【作者】 冯杰；

【导师】 王新卫；

【作者基本信息】 河南农业大学 ， 兽医学， 2020， 硕士

【摘要】 鸡传染性贫血(Chicken infectiousanemia,CIA)是一种严重影响家禽生产的免疫抑制病,此病主要侵染1-3周龄雏鸡,感染鸡群主要表现为明显的生长抑制、雏鸡再生障碍性贫血、骨髓黄化及早期全身淋巴细胞萎缩。鸡传染性贫血病毒(Chicken infectious anemia virus,CIAV)是CIA病原体,在临床上CIA致死率较低,导致养殖户常常忽视对此病的防治往往容易疏忽。自1992年以来,CIA在中国多省份广泛流行,给我国养禽业造成很大经济损失。本研究对我国CIA的流行病学调查相关文献数据进行系统分析,横切面调查市场部分流通禽用弱毒活疫苗和采集河南各地区鸡场病料进行了河南地区CIA的流行病学调查。从河南农业大学禽病研究所收集患病病例,进行病毒分离鉴定和基因组序列测定,对河南地区CIAV流行毒株进行系统分析并进行了部分生物学特性研究。具体研究内容如下:1.我国CIA感染率Meta分析为了解CIAV在国内的流行情况,从各种中英文文献平台下载了 CIAV感染率调查相关文献,并使用3.5.3版本R软件内的Meta软件包对文献内数据进行荟萃分析。分析结果显示,中国CIAV整体阳性率为48%(95%CI:36%-61%),暗示CIAV在国内鸡群存在普遍感染。因文献存在异质性,本研究进一步对文献进行了亚组分析结果发现,在地理分布上,华东地区整体阳性率56%(95%C1:43%-68%)明显高于其他地区38%(95%CI:24%-52%);2010年以前的文献整体阳性率52%(95%CI:38%-66%)与2010年后发表的文献整体阳性率45%(95%C1:31%-60%)差异不大;使用ELISA方法检测病毒整体阳性率66%(95%CI:54%-77%)明显高于PCR 方法 26%(95%CI:15%-42%)及其他方法 30%(95%CI:14%-53%);小于60日龄的育雏阶段的鸡只整体感染率37%(95%CI:28%-47%)小于250日龄的成年鸡感染率80%(95%CI:49%-98%),暗示成年鸡普遍存在CIAV感染。2.河南地区CIA流行病学研究本研究为了解CIA在河南地区的流行情况,采用横断面调查的方法,搜集2017-2019年河南地区临床病料,随机采购河南市场上售卖的弱毒疫苗,通过PCR方法进行组织病料及疫苗分子流行病学调查。结果共收集组织病料437份,其中CIAV核酸阳性41份,阳性率为9.38%(95%(CI:6.8%-12.5%);共采集24个厂家,5个不同种类不同批次120份冻干活疫苗,其中新城疫、支气管炎二联活疫苗53份,鸡痘活疫苗31份,新城疫活疫苗34份,鸡毒支原体活疫苗1份,传喉重组鸡痘病毒基因工程苗1份。最终CIAV检测阳性疫苗4份,总体CIAV核酸阳性率为3.33%(95%Cl:0.9%-8.3%)。4份阳性疫苗来自3个生物制品厂,疫苗厂家阳性检出比例为3/24,整体阳性率为12.5%(95%CI:2.7%-32.4%)。以上结果显示,当前存在鸡用活疫苗的CIAV污染,除自然感染外,鸡群感染此病与疫苗中CIAV污染也可能有关联。3.CIAV河南株的分离鉴定与全基因组分析本研究从河南农业大学禽病研究所收集患病鸡只肝脏及脾脏,进行病毒分离并测定12株分离株的全基因组序列并对其进行分析。结果显示,12个河南分离株之间相似性很高,范围在97.1%-99.3%,与疫苗株Cux-1和Del-Ros相似性范围在97.8%-98.6%。在全基因进化树上河南12个分离株分为三个小的亚组,HN-3,HN-5,HN-7,HN-10,HN-11与日本经典弱毒株C369同一小分支上,可能是由此弱毒株传播进化而来。HN-2、HN-4、HN-6这三株病毒与之前河南HN1405毒株处在同一分支上,HN-1,HN-8,HN-9,HN-12与河南HN1504毒株同一分支上,这两个分支上的毒株较为集中且与之前河南地区分离的毒株都在同一分支,氨基酸位点差异也不大,表明这些毒株可能是在河南本土地区流行的优势毒株。分别对12个分离病毒编码的三个完整蛋白进行分析后发现,衣壳蛋白VP1蛋白氨基酸位点变异最多,总体变异为0-3.4%,其中HN-1,HN-8,HN-9,HN-12毒株VP1蛋白都带有Q139和Q144,其他8个河南分离株携带残基K139和E144。HN-4,HN-6,HN-8在290位存在A290P突变,这是之前从未报道过的。VP2和VP3蛋白与其他毒株相比没有明显的变化。利用RDP4软件对所分离到的12株CIAV毒株进行重组检测发现,HN-4和HN-8毒株存在基因重组。整体而言,河南毒株与亚洲其他地区韩国、日本和美国毒株存在相互重组演化,也存在一定的基因重组事件,推测重组后的毒株适应性、流行性和传播水平会更高。4.CIAV河南分离株HN-4生物学特性初步研究为进一步了解CIAV河南分离株HN-4毒株的生物学特性,利用SYBR Green Ⅰ实时荧光定量PCR和q-PCR测定病毒效价的方法,研究该毒株在马立克氏病淋巴母细胞系(MDCC-MSB1)中的生长特性和增殖规律。结果显示HN-4毒株在MSB1细胞系中能够很好增殖,感染病毒48h后能产生非常明显的CPE,感染后72h细胞裂解物中基因组拷贝数达到峰值(1×107.25copies/μL)此后保持稳定。对1日龄SPF鸡肌肉注射1mL从细胞培养接种物中分离的HN-4毒株(1×107copies/μL),在鸡体上复制出典型的发病症状,感染后7天可从感染组鸡体内可以分离到接种毒株,最终鸡只死亡率为50%。与对照组相比,感染组鸡只在感染后7天体重明显降低,感染后21天时两组鸡体重差异显著(P<0.05)。与对照组相比,感染组在感染后16天时鸡只胸腺、脾脏、法氏囊都有不同程度的萎缩,差异显著(P<0.05)。肾脏萎缩不明显,肝脏轻微肿大,感染组鸡骨髓变为淡黄色,骨质脆。采集组织器官制备病理切片进行HE染色,结果显示感染组鸡各组织器官均有典型CIA引起的病理损伤。以上结果显示HN-4毒株致病性强。更多还原

【Abstract】 Chicken infectious anemia(CIA)is an immunosuppressive disease that severely affects poultry production.This disease mainly affects 1-3 week old chicks.The infected chickens mainly showed obvious growth inhibition,chick aplastic anemia,yellowing of bone marrow,and early systemic lymphocyte atrophy.Chicken infectious anemia virus(CIAV)is the pathogen of CIA,and the clinical mortality rate of CIA is relatively low.As a result,farmers often neglect to prevent and treat the disease.Since 1992,CIA has been widespread in many provinces in China,causing great economic losses to China’s poultry industry.This study combines the epidemiology of CIA in China and the situation in Henan to conduct a systematic analysis.The disease cases were collected from the Poultry Disease Research Institute of Henan Agricultural University,and the virus was isolated and identified,and the genome sequence was determined and analyzed;A cross-section survey of live attenuated live attenuated vaccines for poultry in the market and collection of disease data from chicken farms in various regions of Henan conducted an epidemiological survey of CIA in Henan.The specific research content is as follows:1.Meta analysis of CIA infection rate in ChinaIn order to understand the prevalence of CIAV in China,the relevant documents of the CIAV infection rate survey were downloaded from various Chinese and English literature platforms,and a meta-analysis of the data in the literature was performed using the Meta software package in version 3.5.3 R software.The analysis showed that the overall positive rate of CIAV in China was 0.48(95%CI:0.36-0.61),suggesting that CIAV is commonly infected in domestic flocks.Due to the heterogeneity of the literature,this study further performed a subgroup analysis of the literature.The results showed that geographically,the overall positive rate of 0.56(95%CI:0.43-0.68)in East China was significantly higher than 0.38(0.24-0.52)in other regions;The overall positive rate of literature before 2010 was 0.52(95%CI:0.38-0.66)and the overall positive rate of literature published after 2010 was 0.45(95%CI:0.31-0.60);The overall positive rate of virus detection by ELISA method was 0.66(95%CI:0.54-0.77),which was significantly higher than that of PCR method 0.26(95%CI:0.15-0.42)and 0.30(95%CI:0.14-0.53)of other methods;The overall infection rate of chickens at the brooding stage of younger than 60 days is 0.37(95%CI:0.28-0.47).The infection rate of adult chickens older than 250 days is 0.80(95%CI:0.49-0.98).It is suggested that CIAV infection is widespread in adult chickens.2.Epidemiological study of CIA in HenanIn order to understand the prevalence of CIA in Henan,this study collected clinical data from 2017-2019 in Henan and randomly purchased attenuated vaccines sold in the Henan market.Epidemiological investigation of tissues and vaccines using PCR..A total of 437 tissue disease materials were collected,of which 41 were positive for CIAV nucleic acid,with a positive rate of 9.38%(95%CI:6.8-12.5);A total of 120 lyophilized vaccines were collected from 24 manufacturers,5 different types and different batches.of which 53 were Newcastle disease and bronchitis live vaccines,31 were chickenpox live vaccines,34 were Newcastle disease live vaccines,I were Mycoplasma live vaccines,1 were laryngeal and fowlpox virus genetically engineered vaccines.In the end,4 CIAV-positive vaccines were detected,and the overall CIAV nucleic acid positive rate was 3.33%(95%CI:0.9-8.3).The 4 positive vaccines came from 3 biological product factories.The positive rate of the vaccine manufacturers was 3/24,and the overall positive rate was 12.5%(95%CI:2.7-32.4).The above results show that there is currently CIAV contamination of live vaccines for chickens.In addition to natural infections.chickens infected with this disease may also be associated with CIAV contamination in vaccines.3.Isolation and identification of CIAV Henan strain and whole genome analysisIn this study.diseased chicken tissues were collected from the Institute of Poultry Diseases of Henan Agricultural University,and the 12 virus strains were isolated from the tissues and their entire genome sequences were determined and analyzed.The results showed that the similarity between the 12 Henan isolates was high,ranging from 97.1%to 99.3%,and the similarity to the vaccine strains Cux-1 and Del-Ros,ranging from 97.8%to 98.6%.The 12 isolates of Henan are divided into three small subgroups on the whole gene phylogenetic tree.It may have evolved from the transmission of this weak strain.The three viruses HN-2,HN-4,and HN-6 are on the same branch as the previous Henan HN1405 strain,and HN-1,HN-8,HN-9,and HN-12 are on the same branch as the Henan HN1504 strain.The strains on these two branches are more concentrated and are in the same branch as the previous isolates from Henan,and the amino acid loci are not much different,indicating that these strains may be the dominant strains that are prevalent in the native area of Henan.After analyzing three complete proteins encoded by 12 isolated viruses,it was found that the amino acid locus of the capsid protein VP1 protein has the most variation,and the overall variation is 0-3.4%.Among them,the VP1 proteins of HN-1,HN-8,HN-9,and HN-12 all carried Q139 and Q144,and the other eight Henan isolates carried residues K139 and E144.HN-4,HN-6,and HN-8 have A290P mutations at position 290,which has never been reported before.VP2 and VP3 proteins have no obvious changes compared with other strains.Recombinant detection of 12 isolated CIAV strains using RDP4 software revealed that HN-4 and HN-8 strains had genetic recombination.In general,Henan strains have undergone recombination and evolution with other strains of South Korea,Japan,and the United States,and there have been certain genetic recombination events.It is speculated that the recombined strains will have higher levels of adaptability,prevalence and spread.4.Biological characteristics of CIAV Henan isolate HN-4In order to understand the biological characteristics of CIAV Henan isolate HN-4 strain,SYBR Green I real-time quantitative PCR and q-PCR were used to determine the virus titer.The virus was studied in Marek’s disease lymphoblast cell line(MDCC-MSB1)growth characteristics and proliferation rules.The results show that the HN-4 strain can proliferate well in the MSB1 cell line,produce very obvious CPE 48 hours after infection with the virus,and the genomic copy number in the cell lysate reaches a peak 72 hours after infection(1×107.215 copies/μL).stable.One-day-old SPF chickens were intramuscularly injected with 1 mL of the HN-4 strain(1 × 107 copies/μL)isolated from the cell culture inoculum to replicate the typical symptoms on the chicken body.7 days after infection,the vaccinated strains can be isolated from the infected chickens,and the final mortality of the chickens is 50%.Compared with the control group,the weight of chickens in the infected group was significantly reduced at 7 days after infection,and the body weight of the chickens in the two groups was significantly different at 21 days after infection(P<0.05).Compared with the control group,the thymus,spleen,and bursa of chickens in the infected group were atrophied to varying degrees at 16 days after infection(P<0.05).Renal atrophy is not obvious,and the liver is slightly swollen.The bone marrow of the infected group turned pale yellow and the bone was brittle.Tissues and organs were collected to prepare pathological sections for HE staining.The results showed that the tissues and organs of infected chickens had typical CIA-induced pathological damage.The above results show that the HN-4 strain is highly pathogenic.更多还原