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.

Generation of protoplasts provides a powerful experimental research tool for biological and pathogenicity studies of Pythium insidiosum.

INTRODUCTION: Pythiosis is a high-mortality infectious condition in humans and animals. The etiologic agent is Pythium insidiosum. Patients present with an ocular, vascular, cutaneous/subcutaneous, or gastrointestinal infection. Antifungal medication often fails to fight against P. insidiosum. The effective treatment is limited to radical surgery, resulting in organ loss. Fatal outcomes are observed in advanced cases. Pythiosis needs to be studied to discover novel methods for disease control. Genome data of P. insidiosum is publicly available. However, information on P. insidiosum biology and pathogenicity is still limited due to the lack of a cost-effective animal model and molecular tools. MATERIALS AND METHODS: We aimed to develop a high-efficiency protocol for generating P. insidiosum protoplast, and used it to set up an animal model, in vitro drug susceptibility assay, and DNA transformation for this pathogen. RESULTS: P. insidiosum protoplast was successfully generated to establish a feasible pythiosis model in embryonic chicken eggs and an efficient in vitro drug susceptibility assay. DNA transformation is a critical method for gene manipulation necessary for functional genetic studies in pathogens. Attempts to establish a DNA transformation method for P. insidiosum using protoplast were partly successful. Significant work needs to be done for genetically engineering a more robust selection marker to generate stable transformants at increased efficiency. CONCLUSION: This study is the first to report an efficient P. insidiosum protoplast production for clinical and research applications. Such advances are crucial to speeding up the pathogen's biology and pathogenicity exploration.

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.

Diversity of midgut microbiota in laboratory-colonized and field-collected Aedes albopictus (Diptera: Culicidae): A preliminary study.

Aedes (Ae.) albopictus is an important vector for many pathogens. Previous studies have revealed a role for midgut bacteria during pathogen infection in mosquitoes; however, studies of Ae. albopictus midgut bacteria are limited. We examined the diversity of midgut bacteria in female laboratory-colonized and field-collected Ae. albopictus. A total of 31 bacterial genera were identified representing 10 and 28 genera of laboratory-colonized and field-collected Ae. albopictus, respectively. The predominant bacterial genera in the laboratory-colonized Ae. albopictus were Staphylococcus and Micrococcus, whereas the bacterial diversity in the field-collected Ae. albopictus exhibited a higher proportion of Rhizobium and Agrobacterium as the dominant genera. However, only Staphylococcus showed a significant difference between laboratory-colonized and field-collected Ae. albopictus. The midgut bacterial species were identified from 30 laboratory-colonized Ae. albopictus mosquitoes. A total of 16 bacterial species were identified and the predominant bacterial species was Micrococcus luteus, followed by Staphylococcus epidermidis and Agrobacterium tumefaciens. Field mosquitoes were collected from the Sing Buri, Chumphon, and Yala Provinces of Thailand. The midgut bacterial species identified from the 10 Ae. albopictus collected from the Sing Buri Province included Bacillus subtilis, Staphylococcus haemolyticus, Staphylococcus hominis, and Serratia marcescens. Serratia marcescens was the only bacteria identified from this area. Midgut bacterial species were identified from 40 filed-collected Ae. albopictus from Chumphon Province. A total of 25 bacterial species were identified and the predominant species were Enterobacter cloacae, Micrococcus luteus, and Providencia rettgeri. Only 15 bacterial species were identified from the mosquitoes collected from Chumphon Province. A total of 18 bacterial species were identified from 30 Ae. albopictus collected from Yala Province and the predominant species were Rhizobium pusense and Agrobacterium tumefaciens. Only 12 bacterial species were found in mosquitoes collected from Yala Province. These findings indicate changes in the midgut bacteria population in Ae. albopictus from various locales, which may result from variability in the blood-meal source, diet, or habitat. A comprehensive survey of the midgut bacteria community prevalence in wild populations is critical for not only gaining a better understanding of the role of this bacterium in shaping the microbial community in Ae. albopictus, but also for informing current and future mosquito and disease control programs.

Interactions of duck Tembusu virus with Aedes aegypti and Aedes albopictus mosquitoes: Vector competence and viral mutation.

Duck Tembusu virus (DTMUV) is an emerging flavivirus that causes severe disease in avian hosts, while also affecting mammalian hosts; however, information on viral interaction with mosquito vectors for mammalian hosts is limited. Vector competence of Aedes (Ae.) aegypti and Aedes albopictus mosquitoes for DTMUV were investigated. Both Aedes mosquito species were orally infected with DK/TH/CU-1 strain of Thai DTMUV and isolated DTMUV from BALB/c mouse. Genomes of the viruses isolated from hosts and vectors were analyzed and compared with the positive virus. Findings showed that both Aedes mosquito species could serve as vectors for DTMUV with minimum viral titer in blood meal of 106 TCID50/mL. After taking blood meal with viral titer at 107 TCID50/mL, vector competence of the mosquitoes was significantly different from the lower titer in both species. Both Aedes species did not support development of the isolated viruses from mouse. A point mutation of nucleotide and amino acid was found in all isolated DTMUV from Ae. aegypti saliva, while other viruses were similar to the positive virus. Our findings demonstrated that both Ae. aegypti and Ae. albopictus had potential to transmit the virus and play important roles in the viral transmission cycle in mammalian hosts, while viral mutation occurred in Ae. aegypti mosquitoes.

Pathogenesis of Thai duck Tembusu virus in BALB/c mice: Descending infection and neuroinvasive virulence.

Duck Tembusu virus (DTMUV) is an emerging flavivirus that causes systemic disease in an avian host. The predominant cluster of DTMUV circulating in Thailand was recently classified as cluster 2.1. The pathogenesis of this virus has been extensively studied in avian hosts but not in mammalian hosts. Six-week-old BALB/c mice were intracerebrally or subcutaneously inoculated with Thai DTMUV to examine clinical signs, pathological changes, viral load and virus distribution. Results demonstrated that the virus caused disease in BALB/c mice by the intracerebral inoculation route. Infected mice demonstrated both systemic and neurological symptoms. Pathological changes and virus distribution were observed in all tested organs. Viral load in the brain was significantly higher than in other organs (p < .05), and the virus caused acute death in BALB/c mice. The virus was disseminated in all parts of the body, but no virus shedding was recorded in saliva and faeces. Findings highlighted the potential of Thai DTMUV to transmit disease in mammalian hosts.

Vector competence of Culex tritaeniorhynchus and Culex quinquefasciatus (Diptera: Culicidae) for duck Tembusu virus transmission.

Duck Tembusu virus (DTMUV), an emerging infectious disease in ducks, was detected in Culex (Cx.) tritaeniorhynchus mosquitoes collected from a duck farm; however, the exact role of mosquitoes in the ecology of DTMUV in Thailand remains unclear. Vector competence of Cx. tritaeniorhynchus and Cx. quinquefasciatus was examined for DTMUV. Cx. tritaeniorhynchus mosquitoes were allowed to feed on four levels (102, 103, 104, and 105 TCID50/mL) of DTMUV, while Cx. quinquefasciatus were allowed to feed on two levels (104 and 105 TCID50/mL) of DTMUV. Infection rates in Cx. tritaeniorhynchus were 1.6, 10.2, 35.8, and 59.3% after feeding on 102, 103, 104, and 105 TCID50/mL of DTMUV, respectively, while dissemination and transmission were 20.3 and 16.9% after feeding on 105 TCID50/mL of DTMUV. Infection rates in Cx. quinquefasciatus were 2.5 and 2.3% after feeding on 104 and 105 TCID50/mL of DTMUV, respectively, with no virus dissemination and transmission found in all tested mosquitoes. Another study was conducted to examine the transovarial transmission of DTMUV in Cx. tritaeniorhynchus. Mosquitoes were allowed to feed on blood meal infected with 105 TCID50/mL of DTMUV. Each blood-fed mosquito was isolated and allowed to lay eggs. After oviparity, the mosquitoes were tested for DTMUV infection; 43 DTMUV infected and 37 non-infected female mosquitoes with eggs were included. A total of 182 F1 progeny from DTMUV infected mosquitoes and 145 F1 progeny from non-infected mosquitoes were tested for DTMUV but all were negative. Findings indicated the potential role of Cx. tritaeniorhynchus in the DTMUV transmission cycle in duck farms in Thailand. No transovarial transmission of DTMUV was found in Cx. tritaeniorhynchus.

Duck Tembusu virus detection and characterization from mosquitoes in duck farms, Thailand.

Duck Tembusu virus (DTMUV), an emerging infectious disease in ducks, belongs to the Flavivirus genus and Flaviviridae family. The transmission of DTUMV involves mosquito vectors; however, the exact role of mosquitoes in the ecology of DTMUV in Thailand remains unclear. This study was conducted to examine DTMUV detection and characterization from mosquitoes in duck farms in central Thailand. Mosquitoes were collected from two duck farms in Sing Buri Province and two duck farms in Ang Thong Province from September 2015 to July 2016 using four CDC-light traps. A total of 30,841 mosquitoes were collected and identified to seven species (Anopheles (An.) barbirostris, An. stephensi, Culex (Cx.) gelidus, Cx. quinquefasciatus, Cx. tritaeniorhynchus, Mansonia (Ma.) annulifera and Ma. uniformis). The most common collected species from each duck farm and each collection time was Cx. tritaeniorhynchus. Mosquitoes were pooled according to species, location, and collection time and then examined for DTMUV by RT-PCR. A total of 273 mosquito pools were examined, with only one pool of Cx. tritaeniorhynchus collected from Sing Buri Province in November 2015 testing positive for DTMUV. Phylogenetic analysis of the polyprotein genes demonstrated that a mosquito-derived Thai DTMUV was grouped into subcluster 2.1 and most closely related to the 2013 Thai DTMUVs. Thus, this study indicated that Cx. tritaeniorhynchus may play a role as a vector in the transmission of DTMUV in Thailand. However, additional studies concerning the vector competence of this mosquito for DTMUV are needed.

Vector competence of Aedes albopictus (Skuse) and Aedes aegypti (Linnaeus) for Plasmodium gallinaceum infection and transmission.

Avian malaria caused by Plasmodium gallinaceum is an important mosquito-borne disease. Eradication of this disease remains problematic since its competent vectors are diverse and widely distributed across the globe. Several mosquito species were implicated as competent vectors for this parasite. However, studies on vector competence for P. gallinaceum remain limited. In this study, vector competence in the two most predominant mosquito vectors in tropical countries, Aedes albopictus and Ae. aegypti, was compared. In order to determine their infection rates, Ae. albopictus (>F10), Ae. aegypti (>F10), and Ae. aegypti ( F10) and Ae. aegypti (> F10) and 40-60% by infected Ae. aegypti (F10) and Ae. aegypti (>F10) were highly competent vectors for P. gallinaceum infections. These mosquitoes play a crucial role in the transmission cycle of this parasite in nature.