Abstract：
BACKGROUND: Rabbit coccidiosis is a parasitism caused by either one or multiple co-infections of Eimeria species. Among them, Eimeria intestinalis is the primary pathogen responsible for diarrhea, growth retardation, and potential mortality in rabbits. Concerns regarding drug resistance and drug residues have led to the development of recombinant subunit vaccines targeting Eimeria species as a promising preventive measure. The aim of this study was to assess the immunoprotective efficacy of recombinant subunit vaccines comprising EiROP25 and EiROP30 (rhoptry proteins (ROPs)) against E. intestinalis infection in rabbits.
METHODS: Cloning, prokaryotic expression, and protein purification were performed to obtain EiROP25 and EiROP30. Five groups of fifty 35-day-old Eimeria-free rabbits were created (unchallenged control group, challenged control group, vector protein control group, rEiROP25 group, and rEiROP30 group), with 10 rabbits in each group. Rabbits in the rEiROP25 and rEiROP30 groups were immunized with the recombinant proteins (100 μg per rabbit) for primary and booster immunization (100 μg per rabbit) at a two-week intervals, and challenged with 7 × 104 oocysts per rabbit after an additional two-week interval. Two weeks after the challenge, the rabbits were euthanized for analysis. Weekly collections of rabbit sera were made to measure changes in specific IgG and cytokine level. Clinical symptoms and pathological changes after challenge were observed and recorded. At the conclusion of the animal experiment, lesion scores, the relative weight increase ratio, the oocyst reduction rate, and the anticoccidial index were computed.
RESULTS: Rabbits immunized with rEiROP25 and rEiROP30 exhibited relative weight gain ratios of 56.57% and 72.36%, respectively. Oocysts decreased by 78.14% and 84.06% for the rEiROP25 and rEiROP30 groups, respectively. The anticoccidial indexes were 140 and 155. Furthermore, there was a noticeable drop in intestinal lesions. After the primary immunization with rEiROP25 and rEiROP30, a week later, there was a notable rise in specific IgG levels, which remained elevated for two weeks following challenge (P < 0.05). Interleukin (IL)-2 levels increased markedly in the rEiROP25 group, whereas IL-2, interferon gamma (IFN-γ), and IL-4 levels increased substantially in the rEiROP30 group (P < 0.05).
CONCLUSIONS: Immunization of rabbits indicated that both rEiROP25 and rEiROP30 are capable of inducing an increase in specific antibody levels. rEiROP25 triggered a Th1-type immune protection response, while rEiROP30 elicited a Th1/Th2 mixed response. EiROP25 and EiROP30 can generate a moderate level of immune protection, with better efficacy observed for EiROP30. This study provides valuable insights for the promotion of recombinant subunit vaccines targeting rabbit E. intestinalis infection.
METHODS: Cloning, prokaryotic expression, and protein purification were performed to obtain EiROP25 and EiROP30. Five groups of fifty 35-day-old Eimeria-free rabbits were created (unchallenged control group, challenged control group, vector protein control group, rEiROP25 group, and rEiROP30 group), with 10 rabbits in each group. Rabbits in the rEiROP25 and rEiROP30 groups were immunized with the recombinant proteins (100 μg per rabbit) for primary and booster immunization (100 μg per rabbit) at a two-week intervals, and challenged with 7 × 104 oocysts per rabbit after an additional two-week interval. Two weeks after the challenge, the rabbits were euthanized for analysis. Weekly collections of rabbit sera were made to measure changes in specific IgG and cytokine level. Clinical symptoms and pathological changes after challenge were observed and recorded. At the conclusion of the animal experiment, lesion scores, the relative weight increase ratio, the oocyst reduction rate, and the anticoccidial index were computed.
RESULTS: Rabbits immunized with rEiROP25 and rEiROP30 exhibited relative weight gain ratios of 56.57% and 72.36%, respectively. Oocysts decreased by 78.14% and 84.06% for the rEiROP25 and rEiROP30 groups, respectively. The anticoccidial indexes were 140 and 155. Furthermore, there was a noticeable drop in intestinal lesions. After the primary immunization with rEiROP25 and rEiROP30, a week later, there was a notable rise in specific IgG levels, which remained elevated for two weeks following challenge (P < 0.05). Interleukin (IL)-2 levels increased markedly in the rEiROP25 group, whereas IL-2, interferon gamma (IFN-γ), and IL-4 levels increased substantially in the rEiROP30 group (P < 0.05).
CONCLUSIONS: Immunization of rabbits indicated that both rEiROP25 and rEiROP30 are capable of inducing an increase in specific antibody levels. rEiROP25 triggered a Th1-type immune protection response, while rEiROP30 elicited a Th1/Th2 mixed response. EiROP25 and EiROP30 can generate a moderate level of immune protection, with better efficacy observed for EiROP30. This study provides valuable insights for the promotion of recombinant subunit vaccines targeting rabbit E. intestinalis infection.
摘要：
背景：兔球虫病是一种由一种或多种艾美球虫共同感染引起的寄生虫。其中,肠埃美球虫是引起腹泻的主要病原体，生长迟缓,和兔子的潜在死亡率。对耐药性和药物残留的担忧已导致开发针对艾美球虫物种的重组亚单位疫苗作为有希望的预防措施。这项研究的目的是评估包含EiROP25和EiROP30（跳跳蛋白（ROP））的重组亚单位疫苗对兔肠球菌感染的免疫保护功效。
方法：克隆，原核表达，并进行蛋白质纯化以获得EiROP25和EiROP30。创建五组50只35天龄的无艾美球虫兔（未攻击的对照组，挑战对照组，载体蛋白对照组，rEiROP25组，和REiROP30组），每组10只兔子。rEiROP25和rEiROP30组中的兔子用重组蛋白（每只兔子100μg）免疫，以两周的间隔进行初次和加强免疫（每只兔子100μg），再间隔两周后，每只兔子用7×104个卵囊攻击。挑战两周后，将兔子安乐死用于分析。每周收集兔血清以测量特异性IgG和细胞因子水平的变化。观察并记录激发后的临床症状和病理变化。在动物实验结束时，病变评分,相对重量增加率，卵囊减少率，并计算了抗球虫指数。
结果：用rEiROP25和rEiROP30免疫的家兔表现出56.57％和72.36％的相对增重率，分别。rEiROP25和rEiROP30组的卵囊减少了78.14%和84.06%,分别。抗球虫指数分别为140和155。此外，肠道病变明显下降。在用rEiROP25和rEiROP30进行初次免疫后，一周后，特异性IgG水平显着上升，在攻击后两周内保持升高（P<0.05）。白细胞介素（IL）-2水平在rEiROP25组中明显升高，而IL-2，干扰素γ（IFN-γ），rEiROP30组IL-4水平显著升高(P<0.05)。
结论:兔的免疫表明rEiROP25和rEiROP30都能够诱导特异性抗体水平的增加。rEiROP25引发Th1型免疫保护反应，而rEiROP30引起Th1/Th2混合反应。EiROP25和EiROP30可以产生中等水平的免疫保护，对于EiROP30观察到更好的疗效。本研究为促进重组亚单位疫苗靶向兔肠球菌感染提供了有价值的见解。
方法：克隆，原核表达，并进行蛋白质纯化以获得EiROP25和EiROP30。创建五组50只35天龄的无艾美球虫兔（未攻击的对照组，挑战对照组，载体蛋白对照组，rEiROP25组，和REiROP30组），每组10只兔子。rEiROP25和rEiROP30组中的兔子用重组蛋白（每只兔子100μg）免疫，以两周的间隔进行初次和加强免疫（每只兔子100μg），再间隔两周后，每只兔子用7×104个卵囊攻击。挑战两周后，将兔子安乐死用于分析。每周收集兔血清以测量特异性IgG和细胞因子水平的变化。观察并记录激发后的临床症状和病理变化。在动物实验结束时，病变评分,相对重量增加率，卵囊减少率，并计算了抗球虫指数。
结果：用rEiROP25和rEiROP30免疫的家兔表现出56.57％和72.36％的相对增重率，分别。rEiROP25和rEiROP30组的卵囊减少了78.14%和84.06%,分别。抗球虫指数分别为140和155。此外，肠道病变明显下降。在用rEiROP25和rEiROP30进行初次免疫后，一周后，特异性IgG水平显着上升，在攻击后两周内保持升高（P<0.05）。白细胞介素（IL）-2水平在rEiROP25组中明显升高，而IL-2，干扰素γ（IFN-γ），rEiROP30组IL-4水平显著升高(P<0.05)。
结论:兔的免疫表明rEiROP25和rEiROP30都能够诱导特异性抗体水平的增加。rEiROP25引发Th1型免疫保护反应，而rEiROP30引起Th1/Th2混合反应。EiROP25和EiROP30可以产生中等水平的免疫保护，对于EiROP30观察到更好的疗效。本研究为促进重组亚单位疫苗靶向兔肠球菌感染提供了有价值的见解。
