Isolation and genomic characterization of one novel goose astrovirus causing acute gosling gout in China.

Novel goose astrovirus (NGAstV) is a member of the genus Avain Avastrovirus (AAstV) and the family Astroviridae. NGAstV-associated gout disease has caused huge economic losses to the goose industry worldwide. Since early 2020, NGAstV infections characterized by articular and visceral gout emerged continuously in China. Herein, we isolated a GAstV strain from goslings with fatal gout disease and sequenced its complete genome nucleotide sequence. Then we conducted systematic genetic diversity and evolutionary analysis. The results demonstrated that two genotypic species of GAstV (GAstV-I and GAstV-II) were circulating in China, and GAstV-II sub-genotype IId had become the dominant one. Multiple alignments of amino acid sequences of GAstV capsid protein revealed that several characteristic mutations (E456D, A464N, and L540Q) in GAstV-II d strains, as well as additional residues in the newly identified isolate which varied over time. These findings enrich the understanding of the genetic diversity and evolution of GAstV and may facilitate the development of effective preventive strategies.

Identification and molecular characterization of novel duck reoviruses in Henan Province, China.

Novel Duck reovirus (NDRV) is an ongoing non-enveloped virus with ten double-stranded RNA genome segments that belong to the genus Orthoreovirus, in the family Reoviridae. NDRV-associated spleen swelling, and necrosis disease have caused considerable economic losses to the waterfowl industry worldwide. Since 2017, a significant number of NDRV outbreaks have emerged in China. Herein, we described two cases of duck spleen necrosis disease among ducklings on duck farms in Henan province, central China. Other potential causative agent, including Muscovy duck reovirus (MDRV), Duck hepatitis A virus type 1 (DHAV-1), Duck hepatitis A virus type 3 (DHAV-3), Newcastle disease virus (NDV), and Duck tembusu virus (DTMUV), were excluded by reverse transcription-polymerase chain reaction (RT-PCR), and two NDRV strains, HeNXX-1/2021 and HNJZ-2/2021, were isolated. Sequencing and phylogenetic analysis of the σC genes revealed that both newly identified NDRV isolates were closely related to DRV/SDHZ17/Shandong/2017. The results further showed that Chinese NDRVs had formed two distinct clades, with late 2017 as the turning point, suggesting that Chinese NDRVs have been evolving in different directions. This study identified and genetic characteristics of two NDRV strains in Henan province, China, indicating NDRVs have evolved in different directions in China. This study provides an insight into the ongoing emerged duck spleen necrosis disease and enriches our understanding of the genetic diversity and evolution of NDRVs.

Assessment of the Endophytic Fungal Composition of Lactobacillus plantarum and Enterococcus faecalis-Fermented Astragalus membranaceus Using Single-Molecule, Real-Time Sequencing Technology.

Endophytic fungus represents microorganisms existing within the healthy plant organs, which can significantly influence metabolic product production in plants, a process with great research value and broad prospects for development. To investigate the effect of fermentation with probiotic cultures on the endophytic fungal diversity and composition of Astragalus membranaceus, we used single-molecular, real-time sequencing (Pacific Biosciences) for 18S ribosomal RNA (rRNA) sequencing. The results showed that the endophytic fungi of A. membranaceus mainly belonged to Aspergillus, Penicillium, Cystofilobasidium, Candida, Guehomyces, and Wallemia. Furthermore, the endophytic fungal diversity and abundance of A. membranaceus were more variable after fermentation with Enterococcus faecium and/or Lactobacillus plantarum. Our data lays a solid and comprehensive foundation for further exploration of endophytic fungi from A. membranaceus as potential sources of functional compounds.

Core antigenic advantage domain-based ELISA to detect antibody against novel goose astrovirus in breeding geese.

Goose astrovirus (GAstV), the major causative agent of visceral and joint gout in goslings, is a novel pathogen greatly threatening waterfowl industry. Importantly, the horizontal and vertical transmissibility of GAstV posed a great challenge for disease prevention and control. Given the absence of commercial vaccine, restricting vertical transmission and protecting susceptible goslings must be a priority. Although many detection methods have been established, there is no serological method to detect GAstV-specific antibody, greatly limiting inspection and elimination of infected breeding bird. In this study, the B-cell epitopes of GAstV capsid protein were predicted, and its core antigenic advantage domain (shCAP) was expressed and purified. After authenticating the antigenicity, the recombinant shCAP protein was taken as the coating antigen, and an easily accessible indirect enzyme-linked immunosorbent assay (ELISA) was established to detect GAstV-specific antibody. The working conditions, including antigen concentration, serum dilution and incubation time, blocking buffer concentration, and color developing time, were gradually optimized by checkerboard titration. The cut-off OD450 value of the indirect ELISA for positive sample was 0.379, and the analytical sensitivity was 1:800. There was no cross-reaction with sera against goose parvovirus (GPV), Tembusu virus (TUMV), H5 and H7 subtype avian influenza virus (AIV H5 + H7), and Newcastle disease virus (NDV). The assay was further applied to examine 73 breeding goose serum samples and shared excellent agreement of 93.5% (68/73) with western blot, which also suggested that GAstV is circulating in the goose population in China. In conclusion, the developed indirect ELISA is simple, specific, and sensitive, which will be greatly useful to screen GAstV infection and block vertical transmission. KEY POINTS: • B-cell epitopes of GAstV capsid protein were predicted and expressed as immunogen • A core antigenic advantage domain-based ELISA was established to detect GAstV-specific antibody • The established ELISA will contribute to inspection and elimination of infected breeding geese and provide a useful tool for large scale serological testing of GAstV in geese.

Identification and genomic characterization of emerging goose astrovirus in central China, 2020.

Astroviruses are a non-enveloped virus with large host range breadth. AstV-associated gastroenteritis in human and animal, nephritis in chicken, gout in gosling and hepatitis in duckling pose great threats to public health and poultry industry. Since early 2020, continuous emergence of fatal goose astrovirus (GAstV) infections characterized by articular and visceral gout was reported in China. Here, we described two outbreaks of emerging gout disease in two different goose farms of central China. Two virulent GAstV strains, designated as HNKF-1/China/2020 and HNSQ-6/China/2020, were isolated, and the fifth passage of the isolates could cause urate crystals accumulated in the allantoic fluid and even deposited around great vessels and embryo bodies. Meanwhile, the source of these GAstV outbreaks was tracked to goose hatcheries. The prevalence of GAstV in the goose embryos with hatch failure was confirmed, and embryo-origin HNXX-6/China/2020 was further isolated. The complete genome of these three newly isolates was then sequenced and analysed. The results showed that Chinese GAstVs have formed two distinct groups, and the three GAstV isolates, as well as most of the Chinese GAstVs, belong to the G-I group. There are several amino acid mutations in the three newly identified GAstVs, such as A520T, S535R, V555I and A782T in ORF1a and Q229P in ORF2, suggesting the field stains, HNKF-1/China/2020 and HNSQ-6/China/2020, might derive from the weak goose embryo via vertical transmission. Moreover, the phylogenetic analysis of the complete viral genome and individual viral proteins revealed that Chinese GAstV strains have been constantly evolving towards more complicated and various directions. Our study reported the recently emerging GAstV outbreaks in central China, and further analysed the genetic characteristics of three virulent G-I GAstV isolates from commercial goose farms and goose hatchery, indicating the diverse transmission of the virus and providing a basis for developing effective preventive measures and control strategies.

Fermented Astragalus in diet improved laying performance, egg quality, antioxidant and immunological status and intestinal microbiota in laying hens.

In the era of increased antibiotic resistance and ever-stricter control on antibiotic use, it is urgent to develop green, safe, and non-residue alternatives to antibiotics applied to the poultry industry. To this end, we supplied the potential Lactobacillus plantarum (L. plantarum) fermented Astragalus in the diet of laying hens, with a final addition of 3‰. Its effects have been assessed on laying performance, egg quality, antioxidant and immunological status, and intestinal microbiota, and are compared to the control group, to the Astragalus group containing 3‰ unfermented Astragalus, and to the L. plantarum group containing 2% L. plantarum [5 × 108 colony-forming unit (CFU) per milliliter (mL)]. During the second half of the experimental period (15 to 28 days), the egg production rate was considerably higher in the fermented Astragalus group than that in the other groups, with the fermented Astragalus group having the lowest feed conversion ratio. No significant difference (P > 0.05) was noted among treatments on egg quality. Fermented Astragalus-treated hens exhibited significantly increased catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in serum, and reduced malondialdehyde (MDA) in serum. Furthermore, fermented Astragalus supplementation resulted in a significant increase in ileal microbiota abundance relative to control. In conclusion, feeding laying hens with L. plantarum fermented Astragalus has beneficial effects on production, antioxidant potential, immunity, and ileal microbiota. L. plantarum fermented Astragalus is expected to be a novel feed additive used in poultry production.

Development of an improved dual-promoter-based reverse genetics system for emerging Senecavirus A.

Senecavirus A (SVA), a recently emerging picornavirus, poses a great threat to the swine industry because it causes swine idiopathic vesicular disease and epidemic transient neonatal losses. Thus far, the progress in SVA viral pathogenesis studies and vaccine development remains sluggish, and an available and convenient reverse genetics system would undoubtedly promote relevant research. Herein, we established an improved universal dual-promoter reverse genetics system with an SVA-specific hammerhead ribozyme and hepatitis delta virus ribozyme at both terminals of the viral genome; this system could be applied to rescue all SVA strains by both eukaryotic and prokaryotic RNA polymerase systems. The genome of the clone-derived Chinese field strain CH/HeN-2018 was assembled into the universal vector pcDNA-rSVAuni through the Gibson assembly technique. Moreover, two silent mutations, G6848C and C7163 G, were separately engineered into the full-length cDNA clone with one step site-directed mutagenesis to create a KpnI restriction enzyme site, which served as a unique genetic marker. The viruses, designated rCH/HeN-2018-T7, rCH/HeN-2018-CMV, rCH/HeN-2018-6484 m and rCH/HeN-2018-7163 m, were successfully rescued through both CMV- and T7-dependent pathways, and their biological properties were further evaluated. The results showed that all four viruses grew rapidly in PK-15 cells and exhibited viral titers and growth kinetics similar to those of parental wtCH/HeN-2018. The established reverse genetics system is easily operated and can be applied to rescue all SVA strains in a short time, which will be helpful for studying SVA biology, including viral pathogenesis, antiviral therapies and vaccine development.

Molecular characterization of variant infectious bronchitis virus in China, 2019: Implications for control programmes.

Infectious bronchitis virus (IBV), an ongoing emergence enveloped virus with a single-stranded positive-sense RNA genome, belongs to the Gammacoronavirus genus in the Coronaviridae family. IBV-associated tracheitis, nephritis, salpingitis, proventriculitis and egg drop have caused devastating economic losses to poultry industry worldwide. Since the end of 2018, a remarkably increasing number of commercial broilers and layers, vaccinated or not, were infected with IBV in China. Here, we described two IB outbreaks with severe respiratory system or kidney injury in IBV-vaccinated commercial poultry farms in central China. Other possible causative viral pathogens, including avian influenza virus (AIV), Newcastle disease virus (NDV) and Kedah fatal kidney syndrome virus (KFKSV), were excluded by reverse transcription-polymerase chain reaction (RT-PCR), and three virulent IBV strains, HeN-1/China/2019, HeN-2/China/2019 and HeN-101/China/2019, were identified. Although the gross pathologic appearance of these two IB outbreaks was different, the newly identified IBV strains were all closely related to the ck/China/I0529/17 strain and grouped into GI-19 genotype clade based on the sequencing and phylogenetic analysis of the complete S1 genes. Moreover, there are still some evolutionary distance between the newly identified IBV strains, HeN-101/China/2019 in particular, and other GI-19 strains, suggesting that Chinese IBV strains constantly emerge and evolve towards different directions. In conclusion, this study provided an insight of the recently emerging IBV outbreaks in IBV-vaccinated commercial poultry farms and identified the genetic characteristics of three virulent GI-19 IBV strains, which shows the need to carry out proper preventive measures and control strategies.

Effect of Lactobacillus Plantarum Supplementation on Production Performance and Fecal Microbial Composition in Laying Hens.

The present study was performed to investigate the effects of dietary supplementation with Lactobacillus plantarum (CGMCC1.557) on egg production and fecal microbiota composition in laying hens. Sixty Hy-Line Brown laying hens (18 weeks old) were randomly divided into two groups. The control group was fed a basal diet only, and the test group was fed basal diet supplemented with a final concentration of 1.0 × 109 CFU/mL during the 10-week experimental period. Egg production and fecal microbiota composition were both assessed in 28-week-old hens using high-throughput sequencing technology. The results showed that, compared with the control group, the test group exhibited increased laying and feed intake rates (p < 0.05). At the genus level, Lactobacillus was more abundant in the test group compared with the control group (p < 0.05). Conversely, Romboutsia was more abundant in the control group compared with the test group (p < 0.05). This study provides us with an insight into the potential use of L. plantarum as a food supplement in the laying hen industry. the study also provides us with a better understanding of the interplay between L. plantarum and the fecal microbiota of laying hens.

Fermented Astragalus in diet altered the composition of fecal microbiota in broiler chickens.

The composition and function of the intestinal microbiota play important roles in digestion and degradation of herbal medicines (HMs). However, few studies have examined the relationship between the fecal microbiota and HMs. In this study the effect of unfermented Astragalus (UA) and fermented Astragalus (FA) on growth performance, serum biochemical parameters, and fecal microbiota was evaluated in broiler chickens. In total, 180 one-day-old broiler chickens (Avian breeds) were randomly assigned to a control (C) group fed a basal diet, an unfermented (U) group fed a basal diet containing 0.5% UA, or a fermented (F) group fed a basal diet containing 0.5% FA, for 42 days. The F/G ratio was lower in F and U groups than in C group from 22 to 42 days (P < 0.05). Glutathione superoxide dismutase, antioxidant capacity, and total superoxide dismutase were higher, whereas malondialdehyde was lower in F group than in C and U groups from 1 to 21 days and from 22 to 42 days (P < 0.05). Fecal microbiota were profiled on an Illumina MiSeq platform following PCR amplification of the V4 region of the 16S rRNA gene. At the genus level Lactobacillus was the most abundant genus on day 7 in F group. Importantly, a potentially pathogenic genus, Enterococcus, was less abundant in the U and F groups than in the C group on day 35 (P < 0.05). These results indicate that dietary supplementation with 0.5% FA has beneficial effects on growth performance, serum biochemical parameters and fecal microbiota of broiler chickens.

Assessment of the physicochemical properties and bacterial composition of Lactobacillus plantarum and Enterococcus faecium-fermented Astragalus membranaceus using single molecule, real-time sequencing technology.

We investigated if fermentation with probiotic cultures could improve the production of health-promoting biological compounds in Astragalus membranaceus. We tested the probiotics Enterococcus faecium, Lactobacillus plantarum and Enterococcus faecium + Lactobacillus plantarum and applied PacBio single molecule, real-time sequencing technology (SMRT) to evaluate the quality of Astragalus fermentation. We found that the production rates of acetic acid, methylacetic acid, aethyl acetic acid and lactic acid using E. faecium + L. plantarum were 1866.24 mg/kg on day 15, 203.80 mg/kg on day 30, 996.04 mg/kg on day 15, and 3081.99 mg/kg on day 20, respectively. Other production rates were: polysaccharides, 9.43%, 8.51%, and 7.59% on day 10; saponins, 19.6912 mg/g, 21.6630 mg/g and 20.2084 mg/g on day 15; and flavonoids, 1.9032 mg/g, 2.0835 mg/g, and 1.7086 mg/g on day 20 using E. faecium, L. plantarum and E. faecium + L. plantarum, respectively. SMRT was used to analyze microbial composition, and we found that E. faecium and L. plantarum were the most prevalent species after fermentation for 3 days. E. faecium + L. plantarum gave more positive effects than single strains in the Astragalus solid state fermentation process. Our data demonstrated that the SMRT sequencing platform is applicable to quality assessment of Astragalus fermentation.

Astragalus affects fecal microbial composition of young hens as determined by 16S rRNA sequencing.

The gut microbiota play important roles in the degradation of chemical compounds of herbal medicines (HMs). However, little information regarding the interplay between HMs and the gut microbiota is available. Thus, the aim of this study was to investigate the composition of the fecal microbiota of young (age, 11 weeks) hens fed a conventional diet containing a crude Astragalus (0.5%) additive for 21 days (group A) vs. controls (group B) that were fed only conventional feed. The fecal contents of 14-week-old hens were collected for DNA extraction, and then the V3 and V4 hyper-variable regions of the 16S rRNA gene were amplified and analyzed using high-throughput sequencing technology. A distinctive difference in microbial diversity was observed between the two groups. The microbial composition of hens fed a diet supplemented with Astragalus was greater than that of the control group. At the genus level, Lactobacillus was more abundant in group A than group B (p < 0.05). Importantly, this study is the first to report the observation of a novel Romboutsia sp. in the feces of hens. However, Romboutsia was less abundant in group A than group B (17.94 vs. 33.98%, respectively, p < 0.05). The microbial community differed significantly between the two groups at the genus level, suggesting that Astragalus modulates the composition of the fecal microbiota. Based on these differences, these findings provide fresh insights into the application of Astragalus in the poultry industry, as well as a better understanding of the interplay between HMs and the gut microbiota.

Identification of neutralizing epitopes on the VP2 protein of infectious bursal disease virus by phage-displayed heptapeptide library screening and synthetic peptide mapping.

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease, which is one of the most important and widespread infectious diseases in commercial chickens. Conformational epitopes have been reported in the highly variable region of the VP2 protein of IBDV. In the present study, a random heptapeptide library was screened by using monoclonal antibodies (mAbs), YNW17 and YNW29, directed to the VP2 of IBDV and two peptide motifs, D-X-P-R and A-R-G, were identified. The motifs are present on the N and C terminal sequences of the highly variable region of VP2. Synthetic overlapping peptides covering the motifs on VP2 were analyzed by Dot- ELISA with the mAbs and two epitopes 197CDSSDRPRVYTIT209 and 329ARGSLAVTI337 identified. The above epitopes were also recognized by chicken anti-IBDV sera and shown to inhibit the binding of their mAbs to recombinant VP2. Both mAbs and sera from mice immunized with the conjugated epitope-peptides were able to neutralize serotype I IBDV. These results indicated that the epitopes are two neutralizing linear B-cell epitopes and would be useful for the development of peptide-based IBD vaccines.