| Co-Investigator(Kenkyū-buntansha) |
KURAZONO Hisao Okayama University, Professor, 医学部, 教授 (90186487)
WATARAI Masahisa Obihiro University, Research Assistant, 畜産学部, 助手 (40312441)
SHIRAHATA Toshikazu Obihiro University, Professor, 畜産学部, 教授 (90003110)
ERDENEBAATAR Janchivdorjiin Mongolu University, Lecturer
YONDONDORJ Ajchbazarii Mongolu University, Professor
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| Budget Amount *help |
¥15,400,000 (Direct Cost: ¥15,400,000)
Fiscal Year 2002: ¥5,200,000 (Direct Cost: ¥5,200,000)
Fiscal Year 2001: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 2000: ¥5,300,000 (Direct Cost: ¥5,300,000)
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| Research Abstract |
Pathogenic bacteria are divided into dangerous level-1 to -3, especially infectious diseases by level-3 their have been decreasing drastically in Japan. Therefore, when they occur in our country, a big panic will occur because of no good detection/diagnosis methods. In addition, when they occur as the global scale, it should be a bigger problem. Therefore, we studied on the following matters.
1. Rapid detection system for anthrax: An Aim is to detect Bacillus anthracis DNA from soil using rapid and simple procedures. Various amounts of B. anthracis Pasteur II spores were added artificially to 1 g of soil, which was then washed with ethanol and sterile water. Enrichment of the samples in trypticase soy broth was performed twice. A DNA template was prepared from the second enrichment culture using a FastPrep instrument. The template was then used for nested and real-time PCR with B. anthracis-specific primers, to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasm
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ids. Finally, one cell of B. anthracis in 1 g of soil could be detected by nested and real-time PCR. We also confirmed the usefulness of the PCR method using field samples. Our results indicate that this could be a useful method for detecting anthrax-spore contaminated soil with high sensitivity. Its application could have great impact on the progress of epidemiological surveillance.
2. Vaccine development for anthrax: The development of the new vaccine using the safe and effective protective antigen was planned, using lactic acid bacteria. We made a recombinant strain, but I have not tried yet.
3. Diagnosis of brucellosis: Brucellosis, which is caused by Brucella spp., is a worldwide infectious disease in domestic and wild animals, and also in humans by contact with infected animals or contaminated dairy products. In order to control and eradicate brucellosis, the vaccination have generally been doing for animals. Also the detection and culling of infected animals have been done by the conventional serological tests such as Rose Bengal (RB), tube agglutination (TA) and compliment fixation (CF) tests using inactivated whole bacterial cell or bacterial lipopolysaccharide (LPS) antigens. However, a strong cross-reaction between Brucella spp. And Y. enterocolitica O9 in those tests has been allowed to seriously complicate the diagnosis of animal brucellosis because Brucella has the common antigenic determinants with Y. enterocolitica O9 in the smooth LPS region. Also, the discrimination between infected and vaccinated animals is hardly possible because both animals present a high tittered antibody raised against Brucella. In Mongolia, since the economic loss caused by Brucella infection has been one of the most serious and important problems in the nomadic animal breeding, the government imposed vaccination against all domestic cattle, sheep and goats. In addition, large numbers of domestic animals had been diagnosed as brucellosis by the above mentioned serological tests, meaning that a certain number of animals diagnosed as brucellosis would not be real brucellosis when the cross-reaction was considered. Therefore, a simple detection method to detect real Brucella infected animals must be established. Therefore, we established enzyme-linked immunosorbent assay (ELISA) using soluble antigen extracted from B. abortus 544 by n-lauroylsarcosine, which had an ability to differentiate naturally infected animals from vaccinated and Y. enterocolitica O9 infected ones and that would have the high diagnostic specificity. From serological survey for brucellosis in the nomadic animal husbandry in Mongolia, we validified the method in the field. Less
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