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.

Perspectives on veterinary education in Thailand.

Veterinary education is the foundation of veterinary services in the country. Starting from the service sector in the army, veterinary education and practice in Thailand have been standardized and progressed toward international veterinary standards. The 6-year Doctor of Veterinary Medicine core curriculum is deployed to develop the curriculum for each Veterinary Education Establishment (VEE). The challenges for veterinary education and practices reflect the country's expectations of veterinary services. With regional and global collaboration, the VEEs have been developing tools and learning platforms for delivering qualified veterinary graduates that fit fast-growing society needs.

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.

First Report of Anuran Trypanosoma DNA in Flat-Tailed House Geckos (Reptilia: Gekkonidae) Collected from Southern Thailand: No Evidence as a Reservoir for Human Trypanosomatids.

Over the years, cases of autochthonous leishmaniasis have been dramatically increasing in Thailand. Recently, several publications have claimed certain species of the phlebotomine sand flies and biting midges potentially serve as natural vectors of Leishmania and Trypanosoma species in this country. However, more information regarding the vector-parasite relationships, as well as their natural reservoirs in the country, still needs to be explored. Herein, we hypothesized that synanthropic reptiles in the leishmaniasis-affected area might be a natural reservoir for these parasites. In this present study, a total of nineteen flat-tailed house geckos were collected from the house of a leishmaniasis patient in Songkhla province, southern Thailand, and then dissected for their visceral organs for parasite detection. Small subunit ribosomal RNA (SSU rRNA) gene and internal transcribed spacer 1 (ITS-1)-specific amplifications were conducted to verify the presence of Trypanosoma and Leishmania parasites, respectively. Only Trypanosoma DNA was screened positive in eight gecko individuals by SSU rRNA-PCR in at least one visceral organ (4, 4, and 6 of the heart, liver, and spleen, respectively) and phylogenetically related to the anuran Trypanosoma spp. (An04/Frog1 clade) previously detected in three Asian sand fly species (Phlebotomus kazeruni, Sergentomyia indica, and Se. khawi). Hence, our data indicate the first detection of anuran Trypanosoma sp. in the flat-tailed house geckos from southern Thailand. Essentially, it can be inferred that there is no evidence for the flat-tailed house gecko (Hemidactylus platyurus) as a natural reservoir of human pathogenic trypanosomatids in the leishmaniasis-affected area of southern Thailand.

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.

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.

Development of a novel multiplex PCR assay for the detection and differentiation of Plasmodium caprae from Theileria luwenshuni and Babesia spp. in goats.

Intraerythrocytic parasites are traditionally identified by the microscopic examination of Giemsa-stained blood smears. However, this method does not always allow for the identification of individual species in goat's RBCs. Moreover, its unreliability in detecting low levels of parasitemia makes it unsuitable for epidemiological investigations and leaves goat farms vulnerable to potential outbreaks. In the present study, a novel multiplex PCR (mPCR) targeting the cytochrome c oxidase subunit I (COI) gene was developed to detect and subsequently differentiate Plasmodium caprae, Theileria luwenshuni, and Babesia spp. The specificity of each primer set was assessed both in silico and with a panel of DNA samples from the hosts themselves and other goat hemoparasites. Amplicons generated from each pair of primers were 664, 555, and 320-bp for P. caprae, Babesia spp., and T. luwenshuni, respectively. These products were further confirmed by sequencing. Our novel mPCR reactions successfully demonstrated the accurate and simultaneous amplification of the three parasites' DNA samples. The current mPCR method showed no cross-amplification with unintended targets. The detection limit of the mPCR in this study was 108 parasites' DNA copies per reaction. The current mPCR was able to detect the minimum parasitemia of approximately 0.001%, 0.000005%, 0.00001% for P. caprae, Babesia spp. and T. luwenshuni, respectively. The diagnostic specificity in the detection of P. caprae and T. luwenshuni ranged from 94.9 to 100 %. The mPCR was further applied to a collection of field blood samples from five provinces in Thailand to validate its reliability and applicability. The results demonstrated the successful detection of P. caprae, Babesia spp. and T. luwenshuni in goat samples with the same sensitivity levels as conventional PCR methods. This study also confirmed the presence of T. luwenshuni and Babesia spp. in Thai goats. The current mPCR method offers an alternative for the diagnosis of P. caprae, T. luwenshuni, and Babesia spp., either single or under co-infection conditions, and for large-scale surveillance.

Development and application of a novel multiplex PCR assay for the differentiation of four haemosporidian parasites in the chicken Gallus gallus domesticus.

Haemosporidian infections in domestic chickens (Gallus gallus domesticus) are not only widely prevalent but also cause economic loss. Diagnosis is usually made by microscopic examination; however, the method has several drawbacks such as requiring an experienced microscopist, being unreliable when parasitemia is low and being unable to accurately differentiate between co-infections from multiple parasite species. Therefore, the current extent of haemosporidian infections might be underestimated and neglected. We have developed a novel multiplex PCR assay to simultaneously detect and differentiate between four haemosporidian parasites: Leucocytozoon caulleryi, Leucocytozoon sabrazesi, Plasmodium juxtanucleare and Plasmodium gallinaceum. Primers in the present study specifically amplified the corresponding targets with no cross-species amplification detected. The multiplex PCR exhibited a significantly greater detection rate when compared with microscopic examination (p = 0.0001). The results demonstrate that the detection rate of multiplex PCR for L. sabrazesi, P. juxtanucleare, and P. gallinaceum are all greater than that of microscopic examination with p = 0.002, 0.0001 and 0.004, respectively. Co-infections were also detected more effectively by multiplex PCR. We applied the current method to field samples originating from Nan, Prachinburi, and Chachoengsao Provinces. The current study revealed that positive rates of haemosporidian parasites in chickens in the three study sites ranging from 39.5%-93.8%. The present assay offers a timesaving option for molecular diagnosis instead of using singleplex PCRs for detecting the parasites individually. Within a single reaction, this assay would be a useful tool for the detection of avian haemosporidian parasites either single or under co-infection conditions and for large-scale epidemiology studies.

Patterns of duck Tembusu virus infection in ducks, Thailand: a serological study.

Duck Tembusu virus (DTMUV), a mosquito-borne flavivirus, has been identified as a causative agent of an emerging viral disease in ducks, causing significant economic losses to the duck-producing industry. In Thailand, DTMUV has been detected sporadically in ducks since the first report in 2013. However, information on the patterns of DTMUV infection in ducks in Thailand is limited. In this study, a serological survey of DTMUV on ducks raised in farming and free-grazing systems was conducted during 2015-2016. Blood samples of farm ducks (n = 160) and free-grazing ducks (n = 240) were collected in the summer, rainy, and winter seasons during 2015-2016 and tested for DTMUV infection. Our results showed that DTMUV infection in ducks in Thailand occurred all year-round; however, the patterns of DTMUV infection varied between 2 duck-raising systems. Significant seasonal pattern was found in free-grazing ducks, whereas no seasonality was observed in farm ducks. Notably, DTMUV infection in ducks in Thailand was highest in the winter season. In conclusion, our data indicate distinct patterns of DTMUV infection between farm and free-grazing ducks, and the year-round circulation of DTMUV in ducks in Thailand, with peaks in the winter season. This information will help reduce the risk of DTMUV transmission through prevention and control strategies focusing on the peak period. Routine surveillance of DTMUV in ducks is essential for early detection of DTMUV allowing the implementation of control measures in a timely manner.

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.

Molecular detection and genetic characterization of Anaplasma marginale and Anaplasma platys-like (Rickettsiales: Anaplasmataceae) in water buffalo from eight provinces of Thailand.

BACKGROUND: Anaplasmosis, an animal disease caused by rickettsial bacteria in the genus Anaplasma, is of considerable economic importance in livestock animals in many countries worldwide. The objectives of this study were to determine the identity, prevalence, and geographic distribution of Ehrlichia and Anaplasma in naturally infected water buffalo in Thailand using PCR amplification and sequencing of the 16S ribosomal RNA and heat shock protein groEL genes. A total of 456 buffalo blood samples from Thailand were investigated. Species identification and genetic differentiation of intra-population and inter-population with the global isolates were conducted based on nucleotide sequences. Interplay between the infection and host factors was also assessed. RESULTS: Overall, 41% of water buffalo were found to be infected with rickettsial organisms in the family Anaplasmataceae, but Ehrlichia spp., Neorickettsia spp., and Wolbachia spp. were not found in any of the sequenced samples in this study. Female buffalo were more frequently infected with bacteria in the family Anaplasmataceae than males [71 out of 176 females (40.3%) versus 11 out of 47 males (23.4%)]. The Odds Ratio value indicated that the risk of infection for female buffalo was 2.2-fold higher than that for males (p < 0.05). We detected three haplotypes of A. marginale 16S rRNA gene and they were placed in a clade that was closely related to the A. marginale in buffalo in China; and cattle in Thailand, Uganda, and China. Homology searching of groEL sequences against the GenBank™ database using the BLASTn algorithm revealed that the obtained sequences had a high percentage similarity (98.36-99.62%) to A. platys sequences. The groEL sequences of three A. platys-like isolates were clustered in the same clade as the A. platys from the tick Rhipicephalus microplus in China. CONCLUSIONS: Our data showed that the apparently healthy buffalo were naturally infected by bacteria in the family Anaplasmataceae at a relatively high prevalence. We also report the finding of A. platys-like infections in water buffalo in Thailand for the first time. Water buffalo serving as the reservoir host of anaplasmosis is of concern for managing the disease control and prevention in ruminants.

Synergistic infection of Ichthyophthirius multifiliis and Francisella noatunensis subsp. orientalis in hybrid red tilapia (Oreochromis sp.).

Francisella noatunensis subsp. orientalis (Fno) and Ichthyophthirius multifiliis (Ich) are deadly infectious pathogens in farmed tilapia, particularly during cold season when the water temperature drops to under 25 °C. We hypothesized that infection of the ectoparasite Ich might enhance susceptibility of hybrid red tilapia (Oreochromis sp.) to the facultative intracellular bacterium Fno. To prove the hypothesis, the experiment was designed as follows. Hybrid red tilapia naturally infected by Ich at 9 ± 6 theronts/fish gills and 4 ± 3 theronts/fish skin were distributed into 5 distinct groups exposed to different concentrations of Fno. In parallel, the same number of Ich-free tilapia were challenged to only Fno in the same manner. The results showed that cumulative mortality in the Fno single infection with 2.88 × 106 CFU mL-1 of water was 25 ± 7%, whereas 100% mortality was found in the coinfection treatment at dose of 1.93 × 105 CFU mL-1 of water. No mortality was observed in both control groups (Ich-infected and Ich-free fish). The coinfected fish revealed typical clinical signs and histopathological manifestations of francisellosis and ichthyophthiriasis. This study revealed synergistic effect of the Ich and Fno infection in hybrid red tilapia leading to the exacerbated mortality. Thus, farming management of fish to be free from the Ich ectoparasite might reduce risk of francisellosis and probably other bacterial diseases in farmed tilapia.

Low level of genetic diversity and high occurrence of vector-borne protozoa in water buffaloes in Thailand based on 18S ribosomal RNA and mitochondrial cytochrome b genes.

Vector-borne pathogens (VBPs) pose a great risk to ruminant production through significant economic losses. Several previous studies in Thailand have mainly been focused on the health of dairy and beef cattle. Water buffaloes are one of the important ruminants in the country, but studies on their infection with VBPs remains limited. We conducted a molecular survey on blood samples from 456 buffaloes obtained from eight provinces across different geographical locations of Thailand. The PCR diagnostics indicated that 116 (25.4%) and 59 (12.9%) of these 456 samples were positive for piroplasm and Plasmodium spp., respectively, and were found in six and all eight regions, respectively, across Thailand. Co-infections of piroplasm and Plasmodium spp. were observed in 24 cases (5.26%). Babesia spp. was not detected in any of the 12 sequenced piroplasm-positive samples in the present study. Genetic comparisons and phylogenetic analyses of within and between parasite populations, based on the 18S ribosomal (r)RNA and cytochrome b (cytb) genes for T. orientalis and P. bubalis, respectively, revealed that T. orientalis shared a high similarity within its population and could be divided into four distinct haplotypes. Haplotypes 1 and 4 were placed in the same clade with the samples previously isolated from cattle in Korea, Japan, Australia, and the USA. Haplotypes 2, and 3 were novel and were placed in a separate clade not shared with the other isolates. We also confirmed our previous investigation that at least three cytb haplotypes of P. bubalis were distributed in the country with a relatively high degree of genetic polymorphisms within its population (based on cytb sequences). Type II P. bubalis was phylogenetically closely related to P. caprae in goats in Zambia and Thailand. This study improves our current understanding on the distribution, intra- and inter-population genetic diversity, and genetic relationship of piroplasms and Plasmodium spp. in water buffaloes.