Studies on Comparative Genome Analysis of Theileria Parasites and Vaccine Developement against T. orientalis : Theileria原蟲の比較ゲノム解析とT. orientalisに對するワクチン開發する關する硏究

다국어 초록 (Multilingual Abstract)

1-5. Summary
In the present study, a novel antigenic protein expressed in the piroplasm stage of Theileria orientalis was characterized. A 4,707 by genomic fragment amplified by PCR contained two ORFs. The deduced amino acid sequence of the first ORF showed significantly high similarlity to the ubiquitin carboxy terminal hydrolases/proteases while the second ORF (ToORF2) showed homology to several surface antigens of plasmodia. TbORF2 was expressed to determine whether the protein product is expressed by the parasite. In western blot analysis, bovine antiserum from a T. orientalis-infected calf recognized the recombinant protein containing a C-terminal part of the ORF expressed by baculovirus system. Western blot analysis with the anti-ToORF2 mouse serum recognized a 48 kDa protein in T. orientalis piroplasm lysates. Immunofluorescence antibody test by confocal scanning laser microscopic analysis showed that antisera against the recombinant protein recognized T. orientalis piroplasm in the infected erythrocyte. The results from this study indicate that ToORF2 protein is expressed at the piroplasm stage and is immunogenic. This novel antigenic ToORF2 protein could be exploited for vaccine development against bovine piroplasmosis.
2-5. Summary
The product of an open reading frame found in a fragment of Theileria orientalis genomic DNA was characterized. Homology analysis of the predicted product showed that it encodes a 35 kDa polypeptide (P35) that bears stretches ofa-helical regions and is similar to MESA of Plasmodium falciparum. Since the recombinant product expressed by a baculovirus expression system was recognized by antiserum obtained from a T. orientalis-infected calf, it is immunogenic during T. orientalis infection. Western blot analysis revealed that a murine serum raised against the recombinant product detected a 35 kDa protein in T. orientalis piroplasm lysates. Confocal laser scanning microscopy analysis showed that the antiserum mainly recognized piroplasms in erythrocytes that were losing their intact structures. Triton X-100-treated Sf9 cells expressing the recombinant product were detected by the murine antiserum in immunofluorescence antibody test, which indicates that this molecule is associated with the cytoskeleton of insect cells. P35 was also found to bind specifically to the Triton X-100-insoluble membrane fraction of uninfected bovine erythrocytes. These findings suggest that the novel P35 protein of T. orientalis is predominantly expressed in the late stage of intra-erythrocytic development and that it can interact with the erythrocyte membrane skeleton.
3-5. Summary
Theileria orientalis infects cattle and causes various disease symptoms, including anemia and icterus. The erythrocytic stages are responsible for these symptoms but the molecular events involved in these stages have not yet been fully elucidated. In this study, a T. orientalis cDNA that encodes a polypeptide related to identity to the microneme-rhoptry protein of T. parva wasidentified. Analysis of microneme-rhoptry protein of T. orientalis (ToMRP) by indirect fluorescent-antibody test revealed that it is specifically expressed at the early erythrocytic stage after invasion. This expression disappears during the intermediate stages of intra-erythrocytic development. Its expression then reappears at the late stages after the parasite has divided by binary fission into diad or tetrad forms and before these forms are released from the host erythrocyte. In vitro erythrocyte binding assays showed that ToMRP associates with the Triton-X insoluble fraction of erythrocytes membrane but not with intact erythrocytes. Cosedimentation and western blot analyses revealed that ToMRP binds to band 3, a membrane component of bovine erythrocytes. These observations suggest that ToMRP may be involved in the parasite's egress from and/or invasion into the host erythrocytes by interacting with a protein in the membrane skeleton of the erythrocyte and thereby modifying the structure and function of the cell.
4-4. Summary
Benign Theileria species of cattle are found in most parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a maker for epidemiological and phylogenetical studies of benign Theileria species. Parasites with Ikeda- or Chitose-type MPSP genes are dominant in Japan, but it was reported here mixed infection cases of Theileria_parasites with an additional MPSP type parasite infecting cattle in Abashiri District, Hokkaido. The MPSP gene sequence found in the additional type was closely related to MPSP genes of Theileria_parasites found in Southeast Asian countries, including Thailand (Narathiwat) and Indonesia (Java). Theileria parasites from the blood sample were also distinguishable from the Ikeda or Chitose type parasites by small subunit ribosomal RNA (SSU rRNA) gene sequence analysis, and they are grouped into the SSU rRNA types C/D found in Korea, North America, and Spain. The present finding of mixed infections of cattle with three different types of Theileria makes epidemiological feature of bovine theileriosis in Japan more complex. I have designed a set of primers specific to this MPSP type in order to conduct further epidemiological study.

목차 (Table of Contents)

• Contents
• Contents = I
• Abbreviations = IV
• General Introduction = 1
• 1. The importance of Theileria parasites = 1
• 2. Life cycle and the pathology of Theileria parsites = 2
• 3. Piroplasm stage of T. orientalis = 3
• 4. Vaccine development against Theileria parasites = 4
• 5. Epidemiologic study on T. orientalis = 6
• 6. Aims of the present study = 7
• Chapter 1 Cloning and expression of novel antigenic protein gene (ToORF2) of Theileria orientalis and its immunological characterization = 9
• 1-1. Introduction = 9
• 1-2. Materials and Methods = 10
• 1-3. Results = 15
• 1-4. Discussion = 16
• 1-5. Summary = 19
• Chapter 2 Molecular characterization of a 35 kDaα-helical protein expressed in the piroplasm stage of Theileria orientalis and its interaction with the erythrocyte membrane = 24
• 2-1. Introduction = 24
• 2-2. Materials and Methods = 25
• 2-3. Results = 29
• 2-4. Discussion = 31
• 2-5. Summary = 35
• Chapter 3 Identification of a piroplasm protein of Theileria orientalis that binds to bovine erythrocyte band 3 = 41
• 3-1. Introduction = 41
• 3-2. Materials and Methods = 43
• 3-3. Results = 47
• 3-4. Discussion = 49
• 3-5. Summary = 55
• Chapter 4 Molecular epidemiological survey of benign Theileria parasites of cattle in Japan: Detection of a new type of major piroplasm surface protein gene. = 61
• 4-1. Introduction = 61
• 4-2. Materials and Methods = 62
• 4-3. Results and Discussion = 63
• 4-4. Summary = 68
• General discussion = 75
• Conclusion = 81
• Acknowledgments = 83
• REFERENCES = 86