2019 Fiscal Year Annual Research Report
Development of rapid immunochromatographic assay as a diagnostic tool for bovine cryptosporidiosis
| Project/Area Number |
19F19107
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| Research Institution | Obihiro University of Agriculture and Veterinary Medicine |
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Principal Investigator |
西川 義文 帯広畜産大学, 原虫病研究センター, 教授 (90431395)
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| Co-Investigator(Kenkyū-buntansha) |
FEREIG RAGAB 帯広畜産大学, 原虫病研究センター, 外国人特別研究員
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| Project Period (FY) |
2019-04-25 – 2021-03-31
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| Keywords | クリプトスポリジム / ウシ / 下痢症 / 診断 |
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| Outline of Annual Research Achievements |
During one year of our study related to JSPS theme; Development of rapid immunochromatographic assay as afield diagnostic tool for bovine cryptosporidiosis, we could achieve a number of novel findings. For this purpose, we could collect a number of fecal samples from naturally infected calves with Cryptosporidium parvum from different farms in Tokachi sub-prefecture, Japan. All of these samples have been confirmed for C. parvum infection by commercial immunochromatographic test (ICT). We developed an efficient method for oocyst purification with obtaining high numbers and purity. For screening of C. parvum antibodies (target to develop ICT to detect antigen), we have established ELISA based on crude antigens (soluble antigen from purified oocyst lysate) that could detect specific antibodies from infected mice. This assay was confirmed against reactivity of recombinant antigen CpGP15 and CpP2. Moreover, we developed an efficient and safe IFAT system which firstly applied in C. parvum studies. This system based on using chemical fixation (methanol and ether), and brief autoclaving of oocysts to avoid potential hazards of oocyst spreading during processing. High ability to detect antibody was obtained as validated against commercial monoclonal anti-mouse antibody and our prepared polyclonal anti-rabbit antibody (CpGP15). Results of ELISA and IFAT indicate their usefulness for antibody detection. Moreover, we could prepare a lysis buffer that could replace the buffer from commercial kits to prepare fecal antigen for testing by ICT.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Currently, we are working on production of monoclonal antibodies that have the ability to detect C. parvum antigens in fecal samples of calves. In order to prepare these antibodies, numerous antigen preparations have been used including soluble lysate, whole lysate, heat inactivated whole oocyst, viable whole oocyst antigens. Each antigen preparation was mixed equally in TiterMax adjuvant, and then injected intraperitoneally in BALB/c mice (n = 7). After three times of immunization, specific antibodies in mouse sera of all groups were detected using our developed ELISA and IFAT. These results revealed our success in production of polyclonal antibodies from different antigens to detect C. parvum oocysts collected from the field. Currently, hybridoma from spleen of one mouse (immunized with viable oocysts) was prepared after fusion with SP2 cells. After initial screening test, at least one clone gives positive response against both IgG-based ELISA and IFAT. In addition, a number of clones give high antibody titers against IgG-based ELISA using soluble oocyst lysate antigen and also recombinant CpGP15. Further analyses are currently applied for some candidates including above-mentioned clone prior to hybridoma expansion and large scale production of monoclonal antibody. In addition, spleen cells from other immunized mice are collected for further fusion and screening.
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| Strategy for Future Research Activity |
The final goal of our study is the development of ICT that can detect specific antigens of C. parvum in fecal samples of naturally infected calves. Because currently we are analyzing some hybridoma for antibody production; the most critical step, further analyses will be performed using currently produced hybridoma or testing another mouse until obtaining some potent hybridoma candidates with higher level of antibody secretion. Later, hybridoma candidates with potent monoclonal antibody in antigen detection using ELISA, IFAT and western blotting will be selected. Concentration of monoclonal antibodies will be performed using intraperitoneal injection of selected hybridoma in SCID mice. Isotyping the type of antibody of our prepared monoclonal antibodies will be conducted also using isotyping kit. A number of ICTs using each one of these monoclonal antibodies as a test line, and soluble crude antigen as a control line will be prepared. The designation and preparation of these ICTs will be performed as we applied previously (Fereig et al., 2018. Acta Tropica). The developed ICTs will be tested in detection of fecal antigen of C. parvum from naturally infected calves. We already have a number of control positive and negative control fecal samples from calves (tested by commercial ICT and IFAT). The performance of our developed ICTs will be compared against relevant ELISA and commercial ICT. Regardless of commercial ICTs which are expensive and unavailable in many countries, our developed ICT if succeed will be a substantial step in Cryptosporidium research and control.
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| Remarks |
2019年9月10日に第10回獣医寄生虫学奨励賞を受賞
氏名：Ragab FEREIG、受賞テーマ：Development of potent and safe vaccine candidates against Neospora caninum infection
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Research Products
(2 results)
