Serological and Molecular Surveillance for SARS-CoV-2 Infection in Captive Tigers (Panthera tigris), Thailand.

Coronavirus disease (COVID-19) is an emerging infectious disease caused by SARS-CoV-2. Given the emergence of SARS-CoV-2 variants, continuous surveillance of SARS-CoV-2 in animals is important. To monitor SARS-CoV-2 infection in wildlife in Thailand, we collected 62 blood samples and nine nasal- and rectal-swab samples from captive tigers (Panthera tigris) in Ratchaburi province in Thailand during 2020-2021. A plaque reduction neutralization test (PRNT) was employed to detect SARS-CoV-2 neutralizing antibodies. A real-time RT-PCR assay was performed to detect SARS-CoV-2 RNA. Our findings demonstrated that four captive tigers (6.5%, 4/62) had SARS-CoV-2 neutralizing antibodies against Wuhan Hu-1 and the Delta variant, while no SARS-CoV-2 RNA genome could be detected in all swab samples. Moreover, a low-level titer of neutralizing antibodies against the Omicron BA.2 subvariant could be found in only one seropositive tiger. The source of SARS-CoV-2 infection in these tigers most likely came from close contact with the infected animals' caretakers who engaged in activities such as tiger petting and feeding. In summary, we described the first case of natural SARS-CoV-2 infection in captive tigers during the COVID-19 outbreak in Thailand and provided seroepidemiological-based evidence of human-to-animal transmission. Our findings highlight the need for continuous surveillance of COVID-19 among the captive tiger population and emphasize the need to adopt a One Health approach for preventing and controlling outbreaks of COVID-19 zoonotic disease.

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.

Zoonotic pathogens survey in free-living long-tailed macaques in Thailand.

Long-tailed macaques (Macaca fascicularis) are known to harbour a variety of infectious pathogens, including zoonotic species. Long-tailed macaques and humans coexist in Thailand, which creates potential for interspecies pathogen transmission. This study was conducted to assess the presence of B virus, Mycobacterium spp., simian foamy virus (SFV), hepatitis B virus (HBV), and Plasmodium spp. in 649 free-living Thai long-tailed macaques through polymerase-chain reaction. DNA of SFV (56.5%), HBV (0.3%), and Plasmodium spp. (2.2%) was detected in these macaques, whereas DNA of B virus and Mycobacterium spp. was absent. SFV infection in long-tailed macaques is broadly distributed in Thailand and is correlated with age. The HBV sequences in this study were similar to HBV sequences from orangutans. Plasmodium spp. DNA was identified as P. inui. Collectively, our results indicate that macaques can carry zoonotic pathogens, which have a public health impact. Surveillance and awareness of pathogen transmission between monkeys and humans are important.

Occurrence of Chlamydia spp. In wild birds in Thailand

Objective: To determine the occurrence of Chlamydia spp. in wild birds in Thailand. Methods: Cloacal and tracheal swabs of 313 wild birds from 11 orders, 27 families, and 51 species were tested to determine the occurrence of Chlamydia infection. The outer membrane protein A (ompA) gene was amplified from positive samples to construct a phylogenetic tree. Results: At the time of sample collection, none of the birds showed clinical signs of any disease. Of 313 wild birds, two Asian openbill stork (Anastomus oscitans) were positive for Chlamydia spp., representing 0.64% (2/313) and 4.9% (2/41) occurrence for birds overall and for the Asian openbill stork, respectively. Phylogram analysis based on deduced amino acid of the ompA gene showed that Chlamydia spp. in Asian openbill storks was closely related to that in wildfowl (Pica pica and Cygnus olor) from Poland in a different branch with a 95% bootstrap value and had a shorter evolutionary distance to Chlamydia abortus. Conclusions: Asymptomatic Asian openbill storks could be a potential source of Chlamydia infection in domestic animals, poultry, and humans who share their habitat.

Occurrence of Chlamydia spp. In wild birds in Thailand

Objective: To determine the occurrence of Chlamydia spp. in wild birds in Thailand. Methods: Cloacal and tracheal swabs of 313 wild birds from 11 orders, 27 families, and 51 species were tested to determine the occurrence of Chlamydia infection. The outer membrane protein A (ompA) gene was amplified from positive samples to construct a phylogenetic tree. Results: At the time of sample collection, none of the birds showed clinical signs of any disease. Of 313 wild birds, two Asian openbill stork (Anastomus oscitans) were positive for Chlamydia spp., representing 0.64% (2/313) and 4.9% (2/41) occurrence for birds overall and for the Asian openbill stork, respectively. Phylogram analysis based on deduced amino acid of the ompA gene showed that Chlamydia spp. in Asian openbill storks was closely related to that in wildfowl (Pica pica and Cygnus olor) from Poland in a different branch with a 95% bootstrap value and had a shorter evolutionary distance to Chlamydia abortus. Conclusions: Asymptomatic Asian openbill storks could be a potential source of Chlamydia infection in domestic animals, poultry, and humans who share their habitat.

Molecular characterization of Hepatozoon felis in Rhipicephalus sanguineus ticks infested on captive lions (Panthera leo).

Hepatozoon spp. are protozoan parasites that infect a wide range of domestic and wild animals. The infection occurs by ingestion of an infected tick. This study was carried out to detect and characterize Hepatozoon spp. in ticks collected from captive lions (Panthera leo) in Thailand based on the partial 18S rRNA gene sequence. A total of 30 ticks were collected and identified as Rhipicephalus sanguineus. The collected ticks were separated into 10 tick pools by sex and life stages. Of the 10 tick pools examined, only one (10%) was found to be infected with the Hepatozoon species. Sequencing and phylogenetic analysis showed a clustering of the partial 18S rRNA gene sequence like that of H. felis from the GenBank database. This is the first report of H. felis in R. sanguineus ticks collected from captive lions in Thailand. Our results indicated that R. sanguineus may be a possible vector of feline Hepatozoon in Thailand.

Molecular study of feline hemoplasmas in free- ranging fishing cats (Prionailurus viverrinus) in Thailand.

Feline hemoplasmas, consisting of Mycoplasma haemofelis (M. haemofelis), Candidatus Mycoplasma haemominutum (Ca. M. haemominutum), and Candidatus Mycoplasma turicensis (Ca. M. turicensis), cause feline infectious anemia and zoonoses. Using multiplex PCR and phylogenetic analysis based on 16S rRNA, 22 blood samples from fishing cats (Prionailurus viverrinus) living in Khao Sam Roi Yot National Park, Thailand were determined positive for M. haemofelis (13.6%)and Ca. M. haemominutum (22.7%). M. haemofelis and Ca. M. haemominutum infection can result severe anemia and asymptomatic, respectively. However, not all positive cases exhibit anemia symptoms. Future study of hemoplasma infection in wild felids is necessary for conservation and the preservation of public health in Thailand.

Seasonal abundance and potential of Japanese encephalitis virus infection in mosquitoes at the nesting colony of ardeid birds, Thailand.

OBJECTIVE: To investigate the abundance and seasonal dynamics of mosquitoes, and to detect Japanese encephalitis virus (JEV) in these mosquitoes at the nesting colony of ardeid birds. METHODS: Mosquitoes were collected bimonthly from July 2009 to May 2010 by Centers for Disease Control. Light traps and dry ice, as a source of CO2, were employed to attract mosquitoes. Mosquitoes were first identified, pooled into groups of upto 50 mosquitoes by species, and tested for JEV infection by viral isolation and reverse transcriptase polymerase chain reaction. RESULTS: A total of 20 370 mosquitoes comprising 14 species in five genera were collected. The five most abundant mosquito species collected were Culex tritaeniorhynchus (95.46%), Culex vishnui (2.68%), Culex gelidus (0.72%), Anopheles peditaeniatus (0.58%) and Culex quinquefasciatus (0.22%). Mosquito peak densities were observed in July. All of 416 mosquito pools were negative for JEV. CONCLUSIONS: This study provides new information about mosquito species and status of JEV infection in mosquitoes in Thailand. Further study should be done to continue a close survey for the presence of this virus in the ardeid birds.

Identification of Ehrlichia spp in canines in Thailand.

Canine ehrlichiosis is an endemic parasitic disease widely found in Thailand. The causative microorganism is tick-borne Ehrlichia spp, an obligate intracellular rickettsia residing in leukocytes. Ehrlichia spp in morulae-positive canine blood samples were identified using polymerase chain reaction amplification and direct sequencing of Ehrlichia spp. 16S rDNA 396 bp fragment and 36 of 59 were positive for E. canis. E. chaffeensis and E. ewingii were not detected. Sequencing alignment and phylogenetic analysis showed that 16S rDNA sequences of E. canis strains are 99.1-100% identical among E. canis strains from different countries worldwide. Further studies are required in order to determine new target sequence for genotyping of E. canis strains in the dog population in Thailand.

Detection and phylogenetic characterization of hepatitis E virus genotype 3 in a captive wild boar in Thailand.

Hepatitis E virus (HEV) was studied in different types of wild boar captive settings in Thailand, including a wildlife breeding research station, zoo, and commercial wild boar farm, which were located in different locations of Thailand. Fifty-one fecal samples were collected and screened for HEV RNA and then analyzed. One sample obtained from a wildlife breeding research station in Ratchaburi province was HEV positive. Phylogenetic characterization revealed that the virus was HEV genotype 3 and belongs to subgroup 3e, which is closely related to HEV recently isolated from domestic pigs and humans in the country. It was hypothesized that HEV is shared among wild boars, domestic pigs, and humans in Thailand.

Development of a SYBR Green I-based real-time PCR for detection of elephant endotheliotropic herpesvirus 1 infection in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus 1 (EEHV1) can cause fatal hemorrhagic disease in Asian elephants (Elephas maximus). Several studies have described this virus as a major threat to young Asian elephants. A SYBR Green I-based real-time polymerase chain reaction (PCR) was developed to identify EEHV1 on trunk swabs and necropsied tissues. Two of 29 (6.9%) trunk swab samples from healthy Asian elephants were positive for EEHV1. The viruses were analyzed and classified as EEHV1A based on 231 nucleotides of the terminase gene. Necropsied spleen and heart tissue showed the highest level and second highest levels of DNA virus copy accumulation, respectively. The detection limit of the test was 276 copies/µl of DNA. There was no cross-reaction with other mammalian herpesviruses, such as herpes simplex virus 1 and equine herpesvirus 2. Inter- and intra-assay showed low coefficients of variation values indicating the reproducibility of the test. The results indicated that the test can be practically used for epidemiological study, clinical diagnosis, and management and control of EEHV1.

Development of multiplex polymerase chain reaction for detection of feline hemotropic mycoplasma in blood and tissue specimens.

A multiplex polymerase chain reaction (PCR) was developed for the detection of feline hemotropic mycoplasmas which simultaneously differentiates infections of Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm) and Candidatus Mycoplasma turicensis (CMtc) in feline blood and spleen. These organisms are responsible for the cause of various pathogenicity of feline infectious anemia. These infections are difficult to be detected by microscopic examination, the most commonly used method for general laboratory diagnoses. Specific primers were designed by selected consensus 16S rDNA sequences of three distinct species. The multiplex PCR assay developed in this study was sensitive and specific with detection limit 100 copies/microl DNA of Mhf and CMhm and 10 copies/microl DNA of CMtc. No amplicons could be amplified for other blood parasites or bacterial pathogens. This multiplex PCR will allow studies of pathogenicity and the monitoring of clinical treatment.

Development of multiplex polymerase chain reaction for detection of Ehrlichia canis, Babesia spp and Hepatozoon canis in canine blood.

A multiplex polymerase chain reaction (PCR) has been developed for simultaneous detection of canine blood parasites, Ehrlichia canis, Babesia spp and Hepatozoon canis, from blood samples in a single reaction. The multiplex PCR primers were specific to E. canis VirB9, Babesia spp 16S rRNA and H. canis 16S rRNA genes. Specificity of the amplicons was confirmed by DNA sequencing. The assay was evaluated using normal canine and infected blood samples, which were detected by microscopic examination. This multiplex PCR offers scope for simultaneous detection of three important canine blood parasites and should be valuable in monitoring parasite infections in dogs and ticks.