Tissue engineered ultra-thin descemet stripping corneal endothelial layers using porcine cornea and stem cells.

Due to their very poor proliferative capacity, the dysfunction of corneal endothelial cells can sometimes lead to incurable eye diseases that require corneal transplantation. Although many studies have been performed to reconstruct corneal endothelial cells, corneal transplantation is still considered to be the established approach. In this study, we developed bio-engineered Descemet stripping endothelial (DSE) layers, using porcine cornea and induced pluripotent stem cell (iPSC)-derived corneal endothelial cells (iCECs). First, we optimized a protocol to prepare an ultra-thin and decellularized Descemet stripping (DS) scaffold from porcine cornea. Our DS layers show over 90% transparency compared to the control. Porcine-derived cells and xenogenic antigens disappeared, whereas the collagen matrix remained in the graft. Next, corneal endothelial cell lines or iCECs were seeded on the decellularized DS graft and cultured for 7 days. The drying method reduced graft rolling and edema, and increased transparency during culture. The reseeded cells were evenly distributed over the graft, and most of the cells survived. Although future clinical studies are warranted, engineered DSE tissues using xenogenic tissues and stem cells will be useful tools for the treatment of incurable corneal diseases.

Amino acid substitutions in low pathogenic avian influenza virus strains isolated from wild birds in Korea.

Wild birds are natural hosts and reservoirs for influenza A viruses. However, many species, such as many waterfowl, are asymptomatic when infected and so facilitate the generation of viral genetic diversity. Mutations of key genes affect the replicability, pathogenicity, transmissibility, and antiviral resistance of influenza A viruses. In this study, we isolated avian influenza (AI) viruses from wild bird fecal samples and analyzed changes in amino acids over time and geographic region to monitor the biological change of the AI virus. Between 2014 and 2016, we collected 38,921 fresh fecal samples from major wild bird habitats located throughout Korea and isolated 123 AI viruses. We subsequently selected 22 amino acid sites to analyze for changes. These sites included ten sites associated with replication, ten sites associated with pathogenicity, three sites associated with transmission, and seven sites associated with antiviral resistance. We found substitution rates of 71.7% at the C38Y amino acid site within the polymerase basic protein 1 (PB1) gene, 66.7% at the D222G site within the hemagglutinin (HA) 1 gene, and 75.6% at the A184 site within the nucleoprotein (NP) gene. Alterations of the PB1, HA1, and NP genes are closely associated with increased pathogenicity in chickens and mammals. The remaining sites of interest exhibited few modifications. In this study, we confirmed that AI viruses circulating among wild birds in Korea consistently exhibit modifications at amino acid sites linked with replication and pathogenicity.

Genetic characterization of three novel chicken parvovirus strains based on analysis of their coding sequences.

Chicken parvovirus (ChPV) is one of the causative agents of viral enteritis. Recently, the genome of the ABU-P1 strain of ChPV was fully sequenced and determined to have a distinct genomic composition compared with that of vertebrate parvoviruses. However, no comparative sequence analysis of coding regions of ChPVs was possible because of the lack of other sequence information. In this study, we obtained the nucleotide sequences of all genomic coding regions of three ChPVs by polymerase chain reaction using 13 primer sets, and deduced the amino acid sequences from the nucleotide sequences. The non-structural protein 1 (NS1) gene of the three ChPVs showed 95.0 to 95.5% nucleotide sequence identity and 96.5 to 98.1% amino acid sequence identity to those of NS1 from the ABU-P1 strain, respectively, and even higher nucleotide and amino acid similarities to one another. The viral proteins (VP) gene was more divergent between the three ChPV Korean strains and ABU-P1, with 88.1 to 88.3% nucleotide identity and 93.0% amino acid identity. Analysis of the putative tertiary structure of the ChPV VP2 protein showed that variable regions with less than 80% nucleotide similarity between the three Korean strains and ABU-P1 occurred in large loops of the VP2 protein believed to be involved in antigenicity, pathogenicity, and tissue tropism in other parvoviruses. Based on our analysis of full-length coding sequences, we discovered greater variation in ChPV strains than reported previously, especially in partial regions of the VP2 protein.

Pathological Evaluation of Natural Cases of a Highly Pathogenic Avian Influenza Virus, Subtype H5N8, in Broiler Breeders and Commercial Layers in South Korea.

Outbreaks of highly pathogenic avian influenza (HPAI) virus, subtype H5N8, were observed in two different flocks of local broiler breeder farms and a commercial layer farm in South Korea. Clinically, the cases were characterized by a gradual increase in mortality, slow transmission, and unrecognizable clinical signs of HPAI. Gross observations in both cases included hemorrhagic or necrotic lesions in internal organs, such as serosal and mucosal membranes, spleen, and pancreas. Both cases exhibited similar histopathologic lesions, including multifocal malacia in the brain and multifocal or diffuse necrosis in the spleen and pancreas. Immunohistochemical results indicated that neurons and glial cells in the brain, myocytes in the heart, acinar cells in the pancreas, and mononuclear phagocytic cells in several visceral organs were immunopositive for avian influenza viral antigen. To experimentally reproduce the low pathogenicity and the mortality observed in these two cases, 18 specific-pathogen-free chickens and 18 commercial layers were divided into an H5N8 virus-inoculated group and a contact-exposed group. The mortality of the chickens in the inoculation group was 50%-100%, whereas the mean time to death was delayed or death did not occur in the contact-exposed group. The distributions of the viral antigens and histopathologic lesions in the experimental study were similar to those observed in the field cases. These findings suggest that the H5N8 virus induces a different pattern of pathobiology, including slow transmission and low mortality, compared with that of other HPAI viruses. This is the first pathologic description of natural cases of H5N8 in South Korea, and it may be helpful in understanding the pathobiology of novel H5N8 HPAI viruses.

Altered pro-inflammatory cytokine mRNA levels in chickens infected with infectious bronchitis virus.

Infectious bronchitis virus (IBV) replicates primarily in the respiratory tract and grows in various organs in chickens, with or without pathological effects. The diversity of this virus has been verified by sequence analysis of the S1 glycoprotein gene, but this method must be supplemented with further analysis for characterization of the agent. To increase our understanding of the pathogenesis of the disease caused by this virus, we investigated the response of chickens to 2 IBV with different genotypes, KIIa and ChVI. The clinical signs induced by the viruses were observed. In addition, the mRNA levels of the pro-inflammatory cytokines, IL-6, IL-1ß, and lipopolysaccharide-induced tumor necrosis factor-α factor and the serum levels of α1-acid glycoprotein, which is a major acute phase protein, were measured. The KIIa genotype (Kr/ADL110002/2011) induced clinical signs accompanied by the excessive production of pro-inflammatory cytokines and a higher viral load. In chickens infected with this isolate, simultaneous peaks in the viral copy number and cytokine production were observed at 7 dpi in the trachea and 9 d postinoculation in the kidney. On the other hand, the chickens infected with the ChVI genotype (Kr/ADL120003/2012) did not show a response other than a mild upregulation of cytokines at 1 d postinoculation, which appears to indicate the invasion of the virus. In summary, we confirmed a differential innate response following infection with distinct IBV. We hypothesize that an excessive innate response contributes to the scale of the pathophysiologic effect in chickens.

Genetic evolution of the H9N2 avian influenza virus in Korean poultry farms.

We performed whole genome sequencing of 22 H9N2 avian influenza viruses (AIV) isolated from domestic laying hens on farms between 2005 and 2008, and compared the sequences with viruses previously reported in Asia. A previous study revealed that two antigenically distinct sublineages were established within the MS96 lineage by antigenic drift since the first H9N2 AIV outbreak in South Korea. We designated them as the 01310-like lineage and the 116/04-like lineage. Since late 2004, most identified isolates in Korea have belonged to the 116/04-like lineage, however, in this study we found that six among twenty-two isolates were belonged to 01310-like lineage, indicating that the genetic divergence is still occurring after 2004. Furthermore, it is noteworthy that five isolates among the defined 01310-like lineage had a 24 amino acid deletion in the neuraminidase stalk region, which were not in any other H9N2 isolates previously reported. The internal genes analysis demonstrated extensive reassortment events among isolates from poultry farms, live bird markets, and wild birds, and multiple new genotypes were identified. We identified several features of gene evolution in H9N2 AIV suggesting that the long-term H9N2 AIV surveillance study should be continued in South Korea.

Characterization of avian paramyxovirus type 1 from migratory wild birds in chickens.

Newcastle disease virus (NDV) is one of the most important infectious agents in the poultry industry, and vaccines against it have been widely used for prevention and control. Live vaccines, which can replicate in the respiratory and digestive systems, have been especially needed in areas with outbreaks of viscerotropic velogenic Newcastle disease. Towards the goal of searching for a new live vaccine candidate, avian paramyxovirus type 1 (APMV-1) was isolated from the faeces of wild birds. Three APMV-1 strains thus isolated were characterized in terms of phylogeny, pathogenicity, immunogenicity and tissue tropism, and on the basis of these analyses were classified as lentogenic genotype I NDV. CBU2179, one of the three APMV-1 strains, was selected and was evaluated in terms of its efficacy and safety in specific pathogen-free chickens and commercial broilers. The manufactured trial vaccine from this strain, also called CBU2179, induced similar immune responses to those of VG/GA and B1 commercial vaccines, and provided 100% protection against challenge from viscerotropic velogenic NDV, KJW/49 strain (the official challenge strain in Korea). Also, the CBU2179 virus was re-isolated and persisted as long as or longer than other vaccine strains in both the respiratory and alimentary tracts. Therefore, the CBU2179 strain may represent a good candidate for a live Newcastle disease vaccine to protect chickens against viscerotropic velogenic NDV.

Prevalence, biosecurity factor, and antimicrobial susceptibility analysis of Salmonella species isolated from commercial duck farms in Korea.

Duck meat consumption in South Korea has increased in recent years, but no standard about duck farm-specific biosecurity and hygiene guidelines have yet been established. We here investigated Salmonella contamination levels in duck farms to evaluate biosecurity and hygiene practices. We collected 1,116 environmental samples from 31 duck farms in Jeonnam Province, South Korea. The Salmonella-positive farm rate dramatically increased, from 22.6 to 71.0%, on introduction of ducklings. As the ducklings aged 4-6 wk, the positive rate slightly decreased to 64.5%. The Salmonella detection rate on each sampled surface, such as the feed pan (34.4%), wall (33.9%), litter (32.3%), and nipples (24.2%), was highest at 3 wk of age. The most frequently detected Salmonella serovars were Salmonella London (22.2%), Salmonella Albany (21.6%), Salmonella Bareilly (17.0%), and Salmonella Indiana (16.5%). Implementation of cleaning and disinfection procedures, rodent control, and metal house walls significantly lowered the prevalence of Salmonella (P < 0.001, P < 0.01, and P < 0.05, respectively). A high proportion of Salmonella isolates exhibited antimicrobial resistance: 100 and 62.9% exhibited resistance to erythromycin and nalidixic acid, respectively. Furthermore, a majority of S. Albany and all Salmonella Enteritidis isolates were multidrug resistant. These results indicate the level of Salmonella contamination in duck farm environments in Korea is high. Good biosecurity and hygiene practices are the most effective measures for controlling Salmonella contamination.

Evaluation of neuraminidase antigen based competitive enzyme-linked immunosorbent assay in chickens vaccinated with avian influenza inactivated vaccine.

A competitive enzyme-linked immunosorbent assay (c-ELISA) was developed as a serologic diagnostic tool to detect antibodies against NA subtype 3 of avian influenza virus (AIV). The NA antigen used in this c-ELISA was obtained by pronase treatment of allantoic fluid of specific-pathogen-free (SPF) eggs infected with AIV. The NA specific monoclonal antibodies were produced from purified NA. The N3 c-ELISA was carried out on serum samples collected from both SPF chickens and commercial layers to confirm whether the N3 c-ELISA was capable of detecting specific N3 antibodies. The positive cutoff percentage inhibition value was 6.13%. The sensitivity and specificity of the N3 c-ELISA were 83.7% and 95.6%, respectively, which indicated that N3 c-ELISA can detect the antibodies from SPF chickens or commercial chickens vaccinated with H9N3 subtype of AIV.

Infection and Rapid Transmission of SARS-CoV-2 in Ferrets.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China and rapidly spread worldwide. To prevent SARS-CoV-2 dissemination, understanding the in vivo characteristics of SARS-CoV-2 is a high priority. We report a ferret model of SARS-CoV-2 infection and transmission that recapitulates aspects of human disease. SARS-CoV-2-infected ferrets exhibit elevated body temperatures and virus replication. Although fatalities were not observed, SARS-CoV-2-infected ferrets shed virus in nasal washes, saliva, urine, and feces up to 8 days post-infection. At 2 days post-contact, SARS-CoV-2 was detected in all naive direct contact ferrets. Furthermore, a few naive indirect contact ferrets were positive for viral RNA, suggesting airborne transmission. Viral antigens were detected in nasal turbinate, trachea, lungs, and intestine with acute bronchiolitis present in infected lungs. Thus, ferrets represent an infection and transmission animal model of COVID-19 that may facilitate development of SARS-CoV-2 therapeutics and vaccines.

Genetic Characterization of Fluoroquinolone Resistance in Salmonella enterica Serovar Gallinarum Isolates from Chicken in Korea.

Salmonella enterica serovar Gallinarum is a nonmotile host-adapted Salmonella that causes fowl typhoid (FT), and an outbreak of FT is characterized by anorexia, greenish-yellow diarrhea, paleness, and sudden death with high mortality in poultry. To control and treat FT in commercial chickens, fluoroquinolones are widely used in Korea. This study aimed to investigate the genetic characteristics of fluoroquinolone-resistant Salmonella Gallinarum isolates from 2014-18 from chicken in Korea. A total of 35 ciprofloxacin (CIP)-resistant Salmonella Gallinarum was tested, and 22 (62.9%) isolates were observed to have multidrug resistance. All isolates had a mutation at the Ser83 or Asp87 codon in the gyrA gene, whereas three isolates had only double mutations at Ser83 → Phe and Asp87 → Asn or Ser83 → Phe and Asp87 → Gly. Minimum inhibitory concentrations of isolates with double mutations were relatively higher (≥8 mg/L for CIP and ≥16 mg/L for enrofloxacin) than those of other isolates with a single mutation in gyrA. Among 35 CIP-resistant Salmonella Gallinarum, plasmid-mediated quinolone resistance genes were detected in six (17.1%) isolates, and qnrB and qnrS were detected in four and two isolates, respectively. In the distribution of antimicrobial resistance genes in 35 CIP-resistant Salmonella Gallinarum, ant(2″)-I (54.3%) was the most prevalent gene, followed by TEM-1 (14.3%), sul1 (11.4%), and cmlA (5.7%). Fifteen (42.9%) of the 35 CIP-resistant Salmonella Gallinarum also carried class 1 integrons, which showed five types of resistance gene cassettes: aadA2 (7 isolates), aadA2 + dfrA12 (5 isolates), and aadA1 + aad A2 (3 isolates). Among plasmid replicons, 23 isolates (65.7%) carried five different plasmid replicons: Frep (9 isolates), FIB (7 isolates), FIIA (6 isolates), B/O (4 isolates), and I1 (3 isolates). These results suggest that continued monitoring of fluoroquinolone resistance is necessary to preserve the effectiveness of fluoroquinolones in poultry and to surveil the transmission to humans through the food chain.

Caracterización genética de la resistencia a las fluoroquinolonas en aislados de Salmonella enterica Serovar Gallinarum de pollo en Corea. Salmonella enterica serovar Gallinarum es una Salmonella adaptada al huésped no móvil que causa la tifoidea aviar (FT), y un brote de tifoidea aviar se caracteriza por anorexia, diarrea de color amarillo verdoso, palidez y muerte súbita con alta mortalidad en aves comerciales. Para controlar y tratar la tifoidea aviar en pollos comerciales, las fluoroquinolonas se usan ampliamente en Corea. Este estudio tuvo como objetivo investigar las características genéticas de los aislados de Salmonella Gallinarum resistentes a las fluoroquinolonas entre los años 2014 al 2018 de pollos en Corea. Se evaluaron un total de 35 Salmonella Gallinarum resistentes a la ciprofloxacina (CIP), y se observó que 22 aislamientos (62.9%) tenían resistencia a múltiples fármacos. Todos los aislamientos tenían una mutación en el codón de Serina (Ser) 83 o de aspartato (Asp) 87 en el gene gyrA, mientras que tres aislamientos solo tenían mutaciones dobles Ser83 → fenilalanina (Phe) y Asp87 → asparagina (Asn) o Ser83 → Phe y Asp87 → glicina (Gly). Las concentraciones inhibitorias mínimas de los aislamientos con mutaciones dobles fueron relativamente más altas (≥8 mg/L para ciprofloxacina y ≥16 mg/L para enrofloxacina) en comparación con otros aislamientos con una sola mutación en gyrA. Entre los 35 aislados de Salmonella Gallinarum resistentes a ciprofloxacina, se detectaron genes de resistencia a quinolonas mediados por plásmidos en seis (17.1%) aislamientos, y los genes qnrB y qnrS se detectaron en cuatro y dos aislamientos, respectivamente. En la distribución de genes de resistencia a los antimicrobianos en 35 aislamientos de Salmonella Gallinarum resistentes a ciprofloxacina, el gene ant(2″)-I (54.3%) fue el más prevalente, seguido de TEM-1 (14.3%), sul1 (11.4%) y cmlA (5.7%). Quince (42.9%) de los 35 aislados de Salmonella Gallinarum resistentes a ciprofloxacina también portaban integrones de clase 1, que mostraron cinco tipos de casetes genéticos de resistencia: aadA2 (siete aislamientos), aadA2 + dfrA12 (cinco aislamientos) y aadA1 + aadA2 (tres aislamientos). Entre los replicones de plásmidos, 23 aislamientos (65.7%) portaban cinco replicones de plásmidos diferentes: Frep (nueve aislamientos), FIB (siete aislamientos), FIIA (seis aislamientos), B/O (cuatro aislamientos) e I1 (tres aislamientos). Estos resultados sugieren que el monitoreo continuo de la resistencia a las fluoroquinolonas es necesario para preservar la efectividad de las fluoroquinolonas en las aves comerciales y para vigilar la transmisión a los humanos a través de la cadena alimentaria. Key words: Salmonella enterica serovar Gallinarum, fowl typhoid, chicken, fluoroquinolone resistance.

Molecular characteristic of antimicrobial resistance of Salmonella Gallinarum isolates from chickens in Korea, 2014 to 2018.

Fowl typhoid (FT), which is caused by Salmonella enterica serovar Gallinarum (S. Gallinarum), leads to high morbidity and acute or subacute mortality in chickens of all ages. Although a live S. Gallinarum 9R vaccine was introduced in 2001 for commercial layer chickens in Korea, until recently, a variety of antimicrobials were widely used to prevent or treat FT. In this study, we investigated antimicrobial resistance in S. Gallinarum strains isolated from 2014 to 2018 and characterized the multidrug-resistant (MDR) strains to better understand the resistance trends in recent isolates. A total of 130 S. Gallinarum isolates were collected from chickens with FT, and the isolates showed highest rates of resistance to nalidixic acid (78.5%), followed by gentamicin (52.3%), ciprofloxacin (26.9%), and ampicillin (14.6%). Particularly, significant increases (P < 0.05) in the frequencies of resistance to the following antimicrobials were observed: ampicillin (from 7.7 to 28.6%), amoxicillin-clavulanate (from 0.0 to 10.7%), nalidixic acid (from 69.2 to 100.0%), ciprofloxacin (from 15.4 to 50.0%), chloramphenicol (from 0.0 to 17.9%), and colistin (from 0.0 to 14.3%). The prevalence of MDR isolates also rapidly increased from 23.1% in the 2014 to 60.7% in the 2018 (P < 0.05). The distribution of antimicrobial resistance genes in the 39 MDR S. Gallinarum isolates was as follows: ant(2")-I gene (22 isolates), blaTEM-1 gene (13 isolates), sul1 (9 isolates), sul2 (3 isolates), cmlA (3 isolates), and qnrB (3 isolates). Of 39, 25 (64.1%) MDR S. Gallinarum isolates also carried class 1 integrons, and these showed 5 types of resistance gene cassettes: dfrA12+aadA2 (36.0%), aadA2 (36.0%), aadA1-aadA2 (20.0%), dfrA12+catB3+aadA2 (4.0%), and dfrA12 (4.0%). Among the plasmid replicons, B/O (33.3%) was more prevalent than the other replicon types, followed by Frep (25.0%), FIIA (19.4%), FIB (13.9%), and I1 (8.3%). Antimicrobial resistance may become a serious problem because many drugs are likely ineffective for the treatment of FT. Therefore, these data support the critical need for comprehensive surveillance of antimicrobial resistance in poultry.

The difference of detection rate of avian influenza virus in the wild bird surveillance using various methods.

Korea is located within the East Asian-Australian flyway of wild migratory birds during the fall and winter seasons. Consequently, the likelihood of introduction of numerous subtypes and pathotypes of the Avian influenza (AI) virus to Korea has been thought to be very high. In the current study, we surveyed wild bird feces for the presence of AI virus that had been introduced to Korea between September 2017 and February 2018. To identify and characterize the AI virus, we employed commonly used methods, namely, virus isolation (VI) via egg inoculation, real-time reverse transcription-polymerase chain reaction (rRT-PCR), conventional RT-PCR (cRT-PCR) and a newly developed next generation sequencing (NGS) approach. In this study, 124 out of 11,145 fresh samples of wild migratory birds tested were rRT-PCR positive; only 52.0% of VI positive samples were determined as positive by rRT-PCR from fecal supernatant. Fifty AI virus specimens were isolated from fresh fecal samples and typed. The cRT-PCR subtyping results mostly coincided with the NGS results, although NGS detected the presence of 11 HA genes and four NA genes that were not detected by cRT-PCR. NGS analysis confirmed that 12% of the identified viruses were mixed-subtypes which were not detected by cRT-PCR. Prevention of the occurrence of AI virus requires a workflow for rapid and accurate virus detection and verification. However, conventional methods of detection have some limitations. Therefore, different methods should be combined for optimal surveillance, and further studies are needed in aspect of the introduction and application of new methods such as NGS.

Immune responses and pathogenesis in immunocompromised chickens in response to infection with the H9N2 low pathogenic avian influenza virus.

The H9N2 low pathogenic avian influenza (LPAI) viruses have often caused moderate mortality with severe clinical signs in domestic poultry in many Eurasian countries and have occasionally caused clinical respiratory diseases in humans, but the basis for their pathogenesis remains unclear especially in chickens. To better understand the effect of immunosuppression on the risk of H9N2 viral infection, the pathogenesis and host immune responses of the H9N2 LPAI virus in a T-cell-suppressed chicken model were investigated. Cyclosporin A (CsA) treatment led to suppression of cell-mediated immunity such as CD8+ T-cells and reduced expression of IFN-gamma mRNA. T-cell suppression correlated with high viral load in the oropharynx and cloaca of H9N2 LPAI virus-infected specific pathogen free (SPF) chickens. Elevated level of viral RNA in the peripheral blood lymphocytes was found only in immunocompromised chickens. Viral protein and associated cellular apoptosis were observed only in the kidney of the immunocompromised chickens, particularly in those that had died. Our findings suggest that T-cell-mediated responses are important in influenza viral clearance and may help to explain in part the reasons for the increased mortality in chickens infected with H9N2 LPAI viruses in domestic poultry farms.

Pemphigus-like immune-mediated dermatosis in a Korean native black-bone fowl.

A 3-yr-old female Korean native black-bone fowl weighing 1 kg presented with refractory dermatosis characterized by hyperkeratosis and alopecia on the head, neck, and scapular region. Antibiotic therapy before referral had not reduced the severity of lesions. Histopathologic findings revealed hyperkeratosis, acantholysis, heterophil infiltration, and ballooning degeneration in the epidermis. Immunohistochemical analysis showed immunoglobulin G deposits in the intercellular spaces of the intermediate layer of the epidermis. Based on these observations, this case was diagnosed as a pemphigus-like immune-mediated dermatosis. The administration of prednisolone eliminated the lesions, and the feathers grew back partially.

Outbreaks of hydropericardium syndrome and molecular characterization of Korean fowl adenoviral isolates.

Outbreaks ofhydropericardium syndrome (HPS), caused by fowl adenovirus serotype 4 (FAdV-4), have occurred in Korea and caused severe economic loss due to mortality and weight loss. From these outbreaks, several adenoviruses were isolated and identified in samples from broilers, layers, breeders, and native Korean fowl. In pathologic examinations, hydropericardium and multifocal hepatic necrosis, with an intranuclear inclusion body in hepatocytes, were observed. Specific adenovirus particles were also observed in the nucleus of hepatocytes, by electron microscopic examination. Polymerase chain reaction (PCR) analysis of the hexon gene identified all of the isolates as FAdV, serotype 4 and genotype C. To reproduce FAdV-4 field cases, 8- and 52-week-old specific pathogen free (SPF) chicks were infected intramuscularly with the field isolate CBU070244. The mortality rate of infected chicks ranged from 10%-40%, and specific pathologic lesions, such as swollen livers and hydropericardium, were observed. Further studies to determine the prevalence of infection, and analysis of the economic impact to the poultry industry, are needed in the near future.

Juvenile diabetes mellitus accompanied by exocrine pancreatic insufficiency in a dog.

A 6-month-old male crossbred dog weighing 0.78 kg was presented with acute bilateral immature cataracts, intermittent diarrhea and growth retardation. The clinical manifestations and laboratory findings were suggestive of concurrent juvenile diabetes mellitus (DM) and exocrine pancreatic insufficiency (EPI). Moreover, the DM was associated with a decreased level of serum insulin-like growth factor I. Histological examination revealed a markedly lower number of pancreatic islets and acinar cells. This case shows that juvenile-onset DM can occur simultaneously with EPI and result in growth retardation, acute cataract formation and a high cortisol concentration.

Comparison of the pathogenic potential of highly pathogenic avian influenza (HPAI) H5N6, and H5N8 viruses isolated in South Korea during the 2016-2017 winter season.

Highly pathogenic avian influenza (HPAI) A(H5N6) and A(H5N8) virus infections resulted in the culling of more than 37 million poultry in the Republic of Korea during the 2016/17 winter season. Here we characterize two representative viruses, A/Environment/Korea/W541/2016 [Em/W541(H5N6)] and A/Common Teal/Korea/W555/2017 [CT/W555(H5N8)], and evaluate their zoonotic potential in various animal models. Both Em/W541(H5N6) and CT /W555(H5N8) are novel reassortants derived from various gene pools of wild bird viruses present in migratory waterfowl arising from eastern China. Despite strong preferential binding to avian virus-type receptors, the viruses were able to grow in human respiratory tract tissues. Em/W541(H5N6) was found to be highly pathogenic in both chickens and ducks, while CT/W555(H5N8) caused lethal infections in chickens but did not induce remarkable clinical illness in ducks. In mice, both viruses appeared to be moderately pathogenic and displayed limited tissue tropism relative to HPAI H5N1 viruses. Em/W541(H5N6) replicated to moderate levels in the upper respiratory tract of ferrets and was detected in the lungs, brain, spleen, liver, and colon. Unexpectedly, two of three ferrets in direct contact with Em/W541(H5N6)-infected animals shed virus and seroconverted at 14 dpi. CT/W555(H5N8) was less pathogenic than the H5N6 virus in ferrets and no transmission was detected. Given the co-circulation of different, phenotypically distinct, subtypes of HPAI H5Nx viruses for the first time in South Korea, detailed virologic investigations are imperative given the capacity of these viruses to evolve and cause human infections.

MOLECULAR EPIDEMIOLOGY OF AVIAN POXVIRUS IN THE ORIENTAL TURTLE DOVE (STREPTOPELIA ORIENTALIS) AND THE BITING MIDGE (CULICOIDES ARAKAWAE) IN THE REPUBLIC OF KOREA.

A total of 600 wild birds were analyzed for the causes of mortality in the Republic of Korea (ROK) from 2011 to 2013. Avian poxvirus (APV) infections were identified as the primary cause of mortality in 39% (29/74) Oriental Turtle Doves (Streptopelia orientalis). At necropsy, all 29 S. orientalis birds, of which, 76% (22/29) were juveniles, had severe diphtheritic lesions in their oral and nasal cavities and on their eyelids, which were the lesions of APV that resulted in mortality. We detected APV infection by chorioallantoic membrane inoculation and molecular study of the partial region of the P4b gene. All isolates belonged to the same APV strain and were identical to strains isolated from several different pigeon species in South Africa. Phylogenetically, the APV strain identified in S. orientalis belonged to subclade A2, which includes isolates from several species of pigeons from different parts of the world, including the United Kingdom, Germany, India, Egypt, Hawaii, Georgia, Hungary, South Africa, Tanzania, and the ROK. This identity indicated that this diphtheritic APV strain may be a potential pathogen of other pigeon species in the ROK and neighboring countries throughout the range of S. orientalis. However, reticuloendotheliosis virus insertion into the APV genome was not detected by PCR in any of the 29 APV infections. An identical strain of APV observed in S. orientalis was also detected in Culicoides arakawae (biting midge), with annual peak populations corresponding to the presence of APV in S. orientalis. Culicoides arakawae may be a primary vector of APV in S. orientalis. Active surveillance of APVs in wild birds and C. arakawae is needed to better understand the epidemiology of APVs, host-vector relationships, and its ecological effects on S. orientalis in the ROK.

Assessment of the safety and efficacy of low pathogenic avian influenza (H9N2) virus in inactivated oil emulsion vaccine in laying hens.

In Korea, several outbreaks of low pathogenic AI (H9N2) viral infections leading to decreased egg production and increased mortality have been reported on commercial farms since 1996, resulting in severe economic losses. To control the H9N2 LPAI endemic, the Korea Veterinary Authority has permitted the use of the inactivated H9N2 LPAI vaccine since 2007. In this study, we developed a killed vaccine using a low pathogenic H9N2 AI virus (A/chicken/Korea/ADL0401) and conducted safety and efficacy tests in commercial layer farms while focusing on analysis of factors that cause losses to farms, including egg production rate, egg abnormality, and feed efficiency. The egg production rate of the control group declined dramatically 5 days after the challenge. There were no changes in feed consumption of all three groups before the challenge, but rates of the control declined afterward. Clinical signs in the vaccinated groups were similar, and a slight decline in feed consumption was observed after challenge; however, this returned to normal more rapidly than the control group and commercial layers. Overall, the results of this study indicate that the safety and efficacy of the vaccine are adequate to provide protection against the AI field infection (H9N2) epidemic in Korea.