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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-1β, 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.

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.

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.

This report describes the different responses to dapsone treatment in two cases of sterile nodular panniculitis (SNP). Two dogs were presented with ulcerative skin lesions, painful and erythematous papules, and nodules. History and physical examination revealed systemic signs such as pyrexia, lethargy, depression, and anorexia, in addition to ulcerated and ruptured nodules on the skin. The dermatological diagnostics included clear taping, trichogram, skin scraping, impression smears, fungal and bacterial cultures, and histopathology and special stainings of multiple punch biopsies obtained from the skin lesions. Based on the clinical and histopathologic findings, the absence of microbiological infection, and the positive response to immunosuppressive therapy, both the dogs were diagnosed with SNP. Although both dogs had been treated with various immunosuppressive drugs including prednisolone, cyclosporine, azathioprine, and triamcinolone, therapy was switched to dapsone due to recurrent dermatological signs and presumed steroidinduced hepatotoxicity. The clinical responses to dapsone were opposite in the two cases. In the first case, combination therapy with prednisolone and cyclosporine was effective in attenuating ulcerative lesions, while dapsone alone did not control the clinical signs. In contrast, in the second case, the therapeutic response to the common immunomodulatory drugs such as prednisolone, triamcinolone, and azathioprine was inadequate. Interestingly, dapsone alone was effective in controlling the clinical signs without causing undue side effects. Although the usefulness of dapsone for the treatment of canine SNP is unknown, it may be considered in mild to moderate cases of SNP when the use of steroids is not recommended due to its low efficacy or side effects.

Clostridium perfringens is ubiquitous in the environment and the gastrointestinal tract of warm-blooded animals. While part of the gut microbiome, abnormal growth of C. perfringens causes histotoxic, neurologic, and enteric diseases in a variety of animal species, including humans, due to the production of toxins. There is extremely limited information on C. perfringens infection in non-human primates. Presently, 10 strains were successfully isolated from 126 monkeys and confirmed by molecular and biochemical analyses. All isolates were genotype A based on molecular analysis. Alpha toxin was identified in all isolates. Beta 2 toxin was detected in only three isolates. No other toxins, including enterotoxin, beta, iota, epsilon, and net B toxin, were identified in any isolate. All isolates were highly susceptible to β-lactam antibiotics. Double hemolysis and lecithinase activity were commonly observed in all strains. Biofilm formation, which can increase antibiotic resistance, was identified in 90% of the isolates. The data are the first report the prevalence and characteristics of C. perfringens isolated from captive cynomolgus monkeys.

Caracterización genética de la resistencia a las fluoroquinolonas en aislados de

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.

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

The study of ecosystem assessment(ES) is based on land cover information, and primarily it is performed at the global scale. However, these results as data for decision making have a limitation at the aspects of range and scale to solve the regional issue. Although the Ministry of Environment provides available land cover data at the regional scale, it is also restricted in use due to the intrinsic limitation of on screen digitizing method and temporal and spatial difference. This study of objective is to generate UAV land cover map. In order to classify the imagery, we have performed resampling at 5m resolution using UAV imagery. The results of object-based image segmentation showed that scale 20 and merge 34 were the optimum weight values for UAV imagery. In the case of RapidEye imagery;we found that the weight values;scale 30 and merge 30 were the most appropriate at the level of land cover classes for sub-category. We generated land cover imagery using example-based classification method and analyzed the accuracy using stratified random sampling. The results show that the overall accuracies of RapidEye and UAV classification imagery are each 90% and 91%.

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.

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.

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.

The need for coastal forests has been emphasized by the increased risk of natural disasters in coastal areas in recent years. To identify the effect of coastal forests on the foredune plant ecosystem, plant species numbers, cation distribution, wind speed, temperature and humidity were compared at the grass-covered dune (D1) and the planting site of Pinus thunbergii (D2) in Sohwang dune field, South Korea. The number of plant species at D2 turned out to be 2.1 times larger than at D1, with a total of 44 taxa, including 29 families, 40 genera, 35 species, 7 varieties, and 2 forms. Terrestrial plants such as Artemisia princeps and Lonicera japonica were commonly found at D2, whereas D1 was covered by typical dune plant species such as Elymus mollis and Carex kobomugi. Decreased wind velocity and changes in nutrients due to the forest of P. thunbergii have resulted in these differences. This study demonstrates that developing coastal forests on foredune environments reduces wind velocity, salt spray and blown sand, and thus, disturbs dune vegetation due to the accelerated introduction of terrestrial and alien plants.

While use of mass rearing systems improved poultry production, chances of exposing to contagious diseases have been increased, making flocks more vulnerable to diseases. Diseases of interest which affects egg production adversely include Low pathogenic avian influenza (LPAI), Infectious bronchitis (IB), Avian meta-pneumoviral infection (aMPV) and Egg drop syndrome’76 (EDS’76). This report collected and analyzed 5,385 serum samples, which were collected from 1,330 different chicken flock, provided by Chungbuk National University, Avian Disease Laboratory at 2009. Serums were analyzed based on rearing stages; 0∼1.3weeks (wks) (maternal antibody period), >1.3∼3 wks (starting period), >3∼10 wks (growing period), >10∼22 wks (developing period), >22∼40 wks (peak laying period), >40∼60 wks (late laying period) and over 60 wks (post-molting period). Results showed the 99.7% of the tested flocks were immunized against ND and73.8%, 97.1%, 78,2% and 78% of the flocks were immunized against other 4 agents (LPAI, IB, EDS’76, aMPV). Maternal antibody was transferred to enough quantity for NDV. Generally, antibody titers which were developed at 22 weeks were stabilized permanently for life. In case of IB and aMPV, infection titer emerged as early as 10 weeks and the titer was increased from 99.4% to 100% for life. EDS76 showed increase in titers, reflecting decreased frequency of vaccination programs. Overall, this study displayed general trends of major viral disease in layers, but considering the trend of development of preventive measures and evolution of pathogens, conducting serological surveys on a regular basis is important.

A captive mute swan ( Cygnus olor ) in the brooding period was found dead without any specific signs. On necropsy, the entire digestive tract was severely necrotized with loosely adherent pseudomembranes, and Clostridium perfringens (CP) colonies isolated from the intestinal contents were identified as CP type A by polymerase chain reaction. Microscopically, the intestine and the liver were necrotized and intralesional bacterial clusters were observed. In this case, there were no other mortalities, so the con-dition of a single bird, in particular, stress from incubation effort, could act as the predisposing factor for necrotic enteritis. In this study, we report a unique case of necrotic enteritis associated with in-cubation in a captive mute swan. Key words : Cygnus olor , Incubation effort, Mute swan, Necrotic enteritis, Stress factor INTRODUCTION Necrotic enteritis (NE) is a clinically dramatic enteric disease afflicting birds which is characterized by an in-testinal necrotic fibrinous pseudomembrane with a “Turkish towel” appearance (Opengart and Songer, 2013). NE occurs in the presence of predisposing fac-tors, such as feed ingredients, stress or intestinal para-sites (Timbermont et al, 2011) that cause a microbial imbalance favorable for the overgrowth of the causal agent

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.

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.

Avian paramyxovirus (APMV) type 4 and 6 were isolated during an avian influenza (AI) surveillance program of wild birds. This study also conducted experimental infection of wild-bird-origin APMV type 4 and 6 in specific pathogen free (SPF) chickens to study pathogenicity and transmission within domestic flocks. In addition, serological prevalence data of APMV type 4 and 6 in domestic fowls was conducted with chicken sera collected from 2007 to 2009 in order to understand infection status. The results of the animal experiment showed that APMV type 4 and 6 had the ability to infect chickens with sero-conversion and to transmit the virus from infected birds to contacted birds, but showed low pathogenicity. Serological tests revealed that APMV type 4 was widespread in the poultry industry, especially in layer flocks, but the positive rate for APMV type 6 was very low. This study concluded that wild bird-origin APMV type 4 and 6 could infect the chickens by inter-species transmission and the seroprevalence of APMV type 4 was quite high in Korean poultry. However, since almost all the chicken flocks had a high level of antibody titer against APMV type 1, there was possibility of cross reaction between APMV type 1 and 4, which made the interpretations more complicated. In order to understand infection status in the natural environment, additional study is necessary regarding the seroprevalence of APMV type 4 and 6 in the wild bird population.

An avian rotavirus (AvRV-2) was isolated from feces of broilers suffering from acute gastroenteritis in 2011. It was the first avian rotavirus isolated in Korea. To investigate the molecular characteristics of AvRV-2, the VP4, VP6, VP7 and NSP4 gene nucleotide sequences were determined and compared with those of rotavirus strains available in the GenBank database. The phylogenetic tree of VP7 gene showed that AvRV-2 had a high degree of nucleotide sequence homology (93.4% to 94.7%) with those of rotaviruses belonging to genotype G19 cluster. The phylogenetic tree of the VP4 gene revealed a high degree of nucleotide sequence homology (95.8% to 95.9%) with genotype P[30] rotaviruses isolated from chickens. The VP6 and NSP4 gene nucleotide sequences showed the highest identities with those of avian strains with 95.3% to 96.4% and 90.3% to 92.2%, respectively. Genetic characterization of the VP4, VP6, VP7 and NSP4 showed that AvRV-2 strain was most closely related to chicken rotavirus strains from Germany and Japan. Comparative nucleotide sequences and phylogenetic analysis indicated that avian rotavirus isolated from broilers belonged to genotype G19P[30] and it was the first report on avian rotavirus infection in Korea.

Choi K.H., Kim, Y.M., and Jung, P.M., 2013. Adverse effect of planting pine on coastal dunes, Korea. Forestation on the coastal dunes has been regarded as one of the best programs that help to stabilize the dune landscapes and protect the residential area from strong winds, blown sands, and salt spray. Introducing exotic species for dune stabilization was very common in the past. However, it could deprive coastal land of its ability to naturally recover, causing a retreat of coastline as well. As a typhoon passed through the Korean Peninsula early September in 2010, coastal dunes along the west coast of the peninsula were severely damaged during the storm. Some dunes retreated landward more after erosion, while others recovered their original profile through aeolian transport processes mainly in winter season. Vegetated dunes with pine trees recovered relatively less after the erosion than grass-covered dunes. Planting pine trees may interfere in these interactions, weakening natural recuperative...

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.

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.

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.

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.

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.

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.

The entrance of influenza virus into host cells is facilitated by the attachment of the globular region of viral hemagglutinin to the sialic acid receptors on host cell surfaces. In this study, we have cloned the cDNA fragment encoding the entire globular region (residues 101-257) of hemagglutinin of the H9N2 type avian influenza virus (A/ck/Korea/ ms96/96). The protein segment (denoted as the H9 peptide), which was expressed and purified in E. coli, was used for the immunization of BALB/c mice to obtain the anti-H9 antiserum. To identify specific DNA aptamers with high affinity to H9 peptide, we conducted the SELEX method; 19 aptamers were newly isolated. A random mixture of these aptamers showed an increased level of binding affinity to the H9 peptide. The sequence alignment analysis of these aptamers revealed that 6 aptamers have highly conserved consensus sequences. Among these, aptamer C7 showed the highest similarity to the consensus sequences. Therefore, based on the C7 aptamer, we synthesized a new modified aptamer designated as C7-35M. This new aptamer showed strong binding capability to the viral particles. Furthermore, it could prevent MDCK cells from viral infection by strong binding to the viral particles. These results suggest that our aptamers can recognize the hemagglutinin protein of avian influenza virus and inhibit the binding of the virus to target receptors required for the penetration of host cells.

Avian reovirus (ARV) and fowl adenovirus (FAdV) were evaluated for pathogenicity in specific pathogen free (SPF) chickens. ARV was isolated from the broilers with history of malabsorption syndrome (MAS). FAdV was isolated from the layer breeders with inclusion body hepatitis and hydropericardium syndrome. Total 6 inoculated groups including 1 un-inoculated group were organized and inoculated with the ARV and/or FAdV by oral route. The minimal pathological lesions and lower viral gene detection rates were present in the ARV inoculated groups compared to those of FAdV or ARV/FAdV inoculated groups. Common gross lesions in the ARV inoculated group were distended intestine with foamy contents and in the FAdV group there were foamy cecal contents and hydropericardium among the evaluation methods such as gross and histological lesion, viral gene detection, body weight and serum chemistry, histopathological lesion score was reliable especially in the liver lesions such as hepatic necrosis and lymphocytic infiltration. However, we did not success to evaluate the synergetic effect of mixed infection of ARV and FAdV in this study. Therefore, we need further study to reproduce malabsorption syndrome of ARV infection using different viral agent such as rotavirus and using different dose of virus.

ChoongAng Vaccine Laboratories, Daejeon 305-348, KoreaABSTRACT A case of aspergillosis in 39-day-old layer chickens having a history of gradual emaciation and subsequently death with nervous signs such as torticollis and lack of equilibrium was documented. Based on the results from serology and polymerase chain reaction (PCR) test, this flock was not affected with known viral or bacterial diseases. On postmortem examination of the affected birds, multiple white to yellow nodules measuring 15 mm in diameter were observed in the lungs, cerebrum, liver and kidney. Microscopically, these nodules were identified as granulomatous lesions characterized by mixed population of multinucleated giant cells and lymphocytes. By periodic acid-schiff staining and nucleotide sequencing analysis, Aspergillus flavus with charac-teristic septate and branched hyphae were identified in the granuloma of lung and cerebrum. This case was a chronic and multisys-temic aspergillosis specialized to central nervous system caused by Aspergillus flavus infection in the layer flocks. (Key words : Aspergillus flavus, multisystemic infection, granuloma, layer chickens, central nervous system)To whom correspondence should be addressed : moip@cbu.ac.kr ! "(Aspergillosis)#Aspergillus $%&'()* +,-,./* 012+345'67'8, 69 *:( ;' ?, @A , BCD EFGHIJ K*:LMNMOI(Chutt and Richard, 1997). -PQC"C+,4.RS, TUVW, . "H'O+8, XY'Z*[\), ] ), ^), _)H` ; 0'abc OI. ! "* Xde#2f*ghIJi, -,jk2f*:aba4l;m no )‡£¤‰1n ,¥¦Va\§x¨t*>)NjO4no\§‡ ?()© OI4LM OI(Clark et al., 1954). Ž *: *: ! "#Œa=(«¬ H, 1997), «­(®n¯, 2000; 2002), b ( n>H, 2001), °±G( ²³H, 2004), ´µ(¶·¸H, 2004) E¹Mo(¶·¸H, 2006) H*:./, »¼š½¾4?, š, yšH š/ « K; 0'67~+,ab¨5+,LMNAI. 69«­*: ! "#42f\A20% Xde'l;+,pq8,  ?E» "'BA‡5+,LMNAI(®n¯, 2000; 2002). 'AA ! " p 'AEC9LMNMO4E i, E"E*:4392f Ž}S­Eš*:_vi`

In January 2010, high mortalities of ducklings occurred in two adjacent farms. Ducklings in farm A showed neurologic signs including paddling, opisthotonus and lameness, but ducklings from farm B had no specific clinical signs. At post mortem, diffused hemorrhagic spots in the liver and hemorrhages in the small intestine were observed in ducklings from farm A, and ducklings from farm B had only proventricular ulceration. Microscopically, multiple necrosis of hepatocytes, hyperplasia of the bile ducts, hemorrhages, infiltration of lymphocytes and bacterial colonies were commonly observed in the liver of ducklings from both farms. Also, type A and C duck hepatitis virus (DHV) were isolated from farm A and farm B, respectively and Salmonella typhimurium was commonly isolated and identified serologically and biochemically. To our knowledge, this is the first report about the co-infection of DHV and Salmonella typhimurium in ducklings, and co-circulation of type A and C duck hepatitis viruses in Korea.

Infectious bursal disease (IBD) caused by infectious bursal disease virus (IBDV) is a highly contagious viral disease in chicken. It causes heavy economic loss by immune suppression and high mortality. The IBDV, designated Avibirnavirus in the Family Birnaviridae, has a double-stranded RNA genome formed by two segments, segment A and segment B. Segment A encodes a 108 KDa polypeptide that is self-cleaved to produce pVP2, VP3 and VP4, and later pVP2 is cleaved to VP2. The VP2 contains the antigenic regions responsible for elicitation of neutralizing antibodies and VP3 is a major immunogenic protein of IBDV. In this study, monoclonal antibodies (MAbs) specific for IBDV were produced and characterized. All 15 MAbs were specific for IBDV and did not react with other viruses used in this study. The protein specificity of MAbs was determined by comparing the reactivity patterns of each MAb with IBDV VP2 and VP234 recombinant baculoviruses and Western blot analysis. As a result, 7 MAbs (1F5, 2C8, 2F4, 3C7, 4C3, 6F11, 6G5) and 5 MAbs (2A4, 2G2, 3F5, 3G2, 4F10) were specific for VP2 and VP3, respectively. The protein specificity of 3 MAbs (2B8, 3F7, 3F8) were not determined. Five (2C8, 2F4, 4C3, 6F11, 6G5) of the VP2-specific MAbs had a neutralizing activity against IBDV. Some MAbs reacted with IBDV-infected bursa of Fabricius by indirect fluorescence antibody (IFA) and immunohistochemistry (IHC) assay. The MAbs produced in this study would be used for diagnostic reagents for the detection of IBDV infection.

Serological evaluation for the poultry is important for various reasons, such as designing and assessing the vacci- nation program and diagnosing diseases and for this reason, serologic tests for the layer flocks have been conducted on a regular basis. Moreover, the nationwide serological survey and analysis are essential to understand the epidemiological status of national poultry industry. In this sense, the study was conducted to evaluate the immune status of the layer flocks with the sera submitted to Avian Disease Laboratory, Chungbuk National University in 2009, and several important viral diseases were selected for evalua- tion including low pathogenic avian influenza (LPAI), Newcastle disease (ND), infectious bronchitis (IB) and avian metapneumovirus (aMPV). For LPAI and ND, the age-related patterns of geometric mean titer (GMT) changes were similar but there were differences in the flock positive rate and the level of GMT due to the different vaccination policy. In the case of IB, the values of GMT showed that the field infection was more prevalent than expected. For aMPV, positive birds in a flock increased as the layers got older, which reflected the course of field infection because vaccination against aMPV was not allowed in 2009. From this study, the immune status for the main viral diseases in layers became more clarified but this information was limited because of only one year study. Therefore, serological survey needs to be conducted on a yearly basis and furthermore include broilers and breeders for a better understanding of the health status in the national poultry industry.

Inclusion body hepatitis-hydropericardium syndrome (IBH-HPS) is an acute viral disease usually found in broilers aged from 3 to 5 weeks and causes up to 75% mortality. Among the 12 serotypes of fowl adenovirus group 1, serotype-4 (FAdV-4) was identified as a primary agent of IBH-HPS and was usually isolated in IBH-HPS cases in Korea since 2007. To prevent these IBH-HPS outbreaks in Korea, we developed the FAdV-4 inactivated vaccine using Korean isolate (ADL070244) and evaluated the efficacy of this vaccine. For the efficacy test, 2-week-old specific-pathogen-free (SPF) chickens intramuscularly inoculated with 1 or 2 dose of inactivated vaccine were used and challenged with FAdV-4 through either intramuscular or oral route at 2 weeks after vaccination. The vaccine induced good seroconversion which was confirmed by agar gel precipitation (AGP) test. In addition, the vaccine could decrease the FAdV-4 detection rate and histological lesion severity such as lymphocyte infiltration and necrosis in the liver comparing with those of non-vaccination group. Based on the current results, the developed FAdV-4 inactivated vaccine in this study was effective in the terms of reduction of virus detection rate and histological lesions severity. However, it was difficult to confirm the efficacy of the vaccine clearly because of no mortality and clinical signs in the non-vaccinated group after challenge. Therefore, we need further study to develop a standard challenged model system which could clearly evaluate the efficacy of the vaccines for FAdV-4.

Department of QA/R&D, Choongang Vaccine Laboratory, Daejeon 305-348, KoreaABSTRACT Mycoplasma gallisepticum (MG) infection results in severe economic loss in the poultry industry. In the previous reports, F strain, one of the MG live vaccine strains, could protect the bird from field infection of MG strains. In this study, efficacy of PoulShot MG-F vaccine againset mycoplasma gallisepticum infection was evaluated for filed application in commercial layers. Commercial layers from two different farms received with PoulShot MG-F, MG-F live vaccine at 914 weeks of age. Serological immune response to MG vaccine, the persistency of MG vaccine strain in the upper respiratory tracts and egg production rate were evaluated in the vaccinated, contacted or nonvaccinated flocks. The serological response was first detected at 3 weeks after vaccination (WAV) and persisted for 31 WAV. The MG vaccine strains were also persisted for 31 WAV based on the reisolation and PCR detection. There was no difference between the vaccinated or non-vaccinated flocks in the egg production rate but in the abnormality rate of eggs. Based on the above results, we suggested that the PoulShot, MG-F live vaccine was fully immunogenic and had characteristics of long persistence in the upper respiratory trachea which will reduce economic loss caused by MG infection in the layer farms. (Key words : Mycoplasma gallisepticum, F strain, live vaccine)To whom correspondence should be addressed : moip@cbu.ac.kr

College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Choongbuk 361-763, South Korea ABSTRACT Fatty liver-hemorrhagic syndrome (FLHS) is a common nutritional disease in commercial layers and breeders. The most important clinical sign of FLHS is a sudden drop in egg production and increased mortality which causes significant economic loss in the poultry industry. However, the current diagnostic method for FLHS is based on the gross findings at necropsy which is not helpful to reduce the economic loss because of lateness of diagnosis. Therefore, we need early diagnosis and diagnostic methods before chickens were affected by FLHS. In this study we tried to evaluate the effectiveness of clinical pathology including blood chemistry as an early diagnostic method for FLHS in commercial chickens. Profiles of blood biochemistry were compared between two flocks selected in the same commercial layer farm based on the presence of FLHS clinical sings. A flock with clinical signs of FLHS was designated as FLHS and other flock without clinical signs of FLHS as Non-FLHS. Several parameters of blood biochemistry were selected and compared between FLHS and Non-FLHS to evaluate the possibility of early diagnosis. Average concentrations of serum cholesterol, serum calcium, aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK) were 139.4±87.2 (mg/dL), 24.5±5.4 (mg/dL), 153.6±23.1 (IU/L), 1238.3±475.2 (IU/L) and 1107.3±422.8 (IU/L) in Non-FLHS flock, respectively, and 210.2±173.2 (mg/dL), 25.2±4.1 (mg/dL), 174.3±53.5 (IU/L), 1694.9±691.3 (IU/L) and 1104.9±472.9 (IU/L) in FLHS flock, respectively. The activities of serum cholesterol, AST and LDH except CK, were significantly higher in FLHS than those in Non-FLHS flock (p

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.