Recombinant Attenuated Edwardsiella piscicida Vaccine Displaying Regulated Lysis to Confer Biological Containment and Protect Catfish against Edwardsiellosis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains, Plasmids and Growth Conditions
2.2. Fish Husbandry
2.3. Sequence Analysis
2.4. Construction of E. piscicida Deletion–Insertion Mutation of ΔPmurA
2.5. Growth Curve Analysis
2.6. Attachment, Internalization and Intracellular Replication Assays
2.7. Activation of TLRs and NLRs by E. piscicida ΔPmurA Strain
2.8. Colonization and Lysis of χ16016 in Catfish Tissues
2.9. Determination of Lethal Dose 50 (LD50) χ16016 via Bath Immersion and IC Injection
2.10. Evaluation of Anti-E. piscicida IgM in Catfish Serum and Skin Mucus via Enzyme-Linked Immunosorbent Assay (ELISA)
2.11. Immunization and Challenge
2.12. Isolation of RNA and Synthesis of First-Strand cDNA & Quantitative Real-Time PCR Analysis
2.13. Statistical Analysis
3. Results
3.1. Analysis of Sequences, Construction of Phylogenetic Tree and Generation of Homology 3D Structure of MurA
3.2. Construction and Evaluation of E. piscicida murA Mutant
3.3. Principle of Arabinose-Regulated MurA Synthesis
3.4. Attachment, Internalization and Persistence of E. piscicida Wild-Type and ΔPmurA (χ16016) Strains in Fish Cell Line
3.5. Activation of NF-κB Pathway through TLRs and NLRs by E. piscicida Strains
3.6. Determination of Lethal Dose 50 (LD50) Dose of Wild-Type and Vaccine Candidate via IC Injection and Bath Immersion
3.7. Colonization and Lysis of χ16016 in Catfish Tissues
3.8. Expressional Modulation of il-8, il-1β, tnf-a, il-6 and ifn-γ Genes in χ16016-Vaccinated and Control Catfish
3.9. Expressional Modulation of the T Cell-Related Genes in χ16016 Vaccinated and Control Catfish
3.10. Analysis of E. piscicida Anti-LPS Antibody Responses in Control and Immunized Catfish
3.11. Survival Rate of Vaccinated Fish after Wild-Type E. piscicida Challenge
3.12. The Clinical Observation of Catfish after Challenge with E. piscicida
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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Strain or Plasmid | Genotype and Relevant Characteristics | Source or Reference(s) |
---|---|---|
Strains | ||
Escherichia coli | ||
χ7213 | thi-1 thr-1 leuB6 glnV44 fhuA21 lacY1 recA1 RP4-2-Tc::Mu λpir ∆asdA4 ∆zhf-2::Tn10 | [18] |
E. piscicida | ||
J118 | Wild-type E. piscicida EIB202, highly virulent, fish isolated, Colr | [19] |
χ16016 | J118 derivative; ΔPmurA180::TT araC ParaBAD murA | This study |
Plasmids | ||
pRE112 | Suicide vector; sacB mobRP4 R6K ori; Cmr | [20] |
pYA3700 | Cloning vector containing TT araC ParaBAD cassette | [21] |
pG8R8025 | ΔPmurA180::TT araC ParaBAD murA, pRE112 | This study |
Primer | Sequence (5′-3′) |
---|---|
MurA1-SphI | F-CATGCATGCCGGGCATCACGTGTGTGGATATC |
MurA2-BglII | R-CATAGATCTTCACAGACCGCTCAGCTTGCG |
MurA3-KpnI | F-CATGGTACC AGGAGGAATTAAATGGATAAATTTCGT |
MurA4-EcoRI | R-CCGGAATTCCGCTGACCTTGTCCATCACGATA |
MurA5-XmaI | R-CATCCCGGGCGCTGACCTTGTCCATCACGATA |
pYA3700 | F-ATAACCTTTCATTCCCAGCGG |
(A) | |||||
J118 (Wild-Type E. piscicida) | |||||
Dose CFU/Fish | Mortality | Deaths | Survivals | Mortality % | LD50 |
9 × 102 | 1/10 | 1 | 9 | 5 | 1.7 × 104 |
9 × 103 | 4/10 | 4 | 6 | 38 | |
9 × 104 | 8/10 | 8 | 2 | 87 | |
9 × 105 | 10/10 | 10 | 0 | 100 | |
9 × 106 | 10/10 | 10 | 0 | 100 | |
χ16016 (ΔPmurA180::TT araC ParaBAD murA) | |||||
4 × 104 | 0/10 | 0 | 10 | 0 | 5 × 106 |
4 × 105 | 0/10 | 0 | 10 | 0 | |
4 × 106 | 4/10 | 4 | 6 | 40 | |
4 × 107 | 10/10 | 10 | 0 | 100 | |
4 × 108 | 10/10 | 10 | 0 | 100 | |
(B) | |||||
J118 (Wild-Type E. piscicida) CFU/mL | |||||
9 × 105 | 0/10 | 0 | 10 | 0 | 4.8 × 107 |
9 × 106 | 2/10 | 2 | 8 | 20 | |
9 × 107 | 10/10 | 10 | 0 | 100 | |
χ16016 (ΔPmurA180::TT araC ParaBAD murA) | |||||
4 × 106 | 0/10 | 0 | 10 | 0 | - |
4 × 107 | 0/10 | 0 | 10 | 0 | |
4 × 108 | 0/10 | 0 | 10 | 0 |
Gene | Sequence 5′-3′ | PCR Product Size in bp | GenBank # |
---|---|---|---|
18S rRNA | F-GAGAAACGGCTACCACATCC | 128 | AF021880.1 |
R-GATACGCTCATTCCGATTACAG | |||
CD4-1 | F-GGGGATTTTTGCACAGTTCGG | 243 | DQ435305.1 |
R-TCCACAAGGTGATGCTGTTCA | |||
CD4-2 | F-TGAACATCGGTGTTGTTGGTC | 238 | DQ435304.1 |
R-TCCTGTTCAGTGAAGGTGACTT | |||
CD8-α | F-TCGTACAGCAAGACTAAGAAATCA | 198 | HQ446239.1 |
R-TTTGATTGTTGTTGGGACTGTCT | |||
CD8-β | F-CTTAAGAACAGAGACACACCGGA | 299 | HQ446240.1 |
R-ACACGCTTTGGATTTTTCACTG | |||
IL-1β | F-TGCTGGAAAGGTACTTTGACAG | 296 | DQ157743.1 |
R-GCACTTCAAATTGATCACCATGC | |||
IL-8 | F-GCTGCCCTTGATCATCTTTGC | 219 | AY145142.1 |
R-GGATCCAGGCAGACCTTCATTC | |||
IFN-γ | F-TACTGGACATGAGATTCACAACCT | 257 | NM_001200217.1 |
R-CTTTCACACTTTCCTGGAGTTC | |||
MHC-II | F-CACTGAAGCACAGATCAAACACC | 236 | AF103002.2 |
R-CTGTTCTGTACACTCCTAGGTTGG | |||
TNF-α | F-TCTTCAGGAGTTTGGAGAACCTG | 255 | AJ417565.2 |
R-CAGTTTCAAGCCTGAAGAGAAGG | |||
IL-6 | F-GAAACATCTGGAGTCGAGCTGC | 214 | XM_017455306.1 |
R-GACATTTTTGCGGGCTGAAGTC |
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Swain, B.; Campodonico, V.A.; Curtiss, R., III. Recombinant Attenuated Edwardsiella piscicida Vaccine Displaying Regulated Lysis to Confer Biological Containment and Protect Catfish against Edwardsiellosis. Vaccines 2023, 11, 1470. https://doi.org/10.3390/vaccines11091470
Swain B, Campodonico VA, Curtiss R III. Recombinant Attenuated Edwardsiella piscicida Vaccine Displaying Regulated Lysis to Confer Biological Containment and Protect Catfish against Edwardsiellosis. Vaccines. 2023; 11(9):1470. https://doi.org/10.3390/vaccines11091470
Chicago/Turabian StyleSwain, Banikalyan, Vanessa A. Campodonico, and Roy Curtiss, III. 2023. "Recombinant Attenuated Edwardsiella piscicida Vaccine Displaying Regulated Lysis to Confer Biological Containment and Protect Catfish against Edwardsiellosis" Vaccines 11, no. 9: 1470. https://doi.org/10.3390/vaccines11091470