High-throughput DNA sequence analysis elucidates novel insight into the genetic basis of adaptation in local sheep.

Understanding how evolutionary factors related to climate adaptation and human selection have influenced the genetic architecture of domesticated animals is of great interest in biology. In the current study, by using 304 whole genomes from different geographical regions (including Europe, north Africa, Southwest Asia, east Asia, west Africa, south Asia, east Africa, Australia and Turkey), We evaluate global sheep population dynamics in terms of genetic variation and population structure. We further conducted comparative population analysis to study the genetic underpinnings of climate adaption to local environments and also morphological traits. In order to identify genomic signals under selection, we applied fixation index (FST) and also nucleotide diversity (θπ) statistical measurements. Our results revealed several candidate genes on different chromosomes under selection for local climate adaptation (e.g. HOXC12, HOXC13, IRF1, FGD2 and GNAQ), body size (PDGFA, HMGA2, PDE3A) and also morphological related traits (RXFP2). The discovered candidate genes may offer newel insights into genetic underpinning of regional adaptation and commercially significant features in local sheep.

Gene-expression profiling of endomyocardial biopsies from dogs with dilated cardiomyopathy phenotype.

INTRODUCTION: The employment of advanced molecular biology technologies has expanded the diagnostic investigation of cardiomyopathies in dogs; these technologies have predominantly been performed on postmortem samples, although the recent use of endomyocardial biopsy in living dogs has enabled a better premortem diagnostic approach to study the myocardial injury. ANIMALS, MATERIALS, AND METHODS: Endomyocardial biopsies were collected in nine dogs with a dilated cardiomyopathy phenotype (DCM-p) and congestive heart failure and submitted to histologic examination, next-generation sequencing (NGS), and polymerase chain reaction analysis. Data from three healthy dogs (Fastq files) were retrieved from a previously approved study and used as a control group for ribonucleic acid sequencing. RESULTS: Histologic examination revealed endocardial fibrosis in six of nine dogs, whereas lymphocytic interstitial infiltrates were detected in two of nine dogs, and lymphoplasmacytic and macrophage infiltrates were detected in one of nine dogs. On polymerase chain reaction analysis, two dogs tested positive for canine parvovirus two and one dog for canine distemper virus. Gene-expression pathways involved in cellular energy metabolism (especially carbohydrates-insulin) and cardiac structural proteins were different in all DCM-p dogs compared to those in the control group. When dogs with lymphocytic interstitial infiltrates were compared to those in the control group, NGS analysis revealed the predominant role of genes related to inflammation and pathogen infection. CONCLUSIONS: Next-generation sequencing technology performed on in vivo endomyocardial biopsies has identified different molecular and genetic factors that could play a role in the development and/or progression of DCM-p in dogs.

In-herd prevalence of Fasciola hepatica and Calicophoron / Paramphistomum spp. infections in German dairy cows with comparison of two coproscopical methods and establishment of real-time pyrosequencing for rumen fluke species differentiation.

Infections with liver and rumen flukes are among the most frequent parasitic diseases in cattle worldwide. In Europe, the predominant liver fluke species is Fasciola hepatica, and the recently rapidly spreading rumen flukes are mostly Calicophoron daubneyi and occasionally Paramphistomum leydeni. In this study, 1638 faecal samples from individual dairy cows from 24 northern and 18 southern German farms as well as one central German farm, all preselected for potential F. hepatica infection, were examined to determine in-herd prevalences of liver and rumen fluke infections. Furthermore, individual faecal egg counts (FECs) were determined in the northern and central German cows. On farms with patent F. hepatica infections, the mean in-herd prevalence was 15.8% in northern Germany, 41.6% in southern Germany and 14.0% in the central German farm. Rumen fluke infections resulted in high in-herd prevalences in all regions with a mean prevalence of 46.0% in northern, 48.4% in southern and 40.0% in central Germany. Individual FECs varied between 0.1 and 4.1 (mean 0.4) eggs per gram faeces (EPG) for F. hepatica and between 0.1 and 292.4 (mean 16.9) EPG for rumen flukes. Mean in-herd prevalence and mean FECs did not differ significantly between mono- and coinfected farms for either fluke species. Comparison of the classical sedimentation technique and the Flukefinder® method on a subset of 500 faecal samples revealed a similar number of positive samples, however, Flukefinder® mean FECs were three to four times higher for liver and rumen fluke eggs, respectively, with an increasing gap between EPG levels with rising egg counts. Fluke egg size measurement confirmed P. leydeni eggs on average to be larger in length and width (161.0 µm x 87.1 µm) than those of C. daubneyi (141.8 µm x 72.9 µm). However, due to overlap of measurements, morphological species identification based on egg size proved unreliable. For accurate identification, a real-time pyrosequencing approach was established, offering the advantage over classical Sanger sequencing of unambiguously identifying rumen fluke mixed species infections. Real-time pyrosequencing confirmed C. daubneyi (78.1% [50/64]) as the predominant rumen fluke species in Germany, while P. leydeni was detected in 12.5% (8/64) of sampled cows. A total of 9.4% (6/64) cows were infected with both C. daubneyi and P. leydeni, representing the first finding of a mixed infection in domestic ruminants in Europe to date.

Development of a next-generation sequencing protocol for the canine T cell receptor beta chain repertoire.

Profiling the T cell receptor (TCR) repertoire using next-generation sequencing has become common in both human and translational research. Companion dogs with spontaneous tumors, including canine melanoma, share several features, e.g., natural occurrence, shared environmental exposures, natural outbred population, and immunocompetence. T cells play an important role in the adaptive immune system by recognizing specific antigens via a surface TCR. As such, understanding the canine T cell response to vaccines, cancer, immunotherapies, and infectious diseases is critically important for both dog and human health. Off-the-shelf commercial reagents, kits and services are readily available for human, non-human primate, and mouse in this context. However, these resources are limited for the canine. In this study, we present a cost-effective protocol for analysis of canine TCR beta chain genes. Workflow can be accomplished in 1-2 days starting with total RNA and resulting in libraries ready for sequencing on Illumina platforms.

Targeted next-generation sequencing assay to detect 3 Moraxella spp. directly from bovine ocular swabs.

Infectious bovine keratoconjunctivitis (IBK) is associated with 2 species of Moraxella: M. bovis and M. bovoculi. A third novel Moraxella spp., designated tentatively as M. oculi, has been identified from the eyes of cattle with and without pinkeye. These 3 Moraxella spp. can be found in various combinations within the same clinical sample, making speciation of this genus directly from a sample impossible with Sanger sequencing. Assessing Moraxella diversity found in IBK- and non-IBK-affected cattle eyes, independent of culture, may provide additional information about IBK by avoiding the selectivity bias of culturing. We developed a targeted NGS panel to detect and speciate these 3 Moraxella spp. directly from bovine ocular swabs. Our targeted panel amplifies bacterial essential genes and the 16S-23S ribosomal RNA intergenic spacer region (ITS) of the 3 Moraxella spp. and speciates based on these sequences. Our panel was able to differentiate the 3 species directly from DNA extracted from 13 swabs (6 from healthy animals, 7 from animals with IBK), and every swab except one (clinically healthy eye) had the 3 Moraxella spp. Targeted NGS with sequencing of Moraxella spp. housekeeping genes appears to be a suitable method for speciation of Moraxella directly from ocular swabs.

Development of ion torrent-based targeted next-generation sequencing panel for identification of animal species in pet foods.

Manufacturers may intentionally or unintentionally incorporate ingredients not specified on the label of canned pet foods. Including any unacknowledged ingredients in a food product is considered food fraud or misbranding. Contamination of pet foods may occur in the processing of the foods, including potential cross-contamination in packaging facilities. Of the methods available to identify meat species in food products, Sanger sequencing and several next-generation sequencing methods are available, but there are limitations including the number of targets analyzed at a time and the method specificity. In this study, we developed a targeted next-generation sequencing panel to detect meat species in canned pet foods using Ion Torrent technology. The panel contains multiple primers targeting mitochondrial genes from as many as 27 animal species, of which 7 major animal species were validated. The meat species targets could be identified from samples spiked with as low as 0.01% w/w of the contaminating meat species in a vegetarian food matrix material. Targeted NGS in the current study enriches species-specific multiple target areas in the mitochondrial genome of the target material, which gives high accuracy in the sequencing results.

Unmapped short reads from whole-genome sequencing indicate potential infectious pathogens in german black Pied cattle.

When resequencing animal genomes, some short reads cannot be mapped to the reference genome and are usually discarded. In this study, unmapped reads from 302 German Black Pied cattle were analyzed to identify potential pathogenic DNA. These unmapped reads were assembled and blasted against NCBI's database to identify bacterial and viral sequences. The results provided evidence for the presence of pathogens. We found sequences of Bovine parvovirus 3 and Mycoplasma species. These findings emphasize the information content of unmapped reads for gaining insight into bacterial and viral infections, which is important for veterinarians and epidemiologists.

Quantification of the bacterial flora and its major constituents on the abdominal skin of clinically healthy dogs.

OBJECTIVE: To report the density, and the major constituents, of the bacteria on the skin surface of healthy dogs and to assess if scraping the skin before sampling was necessary. ANIMALS: 20 healthy dogs were recruited for the study, with informed consent in all cases. METHODS: Flocked swabs were used to sample the skin surface and to sample the skin surface after superficial scraping with a blunted spatula. Both samples were taken within a brass guide of 3.5 cm-2 area. Next-generation 16S rRNA sequencing was used to identify and quantify components of the bacterial microbiome. RESULTS: The median density of the bacterial microbiome on the ventral abdomen of 20 healthy dogs was approximately 1.1 X 105 cm-2 (IQR 1.22 X 104, 1.6 X 105 cm-2). Sphingomonas species were isolated on 17 of the 20 dogs and Corynebacterium kroppstedtii from 15. CLINICAL RELEVANCE: This is the first study to report the density of the canine skin microbiome. Superficial scraping of the skin before swabbing does not affect the result of sampling the microbiome in healthy dogs. These results will increase our understanding of the biology of canine skin.

Isolation, full sequence analysis, and in situ hybridization of pigeon paramyxovirus-1 genotype VI.2.1.1.2.2 from oriental turtle doves (Streptopelia orientalis).

Pigeon paramyxovirus-1 (PPMV-1), a genetic variant of avian paramyxovirus-1 (APMV-1), has been identified in Columbiformes and is the primary cause of diseases in captive and free-ranging pigeons. However, it has also been reported that PPMV-1 can infect chickens naturally and experimentally, thus posing a potential threat to the poultry industry. This study investigated a lethal outbreak of paramyxovirus infection that occurred among 16 oriental turtle doves (Streptopelia orientalis) in a walk-in aviary at a zoo from March to April 2021. Necropsies were performed, and histopathological findings revealed mild to moderate lymphoplasmacytic infiltration in several organs, such as the pancreas, liver, kidneys, and lungs. Reverse transcription polymerase chain reaction (RT-PCR) using formalin-fixed paraffin-embedded tissue blocks, virus isolation from fresh tissue, and in situ hybridization against the fusion (F) protein confirmed the diagnosis for PPMV-1 infection. The isolated strain NTU/C239/21 was fully sequenced by next-generation sequencing, and the results of phylogenetic analyses revealed that the F protein of NTU/C239/21 shared 98.8% nucleotide sequence identity with Pigeon/Taiwan/AHRI121/2017, which was isolated from a feral pigeon in Taiwan. The present study is the first to identify PPMV-1 infection in Streptopelia orientalis and suggests that Streptopelia orientalis may also play an important role in spreading the infection, similar to pigeons in APMV-1 spreading.

Next-generation DNA sequencing offers diagnostic advantages over traditional culture testing.

OBJECTIVE: While the clinical utility of next-generation DNA sequencing (NGS) as a diagnostic tool for infections in humans and traditional pets has been demonstrated, there is a lack of data regarding its utility for exotic animals. For exotic patients, traditional culturing is especially challenging for anaerobic and fungal pathogens. Therefore, diagnosis often relies on PCR, which provides a high degree of sensitivity and specificity, although it targets only a predetermined, finite pathogen panel. NGS provides the same benefits as PCR, while also offering de novo identification and quantification of all bacteria and fungi present in a clinical sample, including novel pathogen discovery. PROCEDURES: Clinical samples from 78 exotic animal patients were collected simultaneously for conventional culture testing and NGS analysis. Results provided by each laboratory were compared for the presence and absence of bacterial and fungal pathogens and commensals. RESULTS: Results showed large bacterial and fungal species diversity in the study cohort and a lack of sensitivity of microbial culture testing. Culture failed to grow 15% of putative bacterial and 81% of putative fungal pathogens that were identified by NGS. The probability of a "no growth" diagnosis was 14% higher for bacteria and 49% higher for fungi with culture versus NGS testing if fungal culture was conducted. CLINICAL RELEVANCE: Culture testing failed to diagnose a substantial number of both bacterial and fungal pathogens, which were detected by NGS. This highlights the limitations of traditional culture-based testing and displays the clinically advanced utility of NGS-based diagnostics in exotic animal medicine.

Management and analysis of high-throughput sequence data for infectious animal diseases.

Advances in technology and decreasing costs have accelerated the use of high-throughput sequencing (HTS) for both diagnosis and characterisation of infectious animal diseases. High-throughput sequencing offers several advantages over previous techniques, including rapid turnaround times and the ability to resolve single nucleotide changes among samples, both of which are important for epidemiological investigations of outbreaks. However, due to the plethora of genetic data being routinely generated, the storage and analysis of these data are proving challenging in their own right. In this article, the authors provide insight into the aspects of data management and analysis that should be considered before adopting HTS for routine animal health diagnostics. These elements fall largely into three interrelated categories: data storage, data analysis and quality assurance. Each has numerous complexities and may need to be adapted as HTS evolves. Making appropriate strategic decisions about bioinformatic sequence analysis early on in project development will help to avert major issues in the long term.

Les avancées technologiques dans le domaine du séquençage à haut débit (SHD) et la diminution des coûts liés à cette technique en ont accéléré l'utilisation à des fins de diagnostic et de caractérisation des maladies animales infectieuses. Le séquençage à haut débit offre plusieurs avantages par rapport aux techniques antérieures, en particulier la rapidité de son exécution et une résolution de l'ordre d'un seul changement de nucléotide parmi plusieurs échantillons, ce qui présente un grand intérêt lors des enquêtes épidémiologiques sur les foyers. Néanmoins, la pléthore de données génétiques générées en routine par le SHD devient un véritable problème en termes de stockage et d'analyse de ces données. Les auteurs apportent un éclairage sur les aspects de la gestion et de l'analyse des données qu'il convient de prendre en compte avant d'adopter le SHD pour le diagnostic de routine en santé animale. Ces éléments relèvent de trois catégories étroitement reliées : le stockage de données, l'analyse de données et l'assurance qualité. Chacun de ces aspects présente de nombreuses complexités et nécessitera sans doute d'être adapté à mesure que le SHD évolue. Lorsqu'elles sont prises dès la phase initiale d'un projet, des décisions stratégiques appropriées en matière d'analyse bio-informatique de séquences peuvent contribuer à éviter des problèmes majeurs sur le long terme.

Los avances tecnológicos y la reducción de los costos han acelerado el uso de la secuenciación de alto rendimiento (SAR) con fines de diagnóstico y caracterización de enfermedades animales infecciosas. La secuenciación de alto rendimiento presenta varias ventajas en comparación con otras técnicas anteriores, en particular ciclos más rápidos y una resolución que permite detectar diferencias de un solo nucleótido entre las muestras, aspectos ambos de gran importancia para el estudio epidemiológico de brotes infecciosos. Sin embargo, debido al sinnúmero de datos genéticos que constantemente se generan, no es de extrañar que esté resultando problemático almacenar y analizar los datos obtenidos. Los autores arrojan luz sobre los aspectos de la gestión y el análisis de datos que conviene tener en cuenta antes de aplicar la SAR a las labores sistemáticas de diagnóstico en sanidad animal. Estos elementos corresponden a grandes líneas a tres categorías relacionadas entre sí: el almacenamiento de datos; el análisis de datos; y la garantía de calidad. Cada una de ellas presenta multitud de complicaciones y exige un proceso permanente de adaptación a medida que la técnica de secuenciación va evolucionando. El hecho de adoptar las buenas decisiones estratégicas sobre el análisis bioinformático de secuencias en los primeros momentos de la concepción de un proyecto ayudará a evitar importantes problemas a largo plazo.

Detection of domestic cat hepadnavirus by next-generation sequencing and epidemiological survey in Japan.

The novel domestic cat hepadnavirus (DCH), a member of the Hepadnaviridae, was first detected in Australia and has recently been identified in more countries. In this study, we explored the DCH genome using next-generation sequencing of a plasma sample from a cat with a fever of unknown cause. Nucleotide sequence analysis showed the virus to be relatively genetically distant from the first reported DCH in Australia, showing 89% homology. Then we conducted an epidemiological survey by PCR of plasma samples collected from 203 cats that visited a veterinary hospital for diagnosis and treatment. Two of the 203 surveyed cats a were positive for DCH. One of the two positive cases had elevated liver enzymes of unknown etiology, and the other had hepatocellular adenoma. Our study indicated that DCH infection was observed in domestic cats in the Tokyo area of Japan as well as other reported areas in the world. Further investigations are needed to define the clinical importance of DCH.

Genomic Characterization of Mosquito Isolates of Chikungunya Virus (Outbreak Strains 2022) Using Next-Generation Sequencing.

Background: A massive outbreak of dengue-like illness was reported from Pune district of Maharashtra, India during May-June 2022. Isolation and characterization of the etiological agent at genomic level for possible mutations that led to higher transmissibility is the topic of the study. Methods: Entomological investigations were carried out by ICMR-National Institute of Virology (Pune, India); Aedes aegypti mosquitoes were collected and processed for virus detection by molecular techniques. Positive mosquito pools were processed for virus isolation in cell culture. Sanger sequencing and whole-genome sequencing (WGS) using Oxford Nanopore Technology platform were used for genomic characterization. Results: Reverse transcriptase RT-PCR and qRT-PCR analysis detected chikungunya virus (CHIKV) in mosquito samples. Six CHIKV isolates were obtained. WGS revealed four nonsynonymous mutations in the structural polyprotein region, and five in the nonstructural polyprotein encoding region when compared with Yawat-2000 and Shivane-2016 strains. Sixty-four nucleotide changes in the nonstructural polyprotein region and 35 in the structural polyprotein region were detected. One isolate had an exclusive amino acid change, T1123I, in the nsP2 (protease) region. Conclusion: Abundant Ae. aegypti breeding and detection of CHIKV RNA in mosquitoes confirmed it as a chikungunya outbreak. Novel mutations detected in the epidemic strain warrants investigations to address their role in disease severity, transmission, and fitness.

Investigation of the pathogens contributing to naturally occurring outbreaks of infectious bovine keratoconjunctivitis (pinkeye) using Next Generation Sequencing.

Infectious bovine keratoconjunctivitis (IBK), commonly known as pinkeye, has a marked negative impact on the economy of the cattle industry. Moraxella species, including Mor. bovis and Mor. bovoculi, which have been associated with this disease, colonize clinically healthy eyes as well, suggesting that there are intrinsic changes that may occur to the ocular microbiota or the involvement of additional unrecognized organisms that contribute to IBK. To evaluate this, 104 ocular swabs collected from eyes with IBK or clinically healthy eyes from 16 different cattle herds were subjected to 16 S rRNA gene PCR and next generation sequencing (NGS) analysis. Organisms detected were similar across the herds and there was no difference in the total number of bacterial groups detected among IBK cases and controls. However, the percentages of the different organisms detected varied between the two groups, including Moraxella spp., with more Moraxella spp. in eyes with IBK than controls. Further, using culture and whole genome NGS, a new species of Moraxella (suggested name Mor. oculobovii) was detected from the eyes of cattle from two farms. This strain is non-hemolytic on blood agar, is missing the RTX operon, and is likely a non-pathogenic strain of the bovine ocular microbiome. Alteration of the ocular microbiota composition may have a predisposing role, enhancing bacterial infection and the occurrence of clinical IBK. Future studies are required to evaluate if these changes are permanent or if there is a shift in the microbiome following recovery from the infection and how antibiotics might affect the microbiome.

Clinical experience with next-generation sequencing-based liquid biopsy testing for cancer detection in dogs: a review of 1,500 consecutive clinical cases.

OBJECTIVE: To review ordering patterns, positivity rates, and outcome data for a subset of consecutive samples submitted for a commercially available, blood-based multicancer early-detection liquid biopsy test for dogs using next-generation sequencing at 1 laboratory. SAMPLE: 1,500 consecutively submitted blood samples from client-owned dogs with and without clinical suspicion and/or history of cancer for prospective liquid biopsy testing between December 28, 2021, and June 28, 2022. PROCEDURES: We performed a retrospective observational study, reviewing data from 1,500 consecutive clinical samples submitted for liquid biopsy testing. Outcome data were obtained via medical record review, direct communication with the referring clinic, and/or a patient outcome survey through October 16, 2022. RESULTS: Sixty-four percent (910/1,419) of reportable samples were submitted for cancer screening, 26% (366/1,419) for aid in diagnosis, and 10% (143/1,419) for other indications. The positivity rate was 25.4% (93/366) in aid-in-diagnosis patients and 4.5% (41/910) in screening patients. Outcome data were available for 33% (465/1,401) of patients, and outcomes were classifiable for 428 patients. The relative observed sensitivity was 61.5% (67/109) and specificity was 97.5% (311/319). The positive predictive value was 75.0% (21/28) for screening patients and 97.7% (43/44) for aid-in-diagnosis patients, and the time to diagnostic resolution following a positive result was < 2 weeks in most cases. CLINICAL RELEVANCE: Liquid biopsy using next-generation sequencing represents a novel tool for noninvasive detection of cancer in dogs. Real-world clinical performance meets or exceeds expectations established in the test's clinical validation study.

Evaluation of a commercial NGS service for detection of bacterial and fungal pathogens in infectious ulcerative keratitis.

OBJECTIVES: To compare results from a commercial next-generation sequencing (NGS) service to corneal cytology and culture for identification of causative organisms in veterinary patients presenting for infectious ulcerative keratitis (IUK). PROCEDURE: Swabs for corneal aerobic and fungal cultures and DNA swabs for NGS were submitted for canine and equine normal controls (n = 11 and n = 4, respectively) and IUK patients (n = 22 and n = 8, respectively) for which microbrush cytology specimens confirmed the presence of infectious organisms. The sensitivity of the NGS results was compared with bacterial and fungal culture results. Concordance between the NGS and culture results was determined. RESULTS: The NGS results were positive for bacterial and fungal organisms in 5 and 1 normal and 18 and 1 IUK cases, respectively. Bacterial and fungal cultures were positive for 7 and 2 normal and 20 and 5 IUK cases, respectively. Sensitivity of NGS was 82.14% (95% confidence interval (CI), 63.11% to 93.94%) and specificity was 76.47% (95% CI, 50.10% to 93.19%). Concordance (complete and partial) between identified bacterial and fungal organisms was found in 79% and 100% of cases, respectively. NGS identified organisms in 3 culture-negative IUK samples. CONCLUSION: A commercial NGS service may be useful in the identification of causative agents in IUK cases with a sensitivity greater than the sensitivity previously reported for aerobic culture. Further testing is needed to determine the clinical significance of additional organisms isolated by NGS from infected cases, as well as organisms isolated from normal corneas.

Next-Generation Diagnostics for Pathogens.

Next-generation sequencing (NGS) was initially developed to aid sequencing of the human genome. This molecular method is cost effective for sequencing and characterizing genomes, not only those of humans or animals but also those of bacteria and other pathogens. However, rather than sequencing a single organism, a targeted NGS method can be used to specifically amplify pathogens of interest in a clinical sample for detection and characterization by sequencing. Targeted NGS is an ideal method for ruminant syndromic testing due to its ability to detect a variety of pathogens in a sample with a single test.

An Update on the Ocular Surface Bacterial Microbiota in Small Animals.

High-throughput sequencing (HTS) techniques have revolutionized the way we understand microbial communities in both research and clinical settings and are bringing new insights into what constitutes a healthy ocular surface (and a diseased one). As more diagnostic laboratories incorporate HTS into their technique repertoire, practitioners can expect this technology to become increasingly accessible for clinical practice, potentially becoming the new standard. However, particularly regarding ophthalmic microbiota, considerable research remains to render HTS accessible and applicable.

Small RNA deep sequencing revealed microRNAs' involvement in modulating cellular senescence and immortalization state.

Unlike rodent cells, spontaneous immortalization of avian cells and human cells is a very rare event. According to patent publications and current literature, there are no more than 4 spontaneously immortalized chicken embryo fibroblast (CEF) cell lines established up to date. One of those cell lines is ADOL (Avian Disease and Oncology Laboratory) ZS-1 cell line, which was established by continuous passaging of the CEFs derived from the specific pathogen free (SPF) 0.TVB*S1 (commonly known as rapid feathering susceptible or RFS) genetic line of chickens. The RFS genetic line of chickens was developed and has been maintained on the SPF chicken farm of USDA-ARS facility, ADOL, in East Lansing, Michigan, which is known as one of a few lines of chickens that are free of any known avian endogenous virus genes. To explore potential roles that epigenetic factors may play in modulating cellular senescence processes and spontaneous immortalization state, total RNAs extracted from samples of the RFS primary CEFs, RFS CEFs reached the 21st passage, and the ZS-1 cells were subjected to small RNA sequencing. Collectively, a total of 531 miRNAs was identified in the 3 types of samples. In contrast to the primary CEF samples, 50 miRNAs were identified with significantly differential expression only in the 21st passage samples; a different subset of 63 differentially expressed miRNAs was identified only in the ZS-1 samples; the majority of differentially expressed miRNAs identified in both the 21st passage CEF and the ZS-1 samples were more or less directionally consistent. Gene Ontology analysis results suggested that the epigenetic factor, miRNAs, plays a role in modulating the cellular senescence and spontaneous immortalization processes through various bioprocesses and key pathways including ErbB and MAPK signaling pathways. These findings provided the experimental and bioinformatic evidence for a better understanding on the epigenetic factor of miRNAs in association with cellular senescence and spontaneous immortalization process in avian cells.

Illumina high-throughput sequencing for the genome of emerging fowl adenovirus D species and C species simultaneously.

In recent years, clinical cases of inclusion body hepatitis (IBH) and hydropericardium syndrome (HPS) have been emerging and increasing in chicken flocks worldwide. Mixed infections with 2 or more fowl adenovirus (FAdV) serotypes were common in these cases. Herein, we collected a clinical sample that was positive for FAdV from 40-day-old broilers with IBH and HPS symptoms in Shandong province of China and determined the complete genome of FAdVs on the Illumina HiSeq4000 platform. The results showed that the sample contained 2 FAdV strains of D species and C species and named SD1763-1 and SD1763-2 respectively. The genome of SD1763-1 strain was 43,913 nt in length, with a G+C content of 53.51%, whereas SD1763-2 strain was 43,721 nt in length, with a G+C content of 54.87%. Sequence alignment and phylogenetic analysis revealed that strain SD1763-1 was clustered together with serotype 2/11 of FAdV-D, and SD1763-2 was clustered together with FAdV-4. There is no recombination between the genomes of the 2 viruses of FAdV-D and FAdV-C in the present study. This is the first report of obtaining 2 genomic sequences of FAdV strains simultaneously by direct use of deep sequencing in one clinical individual chicken sample, which provided direct evidence for mixed infections of adenovirus serotypes in the clinic and enriched the genome data to explore the geographic biomarkers and virulence signatures of the genus Aviadenovirus.