猪圆环病毒2型（WH株）灭活疫苗的研究
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摘要
猪圆环病毒2型(PCV2)感染是造成断奶仔猪多系统衰竭综合征(PMWS)的主要原因,其它协同因素可增强疾病的严重程度。目前PCV2已呈全球分布,猪群感染率越来越高。至目前为止,国内外的试验表明,疫苗是预防此病的主要措施之一,但我国还没有商业化的疫苗问世。
本研究利用本实验室从临床发病猪中分离鉴定的增殖滴度高的猪圆环病毒2型WH株,进行了灭活苗的研究,为我国预防和控制该病奠定了基础,具体研究内容如下:
1.PCV2灭活苗的制备
猪圆环病毒2型(PCV2)WH株同步接种到PK-15细胞悬液中,细胞形成单层后,加入适量300mmol/L D-氨基葡萄糖,于37℃作用30min,弃去D-氨基葡萄糖,加入DMEM维持液继续培养48h,收获病毒,经0.3%甲醛灭活后与适量的美国埃索MARCOL52油佐剂,制备灭活苗并进行无菌检验及物理性状检验。
2.PCV2灭活苗最小免疫剂量测定
用含10~(5.0)TCID_(50)、10~(6.0)TCID_(50)和10~(7.0)TCID_(50)等不同抗原含量的疫苗进行猪体免疫和攻毒保护性试验,通过ELISA方法检测抗体、攻毒后体温变化、临床表现、病毒血症及病毒在组织中的分布等指标对疫苗免疫效果进行评价。进一步将最小免疫量的灭活苗按1、2、3ml免疫猪,按上述方法进行评价。结果显示:①10~(7.0)TCID_(50)组免疫猪三周后,ELISA抗体水平达到最高,5头猪全达到1:80,其中3头猪达到1:160,2头达到1:320,而10~(5.0)TCID_(50)和10~(6.0)TCID_(50)抗原含量的灭活苗免疫猪后未检测到特异性抗体。②最小免疫量的灭活苗1ml、2ml、3ml免疫猪均能产生较好的ELISA抗体,但2ml组效果明显优于其它各组。③免疫四周后攻毒,对照组体温高于免疫组,病毒血症检测及病毒DNA在组织中的分布检测结果表明该疫苗能够有效降低猪体的带毒,但是10~(7.0)TCID_(50)抗原含量的灭活苗2ml组效果最佳。
3.PCV2灭活苗免疫效力试验
用灭活苗2ml免疫猪群,于免疫后1、2、3、4周分别进行攻毒保护性试验,通过攻毒后体温变化、临床表现、病毒血症及病毒在组织中的分布等指标对疫苗免疫不同时间的效力进行评价。结果显示,免疫一周和两周后攻毒,免疫组和对照组各指标基本没有差异;免疫三周和四周后攻毒,免疫组均只有两头出现病毒血症及解剖组织检测到病毒,但对照组每头都检测到病毒血症及解剖组织检测到病毒。说明该疫苗免疫三周后可以对猪坚强保护。
4.PCV2灭活苗安全性试验
用单倍剂量和双倍剂量灭活苗免疫仔猪,测定免疫前后的体温,观察猪群的采食情况,触摸局部,有无肿块。免疫后28天,每组解剖2头猪,观察疫苗的吸收状况。结果,免疫前后猪群没有体温升高的现象,采食和精神正常,注射部位未见肿块出现。解剖后,注射部位也未见有白色油乳剂样物质存在。说明单倍和2倍剂量接种后对猪是安全的。
Porcine circovirus type 2 ( PCV2) infection is recognized as the major factor in the development of post-weaning multisystemic wasting syndrome (PMWS),although other factors may enhance the severity of the disease. At present, PCV2 is widely spreaded in the world and maintain high infection rate in herd. Vaccine inoculation is the main method to prevent and control PCV2 infection; however, there is no commercial vaccine against PCV2 in China yet.
An inactivated vaccine was developed based on porcine circovirus type 2 WH strains, a wild strain separated in pig farm Wuhan. To fight against porcine circovirus type 2, the following works were performed aiming to prepare a novel vaccine.
1. Development of inactivated vaccine against porcine circovirus type 2
PCV-2 WH strain was inoculated into PK-15 cell by the way of one-stepinoculation. After forming a single layer, the cells were treated with 300mmol/L D-glucosamine, at 37℃for 30min, discarded the D-glucosamine, then added DMEM with 2% newborn bovine serum and cultured for another 48h, harvesting virus. PCV2 was inactivated with 0.3% formalin. The inactivated vaccine was prepared with inactivated virus and appropriate amount of American oil-adjuvant.Then sterility and physical character of the vaccine was detected.
2. Determination of minimum immune dosage of the inactivated vaccine
Piglets immunized with 10~(5.0)TCID_(50)、10~(6.0)TCID_(50) and 10~(7.0)TCID_(50) antigen doserespectively, then the immune efficacies of the vaccines against PCV2 were evaluated by ELISA antibody detection, clinical signs (fever), viremia and distribution of viral DNA in different tissues. Further Piglets were immunized with minimum immune dosage by 1ml, 2ml and 3ml, respectively, and then the im mune efficacy was evaluated according to the method mentioned above. The results were as follows:①The efficient immune response could be induced with the vaccine of 10~(7.0) TCID_(50) PCV2 in piglets, while specific antibody wasn't detected with the vaccine of 10~(6.0) TCID_(50)PCV2 and 10~(5.0) TCID_(50) PCV2 in piglets.②High ELISA antibody titers of the vaccines with 10~(7.0) TCID_(50) 1ml、2 ml and 3ml could be detected in piglets. The immune efficacy of 10~(7.0) TCID_(50) PCV2 2ml was obviously higher than the other 2 groups.③After PCV2 challenge the rectal temperature of all piglets were recorded. Average temperature of piglets in challenge control group was higher than that of immunized piglets. The fact that vaccines could reduce the persistence of PCV2 in piglets was indicated by detection of viremia and PCV2 in different tissues of challenged piglets. The immune efficacy of 10~(7.0)TCID_(50)PCV2 2ml dosage group was the best in all groups.
3. Evaluation of the efficacy of the inactivated vaccine
After piglets immunized with vaccine 2ml for one week, two weeks, three weeks and four weeks respectively, the immune efficacies of the vaccines against PCV2 were evaluated by clinical signs (fever), viremia and distribution of viral DNA in the tissues. The results showed that challenge with porcine circovirus type 2 strains WH after post immunized for one week and two weeks, there was no difference between the control group and the immunized group. Challenge with porcine circovirus type 2 strains WH after post immunized for three weeks and four weeks, viremia and viral DNA in the tissues were detected in two piglets of the immunized group, while viremia and viral DNA in the tissues were detected in five piglets of the control group. It could be concluded that the inactivated vaccine could protect piglets from PCV2 infection after immunized for three weeks.
4. Evaluation of the safety of the inactivated vaccine
Piglets were inoculated with single dose and double doses of the inactivated vaccine and the rectal temperature of all piglets were recorded before and after inoculation. Then clinical symptoms such as appetite were observed. 28 days after inoculated, the situation of the vaccine absorption was observed. According to clinical symptoms, there was no significant difference between before and after inoculation. After autopsy, there was no residual vaccine in the injection site. Both single dose and double dose of the vaccine are safe to the piglets. It could be concluded that the inactivated vaccine is safe in piglets.
本研究利用本实验室从临床发病猪中分离鉴定的增殖滴度高的猪圆环病毒2型WH株,进行了灭活苗的研究,为我国预防和控制该病奠定了基础,具体研究内容如下:
1.PCV2灭活苗的制备
猪圆环病毒2型(PCV2)WH株同步接种到PK-15细胞悬液中,细胞形成单层后,加入适量300mmol/L D-氨基葡萄糖,于37℃作用30min,弃去D-氨基葡萄糖,加入DMEM维持液继续培养48h,收获病毒,经0.3%甲醛灭活后与适量的美国埃索MARCOL52油佐剂,制备灭活苗并进行无菌检验及物理性状检验。
2.PCV2灭活苗最小免疫剂量测定
用含10~(5.0)TCID_(50)、10~(6.0)TCID_(50)和10~(7.0)TCID_(50)等不同抗原含量的疫苗进行猪体免疫和攻毒保护性试验,通过ELISA方法检测抗体、攻毒后体温变化、临床表现、病毒血症及病毒在组织中的分布等指标对疫苗免疫效果进行评价。进一步将最小免疫量的灭活苗按1、2、3ml免疫猪,按上述方法进行评价。结果显示:①10~(7.0)TCID_(50)组免疫猪三周后,ELISA抗体水平达到最高,5头猪全达到1:80,其中3头猪达到1:160,2头达到1:320,而10~(5.0)TCID_(50)和10~(6.0)TCID_(50)抗原含量的灭活苗免疫猪后未检测到特异性抗体。②最小免疫量的灭活苗1ml、2ml、3ml免疫猪均能产生较好的ELISA抗体,但2ml组效果明显优于其它各组。③免疫四周后攻毒,对照组体温高于免疫组,病毒血症检测及病毒DNA在组织中的分布检测结果表明该疫苗能够有效降低猪体的带毒,但是10~(7.0)TCID_(50)抗原含量的灭活苗2ml组效果最佳。
3.PCV2灭活苗免疫效力试验
用灭活苗2ml免疫猪群,于免疫后1、2、3、4周分别进行攻毒保护性试验,通过攻毒后体温变化、临床表现、病毒血症及病毒在组织中的分布等指标对疫苗免疫不同时间的效力进行评价。结果显示,免疫一周和两周后攻毒,免疫组和对照组各指标基本没有差异;免疫三周和四周后攻毒,免疫组均只有两头出现病毒血症及解剖组织检测到病毒,但对照组每头都检测到病毒血症及解剖组织检测到病毒。说明该疫苗免疫三周后可以对猪坚强保护。
4.PCV2灭活苗安全性试验
用单倍剂量和双倍剂量灭活苗免疫仔猪,测定免疫前后的体温,观察猪群的采食情况,触摸局部,有无肿块。免疫后28天,每组解剖2头猪,观察疫苗的吸收状况。结果,免疫前后猪群没有体温升高的现象,采食和精神正常,注射部位未见肿块出现。解剖后,注射部位也未见有白色油乳剂样物质存在。说明单倍和2倍剂量接种后对猪是安全的。
Porcine circovirus type 2 ( PCV2) infection is recognized as the major factor in the development of post-weaning multisystemic wasting syndrome (PMWS),although other factors may enhance the severity of the disease. At present, PCV2 is widely spreaded in the world and maintain high infection rate in herd. Vaccine inoculation is the main method to prevent and control PCV2 infection; however, there is no commercial vaccine against PCV2 in China yet.
An inactivated vaccine was developed based on porcine circovirus type 2 WH strains, a wild strain separated in pig farm Wuhan. To fight against porcine circovirus type 2, the following works were performed aiming to prepare a novel vaccine.
1. Development of inactivated vaccine against porcine circovirus type 2
PCV-2 WH strain was inoculated into PK-15 cell by the way of one-stepinoculation. After forming a single layer, the cells were treated with 300mmol/L D-glucosamine, at 37℃for 30min, discarded the D-glucosamine, then added DMEM with 2% newborn bovine serum and cultured for another 48h, harvesting virus. PCV2 was inactivated with 0.3% formalin. The inactivated vaccine was prepared with inactivated virus and appropriate amount of American oil-adjuvant.Then sterility and physical character of the vaccine was detected.
2. Determination of minimum immune dosage of the inactivated vaccine
Piglets immunized with 10~(5.0)TCID_(50)、10~(6.0)TCID_(50) and 10~(7.0)TCID_(50) antigen doserespectively, then the immune efficacies of the vaccines against PCV2 were evaluated by ELISA antibody detection, clinical signs (fever), viremia and distribution of viral DNA in different tissues. Further Piglets were immunized with minimum immune dosage by 1ml, 2ml and 3ml, respectively, and then the im mune efficacy was evaluated according to the method mentioned above. The results were as follows:①The efficient immune response could be induced with the vaccine of 10~(7.0) TCID_(50) PCV2 in piglets, while specific antibody wasn't detected with the vaccine of 10~(6.0) TCID_(50)PCV2 and 10~(5.0) TCID_(50) PCV2 in piglets.②High ELISA antibody titers of the vaccines with 10~(7.0) TCID_(50) 1ml、2 ml and 3ml could be detected in piglets. The immune efficacy of 10~(7.0) TCID_(50) PCV2 2ml was obviously higher than the other 2 groups.③After PCV2 challenge the rectal temperature of all piglets were recorded. Average temperature of piglets in challenge control group was higher than that of immunized piglets. The fact that vaccines could reduce the persistence of PCV2 in piglets was indicated by detection of viremia and PCV2 in different tissues of challenged piglets. The immune efficacy of 10~(7.0)TCID_(50)PCV2 2ml dosage group was the best in all groups.
3. Evaluation of the efficacy of the inactivated vaccine
After piglets immunized with vaccine 2ml for one week, two weeks, three weeks and four weeks respectively, the immune efficacies of the vaccines against PCV2 were evaluated by clinical signs (fever), viremia and distribution of viral DNA in the tissues. The results showed that challenge with porcine circovirus type 2 strains WH after post immunized for one week and two weeks, there was no difference between the control group and the immunized group. Challenge with porcine circovirus type 2 strains WH after post immunized for three weeks and four weeks, viremia and viral DNA in the tissues were detected in two piglets of the immunized group, while viremia and viral DNA in the tissues were detected in five piglets of the control group. It could be concluded that the inactivated vaccine could protect piglets from PCV2 infection after immunized for three weeks.
4. Evaluation of the safety of the inactivated vaccine
Piglets were inoculated with single dose and double doses of the inactivated vaccine and the rectal temperature of all piglets were recorded before and after inoculation. Then clinical symptoms such as appetite were observed. 28 days after inoculated, the situation of the vaccine absorption was observed. According to clinical symptoms, there was no significant difference between before and after inoculation. After autopsy, there was no residual vaccine in the injection site. Both single dose and double dose of the vaccine are safe to the piglets. It could be concluded that the inactivated vaccine is safe in piglets.
引文
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