小鹅瘟病毒、鹅星状病毒Ⅰ型与鸭疫里默氏杆菌混合感染的诊断及病原分析

Diagnosis and Pathogen Analysis of Co-infection of Goose Parvovirus,GenotypeⅠGoose Astrovirus and Riemerella anatipestifer

【目的】2023年4月,河南新乡某地鹅场雏鹅群急性发病,死亡率高达25%,为确定引起该鹅场雏鹅发病的病因,本研究对送检的病死雏鹅进行剖检和实验室诊断。【方法】无菌采集病死雏鹅肝脏、脾脏、肾脏和小肠等组织样品及心血和肝脏外层纤维素性渗出物,通过对心血及肝脏外层纤维素性渗出物样品进行细菌分离培养、革兰染色镜检观察、16S rRNA基因及药物敏感性试验鉴定病鹅感染细菌及感染菌株的药物敏感性情况;通过PCR/RT-PCR方法对其常见雏鹅病毒性传染病病原核酸进行检验,并对阳性病原的主要结构蛋白基因进行测序分析,确定病鹅感染的病毒性病原及其分子流行病学情况。【结果】细菌学试验结果显示,分离菌株在血平板上呈半透明圆形突起、边缘整齐、表面光滑菌落,形态学观察显示,该菌为单个和成对的革兰阴性短杆菌,符合鸭疫里默氏杆菌(Riemerella anatipestifer,RA)的特性。16S rRNA基因扩增测序与BLAST分析进一步证实该菌为RA。药敏试验结果显示,该菌株对头孢曲松和头孢噻肟敏感,对阿莫西林、四环素和多黏菌素B耐药。常见雏鹅病毒性传染病病原核酸PCR/RT-PCR检测发现,该鹅场样品小鹅瘟病毒(Goose parvovirus,GPV)和鹅星状病毒Ⅰ型(genotypeⅠGoose astrovirus,GAstV-1)核酸呈阳性,GAstV-2、禽流感病毒(Avian influenza virus,AIV)和鹅呼肠孤病毒(Goose reovirus,GRV)核酸阴性,将致病毒株分别命名为GPV/HN-2023和GAstV-1/HN-2023。进一步对GPV/HN-2023和GAstV-1/HN-2023的主要结构蛋白基因分析发现,GPV/HN-2023与DY-16株的亲缘关系较近,属于DY-16-like毒株,且VP3蛋白存在2个特有的氨基酸位点突变D248E和V314L;GAstV-1/HN-2023与GAstV-1毒株亲缘关系较近,属于GAstV-1分支,ORF2蛋白存在3个特有的氨基酸位点突变G47R、S207G和A628T。【结论】本研究通过综合诊断方法明确了GPV、GAstV-1与RA混合感染是引起该鹅场雏鹅发病的病因,分析了致病菌RA的耐药性和病毒性致病原GPV和GAstV-1的遗传演化及变异特点,为河南地区雏鹅疫病的科学防控提供理论参考。

【Objective】In April 2023,an acute outbreak of infectious disease in goslings with mortality up to 25%occurred in a commercial goose farm in Xinxiang city of Henan province.To determine the potential causative agent of this disease,anatomical examination and laboratory pathogen diagnosis of dead goslings were performed.【Method】The tissue samples of livers,spleen,kidneys,small intestine and cardiac blood and fibrous exudate from the outer layer of the liver were collected.Bacterial isolation and cultivation,Gram staining microscopy observation,16S rRNA gene identification and drug sensitivity testing were performed on samples of cardiac blood and fibrous exudate from the outer layer of the liver to determine the bacterial infection and drug sensitivity of infected geese.PCR/RT-PCR method was used to test the nucleic acid of common gosling viral infectious disease pathogens,and sequencing analysis was performed on the main structural protein genes of positive pathogens to determine the viral pathogens and their molecular epidemiology in infected geese.【Result】In the bacterial test,the results showed that the isolated bacteria could grow as the morphology of translucent,neat edges,smooth colonies on blood agar medium.Gram-negative single and paired bacteria could be observed after Gram staining,which were consistent with the characteristics of Riemerella anatipestifer(RA).PCR amplification of 16S rRNA gene and BLAST analysis were further confirmed the isolate was RA.The drug sensitivity test revealed that the RA strain was highly sensitive to ceftriaxone and cefotaxime,and resistance to amoxicillin,tetracycline and polymyxin B.PCR and RT-PCR detection results for the common pathogens of goslings showed that all the samples were positive for Goose parvovirus(GPV)and genotypeⅠGoose astrovirus(GAstV-1),and no corresponding nucleotide fragments were observed for GAstV-2,Avian influenza virus(AIV)and Goose reovirus(GRV).The two identified virus strains were designated as GPV/HN-2023 and GAstV-1/HN-2023,respectively.Furthermore,the analysis of the main structral protein gene of GPV/HN-2023 and GAstV-1/HN-2023 revealed that GPV/HN-2023 was closely related to DY-16 strain and belonged to DY-16-like strain,with two unique amino acid mutations D248E and V314L in VP3 protein.GAstV-1/HN-2023 was closely related to GAstV-1 strains and grouped into GAstV-1 branch,with three unique amino acid mutations G47R,S207G and A628T in ORF2 protein.【Conclusion】This study identified the mixed infection of GPV,GAstV-1,and RA as the cause of gosling disease in the goose farm through comprehensive diagnostic methods.The drug resistance of pathogenic bacteria RA and the genetic evolution and variation characteristics of viral pathogens GPV and GAstV-1 were analyzed,providing theoretical reference for the scientific prevention and control of gosling disease in Henan region.