Haemosporidian Parasites of White-Breasted Waterhens (Amaurornis phoenicurus), with a Report and Molecular Characterization of Haemoproteus gallinulae in Thailand.

Haemosporidian parasites are vector-borne parasites infecting terrestrial vertebrates as well as avian species, such as the White-breasted Waterhen, a Gruiformes waterbird found in lowlands near wetlands and distributed throughout Thailand. However, information regarding haemosporidia infection in this species is lacking. To establish regional information, 17 blood samples were collected from White-breasted Waterhens. Four haemoparasite lineages were identified in six blood samples: Haemoproteus gallinulae, Plasmodium collidatum, Plasmodium elongatum, and an unidentified Plasmodium species. H. gallinulae was characterized with morphological features in White-breasted Waterhens for the first time; the morphological characteristics were consistent with previous descriptions. H. gallinulae was more closely related to Haemoproteus species of Passeriformes birds than to those of Gruiformes birds. The Plasmodium parasites infecting these White-breasted Waterhens previously caused severe avian malaria in other host species. The unidentified Plasmodium species had rarely been documented, although it was reported in the Culex vector and was possibly associated with specialist parasites either as host or habitat. Our findings reveal multiple haemosporidian species reflecting the role of this avian host as a carrier of haemosporidians. This study offers species records and molecular materials that may provide critical information for further targeted research into these haemosporidia.

Baseline study of the morphological and genetic characteristics of Haemoproteus parasites in wild pigeons (Columba livia) from paddy fields in Thailand.

Haemoproteus columbae is a common haemosporidian parasite of wild pigeons (Columba livia) reported worldwide. In Thailand, the wild pigeon population is increasing due to paddy field monoculture. However, there are limited reports on the presence of H. columbae in these pigeon populations. The aim of the study was to characterize H. columbae in wild pigeons. A total of 87 wild pigeons were examined using microscopic and molecular methods. Haemoproteus columbae was detected in approximately 27.6% of pigeons and their morphological characteristics were described. The partial cytochrome b (cyt b) gene sequence of H. columbae was then characterized into three common lineages (HAECOL1, COLIV03, and COQUI05). By highlighting the morphologic and genetic characteristics of H. columbae commonly found in this population of pigeons, this study provides essential regional knowledge about haemosporidian parasites that could benefit future taxonomic and phylogeographic studies.

Performance of the onstructural 1 Antigen Rapid Test for detecting all four DENV serotypes in clinical specimens from Bangkok, Thailand.

BACKGROUND: Dengue is an arboviral disease that has a large effect on public health in subtropical and tropical countries. Rapid and accurate detection of dengue infection is necessary for diagnosis and disease management. We previously developed highly sensitive immunochromatographic devices, the TKK 1st and TKK 2nd kits, based on dengue virus (DENV) nonstructural protein 1 detection. However, these TKK kits were evaluated mainly using DENV type 2 clinical specimens collected in Bangladesh, and further validation using clinical specimens of other serotypes was needed. METHODS: In the present study, one of the TKK kits, TKK 2nd, was evaluated using 10 DENV-1, 10 DENV-2, 4 DENV-3, 16 DENV-4, and 10 zika virus-infected clinical specimens collected in Bangkok, Thailand. RESULTS: The TKK 2nd kit successfully detected all four DENV serotypes in patient serum specimens and did not show any cross-reactivities against zika virus serum specimens. The IgM and/or IgG anti-DENV antibodies were detected in seven serum specimens, but did not seem to affect the results of antigen detection in the TKK 2nd kit. CONCLUSION: The results showed that the TKK 2nd kit successfully detected all four DENV serotypes in clinical specimens and confirmed the potential of the kit for dengue diagnosis in endemic countries.

Overview of avian influenza virus in urban feral pigeons in Bangkok, Thailand.

This survey assessed the presence of avian influenza virus (AIV) in urban feral pigeons (UFPs) in Bangkok, Thailand. A total of 485 UFPs were collected from eight study sites, and blood, tracheal, and cloacal samples were collected from each bird. Virus isolation and molecular methods did not detect AIV in any of the birds tested. A hemagglutination inhibition test was used to test for antibodies to high and low pathogenicity AIV subtypes. AIV subtype H9 antibodies were the only antibodies detected. The overall seroprevalence of AIV subtype H9 antibodies was 6.9%, and subtype H9 antibodies were found in UFPs at all eight sites. The overall geometric mean titer was 11.07 (range: 8-64). These results reveal that UFPs in Bangkok do not currently pose a risk of transmitting AIV to humans. However, monitoring of AIV in UFPs is necessary for disease control and to minimize the possibility of influenza outbreaks.

Development of a Dengue Virus Serotype-Specific Non-Structural Protein 1 Capture Immunochromatography Method.

Four serotypes of dengue virus (DENV), type 1 to 4 (DENV-1 to DENV-4), exhibit approximately 25-40% of the difference in the encoded amino acid residues of viral proteins. Reverse transcription of RNA extracted from specimens followed by PCR amplification is the current standard method of DENV serotype determination. However, since this method is time-consuming, rapid detection systems are desirable. We established several mouse monoclonal antibodies directed against DENV non-structural protein 1 and integrated them into rapid DENV detection systems. We successfully developed serotype-specific immunochromatography systems for all four DENV serotypes. Each system can detect 104 copies/mL in 15 min using laboratory and clinical isolates of DENV. No cross-reaction between DENV serotypes was observed in these DENV isolates. We also confirmed that there was no cross-reaction with chikungunya, Japanese encephalitis, Sindbis, and Zika viruses. Evaluation of these systems using serum from DENV-infected individuals indicated a serotype specificity of almost 100%. These assay systems could accelerate both DENV infection diagnosis and epidemiologic studies in DENV-endemic areas.

Genetic Diversity of Dengue Virus in Clinical Specimens from Bangkok, Thailand, during 2018-2020: Co-Circulation of All Four Serotypes with Multiple Genotypes and/or Clades.

Dengue is an arboviral disease highly endemic in Bangkok, Thailand. To characterize the current genetic diversity of dengue virus (DENV), we recruited patients with suspected DENV infection at the Hospital for Tropical Diseases, Bangkok, during 2018-2020. We determined complete nucleotide sequences of the DENV envelope region for 111 of 276 participant serum samples. All four DENV serotypes were detected, with the highest proportion being DENV-1. Although all DENV-1 sequences were genotype I, our DENV-1 sequences were divided into four distinct clades with different distributions in Asian countries. Two genotypes of DENV-2 were identified, Asian I and Cosmopolitan, which were further divided into two and three distinct clades, respectively. In DENV-3, in addition to the previously dominant genotype III, a cluster of 6 genotype I viruses only rarely reported in Thailand was also observed. All of the DENV-4 viruses belonged to genotype I, but they were separated into three distinct clades. These results indicated that all four serotypes of DENV with multiple genotypes and/or clades co-circulate in Bangkok. Continuous investigation of DENV is warranted to further determine the relationship between DENV within Thailand and neighboring countries in Southeast Asia and Asia.

A Novel Sub-Lineage of Chikungunya Virus East/Central/South African Genotype Indian Ocean Lineage Caused Sequential Outbreaks in Bangladesh and Thailand.

In recent decades, chikungunya virus (CHIKV) has become geographically widespread. In 2004, the CHIKV East/Central/South African (ECSA) genotype moved from Africa to Indian ocean islands and India followed by a large epidemic in Southeast Asia. In 2013, the CHIKV Asian genotype drove an outbreak in the Americas. Since 2016, CHIKV has re-emerged in the Indian subcontinent and Southeast Asia. In the present study, CHIKVs were obtained from Bangladesh in 2017 and Thailand in 2019, and their nearly full genomes were sequenced. Phylogenetic analysis revealed that the recent CHIKVs were of Indian Ocean Lineage (IOL) of genotype ECSA, similar to the previous outbreak. However, these CHIKVs were all clustered into a new distinct sub-lineage apart from the past IOL CHIKVs, and they lacked an alanine-to-valine substitution at position 226 of the E1 envelope glycoprotein, which enhances CHIKV replication in Aedes albopictus. Instead, all the re-emerged CHIKVs possessed mutations of lysine-to-glutamic acid at position 211 of E1 and valine-to-alanine at position 264 of E2. Molecular clock analysis suggested that the new sub-lineage CHIKV was introduced to Bangladesh around late 2015 and Thailand in early 2017. These results suggest that re-emerged CHIKVs have acquired different adaptations than the previous CHIKVs.

Rickettsiae exposure related to habitats of the oriental house rat (Rattus tanezumi, Temminck, 1844) in Salaya suburb, Thailand.

Rickettsial zoonotic diseases, in particular scrub typhus, murine typhus, and tick typhus, are caused by Orientia tsutsugamushi, Rickettsia typhi, and Rickettsia honei infections. Rickettsiae exposure is typically related to a rodent host in various habitats of marginal regions, including between rural areas and communities such as the Salaya suburb. This allows the oriental house rat (OHR), a highly adaptive species, to live in close proximity to the community and possibly introduce rickettsial diseases. To understand rickettsial exposure in the OHR from different habitats, knowledge of disease exposure can serve as baseline information for disease management and prevention. A total of 185 OHRs from three unrelated habitats were assessed using a standard indirect immunofluorescence assay (IFA) for specific antibody reaction to O. tsutsugamushi, R. typhi, and R. honei. The presence of antibody in the OHR to rickettsiae, either scrub or murine typhus, was associated with the habitat, whereas tick typhus had general exposure. This finding shows the OHR to be a potential reservoir host for rickettsial diseases along the borders of geographic regions in the suburban environment.

Serological evidence of influenza virus infection in captive wild felids, Thailand.

Influenza virus is known to affect wild felids. To explore the prevalence of influenza viruses in these animal species, 196 archival sera from 5 felid species including Panthera tigris (N=147), Prionailurus viverrinus (N=35), Panthera leo (N=5), Pardofelis temminckii (N=8) and Neofelis nebulosa (N=1) collected between 2011 and 2015 in 10 provinces of Thailand were determined for the presence of antibody to avian and human influenza viruses. Blocking enzyme-linked immunosorbent (ELISA) assay and hemagglutination inhibition (HI) assay were employed as the screening tests, which the serum samples with HI antibody titers ≥20 were further confirmed by cytopathic effect/hemagglutination based-microneutralization (CPE/HA-based microNT) test. Based on HI and microNT assays, the seropositive rates of low pathogenic avian influenza (LPAI) H5 virus, highly pathogenic avian influenza (HPAI) H5 virus and human H1 virus were 1.53% (3/196), 2.04% (4/196) and 6.63% (13/196), respectively. In addition, we also found antibody against both LPAI H5 virus and HPAI H5 virus in 2 out of 196 tested sera (1.02%). Evidences of influenza virus infection were found in captive P. tigris in Kanchanaburi, Nakhon Sawan and Ratchaburi provinces of Thailand. The findings of our study highlights the need of a continuous active surveillance program of influenza viruses in wild felid species.

Antibody reaction of leptospirosis in asymptomatic feral boars, Thailand.

AIMS: This study aimed to determine the proportion of exposure to leptospirosis and evaluate the degree of serovar antibody reaction in feral boars. MATERIALS AND METHODS: A total of 58 sera obtained from feral boars in Khao Prathab Chang Wildlife Breeding Center, Ratchaburi, Thailand, were screened for leptospirosis exposure by microscopic agglutination test, conducted with a reference panel of 23 pathogenic serovars and a non-pathogenic serovar. RESULTS: Overall exposure rate of 62.07% was found in the studied population. An antibody reaction presented in 18 of 24 leptospiral serovars. Among the seropositive, Ballum serovar showed predominant exposure in the feral boar population. CONCLUSION: The results show a relatively high exposure to leptospirosis and the predominant serovar was Ballum followed by Canicola, the first finding in feral boars in Thailand. It has been revealed that feral boars act as a natural reservoir host of leptospirosis. There should be more concern about public health problems in leptospirosis arising where feral boars appear.

Optimisation of a serum albumin removal protocol for use in a proteomic study to identify the protein biomarkers for silent gastric ulceration in horses.

Silent gastric ulceration occurs without evidence of clinical signs and is common in horses. There is currently no a simple and effective method to diagnose this disease. Proteomics can be used to identify serum biomarkers, but the most abundant serum protein, albumin, could conceal candidate biomarkers. Therefore, it is recommended to remove albumin before a proteomic study; however, there is no specific albumin depletion kit or standard protocol available for horse samples. The objectives of this study were to optimise a protocol to remove equine serum albumin and to use albumin-depleted serum to identify the protein biomarkers for silent gastric ulceration. Gastroscopy was used to identify gastric ulceration, and serum was obtained from horses with either a healthy gastric mucosa or gastric ulceration. Serum albumin was removed using the trichloroacetic acid (TCA) protein precipitation method, and this protocol was optimised by varying the concentration of TCA, type of organic solvents, ratio of serum to protein precipitation solution, and incubation times. Electrophoresis and image analysis were used to compare the amounts of albumin, immunoglobulins G (IgG), and protein degradation before and after TCA precipitation. The best protocol was chosen to remove albumin for a proteomic study (electrophoresis and mass spectrometry). The results revealed that protocol 2 (ratio of serum to solution 1:5, 10% TCA in acetone, and 90 min incubation) was the most efficient protocol to remove albumin (98%) and IgG heavy (80%) and light (98%) chains without degrading other proteins. After electrophoresis and mass spectrometry analysis, KRT1, KRT6A and KRT18 were identified as potential markers for silent gastric ulceration.

A LONG-TERM SEROSURVEY OF AVIAN INFLUENZA H5 AMONG WILD BIRDS IN NAKHON SAWAN PROVINCE, THAILAND.

An outbreak of HPAIV H5N1 in Nakhon Sawan province, Thailand, in 2004 caused sporadic deaths of Asian openbill storks ( Anastomus oscitans). An investigation was undertaken to determine if this virus occurs and circulates in wild birds in Nakhon Sawan province. Following the outbreak, a widespread serosurvey was conducted using the hemagglutination inhibition assay and microneutralization assay to detect antibodies against AIV H5. From 2007 to 2014, blood was collected from a total of 753 wild birds, representing 10 orders and 44 species. The results reveal that 10 serum samples were positive for AIV H5 antibodies. These seropositive results, found in the orders Ciconiiformes and Anseriformes, demonstrate that waterfowl serve as a reservoir host of AIV. Moreover, the seroprevalences in streak-eared bulbul showed habitat sharing with waterfowl or duck.

Serosurveillance for pandemic influenza A (H1N1) 2009 virus infection in domestic elephants, Thailand.

The present study conducted serosurveillance for the presence of antibody to pandemic influenza A (H1N1) 2009 virus (H1N1pdm virus) in archival serum samples collected between 2009 and 2013 from 317 domestic elephants living in 19 provinces situated in various parts of Thailand. To obtain the most accurate data, hemagglutination-inhibition (HI) assay was employed as the screening test; and sera with HI antibody titers ≥20 were further confirmed by other methods, including cytopathic effect/hemagglutination based-microneutralization (microNT) and Western blot (WB) assays using H1N1pdm matrix 1 (M1) or hemagglutinin (HA) recombinant protein as the test antigen. Conclusively, the appropriate assays using HI in conjunction with WB assays for HA antibody revealed an overall seropositive rate of 8.5% (27 of 317). The prevalence of antibody to H1N1pdm virus was 2% (4/172) in 2009, 32% (17/53) in 2010, 9% (2/22) in 2011, 12% (1/8) in 2012, and 5% (3/62) in 2013. Notably, these positive serum samples were collected from elephants living in 7 tourist provinces of Thailand. The highest seropositive rate was obtained from elephants in Phuket, a popular tourist beach city. Young elephants had higher seropositive rate than older elephants. The source of H1N1pdm viral infection in these elephants was not explored, but most likely came from close contact with the infected mahouts or from the infected tourists who engaged in activities such as elephant riding and feeding. Nevertheless, it could not be excluded that elephant-to-elephant transmission did occur.

Detection and characterization of Chlamydophila psittaci in asymptomatic feral pigeons (Columba livia domestica) in central Thailand.

OBJECTIVE: To detect and characterize Chlamydophila psittaci (C. psittaci) in asymptomatic feral pigeons in central Thailand. METHODS: A total 814 swabs from the trachea and cloacae of 407 non-clinical feral pigeons in central Thailand were collected and tested for the presence of C. psittaci. RESULTS: A 10.8% of feral pigeons in the sample group were positive as determined by nested PCR primer specific to C. psittaci. The outer membrane protein A (ompA) gene of positive samples exhibited amino acid identity of C. psittaci ranging from 71 to 100% and were grouped in genotype B. Exceptionally, BF1676-56 isolate was closely related to Chlamydia avium with 99% identification of the 16S ribosomal (r) RNA gene. CONCLUSIONS: This is the first report on C. psittaci isolated from asymptomatic feral pigeons in Thailand, which provides knowledge for the disease status in pigeon populations in Thailand.