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.

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.

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.

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.

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.

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.

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.

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.

Evaluation of Haemagglutinin Content by RP-HPLC to Generate Pandemic Influenza Vaccine.

The potency of influenza vaccine is determined based on its hemagglutinin (HA) content. In general, single radial immunodiffusion (SRID) assay has been utilized as the standard method to measure HA content. However, preparation of reagents for SRID such as antigen and antibody takes approximately 2~3 months, which causes delays in the development of influenza vaccine. Therefore, quantification of HA content by other alternative methods is required. In this study, we measured HA contents of H1N1 antigen and H1N1 influenza vaccine by reverse phase-high performance liquid chromatography (RP-HPLC) methods. The presence of HA1 and HA2 was investigated by silver staining and Western blot assay. In addition, accuracy and repeatability of HA measurement by RP-HPLC were evaluated. Comparison of HA concentration by SRID and RP-HPLC revealed a precise correlation between the two methods. Our results suggest that RP-HPLC assay can replace SRID in the event of a pandemic flu outbreak for rapid vaccine development.

Pathology and molecular characterization of recent Leucocytozoon caulleryi cases in layer flocks.

Leucocytozoonosis was found in three layer farms in chickens with suspected fatty liver or fatty liver hemorrhagic syndrome in Korea between 2009 and 2011. These layer chicken flocks showed both mortality and decreased egg production for one or two weeks when they were between 59 and 82 weeks old. At the necropsy, the most prominent gross lesions were found in the liver, which was enlarged, had a fragile texture, exhibited yellowish discolorations, and had various hemorrhagic lesions. Tissue reactions associated with megaloschizonts specific for Leucocytozoon caulleryi were prominent upon microscopic examination of the liver without significant lipidosis. In addition, the ovaries and uterus were the most affected organs for Leucocytozoon caulleryi multiplication, which led to decreased egg productions. Molecular studies with formalin-fixed, paraffin-embedded tissues were performed in search of a partial region of the cytochrome b gene for hemosporidian parasites. Based on these results, the causal agent was determined to be closely related to Leucocytozoon caulleryi reported in Japan and Malaysia. In this study, we describe recently re-occurring leucocytozoonosis in layer chickens, which required histopathology for disease diagnosis. To prevent outbreaks and maintain chicken health and egg production, layer chickens need to be monitored for symptoms of leucocytozoonosis.

Review of Avian Influenza Outbreaks in South Korea from 1996 to 2014.

Since the first outbreak of low pathogenic avian influenza (LPAI) in 1996, outbreaks of LPAI have become more common in Korea, leading to the development of a nationwide mass vaccination program in 2007. In the case of highly pathogenic avian influenza (HPAI), four outbreaks took place in 2003-04, 2006-07, 2008, and 2010-11; a fifth outbreak began in 2014 and was ongoing at the time of this writing. The length of the four previous outbreaks varied, ranging from 42 days (2008) to 139 days (2010-11). The number of cases reported by farmers that were subsequently confirmed as HPAI also varied, from seven cases in 2006-07 to 53 in 2010-11. The number of farms affected by the outbreaks varied, from a low of 286 (2006-07) with depopulation of 6,473,000 birds, to a high of 1500 farms (2008) with depopulation of 10,200,000 birds. Government compensation for bird depopulation ranged from $253 million to $683 million in the five outbreaks. Despite the damage caused by the five HPAI outbreaks, efficient control strategies have yet to be established. Meanwhile, the situation in the field worsens. Analysis of the five HPAI outbreaks revealed horizontal farm-to-farm transmission as the main factor effecting major economic losses. However, horizontal transmission could not be efficiently prevented because of insufficient transparency within the poultry industry, especially within the duck industry, which is reluctant to report suspicious cases early. Moreover, the experiences and expertise garnered in previous outbreaks has yet to be effectively applied to the management of new outbreaks. Considering the magnitude of the economic damage caused by avian influenza and the increasing likelihood of its endemicity, careful and quantitative analysis of outbreaks and the establishment of control policies are urgently needed.

Tracking Mallards (Anas platyrhynchos) with GPS Satellite Transmitters Along Their Migration Route Through Northeast Asia.

In this study, Global Positioning System satellite transmitters were attached to three mallards (Anas platyrhynchos) wintering in South Korea to track their migration routes, stopover sites, breeding sites, and migration patterns. We successfully tracked only one mallard (no. 108917) from November 15, 2011, to November 29, 2013, and determined separate migration routes in two cases of spring migration and one case of fall migration. The mallard repeatedly migrated to the same final destination, even though the travel path varied. We identified six stopover sites: Hunhe River, Liaohe River, Yinma River, Yalu River, Songjeon Bay, and Dahuofang Reservoir in China and South Korea. The wintering sites of two migration cases were discovered to be identical (Gokgyo River in Asan, South Korea). The terminal sites, which were presumed to be breeding grounds, were the same in both cases (Hinggan League in Inner Mongolia Autonomous Region, China). On the basis of the migration routes identified in this study, we suggest that future efforts to control highly pathogenic avian influenza (HPAI) should not only include avian influenza surveillance but also implement flyway-based strategies, with regard to all countries affected by potential HPAI outbreaks.

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.

The current epidemiological status of infectious coryza and efficacy of PoulShot Coryza in specific pathogen-free chickens.

Infectious coryza (IC) is an infectious disease caused by Avibacterium (Av.) paragallinarum. IC is known to cause economic losses in the poultry industry via decreased egg production in layers. Between 2012 and 2013, Av. paragallinarum was isolated from seven chicken farms by Chungbuk National University. We identified Av. paragallinarum, the causative pathogen of IC by polymerase chain reaction (PCR) and serovar serotype A, by multiplex PCR. Antibiotic sensitivity tests indicated that a few field-isolated strains showed susceptibility to erythromycin, gentamicin, lincomycin, neomycin, oxytetracycline, spectinomycin, and tylosin. A serological survey was conducted to evaluate the number of flocks that were positive for Av. paragallinarum by utilizing a HI test to determine the existence of serovar A. Serological surveys revealed high positivity rates of 86.4% in 2009, 78.9% in 2010, 70.0% in 2011, and 69.6% in 2012. We also challenged specific pathogen-free chickens with isolated domestic strains, ADL121286 and ADL121500, according to the measured efficacy of the commercial IC vaccine, PoulShot Coryza. We confirmed the effectiveness of the vaccine based on relief of clinical signs and a decreased re-isolation rate of ADL121500 strain. Our results indicate IC is currently prevalent in Korea, and that the commercial vaccine is effective at protecting against field strains.

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.

A hemagglutinin quantification method for development of an influenza pandemic vaccine using size exclusion high performance liquid chromatography.

Single radial immunodiffusion (SRID) assay requires a reference antigen and an antibody to the hemagglutinin (HA) of an influenza vaccine. As it takes 2­3 months to develop the reference antigen, vaccine development is delayed in cases of an influenza pandemic. In the present study, the measurement of the HA content of influenza vaccines was assessed using size exclusion high performance liquid chromatography (SE­HPLC) for the rapid development of a pandemic vaccine. When the 2009 H1N1 reference antigen, pandemic 2009 H1N1 vaccine and 2010 seasonal influenza vaccines were analyzed by SE­HPLC, the HA of the reference antigen and vaccines was specifically separated. The presence and specificity of HA were evidenced with immunoprecipitation and ELISA assays. For the influenza vaccines, the chromatogram pattern and retention time of HA were similar among the antigen types (2009 H1N1, 2010 H3N2 and 2010 B). In addition, when SE­HPLC was applied, the ratio of HA chromatogram to peak area revealed a significant correlation with HA concentration for the reference antigen and vaccine. The result of the HA content calculation based on SE­HPLC exhibited 99.91­100% similarity, compared with that of SRID. These findings suggest that the measurement of peak area ratio/HA content using SE­HPLC may be a substitute for SRID and rapidly measure HA content to enable faster development of a vaccine during an influenza pandemic.

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.

Eggshell apex abnormalities associated with Mycoplasma synoviae infection in layers.

Eggs exhibiting eggshell apex abnormalities (EAA) were evaluated for changes in shell characteristics such as strength, thickness, and ultrastructure. Mycoplasma synoviae (MS) infection was confirmed by serological assay along with isolation of MS from the trachea and oviduct. Changes in eggshell quality were shown to be statistically significant (p < 0.01). We also identified ultrastructural changes in the mammillary knob layer by Scanning Electron Microscopy. While eggs may seem to be structurally sound, ultrastructural evaluation showed that affected eggs do not regain their former quality. In our knowledge, this is the first report describing the occurrence of EAA in Korea.

Pathobiological features of a novel, highly pathogenic avian influenza A(H5N8) virus.

The endemicity of highly pathogenic avian influenza (HPAI) A(H5N1) viruses in Asia has led to the generation of reassortant H5 strains with novel gene constellations. A newly emerged HPAI A(H5N8) virus caused poultry outbreaks in the Republic of Korea in 2014. Because newly emerging high-pathogenicity H5 viruses continue to pose public health risks, it is imperative that their pathobiological properties be examined. Here, we characterized A/mallard duck/Korea/W452/2014 (MDk/W452(H5N8)), a representative virus, and evaluated its pathogenic and pandemic potential in various animal models. We found that MDk/W452(H5N8), which originated from the reassortment of wild bird viruses harbored by migratory waterfowl in eastern China, replicated systemically and was lethal in chickens, but appeared to be attenuated, albeit efficiently transmitted, in ducks. Despite predominant attachment to avian-like virus receptors, MDk/W452(H5N8) also exhibited detectable human virus-like receptor binding and replicated in human respiratory tract tissues. In mice, MDk/W452(H5N8) was moderately pathogenic and had limited tissue tropism relative to previous HPAI A(H5N1) viruses. It also induced moderate nasal wash titers in inoculated ferrets; additionally, it was recovered in extrapulmonary tissues and one of three direct-contact ferrets seroconverted without shedding. Moreover, domesticated cats appeared to be more susceptible than dogs to virus infection. With their potential to become established in ducks, continued circulation of A(H5N8) viruses could alter the genetic evolution of pre-existing avian poultry strains. Overall, detailed virological investigation remains a necessity given the capacity of H5 viruses to evolve to cause human illness with few changes in the viral genome.