Chunhe Wan

Avian metapneumovirus (aMPV) is the main etiological agent of turkey rhinotracheitis, causing respiratory distress and economic losses in turkeys. While its impact in broilers and laying hens is relatively limited, aMPV subtype C (aMPV/C) is an important pathogen in ducks and waterfowl, leading to respiratory distress and severe egg-drop syndrome. To overcome the limitations of current diagnostics, such as heavy reliance on laboratory infrastructure and prolonged turnaround times, we established a rapid detection assay integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a and a lateral flow dipstick (LFD), targeting the conserved N gene of aMPV/C. Reaction parameters were systematically optimized, with 30 nmol/L Cas12a and 40 nmol/L crRNA identified as the optimal concentrations. Rigorous specificity evaluation against seven common avian pathogens revealed no cross-reactivity. The limit of detection (LOD) was determined to be as low as 1.3 × 10¹ copies/μL, outperforming conventional PCR. Validation using clinical samples demonstrated a 98.85% concordance rate with quantitative PCR (qPCR). This platform enables visual detection of aMPV/C within 1 h with minimal equipment requirements. Combining high sensitivity, absolute specificity, and operational simplicity, this assay shows promise as a practical tool for point-of-care diagnostics and epidemiological surveillance of aMPV/C.

Avian metapneumovirus subtype C (aMPV/C) has re-emerged as a widespread pathogen in China, causing hydrosalpinx fluid syndrome (HFS) in laying ducks and resulting in substantial economic losses, highlighting the urgent need for a reliable immunological detection method. In the present study, a non-invasive indirect enzyme-linked immunosorbent assay (iELISA) was developed and optimized for detecting aMPV/C-specific immunoglobulin Y (IgY) in egg yolks of laying ducks, using purified novel aMPV/C as the coating antigen. Checkerboard titration established the optimal conditions as an antigen concentration of 1.25 μg/mL and an IgY dilution of 1:200. The cut-off value, determined by receiver operating characteristic curve analysis, was 0.3268, enabling a detection limit of 1:3200 for IgY antibodies. The assay showed no cross-reactivity with IgY positive for other duck pathogens, and both intra-assay and inter-assay coefficients of variation were less than 10%, demonstrating good specificity and reproducibility. Clinical evaluation of 151 duck egg yolk samples yielded an overall aMPV/C-specific IgY positivity rate of 58.3% (88/151). The concordance rate between the iELISA and an indirect immunofluorescence assay (IFA) was 97.8% (45/46). These results indicate that the established iELISA for detecting aMPV/C-specific IgY is a reliable and practical tool for large-scale detection of aMPV/C exposure in clinical duck egg yolk samples.

The increasing serotypic diversity and multidrug resistance of Riemerella anatipestifer pose a serious threat to the poultry industry, necessitating novel antimicrobial agents. Although phage-derived endolysins offer rapid and specific bactericidal activity with low resistance potential, their efficacy against this Gram-negative pathogen is severely constrained by the outer membrane barrier, driving the need for strategies to enhance endolysin penetration. In this study, the native endolysin NA of phages vB_RanS_CRP2 was verified to lack antibacterial activity. To overcome this barrier, we constructed three chimeric proteins—ALC001, ALC005, and ALC007—by fusing a receptor-binding protein, a cell-penetrating peptide, or a polycationic nonapeptide, respectively. All three chimeras exhibited dose-dependent antibacterial activity, with ALC005 demonstrating the best performance. ALC005 achieved a lytic rate of 72.2% against the tested Riemerella anatipestifer strains, remained stable at 0–40 °C and pH 6–9, and was shown by transmission electron microscopy to exert its bactericidal effect by disrupting the bacterial cell envelope and inducing cell lysis. Collectively, cell-penetrating peptide fusion is a reliable and effective strategy to potentiate endolysin activity against Riemerella anatipestifer.

Waterfowl circovirus (WFCV) is an important member of the genus Circovirus within the family Circoviridae, encompassing duck circovirus (DuCV) and goose circovirus (GoCV). As a pathogen that poses a serious threat to the global waterfowl industry, WFCV induces significant immunosuppression in its hosts, leading to growth retardation, feather abnormalities, and increased susceptibility to other pathogens. Consequently, it results in markedly elevated mortality rates among affected birds and causes substantial economic losses. In recent years, with the advancement of molecular biology techniques, notable progress has been made in research on waterfowl circoviruses. This review aims to systematically consolidate current findings on the genomic characteristics, pathogenic mechanisms, and clinical manifestations of WFCV while also summarizing the existing detection technologies and the status of vaccine development. Future research directions and prevention strategies are proposed, with the goal of providing a scientific reference for the effective control of this disease.

Duck adenovirus 3 (DAdV-3) was firstly identified in Muscovy ducklings exhibiting slight pericardial effusion, swelling, and hemorrhaging in the kidneys, which is a poorly characterized duck virus. This study established a CRISPR/Cas12a coupled with a Lateral flow dipstick (RAA-CRISPR/Cas12a-LFD) detection platform for DAdV-3. The core mechanism of this platform involves a three-step cascade reaction pathway. First, the target sequence of the DAdV-3100 k gene is amplified via RAA isothermal amplification. Second, the amplified products activate the trans-cleavage activity of Cas12a. Finally, signal output is achieved by cleaving probes on the Lateral flow dipstick. This method exhibits high sensitivity, with a minimum detection limit of 2.87 × 10 ² copies/μL for the DAdV-3 positive plasmid. It also demonstrates high specificity for DAdV-3, showing no cross-reactivity with other common duck-origin viruses such as avian influenza virus (AIV), Newcastle disease virus (NDV), duck Tembusu virus (DTMUV), duck plague virus (DPV), Muscovy duck parvovirus (MDPV), duck hepatitis A virus type 1 (DHAV-1), and duck reovirus (DRV). The method enables on-site detection of DAdV-3 within 1 h and 20 min. It does not rely on standardized molecular laboratories or professional operation, provides visualized detection results, allows rapid detection at the grassroots level, and has low detection costs, making it suitable for promotion in small-scale farms. This approach provides a technical means for the detection and epidemiological investigation of DAdV-3.

Riemerella anatipestifer (RA) is the primary causative agent of infectious serositis in ducks, causing significant economic losses. In this study, a rapid and visual loop-mediated isothermal amplification (LAMP) assay targeting the conserved region of the ompA gene was developed. Specific primers and a FAM-labeled probe were designed, and amplification products were visualized using phenol red-based colorimetric detection and a lateral flow dipstick (LFD) system. Among the five candidate primer sets, primer set 2 was selected because it showed the highest amplification efficiency and specificity, with no cross-reactivity detected against 12 common waterfowl pathogens. Under optimal conditions, the phenol red-based LAMP assay yielded visible results after incubation at 65 °C for 30 min, while the LAMP-LFD assay required an additional 3~5 min probe hybridization step, with detection limits of 7.76 × 10² copies/μL for the phenol red-based method and 7.76 × 10⁰ copies/μL for the LAMP-LFD method. Thirty clinical samples suspected of RA infection were analyzed using conventional PCR and the developed visual LAMP assays. The positive detection rates obtained with the LAMP-LFD and phenol red-based LAMP methods were 63.3% and 60%, respectively, showing high concordance with conventional PCR (56.7%). In conclusion, the LAMP assay integrating phenol red visualization and lateral flow dipstick detection is rapid, sensitive, and easy to perform, and both detection formats show potential for point-of-care or on-site applications, and can be used for the early diagnosis and detection of RA.

C. pseudotuberculosis is a Gram-positive pathogenic bacterium that infects various animals, causing diseases such as caseous lymphadenitis, leading to significant economic losses in the livestock industry and posing zoonotic risks. This study targeted the conserved gyrA gene fragment of this bacterium, designed specific primers, optimized the reaction system and conditions, and established a high-resolution melting curve (HRM) detection method with potential utility for preliminary molecular screening. Validation showed that this method exhibits strong specificity, producing specific amplification only for the target biovars. The melting temperatures (Tm values) for the two biovars were 86.16 ± 0.05 °C and 86.92 ± 0.05 °C, respectively, allowing clear differentiation. It demonstrated high sensitivity, with minimum detection limits of 28 copies/μL and 25 copies/μL for standard plasmids of the ovis and equi biovars, respectively. The method also showed good reproducibility, with intra- and inter-batch coefficients of variation both below 1.0%. Applied to 133 clinical nasal swab samples from goats in Fujian Province, the method detected a positivity rate of 19.5% and indicates that a biovar equi-like gene fragment was detected in goat nasal swabs from Fujian Province via molecular screening. The HRM method developed in this study is sensitive, specific, simple, and cost-effective, enabling rapid detection and biovar differentiation of C. pseudotuberculosis. It is suitable for large-scale clinical sample screening and provides an efficient technical approach for epidemiological monitoring and precise control of the disease.

In recent years, pigeon rotavirus A (PiRVA) infection, an important emerging disease, has posed a major threat to the healthy development of the pigeon industry and public health. Therefore, developing an accurate, rapid and convenient detection method for this virus is vital for monitoring and early diagnosis of the disease. In this study, on the basis of the ORF sequence characteristics of the PiRVA VP6 gene, crRNA and reverse transcription recombinase-aided amplification (RT-RAA) primers were designed. On the basis of the CRISPR/Cas12a system, for the first time, the RT-RAA-CRISPR/Cas12a rapid detection method of PiRVA was established by combining RT-RAA and lateral flow strips. This method could specifically detect PiRVA, and there was no cross-reaction with other common viruses originating from pigeons. The minimum detection limit was 16.8 copies/μL, and the results of the intrabatch and interbatch repeated tests were consistent. Moreover, the method established in this study and the previously established common PCR method were used to analyse 56 clinical tissue samples from racing pigeons and domestic pigeons collected in 2025. The positive rates of racing pigeon and domestic pigeon samples detected by PCR were 17.6% and 12.8%, respectively, and the positive rates of racing pigeon and meat pigeon samples detected by the RT-RAA-CRISPR/Cas12a method were 23.5% and 17.9%, respectively, indicating that PiRVA infection occurs in both racing pigeon and domestic pigeon populations in China. In summary, the PiRVA RT-RAA-CRISPR/Cas12a detection method established in this study has good specificity, sensitivity, and reproducibility, and allows visualization of the results, which can be used for field applications. This study provides technical support for epidemiological surveillance and etiological research on PiRVA.

Goose circovirus (GoCV) has been found in waterfowl more than 20 years, causing considerable economic loss worldwide. In the present study, goose circovirus (designated DuGoCV) was identified in Muscovy ducks in China, with complete genomes were sequenced and analyzed. The genetic evolutionary characteristics and recombination patterns of different genotypes were examined. The DuGoCV strain shared genome sequence identities of 89.2–95.9% with the GoCV reference strains and 67.0–67.1% with the DuCV reference strains. Phylogenetic analysis revealed that the DuGoCV strain belongs to GoCV-III. These findings suggest possible cross-species transmission of GoCV between geese and Muscovy ducks, providing important insights for the prevention and control of GoCV infection.

Caseous lymphadenitis (CLA), an infectious disease caused by Corynebacterium pseudotuberculosis (C. pseudotuberculosis), poses a significant economic burden to the global small ruminant industry. This study aimed to investigate genetic variations in the CP40 gene of C. pseudotuberculosis and to develop a rapid detection assay for enhanced pathogen identification. Homology analysis was performed to compare the CP40 gene sequence of the FJ-PN strain with other Corynebacterium species. Specific primers targeting CP40 were designed, and a SYBR Green I-based real-time PCR protocol was optimized. The assay’s specificity, sensitivity, and reproducibility were subsequently validated. The FJ-PN strain exhibited ≥99.65% nucleotide identity and ≥98.94% amino acid identity with C. pseudotuberculosis biovar ovi reference strains, showing 90.18–91.84% nucleotide identity and 88.63–90.77% amino acid identity with C. pseudotuberculosis biovar equi, and ≤82.71% nucleotide identity and ≤78.63% amino acid identity with other Corynebacterium species. The established qPCR assay demonstrated high specificity, the limit of detection was 52 copies/μL, and it demonstrated good reproducibility (intra- and inter-assay CV < 1.0%). Clinical sample testing revealed 18.8% positivity rates in nasal swabs, which was higher than that detected by conventional PCR (16.3%). These results indicate that the CP40 gene is evolutionarily conserved and represents a specific molecular marker for the identification of C. pseudotuberculosis biovar ovis. The developed SYBR Green I real-time PCR assay enables the efficient detection of C. pseudotuberculosis and provides technical support for CLA surveillance and control.