猫细小病毒灭活疫苗的制备及保护性研究
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摘要
猫瘟热又称猫泛白细胞减少症或猫传染性肠炎,是由猫细小病毒(FPV)引起猫的一种接触性急性传染病。临床症状为双相热型、呕吐、腹泻、严重脱水和血液白细胞显著减少,病理上为出血性肠炎,是猫科动物最重要的传染病。FPV在自然状态下可引起猫、水貂、浣熊等动物较高的发病率和死亡率,对各种野生动物和特种经济动物构成极大的威胁。本病目前尚无有效治疗方法,预防的最主要措施是注射疫苗。由于FPV仅有一个血清型,不易产生变异,因此可筛选免疫原性较强的毒株制备疫苗,通过阶段性免疫为动物群提供长期保护。
本研究首先对筛选出来的FPV CC-2型毒株扩大培养,通过试验比较并确定了毒株的最佳生长条件,确定病毒的灭活条件并对灭活剂残留进行检测,实验结果显示经BPL在4℃8小时即可完全灭活,37℃水浴水解2h后BPL检测无残留。参考《兽用生物制品规程》配制了Al(OH)3胶佐剂,主要指标检验合格,与灭活病毒按1:9比例混合,检测铝离子含量为1.6~1.7mg/mL,均在规定范围内。配制的疫苗经无菌检验、支原体检验、外源病毒检验(FCV、FHV)均为阴性,没有任何污染。其中无菌检验为国家质量标准,支原体检验按国家标准需要时间长达28天,本研究采用PCR扩增技术检验,快速、敏感、特异性强,减少了检验周期,外源病毒检验技术由于目前没有国家标准,本试验依靠特异性较好的RT-PCR和PCR技术,无论技术本身还是结果的可靠性,都是值得采纳的方法。
随后对实验室生产制备的疫苗进行安全性检验,分别以一次单剂量、单剂量重复和一次超剂量接种实验猫,通过临床观察、体温测定、抗体水平检测和病理学观察证明,本疫苗具有良好的安全性,同时可诱导机体产生良好抗体水平。免疫保护试验证实本疫苗的免疫效果,对实验猫正常免疫后30d进行攻毒实验,在攻毒后30d内,免疫组试验猫均健活,仅一只猫有轻微病症且最终耐过,保护率为100%;对照组死亡率为80%,经检测死亡猫为FPV阳性。
Feline Parvovirus is also known as Feline panleukopenia virus, cause infectious disease that are characterized primarily by high fever, vomiting, severe reduction of white blood cells and inflammatory. Biphasic fever-type symptoms, vomiting, diarrhea, severe dehydration and reduction of leukocytes in blood are the major clinical symptoms, hemorrhagic enteritis in Pathology, this disease is one of the most important infectious diseases in feline. In natural conditions FPV can cause high morbidity and mortality in cats, mink, raccoons and some other animals, resulting in a grave threat to wild animals、special and economic animals. The disease,which was proved Serological survey, has a wide distribution in all over the world ,also specific anti-FPV antibodies were detected in wild cats of different regions of our country , according to the anatomy and diagnosis of dead animals, FPV is the main reason leading to death. Since there is only one serotype of FPV, all the vaccines in use have long-term effective immunity. However, there is no a fully effective immunization and treatment method to this disease in domestic market, and the approval number of the vaccine yet.
The following aspects of the study:
First, proliferation of the selected FPV CC-2 type strains, the optimal growth conditions is identified , FK81 is cultivated in 37℃,using MEM containing 2% FBS. FPV is inoculatd by MEM containing 5% FBS ,and after 96h, collectting FPV, the viral titer is up to 107/0.1mlTCID50 . Virus inactivation conditions should be determined, and residue of inactivator is tested, results showed that inactivation by BPL at 4℃for 8 hours can be completely inactivated, 37℃, after hydrolysis of 2h detected no residual BPL. Reference to "Veterinary Biological Products standards" prepared the Al (OH) 3 gel adjuvant, the main index are tested and qualified, with inactivated virus mixed by 1:9 ratio, detection of aluminum ion content was 1.6 ~ 1.7mg/mL, within the defined range. The bacteria ,mycoplasma, exogenous virus (FCV, FHV) of the prepared vaccine are tested,which of all are negative, without pollution. Including the sterility test is on the base of the national quality standards, mycoplasma testing would take at least 28 days according to national standards.In this research we used PCR amplification test, it is rapid, sensitive, specific, reducing the waste of human and material resources, because there is no national standard for exogenous virus test, we use good-specific RT-PCR and PCR, the technology and result are both worthy to adopt.
Secondly, safety test of vaccine that produced in laboratory. Respectively, by one single dose, multi-single dose and one single super-dose inoculate cats, through clinical observations, body temperature, detection of Antibody and pathology have shown that the vaccine produced an effective antibody level, with good security. In order to explore the immune effects of the inactivated vaccine, after the normal immune 30d, using virus to inoculate the experimental cats. 30d after inoculating FPV, the immuned experimental cats did not die, only a cat with a slight disease,but finally resisted it. So the protection rate is 100%; control group mortality rate was 80%, death cats are tested positive for the FPV. Using the established ELISA and HA test methods to test antibody levels of cats to determine the law between the time of immune and the level of antibody. The experimental result shows that the antibody level reaches highest in the 4th week after the first immunity. Dynamic law of animal maternal antibodies, vaccines shelf test, field test are also the components of future research.
本研究首先对筛选出来的FPV CC-2型毒株扩大培养,通过试验比较并确定了毒株的最佳生长条件,确定病毒的灭活条件并对灭活剂残留进行检测,实验结果显示经BPL在4℃8小时即可完全灭活,37℃水浴水解2h后BPL检测无残留。参考《兽用生物制品规程》配制了Al(OH)3胶佐剂,主要指标检验合格,与灭活病毒按1:9比例混合,检测铝离子含量为1.6~1.7mg/mL,均在规定范围内。配制的疫苗经无菌检验、支原体检验、外源病毒检验(FCV、FHV)均为阴性,没有任何污染。其中无菌检验为国家质量标准,支原体检验按国家标准需要时间长达28天,本研究采用PCR扩增技术检验,快速、敏感、特异性强,减少了检验周期,外源病毒检验技术由于目前没有国家标准,本试验依靠特异性较好的RT-PCR和PCR技术,无论技术本身还是结果的可靠性,都是值得采纳的方法。
随后对实验室生产制备的疫苗进行安全性检验,分别以一次单剂量、单剂量重复和一次超剂量接种实验猫,通过临床观察、体温测定、抗体水平检测和病理学观察证明,本疫苗具有良好的安全性,同时可诱导机体产生良好抗体水平。免疫保护试验证实本疫苗的免疫效果,对实验猫正常免疫后30d进行攻毒实验,在攻毒后30d内,免疫组试验猫均健活,仅一只猫有轻微病症且最终耐过,保护率为100%;对照组死亡率为80%,经检测死亡猫为FPV阳性。
Feline Parvovirus is also known as Feline panleukopenia virus, cause infectious disease that are characterized primarily by high fever, vomiting, severe reduction of white blood cells and inflammatory. Biphasic fever-type symptoms, vomiting, diarrhea, severe dehydration and reduction of leukocytes in blood are the major clinical symptoms, hemorrhagic enteritis in Pathology, this disease is one of the most important infectious diseases in feline. In natural conditions FPV can cause high morbidity and mortality in cats, mink, raccoons and some other animals, resulting in a grave threat to wild animals、special and economic animals. The disease,which was proved Serological survey, has a wide distribution in all over the world ,also specific anti-FPV antibodies were detected in wild cats of different regions of our country , according to the anatomy and diagnosis of dead animals, FPV is the main reason leading to death. Since there is only one serotype of FPV, all the vaccines in use have long-term effective immunity. However, there is no a fully effective immunization and treatment method to this disease in domestic market, and the approval number of the vaccine yet.
The following aspects of the study:
First, proliferation of the selected FPV CC-2 type strains, the optimal growth conditions is identified , FK81 is cultivated in 37℃,using MEM containing 2% FBS. FPV is inoculatd by MEM containing 5% FBS ,and after 96h, collectting FPV, the viral titer is up to 107/0.1mlTCID50 . Virus inactivation conditions should be determined, and residue of inactivator is tested, results showed that inactivation by BPL at 4℃for 8 hours can be completely inactivated, 37℃, after hydrolysis of 2h detected no residual BPL. Reference to "Veterinary Biological Products standards" prepared the Al (OH) 3 gel adjuvant, the main index are tested and qualified, with inactivated virus mixed by 1:9 ratio, detection of aluminum ion content was 1.6 ~ 1.7mg/mL, within the defined range. The bacteria ,mycoplasma, exogenous virus (FCV, FHV) of the prepared vaccine are tested,which of all are negative, without pollution. Including the sterility test is on the base of the national quality standards, mycoplasma testing would take at least 28 days according to national standards.In this research we used PCR amplification test, it is rapid, sensitive, specific, reducing the waste of human and material resources, because there is no national standard for exogenous virus test, we use good-specific RT-PCR and PCR, the technology and result are both worthy to adopt.
Secondly, safety test of vaccine that produced in laboratory. Respectively, by one single dose, multi-single dose and one single super-dose inoculate cats, through clinical observations, body temperature, detection of Antibody and pathology have shown that the vaccine produced an effective antibody level, with good security. In order to explore the immune effects of the inactivated vaccine, after the normal immune 30d, using virus to inoculate the experimental cats. 30d after inoculating FPV, the immuned experimental cats did not die, only a cat with a slight disease,but finally resisted it. So the protection rate is 100%; control group mortality rate was 80%, death cats are tested positive for the FPV. Using the established ELISA and HA test methods to test antibody levels of cats to determine the law between the time of immune and the level of antibody. The experimental result shows that the antibody level reaches highest in the 4th week after the first immunity. Dynamic law of animal maternal antibodies, vaccines shelf test, field test are also the components of future research.
引文
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