Eun-Ok Jeon

Molecular survey of enteric viruses in commercial chicken farms in Korea with a history of enteritis

Abstract Several enteric viruses have increasingly received attention as potential causative agents of runting-stunting syndrome (RSS) in chickens. A molecular survey was performed to determine the presence of a broad range of enteric viruses, namely chicken astrovirus (CAstV), avian nephritis virus (ANV), chicken parvovirus (ChPV), infectious bronchitis virus (IBV), avian rotavirus (AvRV), avian reovirus (ARV), and fowl adenovirus (FAdV), in intestinal samples derived from 34 commercial chicken flocks that experienced enteritis outbreaks between 2010 and 2012. Using techniques such as PCR and reverse-transcription PCR, enteric viruses were identified in a total of 85.3% of investigated commercial chicken flocks in Korea. Furthermore, diverse combinations of 2 or more enteric viruses were simultaneously identified in 51.7% of chicken farms positive for enteric viruses. The rank order of positivity for enteric viruses was as follows: ANV (44.1%), CAstV (38.2%), ChPV (26.5%), IBV (20.6%), ARV (8.8%), AvRV (5.9%), and FAdV (2.9%). Additionally, other pathogens such as Escherichia coli, Salmonella spp., Eimeria spp., and FAdV were detected in 79% of chicken flocks positive for enteric viruses using PCR, bacterial isolation, and microscopic examination. The results of our study indicate the presence of several enteric viruses with various combinations in commercial chicken farms that experienced enteritis outbreaks. Experimental studies are required to further understand the roles of enteric viruses in RSS in commercial chickens.

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

SUMMARY 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. RESUMEN Reporte de caso- Evaluación patológica de casos clínicos por el virus de la influenza aviar altamente patógeno subtipo H5N8, en reproductores pesados y gallinas de postura comerciales en Corea del Sur. Brotes por el virus de influenza aviar altamente patógena (HPAI), subtipo H5N8, se observaron en dos lotes diferentes de granjas de reproductoras pesadas locales y en una granja de gallinas ponedoras comerciales en Corea del Sur. Clínicamente, los casos se caracterizaron por un aumento gradual de la mortalidad, transmisión lenta y signología no específica de influenza aviar de alta patogenicidad. Las observaciones macroscópicas en ambos casos incluyeron lesiones hemorrágicas o necróticas en los órganos internos, incluyendo membranas serosas y mucosas, el bazo y el páncreas. Ambos casos mostraron lesiones histopatológicas similares, incluyendo malacia multifocal en el cerebro y necrosis multifocal o difusa en el bazo y el páncreas. Los resultados de inmunohistoquímica indicaron reacciones inmunológicas positivas para el antígeno viral de la influenza aviar en las neuronas y células gliales en el cerebro, los miocitos en el corazón, las células acinares del páncreas y en las células mononucleares fagocíticas en varios órganos viscerales. Para reproducir de manera experimental la mortalidad observada en estos dos casos, 18 pollos libres de patógenos específicos y 18 ponedoras comerciales se asignaron en dos grupos, un grupo inoculado con el virus H5N8 y otro grupo de aves expuestas por contacto. La mortalidad de los pollos del grupo inoculado fue del 50% al 100%, mientras que el tiempo promedio de mortalidad se retrasó o no se observó mortalidad en el grupo de aves expuestas por contacto. Las distribuciones de los antígenos virales y las lesiones histopatológicas en el estudio experimental fueron similares a las observadas en los casos de campo. Estos hallazgos sugieren que el virus H5N8 induce un patrón de patobiología diferente, incluyendo una transmisión lenta y baja mortalidad, en comparación con otros virus altamente patógenos de influenza aviar. Esta es la primera descripción patológica de casos clínicos por el subtipo H5N8 en Corea del Sur, y puede ser útil en la comprensión de la biopatología del nuevos virus subtipo de la influenza aviar de alta patogenicidad.

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

Abstract 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.

An unusual case of concomitant infection with chicken astrovirus and group A avian rotavirus in broilers with a history of severe clinical signs.

A molecular study of intestinal samples from 21 broiler flocks with a history of enteritis revealed that 23.8% and 14.3% were positive for chicken astrovirus (CAstV) and avian rotavirus (ARV), respectively. CAstV and group A ARV were simultaneously detected in only one broiler flock. Birds in this group developed the significant intestinal lesions characterized by frothy contents, paleness, and thin intestinal walls. In this report we present an unusual case of runting stunting syndrome (RSS) with a history of high mortality and growth retardation in broiler chickens. We also make the first identification of CAstV and group A ARV in broiler chickens in Korea.

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.

An Outbreak of Lymphomas in a Layer Chicken Flock Previously Infected with Fowlpox Virus Containing Integrated Reticuloendotheliosis Virus

SUMMARY Visceral lymphomas occurred in a 236-day-old layer flock previously diagnosed with reticuloendotheliosis virus (REV)–integrated fowlpox virus (FPV) infection at the age of 77 days. Common pathologic lesions were multiple neoplastic nodules of homogeneous lymphocytes in the livers and spleens of all submitted chickens. All neoplastic tissues were positive for the REV envelope (env) gene by PCR. In a retrospective molecular study of FPV-infected 77-day-old chickens from the same flock, we identified nearly full-length REV provirus integrated into the genome of FPV as well as the REV env gene in trachea samples, whereas only the REV LTR region was present in the FPV strain used to vaccinate this flock. The 622-bp REV env gene nucleotide sequence derived from the trachea and neoplastic tissues was identical. Commercial ELISA of serum samples revealed that all chickens aged between 17 and 263 days in this flock were positive for REV but not for avian leukosis virus. Taken together, the evidence suggests that the visceral lymphomas were caused by a REV-integrated FPV field strain. FPV infections of commercial chickens should be followed up by careful monitoring for manifestations of REV infection, including lymphomas and immune depression, considering the ease with which the REV provirus appears to be able to integrate into the FPV genome. RESUMEN Reporte de Caso—Brote de linfomas en una parvada de aves de postura previamente infectada con un virus de la viruela aviar que contenía al virus de la reticuloendoteliosis integrado. Se produjeron linfomas viscerales en una parvada de aves de postura de 236 días de edad que previamente fue diagnosticada de estar infectada a los 77 días de edad por un virus de la viruela de las gallinas que tenía integrado a un virus de la reticuloendoteliosis (REV). Las lesiones patológicas comunes eran múltiples nódulos tumorales de linfocitos homogéneos en el hígado y el bazo de los pollos enviados al laboratorio. Todos los tejidos neoplásicos fueron positivos a la presencia del gene de la envoltura (env) del virus de la reticuloendoteliosis mediante PCR. En un estudio retrospectivo molecular de las aves infectadas a los 77 días edad con viruela aviar de la misma parvada, se identificó a un provirus de reticuloendoteliosis casi de longitud completa integrado en el genoma del virus de la viruela de las gallinas así como la presencia del gene env del virus de la reticuloendoteliosis en las muestras de tráquea, mientras que sólo las regiones de las repeticiones terminales largas del virus de la reticuloendoteliosis estaban presentes en la cepa de viruela utilizada para vacunar a esta parvada. Las secuencias de nucleótidos de 622 pares de bases del gene env de reticuloendoteliosis derivadas de los tejidos traqueales y neoplásicos eran idénticas. Mediante una prueba de ELISA comercial de muestras de suero, se reveló que todos los pollos con edades comprendidas entre 17 y 263 días de esta parvada eran positivos a la presencia del virus de la reticuloendoteliosis, pero no para el virus de la leucosis aviar. En conjunto, la evidencia sugiere que los linfomas viscerales fueron causados por una cepa de viruela aviar con un virus de reticuloendoteliosis integrado. Las infecciones por el virus de la viruela aviar en pollos comerciales deben ser objeto de seguimiento mediante un control estricto para detectar las manifestaciones de la infección por el virus de la reticuloendoteliosis, incluyendo linfomas e inmunodepresión, teniendo en cuenta la facilidad con la que el provirus del virus de la reticuloendoteliosis puede integrarse en el genoma del virus de la viruela aviar.

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.

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.

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

Abstract 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.

Epidemiological Studies of Avian Paramyxovirus Type 4 and 6 in Commercial Chicken Flocks in Korea

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.