. All sequences were aligned using CLUSTAL X (Conway Institute UCD, Dublin, Ireland) [19] and modified using BioEdit (Informer Technologies Inc., Los Angeles, CA, USA) [20] based on additional reference sequences [21]. Phylogenetic relationships were inferred using the neighbor-joining method in MEGA X (Pennsylvania State University, University Park, PA, USA) [22]. Kimura's two-parameter substitution model was applied, and 1000 replicates were used to estimate neighbor-joining bootstrap values. Bootstrap values of > 70% were considered [23].

3 Results

3.1 Infection rates

The current study included 102 chickens, with an infection rate of 49.01%. The infection rates varied among the different farms, and whereas the third farm had the highest infection rate (40.19%) (Table 1).

Table 1 Percentages of Sticktight flea (E. gallinacea) infection on traditional farms in the Al-Baha region
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3.2 Morphological structures

The sticktight flea acquired its name from the habit of implanting its head, using the broad and serrated laciniae, into the skin of the host. It can continue feeding in this position for more than 19 days (Fig. 1). The fleas were small, less than half the size of cat fleas, and had a dark brown color. Males were smaller than females (Fig. 2a, b). One of the most distinguishing characteristics of the head of this species is that it is flat anteriorly, with a sharp angle inclining caudally from the front, and it has two pairs of hairs behind the antennae. Maxillary laciniae are wide and serrated (Fig. 3a). The thorax narrows dorsally from behind the head. This flea differs from fleas that infect cats and dogs by having no genal or pronotal ctenidia; however, there is a group of spines on the third coxa of the hind legs (Fig. 3b).

Fig. 1
figure 1

Appearance of chicken infection with Sticktight fleas, Echidnophaga gallinacea

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Fig. 2
figure 2

Morphology of fleas, Echidnophaga gallinacean; A female; B male

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Fig. 3
figure 3

A Head of the flea (frons area with strongly angle, mouth parts); B legs of flea (group of spines on 3rd coxa)

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3.3 Molecular structures

3.3.1 Specific DNA sequences blast, alignment, and matching

To determine the genus and species of the flea, the homology of all sequences was evaluated and matched with information from GenBank using the BLAST website. The results in Table 2 indicate that the selected samples with accession number (OR161051, Echidnophaga sp.) had a high similarity rate (95.17–99.82%) with the species of flea, E. gallinacea which was recorded in the GenBank with accession numbers (MW492259, OQ291365, KT376440, and MZ381630). While the similarity ratios were lower (89.27–94.33%) with samples from the same genus (Echidnophaga), but the species are different.

Table 2 Blast results of the cytochrome oxidase subunit 1 gene for the current study of E. gallinacea according to taxonomic data from the Gene Bank
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3.3.2 Analysis of phylogenetic tree for E. gallinacea samples

Based on cox1 phylogenetic analysis, our sample was most closely related to members of the E. gallinacea species (MW492259 and OQ291365) from Thailand, followed by KT376440 and MZ381630 from Australia, with 100% bootstrap support. These data were included for analysis (Fig. 4).

Fig. 4
figure 4

Phylogenetic tree of the flea, Echidnophaga gallinacea from Saudi Arabia and its relatives from other countries, with isolated samples that are registered in the GenBank, using the Mega11 program

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4 Discussion

There is a inconsistency between research conducted to evaluate the prevalence of E. gallinacea infection in chickens. Some of these were high, such as the 49% recorded in thecurrent study, which is similar to the infection rate of 44.36% reported in an Ethiopian study [24]. Some were average, such as those conducted in India, where Udhayavel et al. [1] recorded flea infection rate of 25.54%, while reports from other contries were low, such as those conducted in Egypt, where the infection rate did not exceed 5%. Gharsan and Elhassan [14] descrbed the differences in the infection rate due to poor hygiene, a lack of periodic and early detection of the infection, and treatment of infected chickens. However, Hiluf et al. [25] recognized the difference in infection rates due to seasonal fluctuations. López-Pérez et al., [26] emphasized the widespread presence of E. gallinacea, which is commonly found in association with poultry. In addition, the study by Dobler and Pfeffer [27] demonstrated the potential role of E. gallinacea in disease transmission, such as plague. Hence, it is crucial to have a comprehensive understanding of the prevalence and potential impact of E. gallinacea on various host species, considering its presence in free-living avian species and its pathology in domestic chickens [28]. In addition, the presence of E. gallinacea in backyard domestic chickens in Tunisia and its prevalence in free-range backyard chickens in Iran highlight its extensive distribution and significance as an ectoparasite [29, 30]. The detection of Rickettsia felis and Bartonella henselae in dog and cat fleas in Ethiopia emphasizes the importance of using molecular techniques to identify the pathogens associated with these fleas [7].

This study also addresses the morphological description of the flea (E. gallinacea), with some features explained and illustrated. It was found that this flea differs from the fleas that infect cats and dogs. Important taxonomic characteristics include a distinctive head at a strong angle from the anterior side and a pair of hairs behind the antennae. This species was also characterized by the presence of a group of spines on the coxa of the hind legs, which is consistent with the previous studies [4, 11, 31]. The results showed that this sample was comparable to E. gallinacea samples examined in Thailand and Australia, with a similarity rate of 95.17–99.82%, therefore, confirming that our sample is E. gallinacea type. Moreover, the COI genetic primers utilized were successful in defining the E. gallinacea flea. A similar study was conducted by AbouLaila and Menshawy [11] in Egypt to identify the species that infected chickens. E. gallinacea with accession number (LC500239) is widespread and has a similarity more than 98% with flea specimens in Cameroon (EU169199). In Australia, Huang et al. [32] used genetic analysis to identify fleas that infect cats and dogs. Of all the samples, only one included E. gallinacea, perhaps because this flea prefers avian hosts.

To our knowledge, this study is the first of its kind to address the genetic structure of the chicken fleas, but still there is need to expand the study to include other regions of the Kingdom of Saudi Arabia also. Additionally, expanding molecular studies of other genes of this insect in the future will help in determining the biological patterns of this insect, which in turn will contribute to develop better strategies to control these insects.

5 Conclusion

Accurate genetic identification and DNA barcoding of E. gallinacea are essential for gaining insights into its population genetics, host specificity, and potential involvement in pathogen transmission. Through the use of molecular techniques, researchers can explore the genetic diversity of E. gallinacea populations and their interactions with different host species. This valuable information can be applied to develop more effective control and management strategies. The results of this study reveal that E. gallinacea is prevalent in the Al-Baha region. However, chicken breeders lack information about the appearance and potential threat of this infestation to chickens and other domestic animals. This presents an encouraging area for researchers to explore and identify pathogens transmitted by parasites in Saudi Arabia, and particularly the Al-Baha region.