摘要
为探究猪瘟弱毒标记疫苗候选株RecC-M1(将牛病毒性腹泻病毒1型E^(rns)和猪瘟病毒基因2型流行株E2高变区分别置换弱毒疫苗C株对应区域构建而成)对猪的安全性,本实验以2.0×10^(5)FAID_(50)(50%荧光抗体感染剂量)高剂量的RecC-M1株经颈部肌肉注射接种12头35日龄猪瘟病毒(CSFV)抗体阴性仔猪,另设生理盐水对照组6头。接种后观察各组猪的临床症状,并于接种后不同时间(0、1 d、3 d、5 d、7 d、14 d、28 d和42 d)采血用于细胞计数;接种后7 d和42 d各取6头猪(包括第42 d对照组6头猪),剖杀后观察各组织剖检病变,并取各组织制作切片观察组织病变,采用qPCR检测病毒在各组猪各组织中的分布。根据qPCR结果,选择阳性组织样品(扁桃体、肾脏和淋巴结)的病理切片采用免疫组化法检测RecC-M1株在猪上述各组织中的分布。于接种后0~7 d、14 d、28 d和42 d分别采集各组猪的鼻拭子和肛拭子样品以及不同时间采集的血液,均采用qPCR检测各组猪的病毒血症及排毒;采用间接ELISA分别检测各组猪血清中分别针对BVDV及CSFV相应抗原的抗体水平。结果显示,RecC-M1组猪在42 d的观察期内均未出现临床症状,体温、白细胞数和淋巴细胞数与阴性对照组猪均无明显差异,且均在正常范围内;剖检及组织病变观察结果显示,接种后7 d和42 d猪的各组织均未见淤血或出血;扁桃体、淋巴结、脾、肾、膀胱、回盲瓣等CSFV嗜性组织均未见明显的病理学变化;各组织样品的qPCR结果显示,接种后7 d所有6头猪的肾脏和扁桃体样品和2头猪的淋巴结样品中均检出CSFV核酸,而接种后42 d所有猪的组织样品均为阴性;免疫组化结果显示,接种后7 d猪扁桃体和肾脏样品中CSFV抗原均呈阳性,接种后42 d猪的所有组织样品则均转为阴性;病毒血症和排毒的qPCR结果显示,接种后14 d内部分猪血液、鼻拭子和肛拭子样品中检出CSFV核酸,接种后28 d和42 d所有猪血液、鼻拭子和肛拭子则均为阴性。间接ELISA结果显示,接种后14 d针对BVDV1 E^(rns)抗体开始上升,而针对CSFV E2的抗体则在接种后7 d开始上升,此后两种抗体水平均随时间的推移呈持续上升趋势。阴性对照组猪各组织均未见明显剖检及组织病变,各组织CSFV抗原,各组织、血液及拭子样品均为阴性,也未产生针对相应病毒抗原的抗体。上述结果表明,高剂量的RecC-M1株对猪是安全的,本研究为RecC-M1株作为猪瘟弱毒标记疫苗的产业化开发奠定实验基础。
To examine the safety of a live attenuated marker vaccine candidate RecC-M1(E^(rns)of bovine viral diarrhea virus type 1 and E2 hypervariable region of classical swine fever virus type 2(CSFV) were substituted for the corresponding regions of attenuated C strain, respectively) against classical swine fever in the natural hosts, twelve 35-day-old piglets negative for CSFV antibodies were intramuscularly inoculated in the neck region with a single dose of RecC-M1 at 2.0×10^(5)FAID_(50)(50% fluorescent antibody infective dose) and six pigs were injected with saline as controls. Blood samples were collected on day 0, 1, 3, 5, 7, 14, 28and 42 days post-inoculations(dpi) for determination of leukocyte and lymphocyte counts. A group of six pigs on 7 or 42 dpi,respectively, after inoculation(including six control pigs on 42 dpi) were euthanized and the relevant organs were dissected for gross pathological and histopathological observations, and virus distribution in pig tissues was detected by q PCR. According to qPCR results, the pathological sections of positive samples(tonsils, kidneys and lymph nodes) were selected to detect the distribution of RecC-M1 strain in the above tissues of pigs by immune-histochemistry(IHC). The nasal swab and anal swab samples of pigs in each group collected on 0-7, 14, 28 and 42 dpi and the blood collected at different times were detected by q PCR for the viremia and virus excretion of pigs in each group. Indirect ELISA was adopted to detect the antibody levels against corresponding antigens of BVDV and CSFV in pig serum of each group. The results showed that all pigs inoculated with RecC-M1 did not show any clinical signs during the 42-day observation period. The body temperature and total counts of leukocytes and lymphocytes were comparable between the RecC-M1 group and control group. No congestion or hemorrhage was visually observed, and no obvious histopathological changes were found in tonsils, lymph nodes, spleen, kidney, etc. sampled on 7 or 42 dpi. The viral antigen was found in the tonsil and kidney samples from several RecC-M1 inoculated pigs on 7 dpi, but not from all inoculated pigs on 42 dpi.The IHC results showed CSFV antigens were positive in pig tonsils and kidneys sampled on 7 dpi and negative on 42 dpi. q PCR analysis of viremia and virus excretion revealed presence of viral RNA in blood, nasal and rectal swab samples from some of the pigs inoculated with RecC-M1 within 14 dpi;All pig blood, nasal swabs and anal swabs were negative for viral RNA on 28 and 42dpi. The indirect ELISA results showed that the antibody against BVDV1 E^(rns)began to rise on 14 dpi, while the antibody against CSFV E2 began to rise later on 7 dpi. Since then, the levels of both antibodies have continued to rise over time. In the saline control group, no obvious necropsy or pathological changes were found in pig tissues, CSFV antigen was negative in all tissues, qPCR was negative in all tissues, blood and swab samples, and no antibody against corresponding virus antigen was produced. These findings clearly showed that RecC-M1 is safe to pigs at high inoculum, and this study has laid good foundation for commercial development of RecC-M1-based marker vaccine against classical swine fever.
作者
叶超锋
曹统
王作欢
陈荣
王媛
徐永昊
许翰坤
韩笑
张鹏超
蓝胜芝
吴桃芬
杨香林
李肖梁
方维焕
YE Chao-feng;CAO Tong;WANG Zuo-huan;CHEN Rong;WANG Yuan;XU Yong-hao;XU Han-kun;HAN Xiao;ZHANG Peng-chao;LAN Sheng zhi;WU Tao-fen;YANG Xiang-lin;LI Xiao-liang;FANG Wei-huan(Zhejiang University Institute of Preventive Veterinary Medicine,Hangzhou 310058,China;Zhejiang Ceva Ebvac Biotech Co,Ltd,Hangzhou 310018,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第12期1304-1310,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
浙江省重点研发计划项目(2019C02043)
上海市兽医生物技术重点实验室开放基金(KLAB201711)。
关键词
猪瘟
弱毒标记疫苗候选株
安全性
classical swine fever
live attenuated marker vaccine candidate
safety