关键词: 鸭疫里默氏杆菌; 禽呼肠孤病毒(ARV); 混合感染; 诊断; 病原分析

Abstract: 【Objective】 In December 2024,a flock of 28-day-old broilers in a commercial White-feathered broiler farm in Pingdu city of Shandong province,showed symptoms of swollen hock joints and paralysis,and deaths occurred successively.To determine the cause of the disease in this flock,this study conducted necropsy and laboratory diagnosis on the diseased and dead chickens submitted for examination. 【Method】 Liver,spleen tissues,fibrinous exudate from the serous layer of diseased chicken organs,synovial secretion,and pharyngeal swab were collected for laboratory diagnosis.Real-time PCR/RT-PCR was used to detect nucleic acids of pathogens that cause similar clinical signs including Riemerella anatipestifer (RA),Avian reovirus (ARV) and Mycoplasma synoviae (MS),to identify the etiology of the outbreak.The bacterial characteristics and drug sensitivity of infected chickens were determined through bacterial isolation and culture of joint cavity secretions,Gram staining microscopy observation,serotype identification,16S rRNA gene sequence analysis,and drug sensitivity testing.Tissue homogenates were inoculated into LMH cells for virus amplification.The major structural-protein genes were amplified by PCR and sequenced to define the molecular epidemiology of the viruses infecting the chickens. 【Result】 Real-time quantitative PCR/RT-PCR detection showed that RA and ARV had Ct values＜35,indicating positivity.MS had no Ct value,indicating negativity.The bacteriological test results showed that the isolated strain appeared as semi transparent circular protrusions with neat edges and smooth surface colonies on the blood plate and TSA plate,the bacteria were single and paired Gram negative short rods,suspected to be RA.The serotype identification results showed that the isolated strain from chicken was RA serotype 10.16S rRNA gene amplification sequencing was analyzed by BLAST to be located in the same branch as the RA reference strain that from duck,with a similarity greater than 99%,the isolate was identified as RA.The drug sensitivity test results showed that the strain was sensitive to ceftriaxone,cefotaxime and polymyxin B,but resistant to neomycin,gentamicin,kanamycin,ampicillin,amoxicillin and ciprofloxacin.PCR detection of cell culture showed that the ARV σC gene nucleic acid was positive.Further analysis of the gene fragment revealed that the strain belonged to the GC1-sub genotype，not in the same branch as the classical vaccine strains S1133 and 1733. 【Conclusion】 This study identified the mixed infection of RA and ARV as the cause of the disease in the chicken farm through comprehensive diagnostic methods.The RA isolate exhibited multidrug resistance,whereas the ARV isolate belonged to the GC1-sub cluster.These findings provided a scientific basis for the targeted prevention and control of broiler diseases in Qingdao area.

Key words: Riemerella anatipestifer; Avian reovirus (ARV); co-infection; diagnosis; pathogen analysis