This report describes a one-step real-time polymerase chain reaction assay based on SYBR Green I for detection of a broad range of duck circovirus (DuCV). Align with all DuCV complete genome sequences and other Genus Circovirus download from the GenBank (such as goose circovirus, pigeon circovirus), the primers targets to the replicate gene of DuCV were designed. The detection assay was linear in the range of 1.31 × 10²-1.31 × 10⁷ copies/μL. The reaction efficiency of the assay using the slope (the slope was -3.349) and the Y-intercept was 37.01 from the linear equation was estimated to be 0.99 and the correlation coefficient (R²) was 0.993. A series of experiments were carried out to assess the reproducibility, sensitivity, and specificity of the assay, following by the low intra-assay and inter-assay CVs for CT values obtained with the standard plasmids. The intra-assay CVs were equal or less than 1.89% and the inter-assay CVs were equal or less than 1.26%. There was no cross-reaction occurred with nucleic acids extracted from RA (Riemerella anatipestifer), E. coli (Escherichia coli), Duck Cholera (Pasteurella multocida), Avian influenza virus, avian paramyxovirus, Muscovy duck parvovirus, Duck reovirus, Duck hepatitis A virus as control templates. The nucleic acids extracted from samples of healthy ducks were used as negative controls. The assay was specific and reproducible. The established real time PCR was used to detect 45 DuCV-negative samples, which were tested using conventional PCR under the developed optimal conditions, each 15 for embryonated eggs, non-embryonated budgerigar eggs, newly hatched duck, the mixture of the lung, liver, spleen which were analysis for the presence of DuCV DNA, to conform that whether the DuCV can be transmitted vertically. Meanwhile, no positive result was shown by the real-time PCR method. The SYBR Green I-based quantitative PCR can therefore be practically used as an alternative diagnostic tool and a screening method for ducks infected with duck circovirus.

The viruses of the Parvoviridae family can infect both vertebrate and invertebrate animals. Recently, pigeon parvovirus (PiPV) was detected in the feces of wild urban pigeons. Owing to no specific detection platform for PiPV, studies on the epidemiology of PiPV are still a research gap. To achieve this goal, in this study, a TaqMan-based fluorescence quantitative PCR (TaqMan‒PCR) technique was established. The specific primers and probes used were designed on the basis of the NS gene characterization of PiPV downloaded from GenBank. After optimization, the established TaqMan‒PCR assay provides a sensitive, accurate, reliable and cost-effective platform for PiPV detection. We found that both YDPS and healthy birds can be found to have PiPV infection through field sample investigations, and we also investigated the presence of PiPV in Fujian, mainland China. Owing to the failure to propagate PiPV in embryos and cells, knowledge of PiPV replication mechanisms in birds still needs further study. In conclusion, a TaqMan-based fluorescence quantitative PCR method was developed, with the advantages of sensitivity, specificity, and reproducibility. This method can be used for further epidemiological monitoring of PiPV infection.

The rise of antibiotic-resistant bacterial infections necessitates alternative therapeutic strategies, such as phage therapy. This study investigates the potential of phage vB_PmuM_CFP3 (CFP3) as a therapeutic agent against avian cholera caused by Pasteurella multocida (P. multocida). Phage CFP3 was isolated from the feces and wastewater of a laying hen farm and underwent comprehensive biological characterization, including host range, lytic activity, and environmental stability. Transmission electron microscopy revealed CFP3′s typical myovirus morphology, with a head diameter of approximately 60 nm and a tail length of about 120 nm. CFP3 demonstrated high stability across a pH range of 4–10 and temperatures of 30–40 °C, making it suitable for oral administration in poultry. The phage exhibited a latent period of about 90 min and an optimal multiplicity of infection (MOI) of 1. Despite its narrow host range, with a lysis rate of 28.2% against avian-derived type A P. multocida, CFP3′s specificity minimizes impact on non-target bacteria. Whole-genome sequencing revealed a 32,696 bp linear double-stranded DNA genome with 46 predicted open reading frames (ORFs) and no tRNA or antibiotic resistance genes, enhancing its safety profile. Phylogenetic analysis indicated a close evolutionary relationship with Haemophilus phages HP1, HP2, and Pasteurella phage F108. While CFP3 shows promise as a precision therapeutic tool, further in vivo studies are required to evaluate its efficacy and safety. Future research should focus on expanding the phage library, optimizing phage mixtures, and exploring synergistic effects with other antimicrobial strategies. This study provides foundational data supporting the development of CFP3 as a viable alternative to antibiotics for controlling avian cholera.

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.