The continued evolution of H3 subtype avian influenza virus (AIV)—which crosses the interspecific barrier to infect humans—and the potential risk of genetic recombination with other subtypes pose serious threats to the poultry industry and human health. Therefore, rapid and accurate detection of H3 virus is highly important for preventing its spread. In this study, a method based on real-time reverse transcription recombinase-aided isothermal amplification (RT–RAA) was successfully developed for the rapid detection of H3 AIV. Specific primers and probes were designed to target the hemagglutinin (HA) gene of H3 AIV, ensuring highly specific detection of H3 AIV without cross-reactivity with other important avian respiratory viruses. The results showed that the detection limit of the RT–RAA fluorescence reading method was 224 copies/response within the 95% confidence interval, while the detection limit of the RT–RAA visualization method was 1527 copies/response within the same confidence interval. In addition, 68 clinical samples were examined and the results were compared with those of real-time quantitative PCR (RT–qPCR). The results showed that the real-time fluorescence RT–RAA and RT–qPCR results were completely consistent, and the kappa value reached 1, indicating excellent correlation. For visual detection, the sensitivity was 91.43%, the specificity was 100%, and the kappa value was 0.91, which also indicated good correlation. In addition, the amplified products of RT–RAA can be visualized with a portable blue light instrument, which enables rapid detection of H3 AIV even in resource-constrained environments. The H3 AIV RT-RAA rapid detection method established in this study can meet the requirements of basic laboratories and provide a valuable reference for the early diagnosis of H3 AIV.

The objective of this study was to examine the residue elimination patterns of seven antibiotics in the eggs of Taihang chickens under free-range conditions and develop suitable withdrawal times (WDTs). A total of 240 healthy Taihang chickens, aged 180 days, were randomly divided into eight groups of 30 birds each. The first seven groups were administered oxytetracycline, chlortetracycline, erythromycin, tylosin, tylvalosin, lincomycin, and tiamulin, respectively, in accordance with the maximum dosages and longest durations of treatment recommended by the Veterinary Pharmacopoeia of the People’s Republic of China. Group 8 served as the control group, and the test period continued until no drug residue could be detected. The results demonstrate that the residues of oxytetracycline, chlortetracycline, tylosin, tylvalosin, and tiamulin throughout the test period were below the maximum residue limits (MRLs) outlined in GB 31650-2019, “National food safety standard-Maximum residue limits for veterinary drugs in foods”. The egg yolk was identified as the target tissue for estimating the withdrawal time periods (WDTs) of these seven drugs. It was thus concluded that the recommended withdrawal time for tiamulin should be 0 days. However, the WDTs of oxytetracycline, chlortetracycline, tylosin, tylvalosin, were 2.8, 0.3, 2.4, and 7.4, respectively, when the upper limit of the 95% confidence interval was found to be lower than the MRLs. It was thus determined that the recommended WDTs for oxytetracycline, chlortetracycline, tylosin, and tylvalosin should be 3, 1, 3, and 8 days, respectively. In contrast, erythromycin and lincomycin exhibited concentrations below the permitted MRLs on days 7 and 8, respectively. However, the upper limits of the 95% confidence intervals for erythromycin and lincomycin were found to be below the MRLs when the WDTs were 11 and 8.9, respectively. This suggests that the recommended WDTs for these two antibiotics should be 11 days and 9 days.

Chicken respiratory diseases represent multifactorial conditions resulting from viral, bacterial, mycoplasmal pathogens, and environmental factors, causing significant economic losses within the poultry industry. A specific respiratory disease characterized by breathing difficulties and bronchial occlusion due to caseous exudates is termed chicken bronchial obstruction. However, the absence of rapid, precise, and highly sensitive diagnostic methods for differentiation of primary respiratory disease pathogens or opportunistic pathogens, including avian influenza virus (AIV), infectious bronchitis virus (IBV), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli), constitutes a substantial challenge. This study developed a quadruplex droplet digital polymerase chain reaction (ddPCR) method that targeted the HA gene of H9 subtype AIV, the M gene of IBV, the Pal gene of P. aeruginosa, and the UidA gene of E. coli. Following the optimization of annealing temperature, sensitivity, and repeatability, the minimum detectable concentrations were determined as 3.02 copies/μL for the HA gene of H9 subtype AIV, 3.08 copies/μL for the M gene of IBV, 3.19 copies/μL for the Pal gene of P. aeruginosa, 3.39 copies/μL for the UidA gene of E. coli. No cross-reactivity was observed with Newcastle disease virus (NDV), H5 subtype AIV, H7 subtype AIV, fowl adenovirus serotype 4 (FAdV-4), infectious laryngotracheitis virus (ILTV), Avibacterium paragallinarum, Streptococcus, Salmonella, Pasteurella multocida, and Staphylococcus aureus. The method demonstrated excellent repeatability, with a coefficient of variation (CV) below 9%. The 185 clinical samples collected in Hebei Province China are tested by both quadruplex ddPCR and quadruplex qPCR method and the results compared. The sensitivity of the quadruplex ddPCR method (57.30%; 106/185) slightly exceeded that of the quadruplex qPCR method (49.73%; 92/185). Pathogens or opportunistic pathogens positive rates obtained via the quadruplex ddPCR were 40.00% for H9 subtype AIV, 33.51% for IBV, 24.32% for P. aeruginosa, and 27.57% for E. coli. In comparison, the positive rates of H9 subtypes AIV, IBV, P. aeruginosa, and E. coli from the quadruplex qPCR were 36.22%, 30.81%, 21.62%, and 24.32%, respectively. The coincidence rates between the two methods were 96.22% for H9 AIV, 97.30% for IBV, 97.30% for P. aeruginosa, and 96.76% for E. coli. These results demonstrated that the quadruplex ddPCR method represented a highly sensitive, specific, and rapid technique for identifying H9 subtype AIV, IBV, P. aeruginosa, and E. coli.