【作者】 张博；

【导师】 董世山； 王学静；

【作者基本信息】 河北农业大学 ， 兽医硕士（专业学位）， 2020， 硕士

【摘要】 新型鸭呼肠孤病毒(Novel duckreovirus,NDRV)病,其特征病变是脾脏和肝脏严重的出血和坏死。近几年,NDRV的发病率在5%～35%,死亡率在2%～20%,并且无明显季节性,严重影响了我国养鸭业的健康发展。本研究采集河北某养鸭场肝坏死、脾坏死为特征,疑似NDRV感染的病鸭组织,经处理后,接种9日龄SPF鸡胚进行病毒分离、构建病毒感染模型、NDRV S3基因编码区基因序列分析、NDRV荧光定量RT-PCR方法的建立、制备油乳剂灭活疫苗、制备抗NDRV高免卵黄抗体,并进行抗NDRV高免卵黄抗体的保护效果评价,为NDRV的防控提供技术支持。本研究成功分离出一株NDRV并命名为CH-HB株,分离病毒对鸡胚致病性结果:胚体水肿、出血明显、鸡胚肝脏坏死。病毒生物学特征结果:病毒对鸡胚的半数致死量为1×10-5.5 ELD50/0.2 mL,且不能凝集鸡红细胞。动物回归试验结果表明:感染的雏鸭出现食欲不振,部分鸭眼角出现泪痕,在观察期内,雏鸭无自然死亡,剖检观察发病雏鸭肝脏明显肿大,肝脏伴有不同程度的针尖状坏死点,脾脏三角状区域变硬,表面散在分布大小不同的白色坏死病灶,病理切片结果:脾脏组织散在分布大量坏死灶,坏死灶区域淋巴细胞变性坏死,组织丧失固有结构,肝脏组织中肝细胞脂肪变性,局灶性肝细胞坏死;同源性分析结果显示:分离病毒CH-HB株S3基因编码区基因序列(登录号:MT967492.1)与16株参考病毒S3基因编码区基因序列核苷酸同源性为 66.5%～99.5%,与 NDRV Duck/N-DRV-XT18/China/2018(登录号:MK749405.1)参考株的核苷酸同源性最高为99.5%。遗传进化分析结果:CH-HB株位于NDRV分支,与 Duck/N-DRV-XT18/China/2018 株(登录号:MK749405.1)S3 基因遗传距离最近;与ARV参考毒株S3 基因编码区遗传距离较远;而MDRV参考毒株的S3基因遗传距离最远。NDRV荧光定量RT-PCR方法的建立结果表明:建立的TaqMan荧光定量RT-PCR检测方法标准曲线方程为y=-3.354x+44.818,扩增效率为98.7%,标准曲线的相关系数为0.999;该方法可以特异性检测NDRV,但对MDRV、ARV、TMUV、DHAV均无反应信号;检测质粒标准品最低检测浓度为10拷贝μL;组内与组间变异系数均小于2%;用该方法检测临床样品,结果显示NDRV阳性率为45%,而常规RT-PCR 阳性率为33%,比常规RT-PCR敏感,可为NDRV病实验室诊断和早期流行病学调查的监测提供可靠的技术支撑。NDRV油乳剂灭活疫苗结果:将做好的油乳剂灭活疫苗滴在冷水中,除第一滴出现云雾状扩散外,以后各滴均不扩散,说明制备的疫苗为油包水剂型。吸取制备好的疫苗,垂直放出的平均时间为4.6 s/0.4 mL符合油乳剂灭活疫苗的粘度要求(2s～8s)。将做好的油乳剂灭活疫苗抽取1.5 mL放入2 mL离心管中,3000 rmp/min分离5min后未出现分层现象,说明该疫苗剂型稳定。将疫苗接种LB培养基中,37℃,12 h未发现细菌落生长,说明无菌检验合格。将疫苗接种1日龄樱桃谷鸭5只,每只肌肉注射0.5 mL,观察两周内鸭子状态,除刚打完疫苗有短暂的应激反应外,其他时间的精神状态、采食、饮水与排便等,均没有观察到不良反应,表明本疫苗安全性良好。抗NDRV高免卵黄抗体制备结果:用水稀释法1:7提取卵黄抗体,提取的卵黄抗体用0.1%的甲醛灭活后,未检测到细菌,接种1日龄雏鸭未出现局部炎性反应及全身性不良反应,表明该卵黄抗体安全性良好,并测得卵黄抗体效价约为1:161。抗NDRV高免卵黄抗体对NDRV保护效果研究结果:注射0.2 mL卵黄抗体不能有效阻止NDRV病感染,试验过程中,出现了不同程度的临床症状,在第3、5、7 d均能检测到泄殖腔排毒情况,剖检可观察到脾脏和肝脏有明显出血和坏死;而注射0.5和1 mL卵黄抗体,没有出现明显的NDRV病临床表现,在第3、5、7 d检测排毒情况时,只有0.5 mL卵黄抗体组在第3d时检测到一只有排毒情况,其余均未检测出排毒,剖检观察脾脏和肝脏无明显病变,说明本试验制备的效价为1:161卵黄抗体,0.5 mL以上剂量可以有效抑制NDRV在雏鸭体内的增殖而体阻止雏鸭发病,减小了该病在发病过程中大范围传播的危险。更多还原

【Abstract】 Novel duck reovirus(NDRV)disease is characterized by severe hemorrhage and necrosis of the spleen and liver.In recent years,the incidence rate of NDRV is 5%-35%,and the mortality rate is 2%,20%,with no obvious seasonality,which has seriously affected the healthy development of duck breeding in China.In this study,we collected liver necrosis and splenic necrosis tissues of ducks from a duck farm in Hebei province,which were suspected to be infected with NDRV,and inoculated them with 9-day-old SPF embryos for virus isolation,virus infection model,gene sequence analysis of NDRV S3 gene coding region,RT-PCR method for NDRV fluorescence quantification,preparation of inactivated oil emulsion vaccine,and preparation of anti-NDRV high immunity yolk antibodies.We will also evaluate the protective effect of anti-NDRV high immunity yolk antibodies to provide technical support for the prevention and control of NDRV.In this study,a strain of NDRV was successfully isolated and named as CH-HB strain.The biological characteristics of the virus were as follows:the ELD50 of the virus on chicken embryos was 1 × 10-5.5 ELD50/0.2 mL,and the virus could not agglutinate chicken erythrocytes.The results of the animal regression test showed that:the infected ducklings lost their appetite and some of them had tears in the corners of their eyes;during the observation period,there was no natural death of the ducklings;on autopsy,the liver of the infected ducklings was obviously enlarged,with different degrees of acicular necrosis;the triangular area of the spleen was hardened,and white necrotic foci of different sizes were scattered on the surface.Regional lymphocyte degeneration and necrosis,tissue loss of intrinsic structure,fatty degeneration of hepatocytes in liver tissue,focal hepatocyte necrosis;homology analysis showed that the nucleotide homology of the gene sequence of the S3 gene coding region of the isolated CH-HB strain(registration number:MT967492.1)and the gene sequence of the S3 gene coding region of the 16 reference viruses ranged from 66.5%to 99.5%,with The highest nucleotide homology of the NDRV Duck/N-DRV-XT 18/China/2018(Accession No.:MK749405.1)reference strain was 99.5%.The CH-HB strain was located in the NDRV branch and was the closest genetic distance to the S3 gene of Duck/N-DRV-XT18/China/2018(registration number:MK749405.1),and the most distant genetic distance to the S3 coding region of the ARV reference strain.The results showed that the standard curve equation of the TaqMan fluorescence quantitative RT-PCR method was y=-3.354x+44.818 with an amplification efficiency of 98.7%and a correlation coefficient of 0.999.The method can specifically detect NDRV.but not MDRV,ARV,TMUV,or NDRV.DHAV has no reaction signal;the minimum concentration of plasmid standard is 10 copies/μL;intra-and inter-group coefficient of variation is less than 2%;the results show that the positive rate of NDRV is 45%,while the positive rate of conventional RT-PCR is 33%,which is more sensitive than conventional RT-PCR,and can be used for laboratory diagnosis and early epidemiological investigation of NDRV disease.Provide reliable technical support.NDRV inactivated oleomargarine vaccine results:the prepared inactivated oleomargarine vaccine was put into cold water,except for the first drop,there was no diffusion in the following drops,which indicated that the prepared vaccine was a water-in-oil formulation.The average vertical release time of 4.6 s/0.4 mL of the aspirated prepared vaccine met the viscosity requirements of the inactivated oleomargarine vaccine(2 s-8 s).The inactivated oleomargarine vaccine was extracted 1.5 mL into a 2 mL centrifuge tube and separated at 3000 rmp/min for 5 min without stratification,indicating that the vaccine was stable.Inoculate the vaccine in LB medium at 37℃ for 12 h and no bacterial colonies were found,indicating that the aseptic test was passed.Except for a short period of stress after the vaccination,no adverse reactions were observed in the mental state,feeding,drinking and defecation of the ducks during the two weeks,indicating that the vaccine was safe.Anti-NDRV high immune egg yolk antibody preparation results:the yolk antibody was extracted 1:7 with water dilution method.After the extracted yolk antibody was inactivated with 0.1%formaldehyde,no bacteria were detected.and no local inflammatory reaction and local inflammatory reaction occurred after inoculation with 1-day-old ducklings.Systemic adverse reactions indicate that the yolk antibody is safe,and the measured yolk antibody titer is about 1:161.The results of the study on the protective effect of anti-NDRV high-flux yolk antibodies on NDRV were as follows:0.2 mL of yolk antibodies was not effective in preventing NDRV infection,and clinical symptoms appeared to varying degrees during the test.The clinical signs of NDRV disease were obvious.When the detoxification was detected at 3,5 and 7 days,only one detoxification was detected in the 0.5 mL vitellogenic antibody group at 3 days,but no detoxification was detected in the others.The proliferation of ducklings in the body prevents the disease from developing and reduces the risk of widespread spread of the disease.更多还原