【作者】 丛华；

【导师】 高崧； 彭大新； 田龙；

【作者基本信息】 扬州大学 ， 兽医， 2006， 硕士

【摘要】 按照《中华人民共和国兽用生物制品规程（2000年版）》（以下简称规程）中的《鸡新城疫低毒力活疫苗制造及检验规程》及OIE的标准方法对从中国兽药监察所购买的新城疫病毒（NDV）Lasota株冻干种毒进行种毒鉴定，包括病毒的SPF鸡胚复壮及传代，病毒的平均死亡时间（MDT）和脑内致病指数（ICPI），对鸡胚的毒力测定，病毒含量的测定，安全性，纯净性检验，特异性检验以及免疫原性的测定，结果表明所采用的Lasota毒株是稳定的，符合ND低毒力活疫苗株的要求。按照规程中的《鸡传染性法氏囊病中等毒力活疫苗制造及检验规程》及OIE的标准方法对来自扬州大学农业部畜禽传染病学重点开放实验室的传染性法氏病毒（IBDV）NF₈株进行种毒鉴定，包括毒种的病毒含量的测定、对鸡胚及雏鸡的毒力测定、对雏鸡的安全性试验、毒力稳定性试验、抗体产生期试验、免疫原性的测定、免疫抑制安全性试验、特异性检验以及纯净性检验。结果表明所采用的IBDV．NF₈株毒力稳定、无免疫抑制作用、能突破母源抗体干扰、免疫原性好，是合适的生产鸡传染性法氏囊病中等毒力疫苗的毒种。采用经鉴定合格的鸡新城疫低毒力弱毒株（Lasota株）和鸡传染性法氏囊病中等毒力株（NF₈株），分别接种SPF鸡胚，收获感染Lasota尿囊液和NF₈绒毛尿囊膜及鸡胚组织磨制混合液，进行无菌检验和效价测定，合格后以1:1比例混合，加稳定剂后，经冷冻真空干燥制成一批鸡新城疫（Lasota株）和鸡传染性法氏囊病（NF₈株）二联活疫苗（以下简称二联苗）。检测该批制品，物理形状、无菌检验、外源病毒检验、支原体检验为合格。其新城疫病毒含量分别为10^7.0EID₅₀／羽份，法氏囊病毒含量分别为10^4.50ELD₅₀／羽份。用10个使用剂量及1个使用剂量的二联苗接种SPF雏鸡，同时将不免疫的SPF鸡与免疫鸡关在同一笼内一起饲养21天，进行安全性试验及免疫扩散试验。对于10个使用剂量组，观察14天，全部健活，剖检10个使用剂量组和对照组部分鸡，无新城疫及传染性法氏囊病特异性病理变化，证明该批疫苗安全性良好；分别于免疫后1周、2周、3周采血测NDV的血凝抑制（HI）抗体和IBDV的中和（SN）抗体，于3周时对免疫鸡和非免疫鸡分别攻击ND、IBD强毒，观察免疫鸡和未免疫鸡的保护率。结果表明：当非免疫SPF鸡与二联苗免疫过的SPF雏鸡同居一定的时间后，能使非免鸡感染并产生相应的NDV HI抗体和IBDV SN抗体。分别攻击ND强毒和IBD强毒后不会引起免疫鸡及同笼饲养未免疫鸡产生ND发病症状及法氏囊病变；攻毒后保护指数皆为100％，完全能起到免疫及扩散保护作用。用ND病毒含量分别为10⁴EID₅₀、5000EID₅₀、10³EID₅₀的二联苗免疫SPF雏鸡后，攻击ND强毒；用IBD病毒含量为300ELD₅₀、200ELD₅₀、100ELD₅₀二联苗免疫SPF雏鸡后，攻击IDB强毒进行二联苗的最小免疫量试验。结果表明，二联苗的最小免疫量ND为5000 EID₅₀，IBD为200ELD₅₀。用二联苗、鸡新城疫（Lasota株）单苗和鸡传染性法氏囊病（NF₈株）单苗分组免疫7日龄SPF雏鸡及有母源抗体的雏鸡。对SPF雏鸡免疫后1周、2周、3周分别采血测抗体并在3周后分别攻击ND和IBD强毒。对有母源抗体的雏鸡实验分两部分进行，第一部分（一免组）：一免后1周、2周、3周分别采血测抗体并在3周后分别攻击ND和IBD强毒；第二部分（二免组）：一免后10天进行第二次免疫，其余步骤同一免组。结果表明：用二联活疫苗免疫SPF雏鸡后，NF₈株与Lasota株间不产生明显的相互干扰作用，攻毒保护率与各自的单苗相比无明显差异。用二联活疫苗免疫有母源抗体的雏鸡后，如果只进行一次免疫，新城疫免疫可供完全保护，法氏囊免疫效果稍差，不能完全保护，但攻毒保护率与各自的单苗相比无明显差异；如果二次加强免疫，其抗体水平、攻毒后的保护率与各自的单苗相比无明显差异，具有完全保护效力。用二联苗免疫7日龄SPF鸡，6周、7周、8周、9周后采血测抗体并分别攻击ND强毒、IBD强毒后计算保护指数，其ND和IBD的免疫保护期分别在9周以上和7周左右。更多还原

【Abstract】 The Newcastle disease vires （NDV） strain Lasota bought from China Institute ofVeterinary Drug Control was characterized according to the methods of OIE and "themanufacture and inspection regulations for newcastle disease low virttlence livevaccine" in "Regulations of the people’s republic of china on biological products foranimal use（edition 2000）" （Thereafter called "the regulations"）. The NDV swainLasota was rejuvenated and passaged for several generations by inoculation of SPFchicken embryos. The mean death time of the chicken embryos （MDT）, theintracerbral pathogenicity index （ICPI）, the pathogenicity and viral fiter of the NDVstrain Lasota were determined, and inspection of the safety, purity, specificity andimmunogenicity of the NDV strain Lasota were also performed. The results showed thatNDV strain Lasota we used was steady and suitable for producing NDV live vaccine.The infectious bursal disease virus （IBDV） strain NFs from the key laboratory ofanimal infectious disease, ministry of agriculture, Yangzhou University wascharacterized according to the methods of OIE and "manufacture and inspectionregulations for chicken infections bursal disease middling virulence live vaccine" in theregulations. The tests included determination of the purity, specificity, stability, titer,virulence, immunogenicity, immune suppression of IBDV sWain NF₈. The resultsshowed that the infectious bursal disease vires strain NF₈ we used was steady, and it has no immune suppression, it could break through the maternal antibody, it’simmunogenicity was good enough for producing IBDV live vaccine.The above NDV strain Lasota and IBDV strain NF₈ were inoculated into SPFchicken embryos separately. Then the allantoic fluid from NDV infected embryos andCAM and chick embryos from IBDV infected embryos were collected. After the axenicinspection and titer test, the two kinds of semi-finished products were mixed in a 1:1ratio, and stabilizer were added. We gained a batch of ND-IBD two-combined livevaccine （Thereafter called the two-combined live vaccine） through lyophilization.Whereafter the batch of vaccine was evaluated for physical character, axenic inspection,exogenous virus inspection and mycoplasma inspection, and all test were passed. TheNDV and IBDV titers in the vaccine were 10^7.0EID₅₀/dose and 10^4.5EID₅₀/dose,respectively.Seven-day-old SPF chickens were vaccinated with 10 dose and 1 dose of thetwo-combined live vaccine for safety test, at same time the immunized SPF chickenswere kept in same isolator with unvaccinated SPF chicken for test of contacttransmission. We found them all in good health at 14 days. And there are no specificpathological lession of ND and IBD over all dissected body and the bursal ofimmunized chickens with 10-fold dosage and chickens in same isolator withoutimmunization, as well as in negative control group. Those data indictaed that thetwo-combined live vaccine was safe. Vaccinated chiken were bled for ND HI tests andIBD serum neutralization （SN） antibody tests at 1 week, 2 weeks and 3 weeks intervals.At 3 weeks post-vaccination the vaccinated groups and the nonimmune group werechallenged with virulent NDV or IBDV separately. The results showed: UnvaccinatedSPF chicken could produce relevant IBD SN antibody and ND HI antibody when theunvaccinated SPF chicken were contacted with the chickens vaccinated by thetwo-combined live vaccine for some times. The protection index （PI） of vaccinated andunvaccinated chickens against vNDV or vlBDV are all 100%. So the chickens could be infected by two-combined live vaccine.Seven-day-old SPF chicken were immunised by the two-combined live vaccine ingroups, the dosage of ND was 10⁴EID₅₀, 5000EID₅₀, 10³EID₅₀. respectively; anddosage of IBD was 300 ELD₅₀,200ELD₅₀, and 100ELD₅₀, respectively. The first threevaccinated groups were challenged by vNDV and the last three vaccinated groups werechallenged by vIBDV at 3 weeks post-vaccination. The results showed that theminimum immunal dosage of the two-combined live vaccine was 5000EID₅₀ for ND,and 200ELD₅₀ for IBD respectively.Seven-day-old SPF chicken and chicken with maternal antibody were grouped andimmunised by the two-combined live vaccine, ND monovalent vaccine and IBDmonovalent vaccine, respectively. For the chicken with maternal antibody, the test wasdivided into 2 trials. The 1st trail was same as 7-day-old SPF chicken. The abovevaccinated chicken were bled for ND HI tests and IBD SN antibody tests at 1 week, 2weeks and 3 weeks intervals. At 3 weeks post-vaccination the vaccinated groups and thecontrol group were challenged by vNDV or vIBDV separately. The 2st trail was carriedout using boost immunization at 10 days after prime immunization, and the other stepswas same as the 1st trail. For 7-day-old SPF chickens, there is no siginificant different inantibody response between the two-combined live vaccine group and the correspondingmonovalent vaccine group. For the chickens with maternal antibody, the immuneefficiency was good enough for the NDV strain Lasota, but not so good for IBDV strainNF₈ to protect all chicken. Moreover, referring to the antibody level and protection ratio,there is no significant difference between the two-combined live vaccine group and thecorresponding monovalent vaccine groups if they were boosted at 10 dayspost-vaccination. All groups could provide 100% protection against ND and IBD.Seven-day-old SPF chickens were grouped and immunised by the two-combinedlive vaccine. The vaccinated groups and the control group were challenged for vNDVor vIBDV separately at six weeks, seven weeks, eight weeks, nine weeks post-vaccination. The data indicated the immune duration of the two combinatedvaccine was at least 9 weeks for ND and 7 weeks for IBD, respectively.更多还原