2 MATERIALS AND METHODS
2.1 Case presentation
Case 1. In March 2021, acute outbreaks of spleen necrosis disease occurred on many commercial duck farms in Xinxiang City of Henan province, central China. A duck farm with approximately 30% of 10,000 10-day-old ducklings showed sudden onset and severe symptoms, such as listlessness, white diarrhea, anorexia, and lameness. The outbreak started on 3 March 2021, and antibiotic-traditional veterinary drugs combination therapy did not work. Seven dead ducklings were randomly selected and sent to the laboratory for diagnosis.
Case 2. An epidemic characterized by the sudden death of ducklings emerged on another commercial duck farm in Jiaozuo city of Henan province. The duck flock had approximately 11,000 ducklings of 11-day-old. From 12 August 2021 to 22 August 2021, approximately 350 ducklings per day died acutely. The great majority of the diseased ducklings showed listlessness, white diarrhea, and anorexia. The mortality was about 30%. The antibiotic therapy did not work. Eight dead ducklings were selected randomly to send to the laboratory for diagnosis.
2.2 Molecular diagnosis
To identify the causative agent of the disease, the potential viral and bacterial pathogens were examined. The bacteriological culture was performed as described previously (Wang et al., 2020d). The samples from the same duckling were combined, and processed by extracting RNA using the EasyPure Viral RNA Kit (Takara, Shanghai). Subsequently, the RNA samples were used to detect MDRV, DHAV-1, DHAV-3, NDV, DTMUV, and NDRV using RT-PCR protocols as described previously (Yan et al., 2021).
2.3 Virus isolation
The positive samples of liver and spleen from dead ducklings from the same flock were homogenized in phosphate-buffered saline (PBS, pH 7.2), freeze-thawed three times, and centrifuged at 8000× g for 10 min. The supernatants were filtered through a 0.22 μm filter to remove bacteria and other larger particles. Subsequently, 0.2 mL of each of the two supernatants was separately used to propagate in the allantoic cavity of 10-day-old healthy duck embryos in a 37℃ incubator. If the embryo died at 3-4 days post-inoculation, the allantoic fluid was harvested for another round of inoculation. After three passages in healthy embryonated duck eggs, the allantoic fluids and duck embryos were harvested sterilely and stored at −80°C. The viral RNA extracted from the allantoic fluids were used to detect potential causative agent.
2.4 Gene amplification and sequencing
To analyze the genotype and genetic characteristics of the both newly isolated NDRV strains in this study, the complete σC genes of the NDRV were amplified by using primers as follows: NDRV-S1 forward: 5′-GCTTTTTTCTTCTCTGCCCAT-3′ and DRV-S1 reverse: 5′- GATGAATAGCTCTTCTCATCGC-3′, which were designed based on the S1 gene of NDRV downloaded from NCBI (https://www.ncbi.nlm.nih.gov/). The RT-PCR products were purified and cloned into a pMD18-T vector (Takara, Shanghai) for sequencing with universal M13 forward and reverse primers by Sangon Biotech in Shanghai.