ESTABLISHMENT OF ADAPTIVE IMMUNE THERAPY USING EGG YOLK ANTIBODY ON CRYPTOSPORIDIOSIS

蛋黄抗体对隐孢子虫病适应性免疫治疗的建立

• 批准号: 12356009
• 负责人: NAGASAWA Hideyuki
• 金额: $ 27.51万
• 依托单位: OBIHIRO UNIVERSITY OF AGRICULTURE AND VETERINARY MEDICINE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12356009/
• 关键词: protozoan diseases; virus vector; oocyst; Cryptosporidium parvum; adaptive immunity; transgenic; protective immunity; egg yolk antibody

Cryptosporidium parvum is an intestinal protozoan that causes enteric infection and diarrhea in humans and animals. In immunocompromised patients, diarrhea caused by C.parvum may be chronic and life threatening. Infection with C.parvum in young calves causes severe economic problems. Recently, outbreaks of cryptosporidiosis have been reported, and contamination of water-sheds by cattle operations has been suggested as a source of these outbreaks. Controlling C.parvum infections in cattle is important not only to reduce the economic impact but also to resolve public health concerns. Currently, there are no vaccines or specific therapeutic regimens to control cryptosporidiosis.In order to develop a vaccine against cryptospondiosis in cattle, we constructed a recombinant bovine herpesvirus-1 (BHV-1) expressing an immunodominant surface protein, p23, of C.parvum sporozoites. In the recombinant virus, the p23 gene under the control of a CAG promoter and a gene coding for an enhanced green f … More luorescent protein were integrated into the gG fluorescence of the plaques formed by recombinants. These plaques were among the plaques of the nonfluorescent parental virus. All clones selected for fluorescence also contained the p23 gene. In MDBK cells infected with the recombinant BHV-1, the antibody against the p23 protein recognized the p23 protein as an approximately 23-kDa specific band in Western blotting analysis.Rivers that are polluted with C.parvum and drinking water that is treated for drinking water production from polluted rivers could result in the waterborne disease of cryptospondiosis. We carried out an epidemiological study of natural water supplies in Hokkaido, one of the largest dairy prefectures in Japan. To detect Cryptospondium oocysts in environmental water, the filtration method was used for samples, which were collected from 10 rivers. A method adapted from the United States Environmental Protection Agency filtration method using a cartridge filter has been used for the collection of samples. Oocysts were separated from a pellet by the discontinuous sucrose gradient method. C.parvum oocysts were detected from 10 rivers by iron (III) flocculation method, and identified with the immunofluorescence antibody technique using DIF kit.The viability and infectivity of C.parvum oocysts was investigated using SCID mice. Oocysts were collected from river samples and inoculated orally to SCID mice with 1000 oocysts. Infection was successfully induced, resulting in fecal oocyst shedding. Our results send a warning that Japan may harbor a significant presence of C.parvum oocysts capable of transmitting infection to human host. It needs the establishment to vaccination method and wide range epidemiological study to control cryptospondiosis. Less

隐孢子虫是一种肠道原生动物，可引起人类和动物的肠道感染和腹泻。在免疫功能低下的患者中，由微小隐球菌引起的腹泻可能是慢性的并且危及生命。幼牛感染微小隐孢子虫会导致严重的经济问题。最近，隐孢子虫病的爆发已被报道，并已被认为是这些爆发的来源之一，由牛的操作流域的污染。控制牛的微小隐孢子虫感染不仅对减少经济影响很重要，而且对解决公共卫生问题也很重要。为了研制抗牛隐孢子虫病的疫苗，我们构建了表达微小隐孢子虫（C.parvum）子孢子表面蛋白p23的重组牛疱疹病毒1型（BHV-1）。在重组病毒中，在CAG启动子控制下的p23基因和编码增强的绿色荧光蛋白的基因， ...更多信息 荧光蛋白整合到重组体形成的噬斑的gG荧光中。这些空斑是非荧光亲本病毒的空斑之一。选择用于荧光的所有克隆也含有p23基因。在用重组BHV-1感染的MDBK细胞中，抗p23蛋白的抗体在Western印迹分析中识别p23蛋白为约23-kDa的特异性条带。我们在日本最大的乳制品县之一的北海道进行了一项天然水供应的流行病学研究。为检测环境水体中的隐孢子虫卵囊，采用过滤法对采集自10条河流的样品进行了检测。采用美国环境保护署使用筒式过滤器的过滤方法进行样品采集。通过不连续蔗糖梯度法从沉淀中分离卵囊。采用铁（III）絮凝法对10条河流中的微小隐孢子虫卵囊进行检测，并采用免疫荧光抗体技术对微小隐孢子虫卵囊进行鉴定，采用SCID小鼠对微小隐孢子虫卵囊的存活率和感染性进行了研究。从河流样品中收集卵囊，并用1000个卵囊经口接种给SCID小鼠。成功诱导感染，导致粪便卵囊脱落。我们的研究结果发出了一个警告，日本可能藏有大量的C.parvum卵囊能够传播感染给人类宿主。控制隐孢子虫病需要建立疫苗接种方法和广泛的流行病学研究。少

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