| Co-Investigator(Kenkyū-buntansha) |
OYAMADA Takashi Kitazato University, 獣医畜産学部, 教授 (80050665)
KODAMA Yoshihiro Gen Cooperation, 所長
ISEKI Motohiro Kanazawa University, 医学部, 教授 (20047179)
XUAN Xuanan OBIHIRO UNIVERSITY OF AGRICULTURE AND VETERINARY MEDICINE, National Research Center for Protozoan Diseases, 原虫病研究センター, 助教授 (10292096)
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| Research Abstract |
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
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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
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