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.

Infection, dissemination, and transmission of lumpy skin disease virus in Aedes aegypti (Linnaeus), Culex tritaeniorhynchus (Giles), and Culex quinquefasciatus (Say) mosquitoes.

Lumpy skin disease virus (LSDV) is a transboundary viral disease in cattle and water buffaloes. Although this Poxvirus is supposedly transmitted by mechanical vectors, only a few studies have investigated the role of local vectors in the transmission of LSDV. This study examined the infection, dissemination, and transmission rates of LSDV in Aedes aegypti, Culex tritaeniorhynchus, and Culex quinquefasciatus following artificial membrane feeding of 102.7, 103.7, 104.7 TCID50/mL LSDV in sheep blood. The results demonstrated that these mosquito species were susceptible to LSDV, with Cx tritaeniorhynchus exhibiting significantly different characteristics from Ae. aegypti and Cx. quinquefasciatus. These three mosquito species were susceptible to LSDV. Ae. aegypti showed it as early as 2 days post-infection (dpi), indicating swift dissemination in this particular species. The extrinsic incubation period (EIP) of LSDV in Cx. tritaeniorhynchus and Cx. quinquefasciatus was 8 and 14 dpi, respectively. Ingestion of different viral titers in blood did not affect the infection, dissemination, or transmission rates of Cx. tritaeniorhynchus and Cx. quinquefasciatus. All rates remained consistently high at 8-14 dpi for Cx. tritaeniorhynchus. In all three species, LSDV remained detectable until 14 dpi. The present findings indicate that, Ae. aegypti, Cx. tritaeniorhynchus, and Cx. quinquefasciatus may act as vectors during the LSDV outbreak; their involvement may extend beyond being solely mechanical vectors.

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.

34-kDa salivary protein enhances duck Tembusu virus infectivity in the salivary glands of Aedes albopictus by modulating the innate immune response.

Duck Tembusu virus (DTMUV) is an important flavivirus that can be transmitted to poultry via Aedes albopictus bites. Furthermore, humans residing in the DTMUV epidemic area display activated antiviral immune responses to local DTMUV isolates during the pathogenic invasion, thereby raising the primary concern that this flavivirus may be transmitted to humans via mosquito bites. Therefore, we identified the gene AALF004421, which is a homolog of the 34-kDa salivary protein (34 kDa) of Ae. albopictus and studied the salivary protein-mediated enhancement of DTMUV infection in Ae. albopictus salivary glands. We observed that double-stranded RNA-mediated silencing of the 34 kDa in mosquito salivary glands demonstrated that the silenced 34 kDa impaired DTMUV infectivity, similar to inhibition through serine protease. This impairment occurred as a consequence of triggering the innate immune response function of a macroglobulin complement-related factor (MCR). 34-kDa in the salivary gland which had similar activity as a serine protease, results in the abrogation of antimicrobial peptides production and strong enhance DTMUV replication and transmission. Although the function of the 34 kDa in Ae. albopictus is currently unknown; in the present study, we showed that it may have a major role in DTMUV infection in mosquito salivary glands through the suppression of the antiviral immune response in the earliest stages of infection. This finding provides the first identification of a prominently expressed 34 kDa protein in Ae. albopictus saliva that could serve as a target for controlling DTMUV replication in mosquito vectors.

Establishment of molecular diagnostics targeting the 23S ribosomal RNA gene for the detection of Mycoplasma suis infection in Thai domestic pigs.

Mycoplasma (M.) suis is a pathogenic hemotropic Mycoplasma sp. that causes acute hemolytic anemia or chronic infection in pigs. M. suis infection can be diagnosed using several methods, including molecular diagnosis such as conventional PCR (cPCR) and quantitative PCR (qPCR). In these cases, the common target is the 16S rRNA gene; however, this genetic marker cannot distinguish hemoplasma at the species level owing to high sequence identity. Therefore, the 23S rRNA gene has emerged as another target gene. Other than PCR, the loop-mediated isothermal amplification (LAMP) method can be applied for M. suis. The objective of the present study was to establish cPCR, TaqMan qPCR, and LAMP assays in which the 23S rRNA gene is used to detect M. suis infection in Thai domestic pigs. The analytical sensitivity of cPCR was determined as 7.46 × 104 copies/µl of plasmid DNA, whereas those of qPCR and LAMP were 7.46 × 102 copies/µl. There was no cross reaction with other pathogens in any of the assays. To evaluate the diagnostic performance of the assays, they were tested using 173 samples of genomic DNA. The detection percentage of M. suis infection was 24.86% (43/173; 95% CI: 18.61%-31.89%), 28.32% (49/173; 95% CI: 21.75%-35.66%), and 29.48% (51/173; 95% CI: 22.80%-36.88%) using cPCR, qPCR, and LAMP, respectively. Using qPCR as a reference assay, cPCR showed 81.63% sensitivity, 97.58% specificity, and an almost perfect level of agreement (kappa = 0.823). In comparison, LAMP showed 77.55% sensitivity, 89.52% specificity, and a substantial level of agreement (kappa = 0.662). All assays tested here could be applied in veterinary diagnostic laboratories for monitoring porcine health in the herds. Furthermore, the LAMP assay could be used as a screening test in farm practice without the need for any special equipment.

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.

Effects of Aedes aegypti salivary protein on duck Tembusu virus replication and transmission in salivary glands.

Duck Tembusu virus (DTMUV) infection is an arthropod-borne viral disease that affects many poultry species, including ducks, chickens, and geese. Aedes aegypti mosquito is an important vector of DTMUV. This study sought to determine whether any individual Ae. aegypti salivary protein modulated DTMUV replication in the mosquito salivary gland. Ae. aegypti salivary gland protein of 34 kDa (AaSG34) was found to be expressed explicitly in mosquito salivary glands and was upregulated following DTMUV infection. Thus, AaSG34 was silenced in mosquitoes via RNA interference using double strand RNA (dsRNA), and the mosquitoes were then infected with DTMUV to elucidate their effects on DTMUV replication and transmission. Transcripts of the DTMUV genome in salivary glands and virus titer in saliva were significantly diminished when AaSG34 was silenced, indicating that its presence enhances DTMUV replication in the salivary glands and DTMUV dissemination to saliva. Furthermore, the expression of antimicrobial peptides (AMPs) was upregulated upon AaSG34 silenced. Our results demonstrate that AaSG34 may play a vital role in the suppression of antiviral immune responses to enhance DTMUV replication and transmission. We thus provide new information on the effect of the AaSG34 salivary protein on DTMUV replication in Ae. aegypti as the mechanism of blocking virus transmission to the host.

A simplified method for blood feeding, oral infection, and saliva collection of the dengue vector mosquitoes.

A simple device using folded Parafilm-M as an artificial blood feeder was designed for studying two important dengue vector mosquitoes, Aedes aegypti and Aedes albopictus. The efficiency of the artificial blood feeder was investigated by comparing the numbers of engorged mosquitoes that fed on the artificial blood feeder versus mice as a live blood source. Significantly more engorged females Aedes aegypti fed on the artificial blood feeder than on mice. In addition, the artificial feeder could serve as a useful apparatus for oral infection via artificial blood meals, and for saliva collection in mosquitoes. Our method enabled us to collect saliva from multiple mosquitoes at once, providing sufficient infected saliva for determination of the virus titer by plaque assay analysis. Our artificial feeder has the advantage that it is simple, inexpensive, and efficient.

The Notched-frons Katydids of Taiwan (Orthoptera: Tettigoniidae: Conocephalinae: Copiphorini): redescriptions and keys for identification.

The tribe Copiphorini is one of four tribes in the subfamily Conocephalinae (Orthoptera: Tettigoniidae). It is easily distinguished from other Tettigoniids by the markedly slanting head shape and the notched frons. This study revises the tribe in Taiwan. The specimens, which were examined for this study, were collected in Taiwan from October 1962 to May 2008 and deposited in the Museum of National Chung Hsing University and National Museum of Natural Science. There are five genera and six species distributed in Taiwan such as Euconocephalus nasutus (Thunberg, 1815), Euconocephalus pallidus (Redtenbacher, 1891), Pseudorhynchus gigas Redtenbacher, 1891, Pyrgocorypha formosana Matsumura Shiraki 1908, Ruspolia lineosa (Walker, 1869), and Xestophrys horvathi Bolivar, 1905. These are redescribed and a key is provided to the genera and species from Taiwan. Notes are given on the habitats and distribution of the species.

A salivary protein of Aedes aegypti promotes dengue-2 virus replication and transmission.

Although dengue is the most prevalent arthropod-borne viral disease in humans, no effective medication or vaccine is presently available. Previous studies suggested that mosquito salivary proteins influence infection by the dengue virus (DENV) in the mammalian host. However, the effects of salivary proteins on DENV replication within the Aedes aegypti mosquito remain largely unknown. In this study, we investigated the effect of a specific salivary protein (named AaSG34) on DENV serotype 2 (DENV2) replication and transmission. We showed that transcripts of AaSG34 were upregulated in the salivary glands of Aedes aegypti mosquitoes after a meal of blood infected with DENV2. Transcripts of the dengue viral genome and envelop protein in the salivary glands were significantly diminished after an infectious blood meal when AaSG34 was silenced. The effect of AaSG34 on DENV2 transmission was investigated in Stat1-deficient mice. The intradermal inoculation of infectious mosquito saliva induced hemorrhaging in the Stat1-deficient mice; however, saliva from the AaSG34-silenced mosquitoes did not induce hemorrhaging, suggesting that AaSG34 enhances DENV2 transmission. This is the first report to demonstrate that the protein AaSG34 promotes DENV2 replication in mosquito salivary glands and enhances the transmission of the virus to the mammalian host.