Characterization of natural co-infection with goose astrovirus genotypes I and II in gout affected goslings.

RESEARCH HIGHLIGHTS: Urate tophi were found in the kidneys, liver, spleen and lungs.IFA confirmed the co-expression of GoAstV-I and II antigens in the same kidney.

LAMP assay coupled with a CRISPR/Cas12a system for the rapid and ultrasensitive detection of porcine circovirus-like virus in the field.

A recent outbreak of porcine circovirus-like virus (PCLV), a virus that may be associated with porcine diarrhea, has been reported in swine herds in China. The virus is spreading rapidly, causing huge economic losses to the swine farming industry. To achieve the rapid, inexpensive, and sensitive detection of PCLV, we combined loop-mediated isothermal amplification (LAMP) and the CRISPR/Cas12a system, whose fluorescence intensity readout can detect PCLV ORF4 gene levels as low as 10 copies. To overcome the need for sophisticated equipment, lateral flow strip reading technology was introduced for the first time in a LAMP-Cas12a-based system to detect PCLV. The lateral flow strip (LFS) results were readout by the naked eye, and the method was highly sensitive with a detection limit of 10 copies, with a detection time of about 60 min. In addition, the method is highly specific and has no cross-reactivity with other related viruses. In conclusion, LAMP-CRISPR/Cas12a-based assays have the advantages of rapidity, accuracy, portability, low cost, and visualization of the results. They therefore have great potential, especially for areas where specialized equipment is lacking, and can expect to be an ideal method for early diagnosis and on-site detection of PCLV.

EspE3 plays a role in the pathogenicity of avian pathogenic Escherichia coli.

APEC encodes multiple virulence factors that have complex pathogenic mechanisms. In this study, we report a virulence factor named EspE3, which can be secreted from APEC. This protein was predicted to have a leucine-rich repeat domain (LRR) and may have a similar function to IpaH class effectors of the type III secretion system (T3SS). For further exploration, the regulatory correlation between the espE3 and ETT2 genes in APEC was analysed. We then assessed the pathogenicity of EspE3, detected it in APEC secretion proteins and screened the proteins of EspE3 that interact with chicken trachea epithelial cells. This study provides data on a new virulence factor for further exploring the pathogenic mechanism of APEC.

Visual detection of fowl adenovirus serotype 4 via a portable CRISPR/Cas13a-based lateral flow assay.

The widespread occurrence of fowl adenovirus serotype 4 (FAdV-4)-induced hepatitis-hydropericardium syndrome (HHS) has led to significant economic losses for the poultry industry. A sensitive, accurate, and practical FAdV-4 diagnostic approach is urgently required to limit the incidence of the disease. In the present study, a practical method for detecting FAdV-4 was developed using the CRISPR/Cas13a system and recombinase-aided amplification. The approach was based on 37°C isothermal detection with visible results being achieved. The detection limit of the target gene with this approach was only 101 copies/µl, making it very sensitive and specific. Clinical samples fared well when tested with the Cas13a detection method. For identifying FAdV-4, this novel detection approach was found to be sensitive, specific, and effective.RESEARCH HIGHLIGHTS First study using the CRISPR/Cas13a-based lateral flow detection assay for FAdV-4 detection.The results can be observed by the naked eye.The developed assay could provide an alternative tool for detection of FAdV-4 with minimal equipment.

YqeH contributes to avian pathogenic Escherichia coli pathogenicity by regulating motility, biofilm formation, and virulence.

Avian pathogenic Escherichia coli (APEC) is a pathotype of extraintestinal pathogenic E. coli and one of the most serious infectious diseases of poultry. It not only causes great economic losses to the poultry industry, but also poses a serious threat to public health worldwide. Here, we examined the role of YqeH, a transcriptional regulator located at E. coli type III secretion system 2 (ETT2), in APEC pathogenesis. To investigate the effects of YqeH on APEC phenotype and virulence, we constructed a yqeH deletion mutant (APEC40-ΔyqeH) and a complemented strain (APEC40-CΔyqeH) of APEC40. Compared with the wild type (WT), the motility and biofilm formation of APEC40-ΔyqeH were significantly reduced. The yqeH mutant was highly attenuated in a chick infection model compared with WT, and showed severe defects in its adherence to and invasion of chicken embryo fibroblast DF-1 cells. However, the mechanisms underlying these phenomena were unclear. Therefore, we analyzed the transcriptional effects of the yqeH deletion to clarify the regulatory mechanisms of YqeH, and the role of YqeH in APEC virulence. The deletion of yqeH downregulated the transcript levels of several flagellum-, biofilm-, and virulence-related genes. Our results demonstrate that YqeH is involved in APEC pathogenesis, and the reduced virulence of APEC40-ΔyqeH may be related to its reduced motility and biofilm formation.

Serum resistance factors in avian pathogenic Escherichia coli mediated by ETT2 gene cluster.

Serum resistance is a poorly understood but common trait of some systemic disease pathogenic strains of bacteria. In this study, we analysed the role Escherichia coli type III secretion system 2 (ETT2) of avian pathogenic Escherichia coli (APEC) in serum resistance by bacteria survival number in serum culture, mRNA Seq and Tandem Mass Tag™ (TMT™) detection, lipopolysaccharide (LPS) extraction, and biofilm formation detection. We found that the ETT2 gene cluster deletion strain (ΔETT2) is more resistant to the killing effect of serum than wild-type APEC40. The analysis of ΔETT2 compared to APEC40 in the transcriptomics and proteomics data showed that ETT2 has a negative effect in the ATP-binding cassette (ABC) translator system and quorum sensing system and a positive effect in purine metabolism. ETT2 may affect the LPS, biofilm, flagella, and fimbriae which may affect the serum resistance. These results could lead to effective strategies for managing the infection by APEC. The mRNA Seq data of this study are available in the Sequence Read Archive of the National Center for Biotechnology Information under the BioProject PRJNA757182, and proteomic raw data have been deposited under the accession number IPX0003420000 at iProX.

Hcp2b of T6SS affects colonization of avian pathogenic Escherichia coli and host keratin filament expression.

T6SS (type VI secretion system) is a type of nano-syringe that exists in APEC (avian pathogenic Escherichia coli). Hcp (haemolysin-coregulated protein) of T6SS participates in the regulation of virulence during APEC infection. However, whether hcp plays a role in bacterial colonization by expression in host cells remains unclear. In this study, we analysed the biological characteristics of the mutant hcp2b strain. Our results showed that the hcp2b gene was involved in the regulation of bacterial motility, biofilm formation, anti-serum and anti-oxidative stress. Moreover, our data indicate that the colonization of the hcp2b mutation strain (Δhcp2b) in the lung, liver and kidney of chickens decreased significantly. Hence, overexpression of Hcp2b protein in DF-1 cells was used to analyse the effect of Hcp2b on colonization of APEC. Proteomics analysis showed that overexpression of Hcp2b induced differentially expressed proteins in DF-1 cells (230 were significantly upregulated and 96 were significantly downregulated) and differentially expressed proteins were enriched in keratin filament. In conclusion, our data indicated that hcp2b promoted the colonization of APEC by affecting the expression of keratin filament.

Effect of nutritional and environmental conditions on biofilm formation of avian pathogenic Escherichia coli.

AIMS: To study the effects of environmental stress and nutrient conditions on biofilm formation of avian pathogenic Escherichia coli (APEC). METHODS AND RESULTS: The APEC strain DE17 was used to study biofilm formation under various conditions of environmental stress (including different temperatures, pH, metal ions, and antibiotics) and nutrient conditions (Luria-Bertani [LB] and M9 media, with the addition of different carbohydrates, if necessary). The DE17 biofilm formation ability was strongest at 25°C in LB medium. Compared to incubation at 37°C, three biofilm-related genes (csgD, dgcC, and pfs) were significantly upregulated and two genes (flhC and flhD) were downregulated at 25°C, which resulted in decreased motility. However, biofilm formation was strongest in M9 medium supplemented with glucose at 37°C, and the number of live bacteria was the highest as determined by confocal laser scanning microscopy. The bacteria in the biofilm were surrounded by a thick extracellular matrix, and honeycomb-like or rough surfaces were observed by scanning electron microscopy. Moreover, biofilm formation of the DE17 strain was remarkably inhibited under acidic conditions, whereas neutral and alkaline conditions were more suitable for biofilm formation. Biofilm formation was also inhibited at specific concentrations of cations (Na+ , K+ , Ca2+ , and Mg2+ ) and antibiotics (ampicillin, chloramphenicol, kanamycin, and spectinomycin). The real-time quantitative reverse transcription PCR showed that the transcription levels of biofilm-related genes change under different environmental conditions. CONCLUSIONS: Nutritional and environmental factors played an important role in DE17 biofilm development. The transcription levels of biofilm-related genes changed under different environmental and nutrient conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings suggest that nutritional and environmental factors play an important role in APEC biofilm development. Depending on the different conditions involved in this study, it can serve as a guide to treating biofilm-related infections and to eliminating biofilms from the environment.

Hcp2a of APEC affects mRNA splicing and protein quality control in DF-1 cells.

BACKGROUND: Bacteria deliver effector proteins into the host cell via a secretory system that can directly act on the target to cause disease. As an important pipeline structural protein of the type VI secretion system (T6SS) complex, Hcp acts together with other virulence factors in the target cell. There is growing evidence that T6SS plays a key role in the pathogenic mechanism of APEC. However, the regulatory function played by the effector protein Hcp during its interaction with host cells is not clear. Here, tandem mass tag (TMT) analysis was used to quantify the proteins affected by increased expression of Hcp2a in DF-1 cells. RESULTS: The host response was significantly different between the overexpression and null groups at the protein level. A total of 195 differentially expressed proteins (DEPs) were detected in the overexpression group (upregulated, n = 144, downregulated, n = 51). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the biological functions and pathways of differentially expressed proteins. The results showed that these DEPs were mainly enriched in RNA degradation, spliceosome, and mRNA surveillance pathways. CONCLUSIONS: This study suggests that Hcp2a, the effector protein of APEC, plays an important role in regulating mRNA splicing and protein quality control in DF-1 cells. These findings provide useful clues to elucidate the pathogenic mechanism of effector protein Hcp2a on host target cells.

Identification of novel biofilm genes in avian pathogenic Escherichia coli by Tn5 transposon mutant library.

Avian pathogenic Escherichia coli (APEC) is the main pathogens that inflict the poultry industry. Biofilm as the pathogenic factors of APEC, which can enhance the anti-host immune system of APEC and improve its survival in the environment. In order to screen for new genes related to APEC biofilm. The APEC strain APEC81 was used to construct a mutant library by Tn5 insertion mutagenesis. Moreover the 28 mutant strains with severely weakened biofilm were successfully screened from 1500 mutant strains by crystal violet staining, in which 17 genes were obtained by high-efficiency thermal asymmetric interlaced PCR. The reported genes include 3 flagella genes (fliS, fliD, and fliR), 4 curli fimbriae genes (csgD, csgA, csgF, and csgG) and 3 type 1 fimbriae genes (fimA, fimD, and fimC). The novel genes include 3 coenzyme genes (gltA, bglX, and mltF) and 4 putative protein genes (yehE, 07045, 11735, 11255). To investigate whether these 17 genes co-regulate the biofilm, the 17 identified genes were deleted from APEC strain APEC81. The results showed that except for the 11735 and 11255 genes, the deletion of 15 genes significantly reduced the biofilm formation ability of APEC81 (P < 0.05). The result of rdar (red, dry and rough) colony morphology showed that curli fimbriae genes (csgD, csgA, csgF, and csgG) and other functional genes (fimC, glxK, yehE, 07045, and 11255) affected the colony morphology. In particular, the hypothetical protein YehE had the greatest influence on the biofilm. It was predicted to have the same structure as the type 1 fimbria protein. When yehE was deleted, the fimE transcription was up-regulated, and the fimA and fimB transcription were down-regulated, resulting in a decrease in type 1 fimbriae. Hence, the yehE mutant significantly reduced the biofilm and the adhesion and invasion ability to cells (P < 0.05). This study identified 5 novel genes (gltA, bglX, mltF, yehE, and 07045) related to biofilm formation and confirmed that yehE affects biofilm formation by type 1 fimbriae, which will benefit further study of the mechanism of biofilm regulation in APEC.

The ETT2 transcriptional regulator EivF affects the serum resistance and pathogenicity of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC), a pathotype of extraintestinal pathogenic Escherichia coli (ExPEC), can cause serious systemic infectious diseases in poultry. Escherichia coli type III secretion system 2 (ETT2) is widely distributed in E. coli strains, including ExPEC and Enterohemorrhagic Escherichia coli (EHEC). The transcriptional regulator EivF, which is located at the ETT2 cluster, affects the secretion of LEE-encoded proteins and increases bacterial adhesion to human intestinal epithelial cells in EHEC O157:H7. In a previous study, we demonstrated the transcriptional regulator can affect APEC's motility and biofilm formation. Here, we evaluated whether EivF is involved in the pathogenicity of APEC, and we found that inactivation of eivF significantly enhanced resistance to the serum, adherence to chicken embryo fibroblast (DF-1) cells, and the colonization ability of APEC in chicks. To further clarify the regulation mechanism of transcriptional regulator EivF, we performed transcriptome sequencing to analyze the differentially expressed genes and pathways, showing that EivF regulates membrane, adhesion, environmental stress, and secretion protein genes, and EivF is involved in the localization, biological adhesion, biological regulation, membrane, and toxin activity. These findings indicated that the ETT2 transcriptional regulator EivF plays a crucial role in the pathogenicity of APEC as a negative repressor.

Flagellar rotor protein FliG is involved in the virulence of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC), a type of extraintestinal pathogenic E. coli, causes avian colibacillosis, a disease of significant economic importance to poultry producers worldwide, which is characterized by systemic infection. However, the pathogenesis of avian pathogenic E. coli strains is not well defined. Here, the role of a flagellar rotor protein encoded by the fliG gene of avian pathogenic E. coli strain AE17 was investigated. To study the role of FliG in the pathogenicity of APEC, fliG mutant and complemented strains were constructed and characterized. The inactivation of fliG had no effect on APEC growth, but significantly reduced bacterial motility. Compared with the wild type, the fliG mutant was highly attenuated in a chick infection model and showed severe defects in its adherence to and invasion of chicken embryo fibroblast DF-1 cells. The fliG mutant also showed reduced resistance to serum in chicks. The expression of the inflammatory cytokines interleukin 1ß (IL1ß), IL6, and IL8 was reduced in HD-11 macrophages infected with the fliG mutant strain compared with their expression in the wild-type strain. These results demonstrate that the FliG contributes to the virulence of APEC.

Enzymatic recombinase amplification coupled with CRISPR-Cas12a for ultrasensitive, rapid, and specific Porcine circovirus 3 detection.

Porcine circovirus type 3 (PCV3) is a disease associated with porcine dermatitis and nephrotic syndrome (PDNS) that has caused significant economic losses to swine herds since its discovery in 2016. To develop a simple, on-site, rapid, and sensitive assay to combat the spread of PCV3, we optimized the CRISPR/Cas12a (also known as Cpf1) system combined with enzymatic recombinase amplification (ERA) nucleic acid amplification to diagnose PCV3. The results showed that the ERA-CRISPR/Cas12a reaction could detect PCV3 within 1 h in genomic DNA harboring a minimum of seven copies. Additionally, we confirmed no cross-reactivity with PCV2, PCV4, or other porcine viruses, revealing the good specificity of this technique. These results demonstrated the ability of ERA-CRISPR/Cas12a to detect DNA at the single-molecule level and provide a rapid, simple, ultrasensitive, one-pot point-of-care test for PCV3 and suggest its potential for a variety of nucleic acid detection applications.

Reverse transcription-enzymatic recombinase amplification coupled with CRISPR-Cas12a for rapid detection and differentiation of PEDV wild-type strains and attenuated vaccine strains.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute watery diarrhea and vomiting in unweaned piglets, and is associated with high mortality, thus causing severe economic losses in the pig industry. Currently, although attenuated vaccines are commonly used in commercial pig farms in China, they do not completely protect against all mutated wild-type strains. Existing nucleic acid assays have high sensitivity and specificity, but the complexity of the assay process and expensive instrumentation hinder disease detection. Here, reverse transcription-enzymatic recombinase amplification (RT-ERA) was combined with the CRISPR-Cas12a system to develop a rapid diagnostic method to distinguish PEDV wild-type strains from attenuated vaccine strains. The protocol used crRNA and RT-ERA amplification primers against open reading frame 3 (ORF3), followed by Cas12a/crRNA complex detection of predefined target sequences at 37 °C for 30 min, thus producing results visible to the naked eye under LED blue light. The assay is highly sensitive and specific, detecting as few as two copies of the target gene per test and showing no cross-reactivity with other porcine pathogens. Overall, this integrated RT-ERA pre-amplification and Cas12a/crRNA cleavage assay is a practical tool for reliable and rapid detection of PEDV for diagnostic differentiation.

Development of a SERS-based lateral flow immunoassay for rapid and ultra-sensitive detection of anti-SARS-CoV-2 IgM/IgG in clinical samples.

The accurate and rapid screening of serum antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the key to control the spread of 2019 coronavirus disease (COVID-19). In this study, we reported a surface-enhanced Raman scattering-based lateral flow immunoassay (SERS-LFIA) for the simultaneous detection of anti-SARS-CoV-2 IgM/IgG with high sensitivity. Novel SERS tags labeled with dual layers of Raman dye were fabricated by coating a complete Ag shell on SiO2 core (SiO2@Ag) and exhibited excellent SERS signals, good monodispersity, and high stability. Anti-human IgM and IgG were immobilized onto the two test lines of the strip to capture the formed SiO2@Ag-spike (S) protein-anti-SARS-CoV-2 IgM/IgG immunocomplexes. The SERS signal intensities of the IgM and IgG test zones were easily recorded by a portable Raman instrument and used for the high-sensitivity analysis of target IgM and IgG. The limit of detection of SERS-LFIA was 800 times higher than that of standard Au nanoparticle-based LFIA for target IgM and IgG. The SERS-LFIA biosensor was tested on 19 positive serum samples from COVID-19 patients and 49 negative serum samples from healthy people to demonstrate the clinical feasibility of our proposed assay. The results revealed that the proposed method exhibited high accuracy and specificity for patients with SARS-CoV-2 infection.

Effects of probiotics on cecal microbiome profile altered by duck Escherichia coli 17 infection in Cherry Valley ducks.

Avian colibacillosis is one of the most serious infectious bacterial diseases that endanger the modern poultry industry. Lactobacillus is believed to inhibit intestinal pathogens and maintain a healthy gut microbiota. This study aimed to investigate Lactobacillus supplementation in Cherry Valley ducks to prevent the intestinal flora dysbiosis caused by Duck Escherichia coli 17. One hundred and twenty healthy one day old Cherry Valley ducks were randomized to three study groups (Group I = the control group; Group II = duck Escherichia coli 17 challenge group and Group III = DE17 challenge group supplemented with lactic acid bacteria composite preparation). Cherry Valley ducks in Group II and Group III were gavage challenged with DE17 (1 × 105 CFU/mL) on day 14. Pyrosequencing of the V3/V4 variable regions of the genes encoding for 16S rRNA was used for sequence analysis. The results showed that the normal intestinal microecology was affected by DE17, including a relative increase in proteobacteria. At the same time, the Lactobacillales were increased and harmful bacteria were decreased in different intestinal segments of ducks in Group III, compared to those in Group II. Network analysis showed that dietary lactic acid bacteria addition improved the interaction pattern within the cecal microbiota of ducks and the result showed that in Ruminococcus_2 was independently present in the group III and Lachnospiraceae_NK4A136_group species correlation existed between group I and group III. This study proved that oral supplementation with Lactobacillus casei 1.2435, Lactobacillus rhamnosus 621 and Lactobacillus rhamnosus A4 can mitigate DE17 induced intestinal flora dysbiosis.

Molecular characterization and pathogenicity of highly pathogenic fowl adenovirus serotype 4 isolated from laying flock with hydropericardium-hepatitis syndrome.

Hydropericardium-hepatitis syndrome (HHS) is an important emerging disease responsible for huge economic losses to the poultry industry in China. HHS primarily affects 20 to 60-day-old broilers and rarely occurs in laying flock. In this study, the highly pathogenic fowl adenovirus (FAdV) strain, AH-F19, was isolated from the liver samples of 120-day-old laying flock with HHS and its phylogenetic information, genetic mutations, and pathogenicity was evaluated. The phylogenetic analysis revealed that AH-F19 belonged to the FAdV serotype 4 (FAdV-4) cluster, however, 100K differs from the other FAdV-4 strains and is divided into different branches. Amino acid variations in fiber-2 for pathogenic isolates and non-pathogenic isolates indicated that D219, T300, and T380 may not be responsible for virulence. Animal experiments revealed AH-F19 to be a highly pathogenic isolate that can cause 100% mortality in three-week-old specific pathogen-free (SPF) chickens, which exhibited typical hydropericardium and hepatitis. Microscopically, the presence of basophilic intranuclear inclusion bodies in hepatocytes, fractured heart muscle fibers, as well as kidney degeneration and necrosis was observed. Collectively, these findings enriched our understanding of FAdV-4 pathogenicity and provided a reference for further exploration into its pathogenicity.

Adaptation to host-specific bacterial pathogens drive rapid evolution of novel PhoP/PhoQ regulation pathway modulating the virulence.

The presence of the PhoP-PhoQ system is usually different in various bacterial groups, suggesting that PhoP can control the expression of different genes in species. However, little is known about the evolution of the PhoP-PhoQ system among bacterial pathogens. Here, we study the evolution of PhoP and PhoQ regulation in 15 species of Enterobacteriaceae family. We have determined that the regulatory objectives adopted by PhoP and PhoQ are mainly different, due to the result of horizontal gene transfer events and even the change in the genetic content between closely related species. We have compared many possibilities tests (M1 vs. M2 and M7 with M8) to determine the positive selection. Estimating parameters at M1 and M2, with positive selection in M2 of the two proteins. The proportions of positive selection sites significant with ω = 4.53076 for PhoP and ω = 4.21041 PhQ. M8 was significant for PhoP and PhQ proteins. To further confirm the positive selection results, we used the Selecton server to confer positive selection on individual sites using the Mechanistic-Empirical Combination model, and we noticed that several sites had been identified under selection pressure during the evolution. There was a strong indication for the positive selection in bacterial genes of PhoP and PhoQ showed the results. By the use of REL and IFEL, the positive selection for PhoP was detected 14 and 11 sites respectively at different codon positions. The positively selected sites of amino acids such as Arginine, Alanine, Lysine, and Leucine are more important for the production of signals. Our results suggest that the positive selection of PhoP-PhoQ genes in host adaptation during evolution raises an intriguing possibility causes subtle variations in actions of PhoP-PhoQ and also increases the opportunities that cause modification in protein structure for the evolution of increasing pathogenicity in bacterial pathogens.

Simultaneous differentiation and diagnosis of goose parvovirus and astrovirus in clinical samples with duplex SYBR Green I real-time PCR.

Two pairs of primers were designed to bind conserved genomic regions of goose parvovirus (GPV) and goose astrovirus (GAstV) to establish a simple, sensitive, and highly specific duplex quantitative PCR (qPCR) method to simultaneously detect the two viruses. The duplex qPCR can distinguish GPV (melting point: 82.1 °C) and GAstV (melting point: 79.8 °C) by the peaks of their individual melting curves. Mixed testing with other waterfowl viruses produced no nonspecific peaks. The established standard curves showed good linear relationships (R2 > 0.997) and the limits of detection (LOD) for GPV and GAstV were 5.74 × 101 and 6.58 × 101 copies/µL, respectively. Both intra- and inter-assay coefficients of variation were <2%, indicating that the method has good repeatability. Twenty tissue samples from diseased geese were examined with the duplex qPCR assay and conventional PCR. Duplex qPCR showed positive rates of 25% for GPV and 45% for GAstV, and the positive rate for GPV and GAstV coinfection was 15%, slightly higher than the results for conventional PCR. These results indicated that this duplex qPCR method is highly sensitive, specific, and reproducible, and is suitable for epidemiological studies to effectively control the transmission of GPV and GAstV.

A TaqMan-based quantitative real-time PCR assay for identification of the goose circovirus.

Goose circovirus (GoCV) is a potential immunosuppressive virus that poses a great hazard to the goose industry and has been shown to be widely distributed throughout China. We have established a fast, sensitive and highly specific TaqMan real-time quantitative PCR detection method for this virus. Specific primers and probes were designed against the conserved regions of the genomic GoCV Rep gene. The results showed that the assay was highly specific and sensitive for GoCV and did not cross-react with other non-targeted waterfowl viruses. The established method will be helpful for epidemiological detection and may be effective in the prevention and control of the disease.