Etiology Of Short Beak And Dwarfism Syndrome Of Duck And Transcriptomics On Beaks Of Infected Ducks

Short beak and dwarfism syndrome(SBDS)was originally recognized as a viral disease of mule ducks in 1971 in France.The etiological agent of the disease belongs to the West-European lineage of goose parvovirus(GPV).Since 2014,a new disease,which resembles SBDS of mule ducks,has emerged and circulated in major Pekin duck production areas in China,and has caused huge economic losses.Faced with the outbreaks of the new disease,we firstly characterized the etiological agent of the disease and proposed the prevention and control measures.The disease mainly affected commercial meat-type Pekin ducks.Affected Pekin ducks exhibited clinical signs typical of SBDS.A JS1 isolate of GPV was recovered from typical cases using susceptible goose embryos.Experimental infection of 2-day-old Pekin ducklings with the GPV JS1 isolate resulted in reproduction of SBDS,indicating that GPV is the etiological agent of SBDS in Pekin ducks.Using a waterfowl parvovirus PCR assay,GPV was detected in 77/79 tissue samples of sick Pekin ducks collected from Anhui,Shandong,Jiangsu,and Hebei provinces,and in 35/40 spleen samples of newly hatched Pekin duckling collected from eight hatcheries located in different regions.These results indicate the virus was geographically widespread and that it may be vertical transmitted through eggs.In agar gel precipitin tests,the Pekin duck-origin isolate was found to share identical precipitin antigen with the goose-origin GPV isolate(H strain).Phylogenetic analysis based on partial VP1 and VP3 nucleotide sequences demonstrated that the GPV strains detected from SBDS fell into the West-European lineage of the GPV-related group.Immunization with the yolk antibody preparation of GPV clearly demonstrated that SBDS can be well controlled.To understand the nature of Pekin duck-origin GPV,the genomic sequence of the JS1 isolate was determined,and sequence changes were identified for the Pekin duck-origin GPVs.We also investigated the differences in pathogenicity of JS1 and H viruses to Pekin ducks and goslings.Compared with classical GPV strains,Chinese Pekin duck-origin GPVs contained multiple point mutations and deletions/insertions in the noncoding regions,but these changes did not affect the formation of the hairpin structures.Five VP 1 amino acid substitutions were detected in Chinese variant GPVs,all of which were found to be located in positions distantly from the potential receptor binding sites.Nine amino acid substitutions were detected in NS region.Of the nucleotide substitutions,seven were found to be located within(or in positions close to)regulatory elements.Both JS1 and H isolates influenced the weight gain and the development of beaks and bones of wings and legs,and caused microscopic lesions in internal organs of Pekin ducks.However,the clinical signs typical of SBDS could only be replicated with the JS1 isolate.The findings suggest that both JS1 and H are pathogenic for Pekin ducklings,while the former is more virulent than the latter.Thus,the virus from SBDS of Pekin ducks can be recognized as a virulent mutant of goose parvovirus.Using a quantitative real-time PCR and a GPV VP3-based ELISA,both variant and classical GPVs were shown to infect Pekin ducklings.Infection of goslings with JS1 and H viruses resulted in mortalities 73.3%and 93.3%respectively.The findings suggest that the variant GPV strain,which had specifically adapted to Pekin ducks,still retains high pathogenicity for its original host.RNA-seq was employed to screen gene expression profiles of the beaks among JS1-infected,H-infected and control groups.By pairwise comparison,a total of 452 differentially expressed genes were associated with SBDS.GO analysis provided evidence that these genes may participate in 292 GO terms,including biological processes,cellular component and molecular functions.Using the KEGG searches,110 signaling pathways were significantly enriched,including metabolic pathways,calcium signaling pathway,mTOR signaling pathway,apoptosis,and WNT signaling pathway.These results may provide clues for further studies on the pathogenesis of GPV-caused SBDS.