First detection and genetic characterization of canine bufavirus in domestic dogs, Thailand.

Canine bufavirus (CBuV) was reported in domestic dogs worldwide. We conducted a survey of canine bufavirus in domestic dogs in Thailand from September 2016 to October 2022. Rectal swab samples (n = 531) were collected from asymptomatic dogs and dogs with gastroenteritis signs. The samples were tested for CBuV using PCR with specific primers to the VP1/VP2 gene, and 9.42% (50/531) was CBuV positive. Our findings showed that CBuVs could be detected in both symptomatic and healthy dogs. The Thai CBuVs were found in dogs from different age groups, with a significant presence in those under 1 year (12.60%) and dogs aged 1-5 years (7.34%) (p < 0.05), suggesting a high prevalence of Thai CBuVs in dogs under 5 years of age. We performed complete genome sequencing (n = 15) and partial VP1/VP2 sequencing (n = 5) of Thai CBuVs. Genetic and phylogenetic analyses showed that whole genomes of Thai CBuVs were closely related to Chinese and Italian CBuVs, suggesting the possible origin of Thai CBuVs. The analysis of VP1 and VP2 genes in Thai CBuVs showed that 18 of them were placed in subgroup A, while only 2 belonged to subgroup B. This study is the first to report the detection and genetic characterization of CBuVs in domestic dogs in Thailand. Additionally, surveillance and genetic characterization of CBuVs in domestic animals should be further investigated on a larger scale to elucidate the dynamic, evolution, and distribution of CBuVs.

Using whole blood cultures in interferon gamma release assays to detect Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus).

Elephants are susceptible to Mycobacterium tuberculosis (M. tb) complex (MTBC) infections. Diagnosis of tuberculosis (TB) in elephants is difficult, and most approaches used for human TB diagnosis are not applicable. An interferon gamma release assay (IGRA) to diagnose TB in Asian elephants (Elephas maximus) using peripheral blood mononuclear cells (PBMCs) has been previously developed. Although the assay is shown to be valid in determining MTBC infection status, the laborious PBMC isolation process makes it difficult to use. In this study, we simplified the method by using whole blood cultures (WC) as the starting material. Using PBMC cultures for IGRA, the MTBC infection status of 15 elephants was first confirmed. Among these animals, one has been previously confirmed for M. tb infection by both TB culture and PCR and the other was confirmed for MTBC infection in this study by droplet digital PCR (ddPCR) method. WC for IGRA consisted of an unstimulated sample, a mitogen stimulated sample, and sample stimulated with recombinant M. tb antigens, ESAT6 and CFP10. Using WC for IGRA in the 15 enrolled elephants, the results showed that 7 out of 15 samples yielded MTBC infection positive status that were completely concordant with those from the results using PBMCs. To test this method, WC for IGRA were applied in another elephant cohort of 9 elephants. The results from this cohort revealed a perfect match between the results from PBMC and WC. Responses to ESAT6 or CFP10 by PBMC and WC were not completely concordant, arguing for the use of at least two M. tb antigens for stimulation. Given the ease of sample handling, smaller blood sample volumes and equivalent efficacy relative to the PBMC approach, using WC for IGRA provides a novel, rapid, and user-friendly TB diagnostic method for determining the MTBC infection in elephants.

Coincidence of low genetic diversity and increasing population size in wild gaur populations in the Khao Phaeng Ma Non-Hunting Area, Thailand: A challenge for conservation management under human-wildlife conflict.

The gaur (Bos gaurus) is found throughout mainland South and Southeast Asia but is listed as an endangered species in Thailand with a decreasing population size and a reduction in suitable habitat. While gaur have shown a population recovery from 35 to 300 individuals within 30 years in the Khao Phaeng Ma (KPM) Non-Hunting Area, this has caused conflict with villagers along the border of the protected area. At the same time, the ecotourism potential of watching gaurs has boosted the local economy. In this study, 13 mitochondrial displacement-loop sequence samples taken from gaur with GPS collars were analyzed. Three haplotypes identified in the population were defined by only two parsimony informative sites (from 9 mutational steps of nucleotide difference). One haplotype was shared among eleven individuals located in different subpopulations/herds, suggesting very low genetic diversity with few maternal lineages in the founder population. Based on the current small number of sequences, neutrality and demographic expansion test results also showed that the population was likely to contract in the near future. These findings provide insight into the genetic diversity and demography of the wild gaur population in the KPM protected area that can inform long-term sustainable management action plans.

SARS-CoV-2 delta variant infection in domestic dogs and cats, Thailand.

In June-September 2021, we investigated severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections in domestic dogs and cats (n = 225) in Bangkok and the vicinities, Thailand. SARS-CoV-2 was detected in a dog and a cat from COVID-19 positive households. Whole genome sequence analysis identified SARS-CoV-2 delta variant of concern (B.1.617.2). Phylogenetic analysis showed that SARS-CoV-2 isolated from dog and cat were grouped into sublineage AY.30 and AY.85, respectively. Antibodies against SARS-CoV-2 could be detected in both dog (day 9) and cat (day 14) after viral RNA detection. This study raises awareness on spill-over of variant of concern in domestic animals due to human-animal interface. Thus, surveillance of SARS-CoV-2 in domestic pets should be routinely conducted.

Survey of SARS-CoV-2 in dogs and cats in high-risk areas during the second wave of COVID-19 outbreak, Thailand.

A cross-sectional survey of SARS-CoV-2 in domestic dogs and cats was conducted in high-risk areas, five subdistricts of Samut Sakhon Province, the epicenter of the second wave of the COVID-19 outbreak in Thailand in February 2021. A total of 523 swab samples (nasal, oral, and rectal swabs) and 159 serum samples from dogs (n = 83) and cats (n = 93) were collected and tested for SARS-CoV-2 RNA and antibodies. All swab samples tested negative for SARS-CoV-2 RNA by real-time RT-PCR with three panels of specific primers and probes. Although all dogs and cats were negative for SARS-CoV-2 RNA, 3.14% (5/159) had anti-N-IgG antibodies against SARS-CoV-2 by indirect multispecies ELISA. Our results demonstrated SARS-CoV-2 exposure in domestic animals living in high-risk areas during the second wave of the COVID-19 outbreak in Thailand. Thus, the use of one health approach for monitoring SARS-CoV-2 in domestic animals in high-risk areas of COVID-19 outbreaks should be routinely conducted and will provide benefits to risk communications in communities.

Genetic characterization of influenza A virus subtypes H11N6, H11N7, and H11N9 isolated from free-grazing ducks, Thailand.

Influenza A viruses (IAVs) infect avian species and several mammalian species including humans. Anseriformes water birds are an important reservoir of IAVs. In this study, we identified and characterized IAV subtypes H11N6 (n = 5), H11N7 (n = 3), and H11N9 (n = 3) isolated during the influenza surveillance program in free-grazing ducks from 2012 to 2015 in Thailand. Eleven IAV-H11 viruses were characterized by either whole genome sequencing (n = 5) or HA and NA gene sequencing (n = 6) for phylogenetic and amino acid analyses. Phylogenetic analysis showed that Thai IAV-H11 were grouped into Avian Eurasian lineage. Amino acid analysis showed that all Thai IAV-H11 viruses have low pathogenic avian influenza (LPAI) characteristics and sensitive to Oseltamivir and Amantadine. Novel reassortant viruses (IAV-H11N7 and IAV-H11N9) have been observed. The reassortant viruses contained NP, M, and NS gene segments which originate from intercontinental sources which never been reported in Thai IAVs. In summary, this study demonstrated high genetic diversity of IAV-H11 circulating in free-grazing ducks. Free-grazing ducks infected with IAVs generated novel reassortant IAV-H11. Thus, surveillance of IAVs in free-grazing ducks should be routinely conducted to monitor novel reassortant viruses and subsequently potential virulence viruses.

First cases of SARS-CoV-2 infection in dogs and cats in Thailand.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the coronavirus disease 2019 (COVID-19) pandemic in humans since late 2019. Here, we investigated SARS-CoV-2 infection in dogs and cats during COVID-19 quarantine at private veterinary hospitals in Thailand. From April to May 2021, we detected SARS-CoV-2 in three out of 35 dogs and one out of nine cats from four out of 17 households with confirmed COVID-19 patients. SARS-CoV-2 RNA was detected from one of the nasal, oral, rectal and environmental swabs of dog-A (15 years old, mixed breed, male dog), cat-B (1 year old, domestic shorthair, male cat), dog-C (2 years old, mixed breed, female dog) and dog-D (4 years old, Pomeranian, female dog). The animals tested positive for SARS-CoV-2 RNA from 4 to 30 days after pet owners were confirmed to be COVID-19 positive. The animals consecutively tested positive for SARS-CoV-2 RNA for 4 to 10 days. One dog (dog-A) showed mild clinical signs, while the other dogs and a cat remained asymptomatic during quarantine at the hospitals. SARS-CoV-2 specific neutralizing antibodies were detected in both the dogs and cat by surrogate virus neutralization tests. Phylogenetic and genomic mutation analyses of whole genome sequences of three SARS-CoV-2 strains from the dogs and cat revealed SARS-CoV-2 of the Alpha variant (B.1.1.7 lineage). Our findings are suggestive of human-to-animal transmission of SARS-CoV-2 in COVID-19-positive households and contamination of viral RNA in the environment. Public awareness of SARS-CoV-2 infection in pet dogs and cats in close contact with COVID-19 patients should be raised.

Serological survey of antibodies against SARS-CoV-2 in dogs and cats, Thailand.

Coronavirus disease of 2019 (COVID-19) caused by severe acute respiratory syndrome virus type 2 (SARS-CoV-2) is an emerging severe acute respiratory disease affecting global human health. In this study, a large-scale serological survey of antibodies against SARS-CoV-2 in dogs and cats was conducted during the first and second waves of COVID-19 outbreaks in Thailand, from April to December 2020. A total of 3215 serum samples were collected from dogs (n = 2102) and cats (n = 1113) living in Bangkok and in the vicinities. Serum samples were tested for SARS-CoV-2 antibodies by using an indirect multispecies enzyme-linked immunosorbent assay (ELISA). Positive and suspected samples were additionally tested for neutralizing antibodies by the surrogate virus neutralization test (sVNT). The indirect ELISA results showed that 1.66% (35 out of 2103) of dogs and 0.36% (four out of 1112) of cats were positive for SARS-CoV-2 antibodies. The sVNT results showed that all ELISA-positive and suspected samples were negative for neutralizing antibodies. Positive serum samples (35 dogs and four cats) were obtained from clinically healthy animals and animals with mild respiratory signs aged <1-13 years living in Bangkok and Samutprakarn Provinces. In summary, a serological survey revealed evidence of anti-N-IgG antibodies suggesting SARS-CoV-2 exposure in both dogs and cats during the first and second COVID-19 outbreaks in Thailand.

The first report on internal transcribed spacer region-based characterization of microfilaria in Asian elephants (Elephas maximus) in Thailand.

BACKGROUND AND AIM: Filarial infections can significantly impact the health of both humans and animals. In elephants, filariasis has been associated with cutaneous dermatitis and skin nodules. However, molecular evidence for such infections is limited in Thailand. This study aimed to identify the morphological and molecular characteristics of microfilaria in captive Asian elephants in Thailand. MATERIALS AND METHODS: Whole blood collected from the ear vein of 129 captive Asian elephants was hematologically analyzed, and the blood parasites were evaluated using three standard techniques: The microcapillary test, thin blood smears, and polymerase chain reaction (PCR). RESULTS: Conventional PCR revealed that approximately 17% (22/129) of the sampled elephants were positive for microfilaria. Microscopy revealed that microfilariae are large, unsheathed, with extended nuclei, a short headspace, and a curved tail tapering at the end. Results of internal transcribed spacer region analysis show that the elephant microfilariae are closely related to Onchocerca spp. All of the elephants positive for microfilaria presented with neither skin lesion nor anemic signs. Microfilaria infection was not associated with age; however, microfilariae were more likely to be detected in male elephants due to differences in management systems. CONCLUSION: This is the first study to provide both morphological and molecular evidence of microfilaria in Thai elephants. There is an urgent need to investigate the long-term and large-scale effects of microfilaria on the health of elephants.

Distribution and genetic diversity of Enterovirus G (EV-G) on pig farms in Thailand.

BACKGROUND: Enterovirus G (EV-G) causes subclinical infections and is occasionally associated with diarrhea in pigs. In this study, we conducted a cross-sectional survey of EV-G in pigs from 73 pig farms in 20 provinces of Thailand from December 2014 to January 2018. RESULTS: Our results showed a high occurrence of EV-Gs which 71.6 % of fecal and intestinal samples (556/777) and 71.2 % of pig farms (52/73) were positive for EV-G by RT-PCR specific to the 5'UTR. EV-Gs could be detected in all age pig groups, and the percentage positivity was highest in the fattening group (89.7 %), followed by the nursery group (89.4 %). To characterize the viruses, 34 EV-G representatives were characterized by VP1 gene sequencing. Pairwise sequence comparison and phylogenetic analysis showed that Thai-EV-Gs belonged to the EV-G1, EV-G3, EV-G4, EV-G8, EV-G9 and EV-G10 genotypes, among which the EV-G3 was the predominant genotype in Thailand. Co-infection with different EV-G genotypes or with EV-Gs and porcine epidemic diarrhea virus (PEDV) or porcine deltacoronavirus (PDCoV) on the same pig farms was observed. CONCLUSIONS: Our results confirmed that EV-G infection is endemic in Thailand, with a high genetic diversity of different genotypes. This study constitutes the first report of the genetic characterization of EV-GS in pigs in Thailand.

The occurrence of elephant endotheliotropic herpesvirus infection in wild and captive Asian elephants in Thailand: Investigation based on viral DNA and host antibody.

BACKGROUND AND AIM: Elephant endotheliotropic herpesvirus (EEHV) is a serious disease, threatening the life of young elephants. Many elephants have been infected with no clinical signs and may serve as carriers spreading this disease. It is important to monitor the disease through clinical signs and molecular diagnosis. In this study we investigated the occurrence of EEHV and the efficiency of different techniques used to monitor EEHV infection in various samples and populations of Asian elephants. MATERIALS AND METHODS: Blood and trunk swabs were collected from live elephants, while visceral organs (lung, digestive tract, spleen, lymph nodes, and kidney) were collected from dead elephants. EEHV was detected by polymerase chain reaction (PCR) in whole blood, trunk swabs, and visceral organs as samples, while elephant anti-EEHV immunoglobulin G (IgG) in serum was detected by enzyme-linked immunosorbent assay (ELISA). A total of 162 samples were analyzed in this study: 129 from healthy, 26 from dead, and 7 from sick elephants. RESULTS: The present study showed that the overall incidence of EEHV was 40.1% (n=65/162). Approximately 46.2% (n=12/26) and 85.7% (n=6/7) of dead and sick elephants were positive for EEHV by PCR, respectively. All sick elephants that were young and affected by EEHV clinical disease tested negative for the IgG antibody ELISA, suggesting primary EEHV infection in this group. In addition, 2.3% (n=3/129) of subclinical infections were detected using PCR, and trunk swab samples showed slightly higher sensitivity (5.3%, n=2/38) to detect EEHV than whole blood (1.2%, n=1/84). As many as, 48.4% (n=44/91) of healthy elephants were EEHV seropositive (ELISA-positive), suggesting that many elephants in Thailand had previously been infected. Overall, 30% of dead wild elephants had been infected with EEHV (n=3/10). Moreover, statistical analysis revealed no significant differences in the EEHV detection rate between different age groups or sexes (p>0.05). CONCLUSION: PCR is better than ELISA to detect EEHV active infection in dead/sick elephants and to monitor EEHV in young elephants. ELISA is suitable for detecting previous EEHV infection and carriers, particularly adults. Theoretically, we could use both PCR and ELISA to increase the sensitivity of testing, along with observing abnormal behavior to efficiently monitor this disease. Identification of EEHV carriers within elephant populations is important to prevent transmission to healthy individuals, especially young elephants with high mortality from EEHV. This is the first report from Thailand regarding EEHV infection in wild elephants, showing the importance of preventing disease transmission between captive and wild elephants.

Cerebral cysticercosis in a wild Bengal tiger (Panthera tigris tigris) in Bhutan: A first report in non-domestic felids.

The endangered Bengal tiger (Panthera tigris tigris) is a keystone species playing an essential role in ecology as well as in the social and spiritual lives of the Himalayan people. The latest estimate of the Bengal tiger population in Bhutan accounts for 103 individuals. Infectious organisms, including zoonotic parasites causing high burden in human health, have received little attention as a cause of mortality in tigers. Taeniosis/cysticercosis, caused by the cestode Taenia solium, is considered one of the major neglected tropical diseases in Southeast Asia. We present here a case of neurocysticercosis in a Bengal tiger showing advanced neurological disease outside Thimphu, the capital city of Bhutan. After palliative care, the animal died, and necropsy revealed multiple small cysts in the brain. Here we show the presence of two genetic variants of T. solium in the parasite material collected based on PCR and sequencing of the complete cox1 and cytB genes. The sequences form a discrete branch within the Asia plus Madagascar cluster of the parasite. On other hand, tests for feline morbillivirus, feline calicivirus, canine distemper virus, Nipah, rabies, Japanese encephalitis, feline leukaemia and feline immunodeficiency virus were negative. In contrast, PCR for feline herpesvirus was positive and a latex agglutination test revealed an elevated antibody titer against Toxoplasma gondii (titer 1:256). The molecular examination of taeniid eggs isolated from the tiger faeces produced sequences for which the highest homology in GenBank is between 92% and 94% with T. regis and T. hydatigena. This fatal case of T. solium neurocysticercosis, a disease previously unrecorded in tigers or other non-domestic felids, demonstrates an anthropogenically driven transmission of a deadly pathogen which could become a serious threat to the tiger population.

Genetic characterization of influenza A virus subtypes H1N3 and H1N9 isolated from free-grazing ducks in Thailand.

Influenza A virus (IAV) subtype H1 has been reported to infect birds, pigs and humans. In this study, we characterized IAVs subtype H1N3 and H1N9 isolated from free-grazing ducks in Thailand. Phylogenetic analysis showed that Thai IAV-H1 isolates cluster with avian Eurasian-lineage but not pandemic H1N1 viruses. Analysis of the viruses indicated low-pathogenic avian influenza (LPAI) characteristics. This study is the first report of avian H1N3 and H1N9 in Thailand. Although Thai IAV-H1 viruses do not pose a risk of a pandemic, routine surveillance and genetic monitoring of IAVs should be conducted.

Genetic characterization of influenza A (H7N6) virus isolated from a live-bird market in Thailand.

A one-year influenza A virus (IAV) monitoring program was conducted in a live-bird market (LBM) in Thailand. Using one-step real-time RT-PCR (rRT-PCR), 2.39 % of live birds were found to be IAV positive. Twenty viruses could be identified as IAV subtype H7N6. Eight IAV-H7N6 viruses were subjected to whole-genome sequencing and genetic characterization. Phylogenetic analysis showed that the HA gene of Thai H7N6 is grouped with those of the H7 Eurasian viruses. The NA gene is closely related to those of the N6 Eurasian viruses. This is the first report of IAV subtype H7N6 in Thailand.

Sentinel model for influenza A virus monitoring in free-grazing ducks in Thailand.

Influenza A virus (IAV) can cause influenza in birds and mammals. In Thailand, free-grazing ducks are known IAV reservoirs and can spread viruses through frequent movements in habitats they share with wild birds. In this study, the sentinel model for IAV monitoring was conducted over 4 months in two free-grazing duck flocks. IAV subtypes H4N6 (n=1) and H3N8 (n=5) were isolated from sentinel ducks at the ages of 13 and 15 weeks. Clinical signs of depression and ocular discharge were observed in the infected ducks. Phylogenetic analysis and genetic characterization of the isolated IAVs indicated that all Thai IAVs were clustered in the Eurasian lineage and pose low pathogenic avian influenza characteristics. Serological analysis found that antibodies against IAVs could be detected in the ducks since 9-weeks-old. In summary, our results indicate that the sentinel model can be used for IAV monitoring in free-grazing duck flocks. Since free-grazing ducks are potential reservoirs and transmitters of IAVs, routine IAV surveillance in free-grazing duck flocks can be beneficial for influenza prevention and control strategies.

Genetic diversity of swine influenza viruses in Thai swine farms, 2011-2014.

The pig is known as a "mixing vessel" for influenza A viruses. The co-circulation of multiple influenza A subtypes in pig populations can lead to novel reassortant strains. For this study, swine influenza surveillance was conducted from September 2011 to February 2014 on 46 swine farms in Thailand. In total, 78 swine influenza viruses were isolated from 2,821 nasal swabs, and 12 were selected for characterization by whole genome sequencing. Our results showed that the co-circulation of swine influenza subtypes H1N1, H3N2, and H1N2 in Thai swine farms was observable throughout the 3 years of surveillance. Furthermore, we repeatedly found reassortant viruses between endemic swine influenza viruses and pandemic H1N1 2009. This observation suggests that there is significant and rapid evolution of swine influenza viruses in swine. Thus, continuous surveillance is critical for monitoring novel reassortant influenza A viruses in Thai swine populations.

Genetic characterization of influenza A virus subtype H7N1 isolated from quail, Thailand.

In Thailand, surveillance for the highly pathogenic avian influenza virus H5N1 (HPAI-H5N1) has revealed high prevalence of the virus in quail in live-bird markets. This study monitored avian influenza viruses (AIVs) in quail farms in an area at high risk for HPAI-H5N1 over a 12-month period from 2009 to 2010. One-step real-time RT-PCR (rRT-PCR) results showed that 1.18 % of swab samples (24/2,040) were AIV positive. Among the rRT-PCR positive samples, three samples were identified as subtype H7N1. One Thai H7N1 virus designated "A/quail/Thailand/CU-J2882/2009 (H7N1)" was subjected to whole genome sequencing and genetic characterization. Phylogenetic analysis showed that the HA gene of the Thai H7N1 virus groups with those of the H7 Eurasian viruses. Interestingly, the NA gene of the virus was found to be closely related to those of the HPAI-H5N1 viruses from Vietnam and Thailand. This study constitutes the first report on AIV H7N1 in Thailand. Our results suggest the possibility of genetic reassortment between AIV-H7NX and HPAI-H5N1 in quail. The HA cleavage site of the Thai H7N1 virus contains no multiple amino acid insertions, suggesting low pathogenic characteristics for this virus.

Genetic characterization of canine influenza A virus (H3N2) in Thailand.

In January 2012, several clinical cases of dogs with flu-like symptoms, including coughing, sneezing, nasal discharge, and fever, were reported in a small-animal hospital located in Bangkok, Thailand. One influenza A virus was identified and characterized as an avian-like influenza virus H3N2. The virus was named A/canine/Thailand/CU-DC5299/12. A phylogenetic analysis indicated that the canine virus belonged to an avian Eurasian lineage and was genetically related to the canine influenza viruses H3N2 from China and Korea. This canine virus displays a unique genetic signature with two amino acid insertions in the NA protein, which is similar to the canine influenza viruses from eastern China (Zhejiang and Jiangsu). This study constitutes the first report of H3N2 canine influenza virus infection in a small-animal hospital in Thailand.

Genetic characterization of influenza A virus subtype H12N1 isolated from a watercock and lesser whistling ducks in Thailand.

Monitoring of influenza A virus (IAV) was conducted in wild bird species in central Thailand. Four IAV subtype H12N1 strains were isolated from a watercock (order Gruiformes, family Rallidae) (n = 1) and lesser whistling ducks (order Anseriformes, family Anatidae) (n = 3). All H12N1 viruses were characterized by whole-genome sequencing. Phylogenetic analysis of all eight genes of the Thai H12N1 viruses indicated that they are most closely related to the Eurasian strains. Analysis of the HA gene revealed the strains to be of low pathogenicity. This study is the first to report the circulation of IAV subtype H12N1 in Thailand and to describe the genetic characteristics of H12N1 in Eurasia. Moreover, the genetic information obtained on H12N1 has contributed a new Eurasian strain of H12N1 to the GenBank database.

Influenza A virus surveillance in live-bird markets: first report of influenza A virus subtype H4N6, H4N9, and H10N3 in Thailand.

A one-year influenza A survey was conducted in 10 live bird markets (LBMs) in H5N1 high-risk areas in Thailand from January to December 2009. The result from the survey showed that the occurrence of influenza A virus (IAV) in LBMs was 0.36% (19/5304). Three influenza A subtypes recovered from LBMs were H4N6 (n = 2), H4N9 (n = 1), and H10N3 (n = 16) from Muscovy ducks housed in one LBM in Bangkok. These influenza subtypes had never been reported in Thailand, and therefore such genetic diversity raises concern about potential genetic reassortment of the viruses in avian species in a particular setting. Two influenza A subtypes (H4N6 and H4N9) were isolated from oropharyngeal and cloacal swabs of the same duck, suggesting coinfection with two influenza subtypes and possible genetic reassortment in the bird. In addition, H10N3 infection in ducks housed in the same LBM was observed. These findings further support that LBMs are a potential source of IAV transmission and genetic reassortment.