Genetic characterization of avian polyomaviruses identified from psittacine birds in South Korea.

Budgerigar fledgling disease (BFD) is a contagious disease caused by avian polyomavirus (APV) in psittacine birds and causes high mortality rates. Here, eight APV-positive cases were confirmed from dead parrots or parrot tissue samples by polymerase chain reaction (PCR). Full-length genome sequencing showed high nucleotide identity (98.84-100%) between the APV strains. Phylogenetic analysis revealed that two genogroups were cocirculating in South Korea. The nucleotide sequences of five strains, collected from different parrot species, were identical; however, pathological lesions were observed in only two parrots, both aged 2 months. Pathology included necrotic spots in the liver, subcutaneous haemorrhage, hepatomegaly, ascites, intranuclear inclusion bodies, hepatocyte karyomegaly, hepatic necrosis, and bile duct proliferation. This suggests that the pathogenicity of APV might be host age-dependent regardless of the host species. This study improves our understanding of APV pathogenicity and provides a more detailed genetic characterization of APV strains.RESEARCH HIGHLIGHTS Eight APV strains were identified in South Korea from 2019 to 2021.By phylogenetic analysis, South Korean APV strains were classified into two clades.

Application of metagenomics for diagnosis of broilers displaying neurological symptoms.

BACKGROUND: Thirty-two-day-old broiler chickens at a farm located in northwestern South Korea displayed adverse neurological symptoms including limping, lying down, and head shaking. Approximately 2.1% of chickens died or were culled due to severe symptoms. Five carcasses were submitted to the Avian Disease Division of the Animal and Plant Quarantine Agency (APQA) for disease diagnosis. RESULTS: Broilers displayed severe pericarditis and perihepatitis associated with gross lesions. Broilers also displayed microscopic lesions in the cerebrum and in the granular layer of the cerebellum, which were associated with multifocal perivascular cuffing and purulent necrosis in the cerebrum, and severe meningitis with heterophil and lymphocyte infiltration. Staphylococcus spp. were identified in the liver and heart using bacteriological culture. PCR/RT-PCR assays revealed that broilers were negative for avian Clostridium botulinum, Newcastle disease virus, and avian encephalomyelitis virus. Bacterial and viral metagenomic analysis of brain sample further revealed the presence of Pseudomonas spp. and Marek's disease virus, which are known etiological agents of chicken meningoencephalitis. CONCLUSIONS: This study reports a diagnostic analysis of gross and histopathological lesions from 32-day-old broilers displaying unique neurological symptoms that revealed the presence of the several neurological diseases including meningoencephalitis. The causative agents associated with meningoencephalitis of broilers that had not been identified by routine diagnostic methods could be diagnosed by metagenomics, which proves the usefulness of metagenomics as a diagnostic tool for unknown neurological diseases in broilers.

The Development of Novel Reverse Transcription Loop-Mediated Isothermal Amplification Assays for the Detection and Differentiation of Virulent Newcastle Disease Virus.

Newcastle disease (ND) is a highly pathogenic viral infection of poultry with significant economic impacts worldwide. Despite the widespread use of vaccines, ND outbreaks continue to occur even within vaccinated poultry farms. Furthermore, novel Newcastle disease virus (NDV) genotypes are emerging in poultry, increasing the need for the development of rapid, accurate, and simple diagnostic methods. We therefore developed two novel sets of visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays based on highly conserved regions of the HN and F genes. The limits of detection of the NDV-Common-LAMP assay, for all the NDV strains, were 103.0 EID50/0.1 mL for Kr005 and 102.0 EID50/0.1 mL for Lasota within 35 min. The sensitivity of the NDV-Patho-LAMP assay, used for the strain differentiation of virulent NDV, was 102.0 EID50/0.1 mL for Kr005. No amplification was detected for the non-NDV templates. Next, we probed 95 clinical strains and 7 reference strains with the RT-LAMP assays to assess the feasibility of their use in diagnostics. We observed no cross-reactivity across the 102 strains. Furthermore, there was 100% congruence between the RT-LAMP assays and full-length sequencing of the target genes, indicating the potential for visual RT-LAMP in the identification and differentiation of NDV. These novel RT-LAMP assays are ideally suited for the field or resource-limited environments to facilitate the faster detection and differentiation of NDV, which can reduce or avoid further spread.

Genetic characterization of chicken infectious anaemia viruses isolated in Korea and their pathogenicity in chicks.

Chicken infectious anaemia virus (CIAV) causes severe anemia and immunosuppression through horizontal or vertical transmission in young chickens. Especially, vertical transmission of virus through the egg can lead to significantly economic losses due to the increased mortality in the broiler industry. Here, 28 CIAV complete sequences circulating in Korea were first characterized using the newly designed primers. Phylogenetic analysis based on the complete sequences revealed that CIAV isolates were divided into four groups, IIa (2/28, 7.1%), IIb (9/28, 32.1%), IIIa (8/28, 28.6%) and IIIb (9/28, 32.1%), and exhibited a close relationship to each other. The major groups were IIb, IIIa and IIIb, and no strains were clustered with a vaccine strain available in Korea. Also, for viral titration, we newly developed a quantitative PCR assay that is highly sensitive, reliable and simple. To investigate the pathogenicity of three major genotypes, 18R001(IIb), 08AQ017A(IIIa), and 17AD008(IIIb) isolates were challenged into one-day-old specific-pathogen-free (SPF) chicks. Each CIAV strain caused anaemia, severe growth retardation and immunosuppression in chickens regardless of CIAV genotypes. Notably, a 17AD008 strain showed stable cellular adaptability and higher virus titer in vitro as well as higher pathogenicity in vivo. Taken together, our study provides valuable information to understand molecular characterization, genetic diversity and pathogenicity of CIAV to improve management and control of CIA in poultry farm.

An Amplicon-Based Application for the Whole-Genome Sequencing of GI-19 Lineage Infectious Bronchitis Virus Directly from Clinical Samples.

Infectious bronchitis virus (IBV) causes a highly contagious respiratory disease in chickens, leading to significant economic losses in the poultry industry worldwide. IBV exhibits a high mutation rate, resulting in the continuous emergence of new variants and strains. A complete genome analysis of IBV is crucial for understanding its characteristics. However, it is challenging to obtain whole-genome sequences from IBV-infected clinical samples due to the low abundance of IBV relative to the host genome. Here, we present a novel approach employing next-generation sequencing (NGS) to directly sequence the complete genome of IBV. Through in silico analysis, six primer pairs were designed to match various genotypes, including the GI-19 lineage of IBV. The primer sets successfully amplified six overlapping fragments by long-range PCR and the size of the amplicons ranged from 3.7 to 6.4 kb, resulting in full coverage of the IBV genome. Furthermore, utilizing Illumina sequencing, we obtained the complete genome sequences of two strains belonging to the GI-19 lineage (QX genotype) from clinical samples, with 100% coverage rates, over 1000 × mean depth coverage, and a high percentage of mapped reads to the reference genomes (96.63% and 97.66%). The reported method significantly improves the whole-genome sequencing of IBVs from clinical samples; thus, it can improve understanding of the epidemiology and evolution of IBVs.

Genomic and Phenotypic Characterization of Cutibacterium acnes Bacteriophages Isolated from Acne Patients.

Cutibacterium acnes is a pathogen that can cause acne vulgaris, sarcoidosis, endodontic lesions, eye infections, prosthetic joint infections, and prostate cancer. Recently, bacteriophage (phage) therapy has been developed as an alternative to antibiotics. In this study, we attempted to isolate 15 phages specific to C. acnes from 64 clinical samples obtained from patients with acne vulgaris. Furthermore, we sequenced the genomes of these three phages. Bioinformatic analysis revealed that the capsid and tape measure proteins are strongly hydrophobic. To efficiently solubilize the phage particles, we measured the adsorption rate, one-step growth curve, and phage stability using an SMT2 buffer containing Tween 20. Here, we report the genotypic and phenotypic characteristics of the novel C. acnes-specific phages.

Histopathological Findings and Metagenomic Analysis of Esophageal Papillary Proliferation Identified in Laying Broiler Breeders.

White or pale-yellow nodules 2-7 mm in length were observed in the esophageal lumen in a number of laying broiler breeders with reduced laying rates. Metaplasia of the mucosal epithelial layer and mucous gland, as well as lymphocyte infiltration under the esophageal mucous gland and epithelial cell layer, were observed, which we found were caused by vitamin A deficiency. In one chicken, however, the stratified squamous epithelial cells of the esophagus were completely replaced by increased numbers of ducts/ductules, lymphocytes, and connective tissue, resulting in a papillary morphology. The ducts were surrounded by a fibrous stroma. Multiple hyperplasia of the esophageal gland was also observed and the esophageal glands were lined by a single layer of columnar cells, and a large number of lymphocytes were infiltrated into the submucosal layer. Based on the gross findings, this papillary proliferation was considered papilloma, but histopathologically, a mass composed of squamous epithelium was not observed. Therefore, the papillary lesion appeared as adenoma with squamous metaplasia of the esophageal gland and ectasia, or mucosal epithelial papillary hyperplasia, associated with chronic esophagitis. A metagenomic analysis of the esophagus samples from this chicken was performed to determine the infectious etiology. No viral cause was identified; however, a contributing role of Bradyrhizobium sp. could not be excluded. In this study, we report the first histopathological examination of a rare case of esophageal papillary proliferation in a chicken and highlight the importance of histopathological results for a definitive diagnosis of such cases.

Genetic Diversity of Fowlpox Virus and Putative Genes Involved in Its Pathogenicity.

To determine the genomic variations of fowlpox virus (FPV)-the largest, very ancient, and still harmful avian virus-the complete genomes of 21 FPVs were analyzed. The genomes showed low genetic diversity relative to their overall size. Our studies revealed that FPVs could phylogenetically be divided into two clades, based on their regional distribution, and comparative analysis showed that 40 putative proteins of FPV were associated with geographic differences in viruses, viral pathogenicity, or the onset of diphtheritic lesions. The strain, classified into a subgroup different from others in the genomic analysis, showed relatively low pathogenicity in chickens, and the onset of diphtheritic lesions was observed to be caused only by the specific strain. Despite genetic differences, some commercial vaccines are protective against virulent strains, and intact reticuloendotheliosis virus inserted into field FPV strains was activated but there was no enhancement of the pathogenicity of FPV. These findings will expand our knowledge of the viral proteome and help us understand the pathogenicity of FPV. IMPORTANCE This study aims at determining molecular candidates using comparative genomics to differentiate between the diphtheritic and cutaneous forms of FPV infection, in addition to their association with the pathogenicity of the virus. Full-genomic analyses of multiple fowlpox strains, including field viruses, isolated between 1960s and 2019, and vaccine strains showed the genetic diversity due to regional differences. Comparative genomic analysis offered the clues related to viral virulence. We believe that our study makes a significant contribution to the literature because we are the first to perform such an elaborate study that compares 21 FPVs to study and highlight their diversity, despite the high level of homology between them. Our results shall help provide insights for tackling FPV that has been taking a toll on the poultry for years now.

Research Note: Simultaneous detection of infectious laryngotracheitis virus, fowlpox virus, and reticuloendotheliosis virus in chicken specimens.

Infectious laryngotracheitis (ILT), fowlpox (FP), and reticuloendotheliosis are important poultry diseases caused by gallid herpesvirus 1 (ILTV), fowlpox virus (FWPV), and reticuloendotheliosis virus (REV), respectively. Coinfections with ILTV and FWPV occur naturally in chickens, and FP in its more virulent wet form is characterized by diphtheritic lesions and easily confused with ILT. Moreover, the insertion of only partial REV-LTR or a nearly full-length REV into the FWPV genome, located between the ORF 201 and ORF 203, has increased recently in wild-type field FWPV isolates. Therefore, it is critical to detect ILTV, FWPV, REV-integrated FWPV, and REV early and accurately. In this study, we successfully developed a multiplex PCR assay for the simultaneous detection of ILTV, FWPV, REV-integrated FWPV, and REV, and the detection limits was 1 × 54 copies/tube. When used to test clinical samples, the results of the multiplex PCR were in 100% agreement with singleplex PCRs and sequencing. This new multiplex PCR is a simple, rapid, sensitive, specific, and cost-effective method for detection of 4 viruses in clinical specimens.

Loop-mediated isothermal amplification assays for Enterococcussp., Escherichiacoli and Staphylococcusaureus in chicken.

Bacterial chondronecrosis with osteomyelitis (BCO) is a major cause of lameness in broiler chicken, and results in serious economic losses worldwide. Although the pathogenesis mechanism leading to lameness is not entirely understood, some strains of Enterococcussp., avian pathogenic Escherichia coli or Staphylococcus aureus have been long recognized as important causative pathogens. To prevent the progression of Enterococcussp., avian pathogenic E. coli or S. aureus infections, we developed rapid, sensitive and convenient diagnostic assays using loop-mediated isothermal amplification (LAMP). Entero-Common-LAMP assays were developed for simultaneous detection of eight Enterococcus species. To target specific microorganisms, seven Entero-Specific-LAMP assays for E. faecalis, E. faecium, E. hirae, E. gallinarum, E. avium, E. duransand E. cecorum were developed, as well as E. coli-LAMP and S. aureus-LAMP assays. Considering the prevalence and economic impact of Enterococcussp., E. coli and S. aureus, the 10 different LAMP assays which were developed have considerable potential as routine diagnostic methods in the field or in resource-limited environments.

Multiplex PCR analysis of virulence genes and their influence on antibiotic resistance in Enterococcus spp. isolated from broiler chicken.

Enterococcus spp. are opportunistic pathogens that cause lameness in broiler chickens, resulting in serious economic losses worldwide. Virulence of Enterococcus spp. is associated with several putative virulence genes including fsr, efm, esp, cylA, cad1, ace, gelE, and asa1. In this study, multiplex polymerase chain reaction (PCR) for the simultaneous detection of these virulence genes in Enterococcus spp. was developed, and detection limits for E. faecium, E. faecalis, and E. hirae were 64.0 pg/µL, 320.0 pg/µL, and 1.6 ng/µL DNA, respectively. Among 80 Enterococcus isolates tested, efm and cad1 were detected in all 26 E. faecium samples, and only cad1 was observed in E. hirae. Additionally, the presence of virulence genes in 25 E. faecalis isolates were 100% for cad1, 88.0% for gelE, 64.0% for fsr, 44.0% for asa1, 16.0% for cylA, and 4.0% for esp. No virulence genes were found in E. gallinarum isolates. A total of 49 isolates were resistant to tigecycline and to at least 2 different classes of antibiotics. The most prevalent resistance was to ciprofloxacin (73.5%), quinupristin/dalfopristin (55.1%), and tetracycline (49.0%). No strains were resistant to vancomycin or linezolid. This is the first multiplex PCR assay to simultaneously detect eight virulence genes in Enterococcus spp., and the method provides diagnostic value for accurate, rapid, and convenient detection of virulence genes. Additionally, we report the prevalence of virulence genes and antimicrobial resistance in Enterococcus isolates from commercial broiler chickens suffering lameness.

Loop-mediated isothermal amplification assay for detection of four immunosuppressive viruses in chicken.

Loop-mediated isothermal amplification (LAMP) methods to detect chicken infectious anemia virus (CIAV), reticuloendotheliosis virus (REV), and Marek's disease virus (MDV), and a reverse transcription (RT)-LAMP assay to detect infectious bursal disease virus (IBDV), were developed. The CIAV-LAMP, REV-LAMP, MDV-LAMP, and IBDV-RT-LAMP methods were performed using four sets of six primers targeting the VP1 gene of CIAV, the gp90 gene of REV, the Meq gene of MDV, and the VP2 gene of IBDV. The results (a change in color) were observed visually. The methods showed high specificity and sensitivity. The detection limits were 50 genomic copies of CIAV, 16 genomic copies of REV, 20 genomic copies of MDV, and 250 genomic copies of IBDV. When used to test clinical samples, the results of the LAMP assays were in 100% agreement with a previously described PCR. Therefore, the LAMP assays are simple, rapid, highly sensitive, and specific methods for detecting four immune-suppressive viruses.

Camptothecin acts synergistically with imatinib and overcomes imatinib resistance through Bcr-Abl independence in human K562 cells.

In this study, we have tried to find new targets and effective drugs for imatinib-resistant chronic myelogenous leukemia (CML) cells displaying loss of Bcr-Abl kinase target dependence. The imatinib-resistant K562/R1, -R2 and -R3 cells showed profound declines of Bcr-Abl level and concurrently exhibited up-regulation of Bcl-2 and Ku70/80, and down-regulation of Bax, DNA-PKcs and BRCA1, suggesting that loss of Bcr-Abl after exposure to imatinib might be accompanied by other cell survival mechanism. K562/R3 cells were more sensitive to camptothecin (CPT)- and radiation-induced apoptosis than K562 cells, indicating hypersensitivity of imatinib-resistant cells to DNA damaging agents. Moreover, when K562 cells were treated with the combination of imatinib with CPT, the level of Bax and the cleavage of PARP-1 and DNA-PK were significantly increased in comparison with the effects of each drug. Therefore, our study suggests that CPT can be used to treat CML with loss of Bcr-Abl expression.