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  

Pathogenicity Analysis of Bovine Viral Diarrhea Virus and Immune Effect Evaluation of E2 Protein in New Zealand White Rabbits

作　　者：赵逢淼 郭婷[1] 周雅坪 赵红梅[1] 王宇琛 田广原 孙雅婕 卞宇晨 于佳梁 郝永清[1] ZHAO Fengmiao;GUO Ting;ZHOU Yaping;ZHAO Hongmei;WANG Yuchen;TIAN Guangyuan;SUN Yajie;BIAN Yuchen;YU Jialiang;HAO Yongqing(Laboratory of Veterinary Microbiology and Immunology,Inner Mongolia Agricultural University,Hohhot 010018,China)

机构地区：[1]内蒙古农业大学,兽医微生物学与免疫学实验室,呼和浩特010018

出　　处：《中国畜牧兽医》2022年第9期3549-3558,共10页China Animal Husbandry & Veterinary Medicine

基　　金：牛羊重要疫病免疫与分子快速检测新技术研发(2019GG240);牛支原体感染促进牛病毒性腹泻病毒感染机制的研究(32060790)。

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500μL、耳缘静脉注射500μL),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4.16×10~6 TCID/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【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 17 th 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 28 th 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 4.16×10~6 TCID/mL. Compared with control g

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 

分 类 号：S852.659.6[农业科学—基础兽医学]