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.

Genetic diversity and characterization of Wolbachia endosymbiont in canine filariasis.

Canine filariasis is caused by nematodes from the family Onchocercidae, which is transmitted by arthropod vectors. The disease is commonly found in Southeast Asia and exists worldwide. Some filarial nematodes are associated with intracellular bacteria of the genus Wolbachia, which plays an important role in embryogenesis, molting, and the long-term survival of adult worms. This study aims to characterize Wolbachia sp. and determine the association between Wolbachia and canine filarial nematode species in Thailand. A total of 46 dog blood samples that were naturally infected with filarial nematodes were obtained to identify filarial nematode species by Giemsa stained under a light microscope and confirmed using the molecular technique. In order to characterize Wolbachia sp., the nested PCR assay targeting the 16S rRNA gene showed that all samples of Dirofilaria immitis and fifteen samples of Candidatus Dirofilaria hongkongensis were grouped into Wolbachia supergroup C. In addition, all samples of Brugia spp. and five samples of Candidatus Dirofilaria hongkongensis were classified into Wolbachia supergroup D. The genetic diversity analysis conducted using the 16S rRNA gene revealed a similar result when analyzed through phylogenetic tree analysis. This is the first genetic diversity study of Wolbachia of Candidatus Dirofilaria hongkongensis in infected dogs in Thailand.

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.

Molecular detection of Mycoplasma wenyonii and its closely related hemotropic Mycoplasma sp. in blood-sucking flies from a buffalo farm in Chachoengsao province, Thailand.

Bovine hemoplasmosis is a disease in buffaloes and cattle caused by hemotropic mycoplasmas or hemoplasmas. Only two bovine hemoplasma species, Mycoplasma wenyonii and Candidatus Mycoplasma haemobos, have been described in several countries. Hemoplasmas induce acute hemolytic anemia or chronic infection, leading to production loss. Bovine hemoplasma DNA was also detected in blood-sucking arthropods, suggesting vector transmission in farms. To date, no studies of the molecular detection of bovine hemoplasmas in Thai buffaloes and arthropod vectors have been reported. This study aimed to study the 1-year diversity of hematophagous flies in a buffalo farm located in Chachoengsao province, Thailand, and to investigate the molecular occurrence of bovine hemoplasmas in those flies using a polymerase chain reaction (PCR) assay and sequence analyses. A total of 1,488 mosquitoes, 867 stable flies, and 312 tabanid flies were collected during this study. The most abundant mosquitoes, stable flies, and tabanid flies were Culex tritaeniorhynchus, Stomoxys calcitrans, and Tabanus megalops, respectively. A total of 249 genomic DNA samples of flies were tested using a PCR assay based on the 16S rRNA gene; 23.69% (59/249) of the insect samples were positive in this assay. Positive samples (n = 8) were subjected to bidirectional sequencing. The BLAST results showed that only three samples from Stomoxys calcitrans and two samples from Tabanus megalops showed 99.90% and 99.17% similarities to the M. wenyonii isolate B003 (MG948626/Water buffalo/Cuba) and the M. wenyonii isolate C124 (MG948625/Cattle/Cuba), respectively. This molecular occurrence of bovine hemoplasmas in blood-sucking flies suggested that those flies are the mechanical vectors for bovine hemoplasmas in Thailand. Based on the phylogenetic analysis of the 16S rRNA gene, the sequences of M. wenyonii were likely classified into two subgroups (A and B), suggesting closely related bovine hemoplasma species. Finally, the genetic analysis of the 23S rRNA gene from these two subgroups revealed that subgroup A could be M. wenynoii and subgroup B may be a subspecies of M. wenyonii or another putative novel species. However, further investigation should be conducted in buffaloes, cattle, and blood-sucking flies to gain more 16S rRNA and 23 rRNA gene sequences of bovine hemoplasmas.

Molecular detection and genetic analysis of porcine haemoplasmas in commercial pig farms from Thailand reveal a putative novel species.

Haemoplasma is a trivial name for haemotropic Mycoplasma spp., which can attach to the surface of red blood cells leading to deformity and anaemia in a wide range of mammalian animals, including pigs. In Thailand, there is only one study that reported the occurrence of Mycoplasma suis without other haemoplasma species. In this study, we examined the molecular occurrence and genetic diversity of porcine haemoplasmas in Thai domestic pigs (Sus scrofa domesticus) from commercial farms using a PCR assay targeting the 16S rRNA gene, DNA sequencing, nucleotide sequence type (ntST) analysis and phylogenetic analysis. A total of 665 blood samples were collected from pigs at thirteen farms located in eight provinces of Thailand during 2019-2020. Genomic DNA was extracted from blood samples and tested by PCR. The frequency of haemoplasma infection was 37.1% (247/665, 95% CI: 33.5%-40.9%) in all pigs. Among 247 PCR positive samples, 194 were sequenced and analysed by nucleotide BLAST, ntST diversity, phylogenetic trees and ntST networks. The results of this genetic analysis indicated that at least four species with 27 nucleotide sequence types (Mycoplasma suis, Mycoplasma parvum, Candidatus Mycoplasma haemosuis and a putative novel species) of porcine haemoplasmas were identified. Thus, it appears that haemoplasmas show a high genetic diversity in the Thai pig population. In addition, a putative novel species was genetically characterized by other markers, namely, the 23S rRNA and RNase P RNA (rnpB) genes. For phylogenetic analysis, Candidatus Mycoplasma haemosuis was placed into the Mycoplasma haemofelis group, and the three remaining species were placed into the Mycoplasma suis group in all trees containing the 16S rRNA, 23S rRNA and rnpB genes. Further studies, such as pathobiology and epidemiology, should be conducted to better characterize this putative novel species.