Abstract: 【Objective】 In November 2023,an acute outbreak of yolk peritonitis in laying ducks emerged in a duck farm in Henan province.In order to provide scientific basis for the prevention and control of yolk peritonitis in the duck farm,laboratory diagnosis and pathogen analysis were carried out to determine the cause of disease.【Method】 After pathological anatomical observation,liver,spleen,kidney and blood samples were collected aseptically,and bacterial isolation culture and Gram staining microscopy were performed.The partical genome of the predominant bacterial was amplified by PCR with specific primers and sequenced.The sensitivity of the isolates to common antibiotics were then detected.The pathogens of common viral infectious diseases in laying ducks were detected by PCR/RT-PCR,and the main structural protein genes of positive pathogens were sequenced and analyzed to determine the viral pathogens and molecular epidemiology of infection in laying ducks.【Result】 Bacteriological test results showed that two dominant bacteria with different colony morphology were isolated.One colony showed translucent round protrusive,neat edges,smooth surface,Gram-negative,most of which existed in single or pair,which was consistent with the growth characteristics and bacterial characteristics of Riemerella anatipestifer (RA).The other colony showed a grayish white round protrusion on the blood plate,neat edges,smooth surface,no hemolytic ring,Gram-negative,most of which existed in a single form,in two short blunt circles,which was consistent with the growth characteristics and bacterial characteristics of Escherichia coli (E.coli).The ompA and 16S rRNA genes of the predominant bacterial was amplified and sequenced,respectively.BLAST analysis revealed that the two dominant strains were RA and E.coli. The results of drug susceptibility test showed that the RA isolate was sensitive to ciprofloxacin,amoxicillin-clavulanate potassium and flufenicol.E.coli isolate was sensitive to cefotaxime,ciprofloxacin,ceftriaxone and fosfomycin sodium.PCR/RT-PCR test results showed that the specific primer of Duck circovirus (DuCV) amplified a specific band at 915 bp,which was positive for nucleic acid,while the nucleic acid test results of other pathogens were negative.The isolated strain was named DuCV/HN-2023.Sequencing comparison results showed that the Cap gene of DuCV/HN-2023 had the highest similarity with the nucleotide sequence of duck derived Circovirus ZQ-XYF1010-1 and GZ-GXG0905-X4,which were 99.7% and 99.4%,respectively.The nucleotide sequence similarity with Muscovy circovirus FJFQ315 and FJFQ312 strains was 98.6% and 98.5%,respectively.Phylogenetic tree analysis showed that DuCV/HN-2023 was closely related to ZQ-XYF1010-1 and GZ-GXG0905-X4.【Conclusion】 Through comprehensive diagnosis,it was confirmed that the disease of laying ducks in the duck farm was caused by the mixed infection of RA,E.coli and DuCV.The drug resistance of the isolated strains was analyzed,and the genetic evolution of the isolated strain was performed at the molecular level,and the biological characteristics of the pathogen were further analyzed,which provided a reference for the prevention and control of the disease in clinic.

Key words: Riemerella anatipestifer; Escherichia coli; Duck circovirus; mixed infection; yolk peritonitis