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.