牛病毒性腹泻病毒对新西兰白兔致病性分析及E2蛋白免疫效果的评价

doi:10.16431/j.cnki.1671-7236.2022.09.029

摘要/Abstract

摘要： 【目的】研究牛病毒性腹泻病毒（BVDV）感染对新西兰白兔的致病性以及BVDV E2重组蛋白的免疫效果。【方法】将实验室培养保存的BVDV病毒纯化并按照Reed-Muench法测定其病毒滴度。在致病性试验中，将10只新西兰白兔随机分为感染组和对照组，每组5只。感染组用1 mL纯化的BVDV病毒攻毒（滴鼻500 μL、耳缘静脉注射500 μL），对照组用等体积的生理盐水处理，连续3 d，每天1次，每天观察各组兔的临床症状并测量体温；分别于接种病毒后第6、9、12、15、17天通过耳缘静脉采集血液检测血常规；感染病毒第17天采集鼻拭子进行RT-PCR鉴定，采集后剖杀并采集气管、肺脏、脾脏和小肠组织，制备病理切片观察病理变化。在免疫效果评价试验中，将10只新西兰白兔随机分为免疫组和对照组，每组5只，免疫组用E2重组蛋白（1 mg/只）与佐剂混合后经肌内多点注射免疫新西兰白兔，对照组接种等体积生理盐水；共免疫2次，2次免疫间隔为14 d。在一免后0、7、14、21、28 d采集血清，通过间接ELISA方法检测血清中抗重组蛋白特异性抗体水平；在一免后第28天按致病性试验中方法攻毒，在攻毒第17天采集鼻拭子进行RT-PCR鉴定，采集气管、肺脏、脾脏和小肠组织制备病理切片观察病理变化及免疫组织化学检测。【结果】纯化后BVDV的病毒滴度为4.16×10⁶ TCID₅₀/mL。与对照组相比，感染组部分新西兰白兔6 d内活动减少，采食略微减少，6 d后逐渐恢复正常，在感染第13天出现腹泻症状，从第5天开始体温略微升高，但均在正常范围内波动。与对照组相比，在攻毒第6和9天，感染组白细胞和血小板分别显著和极显著降低（P<0.05；P<0.01）；在攻毒第12、15和17天，感染组白细胞、血小板和淋巴细胞均极显著降低（P<0.01）。鼻拭子RT-PCR检测为阳性，气管、肺脏、脾脏及小肠组织表现出轻度至重度的组织病理学变化。间接ELISA检测结果表明，在一免后7 d时，血清抗体滴度为1：16~1：32；在一免后28 d时，血清抗体滴度为1：256~1：512；免疫攻毒组新西兰白兔鼻拭子经RT-PCR检测为阴性；组织病理学观察显示，免疫攻毒组气管及肺脏表现出轻微的组织病理学变化。免疫组化检测结果显示，免疫组结果均呈阴性，对照组结果均为阳性。【结论】通过滴鼻及耳缘静脉注射BVDV的方式可以构建新西兰白兔致病模型，BVDV E2亚单位疫苗能够刺激机体产生特异性抗体，起到免疫防御的作用。

关键词: 牛病毒性腹泻病毒; 新西兰白兔; 免疫组化; 亚单位疫苗

Abstract: 【Objective】 The aim of this study was to explore the pathogenicity of Bovine viral diarrhea virus (BVDV) and the immune effect of BVDV E2 recombinant protein in New Zealand White rabbits.【Method】 The BVDV virus was purified and its titer was determined by Reed-Muench method. In the pathogenicity analysis test, 10 New Zealand White rabbits were randomly divided into infection group and control group, with 5 rabbits in each group. The infected group was challenged with 1 mL purified BVDV virus (nasal drip 500 μL, ear edge intravenous injection 500 μL), and the control group was treated with equal volume of normal saline, once a day for 3 days. The clinical symptoms and body temperature were observed every day. Blood samples were collected from auricular vein on 6, 9, 12, 15 and 17 days after inoculation. On the 17th day of infection, nasal swabs were collected for RT-PCR identification. After collection, trachea, lung, spleen and small intestine tissues were dissected and pathological sections were prepared to observe the pathological changes. In the immune effect evaluation experiment, 10 New Zealand White rabbits were randomly divided into the immune group and the control group, with 5 rabbits in each group. New Zealand White rabbits were immunized with E2 recombinant protein (1 mg/rabbit) mixed with adjuvant by intramuscular multi-point injection, and the control group was immunized with equal volume of normal saline, two immunizations were given at an interval of 14 days.Serum samples were collected on 0, 7, 14, 21 and 28 d after the first immunization, and the levels of specific antibodies against recombinant protein were detected by indirect ELISA. On the 28th day after the first immunization, nose swabs were collected for RT-PCR identification, and trachea, lung, spleen and small intestine tissues were collected for pathological sections to observe the pathological changes and immunohistochemistry detection.【Result】 The titer of the purified virus was 4.16×10⁶ TCID₅₀/mL. Compared with control group, some New Zealand White rabbits in the infected group had reduced activities and slightly reduced food intake within 6 days, and gradually returned to normal after 6 days. Diarrhea symptoms appeared on the 13th day of infection, and the body temperature increased slightly on the 5th day, but all fluctuated within the normal range. Compared with control group, white blood cells and platelets in infection group were significantly and extremely significantly decreased on the 6th and 9th day of challenge (P<0.05 or P<0.01). On the 12th, 15th and 17th days of challenge, white blood cells, platelets and lymphocytes in the infection group were extremely significantly decreased (P<0.01). RT-PCR test of nasal swabs was positive, and histopathological changes of trachea, lung, spleen and small intestine were mild to severe. The results of indirect ELISA showed that the titer of serum antibody was 1:16 to 1:32 on the 7th day after the first immunization. The nasal swabs of New Zealand White rabbits in the immune challenge group were negative by RT-PCR. Histopathological observation showed slight histopathological changes in trachea and lung in the immune challenge group. On the 28th day after the first immunization, the titer of serum antibody was 1:256 to 1:512. Immunohistochemical test results showed that the results were negative in the immune group and positive in the control group.【Conclusion】 A model of disease in New Zealand White rabbits was established by intranasal and auricular vein injection of BVDV, BVDV E2 subunit vaccine could stimulate the body to produce specific antibodies and play the role of immune defense.

Key words: Bovine viral diarrhea virus(BVDV); New Zealand White rabbits; immunohistochemistry; subunit vaccine

中图分类号:

S852.65⁺9.6

赵逢淼, 郭婷, 周雅坪, 赵红梅, 王宇琛, 田广原, 孙雅婕, 卞宇晨, 于佳梁, 郝永清. 牛病毒性腹泻病毒对新西兰白兔致病性分析及E2蛋白免疫效果的评价[J]. 中国畜牧兽医, 2022, 49(9): 3549-3558.

ZHAO Fengmiao, GUO Ting, ZHOU Yaping, ZHAO Hongmei, WANG Yuchen, TIAN Guangyuan, SUN Yajie, BIAN Yuchen, YU Jialiang, HAO Yongqing. Pathogenicity Analysis of Bovine Viral Diarrhea Virus and Immune Effect Evaluation of E2 Protein in New Zealand White Rabbits[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3549-3558.

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