3.2 Molecular diagnosis
To identify the causative agent of the disease, RT-PCR assays were used to detect the potential viral pathogens. The samples from the dead duckling were positive for NDRV, and no corresponding nucleotide fragments were observed for MDRV, DHAV-1, DHAV-3, NDV, and DTMUV (Figure S1). In addition, Riemerella anatipestifer was also identified from four dead ducklings in case 2 by bacteria isolation.
3.3 Virus isolation
The homogenates of the positive liver and spleen samples were then inoculated into the allantoic cavity of 10-day-old healthy duck embryos. The inoculated duck embryos’ bodies showed varying degrees of hemorrhage and dysplasia (Figure 2b). The allantoic fluids of the duck embryo inoculated with the samples were only positive for NDRV. Eventually, two NDRV strains causing duck spleen necrosis disease were successfully isolated and termed as HNXX-1/2021 and HNJZ-2/2021, respectively.