Title Page
Contents
ABSTRACT 9
General Introduction 12
1. Salmonellosis 12
2. Salmonella in Chickens 13
3. Salmonella Vaccines in Chickens 14
Chapter 1. Construction and Characterization of Temperature-Sensitive Mutant Strains of Salmonella Enteritidis as a Live Vaccine Candidate 16
1.1. Introduction 16
1.2. Materials and Methods 18
1.2.1. Construction of ts Mutant Strains 18
1.2.2. Attenuation Evaluation of the Mutant Strains 18
1.2.3. One-Step Growth Curve Analysis 21
1.2.4. Invasion Assay 21
1.2.5. Complete Genome Sequencing 22
1.2.6. Comparative Genome Analysis 22
1.2.7. Statistical Analysis 22
1.3. Results 24
1.3.1. Construction of the ts Mutant Strains 24
1.3.2. Attenuation of the ts Mutant Strains 24
1.3.3. One-Step Growth Analysis 26
1.3.4. Invasion Assay 29
1.3.5. Complete Genome Sequencing and Comparative Genome Analysis 31
1.4. Discussion 64
Chapter 2. Evaluation of Immune Responses and Protective Efficacy of a Live Attenuated Salmonella Enteritidis Vaccine Candidate in Chickens 68
2.1. Introduction 68
2.2. Material and Methods 70
2.2.1. Animals 70
2.2.2. Bacterial Strains and Culture 70
2.2.3. Vaccination and Challenge Inoculation of S. Enteritidis Strain 70
2.2.4. Serological Assay 73
2.2.5. Preparation of Splenocytes 73
2.2.6. ELISpot Assay 73
2.2.7. S. Enteritidis Bacterial Strain Recovery 74
2.2.8. S. Gallinarum Bacterial Strain Recovery 74
2.2.9. Statistical Analysis 77
2.3. Results 78
2.3.1. Humoral Immune Responses Induced by Vaccination 78
2.3.2. Cellular Immune Responses Induced by Vaccination 80
2.3.3. Re-Isolation of S. Enteritidis Strains 82
2.3.4. Re-Isolation of S. Gallinarum Strains 84
2.4. Discussion 86
General Conclusion 90
References 92
Abstract (in Korean) 106
Table 1-1. Oligonucleotide sequences for Salmonella spp. and S. Enteritidis specific PCR analysis 20
Table 1-2. Re-isolation of S. Enteritidis from the internal organs of chicks 1 week after inoculation 25
Table 1-3. SNPs detected in the S. Enteritidis ts mutant 2S-G10 compared with the parental strain 6NB 33
Table 1-4. The 63 SNPs identified in S. Enteritidis ts mutant 2S-G10 that most likely contributed to the attenuation 55
Table 2-1. Re-isolation of S. Gallinarum from the internal organs of chickens 1 week after challenge 85
Figure 1-1. One-step growth curves based on OD measurement of the mutant and parental strain cultures. Growths of the ts mutant and parental strains were... 27
Figure 1-2. One-step growth curves based on CFU counts of the mutant and parental strains. Growth of the ts mutant 2S-G10 and parental 6NB strains was... 28
Figure 1-3. Invasion rates of the ts mutant strain 2S-G10 and parental strain 6NB into LMH cells. Values indicate the percentages of the recovered bacteria... 30
Figure 1-4. Circular genome of parental strain 6NB. The outer scale represents the size in terms of base pairs. The virulence-related genes with... 63
Figure 2-1. Experimental design diagram for evaluation of immune responses and protective efficacy against S. Enteritidis of a vaccine candidate in chickens. 72
Figure 2-2. Experimental design diagram for evaluation of protective efficacy against S. Gallinarum of a vaccine candidate in commercial brown chickens. 76
Figure 2-3. S. Enteritidis-specific antibody titers induced by vaccination. Blood samples were collected at (a) three weeks after prime vaccination, and (b) two weeks after booster vaccination. Serum was isolated from the blood and S. Enteritidis-... 79
Figure 2-4. ELISpot analysis of stimulated T cells. Splenocytes collected after booster vaccination were added into ChIFN-γ-coated wells and stimulated with... 81
Figure 2-5. Salmonella titers (log₁₀ CFU/g) in cecal contents before and after challenge and in the spleen after the challenge. At two weeks after vaccination... 83