Deer keds of the genus Lipoptena spp. (Diptera: Hippoboscidae) are hematophagous insects that infest mammals (1). The insects become wingless after finding a suitable host and attach to a single host throughout their life span (2–4). Of over 30 species of Lipoptena insects worldwide, L. fortisetosa along with L. cervi, L. depressa, and L. mazamae are the most prevalent and threaten to wildlife, livestock, and pets (5–8). Lipoptena fortisetosa are found on sika deer (Cervus nippon), roe deer (Capreolus capreolus), and red deer (Cervus elaphus) in many countries and incidentally found on dogs (2, 9–12). In addition, humans can be bitten by Lipoptena insects (13). Several molecular epidemiological studies show L. fortisetosa harbors DNA of various pathogens, including Anaplasma phagocytophilum, Babesia spp., Bartonella spp., Borrelia spp., Coxiella-like endosymbionts, Francisella tularensis, Mycoplasma spp., Rickettsia spp., and Theileria spp. (14–16).

The genus Anaplasma includes intracellular gram-negative bacteria transmitted by ixodid ticks (17). Several Anaplasma spp., such as A. marginale, A. centrale, A. ovis, and A. bovis, are obligate bacteria parasitizing blood cells of many ruminants, while A. platys is mainly a pathogen of dogs (17). Anaplasma phagocytophilum is a pathogenic bacterium of a wide range of hosts, including humans and domestic and wild animals (18). In addition, A. phagocytophilum has been detected worldwide in wild ruminants and their ectoparasites (19–23). Although the role of wildlife in circulation of Anaplasma spp. is yet to be clearly defined, several species of wild ruminants are considered important reservoirs (24). In Thailand, studies found evidence of A. platys and A. bovis detection in Dermacentor auratus ticks collected from sambar deer (Cervus unicolor) (25). Because D. auratus ticks are found on humans in Thailand (26, 27), humans infected with Anaplasma bacteria via infected tick bites in addition to other deer ectoparasites is also possible.

Bartonella spp. are intra-erythrocytic gram-negative bacteria mainly transmitted among hosts by arthropod vectors, such as cat fleas (Ctenocephalides felis), body lice (Pediculus humanus), and sand flies (Lutzomyia verrucarum) (28–30). There are 45 Bartonella spp./subspp. Detected or isolated from various animals (31). Bartonella schoenbuchensis, B. capreoli, and B. bovis are detected in wild ruminants in several countries, which are strongly suspected to be transmitted by Lipoptena spp. (32–39). In Thailand, the novel Bartonella spp. was detected and isolated from captive Rusa deer (Rusa timorensis) blood samples (40). Since no ectoparasites are found on these deer, further studies are needed to determine whether ectoparasites transmit Bartonella among deer throughout Thailand. For zoonotic issues, human cases of bartonellosis caused by ruminant-related species, B. schoenbuchensis and B. melophagi, have been previously reported (41, 42). These findings highlight that although Bartonella bacterial infection in animals does not result in serious diseases, this wide range of infected animals could be a reservoir for potential zoonotic infection.

Khao Kheow Open Zoo is located within a wildlife sanctuary in Chon Buri province, eastern Thailand. Several endangered wildlife, as well as Eld’s deer (Rucervus eldii thamin), are bred and raised in the open zoo to increase population numbers. Here, wild animals can freely roam the open zoo and sanctuary areas, increasing the possibility of pathogen transmission among wild animals. Recent evidence shows ruminant-related blood pathogens, including Anaplasma, Babesia, Ehrlichia, and Theileria, in various species of ticks in this surrounding environment (43). Moreover, Tiawsirisup et al. (44) reported the presence of L. fortisetosa on Eld’s deer with Theileria capreoli and T. cervi in these insects in Thailand. Although DNA presence does not guarantee pathogen transmission, it may highlight the potential risk for mechanical transmission of pathogens to humans and healthy animals via bites of infected ectoparasites. Currently, knowledge surrounding epidemiology of pathogens related to endangered wild ruminants in Thailand is limited. This study aims to determine the prevalence and diversity of Anaplasma and Bartonella in the ectoparasite collected from captive Eld’s deer. Our findings may be used to understand the current status of pathogens among Eld’s deer and their ectoparasite, formulate animal welfare policies, and provide valuable information to prevent and control pathogens related to endangered wildlife species in the country.

Lipoptena fortisetosa is a crucial ectoparasite infesting cervids worldwide. This insect is primarily found in sika deer (Cervus nippon) in Japan and has also been reported in Siberian roe deer (Capreolus pygargus) in Korea, Kazakhstan, and Russia (1, 9, 52). The distribution of L. fortisetosa in European countries has been hypothesized by climate change, introduction of alien cervid species into new areas, and adaptation of the insect to different hosts (11, 12). In Thailand, L. fortisetosa was first found on captive Eld’s deer in Chon Buri, as previously reported (44). Lipoptena insects may cause anemia, skin irritation, itching, restlessness, and hair loss in animal hosts (53). However, there were no skin or other symptoms on infested Eld’s deer in Thailand.

To our knowledge, this is the first report on Anaplasma and Bartonella detection in L. fortisetosa in Thailand. The prevalence of Anaplasma spp. detection in L. fortisetosa (46.15%) in the present study was higher than that reported in Poland (8.00%) (54). Although no previous evidence exists of Anaplasma harbored by this insect in the country, the presence of ticks, the primary Anaplasma vector, in the same area of wildlife habitat may enhance the possibility of bacterial infection in other blood-feeding ectoparasites, including Lipoptena insects. This possibility is supported by the results where ntST2 was identical to Anaplasma detected in H. lagrangei ticks in a previous study (43). In addition to Anaplasma, various pathogens, such as Babesia, Ehrlichia, Theileria, and Wolbachia, have also been detected in questing ticks in wildlife habitat in Chon Buri, Thailand (43). This finding highlights that, in the same surrounding area, various ectoparasites may harbor, or transmit the pathogen. Moreover, Anaplasma DNA is found in other species of Lipoptena insects, such as L. cervi and L. depressa (19, 23, 55).

Anaplasma DNA fragments from this study can be clustered with the clades of A. bovis and other ruminant-related Anaplasma. A. bovis has previously been detected in domestic goats (56), but no report on wildlife in Thailand. Interestingly, A. bovis DNA has been found in ticks collected from the Malayan sun bear (Helarctos malayanus), sambar deer (Cervus unicolor), and questing ticks dragged in wildlife habitat in Thailand (25, 43, 47). These findings suggest wild animals may act as a natural reservoir and their ectoparasites may be associated with Anaplasma infection of domestic ruminants. This possibility is supported by the present study showing ntST2 and 4 showed similar genetic material with A. bovis detected in goats in China (OQ132528 and KP062954, respectively). Other ruminant-related Anaplasma, including A. marginale, A. ovis, and A. capra, have been detected in various wild animals, suggesting a broad host range and genetic diversity (57–59). In the present study, using primers for the partial 16S rRNA gene of Anaplasma did not distinguish obtained sequence data from other ruminant-related Anaplasma. Amplification and sequencing of full-length 16S rRNA gene or other specific genes of Anaplasma would be necessary to clarify genetic characterization.

Besides biological transmission of Anaplasma by ticks, other potential vectors have been reported to mechanically transfer bacteria to animal hosts, including biting flies (60) and syringophilid mites (61). For Lipoptena, the insects detach their wings after finding suitable hosts and can only be transferred among hosts via direct contact. Since the insects acquire Anaplasma-infected blood meal on bacteremic hosts, it is possible to transmit bacteria horizontally to noninfected animals via direct contact (55). However, further experimental studies are needed to confirm direct evidence of the vector ability for Anaplasma bacteria transmission.

The prevalence of Bartonella detection in L. fortisetosa (29.67%) in this study was lower than those collected from free-living cervids in Japan (87.87%) and Poland (75.67%) (15, 62). Possible reasons for low prevalence of Bartonella detection are that Eld’s deer are raised in captive areas in Thailand. The semi-wild conditions of deer may confer a lower infestation probability by pathogen-infected ectoparasites compared with free-living wild cervids in Japan and Poland. This possibility is supported by a previous study in Poland showing farm cervids had a lower prevalence of A. phagocytophilum infection than wild individuals (21). Myczka et al. (21) also mentioned the lack of Anaplasma detection in farmed cervids may be due to regular deworming, which strengthens their condition and makes them less susceptible to infection by the bacteria. Notably, novel Bartonella has been detected and isolated with low prevalence (3.60%) from captive Rusa deer in Thailand, including those being regularly dewormed (40). Secondly, despite prior tick presence in the same surrounding area (43), ticks may not be the essential vector for Bartonella transmission among cervids (15, 63). For this reason, we implied that since Eld’s deer are infested by ticks and Lipoptena insects, Bartonella can still be detected, merely not in high prevalence. However, Bartonella detected in captive Eld’s deer and ticks collected from surrounding areas should be analyzed to clarify these reasons.

Bartonella sequences obtained from this study can be grouped with novel Bartonella lineages C, D, and E, which originated from L. fortisetosa collected from deer in Japan (15, 39). In addition, the novel Bartonella lineage B, primarily derived from Japanese sika deer, has previously been found in L. cervi and L. fortisetosa collected from red deer in Poland (62, 64). These findings suggest novel Bartonella stains from wild ruminants in Japan may spread to other countries following Lipoptena vector introduction into these new areas. We also found a new independent lineage of novel Bartonella from collected Lipoptena insects. However, genetic characterization analysis is needed to determine whether these new lineages are Lipoptena insects-specific Bartonella. Obtained Bartonella sequences were clustered with ruminant-related Bartonella. In addition to the report of novel Bartonella isolated from captive Rusa deer in Thailand (40), further reports exist of Bartonella detected or isolated from domestic ruminants in the country. Bai et al. (65) revealed that B. bovis was isolated from water buffalo blood. In 2021, seroprevalence of antibodies against B. henselae, B. vinsonii subspp. Berkhoffii, and B. tamiae in water buffaloes has also been reported (66). These findings support the genetic diversity of Bartonella among ruminants in Thailand. Further molecular surveys of Bartonella in both wild and domestic ruminants and their ectoparasites are needed to clarify the role of bacterial infection among ruminants in the country.

The high prevalence of Bartonella DNA presence in Lipoptena insects raises the question that insects may play an essential role in Bartonella transmission among hosts (5, 15, 34, 37, 38). The evidence of Bartonella survival and propagation in Lipoptena specimens has been reported from previous studies using bacterial isolation from the insects and immunohistochemical analysis (15, 35). In addition, the bacterial DNA detected in both wingless L. cervi females and their offspring suggests the potential for vertical Bartonella transmission (34). However, both in vitro and in vivo studies are required to verify how vector competence of Lipoptena insects facilitates Bartonella transmission.

Co-infections occur in Lipoptena insects but pathogen diversity may vary by species, hosts, and geographic distribution. In the USA, 6.25% of L. cervi removed from white-tailed deer carried both B. burgdorferi s.l. and A. phagocytophilum DNA (19). Further, 50% of L. fortisetosa collected from Korean water deer harbored Coxiella, T. ovis, and T. luwenshuni DNA, but no Rickettsia, Babesia, Bartonella, Borrelia, or Hepatozoon were detected (67). In this study, 11 Lipoptena specimens (12.08%) harbored both Anaplasma and Bartonella DNA. We also found four specimens were additionally infected with Theileria spp. as previously reported by Tiawsirisup et al. (44). The prevalence of more than one pathogen in Lipoptena insects emphasizes their medical and veterinary importance.

In the present study, our findings provide the first molecular detection of Anaplasma and Bartonella on L. fortisetosa in Eld’s deer in the country. Despite no blood samples from Eld’s deer, pathogen DNA detected in insects could represent the health status of animal hosts. Further studies on molecular genetic characterization of related pathogens are needed to investigate correlations of vectors, hosts, and pathogens. In addition, visitors are allowed to have direct contact with animals through petting, feeding, or taking close photos with animals in the zoo. These activities can promote the risks of potential zoonotic infection. Preventive procedures, such as health monitoring, anti-parasite medication, and proper treatments for animals should be regularly conducted. Zoo staff should pay attention to regular hygiene measures before and after working with animals and surrounding areas, such as hand washing, wearing PPE, and foot bathing with an antiseptic solution. Finally, visitors should avoid direct contact with animals and be wary of insects or ectoparasites while visiting the zoo.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/, OQ692407 https://www.ncbi.nlm.nih.gov/, OQ692408 https://www.ncbi.nlm.nih.gov/, OQ692409 https://www.ncbi.nlm.nih.gov/, OQ692410 https://www.ncbi.nlm.nih.gov/, OQ716819 https://www.ncbi.nlm.nih.gov/, OQ716820 https://www.ncbi.nlm.nih.gov/, OQ716821 https://www.ncbi.nlm.nih.gov/, OQ716822 https://www.ncbi.nlm.nih.gov/, OQ716823 https://www.ncbi.nlm.nih.gov/, OQ716824 https://www.ncbi.nlm.nih.gov/, OQ716825 https://www.ncbi.nlm.nih.gov/, OQ716826.

The animal study was approved by the Chulalongkorn University Animal Care and Use Committee (Animal use protocol number: 2231011). The study was conducted in accordance with the local legislation and institutional requirements.

WW contributed data, performed the analysis, and wrote the manuscript. CS-i and KT collected the data and provided technical support. NY, CA, GR, NB, and NS collected samples and data. LB edited the manuscript. UM and PK provided professional support at the sample collecting sites. AS provided technical support. ST supervised the study, edited the manuscript, and corresponded. All authors contributed to the article and approved the submitted version.

This research was funded by the Thailand Science Research and Innovation (TSRI) Fund (CU_FRB640001_01_31_3), the Chulalongkorn University Research Unit (GRU 3310160009), and the Second Century Fund (C2F), Chulalongkorn University.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.