DNA荧光染色和PCR技术在PRRSV冻干疫苗中支原体检测的应用

为完善猪繁殖与呼吸综合征病毒(PRRSV)冻干疫苗生产各阶段的支原体检测体系,本研究分别应用DNA荧光染色法、PCR法和病毒分离培养法检测疫苗生产中的细胞、种毒、半成品、成品,分析方法的可行性和优势.结果显示,DNA荧光染色法检测PRRSV半成品抗原液的支原体污染能够于6d内得到结果,与其余半成品检验项目用时相近,适宜在生产中应用.而采用DNA荧光染色、PCR和病毒分离培养法对我们生产的10批PRRSV冻干疫苗成品的支原体检测结果显示,DNA荧光染色法与病毒分离培养法检测的结果一致,1批疫苗的PCR检测结果与培养法检测结果不符,即PCR法检测阳性,但DNA荧光染色和病毒分离培养法检测为阴性.由此证明,DNA荧光染色法和PCR法检测疫苗中支原体的结果可靠,而PCR法检出率更高.DNA荧光染色法和PCR法操作简便、快速、经济,可以作为疫苗生产质量的内控标准,提高支原体的检出率,保证疫苗质量.     

标准化无菌及支原体检验培养基应用试验

经灵敏度检查合格的标准化无菌及支原体检验培养基已经在4个生物制药公司进行应用实验并且投入使用,分批进行抗原检测,99.5%的培养基经试验检测未被污染,可以投入使用。本文围绕这种无菌及支原体检验培养基进行研究分析,从材料和方法入手,最终进行检测实验以及方法探讨。     

猪瘟活疫苗(脾淋源)污染外源性兔出血症病毒的检测与启示

在进行2批猪瘟活疫苗（脾淋源）效力检验时,出现效力检验家兔突然死亡现象,为了查明家兔死亡原因,采用无菌检验、支原体检验、血凝试验、兔体中和试验、酶联免疫吸附试验（ELISA）对疫苗或注苗后死亡家兔肝脏、脾脏混合病料进行了检测。结果显示,疫苗的无菌检验、支原体检验结果均为阴性;疫苗及注射疫苗死亡家兔肝脏、脾脏混合病料具有较低的血凝价,血凝试验结果均为可疑;在兔体中和试验中,中和组家兔2／2健康存活,未中和的疫苗对照组家兔2／2死亡;疫苗及注射疫苗死亡后家兔肝脏、脾脏混合病料的ELISA检测结果均为阳性。检测结果证实疫苗中含有兔出血症病毒（Rabbit Haemorrhagic Disease Virus,RHDV）。猪瘟活疫苗（脾淋源）污染RHDV的现象启示：应该加强猪瘟活疫苗（脾淋源）抗原制备过程的控制;同时有必要对猪瘟活疫苗（脾淋源）质量标准进行修订使之进一步补充完善。     

禽呼肠孤病毒ZJS株种毒的纯净性鉴定

将禽呼肠孤病毒ZJS株细胞毒(弱毒)在鸡胚成纤维细胞(CEF)上连续传代,对其CI、C2、C3代种毒进行了无菌和支原体污染检验,对C5、C8和C10代种毒进行了外源病毒污染检验,对C3、C8和C10代种毒还进行了特异性鉴定,以确定禽呼肠孤病毒ZJS株细胞毒(弱毒)作为种毒的可行性.结果显示,禽呼肠孤病毒ZJS株细胞毒(...     

禽霍乱荚膜疫苗对SPF鸡免疫保护效果的研究

为了研究粗提的禽霍乱荚膜抗原对SPF鸡的免疫保护效果,试验选取了禽多杀性巴氏杆菌C48-1株,制备了粗提的荚膜抗原,将荚膜抗原和铝胶生理盐水按4∶1的比例混合,制备了两批疫苗(批号分别为2017001、2017002),在制备过程中检验了菌液的活菌数及荚膜抗原的蛋白质、SDS含量,并进行无菌检验。将制备的两批疫苗分别免疫21日龄SPF鸡,并于免疫后第21天用C48-1株进行攻菌,攻菌后连续观察7 d,每天记录鸡只死亡及发病情况。结果表明:经检验,两批疫苗蛋白质含量均在2. 0 mg/m L以上,SDS含量均为0. 03%,符合疫苗的质量标准。两批疫苗对42日龄SPF鸡的保护率为70%～80%。说明该疫苗对预防禽霍乱具有较好的效果。     

禽用活疫苗外源病毒污染情况的调查研究

为对禽用活疫苗污染外源病毒的情况进行彻底摸底调查,抽取了目前使用量大、覆盖面广的禽用活疫苗重点品种共8种28批,涉及18家国内生产企业、7家国外企业,疫苗生产用SPF鸡胚来自国内8家企业。按照《中国兽药典》2010年版三部鸡检查法和《欧洲药典》对抽检的禽病活疫苗进行了外源病毒污染和禽腺病毒污染检验。结果显示所抽检的禽用活疫苗均无外源病毒污染和禽腺病毒污染,表明我国禽用活疫苗的质量控制和安全性良好。     

猪圆环病毒2型灭活疫苗效力检验中参考疫苗的应用

制备了4批不同抗原含量的猪圆环病毒2型（PCV2）灭活疫苗,灭活前PCV2病毒含量分别为107.0、106.5、105.5和104.5TCID50/mL。对此4批PCV2灭活疫苗,利用本实验室建立的小白鼠效力检验标准进行效力检验,即以PCV2参考疫苗作为对照品,测定待检疫苗免疫小白鼠后血清中PCV2抗体效价的高低来判定疫苗效力。3次重复检测结果都显示灭活前病毒含量为107.0、106.5和105.5TCID50/mL批次的疫苗合格,而灭活前病毒含量为104.5TCID50/mL批次的疫苗不合格,这与猪体内的效力检验结果一致,表明建立的针对猪圆环病毒2型灭活疫苗的小白鼠效力检验方法具有良好的准确性和重复性。     

血清3型鸭甲型肝炎病毒阳性血清的制备

通过使用血清3型鸭甲型肝炎疫苗免疫SPF鸡,制备了一批血清3型鸭甲型肝炎诊断用阳性血清,并对其进行了无菌、支原体和特异性检验以及中和效价测定。结果表明,该血清无菌检验合格,无支原体污染,特异性良好,无禽类常见13种外源病毒抗体,血清中和效价为10-2.8。本研究为鸭甲型肝炎病毒血清学鉴定及相关研究提供了重要的物质基础。     

牦牛大肠埃希氏菌病蜂胶疫苗的研制

作者制备了3批牦牛大肠埃希氏菌病蜂胶疫苗。制备了蜂胶佐剂和培养基,进行了牦牛大肠埃希氏菌的菌液培养、纯粹检验、活菌计数、灭活及无菌检验,配苗、分装及成品检验。3批牦牛大肠埃希氏菌病蜂胶疫苗无菌检验为阴性;物理性状检验为：静置后上层是乳黄色液体,下层为土黄色沉淀,振荡后呈均匀混浊液;经家兔安全检验结果为安全;经家兔效力试验,免疫后家兔抗体效价≥12．709lg2,对家兔具有免疫保护作用;蜂胶疫苗经防冻试验证明,随着蜂胶含量的增高,防冻效果越好。本试验为西藏牦牛大肠埃希氏菌病的预防提供依据。     

鸡新城疫和传染性法氏囊病二联灭活疫苗的制备及检验

采用NDV La Sota株毒种接种鸡胚制备NDV抗原液,IBDV BJQ902细胞适应毒接种鸡胚成纤维细胞制备IBDV抗原液,用甲醛溶液灭活后,按一定比例混合,加入油佐剂乳化制成ND-IBD二联灭活疫苗。共制备了3批疫苗,对其进行各项检验。结果表明,疫苗无菌;外观为乳白色均匀乳剂,剂型为W/O/W型,黏度为1.0 s~1.2 s,粒度均匀,99%以上为1μm,个别为2μm~3μm,3 500 r/min离心15 min,管底出水在0.5 mm以下;疫苗安全性检验,接种24日龄~28日龄SPF鸡后,观察14 d,均无任何局部及全身不良反应;疫苗ND部分效检,免疫接种3周后ND HI抗体平均滴度为3.0~3.6 log2,攻毒后保护率分别为9/10、9/10和10/10,IBD部分效检免疫接种后4周,中和抗体滴度分别20 171、24 578及26 616,攻毒保护率均为100%(10/10)。结果证实3批疫苗均符合要求。     

Sensitivity and specificity of Mycoplasma gallisepticum agglutination antigens prepared from medium with artificial liposomes substituting for serum

Three batches of strain A5969 Mycoplasma gallisepticum (MG) serum-plate-agglutination (SPA) antigen grown in regular Frey's medium with 12% swine serum, three batches grown in Frey's medium containing artificial liposomes instead of serum, and one commercial SPA antigen were evaluated for sensitivity and specificity. Sensitivity was measured using chickens exposed to MG by intraocular and intranasal inoculation. Specificity was measured in uninoculated controls and in groups inoculated with the oil-emulsion vaccines Haemophilus paragallinarum, infectious bursal disease inactivated virus vaccine, or Staphylococcus aureus. Sera were tested 1 to 8 weeks postinoculation. All SPA antigens had a perfect sensitivity score, except one liposome-grown antigen batch (LC). The two other liposome-grown antigen batches (LA and LB) maintained significantly higher specificity by yielding significantly (P less than 0.01) fewer false positive (FP) agglutination reactions than did the other antigens. The three antigen batches produced in medium with serum had intermediate levels of FP agglutination reactions. When known MG-negative sera were tested, MG SPA antigens LC and commercial SPA antigen yielded significantly (P less than 0.01) higher numbers of FP agglutination reactions than the other SPA antigens.     

猪支原体肺炎灭活疫苗的免疫效果研究

对猪肺炎支原体（J株）进行培养后,采用新型工艺制备了4批猪支原体肺炎灭活疫苗。并对疫苗进行了安全性、免疫效果、免疫期检验。结果表明该灭活疫苗对猪安全有效,并且可使免疫猪的肺炎减少率达到75％以上,免疫期可达到6个月以上。     

Effect of thimerosal concentration on the efficacy of inactivated Newcastle disease oil-emulsion vaccines

Different quantities of the preservative thimerosal in inactivated Newcastle disease oil-emulsion vaccines were tested to determine the influence on the hemagglutination-inhibition (HI) response of broilers. The effect of thimerosal was measured in vaccines that had been stored for 1, 21, and 52 weeks; HI serology was conducted at 2, 4, and 6 weeks after vaccination. Mean HI titers 4 weeks after vaccination decreased at a significant rate (P less than or equal to 0.001) with increasing concentrations of thimerosal. HI titers 4 weeks after vaccination with 1-week-old vaccine were significantly (P less than or equal to 0.05) higher than those after vaccination with 52-week-old vaccine at all thimerosal concentrations tested. Titers were also significantly higher (P less than or equal to 0.05) after vaccination with 1-week-old vaccine than after vaccination with 21-week-old vaccine at all thimerosal concentrations below about 8.25 mg/ml of antigen. Thimerosal at the levels recommended in commercial vaccines does not significantly decrease vaccine efficacy.     

Evaluation of the the effectiveness of a newly developed tissue-inactivated vaccine against rabies using the NIH test

NIH test was used to verify 12 operation batches of the newly developed inactivated tissue vaccine against rabies. The number of international units per 1 cm3 of vaccine was determined by the volumetric method. The number of international units (I.U.) per 1 cm3 of the newly developed vaccines ranged from 1.08 I.U. to 2.59 I.U. per cm3. The average value of all the 12 batches tested is 1.52 I.U. per cm3. The average value of international units in the vaccination dose for dogs is 4.57. All the tested operation batches of the newly developed vaccines met the activity standard recommended by the World Health Organization for veterinary vaccines of this type.     

鸡痘病毒活疫苗污染禽网状内皮组织 增生症病毒的间接免疫荧光法检测研究

通过向无外源病毒污染的鸡痘病毒活疫苗中添加不同剂量的禽网状内皮组织增生症病毒(REV),然后用间接免疫荧光法(IFA)进行检测,确定了该IFA方法的最低检出量为每500羽份疫苗中污染20 TCID_(50)的REV。使用该方法对国内16家企业生产的60批鸡痘病毒活疫苗进行了检验,结果显示2个企业生产的4批疫苗REV检测为阳性。随机选取5批IFA检测阴性样品和4批IFA检测阳性样品,按鸡检查法进行外源病毒检验,结果两种方法对REV污染的检测结果的符合率为100%。     

纸片微载体培养BHK-21(C-13)细胞制备兽用狂犬冻干活疫苗

为了用激流式生物反应器纸片微载体培养BHK-21(C-13)细胞制备兽用狂犬冻干活疫苗,采用纸片微载体培养BHK-21(C-13)细胞,接种狂犬病病毒Flury株(LEP),测定病毒液滴度.将收获的病毒液制备兽用狂犬冻干活疫苗,按照狂犬病活疫苗规程进行检验.结果显示,3批收获的病毒液滴度平均达1050LD50/0.03 mL;3批实验疫苗检验结果均符合质量标准.实验证明激流式生物反应器纸片微载体培养BHK-21(C-13)细胞制备兽用狂犬冻干活疫苗提高了细胞密度、病毒滴度,增加了病毒液收获体积,减小了批间差,保证了产品均一性,适合工业化大生产.     

A reduction in the duration of infection with Tritrichomonas foetus following vaccination in heifers and the failure to demonstrate a curative effect in infected bulls

Seven batches of 25% water-phase, oil-in-water vaccine were prepared from whole cultures of Tritrichomonas foetus. Two inoculations were given, spaced 6 weeks apart, to virgin heifers and infected bulls. A significant reduction (P less than 0.01) in the duration of infection in vaccinated heifers was seen when they were challenged by being bred to a bull infected with the same isolate as that contained in the vaccine. Only 1/12 vaccinated heifers were pregnant 4.5 months after the end of the breeding season compared to 2/12 in the control group. The vaccine, therefore, has no practical advantage. Vaccine was supplied to 2,724 bulls on properties where the infection was present. From these bulls, 110 reliable results were obtained, where bulls had been infected, been inoculated and tested 1 month later. No curative effect was demonstrable with 69/110 (62.7%) bulls, remaining infected after the course of inoculations. There was also no difference between vaccine batches or between bulls of different ages. Further work on improving the vaccine is indicated. Three media suitable for the culture of T. foetus are described in detail.