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.

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.

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.

Efficacy of Low-Intensity Extracorporeal Shock Wave Treatment in Erectile Dysfunction Following Radical Prostatectomy: A Systematic Review and Meta-Analysis.

Erectile dysfunction (ED) is a well-known complication of radical prostatectomy (RP). Oral 5-phosphodiesterase inhibitors are currently the most widely used penile rehabilitation treatment for ED following RP, but they are less effective than for those with general ED. Low-intensity extracorporeal shock wave treatment (LI-ESWT), causing a biological change that induces neovascularization, has recently been used as a treatment for ED. Therefore, we conducted a systematic review and meta-analysis to investigate the efficiency of LI-ESWT in ED following RP. PubMed, Embase, and the Cochrane Library were searched up until December 2021. The endpoint was the change in IIEF scores after LI-ESWT. Five papers (460 patients) were included in the final analysis. In IIEF scores performed 3-4 months after LI-ESWT, the group receiving LI-ESWT showed statistically significantly better results than the control (WMD = -2.04; 95% CI, -3.72 to -0.35; p = 0.02). However, there were a total of two studies that measured the results after 9-12 months. There was no statistical difference between the two groups (WMD = -5.37; 95% CI, -12.42 to 1.69; p = 0.14). The results of this analysis indicate that LI-ESWT showed a statistically significant effect on early recovery in penile rehabilitation of ED following RP. However, the level of evidence was low. Therefore, careful interpretation of the results is required.

Identification of Campylobacter jejuni and Chlamydia psittaci from cockatiel (Nymphicus hollandicus) using metagenomics.

BACKGROUND: In July 2015, the carcasses of 11 cockatiels were submitted for disease diagnosis to the Avian Disease Division of the Animal and Plant Quarantine Agency of Korea. The cockatiels, which appeared dehydrated and underweight, had exhibited severe diarrhea and 22 % mortality over 2 weeks. Traditional diagnosis did not reveal the causes of these symptoms. METHODS: We conducted metagenomics analysis on intestines and livers from the dead cockatiels using Illumina high-throughput sequencing. To obtain more accurate and longer contigs, which are required for further genetic characterization, we compared the results of three de novo assembly tools (metaSPAdes, MEGAHIT, and IDBA-UD). RESULTS: Sequence reads of Campylobacter jejuni (C. jejuni) and Chlamydia psittaci (C. psittaci) were present in most of the cockatiel samples. Either of these bacteria could cause the reported symptoms in psittaciformes. metaSPAdes (ver.3.14.1) identified the 1152 bp flaA gene of C. jejuni and the 1096 bp ompA gene of C. psittaci. Genetic analysis revealed that flaA of C. jejuni was recombinant between C. jejuni and Campylobacter coli, and that ompA of C. psittaci isolated from cockatiel was closely related to strains isolated from humans. CONCLUSIONS: C. jejuni and C. psittaci were detected in cockatiels in the Republic of Korea using metagenomic analysis. This approach is useful for understanding pathogens of pet birds. Three de novo assemblers were compared to obtain accurate contigs from large quantities of reads, and sequences of C. jejuni and C. psittaci generated by metaSPAdes were analyzed.

Viral Metagenomic Analysis of Japanese Quail (Coturnix japonica) with Enteritis in the Republic of Korea.

We performed viral metagenomics analysis of Japanese quail affected with enteritis to elucidate the viral etiology. Metagenomics generated 21,066,442 sequence reads via high-throughput sequencing, with a mean length of 136 nt. Enrichment in viral sequences suggested that at least three viruses were present in quail samples. Coronavirus and picornavirus were identified and are known as pathogens causing quail enteritis that match the observed morphology. Abundant reads of coronavirus from quail samples yielded four fragment sequences exhibiting six genomes of avian coronavirus. Sequence analysis showed that this quail coronavirus was related to turkey coronavirus and chicken infectious bronchitis virus. Quail picornavirus 8177 bp in size was identified and was similar to the QPV1/HUN/01 virus detected in quails without clinical symptoms in Hungary with 84.6% nucleotide and 94.6% amino acid identity. Our results are useful for understanding the genetic diversity of quail viruses. Further studies must be performed to determine whether quail coronavirus and quail picornavirus are pathogens of the digestive tract of quails.

Artículo regular­Análisis metagenómico viral de la codorniz japonesa (Coturnix japonica) con enteritis en la República de Corea. Se realizó un análisis de metagenómica viral de codornices japonesas afectadas con enteritis para dilucidar la etiología viral. La metagenómica generó 21,066,442 lecturas de secuencia mediante secuenciación de alto rendimiento, con una longitud media de 136 nucleótidos. El enriquecimiento en secuencias virales sugirió que al menos tres virus estaban presentes en las muestras de codorniz. Se identificaron coronavirus y picornavirus que son conocidos como patógenos que causan enteritis de codornices que coinciden con la morfología observada. Las lecturas abundantes de coronavirus de muestras de codorniz produjeron cuatro secuencias de fragmentos que exhibían seis genomas de coronavirus aviar. El análisis de secuencia mostró que este coronavirus de codorniz estaba relacionado con el coronavirus del pavo y con el virus de la bronquitis infecciosa del pollo. Se identificó un picornavirus de codorniz de 8177 pares de bases de tamaño y fue similar al virus QPV1/HUN/01 detectado en codornices sin signos clínicos en Hungría con 84.6% de nucleótidos y 94.6% de identidad de aminoácidos. Estos resultados son útiles para comprender la diversidad genética de los virus de la codorniz. Se deben realizar más estudios para determinar si el coronavirus y el picornavirus de las codornices son patógenos del tracto digestivo de las codornices.

A microfluidic device with 3-d hydrogel villi scaffold to simulate intestinal absorption.

The absorption of drugs via oral route is a subject of a great interest in drug development process. The current in vitro method for measuring the kinetics of drug absorption relies on 2-D monolayer culture of Caco-2 cells on a porous membrane, but physiologically unrealistic environment provided by this method often results in inaccurate drug absorption kinetics. Here we report a novel microfluidic system which better mimics the physiological environment of the human small intestine. Three dimensional geometries of villi of the small intestine were reproduced via novel hydrogel microfabrication technique, and the fluid flow in the apical and basolateral sides of intestinal tract was reproduced with a two-layer microfluidic device. A wide range of flow rates was achieved by using gravity-induced flow, potentially facilitating easier high-throughput implementation. The kinetics of diffusion process through the 3-D villi scaffold in the microfluidic device was measured and mathematically modeled. When combined with intestinal cell culture model, this novel 3-D microfluidic system can serve as an in vitro platform that better mimics the in vivo environment.

Fabrication and characterization of microfluidic liver-on-a-chip using microsomal enzymes.

Biotransformation in the liver plays an important role in determining the pharmacokinetic profile of drugs and food components. Current in vitro platforms for testing the liver metabolism suffers from the lack of resemblance to the human liver metabolism, mainly due to the lost metabolic activity of cultured hepatocytes and the absence of transport phenomena that occurs in the liver tissue. Here we report a microfluidic device with liver microsome encapsulated in 3-D hydrogel matrix, which can mimic the metabolism reaction and the transport phenomena in the liver. Photopolymerization of poly(ethylene glycol) diacrylate (PEG-DA) allows controlling the mass transfer with matrix sizes, and a gravity-induced passive flow can reproduce the blood flow through the liver. We measured the reaction kinetics of P450 enzymes in the device, and simulated the convection-diffusion-reaction characteristics inside the device with a mathematical model. Combination of mathematical analytical tool and the experimental tool allowed us to analyze and optimize the reaction kinetics inside the microfluidic chip. This novel in vitro platform can serve as a tool for screening the liver metabolism of various compounds.