| 摘要 | 第14-16页 |
| Abstract | 第16-18页 |
| Part Ⅰ:Review of Literature | 第19-59页 |
| Chapter 1 Chicken Coccidiosis and Immune Response to Eimeria | 第19-45页 |
| 1.1. Introduction | 第19-20页 |
| 1.2. Economic importance | 第20页 |
| 1.3. The life cycle of an Eimeria parasite | 第20-21页 |
| 1.4. Diagnosis | 第21-22页 |
| 1.4.1. Clinical findings | 第21-22页 |
| 1.4.2. Gross lesions | 第22页 |
| 1.4.3. Sites parasitized by different Eimeria species in the chicken | 第22页 |
| 1.5. Factors that influence the course and severity of coccidial infection | 第22-24页 |
| 1.5.1. Species | 第22-23页 |
| 1.5.2. Extent of contamination | 第23页 |
| 1.5.3. Stocking density of hosts | 第23页 |
| 1.5.4. Extent of dampness of immediate environment | 第23页 |
| 1.5.5. Immune status of the host | 第23页 |
| 1.5.6. Secondary infections with other pathogens | 第23页 |
| 1.5.7. Presence of effective anticoccidial drugs | 第23-24页 |
| 1.6. Chicken coccidial organelles antigen-related functions | 第24-27页 |
| 1.7. Immune response to coccidiosis | 第27-34页 |
| 1.7.1. Immunogenicity of Eimeria | 第27-28页 |
| 1.7.2. Immune response mechanisms in avian coccidiosis | 第28-34页 |
| 1.8. Objectives of the study | 第34-35页 |
| References | 第35-45页 |
| Chapter 2 Control of Chicken Coccidiosis | 第45-59页 |
| 2.1. Management control | 第45-46页 |
| 2.2. Chemoprophylaxis | 第46-48页 |
| 2.3. Feed quality control | 第48页 |
| 2.4. Vaccination | 第48-54页 |
| 2.4.1. Biology of vaccination with special reference to Eimeria | 第49-50页 |
| 2.4.2. Immune responses to vaccination Eimeria infection | 第50页 |
| 2.4.3. Vaccines available against coccidiosis | 第50-54页 |
| References | 第54-59页 |
| Part Ⅱ: Research | 第59-151页 |
| Chapter 3 Isolation and Identification of Eimeria brunetti strain | 第59-69页 |
| 3.1. Introduction | 第59-60页 |
| 3.2. Materials and methods | 第60-63页 |
| 3.2.1. Parasites and chickens | 第60-61页 |
| 3.2.2. Equipments, solutions, chemicals, reagents and buffers | 第61页 |
| 3.2.3. Isolation of oocyst | 第61页 |
| 3.2.4. Identification of Eimeria species by PCR amplification | 第61-63页 |
| 3.3. Results | 第63-65页 |
| 3.4. Discussion | 第65-66页 |
| References | 第66-69页 |
| Chapter 4 Cloning, Expression and Characterization of Eimeria brunetti Microneme-2Protein (EbMIC2) | 第69-91页 |
| 4.1. Introduction | 第69-71页 |
| 4.2. Materials and methods | 第71-76页 |
| 4.2.1. Vectors and bacteria strains | 第71页 |
| 4.2.2. Equipments, solutions, chemicals, reagents and buffers | 第71-72页 |
| 4.2.3. Designing primers | 第72页 |
| 4.2.4. Animals and parasites | 第72页 |
| 4.2.5. Chicken immune sera against E. brunetti | 第72页 |
| 4.2.6. Total RNA extraction | 第72-73页 |
| 4.2.7. Reverse transcription and amplification of cDNA (RT-PCR) | 第73-74页 |
| 4.2.8. Cloning of EbMIC2 gene | 第74页 |
| 4.2.9. Sequence alignments and phylogenetic analysis | 第74-75页 |
| 4.2.10. Construction of recombinant expression vector of EbMIC2 | 第75页 |
| 4.2.11. Expression and purification of recombinant EbMIC2 | 第75-76页 |
| 4.2.12. Immuno-blot for the recombinant EbMIC2 and native protein of EbMIC2 | 第76页 |
| 4.3. Results | 第76-84页 |
| 4.3.1. Cloning of E. brunetti MIC2 gene | 第76-79页 |
| 4.3.2. Percent identity, phylogenetic tree and antigenic analysis | 第79-82页 |
| 4.3.3. Expression and purification of recombinant EbMIC2 protein | 第82-83页 |
| 4.3.4. Immunoblot for the recombinant EbMIC2 and native protein of EbMIC2 | 第83-84页 |
| 4.4. Discussion | 第84-86页 |
| References | 第86-91页 |
| Chapter 5 Immunogenicity of Eimeria brunetti Microneme-2 Protein and ItsProtective Effects on Chicken | 第91-117页 |
| 5.1. Introduction | 第92-93页 |
| 5.2. Materials and methods | 第93-98页 |
| 5.2.1. Equipments, solutions, chemicals, reagents and buffers | 第93页 |
| 5.2.2. Animals and parasites | 第93页 |
| 5.2.3. Cloning of E. brunetti MIC2 gene | 第93页 |
| 5.2.4. Construction of the recombinant eukaryotic expression plasmid | 第93-94页 |
| 5.2.5. Detection of the expressions of EbMIC2 gene in vivo by reverse transcriptionpolymerase chain reaction (RT-PCR) assay and western blot analysis | 第94-95页 |
| 5.2.6. Determination of serum antibody level by enzyme-linked Immuno Sorbent assay(ELISA) | 第95页 |
| 5.2.7. Determination of serum cytokine concentration | 第95-96页 |
| 5.2.8. Production of anti-serum for passive immunization | 第96页 |
| 5.2.9. Immunization and challenge experiment | 第96-97页 |
| 5.2.10. Immunization and challenge experiment in passive immunization | 第97页 |
| 5.2.11. Evaluation of immune protection | 第97-98页 |
| 5.2.12. Statistical analysis | 第98页 |
| 5.3. Results | 第98-106页 |
| 5.3.1. Identification of recombinant plasmid pVAX1-EbMIC2 | 第98-99页 |
| 5.3.2. Detection of the transcription of constructed recombinant plasmid pVAX1-EbMIC2 in vivo | 第99页 |
| 5.3.3. Detection of the expression of EbMIC2 gene in vivo by Western blot | 第99-100页 |
| 5.3.4. IgG and cytokine levels in sera of immunized chickens | 第100-102页 |
| 5.3.5. Protective effects of EbMIC2 against E. brunetti challenge in activeimmunization | 第102-104页 |
| 5.3.6. Protective effects of constructed recombinant plasmid pVAXl-EbMIC2 againstE. brunetti challenge in passive immunization | 第104-106页 |
| 5.4. Discussion | 第106-110页 |
| References | 第110-117页 |
| Chapter 6 Cloning, Expression and Characterization of Eimeria brunetti ApicalMembrane Antigen-1 (EbAMAl) | 第117-133页 |
| 6.1. Introduction | 第117-118页 |
| 6.2. Materials and Methods | 第118页 |
| 6.3. Results | 第118-127页 |
| 6.3.1. Cloning of E. brunetti AMA1 gene | 第118-119页 |
| 6.3.2. Sequence analysis | 第119-122页 |
| 6.3.3. Percent identity, phylogenetic tree and antigenic analysis | 第122-124页 |
| 6.3.4. Construction and identification of the recombinant pET-28a (+)-EbAMA1 | 第124-125页 |
| 6.3.5. Expression and purification of recombinant EbAMAl protein | 第125-126页 |
| 6.3.6. Immunoblot for the recombinant EbAMA1 and native protein of EbAMA1 | 第126-127页 |
| 6.4. Discussion | 第127-129页 |
| References | 第129-133页 |
| Chapter 7 Immunogenicity of Eimeria brunetti Apical Membrane Antigen-1 and ItsProtective Effects on Chicken | 第133-151页 |
| 7.1. Introduction | 第134-135页 |
| 7.2. Materials and Methods | 第135页 |
| 7.3. Results | 第135-143页 |
| 7.3.1. Identification of recombinant plasmid EbAMA1 | 第135-136页 |
| 7.3.2. Detection of the transcription of constructed recombinant plasmid EbAMA1 invivo | 第136页 |
| 7.3.3. Detection of the expression of EbAMA1 gene in vivo by Western blot | 第136-137页 |
| 7.3.4. IgG and cytokine levels in sera of immunized chickens | 第137-139页 |
| 7.3.5. Protective effects of EbAMAl against E. brunetti challenge in activeimmunization | 第139-141页 |
| 7.3.6. Protective effects of recombinant plasmid pVAX1-EbAMA1 against E. brunettichallenge in passive immunization | 第141-143页 |
| 7.4. Discussion | 第143-146页 |
| References | 第146-151页 |
| Conclusion | 第151-153页 |
| Acknowledgements | 第153页 |
