Isolation, Identification, and Pathogenicity of a Goose Astrovirus Genotype 1 Strain in Goslings in China.

Goose astrovirus genotype 1 (GAstV-1) has emerged in goose farms in some provinces of China in recent years and is considered to be one of the pathogens of gout in goslings in China. However, few studies have been conducted on the dynamic distribution, tissue tropism, and pathogenesis of GAstV-1 in goslings. In 2022, an epidemiological investigation of goose astrovirus (GAstV) in goslings was conducted in seven provinces of China. During the investigation, a GAstV-1 designated as GAstV-JSXZ was identified in the kidney of an 8-day-old gosling and was successfully isolated from a goose embryo. The full genome sequence of GAstV-JSXZ was determined using the next-generation sequencing technique. The complete genome of GAstV-JSXZ was 7299-nt-long. Interestingly, the phylogenetic analysis revealed that Chinese GAstV-1 has formed two distinct subgroups based on the ORF 2 genomes, designated GAstV-1 1a and GAstV-1 1b. The GAstV-JSXZ shared the highest identity with GAstV-1 1a strain FLX and TZ03 in nucleotides (ORF1a: 98.3-98.4%; ORF1b: 92.3-99.1%; ORF2: 95.8-98.8%) and amino acid sequences (ORF1a: 99.4-99.5%; ORF1b: 98.2-98.8%; ORF2: 97.0-99.4%). To evaluate the pathogenicity of GAstV-1, 1-day-old goslings were inoculated with the virus by oral and subcutaneous injection routes, respectively. The results revealed that the virus causes extensive pathological organ damage, especially in the kidney, liver, and thymus. Virus-specific genomic RNA could be detected in the cloacal swabs and tissues of infected goslings throughout the experiment. The viral copy numbers examined in the kidney and intestine were the highest, followed by the liver and spleen. These results are likely to provide a new understanding of the pathogenicity of GAstV-1 in geese.

Role of long non-coding RNA DLY6E in regulating TMUV infection.

Long non-coding RNA (lncRNA) is a type of RNA with a length greater than 200 nt and lacking coding ability. In recent years, a considerable number of lncRNAs have been found to have important functions. The lncRNA plays an important role in growth and development, body metabolism, immune function, and regulation of viral replication. A lncRNA, MSTRG8505.2, was screened and named lncRNA DLY6E, which was a new duck-derived lncRNA. The lncRNADLY6E in this study has a complex secondary structure, specifically distributed in the heart, liver and other organs. The expression of lncRNA DLY6E was significantly up-regulated after TMUV infection, which was time-dependent and non-dose-dependent. Overexpression of three structural proteins and seven non-structural proteins of TMUV in DEF cells showed no significant difference in the expression of lncRNADLY6E. Meanwhile, using lipopolysaccharides (LPS) and poly (I:C) to stimulate DEF cells, the results showed that the induced expression of lncRNA DLY6E was associated with the dsRNA-related TLR3/RIG-I/MDA5 pathway rather than the LPS activated signaling pathway. To further explore the function of lncRNA DLY6E, an eukaryotic expression vector was constructed. Overexpression of lncRNA DLY6E in DEF cells can increase the replication of TMUV. After overexpression of lncRNADLY6E, the transcriptional level of its target gene LY6E was detected, and the results showed that lncRNADLY6E did not act through its target gene. Overexpression of lncRNA DLY6E significantly inhibited the mRNA levels of OAS, Mx and PKR, suggesting that lncRNA DLY6E may promote the virus by inhibiting the transcription of antiviral proteins in innate immunity. This phenomenon provides new ideas for the prevention and control of TMUV, which is worth further thinking and exploration.

Epidemiological investigation of fowl adenovirus (FAdV) infections in ducks and geese in Shandong Province, China.

RESEARCH HIGHLIGHTS: Samples of suspected FAdV-infected waterfowl from farms in Shandong Province were collected from 2019 to 2022.Single infections with FAdV were less frequent than mixed infections.477 out of 792 samples (60.23%) tested positive for FAdV nucleic acids.Detection rate of FAdV was 65.47% in fattening duck farms, 55.73% in breeder duck farms and 54.55% in fattening geese farms.

Pathogenicity of duck circovirus 1 in experimentally infected specific pathogen-free ducks.

Ducks infected with duck circovirus (DuCV) show symptoms such as feather loss, growth retardation and low body weight in the flock. The virus induces immunosuppression and increases the prevalence of infection with other pathogens. However, most studies on duck circovirus were focused on coinfection, and fewer studies had been conducted on the pathogenicity of duck circovirus alone. The aim of the present study was to investigate the pathogenesis of DuCV-1 in experimentally infected specific pathogen-free ducks. In this study, we sequenced the whole genome of a strain of duck circovirus and identified the virus genotype as DuCV-1b. This strain of duck circovirus was named SDLH(OR567883). Animal pathogenicity experiments were then conducted, wherein specific pathogen-free ducks were infected by mucosal injection and abdominal injection. Infected ducks were sampled for 4 consecutive weeks after infection and showed symptoms of dwarfism. We further examined the replication of DuCV-1 in the ducks. The highest virus titers in the 2 infection groups were found in the liver and spleen, with different results for the different routes of infection. Pathological sections of duck organs were made and it was found that organs such as the liver and spleen were damaged by DuCV-1. In conclusion, our experimental results indicate that DuCV-1 can infect ducks individually and cause widespread organ damage in infected ducks.

Evidence of vertical transmission of fowl adenovirus 8b in ducks.

Fowl adenovirus mainly causes hydropericardium hepatitis syndrome (HHS), inclusion body hepatitis (IBH) and gizzard erosion (GE), etc. In 2015, the first outbreak of HHS was reported in broiler chickens in central China, followed by an outbreak in waterfowl. The first outbreak of HHS in broiler flocks in central China in 2015, followed by outbreaks in waterfowl, has severely restricted the healthy development of the poultry industry. During the investigation, fowl adenovirus was detected in ducklings from a total of seven hatcheries in Shandong, Inner Mongolia and Jiangsu provinces. In addition, the DNA of fowl adenovirus was detected in breeding ducks and their progeny. To test the hypothesis that FAdV can be transmitted vertically, sixty 250-day-old Cherry Valley breeder ducks were divided equally into three groups for experimental infection. FAdV-8b SDLY isolate (duck/Shandong/SDLY/2021, SDLY) preserved in our laboratory was injected intramuscularly into group A and inoculated orally into group B. FAdV-8b DNA was detected in the yolk membranes, embryos and allantoic fluid of duck embryos in the FAdV-infected group after inoculation. In addition, the FAdV-8b hexon gene isolated from yolk membranes, embryos, allantoic fluid and duck eggs was close to 100% nucleotide homology to the FAdV-8b hexon gene isolated from laying duck ovaries, indicating that fowl adenovirus can be transmitted vertically in ducks. These findings provide evidence for the possible vertical transmission of fowl adenovirus from breeder ducks to ducklings.

Localization and distribution of goose astrovirus 2 antigens in different tissues at different times.

Goose astrovirus 2 (GAstV-2) causes visceral gout in goslings and has resulted in significant economic losses in the goose industry of China since its outbreak in 2017. To further investigate the distribution and localization of GAstV-2 in different tissues at different times, a monoclonal antibody (mAb)-based immunohistochemical (IHC) assay was developed to detect GAstV-2. A total of 80 1-day-old healthy goslings were inoculated with GAstV-2 via the oral (n = 40) and intramuscular routes (n = 40). GAstV-2 in the tissues of interest was detected using the established IHC assay. The results showed that positive signals were detected in most tissues at 1 day post-infection (dpi). Viral antigens were mainly distributed in the cytoplasm, and the staining intensity was higher in the renal tubular epithelial cells than in other cells. Taken together, our data demonstrated that GAstV-2 has a broad tissue tropism and primarily targets the kidneys. These results are likely to provide a scientific basis for further elucidation of the pathogenesis of GAstV-2.

The isolation and characterizations of a duck adenovirus 1 causing Egg Drop Syndrome in ducks, China.

A fowl adenovirus variant designated as DAdV-JSXZ strain was isolated from the tissue specimen of fallopian tubes of a duck case, which was submitted from a 276-day-old Cherry valley breeding duck flock experienced egg-dropping syndromes in March 2022. Full-genome sequence of the DAdV-I JSXZ strain by next-generation sequencing revealed that the complete genome length of DAdV-JSXZ strain was 33,213 nucleotides and shared a high degree of nucleotide identity (97.0-99.4 %) with other DAdV-I reference strains. In pathogenicity studies, this isolated duck JSXZ strain reproduced similar egg-dropping symptoms in healthy breeding ducks, pathologic lesions of follicular hemorrhage, and the laid eggs in low fertilization and hatchability rates. Our research findings demonstrated that DAdV-I JSXZ strain was one of the causative agents of duck egg dropping syndrome in egg-laying ducks and could cause acute respiratory symptoms in ducklings.

Development and biochemical characteristics of a monoclonal antibody against prM protein of Tembusu virus.

Tembusu virus (TMUV), a pathogenic member of the Flavivirus family, is an infectious diseases that seriously jeopardize duck health in 2010 in China. TMUV disease causes significant economic losses to the duck industry. This study aimed to prepare monoclonal antibodies against TMUV prM protein and to identify their epitopes. The 501bp prM gene was amplified to the pET-32a prokaryotic expression vector and expressed as a recombinant protein of size 38 KD in Escherichia coli. The purified recombinant proteins were inoculated into BALB/c mice to generate splenic lymphocytes capable of secreting anti-prM antibodies, and hybridoma cells were obtained after fusion with SP2/0 cells. A new hybridoma cell line named B27, which stably secreted IgG1-antibody against TMUV prM with high antibody titers up to 1:1:3,276,800 was screened. This monoclonal antibody (mAb) is well specific and can be used for ELISA/Western-blot (WB)/indirect fluorescence assay (IFA) etc. The mAb B27 has poor neutralization ability and concentration dependence, with a maximum neutralization degree of 23.87% at antibody dilution 10-6. Next, we truncated prM gene and expressed the truncated protein to screen antigen epitopes. The mAb's linear antigen epitope of the TMUV prM protein was first identified and was accurate to 6 consecutive amino acids 59GYEPED64, which located in the pr protein. Bioinformatic analysis showed that this antigenic epitope was located on the surface of the antigen, which was conducive to the direct contact of antigen antibody and conformed to the properties of antigenic epitopes. In addition, its 6 amino acids are highly homologous among 27 published TMUV strains, indicating that its epitope is stable. This study will help to further understand the protein structure and the function of prM, and lay the foundation for establishing specific prM detection methods and the mechanistic study of TMUV prM protein.

Synergistic pathogenicity of avian orthoreovirus and Staphylococcus aureus on SPF chickens.

Avian arthritis is a relatively common disease in the poultry industry, the cause of which is complex. Bacterial arthritis is often caused by infection of Staphylococcus aureus (S. aureus), whereas viral arthritis is caused by avian orthoreovirus (ARV). To investigate the infection of S. aureus and ARV in cases of avian arthritis, a total of 77 samples characterized by arthritis were collected and detection. The results showed that 68.83% of the samples were positive for ARV, and 66.23% were positive for S. aureus. Among them, the ARV mono-infection rate was 22.08%, the S. aureus mono-infection rate was 19.48%, and ARV and S. aureus co-infection rate was 45.45%, indicating that ARV and S. aureus co-infection is common in arthritis cases. To further investigate the synergistic pathogenicity of ARV and S. aureus, ARV and S. aureus were used to mono-infect, co-infect, and (or) sequential infect SPF chickens and the clinical indications, pathologic changes, ARV load, S. aureus bacterial distribution, and cytokine level of the challenged chickens were evaluated. Decreased weight gain, increased mortality, and difficulties in standing were observed in all dual-infected groups and the singular-infected group. There were significantly more severe macroscopic and microscopic hock lesions, and larger amounts of a wider range of tissue distribution of ARV antigens and S. aureus bacterial distribution in the dual-infected groups compared to the single-infected and control groups. Cytokine detection showed a significant change in IFN-γ, IL-1ß, and IL-6 levels in the infected groups, especially in the ARV-S. aureus co-infection, and (or) sequential infection groups, compared with the control group. Hence, ARV and S. aureus synergistically increased mortality in infected chickens, potentiated the severity of arthritis, and increased the amount of ARV RNA in tendons.

Pathogenicity of goose astrovirus genotype 2 in chickens.

Goose astrovirus genotype 2 (GAstV-2) is the causative agent causing severe visceral gout and joint gout in goslings, with mortality rates of affected flocks up to 50%. To date, continuous GAstV-2 outbreaks still pose a great threat to goose industry in China. Although most researches on GAstV-2 have focused on its pathogenicity to geese and ducks, limited studies have been performed on chickens. Herein, we inoculated 1-day-old specific pathogen-free (SPF) White Leghorn line chickens with 0.6 mL of GAstV-2 culture supernatant (TCID50 10-5.14/0.1 mL) via orally, subcutaneously and intramuscularly, and then assessed the pathogenicity. The results revealed that the infected chickens presented depression, anorexia, diarrhea, and weight loss. The infected chickens also suffered from extensive organ damage and had histopathological changes in the heart, liver, spleen, kidney, and thymuses. The infected chickens also had high viral load in tissues and shed virus after the challenge. Overall, our research demonstrates that GAstV-2 can infect chickens and adversely affect the productivity of animals. And the viruses shed by infected chickens can pose a potential risk to the same or other domestic landfowls.

Co-circulation of multiple genotypes of ARV in poultry in Anhui, China.

ABSTRACTPoultry production in China has been experiencing a high incidence of broiler arthritis /tenosynovitis caused by avian orthoreovirus (ARV) since 2013. In the spring of 2020 severe arthritis cases from broiler flocks were identified in a large-scale commercial poultry company in Anhui Province, China. Diseased organs from dead birds were sent for diagnosis to our laboratory. ARVs, including seven broiler-isolates and two breeder-isolates, were successfully harvested and sequenced. Interestingly, the genotypes of ARVs isolated from infected chickens were inconsistent between different flocks, or even between different houses on the same flocks. Pathogenicity testing in chicks confirmed that the seven broiler-isolates were pathogenic strains, which could cause arthritis in infected chickens. Subsequently, a total of 89.66% serum samples collected from apparently healthy adult broiler flocks not vaccinated against ARV tested positive for ARV antibodies, suggesting that low and high virulence reovirus strains may be co-circulating in the farm. To this end, we collected dead embryos of unhatched chicken eggs for pathogen tracing, and the two ARV breeder-isolates isolated indicated that vertical transmission from breeders to progeny should not be underestimated for the prevalence of ARV within broiler flocks. The findings have implications for the evidenced-based formulation of prevention and control strategies.

Genomic characteristics, pathogenicity and viral shedding of a novel DVEV variant derived from goose.

Duck virus enteritis (DVE), caused by the DVE virus (DVEV), is an acute, septicemic, and contagious disease affecting ducks of different breeds, ages, and sexes. In late spring and summer 2019, several outbreaks of DVE were reported in areas with large waterfowl industries in central and southern China. A goose farm located in Jining County, Shandong Province, was impacted by an acute DVE outbreak in July 2019. The causative DVEV field strain (Goose/DVEV/SDJN/China/2019) was subsequently isolated from the liver specimens collected from acute cases of dead geese, which showed severe hemorrhagic lesions on the esophageal mucosal membranes of specimens collected from all the postmortem cases. Comparison of the genome sequence of this newly isolated field strain (Goose/DVEV/SDJN/China/2019) with the common DVEV strains revealed insertions or mutations in the gB and gC genes, which possibly caused the observed high morbidity and mortality in this acute outbreak. We conducted a trial among geese to evaluate the pathogenicity of this strain. Healthy experimental goslings aged 15 d old were inoculated with 10-5.53 ELD50/0.2 mL doses orally or through intramuscular injection. Clinical signs and esophageal erosion appeared in infected geese. Necropsy revealed hemorrhage and necrosis of the cloacal mucosa and liver. Detection of the virus using real-time PCR in the liver, brain, and spleen indicated that they were the hotspots of DVEV infections. One day after the DVEV infection, virus release and seroconvert were observed in infected geese. Thus, our studies demonstrate that DVEV is highly pathogenic and contagious in geese. To the best of our knowledge, this is the first study on the pathogenicity of mutant duck viral enteritis virus in goslings. This study serves as a foundation for further investigations into the pathophysiology of the recently identified variant DVEV strains.

Genetic characteristics and pathogenicity of avian pox virus for a new host, Cherry Valley breeder ducks in China.

Cherry Valley breeder ducks in Shandong province in northern China have experienced swollen eyes, lachrymation, pox scabs on glabrous or glabrous skin, depression and dysentery since 2021. The spread of this infectious disease has seriously affected the breeder ducks in major Cherry Valley duck farms in China. The virus causing clinical signs in Cherry Valley breeder ducks was isolated by chicken embryo inoculation. We successfully isolated a strain of duck pox virus from diseased breeder ducks by virus replication. We have also successfully conducted experiments for duck pox disease using the isolated strains to infect ducklings. By comparison with 22 pox viruses already published in GenBank, the virus strain obtained in this study was most homologous (about 99.7%) to the strain isolated from infected domestic ducks in Guangxi, China in 2014 (KJ192189), and belonged to the same A5 subtype. Since there were no previous cases of avian pox virus infecting white or Cherry Valley duck breeds, this study identified a new host for avian pox virus infection and provided theoretical support and data for the development of avian pox virus research.RESEARCH HIGHLIGHTS Avian pox virus can infect a new host type - Cherry Valley breeder ducks.The avian pox virus isolated from Cherry Valley breeder ducks showed highest identity with Guangxi hemp duck-derived avian pox virus.Cherry Valley breeder ducks were infected with avian pox virus of subtype A5.

Recombinant characteristics, pathogenicity, and transmissibility of a variant goose orthoreovirus derived from inter-lineage recombination.

Since March 2021, an infectious characterized by white necrotic foci throughout the goose body has appeared in the major goose-producing regions in China. This disease has caused economic hardship for goose farms in many regions of China with approximately 50 % mortality. A novel goose-origin orthoreovirus was isolated from the spleen of diseased geese and designated as N-GRV/HN/Goose/2021/China (N-GRV-HN21) strain. Next-generation sequencing and phylogenetic analysis revealed that the isolate was a reassortant virus containing viral gene segments from three ARV serotypes that infect duck, muscovy duck, and goose. Geese infection test showed that both N-GRV-HN21-infected and contacted geese displayed whole-body white necrotic foci. N-GRV RNA was detected in different organs of both infected and contacted geese, indicating that the N-GRV isolate is pathogenic and transmissible in geese. Seroconversion was also observed in experimentally infected and contacted geese. A prevalence study of 323 goose serum samples collected from different goose breeding areas showed that 86 % of the geese were positive for N-GRV. In conclusion, all results warrant the necessity to monitor orthoreovirus epidemiology and reassortment as the orthoreovirus could be an important pathogen for the waterfowl industry and a novel orthoreovirus might emerge to threaten animal and public health.

Development of an indirect competitive ELISA method based on ORF2 detecting the antibodies of novel goose astrovirus.

Goose astrovirus (GAstV) characterized by articular and visceral gout, is an emerging pathogen with a wide distribution on mainland China, leading to serious economic losses in the goose-raising industry. Because vaccines to prevent GAstV infections are not available currently, early diagnosis is critical when treating symptomatic geese and in preventing GAstV transmission. In this context, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a monoclonal antibody (mAb) to detect GAstV-specific antibodies from geese was developed, and the detections were optimized. A series of experiments proved that the ic-ELISA shows excellent diagnostic performance and discriminatory power with high sensitivity and specificity. The ic-ELISA for GAstV detection was applied on 67 field serum samples, and comparing the detection results with the virus neutralization test verified the accuracy of the ic-ELISA. The correlation coefficient between the ic-ELISA and the virus neutralization test was 80%, demonstrating the proposed ic-ELISA method could be a useful and effective tool for the diagnostic, serological epidemiological investigation and immune monitoring of the GAstV in goose-producing regions.

Genetic and pathogenic characteristics of two novel/recombinant avian orthoreovirus.

In recent years, the emerging avian orthoreovirus (ARV) strains that led to viral arthritis have attracted much attention from the chicken industry worldwide, due to the significant economic losses suffered. In mid-2020, with the assistance of next-generation sequencing technology, we achieved success in characterizing two divergent avian orthoreovirus (ARV) variants (0543/SDYT) and isolating them from the broiler tendons characterized by arthritis. As suggested by the genome characterization of the 0543/SDYT strains, they belong separately to clusters Ⅰ and Ⅳ. As revealed by sequence comparison, phylogenetic, and recombination analysis, for µA, µB, and σNS genes, considerable genetic divergences were also observed in the two new isolates. However, in the case of λA, λB, µNS, σA, and σB genes, very clear clustering patterns were observed for SDYT and 0543 field strains, respectively. In terms of the µA, µNS, p10, p17 genes of SDYT isolate and µNS, p17, σC, σA genes of 0543 isolate, the lower similarity was observed with NCBI stored sequences, with nt highest identity values below 90 %. In addition, there is an intra-fragmental recombination event in the M1 gene of the SDYT strain. In regard, the multiple segmental recombination and accumulation of point mutations play a role in the newly-emerging ARV strains. Not only did the isolates strain exhibit strong replication ability in vivo, but they also displayed strong arthritogenicity in broilers with low neutralizing maternal antibodies, indicating that maternal antibody treatment may not effectively reduce the oral infection of avian orthoreovirus. These findings suggest that it is necessary to develop a new strategy for enhanced effectiveness in preventing and controlling ARV infection.

Genetic evolution of fowl adenovirus serotype 4 and its pathogenicity to Cherry Valley ducks in China.

Hydropericardium hepatitis syndrome (HHS), a novel poultry disease, is caused by fowl adenovirus 4 (FAdV-4). It mainly infects 3-5-week-old broilers. In July 2015, the first outbreak of HHS occurred in the broilers in east China, which caused great economic losses to the poultry industry. In June 2019, infectious disease was detected with suspected HHS symptoms on a duck farm in Linyi City, Shandong Province. The main necropsy lesions included pericardial effusion and hepatitis. In this study, we isolated a strain of FAdV-4 from naturally infected ducks and named it SDLY190604, and the hexon gene sequence was amplified and analyzed using polymerase chain reaction (PCR). In order to study the effect of FAdV-4 on Cherry Valley ducks, we inoculated three-week-old ducks with 0.2 ml of FAdV-4 virus fluid (TCID50 of 10-6.3/0.1 ml) by orally, subcutaneously and intramuscularly. Clinical signs, gross lesions and histopathological changes, cytokines and viral load were detected and recorded within 15 days after infection. The results showed that ducks in the experimental groups exhibited typical symptoms of hydropericardium and hepatitis. The histopathological sections showed multiple-organ damage, including serious liver and kidney damage with elevated levels of inflammatory cytokines, probably due to the infection and innate immune response. Later, immunosuppression occurred, resulting in decreased levels of cytokines. The viral load indicated that the virus could be present in several organs of the ducks, with the highest viral DNA found in the liver, followed by the kidney. Compared to the subcutaneous and oral groups, the intramuscular group exhibited the highest viral load. In summary, this study can increase our understanding of the pathogenicity of FAdV-4 in ducks and provide a basis for further understanding of the virus, imparting new insights into disease research.

Epidemiological investigation of infectious diseases in geese on mainland China during 2018-2021.

Geese play an important role in agricultural economics, with China producing the vast majority of goose meat consumed worldwide annually. The variations in the avian viruses and co-infections result in substantial economic losses to the goose industry in China. To understand the evolutionary characteristics and co-infections of viruses, a broad epidemiological investigation of epizootic viruses of goose was carried out in nine provinces of China during 2018-2021. Here, the results indicated that, among the 1970 clinical samples, 50.81% (1001/1970) were positive for goose astrovirus (GAstV), 18.22% (359/1970) for avian orthoreovirus, 12.74% (251/1970) for goose parvovirus, 11.02% (217/1970) for H9N2 subtype avian influenza virus, 4.01% (79/1970) for Newcastle disease virus, and 2.08% (41/1970) for fowl adenovirus. Among the six viruses, co-infections comprised a large proportion (66.37%) in the field, of which the dual infection was more common. Additionally, phylogenetic analysis of GAstVs indicated that Chinese GAstVs had formed two distinct groups, that is, GAstV-1 and GAstV-2. GAstV-2 sub-genotype II-c had arisen as the dominant genotype in the whole country. Notably, all the H9N2-AIV isolated strains harboured the mammalian adaptation markers I155T, H183N, and Q226L (H3 numbering) in the HA gene, which promotes preferential binding to human-like α2-6-linked sialic acid receptors. And beyond that, we determined that the goose-origin Muscovy Duck Reovirus isolates, showing 51.7%-96% similarities to that of other waterfowl-origin orthoreovirus isolates in sequence homology analysis of the representative part of σC, are a new variant of waterfowl-origin orthoreovirus. These data provide valuable information about the prevalence of infectious diseases in geese on mainland China.

Specific High-Sensitivity Enzymatic Molecular Detection System Termed RPA-Based CRISPR-Cas13a for Duck Tembusu Virus Diagnostics.

In China, drastic losses in the economy have been caused by the Tembusu virus (TMUV), the causative agent of the egg-drop syndrome, to the duck-raising industry. To succeed in preventing and controlling infections, extant techniques must be upgraded to achieve fast detection of viruses. This work is the first attempt to present the development of a recombinase polymerase amplification (RPA)-based clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas13a approach for the TMUV infection diagnosis, where the CRISPR-Cas13a system is exploited, i.e., the programmability of CRISPR RNA (crRNA) and the promiscuous RNase collateral cleavage of Cas13a upon recognition of target RNAs. A prokaryotic expression system was utilized for the expression of LwCas13a soluble protein, while its purification was accomplished by nickel-nitrilotriacetic acid (Ni-NTA) agarose. In the design of a particular crRNA, the target used was the TMUV NS3 RNA transcribed in vitro. The signals used for the Cas13a activity validation were an RNA-bound fluorescent group (single-stranded) and a quenching fluorophore. In the present work, a specific high-sensitivity enzymatic molecular detection system termed RPA-based CRISPR-Cas13a was established by combining Cas13a with T7 transcription and RPA for sensitive detection of TMUV at room temperature. This system can detect 102 copies of the target TMUV DNA standard/µL within 50 min. A comparison revealed that the specificity was superior to that for other avian viruses. Furthermore, the RPA-based CRISPR-Cas13a detection system was successfully applied for clinical samples, and its performance is comparable to the reverse-transcriptase real-time quantitative polymerase chain reaction (RT-qPCR). Being satisfyingly reliable, simple, specific, and sensitive, our RPA-based CRISPR-Cas13a detection system could be expanded and universalized for identifying other viruses, enabling quick detection in the field with a portable lateral flow dipstick.

Isolation, identification and genotyping of Candida albicans from Landes geese.

In May 2018, Landes geese raised in Weifang, Shandong Province, China, developed a disease characterized by thickened oesophageal mucosa and white, round ulcers. Based on pathogen isolation and identification, differential culture and morphological observations, Candida albicans (C. albicans) was identified as the causative pathogen from the oesophagus of infected geese, and artificial infection experiments were then performed using the isolated strains. In experimental reproduction, the symptoms of infected geese were consistent with those of natural infection, and gosling morbidity and mortality were 75% and 60%, respectively. Re-isolation of the strain from the dead goslings confirmed C. albicans as the causative pathogen of oesophageal ulcers. We further performed internal transcribed space rDNA sequence analysis, ABC genotyping and multi-locus sequence typing analysis. We observed 100% sequence similarity between the two strains, designated as WFCL and WFLQ, which were isolated from different regions, with 100% homology between the strains isolated in the present study and the human-origin C. albicans strains isolated previously from China. The goose-origin strains isolated in this study and the human-origin C. albicans isolates were included in the same branch in phylogenetic trees analysis, indicating that the strain responsible for oesophageal ulcer in geese is closely related to human-origin C. albicans. In addition, based on eBURST analysis of sequence types, goose-origin C. albicans strains were relatively independent in terms of population evolution. To the best of our knowledge, this is the first detailed report on goose oesophageal ulceration caused by C. albicans infection in geese. Considering that C. albicans is an important zoonotic pathogen, this study provides a reference for further studies on avian C. albicans infections and is important for ensuring public health and safety.