鸡传染性支气管炎病毒山西株的分离鉴定及致病性分析

Pathogenicity analysis of one avian infectious bronchitis virus stain isolated from Shanxi

为分离鉴定鸡传染性支气管炎病毒(IBV)并研究其致病性,本研究对山西运城疑似传染性支气管炎(IB)感染的送检临床样品研磨后反复冻融,提取基因组进行RT-PCR检测,结果显示其为IBV核酸阳性;进一步对样品研磨液过滤后接种9日龄~11日龄SPF鸡胚,并传至F3代,RT-PCR检测仍为IBV阳性;将阳性样品F11代鸡胚尿囊液接种SPF鸡胚后能引起典型的“侏儒胚”病变,接种气管环能导致气管环纤毛失去活性,表明分离到一株IBV,命名为YC181031。提取分离株基因组进行S1基因的PCR扩增及测序分析,结果显示YC181031株属于致肾病变型的GI-22基因型IBV。将该分离株感染7日龄SPF雏鸡以鉴定其致病性,结果显示YC181031分离株感染能引起雏鸡张口呼吸、精神沉郁、排水样稀粪等临床症状以及死亡,雏鸡死亡率为20%;剖检感染鸡和死亡鸡,能观察到肾脏肿大,并发生明显的尿酸盐沉积等病变;取感染雏鸡和死亡雏鸡的各脏器制备病理切片,观察各脏器的病变,并送公司制备各组鸡各组织的免疫组化切片,检测IBV感染情况。结果显示肾脏、气管、肺脏、腺胃、脾脏、肝脏均能观察到明显病变及病毒感染。对感染鸡每两天采集各组鸡的咽肛拭子,采用RT-PCR检测排毒情况,结果显示感染鸡在试验期14 d内均能持续检测到排毒。采用荧光定量PCR检测各组织器官病毒的载量,结果显示各组织病毒载量由高到低依次为肾脏、气管、肺脏、腺胃、脾脏、肝脏,其中肾脏病毒载量显著高于其他组织的病毒载量(P<0.05)。本研究首次系统研究了GI-22基因型IBV的致病特征,为该病的防制提供了参考依据。

In order to perform the isolation of avian infectious bronchitis virus(IBV)and study the pathogenicity of IBV isolate,the RT-PCR was used to detect nucleic acid extracted from a clinical sample of chickens,which were suspected to be infected with infectious bronchitis virus(IBV)and provided by a farmer in Yuncheng,Shanxi province.And the sample was detected as IBV positive by RT-PCR.Then 9-11-day-old SPF chicken embryonated eggs were inoculated with the sample filtered from the grinding fluid,and the obtained allantoic fluid was blindly passed by three generations(F3)and was also tested as IBV positive;The F11 generation passaged in embryonated eggs caused typical"dwarf embryo"lesions to SPF chicken embryonated eggs,and induced the loss of cilia in tracheal rings.The results showed that an IBV strain was isolated and named as YC181031.The S1 gene amplification and sequencing analysis showed that YC181031 strain belonged to IBV GI-22 genotype,which is also nephropathogenic type IBV.Seven-day-old SPF chicks were used to test the pathogenicity of the isolate.The results showed that several clinical symptoms were showed in chicks infected with YC181031,such as breathing with difficulty,depression,excreting watery droppings and death.The mortality of infected chicks was 20%.Typical pathological changes such as enlargement of kidney and urate deposition in the kidney were observed in infected chicks.The immunohistochemical assay and viral load detection were performed for the tissue samples from infected and dead chicks.The tissue lesions and distribution of virus were observed in the kidney,trachea,lung,glandular stomach,spleen and liver samples of infected chicks.RT-PCR detection of pharyngeal anal swabs showed that the virus shedding by infected chicks could be continuously detected within 14 days of the test period;The viral loads of various tissues were detected by RT-qPCR and the results showed that the viral load from high to low was kidney,trachea,lung,stomach,spleen and liver.The viral load of kidney was significantly higher than that of other tissues(P<0.05).In this study,the pathogenicity characteristics of GI-22 genotype strain were systematically studied for the first time,providing a reference for the prevention and treatment of the disease.