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.