Nairobi sheep disease virus (NSDV) is a typical tick-borne virus belonging to the genus Orthonairovirus in the family Nairoviridae, which causes Nairobi sheep disease (NSD) with mortality rates exceeding 70% in small ruminants such as sheep and goats. This pathogen has also been associated with human infections, highlighting its zoonotic potential. In this study, we developed a TaqMan-based reverse-transcription quantitative PCR (RT-qPCR) assay for NSDV detection in China. The assay indicated high analytical sensitivity; the limit of detection of 10 copies/µL was determined using recombinant plasmid DNA, representing the performance of the qPCR amplification step only, with a standard curve showing excellent linearity (R² = 1.000). The assay exhibited good analytical specificity for NSDV, with no cross-amplification of other related pathogens. Intra-assay and inter-assay coefficients of variation for recombinant plasmid standards were consistently <1%, indicating high reproducibility. We then applied this RT-qPCR assay to quantify viral loads in multiple tissues from sheep experimentally infected with NSDV. Detectable NSDV nucleic acid was found in the heart, liver, spleen, lung, kidney, duodenum, jejunum, rectum, cecum, ileum, and lymph nodes at 3 and 7 days post-infection (dpi). Descriptive analysis showed higher NSDV RNA loads in the spleen and lung relative to other tissues at both 3 dpi and 7 dpi. The maximum RNA load of 10^4.428 copies/mg was detected in the spleen at 3 dpi, indicating that the spleen and lung represent the tissues with the highest NSDV RNA accumulation. Meanwhile, this assay was applied to conduct an epidemiological survey for NSDV nucleic acid in sheep and ticks across selected areas of Heilongjiang Province, China. The NSDV RNA detection proportions were 3.46% (33/955) in sheep and 2.88% (20/694) in ticks, respectively. Phylogenetic analysis revealed that the tick-derived NSDV sequence, designated NSDV/HLJ-1/tick/China/2023, forms a distinct lineage within the Chinese NSDV clade and shares relatively high nucleotide homology with the NSDV SZWH2 strain from China. In conclusion, this accurate and sensitive RT-qPCR assay provides a robust tool for laboratory-based NSDV detection and epidemiological surveillance.

Duck hepatitis B virus (DHBV), Duck adenovirus 3 (DAdV-3) and Streptococcus gallolyticus subsp. pasteurianus (SGSP) are three distinct pathogens associated with infectious diseases in waterfowl. Clinical differentiation among these agents is often difficult due to overlapping signs, underscoring the need for accurate, rapid, and simultaneous diagnostic tools to support early detection and effective disease surveillance. In this study, we developed and validated a triplex TaqMan real-time quantitative PCR (qPCR) assay for the simultaneous detection and quantification of DHBV, DAdV-3, and SGSP. The assay indicated high analytical sensitivity, achieving detection limits of 10 copies/µL for each target, with all standard curves showing excellent linearity (R² = 0.999). The evaluation of specificity showed that the triplex qPCR assay had high specificity for the three target pathogens, as it did not yield any cross-reactive signals with other pathogens examined. Intra-assay and inter-assay coefficients of variation for recombinant plasmid standards were consistently <2%, indicating high reproducibility. Meanwhile, the assay was employed to investigate the epidemiology of the three pathogens in ducks. The positive rates for DHBV, DAdV-3 and SGSP were 73.95% (159/215), 11.16% (24/215) and 37.21% (80/215), respectively. The co-infection rates were 5.58% (12/215) for DHBV and DAdV-3, 27.91% (60/215) for DHBV and SGSP, 2.33% (5/215) for DAdV-3 and SGSP, and 2.33% (5/215) for all three pathogens. In summary, this triplex qPCR assay displays excellent specificity, sensitivity and repeatability, which may serve as a reliable testing tool for routine clinical diagnosis, epidemiological monitoring and co-infection screening of the three target pathogens in locally sampled duck populations.