Tick diversity and molecular detection of Anaplasma, Babesia, and Theileria from Khao Kheow open zoo, Chonburi Province, Thailand.

Ticks are obligate blood-feeding ectoparasites notorious for their role as vectors for various pathogens, posing health risks to pets, livestock, wildlife, and humans. Wildlife also notably serves as reservoir hosts for tick-borne pathogens and plays a pivotal role in the maintenance and dissemination of these pathogenic agents within ecosystems. This study investigated the diversity of ticks and pathogens in wildlife and their habitat by examining ticks collected at Khao Kheow Open Zoo, Chonburi Province, Thailand. Tick samples were collected for 1 year from March 2021 to March 2022 by vegetation dragging and direct sampling from wildlife. A total of 10,436 ticks or 449 tick pools (1-50 ticks per pool) underwent screening for pathogen presence through conventional PCR and DNA sequencing. Out of the 298 samples (66.37%) where bacteria and protozoa were detected, encompassing 8,144 ticks at all stages, 114 positive samples from the PCR screenings were specifically chosen for detailed nucleotide sequencing and comprehensive analysis. Four species of ticks were conclusively identified through the application of PCR, namely, Rhipicephalus microplus, Dermacentor auratus, Haemaphysalis lagrangei, and Haemaphysalis wellingtoni. The highest infection rate recorded was for Anaplasma spp. at 55.23% (248/449), followed by Babesia spp. and Theileria spp. at 29.62% (133/449) and 16.26% (73/449), respectively. Among bacteria identified, three Anaplasma genotypes were closely related to an unidentified Anaplasma spp., A. phagocytophilum, and A. bovis. Among protozoa, only an unidentified Babesia spp. was found, whereas two Theileria genotypes found were closely related to unidentified Theileria spp. and T. equi. Significantly, our findings revealed coinfection with Anaplasma spp., Theileria spp., and Babesia spp. While blood samples from wildlife were not specifically collected to assess infection in this study, the data on the presence of various pathogens in ticks observed can serve as valuable indicators to assess the health status of wildlife populations and to monitor disease dynamics. The findings could be valuable in developing programs for the treatment, prevention, and control of tick-borne illnesses in this area. However, additional research is required to determine the ticks' ability to transmit these pathogens and enhance the current understanding of the relationship among pathogens, ticks, and hosts.

Diversity of Anaplasma and novel Bartonella species in Lipoptena fortisetosa collected from captive Eld's deer in Thailand.

Lipoptena insects are important ectoparasites of cervids and may affect humans that are incidentally bitten. The presence of zoonotic pathogen DNA, such as Anaplasma, and Bartonella, raises the importance of Lipoptena insects in veterinary and human medicine. Eld's deer (Rucervus eldii thamin), an endangered wild ruminant in Thailand, are bred and raised in the open zoo. The semi-wild zoo environment suggests ectoparasite infestation and potential risk for mechanical transmission of pathogens to visitors, zoo workers, or other animals. However, epidemiology knowledge 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 L. fortisetosa collected from captive Eld's deer in Chon Buri, Thailand. Of the 91 Lipoptena DNA samples obtained, 42 (46.15%) and 25 (27.47%) were positive for Anaplasma and Bartonella by molecular detection, respectively. Further, 42 sequences of Anaplasma (4 nucleotide sequence types) showed 100% identity to those detected in other ruminants and blood-sucking ectoparasites. Twenty-five sequences of Bartonella (8 nucleotide sequence types) showed 97.35-99.11% identity to the novel Bartonella species from sika deer and keds in Japan. Phylogenetic trees revealed Anaplasma sequences were grouped with the clusters of A. bovis and other ruminant-related Anaplasma, while Bartonella sequences were clustered with the novel Bartonella species lineages C, D, and E, which originated from Japan. Interestingly, a new independent lineage of novel Bartonella species was found in obtained specimens. We report the first molecular detection of Anaplasma and Bartonella on L. fortisetosa, which could represent infectious status of captive Eld's deer in the zoo. Wild animals act as reservoirs for many pathogens, thus preventive measures in surrounding areas should be considered to prevent pathogen infection among animals or potential zoonotic infection among humans.

Possible role of Lipoptena fortisetosa (Diptera: Hippoboscidae) as a potential vector for Theileria spp. in captive Eld's deer in Khao Kheow open zoo, Thailand.

Eld's deer (Rucervus eldii thamin) is an endangered species endemic to South Asia. Various ectoparasites (hematophagous insects and ticks) and blood parasites (e.g., piroplasms such as Babesia and Theileria) have been reported in this deer. Deer keds of the genus Lipoptena (L.) are wingless hematophagous insects acting as ectoparasites and potential vectors, thereby transmitting diseases to animals and humans. Many Lipoptena species have been reported, including L. fortisetosa; the latter may be a potential vector of several pathogens such as Babesia spp. and Theileria spp. However, the available data regarding Lipoptena in domestic animals and wildlife in Thailand is limited. The aim of this study was to investigate the presence of L. fortisetosa in Eld's deer as well as the role of this insect as a disease vector in Thailand by employing molecular analysis. A total of 91 wingless insects were collected and morphologically identified as L. fortisetosa. A partial fragment of the cytochrome c oxidase subunit I gene (COI) was amplified and successfully sequenced from twelve insects, and the COI nucleotide Basic Local Alignment Search Tool results revealed a 94.28%-94.45% identity to L. fortisetosa (accession number: OL850869/China). The undertaken phylogenetic analysis revealed that the L. fortisetosa samples from Thailand belong to a clade that is distinct from the previously deposited (in GenBank®) L. fortisetosa. As far as the pathogen detection is concerned, 46.2% (42/91) of the deer keds were positive for Theileria, while no Lipoptena was found to be positive for Babesia. Twenty-one sequences of Theileria were obtained and exhibited a 98.84%-100% identity to the Theileria sp. from several hosts. The phylogenetic analysis of Theileria revealed that Theileria capreoli and Theileria cervi were present in our L. fortisetosa samples. It can be implied that L. fortisetosa may serve as a vector of Theileria spp. in the Eld's deers of Thailand. We believe that the particular open zoo (from where the sampling took place) should implement preventive and control strategies for deer keds, other vectors, and vector-borne diseases.