鸭副黏病毒病诊断与同源疫苗的研制及初步应用
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摘要
新城疫是由新城疫病毒引起的一种高死亡率高传染性的疾病，为世界卫生组织公布的必报类动物疫病之一，每年都给养禽业造成严重的经济损失。虽然新城疫病毒只有一个血清型，但根据种系发生的重建，新城疫病毒被分成两个组，classI和class II，class I有9个基因型而class II有10个基因型，并且随着外界环境中的种种压力因素，新城疫病毒也在不断进化。实践中，鸡源新城疫疫苗用来预防鸭源新城疫效果不理想，亟需同源疫苗的创制。所以通过临床及病理解剖等综合诊断来提前快速确诊新城疫非常必要，可以有效的控制新城疫的发展，做到尽早预防，防止新城疫病毒的进一步扩散，把损失降到最低点。
由于新城疫疫苗种类繁多，疫苗使用操作方法不一，往往会因疫苗的选择和使用不当造成免疫失败，给养禽业带来极大的经济损失。DNA疫苗是一种基因工程的疫苗，是通过肌肉注射或者皮下注射含有目的基因的质粒。这个转基因编码来自一个病原的目的蛋白的序列，并存在于真核表达启动子的控制下。基因工程类疫苗比传统的疫苗有很多优势，包括安全性，制作简单，稳定性等方面。DNA疫苗可以诱导长效的细胞免疫应答和体液免疫应答，并且不恢复成强毒株，在安全性上有了保障，所以DNA疫苗是今后疫苗发展的一个重要方向。
本研究以吉林省吉林市经济技术开发区四方养殖场饲养的1500只蛋鸭临床病例为基础，首先通过临床剖检来进行新城疫的初步诊断，然后将疑似病料进行系统的实验室检测，包括细菌学鉴定和病毒学鉴定。对新城疫的初步临床诊断结果显示，该病例为新城疫。通过细菌学鉴定，该病例混有大肠杆菌感染。通过病毒学鉴定，新城疫病毒被分离出来，该病例为新城疫病毒同致病性大肠杆菌混合感染。由于分离毒株与本实验室保存的DP1/02株鸭源副黏病毒株遗传距离相近，均为强毒株，本试验研制了DP1/02株鸭源副黏病毒灭活疫苗，并进行了初步应用研究。
根据本实验室分离的鸭源副黏病毒DP1/02株F基因序列，设计引物，克隆F基因到真核表达载体pCI-neo中，构建重组真核表达质粒pCI-F，并进行分子生物学相应的鉴定。结果表明，表达鸭源副黏病毒F基因的重组真核表达质粒构建成功。将构建好的重组真核表达质粒进行动物免疫试验，通过攻毒保护性试验，运用血凝抑制试验和酶联免疫吸附试验来分别测定体液免疫应答水平和细胞免疫应答的水平，进而来评价免疫效果。血凝抑制试验结果显示，pCI-F重组质粒诱导机体产生较高水平的体液免疫应答反应，ELISA试验结果显示，pCI-F重组质粒可以诱导机体产生较高的细胞免疫应答反应。攻毒保护性试验结果显示，pCI-F重组质粒对NDV强毒的攻击具有一定的保护作用，其保护率为73%。
综上所述，根据实验室诊断证实了临床诊断的有效性，通过临床症状可以提前对感染新城疫的病禽进行隔离、扑杀，对新城疫的早期诊断和防控有非常重要的意义。成功构建的重组真核表达质粒pCI-F可以有效的诱导机体产生细胞免疫和体液免疫，攻毒保护性试验证明该质粒可以有效的保护NDV的攻击。研制了DP1/02株鸭源副黏病毒灭活疫苗，该疫苗免疫保护率为100%。该研究为防控鸭新城疫体系的完善提供了理论基础。
Newcastle disease (ND) is a viral disease of poultry caused by a single-strand,nonsegmented, negative-sense RNA virus known as Avian paramyxovirus1(APMV-1). The disease is present worldwide and affects many species of birds causing severelosses in the poultry sector. Although all NDV isolates belong to a single serotype(APMV-1), there is great genetic variability among different strains. Based uponphylogenetic reconstruction, NDV can be divided into2classes (I and II), each ofthose respectively subdivided into9and10genotypes.Since the environment whichvirus exist become worse and worse, the virus generate genetic variability to accom--modate the new environments. The coinfection between virus and bacteria has beenobserved. The NDV vaccine from chicken can not protect the duck resist NDVinfection, it is necessary to prepare homologous vaccine. The clinical diagnosis notonly identifies the disease in earlier period but also limits the shedding of the virus. Sothe clinical diagnosis is very important.
Since the great genetic variability among different NDV strains and the methodof operation Newcastle Disease vaccine has difference, the failure of immune oftenappear and cause severe losses in the poultry industry.Genetic vaccines havenumerous advantages over traditional vaccines in terms of safety, ease ofmanufacturing and stability. Possibly the best current vaccine platform that inducesboth humoral and cellular immune responses is the use of live-attenuated pathogens;however, this also poses important safety concerns with the risk of reversion of thepathogen to a more virulent form. DNA vaccines can also induce both long-lastingcellular and humoral immune responses but do not revert into virulence, and thereforeraise fewer safety concerns. In addition, clinical trials with DNA vaccines have shownfewer incidences of systemic adverse effects such as redness, swelling, fever and headache. So the research of DNAvaccine is a important purpose in the future.
First of all, clinical diagnosis has been applied on the base of economic technicaldevelopment area JiLin city JiLin province si fang duck nursery for identification theNewcastle Disease. Subsequently, the borderline cases are identified by virusdiagnosis and bacteria diagnosis in laboratory. The result of bacteria diagnosisanalysis shows that the borderline cases are infected by Bacillus coli. The result ofvirus diagnosis analysis shows that the borderline cases are infected by NewcastleDisease Virus. So the ducks are infected by Bacillus coli and Newcastle Disease Virus.In addition, inactivated vaccine of Duck Newcastle Disease Virus were prepared andidentified.
According to the sequence of Duck Newcastle Disease Virus F gene which wasisolated from duck by our laboratory, the F gene primers are designed by primer5.0.The F gene is cloned into eukaryotic expression vector pCI-neo named pCI-F. pCI-Fis identified by molecular biology method as PCR and indirect immunofluorescence.The identification shows that pCI-F is constructed successfully.
Animal immune experiment is performed with pCI-F. In order to assess thecellular immune respond level and the humoral immune respond level, the F geneantibody level and γ-IFN level are measured by HI assay and ELISA. Subsequent, theanimal protection experiment is performed. The HI assay and ELISA results show thatpCI-F induced F gene antibody level less than inactivated vaccine and pCI-F inducedγ-IFN level more than inactived vaccine and similar with attenuated vaccine. Theanimal protection experiment result shows that pCI-F can protect velogenic viruschallenge and the protection rate is73%, the protection rate of inactivated vaccine ofDuck Newcastle Disease Virus is100%.
This research is a basis of the duck NDV DNA vaccine for prevention in thefuture.
由于新城疫疫苗种类繁多，疫苗使用操作方法不一，往往会因疫苗的选择和使用不当造成免疫失败，给养禽业带来极大的经济损失。DNA疫苗是一种基因工程的疫苗，是通过肌肉注射或者皮下注射含有目的基因的质粒。这个转基因编码来自一个病原的目的蛋白的序列，并存在于真核表达启动子的控制下。基因工程类疫苗比传统的疫苗有很多优势，包括安全性，制作简单，稳定性等方面。DNA疫苗可以诱导长效的细胞免疫应答和体液免疫应答，并且不恢复成强毒株，在安全性上有了保障，所以DNA疫苗是今后疫苗发展的一个重要方向。
本研究以吉林省吉林市经济技术开发区四方养殖场饲养的1500只蛋鸭临床病例为基础，首先通过临床剖检来进行新城疫的初步诊断，然后将疑似病料进行系统的实验室检测，包括细菌学鉴定和病毒学鉴定。对新城疫的初步临床诊断结果显示，该病例为新城疫。通过细菌学鉴定，该病例混有大肠杆菌感染。通过病毒学鉴定，新城疫病毒被分离出来，该病例为新城疫病毒同致病性大肠杆菌混合感染。由于分离毒株与本实验室保存的DP1/02株鸭源副黏病毒株遗传距离相近，均为强毒株，本试验研制了DP1/02株鸭源副黏病毒灭活疫苗，并进行了初步应用研究。
根据本实验室分离的鸭源副黏病毒DP1/02株F基因序列，设计引物，克隆F基因到真核表达载体pCI-neo中，构建重组真核表达质粒pCI-F，并进行分子生物学相应的鉴定。结果表明，表达鸭源副黏病毒F基因的重组真核表达质粒构建成功。将构建好的重组真核表达质粒进行动物免疫试验，通过攻毒保护性试验，运用血凝抑制试验和酶联免疫吸附试验来分别测定体液免疫应答水平和细胞免疫应答的水平，进而来评价免疫效果。血凝抑制试验结果显示，pCI-F重组质粒诱导机体产生较高水平的体液免疫应答反应，ELISA试验结果显示，pCI-F重组质粒可以诱导机体产生较高的细胞免疫应答反应。攻毒保护性试验结果显示，pCI-F重组质粒对NDV强毒的攻击具有一定的保护作用，其保护率为73%。
综上所述，根据实验室诊断证实了临床诊断的有效性，通过临床症状可以提前对感染新城疫的病禽进行隔离、扑杀，对新城疫的早期诊断和防控有非常重要的意义。成功构建的重组真核表达质粒pCI-F可以有效的诱导机体产生细胞免疫和体液免疫，攻毒保护性试验证明该质粒可以有效的保护NDV的攻击。研制了DP1/02株鸭源副黏病毒灭活疫苗，该疫苗免疫保护率为100%。该研究为防控鸭新城疫体系的完善提供了理论基础。
Newcastle disease (ND) is a viral disease of poultry caused by a single-strand,nonsegmented, negative-sense RNA virus known as Avian paramyxovirus1(APMV-1). The disease is present worldwide and affects many species of birds causing severelosses in the poultry sector. Although all NDV isolates belong to a single serotype(APMV-1), there is great genetic variability among different strains. Based uponphylogenetic reconstruction, NDV can be divided into2classes (I and II), each ofthose respectively subdivided into9and10genotypes.Since the environment whichvirus exist become worse and worse, the virus generate genetic variability to accom--modate the new environments. The coinfection between virus and bacteria has beenobserved. The NDV vaccine from chicken can not protect the duck resist NDVinfection, it is necessary to prepare homologous vaccine. The clinical diagnosis notonly identifies the disease in earlier period but also limits the shedding of the virus. Sothe clinical diagnosis is very important.
Since the great genetic variability among different NDV strains and the methodof operation Newcastle Disease vaccine has difference, the failure of immune oftenappear and cause severe losses in the poultry industry.Genetic vaccines havenumerous advantages over traditional vaccines in terms of safety, ease ofmanufacturing and stability. Possibly the best current vaccine platform that inducesboth humoral and cellular immune responses is the use of live-attenuated pathogens;however, this also poses important safety concerns with the risk of reversion of thepathogen to a more virulent form. DNA vaccines can also induce both long-lastingcellular and humoral immune responses but do not revert into virulence, and thereforeraise fewer safety concerns. In addition, clinical trials with DNA vaccines have shownfewer incidences of systemic adverse effects such as redness, swelling, fever and headache. So the research of DNAvaccine is a important purpose in the future.
First of all, clinical diagnosis has been applied on the base of economic technicaldevelopment area JiLin city JiLin province si fang duck nursery for identification theNewcastle Disease. Subsequently, the borderline cases are identified by virusdiagnosis and bacteria diagnosis in laboratory. The result of bacteria diagnosisanalysis shows that the borderline cases are infected by Bacillus coli. The result ofvirus diagnosis analysis shows that the borderline cases are infected by NewcastleDisease Virus. So the ducks are infected by Bacillus coli and Newcastle Disease Virus.In addition, inactivated vaccine of Duck Newcastle Disease Virus were prepared andidentified.
According to the sequence of Duck Newcastle Disease Virus F gene which wasisolated from duck by our laboratory, the F gene primers are designed by primer5.0.The F gene is cloned into eukaryotic expression vector pCI-neo named pCI-F. pCI-Fis identified by molecular biology method as PCR and indirect immunofluorescence.The identification shows that pCI-F is constructed successfully.
Animal immune experiment is performed with pCI-F. In order to assess thecellular immune respond level and the humoral immune respond level, the F geneantibody level and γ-IFN level are measured by HI assay and ELISA. Subsequent, theanimal protection experiment is performed. The HI assay and ELISA results show thatpCI-F induced F gene antibody level less than inactivated vaccine and pCI-F inducedγ-IFN level more than inactived vaccine and similar with attenuated vaccine. Theanimal protection experiment result shows that pCI-F can protect velogenic viruschallenge and the protection rate is73%, the protection rate of inactivated vaccine ofDuck Newcastle Disease Virus is100%.
This research is a basis of the duck NDV DNA vaccine for prevention in thefuture.
引文
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