羊传染性脓疱皮炎弱毒苗耐热保护剂初步优化筛选试验研究

试验以羊传染性脓疱皮炎弱毒活疫苗为对象,依据冻干保护剂的保护功能,对耐热保护剂的组成、混合比例进行了初步筛选。通过反复试验,对其物理性状观察、水分含量测定、冻干前后病毒损失量、经37℃处理、25℃保存后病毒损失量等指标的比较进行筛选,优选出了符合要求的A3、A6、C4三个耐热保护剂配方。试验结果表明,使用保护剂的羊口疮疫苗在37℃保存20 d,病毒含量下降0.5~0.7个滴度,常规疫苗下降2.3个滴度;25℃保存60d,病毒含量下降0.3~0.6个滴度,常规疫苗下降2.5个滴度。从而证明了羊orf耐热活疫苗耐热效果优于常规疫苗。     

鸡新城疫、传染性支气管炎二联耐热保护剂活疫苗中试示范项目启动

前不久,国家农业科技成果转化资金"鸡新城疫、传染性支气管炎二联耐热保护剂活疫苗中试示范"项目启动会在山东省滨州市召开。"鸡新城疫、传染性支气管炎二联耐热保护剂活疫苗中试示范"项目计划实施期为2年。该疫苗产品保存条件为2℃～8℃,有效期为24个月,降低了常规疫苗的保存条件,延长了保存期。     

鸡痘耐热保护剂活疫苗的研究

选择不同基质组成保护剂与鸡痘抗原进行冻干试验，试制一种适宜鸡痘弱毒株的耐热冻干保护剂。应用耐热冻干保护剂和专用的冻干曲线，制成鸡痘耐热保护剂活疫苗，经37℃保存10d的耐老化试验，每羽份病毒含量下降0．2—0．4个滴度；疫苗在2—8℃保存24个月后，每羽份病毒含量下降0．2-0．4个滴度，且均≥10^3.0EID50／羽，具有良好的热稳定性。19万羽的田间试验证明，鸡痘耐热保护剂活疫苗安全、有效。     

鸡传染性支气管炎耐热活疫苗的研究

应用鸡传染性支气管炎病毒(H52株)与不同耐热保护剂配方和冻干曲线进行筛选.结果表明:采用冻干曲线2和耐热保护剂WXS冻干鸡传染性支气管炎病毒H52弱毒疫苗效果最佳.试制耐热活疫苗3批,其各项检验均符合要求.耐老化试验表明:经过37℃放置10d的每羽份病毒含量下降0.6～0.8个滴度,每羽份病毒含量达104.7～105.1EID50.保存期试验表明:2～8℃保存24个月疫苗每羽份病毒含量下降0.4～0.6个滴度,每羽份病毒达104.1～104.3EID50.疫苗2～8 ℃保存24个月后效检抗体水平达到1:16～1:32.免疫持续期试验表明:免疫21日龄SPF鸡,6个月后血清中和抗体效价达到1:32～1:64.田间试验表明临床观察无不良反应,安全有效.特异性试验表明耐热活疫苗抗原性不变.耐热保护剂生物学和理化特性表明保护剂安全、稳定,室温保存期达3个月.     

鸡传染性支气管炎活疫苗耐热保护剂的研究

应用冻干制品加速热稳定性试验,测试三组冻干保护剂配方对鸡传染性支气管炎(H120株)活疫苗的耐热保护效果。结果表明A配方的耐热保护剂效果最好,使用该保护剂的疫苗表现为37℃放置10d后,病毒含量下降在0.6~0.8个滴度。     

猪繁殖与呼吸综合征活疫苗耐热冻干保护剂的研究

为了解耐热冻干保护剂在猪繁殖与呼吸综合征活疫苗中的应用效果,将高致病性猪繁殖与呼吸综合征病毒(PPRSV)分别与耐热冻干保护剂和常规冻干保护剂进行配比、冻干,制备疫苗,分别将疫苗置2~8℃保存,定期测定其效价,同时进行37℃、25℃耐老化试验。结果显示,疫苗在2~8℃条件下保存24个月,耐热苗效价下降不超过0.5个滴度,常规苗效价下降超过3.0个滴度;37℃保存10d,耐热苗效价下降不超过0.5个滴度,常规疫苗下降超过2个滴度;25℃常温保存2个月,耐热保护剂疫苗效价下降不超过0.35个滴度,而常规苗效价下降超过2个滴度。试验结果表明,耐热冻干保护剂对病毒的保护效果明显优于常规冻干保护剂,具有良好的热稳定性。     

羊传染性脓疱皮炎弱毒苗耐热保护剂的筛选优化

旨在筛选出羊传染性脓疱皮炎弱毒苗的最佳耐热保护剂。通过初步筛选,选出符合本试验设计要求的3组保护剂A3、A6、C4,以羊传染性脓疱皮炎弱毒为对象,以保存环境、保存期长短、病毒损失量为参数,采用单因子试验对3组耐热保护剂进行优化,优选出对羊传染性脓疱皮炎弱毒苗耐热保护效果最好、且经济实用的耐热保护剂A6组。试验结果显示,使用A6保护剂的羊传染性脓疱皮炎疫苗在37℃保存3周,病毒含量下降0.4个滴度,常规疫苗下降2.4个滴度;25℃保存60 d,病毒含量下降0.3个滴度,常规疫苗下降2.2个滴度;4℃保存9个月,病毒含量下降0.4个滴度,常规疫苗下降3.8个滴度,说明A6可作为羊传染性脓疱皮炎弱毒苗良好的耐热保护剂。     

耐热保护剂冻干活疫苗冷冻干燥工艺研究

以鸡新城疫、传染性支气管炎二联耐热保护剂冻干活疫苗的冷冻干燥试验为例,描述了冻干耐热保护剂冻干活疫苗时的工艺改进过程,获得了较佳冻干工艺曲线.     

羊传染性脓疱病毒耐热冻干保护剂筛选及冻干曲线优化

采用冻干试验筛选出保护剂基础配方,再通过Plackett-Burman设计法筛选主要保护剂基质,然后通过Box-Behnken响应面分析法对羊传染性脓疱病毒(ORFV)耐热保护剂配方进一步优化,用冻干试验进一步验证筛选出1种针对ORFV保护效果最好的耐热冻干保护剂作为疫苗用候选配方。同时,依据前期试验获得的活疫苗冻干曲线,通过进一步优化,获得疫苗最佳冻干曲线。利用该冻干工艺冻干的疫苗,保护剂的保护率可达95.3%,且疫苗各项指标均达到《中华人民共和国兽用生物制品规程》要求。经37℃保存7,15,30d病毒滴度分别下降0.46,0.70,1.00;2~8℃保存3,6,12,24个月病毒滴度分别下降0.10,0.10,0.40,0.60,证明此配方制备的疫苗具有良好的热稳定性。本试验解决了ORFV细胞弱毒疫苗仅能在低温条件下保存和运输的瓶颈技术问题,使疫苗的储存、运输更为方便。     

鸡新城疫耐热冻干保护剂活疫苗试验

用耐热冻干保护剂冻干的鸡新城疫（LaSota系）活疫苗，在37℃环境下放置14d，疫苗的病毒含量EID50由10^9.1下降为10^8.1，在4℃环境下放置1年半，疫苗的病毒含量基本不变。     

Determination of optimal conditions for thawing and diluting cell-bound CVI 988 Marek's disease vaccine and stability of the diluted vaccine

The cell-associated vaccine strain CVI 988, which is the active component of several commercial Marek's disease vaccines, normally is frozen and stored in liquid nitrogen. In order to ascertain good efficacy of the vaccine, it is crucial that the right procedures are followed for thawing and diluting of the virus. In the study presented here, ampoules containing the frozen product were taken from storage in liquid nitrogen and were thawed in a water bath at 27 C, which is similar to a lukewarm bath, and in a water bath at 37 C, with and without agitation. The effect of the thawing procedure on the live virus titer of the vaccine was investigated. Samples of thawed vaccine were diluted in diluent with different temperatures, and live virus titers were determined directly after dilution and after incubation of the diluted vaccine at different temperatures. The results show that directly after thawing in the water baths at 27 C and 37 C, with or without agitation, the live virus titers for CVI 988 were all in the same range. After incubation of the thawed virus at both temperatures for 15 min, the live virus titers were still in the same range. Decreases in live virus titers were observed after incubation for 4 hr. Live virus titration of the vaccine in diluent in different temperatures revealed that the highest titers were found with diluent at a temperature of 30 C to 37 C and the lowest titers in diluent at a temperature of 5 C. In addition, a combination product containing cell-associated CVI 988 and cell-associated herpesvirus of turkeys (HVT) was tested. For this combination product, the titers for HVT also were highest in diluent with a high temperature (i.e., 37 C), whereas the titers for CVI were highest in diluent at a temperature of 22 C. Both strains had relatively low titers in diluent at 5 C. After incubation of the diluted vaccine at the various temperatures for several hours, again, live virus titrations were done. Live virus titers were most stable with diluent at temperatures of 22 C. Vaccine virus diluted in diluent at 37 C could be stabilized by placing the diluted vaccine at 5 C directly after diluting. After evaluation of these data, the following is recommended. For thawing of the vaccine, a water bath at approximately 27 C, which is similar to a lukewarm bath, is preferred. For diluting the vaccine, diluent should be used at a temperature of 22 C or higher. If diluted in diluent at temperatures higher than 22 C, the diluted vaccine should be stored under cooling in order to avoid titer losses.     

鸡产蛋下降综合症油乳剂灭活苗的免疫及攻毒保护试验

对应用当地分离病毒株研制的鸡产蛋下降综合症系列油乳剂灭活苗进行了免疫试验。结果表明,免疫后,鸡产蛋下降综合症（ＥＤＳ７６）油乳剂灭活苗、鸡新城疫（ＮＤ）－产蛋下降综合症二联油乳剂灭活苗及鸡新城疫－鸡传染性支气管炎（ＩＢ）－产蛋下降综合症三联油乳剂灭活苗免疫组的血清ＥＤＳ７６ＨＩ抗体迅速上升,维持４个月后仍在６．８－８（ｌｏｇ２）以上,并且能够抵抗强毒的攻击。证明所研制的ＥＤＳ７６油苗、ＮＤ－ＥＤＳ７６二联油苗、ＮＤ－ＩＢ－ＥＤＳ７６三联油苗对ＥＤＳ７６具有良好的免疫作用,能够抵抗ＥＤＳ７６强毒的攻击并产生高而持久的血清ＨＩ抗体。此外,对ＮＤ－ＥＤＳ７６二联苗、ＮＤ－ＩＢ－ＥＤＳ７６三联苗免疫组的血清ＮＤＨＩ抗体测定结果表明,上述二联苗、三联苗能产生与接种ＮＤ油苗一样良好的ＮＤ免疫效果,在免疫后１３０天时,其血清ＨＩ抗体仍高达９－１１（ｌｏｇ２）以上,与对照组有极显著的差异     

Evaluation in swine of a subunit vaccine against pseudorabies

A subunit vaccine against pseudorabies virus (PRV) was prepared by treating a mixture of pelleted virions and infected cells with the nonionic detergent Nonidet P-40 and emulsifying the extracted proteins incomplete Freund's adjuvant. Three 7-week-old pigs without antibodies against PRV were given 2 IM doses of this vaccine 3 weeks apart. Thirty days after the 2nd vaccination, 10(6) median tissue culture infective doses (TCID50) of a virulent strain of PRV were administered intranasally. Tonsillar and nasal swabs were collected daily between 2 and 10 days after challenge exposure. The pigs vaccinated with the subunit vaccine were not found to shed virulent PRV. Two groups of five 7-week-old pigs vaccinated with commercially available vaccines, either live-modified or inactivated virus, and subsequently exposed to 10(6) TCID50 of virulent PRV, shed virulent virus for up to 8 days. The subunit vaccine induced significantly higher virus-neutralizing antibody titers than either the live-modified or inactivated virus vaccine.     

Immune response of pigs inoculated with virulent pseudorabies virus and pigs inoculated with attenuated or inactivated pseudorabies virus vaccine before and after challenge exposure

Pseudorabies virus (PRV) antibodies, detectable by indirect radioimmunoassay (IRIA), serum-virus neutralization test (NT), or microimmunodiffusion test (MIDT) were developed within 8 days after pigs were inoculated with virulent PRV or attenuated PRV vaccine. Indirect radioimmunoassay and NT titers in pigs inoculated with virulent PRV were developed at the same rate, with IRIA titers being higher than NT titers. Pigs inoculated with attenuated or inactivated PRV vaccine developed peak mean prechallenge NT antibody titers of 4 and 1 (reciprocals of serum dilutions), respectively. Pigs inoculated with attenuated PRV vaccine had peak mean prechallenge IRIA antibody titers of 6, whereas pigs inoculated with inactivated PRV vaccine had mean IRIA antibody titers of 64. Challenge exposure of swine inoculated with attenuated or inactivated PRV vaccine elicited quantitatively equivalent responses, as measured by IRIA or NT, which were higher than prechallenge titers. There were no false-positive IRIA, NT, or MIDT results obtained when sera from nonvaccinated, nonchallenge-exposed pigs were tested. It appears that the PRV infection status of a seropositive swine herd could be ascertained by serologically monitoring several representative animals from a herd, using the NT. If 2 or more tests of representative animals at 14-day intervals were done and the mean NT titer was 4 or less, it could be concluded that the herd was vaccinated against, but not infected with, virulent virus.     

猪伪狂犬病病毒基因缺失灭活疫苗（LA-A株）的最小免疫剂量和制品保存期的研究

本研究将伪狂犬病病毒(Pseudorabies virus,PRV)变异株(PRV AH02LA株)的gE基因缺失株(LA-A株)接种BHK-21细胞,经纯悬浮培养制备抗原,甲醛灭活后制备油乳剂灭活疫苗,并确定该灭活疫苗的最小免疫剂量和效力检验方法,以及在2~8℃保存期。结果显示:猪伪狂犬病病毒基因缺失灭活疫苗(LA-A株)的效力检验方法为以2.0 mL(抗原含量108.20TCID50)接种4~5周龄PRV阴性健康仔猪,颈部肌肉注射,间隔28 d以相同剂量和方法加强免疫,加强免疫后第21 d,免疫猪血清PRV抗体中和指数应不低于10000,攻毒保护率应不低于80%;最小免疫剂量为1.0 mL(抗原含量107.90 TCID50);制品保存期:在2~8℃保存期为18个月。该研究结果为新型疫苗的研制提供了重要的试验依据。     

Efficacy of a pseudorabies virus vaccine based on deletion mutant strain 783 that does not express thymidine kinase and glycoprotein I.

The vaccine efficacy of a genetically engineered deletion mutant strain of pseudorabies virus, strain 783, was compared with that of the conventionally attenuated Bartha strain. Strain 783 has deletions in the genes coding for glycoprotein I and thymidine kinase. In experiment 1, which had a 3-month interval between vaccination and challenge exposure, strain 783 protected pigs significantly (P less than 0.05) better against virulent virus challenge exposure than did the Bartha strain. The growth of pigs vaccinated with strain 783 was not arrested, whereas that of pigs vaccinated with the Bartha strain was arrested for 7 days. Of 8 pigs given strain 783, 4 were fully protected against challenge exposure; none of the pigs given strain Bartha was fully protected. In experiment 2, which had a 3-week interval between vaccination and challenge exposure, the growth of pigs vaccinated with strain 783 was arrested for 3.5 days, whereas that of pigs vaccinated with the Bartha strain was arrested for 6 days. In experiment 3, pigs with moderate titer of maternal antibodies were vaccinated twice IM or once intranasally with either strain 783 or Bartha and were challenge-exposed 3 months after vaccination. Pigs given strain 783 twice IM were significantly (P less than 0.05) better protected than were the other pigs. They had growth arrest of only 6 days, compared with 9 days for pigs of other groups, and shed less virus after challenge exposure. Results of this study indicate that the vaccine based on the deletion mutant strain 783 is more efficacious than is the Bartha strain of pseudorabies virus.     

口蹄疫疫苗效力试验和血清学取代方法研究进展

口蹄疫疫苗效力试验是通过强毒攻击疫苗免疫动物来评价12蹄疫疫苗免疫效果的一种方法,该方法在安全性、经济性、动物福利方面存在问题,寻找更安全的12蹄疫疫苗效果评价方法成了各国口蹄疫疫苗研究的重要任务。许多研究表明,动物免疫后的血清学指标和动物能否抵抗强毒攻击有相关关系,提出了疫苗效力试验的各种血清学替代方法,如“抗体平均值预测”、“bootstrap”和“抗体滴度最佳临界点”等,取得了很多进展,其中用“抗体滴度最佳临界点”方法可以计算出疫苗的PD50（50％ protectivedose）,重复性（re—peatability）为65．8％,再现性（reproducibility）为60．7％,该方法有望标准化。     

Survival and immunization of raccoons after exposure to pseudorabies (Aujeszky's disease) virus gene-deleted vaccines

In a controlled experiment, 16 wild-trapped raccoons were exposed to 1 of 2 genetically modified live pseudorabies virus (PRV) vaccines used in swine. One vaccine had genes deleted for thymidine kinase (TK(-)) and glycoprotein G (gG(-)); the other had an additional deletion for glycoprotein E (gE(-)). These vaccines were administered orally and intranasally at four dose levels: 10(3), 10(4), 10(5), and 10(6) TCID(50). The 21 days survival rate was 37.5% for the gG(-)TK(-) vaccine; all of the survivors developed antibodies to PRV. All animals receiving the gG(-)gE(-)TK(-) vaccine survived; 75% (all except the lowest dose) developed anti-PRV antibodies. Survivors were challenged intranasally with a 3.2x10(3) TCID(50) dose of the virulent wildtype PRV Shope strain. Two of the remaining three gG(-)TK(-) vaccinated raccoons survived the challenge; for the gG(-)gE(-)TK(-) vaccine, the survival rate was 50% (4/8). The raccoons with higher vaccine-induced antibody titers were more likely to survive the challenge with the virulent PRV; there was a 100% mortality rate for raccoons lacking detectable anti-PRV antibodies. This experiment indicates that exposure of raccoons to modified live gene-deleted PRV vaccines may result in an immune response, and that this immunity provides some protection against exposure to virulent virus.     

Evaluation of oral and subcutaneous delivery of an experimental canarypox recombinant canine distemper vaccine in the Siberian polecat (Mustela eversmanni).

We assessed the safety and efficacy of an experimental canarypox-vectored recombinant canine distemper virus (CDV) subunit vaccine in the Siberian polecat (Mustela eversmanni), a close relative of the black-footed ferret, (M. nigripes), an endangered species that is highly susceptible to the virus. Siberian polecats were randomized into six treatment groups. Recombinant canine distemper vaccine was administered s.c. at three dose levels (10(4.5), 10(5.0), and 10(5.5) plaque-forming units [PFU] per dose) and was administered orally by spraying the vaccine into the oropharnyx at two dose levels (10(5.5), 10(8.0) PFU per dose). The sixth group of control animals was not vaccinated. For both routes of administration, two 1-ml doses of reconstituted vaccine were delivered 4 wk apart, followed by live virus challenge 3 wk after the second vaccination. During the challenge, Synder Hill test strain CDV obtained from the National Veterinary Services Laboratory in Ames, Iowa, was administered i.p. Serial blood samples for CDV serology were collected immediately before vaccination and challenge, and 10, 15, and 20 days after challenge. Clinical signs and body weights were recorded up to 32 days after challenge. The survival rate in animals receiving vaccine at the highest oral dose (10(8.0) PFU per dose) was 83.3%. Survival rate was 50.0% in the high s.c. and 60.0% in the medium s.c. groups. All animals in the low-s.c. dose, low-oral dose, and control groups died after exposure. Vaccine dose overall (oral and s.c.) and dose in response to s.c. administration when considered alone were significant predictors of survival (P = 0.006 and P = 0.04, respectively). Among the polecats challenged with virulent virus, those that died became sick sooner than those that survived. Animals that died lost significantly more weight during the 10 days after challenge than did animals that survived (P = 0.02). Survival rates did not differ by sex, founder female status, or breeding pedigree in any of the treatment groups. Survival rates were higher in animals with increasing serum neutralization titers (P = 0.027). This study demonstrates the efficacy of oral delivery of a recombinant CDV vaccine in the Siberian polecat. Further studies are needed to evaluate the safety and efficacy of vectored recombinant vaccines in highly susceptible species and especially in those species in which vaccination with modified live CDV has led to disease.