High Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Virus Infection among the Dog Population in Thailand.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne zoonotic disease caused by the SFTS virus (SFTSV). In Thailand, three human cases of SFTS were reported in 2019 and 2020, but there was no report of SFTSV infection in animals. Our study revealed that at least 16.6% of dogs in Thailand were seropositive for SFTSV infection, and the SFTSV-positive dogs were found in several districts in Thailand. Additionally, more than 70% of the serum samples collected at one shelter possessed virus-neutralization antibodies against SFTSV and the near-complete genome sequences of the SFTSV were determined from one dog in the shelter. The dog SFTSV was genetically close to those from Thailand and Chinese patients and belonged to genotype J3. These results indicated that SFTSV has already spread among animals in Thailand.

Characterization and Homology Modeling of Catalytically Active Recombinant PhaCAp Protein from Arthrospira platensis.

Polyhydroxybutyrate (PHB) is a biocompatible and biodegradable polymer that has the potential to replace fossil-derived polymers. The enzymes involved in the biosynthesis of PHB are ß-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). PhaC in Arthrospira platensis is the key enzyme for PHB production. In this study, the recombinant E. cloni®10G cells harboring A. platensis phaC (rPhaCAp) was constructed. The overexpressed and purified rPhaCAp with a predicted molecular mass of 69 kDa exhibited Vmax, Km, and kcat values of 24.5 ± 2 µmol/min/mg, 31.3 ± 2 µM and 412.7 ± 2 1/s, respectively. The catalytically active rPhaCAp was a homodimer. The three-dimensional structural model for the asymmetric PhaCAp homodimer was constructed based on Chromobacterium sp. USM2 PhaC (PhaCCs). The obtained model of PhaCAp revealed that the overall fold of one monomer was in the closed, catalytically inactive conformation whereas the other monomer was in the catalytically active, open conformation. In the active conformation, the catalytic triad residues (Cys151-Asp310-His339) were involved in the binding of substrate 3HB-CoA and the CAP domain of PhaCAp involved in the dimerization.

Molecular detection of Loxodontofilaria spp. in Asian elephants (Elephas maximus) from elephant training camps in Thailand.

Filarial infection is an important disease in human and animal medicine. Several filarial worms are of importance, especially nematodes in the Onchocercidae. The Asian elephant (Elephas maximus) is an endangered animal and is very important from several socio-economic and ecological aspects in Thailand. Various parasites can be found in elephants; however, data related to filarial infections in elephants is limited. The objective of this study was to detect filaria in the blood of Asian elephants in Thailand, based on a polymerase chain reaction (PCR) technique. Blood samples were collected from 208 Asian elephants and detected for filaria using PCR, targeting the region of the internal transcribed spacer 2 (ITS2), the cytochrome c oxidase subunit 1 (cox1), and the RNA polymerase II large subunit (rbp1). In total, 4.33% (9 out of 208) of the sampled elephants had Loxodontofilaria spp. DNA with 100% query coverage. In addition, the obtained cox1 and rbp1 sequences matched with Loxodontofilaria sp., Onchocerca sp., and Dirofilaria sp. There were no identified risk factors (sex, age, location, and packed cell volume) related to Loxodontofilaria infection in elephants. The analyses of the phylogeny of ITS2 sequences demonstrated that the Loxodotofilaria-positive sequences were closely related to Onchocerca dewittei japonica and Onchocerca dewittei dewittei with 100% query coverage. Notably, the concatenated phylogenetic trees of ITS2 and the cox1 and rbp1 genes were closely similar to Loxodontofilaria sp. To describe in detail the genomic DNA of Loxodontofilaria spp., other genes should be additionally studied using a more discriminatory technique, such as DNA barcoding or whole genome sequencing.

Elucidating structure and dynamics of glutathione S-transferase from Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is tick parasite that affects the cattle industry worldwide. In R. (B.) microplus, acaricide resistance develops rapidly against many commercial acaricides. One of main resistance strategies is to enhance the metabolic detoxification mediated by R. (B.) microplus glutathione-S-transferase (RmGST). RmGST detoxifies acaricides by catalyzing the conjugation of glutathione to acaricides. Although structural and dynamic details of RmGST are expected to elucidate the biologic activity of this molecule, these data have not been available to date. Thus, Molecular Dynamics simulations were employed to study ligand-free RmGST at an atomic level. Like other m-class GSTs, the flexible m loop (m1) of RmGST was observed. M1 seems to shield the active sites from the bulk. A RmGST dimer is stabilized by the lock-and-key motif (F57 as "key") and hydrogen bonds of R82-E91 and R82-D98 at the dimer interface. Without substrates, conserved catalytic Y116 and N209 can interact with V112, G210 (for Y116) and F215 (for N209). Overall, most residues involving in RmGST function and stability are similar to other m-class GSTs. This implies similar structural stability and catalytic activity of RmGST to other GSTs. An insight obtained here will be useful for management of acaricide resistance and tick control.Communicated by Ramaswamy H. Sarma.

Plasmidome in mcr-1 harboring carbapenem-resistant enterobacterales isolates from human in Thailand.

The emergence of the mobile colistin-resistance genes mcr-1 has attracted significant attention worldwide. This study aimed to investigate the genetic features of mcr-1-carrying plasmid among carbapenem-resistant Enterobacterales (CRE) isolates and the potential genetic basis governing transmission. Seventeen mcr-harboring isolates were analyzed based on whole genome sequencing using short-read and long-read platforms. All the mcr-1-carrying isolates could be conjugatively transferred into a recipient Escherichia coli UB1637. Among these 17 isolates, mcr-1 was located on diverse plasmid Inc types, consisting of IncX4 (11/17; 64.7%), IncI2 (4/17; 23.53%), and IncHI/IncN (2/17; 11.76%). Each of these exhibited remarkable similarity in the backbone set that is responsible for plasmid replication, maintenance, and transfer, with differences being in the upstream and downstream regions containing mcr-1. The IncHI/IncN type also carried other resistance genes (blaTEM-1B or blaTEM-135). The mcr-1-harboring IncX4 plasmids were carried in E. coli ST410 (7/11; 63.6%) and ST10 (1/11; 9.1%) and Klebsiella pneumoniae ST15 (1/11; 9.1%), ST336 (1/11; 9.1%), and ST340 (1/11; 9.1%). The IncI2-type plasmid was harbored in E. coli ST3052 (1/4; 25%) and ST1287 (1/4; 25%) and in K. pneumoniae ST336 (2/4; 50%), whereas IncHI/IncN were carried in E. coli ST6721 (1/2; 50%) and new ST (1/2; 50%). The diverse promiscuous plasmids may facilitate the spread of mcr-1 among commensal E. coli or K. pneumoniae strains in patients. These results can provide information for a surveillance system and infection control for dynamic tracing.

Observing How Glutathione and S-Hexyl Glutathione Bind to Glutathione S-Transferase from Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is one of the most widespread ticks causing a massive loss to livestock production. The long-term use of acaracides rapidly develops acaracide resistance. In R. microplus, enhancing the metabolic activity of glutathione S-transferase (RmGST) is one of the mechanisms underlying acaracide resistance. RmGST catalyzes the conjugation of glutathione (GSH) to insecticides causing an easy-to-excrete conjugate. The active RmGST dimer contains two active sites (hydrophobic co-substrate binding site (H-site) and GSH binding site (G-site)) in each monomer. To preserve the insecticide efficacy, s-hexyl glutathione (GTX), a GST inhibitor, has been used as a synergist. To date, no molecular information on the RmGST-GSH/GTX complex is available. The insight is important for developing a novel RmGST inhibitor. Therefore, in this work, molecular dynamics simulations (MD) were performed to explore the binding of GTX and GSH to RmGST. GSH binds tighter and sits rigidly inside the G-site, while flexible GTX occupies both active sites. In GSH, the backbone mainly interacts with W8, R43, W46, K50, N59, L60, Q72, and S73, while its thiol group directs to Y7. In contrast, the aliphatic hexyl of GTX protrudes into the H-site and allows a flexible peptide core to form various interactions. Such high GTX flexibility and the protrusion of its hexyl moiety to the H-site suggest the dual role of GTX in preventing the conjugation reaction and the binding of acaracide. This insight can provide a better understanding of an important insecticide-resistance mechanism, which may in turn facilitate the development of novel approaches to tick control.

The Use of "Tail-Pedometers" to Evaluate the Impact of Dipterans in Feeder Cattle.

Hematophagous flies are a pest for livestock; their direct impact reduces productivity, and they are vectors of parasites, bacteria and viruses. Their control using insecticides is inefficient and highly polluting. The validation of new control tools requires efficacy and cost-effectiveness evaluation. The quantification of hematophagous insects' impact in livestock is a challenging prerequisite. Tail flicks counts can reliably evaluate fly-burden; however, visual records are tedious and time-consuming. In the present study, automation of tail flick counts was made through the use of pedometers attached to the tail, in two groups of feeder cattle. Group A was kept in a pen under the protection of a mosquito net, and Group B was kept in an open-air pen. The fly density of Group B was evaluated using fly traps. The apparent density per trap ranged from 130 to 1700 in the study. The mean pedometer records per 24 h ranged from 957+/-58 bits in Group A to 11,138+/-705 bits in Group B. The night/day records observed in Group A (200/800 bits) were drastically increased in Group B (1000-4000/4000-14,000 bits) and variable along seasons. A very high correlation was observed between fly density and visual records or pedometer records (PR). Two-hour PRs proved to be a reliable predictive tool for fly density. Moreover, the pedometers revealed an unsuspected but significant nuisance of mosquitoes, which should be thoroughly investigated.

Diagnosis of animal trypanosomoses: proper use of current tools and future prospects.

Reliable diagnostic tools are needed to choose the appropriate treatment and proper control measures for animal trypanosomoses, some of which are pathogenic. Trypanosoma cruzi, for example, is responsible for Chagas disease in Latin America. Similarly, pathogenic animal trypanosomoses of African origin (ATAO), including a variety of Trypanosoma species and subspecies, are currently found in Africa, Latin America and Asia. ATAO limit global livestock productivity and impact food security and the welfare of domestic animals. This review focusses on implementing previously reviewed diagnostic methods, in a complex epizootiological scenario, by critically assessing diagnostic results at the individual or herd level. In most cases, a single diagnostic method applied at a given time does not unequivocally identify the various parasitological and disease statuses of a host. These include "non-infected", "asymptomatic carrier", "sick infected", "cured/not cured" and/or "multi-infected". The diversity of hosts affected by these animal trypanosomoses and their vectors (or other routes of transmission) is such that integrative, diachronic approaches are needed that combine: (i) parasite detection, (ii) DNA, RNA or antigen detection and (iii) antibody detection, along with epizootiological information. The specificity of antibody detection tests is restricted to the genus or subgenus due to cross-reactivity with other Trypanosoma spp. and Trypanosomatidae, but sensitivity is high. The DNA-based methods implemented over the last three decades have yielded higher specificity and sensitivity for active infection detection in hosts and vectors. However, no single diagnostic method can detect all active infections and/or trypanosome species or subspecies. The proposed integrative approach will improve the prevention, surveillance and monitoring of animal trypanosomoses with the available diagnostic tools. However, further developments are required to address specific gaps in diagnostic methods and the sustainable control or elimination of these diseases.

Development and evaluation of indirect enzyme-linked immunosorbent assay using recombinant dense granule antigen 7 protein for the detection of Toxoplasma gondii infection in cats in Thailand.

Background and Aim: Toxoplasma gondii is recognized as a zoonosis causing toxoplasmosis in animals globally. Cat is a definitive host of T. gondii and sheds oocyst through feces, which can infect human beings and animals through contaminated food ingestion. A precise diagnostic test is essential to prevent T. gondii infection in both humans and animals. This study aimed to develop and evaluate the pETite-dense granule antigen 7(GRA7)-based indirect enzyme-linked immunosorbent assay (ELISA) to detect T. gondii infection in cats. Materials and Methods: T. gondii-GRA7 was cloned and expressed in the Expresso®small ubiquitin-related modifier (SUMO) T7 Cloning and Expression System. The recombinant pETite-GRA7 was purified using HisTrap affinity chromatography and confirmed using Western blot analysis. The recombinant protein was used to develop and evaluate the indirect ELISA for T. gondii infection detection. In total, 200 cat sera were tested using pETite-GRA7-based indirect ELISA and indirect fluorescent antibody test (IFAT). The statistical analysis based on Kappa value, sensitivity, specificity, positive predictive value, negative predictive value, χ 2 test, and receiver operating characteristic (ROC) curve was used to evaluate the performance of the test. Results: A 606 bp GRA7 polymerase chain reaction (PCR) product was obtained from T. gondii RH strain genomic DNA. The gene was cloned into the pETite™ vector and transformed to HI-Control Escherichia coli BL21 (DE3) for protein expression. Approximately 35 kDa of recombinant pETite-GRA7 was observed and Western blot analysis showed positive bands against anti-6-His antibody and positive-T. gondii cat serum. A sample of 0.5 µg/mL of pETite-GRA7 was subjected to indirect ELISA to detect T. gondii infection in the cat sera. The results showed sensitivity and specificity of pETite-GRA7-based indirect ELISA at 72% and 96%, respectively. An acceptable diagnostic performance was characterized by high concordant results (94%) and substantial agreement (Kappa value=0.65) with IFAT. The seroprevalence levels of ELISA and IFAT were 10% and 9%, respectively, and were not significantly (p>0.05) different. The expected performance of ELISA at different cutoff points using the ROC curve analysis revealed 89% sensitivity and 92% specificity at the cutoff value of 0.146, with a high overall assay accuracy (area under the curve=0.94). Conclusion: In this study, the pETite™ vector, N-terminal 6xHis SUMO fusion tag, was used to improve the solubility and expression level of GRA7. The recombinant pETite-GRA7 showed enhanced protein solubility and purification without special condition requirements. This pETite-GRA7-based indirect ELISA showed high concordant results and substantial agreement with IFAT. ELISA revealed an acceptable sensitivity and specificity. These initial data obtained from cats' sera demonstrated that pETite-GRA7-based indirect ELISA could be a useful method for local serological diagnosis of T. gondii infection in cats in Thailand.

A review on the diagnosis of animal trypanosomoses.

This review focuses on the most reliable and up-to-date methods for diagnosing trypanosomoses, a group of diseases of wild and domestic mammals, caused by trypanosomes, parasitic zooflagellate protozoans mainly transmitted by insects. In Africa, the Americas and Asia, these diseases, which in some cases affect humans, result in significant illness in animals and cause major economic losses in livestock. A number of pathogens are described in this review, including several Salivarian trypanosomes, such as Trypanosoma brucei sspp. (among which are the agents of sleeping sickness, the human African trypanosomiasis [HAT]), Trypanosoma congolense and Trypanosoma vivax (causing "Nagana" or animal African trypanosomosis [AAT]), Trypanosoma evansi ("Surra") and Trypanosoma equiperdum ("Dourine"), and Trypanosoma cruzi, a Stercorarian trypanosome, etiological agent of the American trypanosomiasis (Chagas disease). Diagnostic methods for detecting zoonotic trypanosomes causing Chagas disease and HAT in animals, as well as a diagnostic method for detecting animal trypanosomes in humans (the so-called "atypical human infections by animal trypanosomes" [a-HT]), including T. evansi and Trypanosoma lewisi (a rat parasite), are also reviewed. Our goal is to present an integrated view of the various diagnostic methods and techniques, including those for: (i) parasite detection; (ii) DNA detection; and (iii) antibody detection. The discussion covers various other factors that need to be considered, such as the sensitivity and specificity of the various diagnostic methods, critical cross-reactions that may be expected among Trypanosomatidae, additional complementary information, such as clinical observations and epizootiological context, scale of study and logistic and cost constraints. The suitability of examining multiple specimens and samples using several techniques is discussed, as well as risks to technicians, in the context of specific geographical regions and settings. This overview also addresses the challenge of diagnosing mixed infections with different Trypanosoma species and/or kinetoplastid parasites. Improving and strengthening procedures for diagnosing animal trypanosomoses throughout the world will result in a better control of infections and will significantly impact on "One Health," by advancing and preserving animal, human and environmental health.

Molecular characterization of Anaplasma marginale based on the msp1a and msp1b genes.

Anaplasma marginale is an intracellular rickettsial bacterium causing anaplasmosis in ruminants. A. marginale is transmitted biologically by ticks and mechanically by blood-sucking vectors. Anaplasmosis occurs in tropical and subtropical areas of the world. This disease causes huge economic losses due to decreasing meat yield and milk production. The aims of this study were to determine the genetic diversity and antigenicity of A. marginale based on the msp1a and msp1b genes in cattle in Thailand. The A. marginale msp1a and msp1b genes were amplified by the polymerase chain reaction (PCR). There have been four copies of MSP1a tandem repeats among A. marginale Thailand strain, and thirteen different MSP1a tandem repeats were found including repeats B, 25, 27, M, 3, S, C, H, ß, 80, 4, TH1 and TH2. Notably, this study showed two copies of the novel conserved tandem sequences namely Thailand Type 1 (TH1) and Type 2 (TH2). The phylogenetic analysis revealed that A. marginale msp1a and msp1b genes were genetically diverse and showed 9 and 5 clades with similarity ranging from 98 to 100% and 79.5 to 100%, respectively, when compared within the isolates of this study. The results of diversity analysis showed 18 and 16 haplotypes of the msp1a and msp1b genes, respectively. The entropy analyses of msp1a and msp1b nucleic acid sequences showed 39 and 900 high entropy peaks with values ranging from 0.35 to 0.85 and from 0.41 to 1.48, respectively, while those of MSP1a and MSP1b amino acid sequences exhibited 75 and 72 high entropy peaks with values ranging from 0.35 to 1.06 and from 0.41 to 1.55, respectively. In addition, B-cell and T-cell epitopes have also been investigated in this study. Hence, our results could be employed to improve the insight input of molecular phylogenetics, genetic diversity and antigenicity of A. marginale Thailand strain.

Molecular detection of Giardia duodenalis and Cryptosporidium spp. from stray dogs residing in monasteries in Bangkok, Thailand.

Both Cryptosporidium spp. and Giardia duodenalis are enteric protozoan parasites that infect a wide variety of domestic animals as well as humans worldwide, causing diarrheal diseases. Giardia duodenalis assemblages C and D are specific to canine hosts and zoonotic assemblages A and B are also found in dogs as a reservoir host. In dogs, Cryptosporidium canis is the host-specific species while humans are infected by C. hominis and C. parvum and at least another 16 zoonotic Cryptosporidium species have been reported causing human infections, with C. meleagridis, C. viatorum, and C. ubiquitum being the most frequent. The objective of this study was to determine the prevalence of Cryptosporidium spp. and G. duodenalis from stray dogs in areas of Bangkok and to identify the species and assemblages. Fecal samples (540) were collected from dogs residing in 95 monasteries in 48 districts in the Bangkok metropolitan area. Nested Polymerase Chain Reaction (PCR) was performed using the ssu-rRNA gene for both parasites. In total, 3.0% (16/540) samples were positive for G. duodenalis, with most being G. duodenalis assemblage D (7/16) followed by assemblage C (7/16) and zoonotic assemblage A (2/16). The prevalence of Cryptosporidium spp. was 0.7% (4/540) based on the PCR results and all were the dog genotype C. canis. These results indicated that dogs residing in Bangkok monasteries poses a limited role as source of human giardiosis and cryptosporidiosis.

Decoding the RNA viromes in rodent lungs provides new insight into the origin and evolutionary patterns of rodent-borne pathogens in Mainland Southeast Asia.

BACKGROUND: As the largest group of mammalian species, which are also widely distributed all over the world, rodents are the natural reservoirs for many diverse zoonotic viruses. A comprehensive understanding of the core virome of diverse rodents should therefore assist in efforts to reduce the risk of future emergence or re-emergence of rodent-borne zoonotic pathogens. RESULTS: This study aimed to describe the viral range that could be detected in the lungs of rodents from Mainland Southeast Asia. Lung samples were collected from 3284 rodents and insectivores of the orders Rodentia, Scandentia, and Eulipotyphla in eighteen provinces of Thailand, Lao PDR, and Cambodia throughout 2006-2018. Meta-transcriptomic analysis was used to outline the unique spectral characteristics of the mammalian viruses within these lungs and the ecological and genetic imprints of the novel viruses. Many mammalian- or arthropod-related viruses from distinct evolutionary lineages were reported for the first time in these species, and viruses related to known pathogens were characterized for their genomic and evolutionary characteristics, host species, and locations. CONCLUSIONS: These results expand our understanding of the core viromes of rodents and insectivores from Mainland Southeast Asia and suggest that a high diversity of viruses remains to be found in rodent species of this area. These findings, combined with our previous virome data from China, increase our knowledge of the viral community in wildlife and arthropod vectors in emerging disease hotspots of East and Southeast Asia. Video abstract.

Apigenin induces oxidative stress in mouse Sertoli TM4 cells.

BACKGROUND AND AIM: Apigenin (API) is an estrogenic compound found in many plants. Sertoli cells reside in the testis and are a key target of environmental toxicants. This study aimed to examine the cytotoxicity, especially oxidative stress of API in mouse Sertoli TM4 cells. MATERIALS AND METHODS: Mouse Sertoli TM4 cells were treated with 50 and 100 µM API for 48 h. Cell viability, lactate dehydrogenase (LDH) activities, glutathione reductase (GR) activities, production of reactive oxygen species (ROS), and malondialdehyde (MDA) levels were evaluated using various assays. RESULTS: Treatment with API at both 50 and 100 µM decreased viability and GR activity but increased LDH activity, ROS production, and MDA levels in mouse Sertoli TM4 cells. CONCLUSION: Exposure to API induced oxidative stress in mouse Sertoli TM4 cells.

Detection of specific IgM and IgG antibodies in acute canine monocytic ehrlichiosis that recognize recombinant gp36 antigens.

The efficacy of antibody detection tools for all stages of Ehrlichia canis infections and for various genotypes remains unclear. We produced recombinant gp36 (rgp36) antigens from different isolates of Thai E. canis to confirm the immunoreactivities to these recombinant proteins from naturally infected dogs. Sera and blood samples were taken from 21 dogs naturally infected with E. canis and in the clinical stages of acute phase ehrlichiosis. The expression vectors and competent E. coli produced two isolates of rgp36. These two major rgp36s were recognized by the dogs' sera in Western blotting, with both anti-dog IgM and IgG used as secondary antibodies. The two different genotypes of these local recombinant immunoreactive proteins were gp36 subgroup A (isolate 1055) and subgroup B (isolate 533). The Western blot analyses successfully identified both specific IgM and IgG from the dogs' sera. Of all 21 cases, five dogs presented specific IgM, twenty dogs presented specific IgG, and the commercial test used found fifteen seropositive dogs. There were four dogs that presented both specific IgM and IgG. Only one dog presented specific IgM only. This report is the first identification of a specific IgM in dogs in response to acute infections with E. canis. The recombinant gp36 isolates may be useful as potential antigenic material for subsequent serological tests that have a high possibility for differentiating between acute, chronic, primary, and nonprimary infections with E. canis.

Molecular detection and genetic diversity of Anaplasma marginale based on the major surface protein genes in Thailand.

Anaplasma marginale is the rickettsial agent of anaplasmosis, a tick-borne disease, which affects cattle and other ruminants in tropical and subtropical areas of the world, and causing huge economic losses because of decreasing meat and milk production. In the present study, molecular methods have been used to determine the occurrence and genetic diversity of A. marginale, based on the genes encoding the major surface proteins (msps) genes, in blood samples from 520 cattle and 121 buffaloes in the north and northeastern regions of Thailand. The polymerase chain reaction (PCR) results based on the msp4 gene indicated that 66 (10.30%) cattle were positive for A. marginale, whereas no positive result was obtained from buffaloes. The phylogenetic analysis based on the maximum likelihood method using 13, 29 and 27 nucleotide sequences from msp2, msp4, msp5 clones, respectively, revealed that the sequences detected in this study are obviously distributed in different clusters. The sequence analysis demonstrated that msp2 gene is genetically diverse, while msp4 and msp5 genes are conserved in Thailand. These findings corroborated the diversity analysis of the same sequences, which showed 13, 27 and 27 haplotypes of the msp2, msp4 and msp5 genes, respectively. In addition, the entropy analyses of amino acid sequences exhibited 127, 75 and 51 high entropy peaks with values ranging from 0.27119 to 2.45831, from 0.14999 to 2.17552 and from 0.15841 to 1.05453 for MSP2, MSP4 and MSP5, respectively. Therefore, the results indicate a low molecular occurrence of A. marginale in cattle blood samples in Thailand. From these results; however, a high degree of genetic diversity was observed in the analyzed A. marginale population. Hence, our finding could be used to improve the immunodiagnostics and vaccination programs for anaplasmosis.

Dynamic and structural insights into tick serpin from Ixodes ricinus.

Ixodid ticks have a crucial impact on people and domestic animals worldwide. These parasites also pose a serious threat to livestock. To date, vaccination of hosts against ticks is a safer, more sustainable alternative to chemical control of ticks and the disease agents they transmit. Because of their roles in tick physiology, serpins (serine protease inhibitors) from tick saliva are among the candidates for anti-tick vaccines. Inhibitory serpins employ a suicide inhibition mechanism to inhibit proteases, where the serpin reactive centre loop (RCL) is cleaved, by the targeted protease, and then inserted into the main ß-sheet of the serpin. This causes a massive conformational change called the 'stressed to relaxed' (S→R) transition, leading to the breakdown of serpin into two regions (core domain and cleaved polypeptide). Recently, the first tick serpin crystal structure from Ixodes ricinus in R-state was reported. We thus employed molecular dynamics simulations to better understand serpin structure and dynamics in atomic detail. Overall, R-state serpin showed high rigidity, especially the core domain. The most flexible region is the terminal of the cleaved polypeptide, due to its high-water exposure, while the rest of the cleaved polypeptide is stably trapped behind the core domain. T363, D367 and N375 are found to play a vital role in protein-protein attachment. This finding can be used to explain the high stability of the R-state serpin at the atomic level and provides insight into this tick serpin which will be useful for rational anti-tick vaccine development. AbbreviationsMDMolecular DynamicsRCLReactive centre loopCommunicated by Ramaswamy H. Sarma.

Distribution of Japanese Encephalitis Virus, Japan and Southeast Asia, 2016-2018.

During 2016-2018, we conducted surveillance for Japanese encephalitis virus (JEV) in mosquitoes and pigs in Japan, Thailand, the Philippines, and Indonesia. Phylogenetic analyses demonstrated that our isolates (genotypes Ia, Ib, III, IV) were related to JEV isolates obtained from the same regions many years ago. Indigenous JEV strains persist in Asia.

The Indirect ELISA Trypanosoma evansi in Equids: Optimisation and Application to a Serological Survey including Racing Horses, in Thailand.

Surra, caused by Trypanosoma evansi, is a widely distributed animal trypanosomosis; it affects both domestic and wild mammals with high economic impact. Clinical picture is moderate in bovines but severe in equids. Surra is also an important constraint for international animal trade and movements. Despite its impact, surra remains poorly diagnosed because of low sensitivity tests. To improve epidemiological knowledge of the disease and to secure international movement, efficient diagnosis tools are required. Here, we optimized and applied to equids the OIE-recommended indirect ELISA T. evansi that was validated in other species. Based on 96 positive and 1,382 negative horse reference samples from Thailand, a TG-ROC analysis was conducted to define the cutoff value. ELISA's sensitivity and specificity were estimated at 97.5% and 100%, respectively, qualifying the test to provide a reliable immune status of equids. The test was then applied on 1,961 horse samples from 18 Thai Provinces; the only scarce positives suggested that horses do not constitute a reservoir of T. evansi in Thailand. All samples from racing horses were negative. Conversely, two outbreaks of surra reported to our laboratory, originating from a bovine reservoir, exhibited high morbidity and lethality rates in horses. Finally, posttreatment follow-ups of infected animals allowed us to provide outbreak management guidelines.