# Checklist and diversity of tick fauna associated with domestic animals in Cambodia

Sony Yean, Didot Budi Prasetyo, Theary Ren, Davy Krib, Saoya Sen, Bunthon Chea, Sothyra Tum, Sorn San, Samuth Sum, Sarah Bonnet, et al.

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► To cite this version:

Sony Yean, Didot Budi Prasetyo, Theary Ren, Davy Krib, Saoya Sen, et al.. Checklist and diversity of tick fauna associated with domestic animals in Cambodia. Ticks and Tick-borne Diseases, 2025, 16 (6), pp.102570. 10.1016/j.ttbdis.2025.102570. hal-05424748

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Original article

# Checklist and diversity of tick fauna associated with domestic animals in Cambodia

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Sony Yean<sup>a,e,*</sup>, Didot Budi Prasetyo<sup>a,b</sup>, Theary Ren<sup>b</sup>, Davy Krib<sup>a</sup>, Saoya Sen<sup>a</sup>, Bunthon Chea<sup>c</sup>, Sothyra Tum<sup>b</sup>, Sorn San<sup>b</sup>, Samuth Sum<sup>c</sup>, Sarah Bonnet<sup>d,e</sup>, Sébastien Boyer<sup>a,d</sup>

## ARTICLE INFO

**Keywords:**

Tick fauna
Argasidae
Ixodidae
cox1 gene
Cambodia

## ABSTRACT

The tick fauna in Cambodia is poorly documented, with limited information on the diversity and distribution of tick species. This study aimed to address this knowledge gap by investigating tick species collected from various hosts, and across different habitats and regions of the country. Tick sampling was conducted in urban areas, rural villages, farms, and forest fringes, with additional samples obtained from a wildlife rescue center, caves, and swiftlet nests. Ticks were collected directly from hosts, as well as on vegetation through flagging methods. Tick species were identified morphologically, and selected specimens were subjected to molecular identification using the *cox1* gene. A total of 853 vertebrate hosts representing 13 different animals, including five wildlife, were inspected: buffalo, cats, cattle, chickens, dogs, ducks, goats, goose, pigs, pigeons, pangolins, pythons, and wild pigs. From this first nationwide tick collection, a total of 7,461 ticks were collected, comprising six genera and 14 species of ticks, including seven newly identified species such as *Carios batuensis*, *Dermacentor steini*, *Dermacentor filippovae*, *Haemaphysalis canestrinii*, *Haemaphysalis formosensis*, *Haemaphysalis hystricis*, and *Haemaphysalis wellingtoni*. *Rhipicephalus microplus* and *Rhipicephalus linnaei* were the most predominant tick species collected from cattle and dogs, respectively, whereas no ticks were found on buffaloes, chickens, ducks, goose, pigs or pigeons. Results showed that ticks are widely distributed throughout Cambodia, with particularly rich diversity observed in forest fringes habitats. This study also allowed the number of tick species identified in the country with seven additional species, bringing the total to 26.

## 1. Introduction

Ticks are the primary vectors of various pathogens causing diseases in both animals and humans in the northern hemisphere. In the southern hemisphere, they are the second most important vector after mosquitoes for humans and first for animal health (de la Fuente et al., 2023). Among the most important tick species, some ticks of the *Rhipicephalus* genus have a significant impact due to the transmission of *Babesia* and *Theileria* parasites to both cows and buffaloes (Altay et al., 2008; Alvarez et al., 2019; Cao et al., 2012; El-Alfy et al., 2023). Tick-borne diseases, including those caused by these parasites, result in an estimated economic loss of 22-30 billion USD annually (Singh et al., 2022). Regarding human health, several *Ixodes* spp. serve as vectors for various zoonotic

diseases, including Lyme borreliosis and tick-borne encephalitis in Europe, North America, and Asia (Jenkins et al., 2022). Lyme disease alone is an emerging tick-borne disease that affects over 476,000 individuals annually in the United States, and around 200,000 cases are reported each year in Europe (Kapoor et al., 2024; Marques et al., 2021).

Recently, interest in ticks and tick-borne diseases has grown in Southeast Asia due to their impact on public health. Approximately 980 tick species have been described worldwide, as descriptions of new species have appeared after the two cited reviews, with 760 belonging to the family Ixodidae (Guglielmone et al., 2023) and 220 to Argasidae (Mans et al., 2019). In Southeast Asia, Petney et al. described 97 species from nine genera (Petney et al., 2019). Efforts are underway to expand knowledge about the diversity of tick species and their geographical

<footnote><sup>a</sup> Medical and Veterinary Entomology Unit, Institut Pasteur du Cambodge, No. 5 Monivong Blvd., P.O Box 983, Phnom Penh, 12201, Cambodia</footnote>

<footnote><sup>b</sup> National Animal Health and Production Research Institute, General Directorate of Animal Health and Production, Phnom Penh, Cambodia</footnote>

<footnote><sup>c</sup> Faculty of Veterinary Medicine, Royal University of Agriculture, Dongkor District, Phnom Penh, Cambodia</footnote>

<footnote><sup>d</sup> Ecology and Emergence of Arthropod-borne pathogens Unit, Institut Pasteur, Université Paris Cité, CNRS UMR 2000, INRAE USC 1510, Paris, France</footnote>

<footnote><sup>e</sup> Department of Structure and Dynamics of Living Systems (SDSV), Université Paris-saclay, 91190 Gif-sur-Yvette Paris, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: ysony@pasteur-kh.org (S. Yean).</footnote>
distribution in countries such as Laos (Apanaskevich et al., 2019; Vongphayloth et al., 2022, 2016), Malaysia (Apanaskevich, M.A., and Apanaskevich, D.A., 2015), Singapore (Kwak et al., 2021), Thailand (Apanaskevich, D.A., and Apanaskevich, M.A., 2015), and Vietnam (Apanaskevich, D.A., and Apanaskevich, M.A., 2015; Apanaskevich, M. A., and Apanaskevich, D.A., 2015).

Tick burden and its potential threat refer to the number and intensity of ticks infesting a host, such as animals or humans. This issue is a growing concern in Southeast Asia due to the region's warm, humid climate, abundant vegetation, and frequent interactions among humans, livestock, and wildlife. Ticks from the genera *Amblyomma*, *Dermacentor*, *Rhipicephalus*, and *Haemaphysalis* are known vectors of various pathogens, including *Anaplasma*, *Babesia*, *Coxiella*, *Ehrlichia*, *Hepatozoon*, *Rickettsia*, and *Theileria* (Aziz et al., 2022; Koh et al., 2018; Tan et al., 2021). Species of these genera have been detected in Malaysia, Laos, Thailand, and Vietnam (Huynh et al., 2021; Kaewmongkol et al., 2017; Koh et al., 2018; Laummaunwai et al., 2014; Petney et al., 2019; Sumrandee et al., 2015), where they cause significant health and economic impacts. Wildlife serves as reservoirs for these pathogens, further complicating control efforts. The emergence of severe fever with thrombocytopenia syndrome (SFTS) in humans in Vietnam, Myanmar, and Thailand (Rattanakomol et al., 2022; Tran et al., 2019; Win et al., 2020) highlights the growing public health risk. Climate change worsens the situation, as rising temperatures and changing rainfall patterns are expanding tick habitats, prolonging tick activity seasons, and speeding up their life cycles (Choi and Lee, 2025). These combined factors make the tick burden a major veterinary and public health issue in Southeast Asia. Tackling this threat requires integrated tick management, improved surveillance, public education, and stronger veterinary services. A coordinated One Health approach is essential to reduce the

impact of tick-borne diseases in the region.

In Cambodia, tick fauna remains poorly documented, with limited information on their diversity and geographical distribution (Yean et al., 2024). Notably, only 19 tick species belonging to six genera have been identified (Petney et al., 2019; Yean et al., 2024). This study aimed to collect ticks from domestic animals and livestock, as well as vegetation by using the flagging method to better understand the diversity and distribution of tick species in Cambodia. All specimens were morphologically identified to species level, and a subset of individuals from each species was selected for DNA barcoding using a mitochondrial marker. This effort provides updated records of tick species in Cambodia and contributed valuable DNA sequence data in the public database, enhancing the knowledge of tick diversity in the country.

# 2. Materials and methods

## 2.1. Ethical approval

The study was approved and supported by the General Directorate of Animal Health and Production from the Ministry of Agriculture, Forestry and Fisheries of Cambodia, under permit No. 2981, signed on October 1st, 2021.

## 2.2. Tick sampling sites and collection

Between November 2022 and April 2023, tick sampling was conducted once in each of the 24 provinces of Cambodia. Phnom Penh was excluded from the study due to the absence of forested areas and animal farms. Each collection included sampling from four different habitats: urban area (excluding Phnom Penh), rural areas with four houses

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Maximun-likelihood phylogenetic tree of tick species collected in Cambodia based on a fragment of the cox1 gene. The tree was constructed using IQTree v2.3.5 with TIM+F+I+G4 nucleotide substitution model. Sequences obtained from GenBank are indicated in blue text, while sequence generated from this study are presented in black. Asterisks (*) indicate new distribution records for the country.</figcaption></figure>
sampled per village, livestock farms including eight cattle farms, eight chicken farms, and eight pig farms, and forest fringes (Fig. 1). In urban areas, ticks were collected by veterinarians in each province. Collection sites in villages and livestock farms were primarily selected based on their accessibility. Provinces with the highest number of each farm type (cattle, pig, and poultry) were first identified, after which a representative farm in each province was selected for sampling upon obtaining the owner's permission. Nearby villages were then selected for logistical convenience, within five km distance from the farm. Forest fringes were sampled based on accessibility to suitable areas.

Additional sampling was conducted in more specific habitats, including two caves (one in Battambang and one in Stung Treng province), 17 swiftlet farms (10 swiftlet nests examined per farm) in Banteay Meanchey, Battambang, Kampong Cham, Kampong Chhnang, Kampong Thom, Kep, Koh Kong, Kratie, Oddar Meanchey, Pailin, Preah Vihear, Prey Veng, Pursat, Siem Reap, Svay Rieng, Takeo, and Tboung Khmum. Occasional donations from wildlife rescue centers and forest goers also contributed to the sampling, which included tick collection from two pythons and one pangolin in Takeo, as well as two wild pigs from Koh Kong (Fig. 1).

Ticks were collected from animals selected based on the availability of hosts in urban areas, where veterinarians examined domestic animals such as cats, cattle, and dogs. In rural villages, animals such as buffalo, cats, cattle, chickens, dogs, ducks, goats, goose, pigs, and pigeons were selected according to the presence of hosts in the household, with a maximum of four animals sampled per host type. For livestock farm, ten animals per farm were randomly selected with the permission of the farm owner. All tick sampling from the host, involving a full-body inspection of each animal was conducted for up to 30 minutes per animal. Ticks were collected directly from hosts using fine-tip forceps or tick twisters, ensuring minimal discomfort to the animals during the procedure (Yean et al., 2025).

Flagging was performed along forest fringe paths to collect ticks from vegetation, using a 40×60 cm white cloth mounted on a 1.5 m pole (Espada et al., 2021). The cloth was swept over taller vegetation, with frequent inspections to prevent ticks from falling off. Six collectors performed the flagging for up to 60 minutes in the morning, between 9:00 am to 11:00 am, depending on arrival time and accessibility to forest fringe.

All tick samples were transferred to 15 mL tube containing 70% ethanol and labeled according to location, host, habitat, and date of collection. All specimen collection was performed by trained entomology technicians and local veterinarians and were subsequently sent to the Institut Pasteur du Cambodge (IPC) laboratory for further processing.

In the laboratory, ticks were sorted by sex and life stage and morphologically identified using species descriptions and taxonomic keys from other publication (Apanaskevich and Apanaskevich, 2015; Berry, 2017; Cooley, 1946; Dao et al., 2024; Estrada-Peña et al., 2012; Geevarghese and Mishra, 2011; Hirst, 1929; Kaufman, 1972; Kazim et al., 2022; Tanskul and Inlao, 1989; Voltzit and Keirans, 2002; Walker, 2003; Walker et al., 2000; Wassef and Hoogstraal, 1986, 1984) as well as pictorial keys developed in-house by the Medical and Veterinary Entomology Unit at IPC. Representative specimens from each species, along with unidentified nymphs, were further subjected to molecular identification using the cytochrome c oxidase I (cox1) gene as a molecular biomarker.

## 2.3. DNA extraction

DNA extraction from ticks was performed using the QIAmp DNA Mini Kit (Qiagen, Germany), with slight adjustments to accommodate the non-destructive protocol (Takhampunya et al., 2021). A small incision was made in the tick's abdomen with a surgical blade or fine pin prior to the addition of 180 µL buffer ATL and 20 µL proteinase K. The mixture was incubated overnight at 56°C to facilitate complete

enzymatic digestion of the tissues. Then, 200 µL of the supernatant was transferred to a new 1.5 mL tube and subjected to the next DNA extraction steps according to the manufacturer's instructions. The remaining exoskeletons of the ticks were washed several times with distilled water, preserved in 70% ethanol for morphological examination, and archived for collection.

A 709 bp region of the mitochondrial *cox1* gene was amplified by PCR using established primer pair LCO1490 (5'-GGTCAACAAATCA-TAAAGATATTGG-3') and HCO2198 (5'-TAAACTTCAGGGTGAC-CAAAAAATCA-3') (Folmer et al., 1994). Each 45 µL of PCR mix contained 4 µL of 10x PCR buffer B2 (Solis Biodyne, Estonia), 0.8 µL of each primer (10 µM), 0.4 µL of 20 mM dNTP mix, 5.6 µL of 25 mM MgCl2, 0.4 µL of Hot FirePol DNA polymerase (5 U/µL) (Solis Biodyne, Estonia), 23 µL of nuclease-free water, and 5 µL of DNA template. PCR assays were conducted using a T100 thermal cycler (Bio-Rad, USA) with the following conditions: initial denaturation at 95°C for 15 min; followed by 40 cycles of 95°C for 1 min, 50°C for 1 min, and 72°C for 1 min; and a final extension at 72°C for 10 min.

## 2.4. Sequencing and phylogenetic analysis

PCR products were separated using 1.5% agarose gel electrophoresis and visualized using UV chamber. The amplicons were subsequently sent to Macrogen (Daejeon, South Korea) for purification and DNA sequencing. Sequence reads were analyzed using Unipro UGENE version 50.0, with consensus sequences aligned against reference sequences from the GenBank database using ClustalW for multiple alignment. A maximum likelihood phylogenetic tree was constructed using the optimal nucleotide substitution model with 1,000 bootstrap replicates (Liu et al., 2022; Tian et al., 2022) in IQ-TREE version 2.3.5 (Minh et al., 2020), and the tree was visualized (Krishnamoorthy et al., 2021) and edited using MEGA version 11.0.13 (Tamura et al., 2021).

# 3. Results

## 3.1. Morphological identification, host and distribution by habitat

A total of 853 animals (609 domestic, 239 livestock, and five wildlife animals) were inspected, representing 13 different host species across various habitats. These included five buffaloes, 20 cats, 255 cattle, 188 chickens, 268 dogs, 15 ducks, seven goats, one goose, 86 pigs, and three pigeons from domestic and livestock, along with one pangolin, two pythons, and two wild pigs from wildlife. A total of 7,461 tick specimens were collected including 5,173 adults (2,975 females and 2,198 males), 2,220 nymphs and 68 larvae. Among these, 1,357 ticks obtained from urban areas, 2,991 ticks from livestock farm (cattle), 2,898 from rural villages, 66 ticks from forest fringes and 118 ticks from caves. No ticks were collected from pig, chicken, and swiftlet nest. Additionally, five ticks were collected from wild pigs, 17 from pangolins, and nine from pythons (Tables 1,2).

Morphological identification classified all tick specimens into two families: Argasidae and Ixodidae. The Argasidae family included one genus and one species, *Carios batuensis* (22 females, 35 males, and 60 nymphs). The Ixodidae family contained five genera and 13 species, including *Amblyomma javanense* (three females and 14 males), *Amblyomma varanense* (six females and three males), *Dermacentor auratus* (one female), *Dermacentor filippovae* (two females and two males), *Dermacentor steini* (one female), *Haemaphysalis canestrinii* (one male), *Haemaphysalis hystricis* (two females, one male, and one nymph), *Haemaphysalis formosensis* (one nymph), *Haemaphysalis shimoga* (one female and one male), *Haemaphysalis wellingtoni* (one female, one male, and 15 nymphs), along with unidentified *Haemaphysalis* spp. (35 nymphs), *Ixodes kopsteini* (one nymph), *Rhipicephalus linnaei* (610 females, 824 males, and eight nymphs), *Rhipicephalus microplus* (2,274 females, 1,231 males, and 16 nymphs), and *Rhipicephalus* spp. (52 females, 85 males, 2,083 nymphs, and 68 larvae). Among the collected ticks, seven species
<caption>**Table 1**</caption>

Abundance and relative proportion (%) of ticks collected on animals from urban, farm, and rural environments across 24 provinces in Cambodia.

<table><thead><tr><th rowspan="2">Tick species</th><th colspan="3">Urban</th><th colspan="3">Farm</th><th colspan="4">Rural village</th></tr><tr><th>Cat<br>n=4/4</th><th>Cattle<br>n=11/11</th><th>Dog n=134/<br>144</th><th>Cattle<br>n=63/79</th><th>Chicken<br>n=0/80</th><th>Pig n=0/<br>80</th><th>Cattle n=104/<br>165</th><th>Dog n=56/<br>124</th><th>Goat<br>n=2/7</th><th>other animals<br>n=0/154</th></tr></thead><tbody><tr><td>R. microplus</td><td>-</td><td>170 (85)</td><td>10<br>(1)</td><td>1,848 (62)</td><td>-</td><td>-</td><td>1,467<br>(63)</td><td>7<br>(1)</td><td>7 (88)</td><td>-</td></tr><tr><td>R. linnaei</td><td>44 (96)</td><td>-</td><td>1,045<br>(94)</td><td>-</td><td>-</td><td>-</td><td>21<br>(1)</td><td>331<br>(59)</td><td>-</td><td>-</td></tr><tr><td>Rhipicephalus<br>spp.</td><td>2<br>(4)</td><td>30<br>(15)</td><td>56<br>(5)</td><td>1,143 (38)</td><td>-</td><td>-</td><td>843<br>(36)</td><td>220<br>(40)</td><td>1 (12)</td><td>-</td></tr><tr><td>H. canestrinii*</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td><td>-</td><td>-</td></tr><tr><td>Total</td><td>46</td><td>200</td><td>1,111</td><td>2,991</td><td>0</td><td>0</td><td>2,331</td><td>559</td><td>8</td><td>0</td></tr></tbody></table>

* New record tick species in Cambodia

- Other animals: Buffalo (5), Cat (16), Chicken (108), Duck (15), Goose (1), Pig (6) & Pigeon (3)

Note: The formula below each animal represents the number of animals infested with ticks over the total number of animals examined.

<caption>**Table 2**</caption>

Abundance and relative proportion (%) of ticks collected from various non-domestic habitats, including forest fringes, wildlife rescue centers, and cave environments.

<table><thead><tr><th rowspan="2">Tick species</th><th colspan="2">Forest Fringe</th><th rowspan="2">Bat<br>Cave<br>Cave<br>floor<br>n=2/2</th><th colspan="2">Wildlife rescue center</th></tr><tr><th>Vegetation<br>n=10/24</th><th>Wild<br>pig<br>n=2/2</th><th>Pangolin<br>n=1/1</th><th>Python<br>n=2/2</th></tr></thead><tbody><tr><td>A. javanense</td><td>-</td><td>-</td><td>-</td><td>17 (100)</td><td>-</td></tr><tr><td>A. varanense</td><td>-</td><td>-</td><td>-</td><td>-</td><td>9 (100)</td></tr><tr><td>C. batuensis*</td><td>-</td><td>-</td><td>117<br>(99)</td><td>-</td><td>-</td></tr><tr><td>D. auratus</td><td>-</td><td>1 (20)</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D. filippovae*</td><td>-</td><td>4 (80)</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D. steini*</td><td>1 (2)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>H. hystricis*</td><td>4 (6)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>H. formosensis*</td><td>1 (2)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>H. shimoga</td><td>2 (3)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Haemaphysalis<br>spp.</td><td>35 (53)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>H. wellingtoni*</td><td>17 (26)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>I. kopsteini</td><td>-</td><td>-</td><td>1 (1)</td><td>-</td><td>-</td></tr><tr><td>Rhipicephalus<br>spp.</td><td>6 (9)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Grand Total</td><td>66</td><td>5</td><td>118</td><td>17</td><td>9</td></tr></tbody></table>

* New record tick species in Cambodia

represent new records for Cambodia collected from rural villages, caves, wildlife animals, and forest fringe habitats (Tables 1,2).

The geographical distribution and species diversity of tick were examined at six habitat types (Fig. 2). *Rhipicephalus linnaei* and *R. microplus* were the two species commonly collected in the 24 collection sites in urban areas. *Rhipicephalus linnaei* was the dominant species except in Kratie and Pailin provinces where no *R. linnaei* were found. Rural villages exhibited a higher abundance of three tick species compared to urban areas. *Rhipicephalus microplus* was the predominant species in 17 rural locations, with some regions displaying a more even species distribution. However, *R. microplus* were not collected in Kampong Cham, Kandal, Kratie, Prey Veng, Pursat, Svay Rieng and Tboung Khmum province. A single specimen of *H. canestrinii*, representing a new record for Cambodia, was collected from a dog in Kratie Province.

Cattle farms demonstrated the highest tick abundance among all habitat types. The tick community was primarily composed of *R. microplus*, reflecting the strong association of these species with livestock environments. The forest fringe habitats showed notable species diversity with five species collected from 10 provinces, such as Kampong Cham, Kampong Chhnang, Kampong Speu, Kampong Thom, Kampot, Kep, Koh Kong, Prey Veng, Stung Treng, & Tboung Khmum

provinces. Ticks associated with wildlife animals such as pangolins, pythons, and wild pigs were less abundant but displayed considerable diversity of species. Another habitat, caves harbored a unique tick community and predominated by *C. batuensis* in Stung Streng with smaller populations of *I. kopsteini* in Battambang province.

## 3.2. Molecular analysis of Ixodid ticks

A total of 155 tick specimens from various habitats were selected for molecular analysis, including 108 *Rhipicephalus* spp., 27 *Haemaphysalis* spp., eight *Amblyomma* spp., six *Dermacentor* spp., five *Carios* spp., and one *Ixodes* sp. (Supplementary Table 1). Among the 155 specimens, five *Rhipicephalus* spp. specimens were excluded and not submitted to Gen-Bank, as they had different sequence lengths.

Molecular analysis of ten samples from the *R. microplus* complex revealed over 99% sequence identity with the *R. microplus* clade A. Expanding this investigation to include 93 samples from 21 provinces across Cambodia yielded consistent results, with all samples aligning with *R. microplus* clade A (Supplementary Fig. 1). Amplification of the *cox1* gene failed in *R. linnaei* samples (n=4), except for one nymph, which showed 99% identity with *R. linnaei* from Mexico (accession PP919883). The phylogenetic tree showed that *R. linnaei* from this study aligns in a separate clade from *R. sanguineus* from the U.S.

All *Dermacentor* spp. specimens in this study were collected exclusively from Koh Kong province. The *cox1* sequence from a single specimen of *D. steini* (PQ838766) showed 99.24% identity with *D. steini* from Thailand (accession ON680802) but exhibiting less than 93.21% identity with another *D. steini* sequences available in GenBank. The sequence from a *D. auratus* (PQ838771) specimen demonstrated 99.65% identity with a *D. auratus* from Singapore (accession PP837374). Unlike the other *Dermacentor* species, *D. filippovae* (PQ838767-PQ838770) was identified based solely on morphological characteristics, as no reference sequence is currently available in GenBank.

The *cox1* sequence of *A. javanense* (PQ838776-PQ838779) specimens showed 99.48% similarity to *A. javanense* from China (NC_043872). However, the identification of other *Amblyomma* species, *A. varanense* (PQ838772-PQ838775), relied solely on morphology, as there are no reference sequences for the *cox1* gene available in GenBank.

The *Haemaphysalis* species identified in this study were predominantly nymphs, with some adults belonging to the subgenera *Kaiseriana*, *Aborphysalis*, and *Rhipistoma*. *Cox1* sequences from three representative samples of *H. hystricis* (PQ838800, PQ838801, PQ838802) (displayed over 99.8% identity to *H. hystricis* from China (OQ135148). Additionally, the sequence from one nymph (PQ838782) showed 99.83% similarity to *H. formosensis* from Japan (OL741738), marking Cambodia and Vietnam as the only Southeast Asian countries where this species has been recorded. Identification of other *Haemaphysalis* species, such as *H. shimoga* (PQ838780, PQ838781), *H. wellingtoni* (PQ838783-
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Map of tick species collected in six habitats by province, Cambodia. (A) Tick sampling in urban areas; Tick sampling in rural villages; (C) Tick sampling in cattle farms (no ticks were found in 8 chicken and 8 pig farms); (D) Tick sampling in forest fringes; (E) Tick sampling on wildlife animals; (F) Tick sampling in bat cave floor.</figcaption></figure>

★New record of tick species in Cambodia

Abbreviations: *Amblyomma* (A.), *Carios* (C.), *Dermacentor* (D.), *Haemaphysalis* (H.), *Rhipicephalus* (R.), *Ixodes* (I.).

PQ838799), and *H. canestrini* (PQ838807), was based solely on morphology due to the lack of *cox1* gene reference sequences in the GenBank database. A maximum-likelihood phylogenetic tree revealed that a total of 15 *Haemaphysalis* spp. nymphs clustered together with *H. wellingtoni*, suggesting that all these specimens are *H. wellingtoni* nymphs.

The only *Ixodes* species collected in this study was *I. kopsteini* (PQ838806), while the only tick species belonging to the Argasidae family was identified as *C. batuensis* (PQ838808-PQ838812). Both species were identified solely based on morphological characteristics (Fig. 3).

## 4. Discussion

Our study provides a significant contribution to the knowledge of tick fauna in Cambodia and underscore the country's notable biodiversity. With the addition of seven new species, the total number of recorded tick species in Cambodia has increased to 26 (Table 3). Ticks were predominantly collected from domestic animals like dogs and cattle, with a highest tick density observed at a cattle farm in Banteay Meanchey, surpassing other provinces. Ticks from human-associated habitats, including urban areas, rural settlements, and livestock farms, comprised 98% of the total collection. In contrast, only 2% were collected from non-domestic environments such as forest fringes, caves, and wildlife rescue centers. Despite their smaller proportion, ticks from

these habitats exhibit higher species richness, contributing to six out of the seven newly recorded species in this study.

However, despite this, our findings indicate a relatively low diversity of tick species in Cambodia compared to neighboring countries. For instance, Malaysia has 48 tick species belonging to seven genera, Indonesia has 61 species within seven genera, Thailand has reported 62 species belonging to nine genera, and Vietnam has described 65 species belonging to eight genera (Yean et al., 2024). The relatively lower diversity of tick species in Cambodia could be attributed to the country's limited wildlife population compared to its neighbors. For instance, Cambodia has fewer large mammals and other wildlife hosts that are known to support diverse tick species (National Council for Sustainable Development, 2016). In contrast, countries like Thailand and Vietnam, with richer wildlife populations, likely provide a wider range of ecological niches and host species, supporting higher tick diversity. It would be valuable to consider how host biodiversity, especially the variety of vertebrate species, contributes to the observed differences in tick diversity between these countries. Wildlife species play multiple roles in the increasing prevalence of tick-borne diseases. The distribution and population levels of these species are affected by various interconnected factors, such as natural resource management strategies, weather conditions, terrain, and territorial patterns (Tsao et al., 2021). These elements often work together to amplify their impact. Additionally, limited sampling effort also contributes to the low diversity observed in Cambodia. Although our study presents the first nationwide
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Maximun-likelihood phylogenetic tree of tick species collected in Cambodia based on a fragment of the cox1 gene. The tree was constructed using IQTree v2.3.5 with TIM+F+I+G4 nucleotide substitution model. Sequences obtained from GenBank are indicated in blue text, while sequence generated from this study are presented in black. Asterisks (*) indicate new distribution records for the country.</figcaption></figure>

tick collection, prior research on the subject remains scarce. Neighboring countries have benefited from more extensive research and sampling efforts from various vegetation types and wildlife, which have contributed to a greater diversity of tick fauna. However, we anticipate that expanding sampling efforts to more diverse locations will likely reveal additional tick species.

Among the newly recorded ticks, *C. batuensis*, the only soft tick (family Argasidae) identified in this study, was collected from cave floors in Stung Treng Province. Although it has been well documented in other Southeast Asia countries (Klompen and Oconnor, 1995). The identification of this species was based exclusively on its morphological description, as there was no available *cox1* reference sequence in the GenBank database at the time this manuscript was written. *Carios batuensis* (Supplementary Fig. 2) can be morphologically distinguished by a prominent transverse groove located posterior to the anus. Its legs are relatively long, with the first tarsus bearing a well-developed dorsal projection. The pulvilli are notably large (Supplementary Fig. 2E), as originally described by Hirst (1929) (Hirst, 1929). This species has been predominantly associated with bats located on rocks or in caves across multiple regions as well as bird nesting in Peninsular Malaysia, Malaysian Borneo, Indonesia, the Philippines (Durden et al., 2008; Kazim et al., 2021), Thailand (Petney et al., 2019), and Singapore (Kwak et al., 2024). Despite its widespread distribution, there is limited knowledge regarding the potential role of *C. batuensis* in transmitting tick-borne pathogens in Southeast Asia.

*Amblyomma varanense*, previously recorded in Cambodia, was collected from pythons at a wildlife rescue center in Takeo Province, aligning with observations in Malaysia, Vietnam, Laos, Singapore, and Thailand (Kho et al., 2015; Kolonin, 1995; Kwak et al., 2018; Trinachartvanit et al., 2016; Vongphayloth et al., 2016). This species was previously classified under the genus *Aponomma* (eyeless and often associated with reptiles) and can be recognized by characters described by Kaufman (1972) (Kaufman, 1972): eyes absent, rounded body with iridescent ornamentation on scutum and the presence of two distinct spurs on coxa I, with the external spur longer than the internal (Supplementary Fig. 3). *Amblyomma varanense*, which was once classified under the genus *Aponomma* as *Aponomma varanense*, has been reclassified following recent taxonomic revisions. Molecular phylogenetic studies and morphological analyses have demonstrated that the former genus *Aponomma* shares overlapping characteristics and strong genetic similarity with the genus *Amblyomma*. As a result, the correct and currently accepted name for this species is *A. varanense* (Burger et al., 2012; Khan et al., 2022; Nava et al., 2009; Patra et al., 2016; Pun et al., 2018). This species, known to infest reptiles (snake), has been associated with the transmission of various pathogens including Spotted Fever Group rickettsia and *Borrelia* spp. in Thailand (Kaenkan et al., 2020; Sumrandee et al., 2014; Trinachartvanit et al., 2016).

*Amblyomma javanense*, found on pangolins at the same rescue center, has a broad geographic distribution across South and Southeast Asia such as Pakistan, India, Sri Lanka, Myanmar, Thailand, Vietnam, Malaysia, Singapore, Indonesia, the Philippines, and China (Ali et al., 2024; Hassan et al., 2013; Kwak et al., 2018). The identification of this species follow description by Voltzit & Keirans (2002) (Voltzit and Keirans, 2002): eyes present, no ornamentation on the scutum, a short cervical groove, and a single broadly rounded spur on coxeae II–IV (Supplementary Fig. 4). This species recognized for its role in harboring multiple pathogens such as *Anaplasma*, *Babesia*, *Ehrlichia*, *Rickettsia*, and *Trypanosoma* spp. (Dao et al., 2024; Jiang et al., 2021; Yodsheewan et al., 2021; Zhai et al., 2021).

*Dermacentor auratus* was collected from wild boar in Koh Kong province and is known to infest wild pigs and other large mammals throughout Southeast Asia. This species can be identified by distinct morphological features present in both male and female specimens, as described by Wassef and Hoogstraal (1984). The internal and external spurs on coxa I are subequal in length and moderately separated, with the internal spur being broadly triangular and the external spur slightly
<table><caption>**Table 3**
List of tick species identified in Cambodia and tick presence in our study.</caption><thead><tr><th>Family</th><th>Species</th><th>Descriptor Name and Year</th><th>Stages</th><th>Present in this study</th><th>References</th></tr></thead><tbody><tr><td rowspan="2">Argasidae</td><td>Argas vespertilionis</td><td>(Latreille, 1796)</td><td>M,F,N,<br>L</td><td></td><td>(José, 2012; Petney et al., 2019)</td></tr><tr><td>Carios batuensis*</td><td>Hirst, 1929</td><td>M,F,N</td><td>√</td><td>(Hirst, 1929; Kazim et al., 2021; Kwak et al., 2024)</td></tr><tr><td rowspan="10">Ixodidae</td><td>Amblyomma geoemydae</td><td>(Cantor, 1847)</td><td>M,F,N,<br>L</td><td></td><td>(Amarga et al., 2024; Lah et al., 2022; Voltzit and Keirans, 2002)</td></tr><tr><td>Amblyomma javanense</td><td>(Supino, 1897)</td><td>M,F,N,<br>L</td><td>√</td><td>(Jabin, 2019; Voltzit et al., 2002)</td></tr><tr><td>Amblyomma testudinarium</td><td>Koch, 1844</td><td>M,F,N,<br>L</td><td></td><td>(Voltzit and Keirans, 2002)</td></tr><tr><td>Amblyomma varanense</td><td>(Supino, 1897)</td><td>M,F,N,<br>L</td><td>√</td><td>(Kaufman, 1972)</td></tr><tr><td>Dermacentor auratus</td><td>Supino, 1897</td><td>M,F,N,<br>L</td><td>√</td><td>(Wassef and Hoogstraal, 1984)</td></tr><tr><td>Dermacentor filippovae*</td><td>Apanaskevich & Apanaskevich, 2015</td><td>M,F</td><td>√</td><td>(Apanaskevich and Apanaskevich, 2015)</td></tr><tr><td>Dermacentor steini*</td><td>(Schulze, 1933)</td><td>F</td><td>√</td><td>(Wassef and Hoogstraal, 1986)</td></tr><tr><td>Haemaphysalis arborensis</td><td>Warburton, 1913</td><td>M,F,N,<br>L</td><td></td><td>(Hoogstraal and Kawther, 1971)</td></tr><tr><td>Haemaphysalis anomala</td><td>Warburton, 1913</td><td>M,F,N,<br>L</td><td></td><td>(Hoogstraal et al., 1971)</td></tr><tr><td>Haemaphysalis canestrinii*</td><td>(Supino, 1897)</td><td>M</td><td>√</td><td>(Tanskul and Inlao, 1989)</td></tr><tr><td>Haemaphysalis hystricis*</td><td>Supino, 1897</td><td>F</td><td>√</td><td>(Hoogstraal and Glen, 1965)</td></tr><tr><td>Haemaphysalis lagrangei</td><td>Larrousse, 1925</td><td>M,F,N,<br>L</td><td></td><td>(Hoogstraal et al., 1973; Tanskul and Inlao, 1989)</td></tr><tr><td>Haemaphysalis obesa</td><td>Larrousse, 1925</td><td>M,F,N,<br>L</td><td></td><td>(Hoogstraal and Glen, 1966)</td></tr><tr><td>Haemaphysalis papuana</td><td>Thorell, 1883</td><td>M,F,N,<br>L</td><td></td><td>(Trapido et al., 1964)</td></tr><tr><td>Haemaphysalis formosensis*</td><td>Neumann, 1913</td><td>N</td><td>√</td><td>(Hoogstraal & Kawther, 1971; Yamaguti et al., 1971)</td></tr><tr><td>Haemaphysalis shimoga</td><td>Trapido and Hoogstraal, 1964</td><td>M,F,N,<br>L</td><td>√</td><td>(Trapido et al., 1964)</td></tr><tr><td>Haemaphysalis wellingtoni*</td><td>Nuttall and Warburton, 1908</td><td>M,F</td><td>√</td><td>(Tanskul and Inlao, 1989)</td></tr><tr><td>Haemaphysalis yeni</td><td>Toumanoff, 1944</td><td>M,F,N,<br>L</td><td></td><td>(Saito and Hoogstraal, 1973)</td></tr><tr><td>Ixodes granulatus</td><td>Supino, 1897</td><td>M,F,N,<br>L</td><td></td><td>(Yamaguti et al., 1971)</td></tr><tr><td>Ixodes kopsteini</td><td>(Oudemans, 1926)</td><td>M,F,N,<br>L</td><td>√</td><td>(Kohls and Clifford, 1968)</td></tr><tr><td>Rhipicephalus australis</td><td>Fuller, 1899</td><td>M,F,N,<br>L</td><td></td><td>(Kazim et al., 2022)</td></tr><tr><td>Rhipicephalus<br>haemaphysaloides</td><td>Supino, 1897</td><td>M,F,N,<br>L</td><td></td><td>(Walker et al., 2000)</td></tr><tr><td>Rhipicephalus microplus</td><td>(Canestrini, 1888)</td><td>M,F,N,<br>L</td><td>√</td><td>(Kazim et al., 2022)</td></tr><tr><td>Rhipicephalus linnaei</td><td>(Audouin, 1826)</td><td>M,F,N,<br>L</td><td>√</td><td>(Walker et al., 2000)</td></tr></tbody></table>

Abbreviations: M=Male, F=Female, N=Nymph, L=Larvae

narrower. A key diagnostic characteristic is the uninterrupted median brown patch, which appears as a continuous line on the scutum in females and on the pseudoscutum in males. The genital pore in female has broad U-shaped (Supplementary Fig. 5). This tick is of significant medical and veterinary concern due to its ability to transmit various pathogens including *Anaplasma*, *Bartonella*, *Borrelia*, *Rickettsia*, and several viruses like African swine fever virus, Kyasanur forest diseases virus and Lanjan virus Although widely studied elsewhere, no tick-borne pathogens have yet been reported in *D. auratus* in Cambodia.

*Dermacentor filippovae* and *D. steini* were newly recorded in the country, collected from wild boars and vegetation in Koh Kong Province. While morphologically like *D. auratus*, *D. filippovae* can be differentiated based on several morphological characteristics based on *Apanaskevich and Apanaskevich (2015)* (*Apanaskevich and Apanaskevich, 2015*). The median brown patch is indistinct and incomplete on the scutum of female and pseudoscutum in males. In both sexes, coxa I bears a narrowly triangular internal spur with a tapering apex and a longer tapering external spur. Females also have a moderately broad, U-shaped genital pore with a bulging pretrial fold sloping posteriorly (Supplementary Fig. 6). This species has been linked to pathogens like *Hepatozoon canis*

and *Ehrlichia canis*, and is adapted to both forested and human-modified environments in Thailand and Vietnam (*Apanaskevich and Apanaskevich, 2015*). In addition to being found on animal hosts, *D. filippovae* was frequently collected from vegetation in primary forests, suggesting that it is well adapted to undisturbed ecosystems. At the same time, its presence in areas influenced by human activity points to a degree of ecological flexibility and potential adaptability to anthropogenic environments (*Apanaskevich and Apanaskevich, 2015; Vongphayloth et al., 2018*).

*Dermacentor steini* frequently collected from wild boars and vegetation in Laos (*Vongphayloth et al., 2016*) and Indonesia (*Durden et al., 2008*), as well as from wild boars in Malaysia (*Ernieenor et al., 2020; Khoo et al., 2016*). In a microbiome study, sequences corresponding to *Francisella*, *Coxiella*, and *Rickettsia* were detected in this tick species collected from wild boar (*Lim et al., 2020*). The morphological identification of *D. steini* follows description by *Wassef and Hoogstraal (1986)*. It can be distinguished from *D. auratus* by the external and internal spurs on coxa I, which are not separated. In females, the scutum has angular posterolateral margins, and a prominent brown stripe extends regularly from one eye to the other, following the lateral and posterior margins of

<footnote>* Newly recorded tick species in Cambodia; √ = Species recorded in the present study</footnote>
the scutum. In males, the median brown patch on the pseudoscutum is narrow, distinct, and uninterrupted (Supplementary Fig. 7). The *cox1* sequence from *D. steini* in this study aligned with only one *D. steini* sequences from Thailand (ON680802, percent identity 99.24%) but did not align well with other *D. steini* sequences available in GenBank. It is important to note the limitations associated with species identification based solely on publicly available sequence data. Given the documented taxonomic inconsistencies in public databases such as NCBI, there is a risk of misidentification, particularly among morphologically similar or poorly characterized species. As such, the identification of these species in this study should be interpreted with caution, and we emphasize the importance of combining molecular data with morphological examination and, when possible, expert validation.

*Haemaphysalis canestrinii* was recorded for the first time in Cambodia from a dog in Kratie Province. The identification of *H. canestrinii* follows the description in Tanskul and Inlao (1989) (Tanskul and Inlao, 1989), which shows that the posterodorsal spur of the palpus segment II is located at the midpoint and is shaped like an elongated triangle with pointed tips. Additionally, the lateral grooves extend to the level of coxa III, enclosing the first festoon (Supplementary Fig. 8). This species has been reported from carnivores in neighboring countries such as Myanmar, Thailand, and Vietnam (Petney et al., 2019). Currently, there are no documented reports of tick-borne pathogens associated with this species in Southeast Asia.

*Haemaphysalis formosensis*, identified from a single nymph (Supplementary Fig. 9) collected from vegetation in Kampot Province using molecular techniques. It has a limited presence in Southeast Asia but has been reported in Taiwan, Korea, and Japan, where it infests various wildlife including pangolins and migratory birds (Khatri-Chhetri et al., 2016; Kolonin, 1995; Kuo et al., 2017; Petney et al., 2019). This represents the first record of the species in Cambodia.

Similarly, *H. hystricis*, collected from forest fringes in three provinces, is a newly recorded species. *Haemaphysalis hystricis* is recognized morphologically by a campanulate palpus, dental formula 4/4, and posterodorsal spur on segment III which overlapping 1/5 anterior part of segment II. Posteroventral margin of palpal segment II forming spur-like angle (Hoogstraal and Glen, 1965; Tanskul and Inlao, 1989) (Supplementary Fig. 10). This species known to infest both wild and domestic hosts in other countries (Arthan et al., 2015; Durden et al., 2008; Vongphayloth et al., 2016). This species has been found to carry pathogen DNA, including *Coxiella burnetii*, *Borrelia* spp., *Anaplasma* spp., and *Ehrlichia* spp., highlighting its zoonotic potential (Khatri-Chhetri et al., 2016; Khoo et al., 2016).

*Haemaphysalis shimoga*, previously recorded in Cambodia, was primarily found on vegetation in forest fringe areas. Although *H. shimoga* was regarded as a synonym of *H*. (Kaiseriana) *taiwana* by Kolonin (2009), recent global tick catalogues have recognized *H. shimoga* as a valid species (Guglielmone et al., 2014, 2010). Morphologically, *Haemaphysalis cornigera* *taiwana* is a synonym of *H. cornigera* (Robbins, 2005) and can be distinguished from *H. shimoga* by the length of the coxa IV spurs in male (both external and internal), which are almost equal in *H. shimoga*. In *H. taiwana*, these long spurs meet apically on coxa IV (Trapido and Hoogstraal, 1964). Our phylogenetic analysis further supports this distinction, showing *H. shimoga* and *H. taiwana* in separate clades (Fig. 3). *Haemaphysalis shimoga* is morphologically characterized by truncate palpi with bizarre angles in males, along with distinctly long, externally directed spurs on coxa IV and indistinct V-shaped spurs near the inner margins of the posterodorsal margin of palpal segment III. Females exhibit palpi with basolateral salience, small pointed median dorsobasal spurs on the posterodorsal margin of palpal segment III and typically possess 4–6 infrainternal setae (Supplementary Fig. 11). The remaining morphological characters follow the description by Trapido and Hoogstraal (1964) (Trapido and Hoogstraal, 1964). This species is known to harbor *Coxiella*-like bacteria in Thailand (Arthan et al., 2015; Petney et al., 2019).

*Haemaphysalis wellingtoni*, another new record for Cambodia, was

also collected from forest fringe vegetation. *Haemaphysalis wellingtoni* can be easily recognized by the presence of posterointernally directed retroverted spurs on palpal III in both male and female specimen (Tanskul and Inlao, 1989) (Supplementary Fig. 12). This bird-associated tick has been reported in various Southeast Asian countries and is known to feed on a wide range of hosts, including humans in Singapore, Thailand (Kwak and Ng, 2022; Parola and Sanogo, 2003) and domestic animals such as chickens, dogs, cats, and rodents in rural Malaysia and Indonesia (Durden et al., 2008). Its association with *Anaplasma* suggests it may play a role in pathogen transmission, especially in rural areas (Khoo et al., 2021).

Two unidentified *Haemaphysalis* nymphs collected in Kampot Province did not match any known sequences in GenBank, indicating the potential presence of undocumented species. This highlights the need for further molecular studies. Furthermore, *I. kopsteini* (Supplementary Fig. 13) the only *Ixodes* species collected, was found on a field technician in a Phnom Sampov cave in Battambang province. The identification of *I. kopsteini* followed morphological description by Kohls and Clifford (1968) (Kohls and Clifford, 1968), under its former synonym, *I. paradoxus*. The first record of *I. kopsteini* in Cambodia was from *Chaerephon plicata* bat in Phnom Penh (Robbins et al., 2004). This species commonly associated with bats, and in Southeast Asia has been recorded in Indonesia (Anastos et al., 1973), Philippines, Malaysia (Kohls and Clifford, 1968) and Thailand (Tanskul et al., 1983). To date, there are no reports of tick-borne pathogens from these species affecting animals and humans.

In this study, *R. microplus* (Supplementary Fig. 14) was the most prevalent species found on cattle. Morphological examination revealed two morphotypes present in Cambodia: (1) a character set matching *R. microplus* as described by Walker (2003), and (2) characters matching *R. australis* as described by Estrada-Peña et al (2012) (Estrada-Peña et al., 2012). However, DNA barcoding analysis indicated that all specimens belong to *R. microplus* and only from Clade A (Prasetyo et al., 2025), similar with findings in Vietnam (Hornok et al., 2024). Other countries, such as Thailand and Malaysia, host multiple clades such as *R. microplus* clades A and C (Low et al., 2015; Thinnabut et al., 2023). These results altogether suggest a widespread distribution of *R. microplus* clade A in Laos, Indonesia, Philippine, & Vietnam, excepted Thailand and Malaysia, also harboring *R. microplus* clade C.

*Rhipicephalus linnaei* (Supplementary Fig. 15) was found infesting cat and dog from urban and rural habitats in this study. This species has been frequently reported from domestic dog in Southeast Asia, including Indonesia (Sahara et al., 2019), the Philippines (Marquez et al., 2021), Malaysia (Low et al., 2015), Myanmar (Roy et al., 2018), and Thailand (Thinnabut et al., 2023). This species which was previously considered a form of *R. sanguineus* is now recognized as a distinct species as confirmed by genetic analysis (Slapeta et al., 2021). Morphologically, the male *R. sanguineus* has shorter, non-tapering extension of the spiracular plate. In females, the genital pore is broadly U-shaped in *R. linnaei*, whereas in *R. sanguineus* it forms a narrower U-shape. *Rhipicephalus linnaei* is primarily found in tropical region (Burger et al., 2014; Colella et al., 2020; Marquez et al., 2021; Tan et al., 2021; Yean et al., 2024). This species has been found harboring *Ehrlichia canis*, *Anaplasma platys*, *Hepatozoon canis*, and *Babesia (canis) vogeli* in Thailand (Juasook et al., 2021).

While many of the species discussed are frequently associated with pathogens, it is important to understand that the detection of pathogen DNA in ticks does not inherently demonstrate infectivity or transmission capability. This limitation arises because molecular techniques such as PCR may simply represent residual material from ingested blood rather than evidence of active infection within the tick. Even if a tick has fed on an infected host, the mere presence of pathogen DNA does not confirm that the microbe has survived, replicated, or reached the salivary glands critical steps required for successful transmission. Misinterpreting DNA detection as evidence of vector competence can result in exaggerated risk assessments and incorrect assumptions about tick-pathogen dynamics (Estrada-Peña et al., 2013). Therefore, to accurately assess a
tick's role in disease transmission, additional methods such as culturing viable pathogens, detecting RNA to indicate active replication, or conducting transmission experiments are essential. Without this validation, DNA detection alone should not be equated with the potential for pathogen transmission (Salata et al., 2021).

Interestingly, no ticks were found on domestic pigs and chickens in our study, which aligns with previous research in Thailand (Changbunjong et al., 2009). The absence of ticks may be attributed to the host preferences of the species present in domestic areas, as well as specific management practices, such as the use of drugs or chemical in farm which might limit tick populations. In fact, in Cambodia, pigs are raised in cages with regular daily cleaning, and chickens are treated with commercial insecticide sprays (pyrethroids) to control ectoparasites and other pest in their enclosures. These practices are likely to reduce tick exposure in the farm settings. However, research from other countries presents a different viewpoint. For instance, a study conducted in Thailand identified ticks on domestic chicken, wild chicken, and Siamese fireback (*Lophura diardi*) also known as Diard's fireback (Trinachartvanit et al., 2018) while research from Pakistan found ticks on domestic poultry, and ducks (Zahid et al., 2023). In Laos and Korea, hard ticks have been collected from domestic pigs (Chae et al., 2017). Similarly, in Brazil, ticks have been reported on domestic pigs raised in paddocks (Osava et al., 2016), suggesting that open spaces and free land provide suitable environments for tick infestation.

Additional sampling from diverse habitats, particularly non-
domestic environment such as swiftlet nests, caves, and forest fringes
provided further insights into tick ecology and diversity in Cambodia.
The absence of ticks in edible-nest swiftlet (*Aerodramus fuciphagus*) farm
in Cambodia might be attributed to limited sampling efforts, as only one
farm per province was selected as collection point due to logistical
constraints. The sampling methodology may also play a role, as the tick
inspections were performed on the nests rather than on the birds
themselves. While infestation of lice and mites have been reported in
edible-nest swiftlet, records of tick infestation are scarce. In Indonesia,
an Argasidae tick, *Ornithodoros collocaliae* has been found on swiftlet
nests in a coastal cave areas (Hoogstraal et al., 1974). The host of the
ticks mentioned in Hoogstraal paper are cave swiftlets (*Collocalia linchi*),
which differ from the species examined in our study (edible-nest swift-
let: *Aerodramus fuciphagus*).

This study provides valuable insights into tick diversity across mul-
tiple habitats in Cambodia. Future research should involve more
extensive and repeated sampling effort across a broader range of habi-
tats and seasons to ensure a more comprehensive understanding of tick
biology and distribution in the region would be a good way to frame the
need for further investigation. Overall, our findings enhance the un-
derstanding of tick diversity in Cambodia, revealing both known and
newly recorded species for the country.

5. Conclusion

Our study highlights a contribution to a one-third increase to the previously recorded tick species in the country. Ticks were found to be widely distributed across Cambodia, with different species identified according to the environments in which they were collected. Future research in Cambodia should focus on creating a comprehensive catalog of tick species across various hosts and habitats, while investigating the interactions between ticks, their hosts, and the environment. The absence of reliable reference sequences in GenBank limits accurate comparisons, making it difficult to determine whether some species are new discoveries or unidentified variants. To address this, improving tick sequencing data sharing in GenBank and updating research is crucial for accurate species identification and scientific advancement.

Funding sources

This study was supported by the French Embassy in Cambodia

through its FSPI – The Solidarity Fund for Innovative Projects. The
funder had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.

CRediT authorship contribution statement

**Sony Yean:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis. **Didot Budi Prasetyo:** Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation. **Theary Ren:** Investigation. **Davy Krib:** Investigation. **Saoya Sen:** Investigation. **Bunthon Chea:** Investigation. **Sothyra Tum:** Supervision, Investigation. **Sorn San:** Supervision, Investigation. **Samuth Sum:** Investigation. **Sarah Bonnet:** Writing – review & editing, Validation. **Sébastien Boyer:** Writing – review & editing, Validation, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of interest

The authors declare no conflicts of interest.

Acknowledgements

The author is grateful thanks to the medical and veterinary ento-
mology team for their assistance in tick collection and morphological
identification. We would like to thank the Faculty of Veterinary Medi-
cine, Royal University of Agriculture, for their support in facilitating tick
collection activities. The author also appreciates the excellent coordi-
nation of the National Animal Health and Production Research Institute,
General Directorate of Animal Health and Production. Lastly, appreci-
ation is due to the veterinary departments of both provinces for granting
authorization to collect ticks from animals in city, village, farm, and
vegetation in forest fringe.

**Supplementary materials**

Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.ttbdis.2025.102570.

Data availability

Data will be made available on request.

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