High prevalence of a gene cluster conferring resistance to streptomycin, sulfonamide, and tetracycline in Escherichia coli isolated from indigenous wild birds.

A total of 116 Escherichia coli isolates from cecal contents of 81 indigenous wild birds in Korea were tested for antimicrobial susceptibility. Seventy-one isolates from sparrows (Passer montanus) and one isolate from doves (Columba livia) were resistant to three antimicrobials, including streptomycin, sulfonamide, and tetracycline (SSuT). PCR and subsequent sequence analysis revealed the SSuT gene cluster region (approximately 13 kb) harboring genes encoding resistance to streptomycin (strA and strB), sulfonamide (sul2), and tetracycline (tetB, tetC, tetD, and tetR). In particular, tetracycline resistance genes were located on the transposon Tn10-like element. The SSuT element-harboring E. coli can be an important source of the transmission of antimicrobial resistance to other pathogenic bacteria. Therefore, strict sanitary measures in human and animal environments are necessary to prevent the spread of resistant bacteria through fecal residues of wild birds.

High prevalence and variable fitness of fluoroquinolone-resistant avian pathogenic Escherichia coli isolated from chickens in Korea.

Colibacillosis caused by avian pathogenic Escherichia coli (APEC) is the most common bacterial disease in poultry, resulting in significant economic losses. Resistance to fluoroquinolones has been found to be high in APEC worldwide, which has increased concerns about risks to human health as well as poultry production. In the present study, we determined the prevalence, genetic traits, and fitness traits of fluoroquinolone-resistant APEC isolated from chickens in Korea using a total of 286 APEC isolates collected between 2014 and 2017. The APEC isolates were highly resistant to nalidixic acid (86.0%), ampicillin (71.7%), tetracycline (69.6%), and sulfisoxazole (61.2%), and 132 (46.2%) of the isolates were resistant to both enrofloxacin and ciprofloxacin. These fluoroquinolone-resistant isolates showed eight mutation combinations including single- or double-point mutations in the gyrA, parC, or parE genes. The isolates with double mutations (codons 83 and 87) in gyrA and additional mutations in parC and parE showed high-level fluoroquinolone resistance (minimum inhibitory concentrations, 16-128 µg/ml). The isolates fell into four phylogenetic groups, and groups A (47/132, 35.6%) and B1 (47/132, 36.4%) were the most predominant. Nine isolates (6.8%) belonged to group B2 and included major lineages of extraintestinal pathogenic E. coli, sequence type (ST) 95 (n = 3) and ST69 (n = 2). The isolates varied in their virulence-associated gene content, biofilm formation, and intramacrophage survival. Overall, fluoroquinolone-resistant APEC in poultry poses a potential risk to public health and represents a highly diverse group of the resistant bacteria that varied in their genetic and fitness traits.

Impact of Dermanyssus gallinae infestation on persistent outbreaks of fowl typhoid in commercial layer chicken farms.

Although it has rapidly decreased since the early 2000s, fowl typhoid still occurs in commercial layer chickens, causing a significant economic loss in Korea. There is growing concern about the emergence of new pathogenic strains of the causative agent, Salmonella Gallinarum, which is able to overcome vaccine immunity. It has also been suspected that the poultry red mite, Dermanyssus gallinae, which is commonly found in layer chicken farms, may be an important cause of the recurrence of fowl typhoid in the farms. This study was conducted to examine changes in the virulence of recent isolates of S. Gallinarum obtained from layer farms and estimate the potential of the disease transmission of D. gallinae in the farms. Clinical and environmental samples and mites collected from layer farms affected by fowl typhoid between 2013 and 2018 were tested for S. Gallinarum. The isolates were characterized by genotypic analyses and in vitro virulence assays with chicken-derived cell lines. Vaccine protection against recent isolates was examined in the chickens. A total of 45 isolates of S. Gallinarum were collected and there was no evidence of changes in their virulence. It has also been demonstrated that the S. Gallinarum 9R vaccine strain widely used in Korea is still effective in controlling fowl typhoid if the susceptibility of birds to the disease is not increased by stress. Salmonella Gallinarum isolated from the outer and inner parts of D. gallinae, environmental dust, and dead birds of the same farm showed the same or closely related genotypes. Consequently, the present study indicated that the horizontal transmission and environmental persistence of S. Gallinarum and the increased disease susceptibility of chickens in layer farms could be mediated by D. gallinae, causing persistent outbreaks of fowl typhoid.

Genotypic divergence of Avibacterium paragallinarum isolates with different growth requirements for nicotinamide adenine dinucleotide.

In 2017, for the first time in Asia, we reported the isolation of variants of Avibacterium paragallinarum with atypical NAD dependency. The present study was conducted to characterize the genotypes of 24 isolates of Av. paragallinarum in Korea, including the four variants reported previously. Most of the typical isolates (19/20) showed a unique ERIC-PCR pattern with no ERIC-PCR patterns in common between the typical isolates and the variants. Furthermore, the variants shared no ERIC-PCR patterns among themselves. All the typical NAD-dependent isolates belonged to the same phylogenetic group based on both 16S rRNA and hagA gene sequences. The four variants were placed in several groups distinct from the typical isolates. In the 16S rRNA phylogenetic analysis, two of the variants were not closely aligned to any other Av. paragallinarum, isolate although they were clearly members of the genus Avibacterium. The other variants were clustered together with NAD atypical isolates from geographically diverse global locations. Compared with the Modesto reference strain AY498870, all the variants lacked a TTTTT stretch at positions 182-186 in the 16S rRNA gene and the same deletion was shown in most of the reported variants. The typical isolates and variants shared 97.3-98.2% and 95.2-97.2% nucleotide sequence similarity, for 16S rRNA and hagA, respectively. In addition, the similarities among variants were within 98.3-100% and 96.5-98.4% for the two genes, respectively. Our results indicate that the Av. paragallinarum variants with altered NAD growth requirements were genetically different and highly divergent from the typical NAD-dependent isolates.RESEARCH HIGHLIGHTS NAD variant Korean Av. paragallinarum isolates show genetic diversity, whereas typical Korean Av. paragallinarum isolates do not.The Korean variants were not closely aligned to all other Av. paragallinarum in the 16S rRNA phylogeny.NAD atypical isolates from geographically diverse global locations clustered together.Almost all variants, including all Korean variants of Av. paragallinarum, lack a specific fragment of the 16S rRNA gene.

Sequential Transmission of Salmonella in the Slaughtering Process of Chicken in Korea.

Salmonella is one of the most common foodborne pathogens worldwide. Salmonella infections in humans are mainly associated with consumption of poultry products contaminated with this foodborne pathogen. Therefore, strict sanitary measures are necessary to control Salmonella contamination during the slaughtering process of poultry. The objective of this study was to determine the occurrence and transmission of Salmonella at a series of steps in the slaughtering process of chicken. A total of 601 samples were collected from a series of slaughtering steps (10 sampling sites) of 26 chicken slaughterhouses throughout Korea. Salmonella was isolated from samples and its distribution was analyzed along the slaughtering process. Isolates from each sampling site were tested for susceptibility to 15 antibiotics by the broth microdilution method. They were also genotypically characterized by pulsed-field gel electrophoresis (PFGE). Salmonela was isolated from 168 out of 601 samples. Sixteen serotypes were identified while six isolates were untypable. Salmonella enterica serovars Montevideo (n = 29) and Virchow (n = 27) were the most common serotypes out of 119 nonredundant isolates. Relatively high contamination rates of Salmonella were found in shackles (75.0%), feathers near plucking machine (68.5%), and feces from crates (44.0%). Twenty-three antibiotic resistance patterns were recognized and 40 (33.6%) isolates were resistant to five or more antibiotics. The same serotypes of Salmonella were distributed along the slaughtering process of each Salmonella-positive slaughterhouse. Most of those isolates belonging to the same serotype had identical or closely related PFGE profiles. They also shared common antibiotic resistance patterns. Overall findings of this study indicated that Salmonella were sequentially transmitted through the chicken slaughtering process. PRACTICAL APPLICATION: This study provides useful information on the distribution and transmission of Salmonella serotypes through the chicken slaughtering process. Overall findings indicated the need for routine microbiological monitoring along the slaughtering process. This study also showed that on-farm control of Salmonella is needed to obtain Salmonella-free chicken carcasses.

Isolation and characterization of Avibacterium paragallinarum with different nicotinamide adenine dinucleotide requirements.

Twenty field isolates of Avibacterium paragallinarum were obtained from chickens in South Korea during 2011-2015. The isolates were identified by a HPG-2 PCR assay specific for A. paragallinarum and by biochemical tests. Growth requirements, Page serovars, carbohydrate fermentation patterns, and antimicrobial susceptibility were also examined. Most isolates (16/20) showed the typical requirement for nicotinamide adenine dinucleotide (NAD) and an enriched CO2 atmosphere for growth. One isolate needed increased levels of NAD and serum for good growth. Three isolates showed NAD-independent growth on blood agar under aerobic conditions. In terms of carbohydrate fermentation patterns, three biochemical biovars were recognized; these varied with respect to acid production from maltose and D-xylose. The 16 typical NAD-dependent isolates were serovar A while the variants, both NAD-independent isolates and the isolate with increased NAD dependency were non-typeable. All isolates were sensitive to amoxicillin-clavulanic acid, ceftiofur, gentamicin, and spectinomycin. High rates of resistance, including intermediate resistance, to lincomycin (100%), cloxacillin (75%), and erythromycin (70%) were observed. The four variant strains (the three NAD-independent isolates and the isolate showing unusual growth requirements) were more resistant to antibiotics than the typical NAD-dependent strains. The finding of NAD-independent forms of A. paragallinarum extends the known distribution of this form, previously only reported in South Africa, Mexico and Peru. There is clearly a need for increased caution in the diagnosis and, possibly, the control of infectious coryza.

Prevalence of Campylobacter species in wild birds of South Korea.

Campylobacter species cause human gastrointestinal infections worldwide. They commonly inhabit intestines of avian species including wild birds. They might play a role in the spread of infections to humans and other bird species. The prevalence of Campylobacter species in 2164 faecal samples of wild birds (representing 71 species and 28 families) captured across the Korean peninsula was evaluated in this study. The overall prevalence was 15.3% (332/2164). Bird species belonging to the family Charadriidae had the highest isolation rate (30.0%), followed by those belonging to the families Ardeidae (26.4%), Turdidae (21.9%), and Anatidae (15.3%). The prevalence of Campylobacter spp. differed significantly according to migratory habit. Stopover birds were the most commonly infected (19.0%), followed by winter migratory (16.7%) and summer migratory birds (12.3%). However, indigenous birds showed very low prevalence (2.7%). Antimicrobial susceptibility tests were performed for 213 isolates. Results showed that Campylobacter jejuni isolates (n = 169) exhibited resistance to nalidixic acid (5.3%), ciprofloxacin (3.0%), and tetracycline (1.8%), while Campylobacter lari (n = 1) displayed resistance to nalidixic acid and ciprofloxacin. However, all Campylobacter coli isolates (n = 20) were susceptible to all antimicrobials tested. This is the first report on the prevalence of Campylobacter species in wild birds that seasonally or indigenously inhabit the Korean peninsula. Our results indicate that the overall prevalence of Campylobacter in wild birds is moderate. Therefore, birds might serve as significant reservoirs for Campylobacter pathogens.

Public health significance of major genotypes of Salmonella enterica serovar Enteritidis present in both human and chicken isolates in Korea.

Salmonella enterica serovar Enteritidis is one of the most common serotypes implicated in Salmonella infections in both humans and poultry worldwide. It has been reported that human salmonellosis is mainly associated with the consumption of poultry products contaminated with serovar Enteritidis. The present study was to extensively analyze the public health risk of serovar Enteritidis isolates from chickens in Korea. A total of 127 chicken isolates were collected from clinical cases, on-farm feces, and chicken meat between 1998 and 2012 and 20 human clinical isolates were obtained from patients with diarrhea between 2000 and 2006 in Korea. To characterize the isolates from chickens and humans, we compared the pulsed-field gel electrophoresis (PFGE) patterns and multilocus variable-number tandem-repeat analysis (MLVA) profiles of the isolates. We further characterized representative isolates of different genotypes using a DNA microarray. PFGE revealed 28 patterns and MLVA identified 16 allelic profiles. The DNA microarray showed high genetic variability in plasmid regions and other fimbrial subunits of the isolates although the virulence gene contents of isolates from the same source and/or of the same genotype were unrelated. PFGE and MLVA showed that major genotypes were present in both human and chicken isolates. This result suggests that chickens in Korea pose a significant risk to public health as a source of serovar Enteritidis as has been noted in other countries.

Multiple novel H5N6 highly pathogenic avian influenza viruses, South Korea, 2016.

We report the identification of novel highly pathogenic avian influenza viruses of subtype H5N6, clade 2.3.4.4, that presumably originated from China. In addition, reassortant strains with Eurasian lineage low pathogenic avian influenza viruses were isolated in wild birds and poultry in South Korea. The emergence of these novel H5N6 viruses and their circulation among bird populations are of great concern because of the potential for virus dissemination with intercontinental wild bird migration.

Comparison of the Antimicrobial and Sanitizer Resistance of Salmonella Isolates from Chicken Slaughter Processes in Korea.

Salmonella is a foodborne pathogen worldwide. Outbreaks of Salmonella are commonly associated with consumption of contaminated foods such as poultry products. Therefore, the objective of this study was to determine the occurrence, biofilm formation, antibiotic resistance, and sanitizer resistance of Salmonella enterica isolated from chicken carcasses. A total of 318 samples were collected from 15 chicken slaughterhouses in 8 provinces of Korea. They were then examined for Salmonella contamination. S. enterica isolates were tested for their susceptibilities to 15 antimicrobials by broth microdilution method. Their biofilm formation ability and resistance to sanitizers were also evaluated. Eighty-two isolates of S. enterica were obtained from the 318 samples. There were 14 serotypes and 2 untypable isolates. Fifty-seven (69.5%) isolates were resistant to at least one antibiotic while 30 (36.6%) isolates were resistant to 5 or more antibiotics. Two S. Senftenberg and 3 S. Montevideo isolates exhibited considerable biofilm formation ability (A600 >0.2) following incubation in Luria-Bertani (LB) broth for 48 h. Biofilm cell survival and recovery growth assay after sanitization showed that most isolates were highly susceptible to 2.5% lactic acid and 0.1% cetylpyridinium chloride. Therefore, lactic acid and cetylpyridinium chloride might be alternatively or additionally used in addition to chlorine-based sanitizers that are frequently used to reduce Salmonella contamination of chicken carcasses. Our results provide basic information on the distribution of Salmonella serotypes in chicken slaughterhouses. This study also highlights the necessity to improve farming practices and use antimicrobial agents cautiously. This study also suggests that sanitization during the slaughtering process might be necessary to reduce Salmonella contamination of chicken carcasses.

Plasmid-Mediated Quinolone Resistance in Escherichia coli Isolates from Wild Birds and Chickens in South Korea.

A total of 2,423 nonduplicate isolates of Escherichia coli recovered from wild birds (n=793) and chickens (n=1,630) in South Korea were investigated for plasmid-mediated quinolone resistance (PMQR) genes. Altogether, 56 isolates with PMQR genes were identified, including 25 (3.2%) from wild birds and 31 (1.9%) from chickens, which were further characterized using molecular methods. Among them, qnrS, aac(6')-Ib-cr, qnrB, and qepA genes were detected in 47 (1.9%), 6 (0.24%), 2 (0.08%), and 1 (0.04%) isolates, respectively. The most prevalent gene, qnrS, was identified in 21 (0.9%) and 26 (1.1%) isolates from wild birds and chickens, respectively. The qnrB gene was identified in two chicken isolates, which included qnrB19 and a novel qnrB44 gene. Plasmid isolation and Southern hybridization revealed that qnrS1 was located on a large (>200 kbp) plasmid. The spread of the PMQR genes was attributed to a combination of horizontal dissemination and clonal expansion. The horizontal dissemination of PMQR genes was mostly mediated by IncK plasmids. Molecular typing demonstrated that the majority of the PMQR-positive isolates were genetically diverse. Only one chicken isolate belonged to ST131, which harbored an additional CMY-2 gene. Our findings suggest that the wild birds could serve as reservoirs of PMQR genes and spread them over long distances through migration. To our knowledge, this is the first report of PMQR genes in Korean wild birds. This study also reports qnrS2, qnrB19, qnrB44, and qepA genes for the first time in animal E. coli isolates from South Korea.

Prevalence and characteristics of Salmonella spp. isolated from commercial layer farms in Korea.

Salmonellosis is one of the most prevalent foodborne illnesses. The outbreak of this disease is often associated with eggs. In this study, the prevalence and characteristics of Salmonella was surveyed in layer farms in Korea. In addition, the risk factors affecting the prevalence of Salmonella in these farms were also assessed. Of the 32 farms and 67 flocks examined, 19 farms (59.3%) and 34 flocks (50.7%) were observed to be positive for Salmonella contamination. Salmonella was detected in the surrounding environment such as feces (41.8%), dust (40.3%), egg shells (17.2%), as well as the internal egg contents (5.2%). The incidence of Salmonella positives were tended to increase when the flock size is larger (P = 0.021). Differences in the provinces also affected Salmonella prevalence (P < 0.001). The most frequently observed Salmonella serovars in the flocks were Salmonella Bareilly (41.2%), Salmonella Mbandaka (32.4%), and Salmonella Rissen (17.6%). Twenty of the flocks revealed multi-serovar contamination, with the isolation of 2 to 4 serovars. Antimicrobial susceptibility testing revealed that 93 out of 101 isolates were susceptible to the 17 tested antimicrobial agents. The remaining isolates displayed resistance to ampicillin (4.0%), nalidixic acid (3.0%), tetracycline (1.0%), cephalothin (1.0%), and gentamicin (1.0%). As human salmonellosis has been repeatedly correlated to the consumption of poultry products worldwide, continuous studies are required to effectively minimize the Salmonella contamination in layer farms and egg products.

Arthritis in an egret (Egretta intermedia) caused by Salmonella Typhimurium and its potential risk to poultry health.

A dead Intermediate Egret (Egretta intermedia) was found on the shore of a stream in South Korea in January 2013. Salmonella Typhimurium was isolated from purulent exudates in the foot joints, demonstrating bacterial arthritis. The isolate was similar to a poultry isolate determined by pulsed-field gel electrophoresis.

SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of avian influenza viruses and comparison to standard serological subtyping tests.

Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10(1.5), 10(2.3), and 10(3.1) 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples.

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.

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.

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.

Continuing evolution and interspecies transmission of influenza viruses in live bird markets in Korea.

Live bird markets (LBMs) provide an ideal environment for the evolution and interspecies transfer of avian influenza viruses (AIVs). In this study, we analyzed AIVs present in LBMs in Korea during the winter seasons of 2006-08. Sixty-five AIVs that belong to four hemagglutination (HA) subtypes ofAIV (H3, H4, H6, and H9) were isolated from 644 pooled tissue or swab samples collected in LBMs. Most H9 subtypes of AIVs were isolated from Galliformes (chickens, silky fowls, pheasants, and guinea fowls), and other subtypes were isolated from Anseriformes (Pekin ducks and mallards). In addition, we obtained a single H3N2 virus from nasal swabs of dogs sold in LBMs, and the virus was genetically identical to the canine influenza virus (CIV) isolated from pet dogs in Korea. Phylogenetic analysis suggests that the Korean H9N2 viruses prevalent in chickens have provided their gene segments to AIVs circulating in ducks. These gene transfers facilitated reassortment events among AIVs and likely generated the ancestors of CIV in Korea. An animal challenge study using chickens, quail, mice, and dogs had shown that the H4 and H6 subtypes could replicate in mice and that some H4 and H6 viruses could replicate in chickens without preadaptation. In addition, two H3 subtype viruses (H3N2 and H3N8) induced interstitial pneumonia that accompanied clinical signs and seroconversion in dogs. Our findings indicate that the newly evolved AIVs have been continuously generated by reassortment in ducks, and these reassortments could result in expanding the host range of AIVs.