Pathogenicity in domestic ducks and mice of highly pathogenic H5N1 clade 2.3.2.1 influenza viruses recently circulating in Eastern Asia.

Influenza virus A (H5N1) clade 2.3.2.1 has recently caused widespread outbreaks of disease in domestic poultry and wild birds in Eastern Asia. In the current study, the antigenicity and pathogenicity of three clade 2.3.2.1 viruses (Ck/Kr/Gimje/08, Ws/Mongolia/1/09, and Ws/Mongolia/7/10) were investigated in domestic ducks and mice. The H5N1 influenza viruses in this study were antigenically similar to each other (r-values of 0.35-1.4). The three viruses replicated systemically in all tissues tested in domestic ducks, indicating high pathogenicity. However, the viruses produced different clinical signs and mortality rates: Ck/Kr/Gimje/08 and Ws/Mongolia/1/09 resulted in 100% mortality with severe neurological signs, whereas Ws/Mongolia/7/10 resulted in 50% mortality with relatively mild neurological signs. In mice, infection with Ck/Kr/Gimje/08 and Ws/Mongolia/7/10 resulted in weight loss that peaked at 4 days post-infection (22.3% and 20.8%, respectively), same MLD50 (2.2 Log10 EID50) and systemic replication. The three viruses had K deletion at the -2 position of the HA1-connecting peptide (PQRERRRK-R), which is associated with increased virulence in domestic ducks and harbored NA stalk deletion, NS1 deletion and mutation of P42S in NS1, and full length (90aa) in PB1-F2, which confer increased virulence in mice. Our study shows that clade 2.3.2.1 viruses from Korea and Mongolia are antigenically similar and highly pathogenic in both domestic ducks and mice. Moreover, we provide molecular determinants of the clade 2.3.2.1 viruses associated with the pathogenicity in domestic ducks and mice, respectively.

Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus.

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 µL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4°C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.

Evidence for recent acquisition and successful transmission of bla(CTX-M-15) in Salmonella enterica in South Korea.

We identified two distinct bla(CTX-M)-bearing and five distinct bla(CMY-2)-bearing genetic structures located on plasmids from Salmonella enterica and Escherichia coli isolates (n = 35) collected from chickens in South Korea. All Salmonella plasmids shared a common replicon, bla(CTX-M-15) transposon, and core resistance phenotype, while E. coli bla(CTX-M-15) plasmids included four distinct replicons.

Bacterial pathogens and flora isolated from farm-cultured eels (Anguilla japonica) and their environmental waters in Korean eel farms.

The purpose of this study was to investigate bacterial pathogens and flora in both sick and clinically healthy eels, Anguilla japonica, and the environmental rearing waters of Korean eel farms. Between 2003 and 2010, a total of 621 sick eels were submitted for diagnosis, while 216 healthy eels and 87 environmental water samples were collected during a survey of 26 eel farms in Korea. Seven different bacterial species were obtained from 183 isolates, which were recovered from the internal organs of the 621 sick eels. The most frequently isolated bacterium was Edwardsiella tarda (71.0%), followed by Aeromonas hydrophila (9.3%), Citrobacter freundii (7.7%), Aeromonas veronii (6.0%), Listonella anguillarum (2.7%), Plesiomonas shigelloides (2.2%), and Pseudomonas anguilliseptica (1.1%). From the eel and water samples of the survey, a total of 472 isolates from 34 different species belonging to 15 genera of bacteria were isolated. The most prevalent genus of bacteria was Aeromonas spp. (141/472, 29.8%). Among the 34 types of bacterial species, C. freundii (20.1%) and A. hydrophila (19.9%) were the most frequently isolated. The results of this study indicate that a wide range of bacterial species, which can act as primary or opportunistic pathogens, may be recovered from clinically healthy eels and rearing waters. This study provides baseline information about bacterial pathogens and floral contamination for the control and treatment of bacteria in Korean eel farms.

Protective efficacy of baculovirus-derived influenza virus-like particles bearing H5 HA alone or in combination with M1 in chickens.

Since 2003, the highly pathogenic avian influenza (HPAI) H5N1 has become a serious problem in animals and an increasing threat to public health. To develop effective vaccines for H5 HPAI in chickens, virus-like particles (VLP) were produced using a baculovirus expression system. The particles comprised hemagglutinin (HA) alone (HA-VLP) or HA in combination with a matrix protein (M1; HAM-VLP) derived from a recent clade 2.3.2.1 H5N1 HPAI virus. To compare the immunogenicity and protective efficacy of these VLPs, 10 µg HAM-VLP, the equivalent amounts of HA incorporated HA-VLP or whole inactivated virus (WIV), were emulsified with mineral oil and used to immunize chickens. The serum hemagglutination inhibition antibody levels induced by HA-VLP and HAM-VLP were comparable to WIV. Antibodies to nucleoprotein were detected only in the WIV group. Immunized chickens in each group survived and were protected against a lethal homologous virus challenge, showing no clinical signs of infection. The challenge virus was detected intermittently in some oropharyngeal swabs, but not in cloacal swabs or various organs, which means that VLPs and WIV provide protection against systemic but not local virus replication in chickens. After the challenge, the HA-VLP group showed significantly increased serum antibody levels compared to the HAM-VLP and WIV groups, and some chickens in the HA-VLP group seroconverted with respect to nucleoprotein. Taken together, these results suggest that VLPs may be an effective method for controlling HPAI in chickens. They could be applied to a differentiating infected from vaccinated animals (DIVA) strategy. In addition, it is likely that HAM-VLP is more efficacious than HA-VLP in chickens.

Comparative proteome and transcriptome analyses of wild-type and live vaccine strains of Salmonella enterica serovar Gallinarum.

Salmonella enterica serovar Gallinarum causes fowl typhoid in chickens and is of economic importance to the chicken industry. A serovar Gallinarum live vaccine strain 9R (SG 9R) has been used to control fowl typhoid in many areas where the disease is endemic. Because the attenuation mechanism of SG 9R was not defined, there has been continued concern about reversion to virulence. In this study, we examined the molecular characteristics, which might provide better insight into attenuation of SG 9R, by comparing its proteome and transcriptome with those of two wild-type strains (287/91 and 06Q110). Proteins present in wild-type strains but absent in SG 9R were identified by two-dimensional gel electrophoresis and MALDI-TOF MS. Genes up- or down-regulated in SG 9R compared to wild-type strains were identified using an expression array. The proteome analysis identified nine proteins absent in SG 9R of which one protein had relevance to virulence. The transcriptome analysis revealed 24 up-regulated and 97 down-regulated genes in SG 9R. Approximately one-half of down-regulated genes (42 genes) were associated with virulence mechanisms. This finding suggests that attenuation of SG 9R may be associated with a combination of impaired virulence factors and thus reversion to virulence would not be caused by any single mutation event.

Isolation of a reassortant H13N2 virus from a mallard fecal sample in South Korea.

BACKGROUND: Virus subtype H13N2, A/mallard/Kr/SH38-45/2010 (H13N2), was first isolated from a mallard fecal sample in South Korea. RESULTS: Phylogenetic analysis of all eight viral genes revealed that this virus emerged by genetic mixing between Eurasian and North American gene pools, and possibly between wild ducks and gulls. The H13 and N2 surface genes clustered together in a group with Eurasian isolates from gulls and wild birds, respectively. The PB2, PA, NP, M and NS segments belonged to the Eurasian lineage, whereas the PB1 gene clustered in the North American lineage. Furthermore, they showed a bird-dependent pattern in phylogenetic analysis: the M gene was similar to subtype H13 viruses within gulls, whereas other segments were similar to avian influenza viruses of other subtypes from wild ducks. CONCLUSIONS: The data suggests that the novel reassortant H13N2 virus isolated in South Korea might have emerged by genetic reassortment between intercontinental and interspecies transmission in wild birds.

Isolation and epidemiological characterization of heat-labile enterotoxin-producing Escherichia fergusonii from healthy chickens.

Escherichia fergusonii has been associated with a wide variety of intestinal and extraintestinal infections in both humans and animals. The aim of this study was to demonstrate the presence of heat-labile enterotoxin (LT)-producing E. fergusonii in healthy chickens and its plasmid-mediated LT toxin gene transfer to other Enterobacteriaceae. We tested faecal samples from 184 chicken flocks (consisting of 109 broilers and 75 layers) of 78 commercial chicken farms for the presence of the LT gene using a polymerase chain reaction-based screening and identified samples from 43 flocks (23.4%) as positive for the LT gene. We subsequently isolated and identified E. fergusonii harboring the LT gene from all LT-positive samples and observed 21 various biochemical types. The plasmids encoding LT in 16 (37.2%) of the 43 isolates were conjugally transferred to the recipient strain Escherichia coli J53. Southern hybridization showed that all plasmids from the transconjugants carried the eltAB gene (Ent plasmid) and belonged to the narrow-host-range IncF type. In addition, all the E. fergusonii strains identified were classified into 17 pulsed-field gel electrophoresis (PFGE) types, and it is likely that there was an association between the PFGE types and geographical location or breed of flocks. In conclusion, this is the first study to demonstrate that LT-producing E. fergusonii strains were present in the faeces of healthy chickens and that plasmid-mediated virulence genes can be transferred to E. coli and may have a great potential to cause human disease.

Differential identification of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum and the biovar Gallinarum live vaccine strain 9R.

Salmonella enterica serovar Gallinarum biovar Gallinarum causes fowl typhoid in chickens and has been of economic importance to the chicken industry in many countries. The biovar Gallinarum live vaccine strain 9R (SG 9R) has been used to control fowl typhoid in many areas where the disease is endemic. Therefore, a definitive diagnosis of this disease may require differentiation of wild-type field isolates of biovar Gallinarum from the live vaccine strain SG 9R. Here, we report the development of a triplex polymerase chain reaction (PCR) assay to differentially identify serovar Gallinarum biovars Gallinarum and Pullorum and SG 9R. Sequences specific to SG 9R, which are absent or highly divergent in the fully sequenced biovar Gallinarum strain 287/91, were identified by constructing the suppression subtractive hybridization (SSH) library. A total of 565 nonredundant inserts were obtained from successfully sequenced SSH clones (718 clones). Sequences of 14 inserts were unique to SG 9R, but single nucleotide polymorphisms (SNPs) found in another insert (9R22C9) were more useful for strain discrimination. A new PCR primer set was designed to target SNP regions of the insert and was integrated into a duplex PCR assay developed previously (Kang et al., 2011). Boiled lysates of 138 reference and field strains of Salmonella and other related Gram-negative bacteria were tested to validate the triplex PCR assay. All strains of biovars Gallinarum (n=53) and Pullorum (n=21) and SG 9R (n=7) tested were differentially identified, whereas the other strains (n=57) were PCR negative. This triplex PCR assay will be very useful for rapid differential diagnoses of fowl typhoid and pullorum disease in veterinary laboratories.

Molecular epidemiologic investigation of lentogenic Newcastle disease virus from domestic birds at live bird markets in Korea.

A Newcastle disease surveillance program was conducted at live bird markets in Korea to expand our epidemiologic understanding of the disease in Korea. During the surveillance program, 10 lentogenic Newcastle disease viruses (NDVs) were isolated and identified from apparently healthy chickens and ducks at live bird markets. The lentogenic viruses had sequence motifs of either 112GKQGRL117 (n = 8) or 112GRQGRL117 (n = 2) at the F0 cleavage site. Sequencing and phylogenetic analyses of NDV isolates based on the hypervariable region of the F protein revealed two different genotypes: genotypes I (n = 8) and II (n = 2). Genotype I viruses were most closely related to the NDV V4 strain (n = 7) or the NDV Ulster 2C strain (n = 1). In contrast, genotype II viruses clustered with the NDV vaccine strains (LaSota and VG/GA) that are commonly used as live vaccines in Korea. The epidemiologic importance of NDV at live bird markets in Korea is discussed.

Genetic relationship of H3 subtype avian influenza viruses isolated from domestic ducks and wild birds in Korea and their pathogenic potential in chickens and ducks.

The H3 subtype avian influenza virus (AIV) is one of the most frequently isolated subtypes in domestic ducks, live poultry markets, and wild birds in Korea. In 2002-2009, a total of 45 H3 subtype AIVs were isolated from the feces of clinically normal domestic ducks (n=28) and wild birds (n=17). The most prevalent subtypes in domestic ducks were H3N2 (35.7%), H3N6 (35.7%), H3N8 (25.0%), and H3N1 (3.6%, novel subtype in domestic duck in Korea). In contrast, H3N8 (70.6%) is the most prevalent subtype in wild birds in Korea. In the phylogenetic analysis, HA genes of the Korean H3 AIVs were divided into 3 groups (Korean duck, wild bird 1, and wild bird 2) and all viruses of duck origin except one were clustered in a single group. However, other genes showed extensive diversity and at least 17 genotypes were circulating in domestic ducks in Korea. When the analysis expanded to viruses of wild bird origin, the genetic diversity of Korean H3 AIVs became more complicated. Extensive reassortments may have occurred in H3 subtype influenza viruses in Korea. When we inoculated chickens and ducks with six selected viruses, some of the viruses replicated efficiently without pre-adaptation and shed a significant amount of viruses through oropharyngeal and cloacal routes. This raised concerns that H3 subtype AIV could be a new subtype in chickens in Korea. Continuous surveillance is needed to prepare the advent of a novel subtype AIV in Korea.

Epidemiological investigation of eaeA-positive Escherichia coli and Escherichia albertii strains isolated from healthy wild birds.

Escherichia coli has commonly been associated with diarrheal illness in humans and animals. Recently, E. albertii has been reported to be a potential pathogen of humans and animals and to be carried by wild birds. In the present study, the prevalence and genetic characteristics of intimin-producing E. coli and E. albertii strains were evaluated in wild birds in Korea. Thirty one of 790 Enterobacteriaceae strains from healthy wild birds were positive for the intimin gene (eaeA) and twenty two of the 31 strains were identified as atypical enteropathogenic E. coli (aEPEC) that did not possess both EAF and bfpA genes. A total of nine lactose non-fermenting coliform bacterial strains were identified as E. albertii by PCR and sequence analysis of housekeeping genes. A total of 28 (90.3%) eaeA-positive strains were isolated from waterfowl. Fifteen aEPEC (68.2%) and two E. albertii (22.2%) strains had a ß-intimin subtype and 14 aEPEC strains harboring ß-intimin belonged to phylogenetic group B2. AU eaeA-positive E. albertii and 3 aEPEC strains possessed the cytolethal distending toxin gene (cdtB). The eaeA-positive E. coli and E. albertii strains isolated from healthy wild birds need to be recognized as a potential pathogroup that may pose a potential threat to human and animal health. These findings indicate that eaeA-positive E. coli as well as E. albertii can be carried by wild birds, posing a potential threat to human and animal health.

Characterization of Edwardsiella tarda isolated from farm-cultured eels, Anguilla japonica, in the Republic of Korea.

We surveyed the occurrence of edwardsiellosis on eel farms and investigated the characteristics of Edwardsiella tarda isolated from farm-cultured eels in the Republic of Korea. The occurrence rate of edwardsiellosis was 72% in the investigated samples. Among the edwardsiellosis cases, 46% were found to be mixed infections, with parasites and other kinds of bacteria. Some of the biochemical characteristics of the E. tarda isolates were different from those of the previously reported E. tarda isolated from several kinds of fish from different countries, especially in terms of hydrogen sulfide and indole production. The E. tarda isolated from the eels in the Republic of Korea had the characteristics of two biogroups, the wild-type biogroup and biogroup 1. The enzymatic activity of the E. tarda showed similar patterns to previously reported E. tarda strains and ATCC strains. This is the first it has been reported that E. tarda isolated from farm-cultured eels had some different biochemical characteristics from those of previously reported E. tarda isolated from several kinds of fish.

Genetic characterization of H1 avian influenza viruses isolated from migratory birds and domestic ducks in Korea.

H1 avian influenza viruses (AIVs) isolated from migratory birds and domestic ducks from 2003 to 2007 were analyzed to determine their genetic relationship. Phylogenic analysis with nucleotide sequences of all eight gene segments showed that 13 H1 AIVs from migratory birds and domestic ducks belonged to Eurasian avian lineages and were closely related to each other. Compared with H1 influenza viruses of swine or human origin in Korea, there was no evidence of reassortment among the human, swine, and avian hosts. Our results show that H1 AIVs isolated in Korea from 2003 to 2007 were genetically stable. However, continued surveillance is needed considering the role of migratory birds and domestic duck as a source of AIVs.

Genetic analyses of H5N1 avian influenza virus in Mongolia, 2009 and its relationship with those of eastern Asia.

In May and August 2009, 14 highly pathogenic H5N1 isolates were obtained from migratory birds in central Mongolia. To trace the genetic lineage of the isolates, nucleotide sequences of all eight genes were determined and phylogenetically analyzed. Hemagglutinin and nucleoprotein genes were clustered in clade 2.3.2. The polymerase acidic gene was related to the isolates of South Korea and Japan obtained in 2003 and 2004 outbreaks, and a migratory duck isolate from Jiangxi, China. The neuraminidase and other internal genes were closely related to those of clade 2.3.4 viruses. The results indicate evolving genetic diversity of the hemagglutinin gene and acquisition of different polymerase acidic gene in the 2009 Mongolian isolates, likely via bird migration. Prevention of potentially wider outbreak in domestic poultry and accurate monitoring of H5N1 genetic mutation will require continuous monitoring for H5N1 in both domestic and wild birds, and will necessitate international cooperation with neighboring countries sharing migratory flyways.

Differential identification of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum based on polymorphic regions of glgC and speC genes.

Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum cause fowl typhoid and pullorum disease in avian species, respectively, and have been of considerable economic importance to the poultry industry in parts of the world. The definitive diagnosis of these diseases can be made only by isolation and identification of the causative agent. However, rapid identification of biovars Gallinarum and Pullorum is not easily feasible due to their common antigenic structure and genomic sequence similarity. We developed a duplex polymerase chain reaction (PCR) assay to identify and discriminate between strains of biovars Gallinarum and Pullorum. Duplex PCR primers were designed to target polymorphic regions of glgC and speC genes showing multiple mutations in the sequenced S. enterica subsp. enterica serovar Gallinarum 287/91 genome and were applied to the specific identification of biovars Gallinarum and Pullorum. Boiled lysates of 131 reference and field strains of Salmonella and other related Gram-negative bacteria were tested to validate the duplex PCR assay. All strains of biovars Gallinarum (n=53) and Pullorum (n=21) tested were correctly identified based on this assay (100% sensitivity) while the other strains (n=57) were PCR negative (100% specificity). These results demonstrate that a highly accurate biovar-specific duplex PCR assay can be performed for the rapid identification and discrimination of biovars Gallinarum and Pullorum from field isolates.

Field application of the H9M2e enzyme-linked immunosorbent assay for differentiation of H9N2 avian influenza virus-infected chickens from vaccinated chickens.

Vaccination for control of H9N2 low-pathogenicity avian influenza (LPAI) in chickens began in 2007 in South Korea where the H9N2 virus is prevalent. Recently, an enzyme-linked immunosorbent assay (ELISA) using the extracellular domain of the M2 protein (M2e ELISA) was developed as another strategy to differentiate between vaccinated and infected chickens. Here, an ELISA using the extracellular domain of the M2 protein of H9N2 LPAI virus (H9M2e ELISA) was applied to differentiate infected from vaccinated chickens using the H9N2 LPAI virus M2 peptide. The specificity and sensitivity of the optimized H9M2e ELISA were 96.1% and 83.8% (the absorbance of the sample to the absorbance for the positive control [S/P ratio] ≥ 0.6), respectively, with the cutoff value (S/P ratio = 0.6), and the criterion of avian influenza (AI) infection in a chicken house was established as >20% reactivity of anti-M2e antibody per house with this cutoff value. After infection in naïve chickens and once-vaccinated chickens with a hemagglutination inhibition (HI) assay titer of 9.25 ± 0.75 log(2) units, the sera from infected chickens were confirmed as AI infected when the chickens were 1 week old in both groups, and AI infection lasted for 24 weeks and 9 weeks in naïve and once-vaccinated chickens, respectively, although in twice-vaccinated chickens with a higher HI titer of 11.17 ± 0.37 log(2) units, anti-M2e antibody in infected sera did not reach a level indicating AI infection. In field application, anti-M2e antibody produced in infected chickens after vaccination or in reinfected chickens could be identified as AI infection, although HI test could not distinguish infected from vaccinated sera. These results indicate the utility of H9M2e ELISA as a surveillance tool in control of H9N2 LPAI infections.

DNA barcoding techniques for avian influenza virus surveillance in migratory bird habitats.

Avian influenza virus (AIV) circulates among free-ranging, wild birds. We optimized and validated a DNA barcoding technique for AIV isolation and host-species identification using fecal samples from wild birds. DNA barcoding was optimized using tissue and fecal samples from known bird species, and the method was shown to distinguish 26 bird species. Subsequently, fecal samples (n=743) collected from wild waterfowl habitats confirmed the findings from the laboratory tests. All identified AIV-positive hosts (n=35) were members of the order Anseriformes. We successfully applied the DNA barcoding technique to AIV surveillance and examined AIV epidemiology and host ecology in these wild waterfowl populations. This methodology may be useful in the design of AIV surveillance strategies.

Antigenic and immunogenic investigation of the virulence motif of the Newcastle disease virus fusion protein.

Newcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is a highly contagious viral disease of poultry. Virulent NDVs characteristically have a multibasic amino acid sequence (virulence motif) such as (112)RRQKRF(117) at the cleavage site of the precusor fusion (F0) protein. The antigenic and immunogenic characteristics of the virulence motif (112)RRQKRF(117) in the F0 protein of virulent NDVs were investigated. Epitope mapping analysis revealed that a RRQKRF-specific monoclonal antibody 4G2 recognized the KRF section of the motif. A synthetic peptide bearing the RRQKRF motif reacted strongly with sera from virulent NDV (with RRQKRF motif)-infected chickens. These sera also showed reactivity to peptides bearing other virulence motifs ((112)KRQKRF(117), (112)RRQRRF(117) and (112)RRRKRF(117)) but not an avirulence motif ((112)GRQGRL(117)) by ELISA. The synthetic bearing RRQKRF motif reacted with 60% to 91% of sera taken from surviving chickens on ND outbreak farms but not with sera from vaccinated birds, even though most of the sera had antibody to NDV due to vaccination. This indicates that the virulence motif has the potential to differentiate virulent NDV infected birds from vaccinated birds.

Application of DNA barcoding technique in avian influenza virus surveillance of wild bird habitats in Korea and Mongolia.

In a previous study, we optimized DNA barcoding techniques for avian influenza virus (AIV) isolation and host identification, using fecal samples from wild birds, for high-throughput surveillance of migratory waterfowls. In the present study, we surveyed AIV in Mongolia during the breeding season and, subsequently, in Korea in winter, to compare prevalent AIV subtypes and hosts using DNA barcoding. In Korea, H4 and H5 subtypes were the most abundantly detected HA subtypes, and most AIVs were isolated from the major population (mallards, Anas platyrhynchos) of wild bird habitats. On the other hand, in Mongolia, H3 and H4 subtypes were the most abundantly detected HA subtypes, and most AIVs were isolated from a small population of wild bird habitats that were not visible at the sampling site. In conclusion, AIV isolation using fecal samples, accompanied with DNA barcoding techniques as a host bird species identification tool, could be useful for monitoring major and minor populations of wild bird habitats. Further, continuous, and large-scale surveillance could be helpful for understanding the AIV epidemiology, evolution, and ecology in wild waterfowl.