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.

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.

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 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.

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.

Diagnostic utility of egg yolk for the detection of avian metapneumovirus antibodies in laying hens.

Surveillance and diagnosis of avian metapneumovirus (AMPV) infection typically involve measurement of serum antibodies. In the current study, eggs instead of serum samples were used for the detection of AMPV antibodies in egg-laying chicken hens by enzyme-linked immunosorbent assay (ELISA). AMPV-free commercial layer hens were experimentally challenged with AMPV strain SC1509 through intravenous or oculonasal administration. Antibody levels were determined by ELISA. AMPV antibodies were detected in egg yolks from challenged hens by 7 days postinoculation (dpi), with the peak titer at 16 dpi. Antibody levels in eggs laid at 28 dpi correlated well (r = 0.93) with sera taken 28 dpi from the same hens. In a field trial of the yolk ELISA, six broiler breeder farms were surveyed, and all tested positive for AMPV antibodies in hen eggs, although positivity varied from farm to farm. Abnormal discolored eggs collected from outbreak farms had significantly higher titers of AMPV yolk antibodies than normal eggs from the same farm, unlike clinically healthy farms, where normal and abnormal eggs had similar antibody titers. These results indicate that diagnosis of AMPV infection by yolk ELISA to detect anti-AMPV antibodies may be a suitable alternative to serologic testing.

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.

Molecular epizootiology of infectious bursal disease (IBD) in Korea.

We conducted a molecular epizootiological study of infectious bursal disease (IBD) in Korea by analyzing 85 IBD viruses (IBDVs) obtained from vaccinated or unvaccinated flocks between 1980 and 2007. Phylogenetic analysis of the partial nucleotide sequence of the hypervariable region of the VP2 gene (nucleotides 661-1020) and pathogenicity tests revealed more genetic and phenotypic diversity of IBDV in Korea than has been reported previously. We showed that very virulent IBDVs (vvIBDVs) were already present in Korea in 1986. Moreover, vvIBDVs were repeatedly detected in Korean poultry that had been vaccinated, which casts doubt on the IBD vaccine programs. We also identified novel putative antigenic variant (AV)-like IBDV isolates on the basis of their antigenic indices and the presence of amino acid changes (P222S or P222T-A321D) that are known to affect the antigenicity of VP2. These observations suggest that future studies examining the efficacy of conventional vaccines against atrophy of the bursa of Fabricius and vvIBDV shedding may be useful. Moreover, it will be of interest to determine the prevalence of putative Korean antigenic variants and whether these strains exert immunosuppressive effects in vaccinated birds.

Molecular epidemiological investigation of Newcastle disease virus from domestic ducks in Korea.

To expand the epidemiological understanding of Newcastle disease virus (NDV) found in domestic ducks in Korea, 14 NDV isolates from apparently healthy domestic ducks were biologically and genetically characterized. Thirteen and 1 isolates of NDV were categorized into lentogenic and velogenic viruses, respectively, based on in vivo pathogenicity tests. Twelve lentogenic viruses showed HA activity to horse RBCs, while 1 lentogenic virus and the velogenic virus were negative. Lentogenic viruses (n=13) had sequence motifs of (112)ERQERL(117) (n=1) or (112)GRQGRL(117) (n=12) at the F0 cleavage site, while the velogenic virus (n=1) had a sequence motif of (112)RRQKRF(117) at the same site. Phylogenetic analysis revealed that at least three distinct genotypes may exist in domestic ducks in Korea; one class I genotype (genotype 2), and two class II (genotypes I and VII) genotypes. The class I virus was most closely related to strains of genotype 2 which were isolated in birds from the USA, Germany and Denmark. Twelve lentogenic class II viruses were grouped together in genotype I, and were then divided into at least three clusters, namely Aomori-like, Ulster2C-like, and V4-like. The velogenic class II virus was assigned to genotype VII which represents viruses responsible for recent epidemics in many Asian countries including Korea. The epidemiological importance of domestic duck isolates of NDV in Korea is discussed.

Experimental infection of chickens, ducks and quails with the highly pathogenic H5N1 avian influenza virus.

Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype have spread since 2003 in poultry and wild birds in Asia, Europe and Africa. In Korea, the highly pathogenic H5N1 avian influenza outbreaks took place in 2003/2004, 2006/2007 and 2008. As the 2006/2007 isolates differ phylogenetically from the 2003/2004 isolates, we assessed the clinical responses of chickens, ducks and quails to intranasal inoculation of the 2006/2007 index case virus, A/chicken/Korea/IS/06. All the chickens and quails died on 3 days and 3-6 days post-inoculation (DPI), respectively, whilst the ducks only showed signs of mild depression. The uninoculated chickens and quails placed soon after with the inoculated flock died on 5.3 and 7.5 DPI, respectively. Both oropharyngeal and cloacal swabs were taken for all three species during various time intervals after inoculation. It was found that oropharyngeal swabs showed higher viral titers than in cloacal swabs applicable to all three avian species. The chickens and quails shed the virus until they died (up to 3 to 6 days after inoculation, respectively) whilst the ducks shed the virus on 2-4 DPI. The postmortem tissues collected from the chickens and quails on day 3 and days 4-5 and from clinically normal ducks that were euthanized on day 4 contained the virus. However, the ducks had significantly lower viral titers than the chickens or quails. Thus, the three avian species varied significantly in their clinical signs, mortality, tissue virus titers, and duration of virus shedding. Our observations suggest that duck and quail farms should be monitored particularly closely for the presence of HPAIV so that further virus transmission to other avian or mammalian hosts can be prevented.

Highly pathogenic avian influenza virus (H5N1) in domestic poultry and relationship with migratory birds, South Korea.

During the 2006-2007 winter season in South Korea, several outbreaks of highly pathogenic avian influenza virus (H5N1) were confirmed among domestic poultry and in migratory bird habitats. Phylogenetic analysis showed that all isolates were closely related and that all belong to the A/bar-headed goose/Qinghai/5/2005-like lineage rather than the A/chicken/Korea/ES/2003-like lineage.

Very virulent infectious bursal disease virus isolated from wild birds in Korea: epidemiological implications.

To explore the epidemiological link between infectious bursal disease virus (IBDV) in wild birds and domestic chickens in Korea, we examined 107 free-living wild birds, representing 7 species, that were found dead of apparent natural causes in Korea over the past two years for the presence of IBDV. Five birds were tested positive for IBDV by RT-PCR assay: black-billed magpie (n=1), mallard duck (n=2), bean goose (n=1) and white-fronted goose (n=1). IBDV was isolated from RT-PCR-positive tissues following chicken embryo inoculation. Sequence analysis of the VP2 gene indicated that all of the isolates from the wild birds encode amino acids A222, I242, I256, I294 and S299 of VP2, which are conserved among strains of very virulent IBDV (vvIBDV). Phylogenetic analysis revealed that the wild bird IBDV isolates are closely related to strains of vvIBDV. An IBDV isolate from a magpie showed 60% mortality in SPF chickens and severe bursal atrophy. The epidemiological implications of IBDV in free-living wild birds are discussed. To our knowledge, this is the first report of vvIBDV in free-living wild birds.

Differential diagnosis between type-specific duck hepatitis virus type 1 (DHV-1) and recent Korean DHV-1-like isolates using a multiplex polymerase chain reaction.

Duck hepatitis can be caused by four types of viruses: duck hepatitis virus (DHV) type 1 (DHV-1), DHV-1a (a variant strain of DHV-1), DHV-2 and DHV-3. In Korea, duck hepatitis has been associated with two types of DHV-1, original DHV-1 type-specific strain (DHV-1s) and the recently reported DHV-1 variant strains (DHV-1v). The pathogenicity and pathological findings of ducklings infected with the recent DHV-1v isolates, AP-04114 and AP-04203, were almost identical to those infected with members of the DHV-1s, DHV-HS and the type-specific strain DRL-62. To be able to monitor the epidemiological patterns exhibited by the two Korean types, a specific gene-based differential diagnostic method based on multiplex polymerase chain reaction was developed. The primers selected were designed to bind to and amplify conserved regions within the RNA-dependent RNA polymerase (3D) gene, the complete capsid (P1) region or the 5'-untranslated region to distinguish between the DHV-1s and DHV-1v groups. The described multiplex polymerase chain reaction method was able to selectively recognize ducklings infected with either of the two groups of Korean isolates. The method was also able to distinguish between DHVs and other avian-originated RNA viruses. The detection limit of the diagnostic method was determined to correspond to 10(3) copies viral RNA and 100 pg used as starting template. As a result, the use of this test allows rapid and early diagnosis of two different virus types affecting the commercial duckling industry.

Generation and evaluation of reassortant influenza vaccines made by reverse genetics for H9N2 avian influenza in Korea.

The prevalence and continuous evolution of H9N2 avian influenza viruses in poultry have necessitated the use of vaccines in veterinary medicine. Because of the inadequate growth properties of some strains, additional steps are needed for producing vaccine seed virus. In this study, we generated three H9N2/PR8 reassortant viruses using a total cDNA plasmid-transfection system, as an alternative strategy for developing an avian influenza vaccine for animals. We investigated the vaccine potency of the reassortant viruses compared with the existing vaccine strain which was adapted by the 20th serial passages in embryonated eggs with A/Ck/Kor/01310/01 (H9N2). The H9N2/PR8 reassortant viruses, containing the internal genes of the high-yielding PR8 strain and the surface gene of the A/Ck/Kor/01310/01 strain, could be propagated in eggs to the same extent as existing vaccine strain without additional processing. Similar to vaccine strain, the H9N2/PR8 reassortant viruses induced hemagglutination-inhibiting antibodies in chickens and prevented virus shedding and replication in multiple organs in response to homologous infection. However, due to the continuing evolution and increasing biologic diversity of H9N2 influenza in Korea, the vaccine provided only partial protection against currently isolates. Taken together, our results suggest that the H9N2/PR8 reassortant virus can be used as a seed virus for avian influenza vaccines in poultry farm. Considering the constant genetic changes in H9 strains isolated in Korea, this reverse genetic system may offer a prompt and simple way to change the vaccine seed virus and mitigate the impact of unexpected influenza outbreaks.

Genetic diversity of avian infectious bronchitis virus isolates in Korea between 2003 and 2006.

Thirty-three field isolates of avian infectious bronchitis virus (IBV) were recovered from commercial chicken flocks in Korea between 2003 and 2006 and were characterized phylogenetically by nucleotide sequence analysis of the IBV S1 gene hyper-variable region. Our phylogenetic analysis revealed that recent field isolates of IBV formed at least three distinct phylogenetic types, including K-I, K-II, and K-III. K-I type IBV consisted of indigenous, 13 IBV isolates which evolved from the Kr-EJ/95 strain and then separated into the lineages of type K-Ia and type K-Ib. K-II type IBV isolates (n = 19) were closely related to nephropathogenic IBV variants from China and Japan. The K-III type isolate (Kr/D064/05), first identified by this study, was closely related to enteric IBV variants from the Chinese strains that cause proventriculitis. Sequence comparisons showed amino acid differences of >27.5% between IBV types. The molecular epidemiologic characteristics of IBV field isolates are briefly discussed.

Isolation of a recent Korean epizootic strain of Newcastle disease virus from Eurasian Scops Owls affected with severe diarrhea.

Velogenic Newcastle disease virus (NDV) was recovered from two dead Eurasian Scops Owls (Otus scops) from a wildlife rescue center in Korea during 2005. Phylogenetic analysis based on the sequence of the partial fusion (F) protein revealed that the isolates had the highest level of homology to recent Korean NDV strains from poultry.

An inactivated vaccine to control the current H9N2 low pathogenic avian influenza in Korea.

The H9N2 subtype low pathogenic avian influenza is one of the most prevalent avian diseases worldwide, and was first documented in 1996 in Korea. This disease caused serious economic loss in Korea's poultry industry. In order to develop an oil-based inactivated vaccine, a virus that had been isolated in 2001 (A/chicken/Korea/01310/2001) was selected based on its pathogenic, antigenic, and genetic properties. However, in animal experiments, the efficacy of the vaccine was found to be very low without concentration of the antigen (2 7 to 2 10 hemagglutinin unit). In order to overcome the low productivity, we passaged the vaccine candidate virus to chicken eggs. After the 20th passage, the virus was approximately ten times more productive compared with the parent virus. For the most part, the passaged virus maintained the hemagglutinin cleavage site amino acid motif (PATSGR/GLF) and had only three amino acid changes (T133N, V216G, E439D, H3 numbering) in the hemagglutinin molecule, as well as 18 amino acid deletions (55-72) and one amino acid change (E54D) in the NA stalk region. The amino acid changes did not significantly affect the antigenicity of the vaccine virus when tested by hemagglutination inhibition assay. Though not complete, the vaccine produced after the 20th passage of the virus (01310 CE20) showed good protection against a homologous and recent Korean isolate (A/chicken/Korea/Q30/2004) in specific pathogen- free chickens. The vaccine developed in this study would be helpful for controlling the H9N2 LPAI in Korea.

Molecular characterization and genogrouping of VP1 of aquatic birnavirus GC1 isolated from rockfish Sebastes schlegeli in Korea.

The cDNA nucleotide sequence of genome segment B encoding the VP1 protein was determined for the aquatic birnavirus GC1 isolated from the rockfish Sebastes schlegeli in Korea. The VP1 protein of GC1 contains a 2,538 bp open reading frame, which encodes a protein comprising 846 amino acid residues that has a predicted MW of 94 kDa. The sequence contains 6 potential Asn-X-Ser/Thr motifs. Eight potential Ser phosphorylation sites and 1 potential Tyr phophorylation site were also identified. GC1 contains the Leu-Lys-Asn (LKN) motif instead of the typical Gly-Asp-Asp (GDD) motif found in other aquatic birnaviruses. We also identified the GLPYIGKT motif, the putative GTPbinding site at amino acid position 248. In total, the VP1 regions of 22 birnavirus strains were compared for analyzing the genetic relationship among the family Birnaviridae. Based on the deduced amino acid sequences, GC1 was observed to be more closely related to the infectious pancreatic necrosis virus (IPNV) from the USA, Japan, and Korea than the IPNV from Europe. Further, aquatic birnaviruses containing GC1 and IPNV have genogroups that are distinct from those in the genus Avibirnaviruses and Entomo-birnaviruses. The birnavirusstrains were clustered into 5 genogroups based on their amino acid sequences. The marine aquatic birnaviruses (MABVs) containing GC1 were included in the MABV genogroup; the IPNV strains isolated from Korea, Japan, and the USA were included in genogroup 1 and the IPNV strains isolated primarily from Europe were included in genogroup 2. Avibirnaviruses and entomobirnaviruses were included in genogroup 3 and 4, respectively.

Protective efficacy of commercial inactivated Newcastle disease virus vaccines in chickens against a recent Korean epizootic strain.

Despite the intensive vaccination policy that has been put in place to control Newcastle disease virus (NDV), the recent emergence of NDV genotype VII strains in Korea has led to significant economic losses in the poultry industry. We assessed the ability of inactivated, oil-emulsion vaccines derived from La Sota or Ulster 2C NDV strains to protect chickens from challenge with Kr-005/00, which is a recently isolated Korean epizootic genotype VII strain. Six-week-old SPF chickens were vaccinated once and challenged three weeks later via the eye drop/intranasal route. All vaccinated birds were fully protected from disease, regardless of the vaccine strains used. All vaccinated and challenged groups showed significant sero-conversion 14 days after challenge. However, some vaccinated birds, despite being protected from disease, shed the challenge virus from their oro-pharynx and cloaca, albeit at significantly lower titers than the unvaccinated challenged control birds. The virological, serological, and epidemiological significance of our observations with regard to NDV disease eradication is discussed.