鸡传染性鼻炎(三价)和新城疫二联灭活疫苗的研究

利用Page A型、B型、C型副鸡禽杆菌和新城疫病毒La Sota株,研制鸡传染性鼻炎(三价)和新城疫二联油乳剂灭活疫苗。用3个批次疫苗进行单剂量(0.5mL/次)3次接种和大剂量(2.0mL)单次接种的安全性试验、SPF鸡的免疫效力试验、商品鸡的免疫持续期试验和疫苗的保存期试验。结果表明,3批疫苗对试验鸡安全无副作用;免疫接种SPF鸡只30d后对A、B、C型副鸡禽杆菌攻毒的保护率≥80%,用20μL/只疫苗免疫接种SPF鸡21d后新城疫的平均HI抗体24;商品鸡42日龄首免,110日龄二免,二免后9个月对A、B、C型副鸡禽杆菌攻毒的保护率≥70%,对新城疫病毒强毒100%保护;用4℃~8℃保存12个月和15个月的3批疫苗进行了SPF鸡的近期免疫效力试验,结果对A、B、C型副鸡禽杆菌攻毒的保护率≥70%,用20μL/只疫苗免疫SPF鸡,免疫接种后21d新城疫病毒的平均HI抗体24,对新城疫病毒强毒的攻毒保护率70%。说明研制的疫苗安全有效。     

鸡传染性鼻炎二价油乳剂灭活疫苗免疫持续期的研究

应用3批鸡传染性鼻炎二价油乳剂灭活疫苗进行了免疫持续期的试验。结果表明，鸡群免疫1次后，对A型强致病力菌株的攻毒保护率为83%，对C型强致病力菌株的攻毒保护率为51.5%，在首免后4周左右进行二免，则免疫鸡群对A型菌的保护率在免疫后13个月仍然保持100%，对C型菌的保护率在免疫后9个月内可达到90.5%。免疫鸡血清抗体也均保持较高的水平，其中B-ELISA的A型抗体的检出率为83%-100%、C型的检出率为33%-87%，血凝抑制试验（HI）的A型抗体阳性检出率为87%-100%，C型抗体的检出率为62.8%-93%，血清平板凝集抗体的检出率为62.8%-100%，此结果充分显示了鸡传染性鼻炎二价油乳剂灭活疫苗的良好的免疫效力以及B-ELISA、HI及血清平板凝集试验较高的敏感性的特异性，研究还证实了HI抗体与攻毒保抗力的密切相关。     

鸡新城疫基因Ⅶ型灭活标记疫苗（MG7株）最小有效免疫剂量的研究

为科学指导鸡新城疫基因Ⅶ型灭活标记疫苗（MG7株）的临床免疫剂量,通过测定免疫鸡HI抗体滴度和攻毒保护试验,进行了10日龄和30日龄SPF鸡最小有效免疫剂量的研究。动物实验使用10 μL、20 μL和40 μL三种不同免疫剂量,免疫疫苗后21 d检测HI抗体效价,10日龄SPF鸡001批次疫苗HI抗体平均效价依次为：4.8、6.2和6.5,攻毒保护率依次为80%、100%和100%,30日龄SPF鸡HI抗体平均效价依次为：5.0、6.3和6.5,攻毒保护率依次为70%、100%和100%,结果表明,新城疫灭活标记疫苗（MG7株）HI 抗体效价和保护效力呈正相关, 免疫剂量与HI 抗体效价和保护效力也呈正相关。用NDV基因Ⅶ型强毒G7株进行攻毒保护试验,两种日龄鸡在20 μL的免疫剂量下,疫苗仍能达到良好的免疫保护效果。     

鸡传染性鼻炎三价灭活疫苗安全性试验和效力检验初报

利用PageA、B、C型副鸡禽杆菌,研制鸡传染性鼻炎(IC)三价灭活疫苗。用6批IC疫苗进行了大剂量单次接种和单剂量多次接种的安全性试验,结果表明,IC三价灭活疫苗安全无副作用;用3批疫苗进行SPF鸡和普通鸡的免疫效力试验,结果对A、B、C型副鸡禽杆菌攻毒的保护率≥80%;商品鸡42日龄首免,110日龄二免,二免后约9个月对A、B、C型副鸡禽杆菌攻毒的保护率≥80%;用4℃～8℃保存一年的3批疫苗进行了SPF鸡的近期免疫效力试验,结果对A、B、C型副鸡禽杆菌攻毒的保护率≥80%。     

禽霍乱禽霍乱、新城疫二联灭活疫苗免疫攻毒试验与HI试验的平行关系研究

对8批禽霍乱、新城疫二联灭活疫苗进行新城疫组分的效力检验,在检测新城疫血凝抑制效价(HI)的同时,进行新城疫病毒强毒攻击试验,旨在寻找抗攻毒保护与HI抗体效价之间的平行关系。结果表明:免疫剂量与HI抗体效价和攻毒保护率呈正相关,免疫40、20、10、5μL剂量组攻毒保护率和HI抗体效价分别为94.9%(75/79)、5.2 log_2,87.5%(70/80)、4.4 log_2,29.1%(23/79)、2.7 log_2,11.4%(9/79)、2.1 log_2;对照组攻毒保护率为0(0/10),HI抗体效价≤2.0 log_2;新城疫HI抗体效价与攻毒保护率也呈正相关,当HI3.0 log_2,攻毒保护率为4.1%;HI=3.0 log_2,攻毒保护率为22.9%;HI=4.0 log_2,攻毒保护率为87.7%;HI=5.0 log_2,攻毒保护率为94.4%;HI=6.0 log_2,攻毒保护率为100%。本研究为制定疫苗质量标准中新城疫病毒株的效力检验方法和标准提供实验室理论依据。     

鸡传染性鼻炎二价油乳剂灭活疫苗的保存期检验

应用3批鸡传染性鼻炎二价油乳剂灭活疫苗进行了保存期检验,结果表明,二价苗在4～8℃保存12个月和18个月后免疫鸡群,鸡群对A、C型强致病力菌株的保护率在二免后4周左右分别为100%和88.5%,血清SPA、阻断ELISA和HI抗体阳性检出率较高,但疫苗保存18个月后所免疫鸡的血清C型HI抗体效价的几何平均数有显著的下降.因此,该二价苗的保存期以12个月为宜.     

不同厂家的猪O型口蹄疫疫苗对口蹄疫流行毒株保护性的研究

为了探索不同厂家疫苗对猪O型口蹄疫流行毒株的保护情况,选用当前政府采购的A、B灭活疫苗厂家和C合成肽疫苗厂家生产的猪O型口蹄疫疫苗,按照农业部推荐免疫程序对试验猪分别进行二次免疫后90天,用OZK/93和O/(XJ/10-11/MYA/98)进行攻毒,观察其各自保护率。结果显示,A、B两个厂家的灭活疫苗对OZK/93株的攻毒保护率分别为60%、80%,对O/(XJ/10-11/MYA/98)株的攻毒保护率分别为100%、100%;合成肽疫苗对OZK/93株的保护率为100%,对O/(XJ/10-11/MYA/98)株保护率为60%。说明试验用的3个厂家的猪O型口蹄疫疫苗对毒株的保护有差异。     

鸡新城疫灭活疫苗免疫攻毒试验与血清学(HI)试验的平行关系研究

对19批鸡新城疫(ND)灭活疫苗进行了效力检验,在测定PD50的同时,进行了HI抗体效价的测定,以寻找血清学与抗攻毒保护的平行关系.结果表明,免疫剂量与GMHI抗体效价和攻毒保护率呈正相关:免疫1/12.5剂量组共保护54/60(90%),GMHI抗体效价为5.6 log2;免疫1/25剂量组共保护152/191(79.6%),GMHI抗体效价为4.9 log2;免疫1/50剂量组共保护123/183(67.2%),GMHI抗体效价为4.0 log2;免疫1/100剂量组共保护46/139(33%),GMHI抗体效价为3.0 log2;对照组保护0/89(0),GMHI抗体效价≤2.0 log2.采用PD50测定的18批疫苗中,有15批合格,3批不合格;按照我国现有质量标准,即免疫1/25剂量攻毒的方法进行检验,19批疫苗中有16批合格,3批不合格;血清学方法检验19批疫苗中有17批合格,2批不合格.其中免疫攻毒效检方法与PD50测定方法的不合格疫苗批次完全相同,其中2批为免疫胚制造的疫苗,1批为进口的灭活疫苗,血清学检验不合格的2批疫苗包含在其中,三种检验方法结果基本一致.试验还发现,HI抗体效价和抗攻毒保护呈正相关:未免疫对照组鸡血清HI抗体效价≤2.0 log2,攻毒保护率为0(0/89);免疫ND灭活疫苗后如果血清HI抗体效价＜3.0 log2,攻毒保护率为3.5%(4/113);HI为3.0 log2,攻毒保护率为16.1%(14/87);HI为4.0 log2,攻毒保护率为86.7%(86/99);HI达到5.0 log2,攻毒保护率为97.2%(103/106),HI≥6.0 log2,攻毒保护率为100%(168/168).     

猪乙型脑炎(HW1株)细胞灭活疫苗免疫抗体水平分析

为评估猪乙型脑炎(HW1株)细胞灭活疫苗的免疫原性,用商品化的乙型脑炎鼠脑疫苗、乙型脑炎弱毒疫苗(SA14-14-2株)以及自制乙型脑炎细胞灭活疫苗(HW1株)分别免疫仔猪,通过抗体水平监测试验、中和抗体试验、小鼠攻毒保护试验分别检测了其特异性抗体消长情况、中和抗体效价产生情况及疫苗对小鼠的攻毒保护率。结果显示,3种疫苗均能产生中和抗体,乙型脑炎细胞灭活疫苗免疫组中和抗体水平要高于鼠脑疫苗和弱毒疫苗免疫组,乙型脑炎细胞灭活疫苗和弱毒疫苗免疫组对小鼠的攻毒保护率高于鼠脑疫苗免疫组,但两者之间差异不显著。抗体消长情况显示,至8周观测期结束,鼠脑疫苗免疫组的抗体阳性率为80%,乙型脑炎细胞灭活疫苗和弱毒疫苗免疫组抗体阳性率为100%。结果表明猪乙型脑炎(HW1株)细胞灭活疫苗的免疫原性优于鼠脑灭活疫苗和弱毒疫苗。     

鹿产气荚膜梭菌二价灭活疫苗的制备和免疫效力试验

为了研制鹿产气荚膜梭菌A型和C型二价灭活疫苗,对疫苗的免疫效力和免疫持续期以及疫苗保存期进行测定,从而为开发免疫原性较好的无毒副作用的灭活疫苗奠定基础。将产气荚膜梭菌T5-A和J28-C株的培养液按1∶1混和,混合菌液按9∶1加入灭菌铝胶,经浓缩制备3批灭活疫苗0406-1,0406-2,0406-3。安全检验合格后,采用"血清中和试验"检验技术,进行家兔攻毒与血清抗体效价的平行试验,测定疫苗的免疫效力和免疫持续期以及疫苗保存期。结果是疫苗的安全性良好,免疫效力表明疫苗的免疫保护率为100%,抗体效价显示C型抗体效价高于A型,疫苗对C型菌的保护期长达6个月,而对A型菌的保护期为3个月。疫苗保存期的测定表明,疫苗可以保存10个月以上。表明3批疫苗安全性较好,对家兔有较强的免疫保护,为疫苗的下一步鹿田间试验提供了理论依据。     

鸡传染性鼻炎二介油乳剂灭活疫苗的保存期检验

应用3批鸡传染性鼻炎二价油乳剂灭活疫苗进行了保存期检验，结果表明，二价苗在4－8℃保存12个月和18个月后免疫鸡群，鸡群对A、C型强致病力菌株的保护率在二免后4周左右分别为100％和88．5％，血清SPA、阻断ELISA和HI抗体阳性检出率较高，但疫苗保存18个月后所免疫鸡的血清C型HI抗体效价的几何平均数有显著的下降。因此，该二价苗的保存期以12个月为宜。     

Newcastle disease vaccination of broilers with high- and low-titered commercial vaccines

The virus titers of seven commercial B1 strain Newcastle disease vaccines were evaluated. A 2 log difference in virus content was found between the vaccine with the highest titer (10(8.8) EID 50/ml) and the one with the lowest titer (10(6.8) EID 50/ml). Broiler chickens were vaccinated with the high- and low-titered vaccines to compare hemagglutination-inhibition (HI) antibody and challenge responses. The effect of vaccination at different ages on the HI titers was also examined. There were no significant differences between vaccine groups in HI antibody response or resistance to challenge. However, the high-titered vaccine may provide a margin of safety with the currently used methods of mass vaccination.     

禽流感油乳剂灭活疫苗的研究

将6种不同亚型的禽流感病毒（AIVH2N9、H3N8、H5N1、H5N2、H7N1、H9N2）分别接种鸡胚,收获尿囊液,经甲醛灭活,以矿物油为佐剂制成油乳剂灭活疫苗。疫苗接种4和8周龄SPF鸡,注苗后均无不良反应。每种亚型疫苗免疫后14天和21天攻毒保护率均达90％-100％。分别用H5N1和H9N2亚型灭活疫苗免疫8周龄SPF鸡、25和28周龄健康商品蛋鸡,免疫后7天产生免疫力,14天保护率达100％,21天后抗体达高峰,仔鸡接苗后最高血凝抑制（HI）几何平均滴度（GMT）为7.3-8.0log2,蛋鸡为8.0-10.5log2。免疫后180天,抗体效价不低于6.5log2。免疫后180天分别以AIV攻击,H5亚型疫苗组,用强毒攻击无一发病和死亡,对照鸡全部发病死亡;H9亚型疫苗组,对照鸡在攻毒后72小时停产,免疫鸡无一发病且产蛋正常,对同源攻毒的保护率达100％。     

Antibody responses of guinea-pigs, rabbits and pigs to inactivated porcine parvovirus vaccines

Antibody responses were compared in guinea-pigs, rabbits and pigs following vaccination with inactivated porcine parvovirus (PPV) vaccines. Mean PPV hemagglutination inhibition (HI) antibody titers of 52, 56 and 36 at 1 week after first vaccination and 896, 640 and 512 at 2 weeks after second vaccination were detected in guinea-pigs, rabbits and pigs, respectively. PPV vaccines prepared with greater concentrations of virus, as determined by hemagglutination (HA) units, and of aluminum hydroxide gel adjuvant, induced higher HI antibody titers in guinea-pigs. Optimal concentrations for inducing consistently high antibody titers consisted of vaccine virus with a HA titer of 256/0.1 ml and gel adjuvant at a final concentration of 50%. A second vaccination at 4 weeks compared to 2 or 3 weeks after first vaccination resulted in higher mean HI titers. These data provide preliminary information on the use of guinea-pigs or rabbits as laboratory animal models for testing the potency of PPV vaccines.     

An evaluation of inactivated infectious coryza vaccines containing a double-emulsion adjuvant system.

The efficacy of experimental inactivated infectious coryza vaccines produced by a commercial vaccine manufacturer was evaluated. The vaccines, containing as the adjuvant phase either a double-emulsion mineral oil system or aluminum-hydroxide gel, were administered to 6-week-old chickens as a single dose. Some vaccines were a monovalent product containing a Page serovar C Haemophilus paragallinarum strain, and others were a bivalent product containing both Page serovar A and serovar C strains. After 3 weeks, all chickens were challenged by infraorbital sinus inoculation of virulent H. paragallinarum, either Page serovar C (strain HP31) or Page serovar A (strain HP14). The monovalent serovar C double-emulsion-based vaccines gave significant protection against a serovar C challenge, with the level of protection varying from 60% to 100%. The monovalent serovar C aluminum-hydroxide-gel vaccine also gave significant protection (94%) against a serovar C challenge. The bivalent double-emulsion vaccine gave significant protection against challenge from both serovars (100% for serovar C and 83% for serovar A). Although no major adverse reactions were detected, some chickens receiving both the double-emulsion vaccines and the aluminum-hydroxide vaccine developed relatively minor granulomatous reactions at the site of injection.     

Development of an enzyme-linked immunosorbent assay for the measurement of antibodies against infectious coryza vaccine

Infectious coryza is an acute respiratory disease caused by infection with Avibacterium (Haemophilus) paragallinarum. It is characterized by nasal discharge and facial swelling and is associated with growth retardation and a reduction in egg production. Hemagglutination inhibition (HI) tests are used to estimate vaccine-induced immunity against infectious coryza in vitro; however, these procedures are complicated and their sensitivity is insufficient. To address these problems, an enzyme-linked immunosorbent assay (ELISA) technique using serovar-specific regions of HMTp210 (210 kDa), an outer-membrane protein of A. paragallinarum, was developed to measure the antibodies against infectious coryza. Chickens with an ELISA titer of 0.3 or more did not exhibit clinical signs of infectious coryza against challenge with A. paragallinarum, although their HI antibody titers were negative. On the other hand, chickens with an ELISA titer below 0.3 exhibited clinical signs of the disease with one exception. Antibody prevalence rates on ELISA were 80% and 60% against infection with serovars A and C, respectively, and ELISA also detected antibodies in chickens infected with A. paragallinarum with a sensitivity higher than that of HI tests. Taken together, the ELISA technique developed in this study is a valuable tool for the measurement of antibodies produced against the infectious coryza vaccine or in response to an infection with A. paragallinarum.     

副鸡嗜血杆菌B型分离株免疫原性的比较和疫苗株的筛选

利用分离自我国不同地区的3株B型副鸡嗜血杆菌菌株及0222标准株分别制作油乳剂灭活疫苗免疫无鸡传染性鼻炎病史的鸡群，通过攻毒保护试验和HI抗体测定，分析比较了其免疫原性，以筛选适用于制备疫苗的菌株。结果表明：不同地区的分离株在致病性和免疫原性上存在-定的差异，TJ-1株疫苗免疫鸡对TJ-1和DL-1株的保护作用可达90％和91．7％，对BJ-1株的保护率为63．6％，具有相对较好的保护作用，目前可以作为疫苗株使用。在各免疫组试验鸡中，0222株和BJ-1株免疫鸡群的血清抗体水平较高，但这可能与血凝抑制试验所采用的抗原是由0222株制备的及BJ-1株在抗原性上可能更接近于0222株有关。DL-1和TJ-1株疫苗免疫鸡的抗体水平都很低，但其免疫保护却要强于0222和BJ-1株疫苗免疫鸡。由此说明B型株因菌株之间免疫原性的差异使得HI抗原的选择难度增加。     

Effects of Salmonella enterica subsp. enterica serovar Enteritidis vaccination in layer hens subjected to S. Enteritidis challenge and various feed withdrawal regimens

Levels of Salmonella enterica subsp. enterica serovar Enteritidis infection and serum S. Enteritidis antibodies after experimental S. Enteritidis challenge and feed withdrawal were investigated in S. Enteritidis-vaccinated and unvaccinated hens. The results were used to determine whether formalin-inactivated S. Enteritidis vaccination can protect layer hens from S. Enteritidis challenge during feed withdrawal periods. S. Enteritidis infection rates were evaluated from cloacal swabs, eggs and organs. Serum antibody titers to deflagellated S. Enteritidis whole cells (DEWC) and S. Enteritidis FliC-specific 9-kDa polypeptide (SEp 9) were examined by commercial ELISA kits. Cloacal S. Enteritidis recovery rates were lower in the vaccinated than unvaccinated group. Recovery rates of S. Enteritidis from samples increased after feed withdrawal and decreased after re-introduction of feed. S. Enteritidis counts in cloacal swabs were lower in the vaccinated than in the unvaccinated group (P<0.05). More S. Enteritidis-positive eggs were detected from the unvaccinated group. Before S. Enteritidis challenge, the DEWC ELISA titer of the vaccinated group was higher (P<0.05) than the unvaccinated group; subsequently, the S. Enteritidis DEWC ELISA titers of both groups increased gradually. In contrast, only the vaccinated group elicited high SEp-9 antibody titer during post-challenge and feed withdrawal. Additionally, vaccinated hens yielded negative S. Enteritidis isolation rates from egg contents. There is a correlation between negative S. Enteritidis isolation rates and high SEp 9 titers in vaccinated layer hens challenged with S. Enteritidis and subjected to feed withdrawal regimens. These findings suggest the S. Enteritidis vaccination of pullets may protect against S. Enteritidis infection during forced molting and that SEp 9 titer could be a potential indicator of antibody protection against S. Enteritidis infection. The potential of the SEp 9 peptide as an antigen for S. Enteritidis vaccination in the future is worth noting.     

A cross-protection experiment in pigs vaccinated with Haemophilus parasuis serovars 2 and 5 bacterins, and evaluation of a bivalent vaccine under laboratory and field conditions.

Cross-protection between Haemophilus parasuis serovars 2 and 5 was examined in pigs using a bacterin based vaccine, and subsequently the safety and efficacy of a bivalent vaccine were evaluated. Upon intratracheal challenge of a serovar 2 or 5 strain, pigs immunized with a monovalent vaccine were protected against challenge with a homologous serovar strain, but not with a heterologous serovar strain. Immunization with a bivalent vaccine containing both serovars 2 and 5 bacterins conferred protection in pigs against lethal challenge with each of the serovar strains. A total of 86 pigs from two SPF herds were injected with the bivalent vaccine intramuscularly twice at a four-week interval. No adverse reactions following the vaccination were observed. On day 7 after the second vaccination, vaccinated and non-vaccinated control pigs from herd A were transferred to herd B, where Glasser's disease had broken out. Pigs in the control group developed clinical signs of the disease, and 6 of 8 (75%) pigs died until slaughter, in contrast with only 4 of 46 (9%) pigs in the vaccinated group. In herd C, where there was no outbreak of Glasser's disease, complement fixation antibody titer was raised only in the vaccinated group. A challenge experiment on days 20 and 79 after the second vaccination showed that only the vaccinated pigs were protected. From these findings, the safety and efficacy of the bivalent vaccine were confirmed under laboratory and field conditions.