WO2017209917A1 - Live attenuated catfish vaccine and method of making - Google Patents
Live attenuated catfish vaccine and method of making Download PDFInfo
- Publication number
- WO2017209917A1 WO2017209917A1 PCT/US2017/032345 US2017032345W WO2017209917A1 WO 2017209917 A1 WO2017209917 A1 WO 2017209917A1 US 2017032345 W US2017032345 W US 2017032345W WO 2017209917 A1 WO2017209917 A1 WO 2017209917A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- esc
- ictaluri
- sdhc
- fish
- gcvp
- Prior art date
Links
- 230000002238 attenuated effect Effects 0.000 title claims abstract description 18
- 241001233037 catfish Species 0.000 title abstract description 60
- 229960005486 vaccine Drugs 0.000 title abstract description 40
- 238000004519 manufacturing process Methods 0.000 title description 11
- 241000949274 Edwardsiella ictaluri Species 0.000 claims abstract description 95
- 238000000034 method Methods 0.000 claims abstract description 49
- 230000001580 bacterial effect Effects 0.000 claims abstract description 37
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 17
- 239000000203 mixture Substances 0.000 claims abstract description 16
- 241000588921 Enterobacteriaceae Species 0.000 claims abstract description 12
- 206010040047 Sepsis Diseases 0.000 claims abstract description 9
- 208000013223 septicemia Diseases 0.000 claims abstract description 9
- 230000001900 immune effect Effects 0.000 claims abstract 5
- 108090000623 proteins and genes Proteins 0.000 claims description 114
- 108010012901 Succinate Dehydrogenase Proteins 0.000 claims description 56
- 102000019259 Succinate Dehydrogenase Human genes 0.000 claims description 56
- 230000035772 mutation Effects 0.000 claims description 50
- 102000004169 proteins and genes Human genes 0.000 claims description 32
- 108010014977 glycine cleavage system Proteins 0.000 claims description 26
- 101100175237 Caldanaerobacter subterraneus subsp. tengcongensis (strain DSM 15242 / JCM 11007 / NBRC 100824 / MB4) gcvPB gene Proteins 0.000 claims description 25
- 101150053469 SDHC gene Proteins 0.000 claims description 25
- 101150011909 gcvP gene Proteins 0.000 claims description 25
- 101100023132 Wolinella succinogenes (strain ATCC 29543 / DSM 1740 / LMG 7466 / NCTC 11488 / FDC 602W) sdhE gene Proteins 0.000 claims description 23
- 101150114996 sdhd gene Proteins 0.000 claims description 23
- 108010026217 Malate Dehydrogenase Proteins 0.000 claims description 12
- 241001465754 Metazoa Species 0.000 claims description 10
- 102000013460 Malate Dehydrogenase Human genes 0.000 claims 10
- 208000028774 intestinal disease Diseases 0.000 claims 3
- 241000251468 Actinopterygii Species 0.000 abstract description 87
- 229940124590 live attenuated vaccine Drugs 0.000 abstract description 30
- 229940023012 live-attenuated vaccine Drugs 0.000 abstract description 30
- 241000894006 Bacteria Species 0.000 abstract description 15
- 238000012224 gene deletion Methods 0.000 abstract 1
- 238000007654 immersion Methods 0.000 description 39
- 238000002255 vaccination Methods 0.000 description 35
- 230000000694 effects Effects 0.000 description 26
- 238000011282 treatment Methods 0.000 description 25
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 25
- 210000002966 serum Anatomy 0.000 description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 24
- 210000000440 neutrophil Anatomy 0.000 description 23
- 230000004083 survival effect Effects 0.000 description 23
- 230000001018 virulence Effects 0.000 description 22
- 102000004190 Enzymes Human genes 0.000 description 21
- 108090000790 Enzymes Proteins 0.000 description 21
- 238000003780 insertion Methods 0.000 description 21
- 230000037431 insertion Effects 0.000 description 21
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 20
- 239000013612 plasmid Substances 0.000 description 20
- 241000588724 Escherichia coli Species 0.000 description 17
- 238000011161 development Methods 0.000 description 16
- 230000018109 developmental process Effects 0.000 description 16
- 238000012216 screening Methods 0.000 description 15
- 241000252498 Ictalurus punctatus Species 0.000 description 14
- 241000607142 Salmonella Species 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 12
- 208000015181 infectious disease Diseases 0.000 description 11
- 239000004471 Glycine Substances 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- 230000029918 bioluminescence Effects 0.000 description 9
- 238000005415 bioluminescence Methods 0.000 description 9
- 238000012217 deletion Methods 0.000 description 9
- 230000037430 deletion Effects 0.000 description 9
- 238000001727 in vivo Methods 0.000 description 8
- 229920001817 Agar Polymers 0.000 description 7
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 7
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 7
- 210000004379 membrane Anatomy 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 230000010076 replication Effects 0.000 description 7
- 108010078777 Colistin Proteins 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 229960003346 colistin Drugs 0.000 description 6
- 230000001976 improved effect Effects 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- JORAUNFTUVJTNG-BSTBCYLQSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(3s,6s,9s,12s,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3-[(1r)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-h Chemical compound CC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O.CCC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O JORAUNFTUVJTNG-BSTBCYLQSA-N 0.000 description 6
- 230000008506 pathogenesis Effects 0.000 description 6
- 230000037361 pathway Effects 0.000 description 6
- XDJYMJULXQKGMM-UHFFFAOYSA-N polymyxin E1 Natural products CCC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O XDJYMJULXQKGMM-UHFFFAOYSA-N 0.000 description 6
- KNIWPHSUTGNZST-UHFFFAOYSA-N polymyxin E2 Natural products CC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O KNIWPHSUTGNZST-UHFFFAOYSA-N 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 101150055592 rseB gene Proteins 0.000 description 6
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 6
- 150000003628 tricarboxylic acids Chemical class 0.000 description 6
- 230000037357 C1-metabolism Effects 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 241001129672 Edwardsiella ictaluri 93-146 Species 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 244000052616 bacterial pathogen Species 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 238000003306 harvesting Methods 0.000 description 5
- 238000003384 imaging method Methods 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 4
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 4
- 229930182566 Gentamicin Natural products 0.000 description 4
- 102000002667 Glycine hydroxymethyltransferase Human genes 0.000 description 4
- 108010043428 Glycine hydroxymethyltransferase Proteins 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 241001138501 Salmonella enterica Species 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 229960002518 gentamicin Drugs 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 4
- QYNUQALWYRSVHF-OLZOCXBDSA-N (6R)-5,10-methylenetetrahydrofolic acid Chemical compound C([C@H]1CNC=2N=C(NC(=O)C=2N1C1)N)N1C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 QYNUQALWYRSVHF-OLZOCXBDSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 239000006142 Luria-Bertani Agar Substances 0.000 description 3
- 239000006137 Luria-Bertani broth Substances 0.000 description 3
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 3
- 241000021375 Xenogenes Species 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 230000021615 conjugation Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 210000000981 epithelium Anatomy 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000001802 infusion Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000000543 intermediate Substances 0.000 description 3
- 230000002147 killing effect Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 230000002516 postimmunization Effects 0.000 description 3
- NPCOQXAVBJJZBQ-UHFFFAOYSA-N reduced coenzyme Q9 Natural products COC1=C(O)C(C)=C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)C(O)=C1OC NPCOQXAVBJJZBQ-UHFFFAOYSA-N 0.000 description 3
- 230000035806 respiratory chain Effects 0.000 description 3
- 230000011506 response to oxidative stress Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 108091093088 Amplicon Proteins 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 108090000363 Bacterial Luciferases Proteins 0.000 description 2
- ACTIUHUUMQJHFO-UHFFFAOYSA-N Coenzym Q10 Natural products COC1=C(OC)C(=O)C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UHFFFAOYSA-N 0.000 description 2
- 102100037579 D-3-phosphoglycerate dehydrogenase Human genes 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 241000607473 Edwardsiella <enterobacteria> Species 0.000 description 2
- 102000011687 Electron Transport Complex II Human genes 0.000 description 2
- 108010076322 Electron Transport Complex II Proteins 0.000 description 2
- 101001088154 Escherichia coli Regulatory protein rop Proteins 0.000 description 2
- MBMLMWLHJBBADN-UHFFFAOYSA-N Ferrous sulfide Chemical compound [Fe]=S MBMLMWLHJBBADN-UHFFFAOYSA-N 0.000 description 2
- 108090000698 Formate Dehydrogenases Proteins 0.000 description 2
- 102100033495 Glycine dehydrogenase (decarboxylating), mitochondrial Human genes 0.000 description 2
- 241000590002 Helicobacter pylori Species 0.000 description 2
- 101000998096 Homo sapiens Glycine dehydrogenase (decarboxylating), mitochondrial Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 108010090127 Periplasmic Proteins Proteins 0.000 description 2
- 206010039438 Salmonella Infections Diseases 0.000 description 2
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000191963 Staphylococcus epidermidis Species 0.000 description 2
- 241000607734 Yersinia <bacteria> Species 0.000 description 2
- 101100472686 Yersinia pestis bv. Antiqua (strain Nepal516) rnfB gene Proteins 0.000 description 2
- 239000000370 acceptor Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- XMQFTWRPUQYINF-UHFFFAOYSA-N bensulfuron-methyl Chemical compound COC(=O)C1=CC=CC=C1CS(=O)(=O)NC(=O)NC1=NC(OC)=CC(OC)=N1 XMQFTWRPUQYINF-UHFFFAOYSA-N 0.000 description 2
- 230000001851 biosynthetic effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 description 2
- 235000017471 coenzyme Q10 Nutrition 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 230000008094 contradictory effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 210000001156 gastric mucosa Anatomy 0.000 description 2
- 108091008053 gene clusters Proteins 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 229940037467 helicobacter pylori Drugs 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 101150109249 lacI gene Proteins 0.000 description 2
- 101150066555 lacZ gene Proteins 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- -1 organic acids formate Chemical class 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 101150115898 rseA gene Proteins 0.000 description 2
- 101150049214 rsxB gene Proteins 0.000 description 2
- 206010039447 salmonellosis Diseases 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000035882 stress Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 229940035936 ubiquinone Drugs 0.000 description 2
- WKZGKZQVLRQTCT-ABLWVSNPSA-N (2S)-2-[[4-[(2-amino-4-oxo-5,6,7,8-tetrahydro-3H-pteridin-6-yl)methylamino]benzoyl]amino]-5-formyloxy-5-oxopentanoic acid Chemical compound N1C=2C(=O)NC(N)=NC=2NCC1CNC1=CC=C(C(=O)N[C@@H](CCC(=O)OC=O)C(O)=O)C=C1 WKZGKZQVLRQTCT-ABLWVSNPSA-N 0.000 description 1
- HLXHCNWEVQNNKA-UHFFFAOYSA-N 5-methoxy-2,3-dihydro-1h-inden-2-amine Chemical compound COC1=CC=C2CC(N)CC2=C1 HLXHCNWEVQNNKA-UHFFFAOYSA-N 0.000 description 1
- 230000002407 ATP formation Effects 0.000 description 1
- 108010009924 Aconitate hydratase Proteins 0.000 description 1
- 241000606748 Actinobacillus pleuropneumoniae Species 0.000 description 1
- 241000606731 Actinobacillus suis Species 0.000 description 1
- 241000606749 Aggregatibacter actinomycetemcomitans Species 0.000 description 1
- 108020004306 Alpha-ketoglutarate dehydrogenase Proteins 0.000 description 1
- 102000006589 Alpha-ketoglutarate dehydrogenase Human genes 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 241000606767 Avibacterium paragallinarum Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000588851 Bordetella avium Species 0.000 description 1
- 241000588779 Bordetella bronchiseptica Species 0.000 description 1
- 241000588780 Bordetella parapertussis Species 0.000 description 1
- 241000588832 Bordetella pertussis Species 0.000 description 1
- 241000589567 Brucella abortus Species 0.000 description 1
- 241000722910 Burkholderia mallei Species 0.000 description 1
- 241001136175 Burkholderia pseudomallei Species 0.000 description 1
- 241000589875 Campylobacter jejuni Species 0.000 description 1
- 241000606678 Coxiella burnetii Species 0.000 description 1
- 102100039868 Cytoplasmic aconitate hydratase Human genes 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000016862 Dicarboxylic Acid Transporters Human genes 0.000 description 1
- 108010092943 Dicarboxylic Acid Transporters Proteins 0.000 description 1
- 101100121464 Dictyostelium discoideum gcvH1 gene Proteins 0.000 description 1
- 101100175157 Dictyostelium discoideum gcvH2 gene Proteins 0.000 description 1
- 101100175166 Dictyostelium discoideum gcvH3 gene Proteins 0.000 description 1
- 101100014303 Dictyostelium discoideum gcvH4 gene Proteins 0.000 description 1
- 101100014305 Dictyostelium discoideum gcvH5 gene Proteins 0.000 description 1
- 101100363960 Escherichia coli (strain K12) rseC gene Proteins 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 208000035874 Excoriation Diseases 0.000 description 1
- DGXLYHAWEBCTRU-UHFFFAOYSA-N Fluorocitric acid Chemical compound OC(=O)CC(O)(C(O)=O)C(F)C(O)=O DGXLYHAWEBCTRU-UHFFFAOYSA-N 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 241000589602 Francisella tularensis Species 0.000 description 1
- 108010036781 Fumarate Hydratase Proteins 0.000 description 1
- 102100036160 Fumarate hydratase, mitochondrial Human genes 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 241000606807 Glaesserella parasuis Species 0.000 description 1
- 241000606768 Haemophilus influenzae Species 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 241000606831 Histophilus somni Species 0.000 description 1
- 102000005298 Iron-Sulfur Proteins Human genes 0.000 description 1
- 108010081409 Iron-Sulfur Proteins Proteins 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- 241000589242 Legionella pneumophila Species 0.000 description 1
- 241000186779 Listeria monocytogenes Species 0.000 description 1
- 241001293418 Mannheimia haemolytica Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241000588622 Moraxella bovis Species 0.000 description 1
- 241000588655 Moraxella catarrhalis Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000588652 Neisseria gonorrhoeae Species 0.000 description 1
- 241000588650 Neisseria meningitidis Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 101710116435 Outer membrane protein Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000606856 Pasteurella multocida Species 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 206010037596 Pyelonephritis Diseases 0.000 description 1
- 101150033071 RPO7 gene Proteins 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 241000607762 Shigella flexneri Species 0.000 description 1
- 239000000589 Siderophore Substances 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 108700025695 Suppressor Genes Proteins 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 208000034784 Tularaemia Diseases 0.000 description 1
- 108010069584 Type III Secretion Systems Proteins 0.000 description 1
- 102100029640 UDP-glucose 6-dehydrogenase Human genes 0.000 description 1
- 108030001662 UDP-glucose 6-dehydrogenases Proteins 0.000 description 1
- 238000001793 Wilcoxon signed-rank test Methods 0.000 description 1
- 241000607447 Yersinia enterocolitica Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 241000607477 Yersinia pseudotuberculosis Species 0.000 description 1
- 241001148129 Yersinia ruckeri Species 0.000 description 1
- BUFLLCUFNHESEH-UHFFFAOYSA-N [5-(2-amino-6-oxo-3h-purin-9-yl)-4-hydroxy-2-[[hydroxy(phosphonooxy)phosphoryl]oxymethyl]oxolan-3-yl] phosphono hydrogen phosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(O)=O)C(OP(O)(=O)OP(O)(O)=O)C1O BUFLLCUFNHESEH-UHFFFAOYSA-N 0.000 description 1
- 241000606834 [Haemophilus] ducreyi Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000004523 agglutinating effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000003578 bacterial chromosome Anatomy 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 229960001212 bacterial vaccine Drugs 0.000 description 1
- 239000007621 bhi medium Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229940056450 brucella abortus Drugs 0.000 description 1
- 201000006824 bubonic plague Diseases 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229940074375 burkholderia mallei Drugs 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- NIXDOXVAJZFRNF-UHFFFAOYSA-N di-mu-sulfido-diiron(0) Chemical compound S1[Fe]S[Fe]1 NIXDOXVAJZFRNF-UHFFFAOYSA-N 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000027721 electron transport chain Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 229940118764 francisella tularensis Drugs 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 108010077700 fumarate reductase complex Proteins 0.000 description 1
- 208000018685 gastrointestinal system disease Diseases 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 101150110684 gcvH gene Proteins 0.000 description 1
- 101150022706 gcvT gene Proteins 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000004110 gluconeogenesis Effects 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 244000000059 gram-positive pathogen Species 0.000 description 1
- 239000003673 groundwater Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229940047650 haemophilus influenzae Drugs 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000012750 in vivo screening Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000005732 intercellular adhesion Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000037041 intracellular level Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 229940115932 legionella pneumophila Drugs 0.000 description 1
- 210000004901 leucine-rich repeat Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000001325 log-rank test Methods 0.000 description 1
- 238000007477 logistic regression Methods 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 238000013208 measuring procedure Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 235000020938 metabolic status Nutrition 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 229940051027 pasteurella multocida Drugs 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 238000011533 pre-incubation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000004053 quinones Chemical class 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000031070 response to heat Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 101150034869 rpo5 gene Proteins 0.000 description 1
- 101150040886 rpoE gene Proteins 0.000 description 1
- 101150106872 rpoH gene Proteins 0.000 description 1
- 101150025220 sacB gene Proteins 0.000 description 1
- 241001507086 salmonid fish Species 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 101150077142 sigH gene Proteins 0.000 description 1
- 230000003393 splenic effect Effects 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000013179 statistical model Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000000153 supplemental effect Effects 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000002277 temperature effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- FQZJYWMRQDKBQN-UHFFFAOYSA-N tricaine methanesulfonate Chemical compound CS([O-])(=O)=O.CCOC(=O)C1=CC=CC([NH3+])=C1 FQZJYWMRQDKBQN-UHFFFAOYSA-N 0.000 description 1
- 229940013180 tricaine methanesulfonate Drugs 0.000 description 1
- 229940040064 ubiquinol Drugs 0.000 description 1
- QNTNKSLOFHEFPK-UPTCCGCDSA-N ubiquinol-10 Chemical compound COC1=C(O)C(C)=C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)C(O)=C1OC QNTNKSLOFHEFPK-UPTCCGCDSA-N 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01037—Malate dehydrogenase (1.1.1.37)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
- C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
- C12Y103/01006—Fumarate reductase (NADH) (1.3.1.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
- C12Y103/05—Oxidoreductases acting on the CH-CH group of donors (1.3) with a quinone or related compound as acceptor (1.3.5)
- C12Y103/05001—Succinate dehydrogenase (ubiquinone) (1.3.5.1)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
Definitions
- the present invention is generally directed toward a live attenuated vaccine for fish, and more particularly, it is directed toward mutant strains of Edwardsiella ictaluri as a live attenuated vaccine against enteric septicemia of catfish and methods related to the same.
- E. ictaluri is the causative agent of enteric septicemia of catfish.
- E. ictaluri has the ability to resist killing by professional phagocytes.
- ictaluri is resistant to channel catfish neutrophils, which is an important aspect of pathogenesis because (1) neutrophils are the predominant cell type in channel catfish intestinal tract immune cells, and (2) the intestine is an important site of entry for E. ictaluri. E. ictaluri is also resistant to killing by the alternative complement pathway in channel catfish.
- a commercial live attenuated vaccine is now available and antibiotics are still used against E. ictaluri and ESC, ESC is still a major threat to the catfish industry (Klesius and Shoemaker, 1999). Live attenuated vaccines often offer the best prospect for vaccines by providing good protection against diseases through stimulating cellular immune responses (Id.), but the currently available commercial vaccine leaves much to be desired in performance and safety.
- TCA cycle supplies intermediates and ATP for bacterial biosynthesis, thus transcription of genes encoding the enzymes of the TCA cycle is regulated by the availability of amino acids (Somerville et al., 2003).
- the TCA cycle exerts a metabolic regulation over the synthesis of capsular polysaccharide by gluconeogenesis (Sadykov et al., 2010).
- succinate dehydrogenase and fumarate reductase as part of the TCA cycle are related membrane-bound enzyme complexes (Cecchini et al., 2002).
- Salmonella enterica serovar Typhimurium requires glycolysis and glucose for intracellular replication in macrophage, and the complete TCA cycle is needed for full virulence in the murine infection model (Yimga et al., 2006).
- E. ictaluri some live attenuated vaccine candidates have been developed, including auxotrophic (Lawrence et al., 1997; Thune et al., 1999) and iron- siderophore uptake (Abdelhamed et al., 2013) mutants.
- auxotrophic Lawrence et al., 1997; Thune et al., 1999
- iron- siderophore uptake Abdelhamed et al., 2013
- TCA and one- carbon (C1) metabolism pathways contribute to E. ictaluri pathogenesis (Dahal et al., 2014; Dahal et al., 2013).
- compositions for use as live attenuated vaccines and methods related to the same that overcome the stated deficiencies in the prior art, as well as other benefits.
- the compositions comprise mutant strains of the bacteria Edwardsiella ictaluri or another pathogenic bacterial strain of Enterobacteriaceae.
- the mutant strains are the triple mutants ESC-NDKL1 ( ⁇ gcvP ⁇ sdhC ⁇ frdA) and ESC-NDKL2 ( ⁇ gcvP ⁇ sdhC ⁇ mdh) in the bacteria Edwardsiella ictaluri or another pathogenic bacterial strain of Enterobacteriaceae. More preferably, the mutant strain is the ESC-NDKL1 strain in the bacteria Edwardsiella ictaluri.
- FIG. 1 depicts an overview of mutant screening procedures using BLMS method.
- FIG.2 depicts the results of a vaccine efficacy trial and shows the percent mortalities resulting from vaccination using mutants developed through the BLMS method. Percent mortalities are the mean of four replicate tanks per treatment.
- PBS is saline control
- Wt wild-type
- AQUAVAC-ESC is a commercially available live attenuated vaccine.
- Capital letters above each bar indicate statistical groupings.
- FIG. 3 depicts the results of percent mortalities resulting from challenge with parent strain 93-146 twenty one days post-vaccination using mutants developed through the BLMS method. As in FIG. 2, percent mortalities are the mean of four replicate tanks per treatment. PBS is saline control, Wt is parent strain 93-146, and AQUAVAC-ESC is a commercially available live attenuated vaccine. Capital letters above each bar indicate statistical groupings. Groups marked with the same capital letter do not show statistically significant differences. (P ⁇ 0.05) [0012] FIG.
- FIGS.5A and 5B are bar graphs showing percent mortality of catfish fry.
- FIG. 5A is a bar graph showing percent mortalities of catfish fry immunized with ESC- NKDL1, ESC-NKDL2, AQUAVAC-ESC, and sham control.
- FIG. 5B is a bar graph showing percent mortalities of catfish fry challenged with wild-type E. ictaluri at 21 days post-immunization. (*p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.005) [0014] FIGS.
- FIG. 6A and 6B are bar graphs showing percent mortality of catfish fingerlings.
- FIG. 6A is a bar graph showing percent mortalities of catfish fingerlings immunized with ESC-NKDL1, ESC-NKDL2, AQUAVAC-ESC, and sham control.
- FIG.6B is a bar graph showing percent mortalities of efficacy for catfish fingerlings challenged with wild-type E. ictaluri at 21 days post-immunization. (*p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.005) [0015]
- FIG. 7A is a bar graph showing the mean number of fish remaining in each pen at harvest. This data represents the mean of four replicate pens in each earthen pond.
- FIG.7B is a bar graph showing the probability of survival of each treatment.
- the data represents the mean of four replicate pens in each earthen pond.
- FIG. 8 is a bar graph showing the mean total weight of fish per pen at harvest. This data represents the mean of four replicate pens in each pond.
- FIG. 9 is a bar graph showing the mean individual fish weight for thirty fish from each pen. This data represents the mean of four replicate pens in each pond.
- FIG. 10 is a bar graph showing the mean individual fish length for thirty fish from each pen. The data represents the mean of four replicate pens in each pond. DETAILED DESCRIPTION
- the BLMS method described herein was successfully used in the development of a vaccine for channel catfish against E. ictaluri. Although the following embodiment describes the methods as applied in vaccine development against E. ictaluri, the BLMS method is widely applicable to the screening of any bacterial species. Other methods were employed below to prepare mutant E. ictaluri for development of live attenuated vaccines for channel catfish against E. ictaluri. These other methods can be used to create the equivalent mutants in other bacterial species for live attenuated vaccines in channel catfish and in other fish and/or catfish species. [0020] Technological developments in functional genomics allow detection of molecular phenotypes that evade detection at the physiological or morphological levels.
- BLMS bioluminescence mutant screening
- BLMS bioluminescence mutant screening
- luxCDABE bacterial luciferase operon
- luxCDABE genes could also be incorporated into bacterial chromosomes for a similar BLMS purpose. Chromosomal insertion of luxCDABE operon may require more sensitive instrumentation to alleviate the reduced amount of bioluminescence produced from a single copy lux operon.
- Our BLMS procedure requires use of IPTG because our mutant library expresses lux operon conditionally from a lacZ promoter on pAKgfplux2, which also carries a lacI q suppressor gene.
- ictaluri mutants were first characterized in terms of their virulence and vaccine potential and later mutated genes in each mutant were determined by transposon insertion sites identification. Finally, selected mutants were compared to a commercial vaccine (AQUAVAC-ESC®) to determine their commercial value. Findings from our research suggest that BLMS is a very powerful screening method for development of live attenuated vaccines. Fourteen mutants identified by utilizing BLMS showed reduced virulence as compared to wild-type E. ictaluri and provided increased protection from ESC compared to non-vaccinated controls. Among the 14 mutants, we observed redundant mutations in two genes.
- BLMS can detect novel virulence relevant genes located on native plasmids or show importance of native plasmids in bacterial virulence if random mutation occurs in the origin of replication of native plasmids. While applying BLMS to E. ictaluri, we observed that at least one of the two native plasmids of E.
- ictaluri may be important in E. ictaluri virulence because two different locations were targeted on this plasmid in two different mutants (EiAKMut04 and EiAKMut10).
- EiAKMut04 and EiAKMut10 Two different mutants.
- EiAKMut04 and EiAKMut10 Two different mutants.
- EiAKMut04 and EiAKMut10 Two different mutants.
- Production of Edwardsiella ictaluri mutant library [0026] A library of random transposon insertion mutations in conditionally bioluminescent E. ictaluri strain 93-146 carrying pAKgfplux2 was generated. The library containing approximately 15,000 mutants was created by using a derivative of the mariner transposon Himar1 carried on pMAR2xT7 plasmid. The library consisted of mutants arrayed in 39 384-well plates. A duplicate of the whole library was also prepared.
- the produced mutant library is compatible with genetic footprinting of the mutants with transposon-site hybridization (TraSH) analysis.
- TraSH transposon-site hybridization
- FIG. 1 A general outline of the integrated procedures including in vitro BLMS and in vivo fish screening applied can be seen in FIG. 1.
- Injection of catfish with 100 BLMS-selected mutants resulted in identification of 14 attenuated mutants including 8 mutants susceptible to both serum and neutrophils, 4 susceptible to neutrophils, and 2 susceptible to serum, which were further characterized in vivo.
- Characterization of virulence and vaccine potentials of Edwardsiella ictaluri mutants [0030] In vitro BLMS allowed us to reduce the number of target mutants to an amenable size for in vivo characterization. Fish were infected with the 14 mutants, and their attenuation and vaccine potentials were determined (see TABLE 1).
- MAR2XT7 insertion locations were determined using single primer PCR amplification of transposon ends and nested primer sequencing. We determined the insertion locations of MAR2XT7 and identified disrupted (non-functional) genes from all fourteen mutants (TABLE 2). Gene identification using database searches indicated that glycine cleavage system protein P (gcvP) was disrupted in three of the mutants (EiAKMut02, EiAKMut03, and EiAKMut08) but at different locations within the genetic sequence.
- gcvP glycine cleavage system protein P
- EiAKMut13 performed inferior to commercial vaccine in terms of protection, but EiAKMut13 provided slightly greater protection against wild-type infection as compared to the sham vaccinated fish.
- the following description more particularly discloses the steps used in practicing the BLMS method as applied to the BLMS E. ictaluri live attenuated vaccine.
- E. coli SM10 ⁇ pir was used as the donor strain in conjugations for transfer of pAKgfplux2 and pMAR2XT7 into Edwardsiella ictaluri strain 93-146.
- E. ictaluri and E. coli DH5 ⁇ carrying pAKgfplux2 were used as negative and positive controls in neutrophil and serum screening experiments.
- E. coli strains were grown using Luria-Bertani (LB) broth and agar plates at 37 o C and E. ictaluri was grown using brain heart infusion (BHI) broth and agar plates at 30 o C.
- Antibiotics were added to the following final concentrations: ampicillin (100 ⁇ g/ml), colistin (12.5 ⁇ g/ml), gentamicin (12.5 ⁇ g/ml). 2 mM Isopropyl-ß-D-thiogalactopyranoside (IPTG) was used in growth medium and screening assays to induce expression of bacterial luciferase operon (luxCDABE) from the lacZ promoter in pAKgfplux2. E. ictaluri minimal medium was used to eliminate auxotrophic mutants. [0038] Construction of Edwardsiella ictaluri mutant library. MAR2xT7 insertions were generated by introducing pMAR2xT7 from E. coli SM10 ⁇ pir into E.
- Putative transposants were picked robotically using a Flexsys Colony Picker (GENOMIC SOLUTIONS, Ann Arbor, MI) into 60 ⁇ l of LB broth containing colistin and gentamicin in 384-well microtiter plates and grown overnight in HIGRO shaker (GENOMIC SOLUTIONS) at 30 o C.
- a duplicate library was prepared by the Flexsys Colony Picker before sterile glycerol was robotically added to the cultures at a final concentration of 20%. Plates were sealed with aluminum foil to prevent cross contamination, lids were taped to prevent freeze drying, and libraries were frozen at -80° C. [0039] Catfish serum and neutrophil preparation.
- SPF pathogen free fish facility at the College of Veterinary Medicine, Mississippi State University maintains SPF channel catfish.
- SPF catfish For serum preparation, 1-2 kg SPF catfish were anesthetized in water containing 200 mg/l tricaine methanesulfonate (MS-222) and blood was collected at 1% of body weight. A recovery period of one month was given for subsequent blood collections. Serum was obtained and stored at -80° C as single use aliquots.
- Neutrophils were isolated from the single cell suspensions of anterior kidney cells of SPF catfish (38.63 ⁇ 0.68 cm, 424.20 ⁇ 23.34 g) using discontinuous percoll gradient centrifugation procedure described previously. Purity of neutrophils collected from the 1.060-1.080 interface was determined using flow cytometry.
- Percent light change between the first and last measurement times was determined for each mutant and compared to serum resistant (E. ictaluri 93-146 pAKgfplux2) and serum sensitive (DH5 ⁇ pAKgfplux2) controls included in each plate.
- Neutrophil screening was accomplished by setting up phagocytosis assays including freshly isolated neutrophils with 75% or higher purity, 15% SPF catfish serum, 2 mM IPTG, and mutant bacteria. Neutrophil to bacteria numbers were adjusted to give a ratio between 1:40 and 1:80. Bioluminescence imaging was conducted as described above in the serum screening procedure. Percent bioluminescence change in 2,256 mutants was calculated and compared to those of positive and negative controls.
- Virulence and efficacy of the 14 mutants were characterized by infecting catfish by both intraperitoneal injection and immersion. Each 40 liter flow-through tank contained twenty fish, and four tanks were used for each mutant. Fish were allowed to acclimate for one week. Quadruplicate wild-type and PBS controls were also included in all experiments. Bacteria numbers were adjusted to be equal by determining OD 600 readings and adjusting volumes accordingly. In the first study, fish (13.80 ⁇ 0.26 cm, 25.83 ⁇ 1.49 g) were infected by immersion in water containing 1 x 10 6 CFU /ml for one hour. After 21 days, immunized fish were infected with wild-type E.
- Transposon insertion sites were identified by using a single primer PCR protocol. Mutants were grown for 18 hours and genomic DNA was prepared using WIZARD Genomic DNA Purification Kit (PROMEGA).
- the transposon specific template was amplified linearly for 40 cycles.
- a second round produced specific and non-specific amplicons due to low annealing temperature at 30 o C.
- the final round further amplified the amplicons.
- the 25 ⁇ l PCR reaction contained 0.2 mM dNTPs, 0.2 ⁇ M transposon specific primer, 1.5 mM MgCl2, buffer, and 1.25 units of Taq polymerase (PROMEGA).
- the five ⁇ l single primer PCR reaction was cleaned with 2 ⁇ l of EXOSAP-IT enzyme mix (USB CORP.) according to the manufacturer’s instructions.
- Succinate-ubiquinone oxidoreductase (SQR) encoded by the sdhCDAB gene cluster and menaquinol-fumarate oxidoreductase (QFR) encoded by the frdABCD gene cluster are part of the tri-carboxylic acid (TCA) cycle and are structurally and functionally related membrane-bound enzyme complexes.
- EiAKMut05 has an insertion in the sdhC gene, which encodes one of four subunits of the succinate dehydrogenase complex.
- SdhC is one of the two subunits that anchor the complex in the cytoplasmic membrane.
- Succinate dehydrogenase is part of the aerobic respiratory chain and the Krebs cycle, oxidizing succinate to fumarate while reducing ubiquinone to ubiquinol. It is closely related to fumarate reductase, which catalyzes the reverse reaction.
- Succinate dehydrogenase and fumarate reductase can replace each other at different relative rates and with different apparent substrate affinities.
- sdhCDA mutant of Salmonella enterica serovar Typhimurium were slightly attenuated and complete attenuation was achieved by succinate dehydrogenase/ fumarate reductase double mutation.
- E. ictaluri sdhC is the first gene in a polycistronic operon that encodes the four components of succinate dehydrogenase; therefore, it is possible that the mutation in sdhC has a polar effect on expression of downstream genes.
- Our results indicate that attenuation of E. ictaluri was achieved with sdhC mutation without a need for generating double mutants in frd genes.
- SdhC activity is located in the cytoplasm, and it utilizes artificial electron acceptors; in contrast, wild-type E. coli has membrane-associated SdhC activity with ubiquinone as the electron acceptor.
- fumarate reductase is expressed under anaerobic conditions with glucose as a carbon source.
- SdhC has similar function, hydrophobicity, and protein size to the membrane-binding subunit from fumarate reductase (FrdC), SdhC and FrdC do not share significant sequence identity.
- FrdC fumarate reductase
- SdhC and FrdC do not share significant sequence identity.
- fumarate reductase was found to be essential for colonization of mouse gastric mucosa.
- E. pylori fumarate reductase was found to be essential for colonization of mouse gastric mucosa.
- succinate dehydrogenase is known to contribute to pathogenicity.
- the organic acids formate and succinate have a protective effect in stationary phase cells against killing effects of antimicrobial peptide BPI, which appears to disrupt the bacterial respiratory chain. Maintenance of protective levels of formate and succinate requires the activity of formate dehydrogenase and succinate dehydrogenase, respectively.
- E. ictaluri also encodes the formate dehydrogenase complex in its genome. [0050] Mutants 2, 3, and 8 all had insertions in gcvP, which encodes a protein that is part of the glycine cleavage system.
- the glycine cleavage system is a loosely associated four subunit enzyme complex that catalyzes the reversible oxidation of glycine to form 5, 10- methylenetetrahydrofolate, which serves as a one carbon (“1C”) donor. It is one of two sources of 1C units with serine hydroxymethyltransferase being the other (and is considered the more important source). Expression of the glycine cleavage enzyme system is induced by glycine, and gcv mutants are unable to use glycine as a 1C source and excrete glycine. The glycine cleavage system is also part of the formyltetrahydrofolate biosynthesis system.
- GcvP is a 104 kDa protein that catalyzes the decarboxylation of glycine.
- gcvP is the third gene in a three gene operon; it is located downstream of gcvH and gcvT, which encode subunits of the glycine cleavage system.
- E. ictaluri also has a gene that encodes serine hydroxymethyltransferase.
- the glycine cleavage system has not been linked with virulence previously, and our disclosed composition and method are the first to employ it. [0051]
- Mutant 1 had an insertion in rseB, which encodes one of two negative regulators of sigmaE.
- RseA is considered the major regulator of sigmaE. SigmaE is expressed in response to heat shock and other stresses on membrane and periplasmic proteins, including misfolding of outer membrane proteins, hyperosmotic stress, metal ion exposure, changes in LPS structure, and starvation signal ppGpp. SigmaE is required for heat-induced transcription of rpoH, which encodes heat shock factor sigma32 and other heat shock proteins.
- RseB is a periplasmic protein that interacts with RseA. RseB stimulates binding of RseA to sigmaE, thereby assisting RseA in tethering sigmaE to the cytoplasmic membrane.
- rseA Degradation of RseA releases sigmaE and allows it to interact with the core enzyme of RNA polymerase to initiate transcription.
- mutations in rseA cause increased sigmaE activity
- an rseB mutant shows wild-type sigmaE activity under inducing conditions and exhibits a small increase in sigmaE activity under non-inducing conditions.
- rseB is the third gene in a polycistronic operon. It is downstream of rpoE, which encodes sigmaE, and rseA, and it is upstream of rseC, which encodes a positive regulator of sigmaE.
- Mutant 6 has an insertion in rsxB, which encodes one of six proteins that form a SoxR reducing system in E. coli. SoxR is a regulatory protein that senses superoxide and nitric oxide and induces expression of an oxidative stress response.
- SoxR When SoxR is activated by oxidation of its [2Fe-2S] cluster, it induces expression of SoxS, which is a transcriptional regulator that induces expression of superoxide dismutase and other oxidative response proteins. The SoxR reducing system inactivates SoxR, thereby turning off the oxidative stress response.
- SoxS In E. coli, when any of the six rsx genes are mutated, SoxS is constitutively expressed, leading to induction of oxidative stress response.
- SoxS In Salmonella, SoxS is not essential for virulence, but SoxS was found to contribute to virulence in an E. coli mouse pyelonephritis model. In E.
- Mutant 4 has an insertion in a gene encoding a hypothetical protein located on one of the two E. ictaluri constitutive plasmids, pEI1. The protein has >50% identity with Salmonella effector proteins with leucine rich repeats that are secreted through a type III secretion system. The 618 amino acid protein appears to be in a monocistronic operon.
- EXAMPLE IDENTIFYING MUTANTS THAT FAIL TO ATTACH TO THE HOST EPITHELIUM.
- the BLMS method can also be used to identify bacterial mutants that fail to attach to the host molecules, cells, or surfaces. Attachment and colonization of the host epithelium is an indispensable first step to any bacterial infection and can be achieved through a variety of diverse mechanisms.
- To investigate these attachment mechanisms in Edwardsiella ictaluri we used random insertion of the pMar2xT7 transposon to generate a library of 1728 mutants. Each mutant expressed bioluminescence constitutively from the plasmid pAKlux1. This library was then screened in a high throughput fashion using an IVIS Living Image System (XENOGEN) in a series of nested in vivo challenges using a skin abrasion model we developed.
- IVIS Living Image System XENOGEN
- Combinations of mutations can be constructed using the pathways we have disclosed. Specifically, in-frame deletions in TCA cycle enzymes and glycine cleavage system protein can be constructed to create greater attenuation while retaining antigenicity. Mutation of the glycine cleavage system as a vaccine strategy is a new strategy that has never been previously reported. EiAKMut2 has a mutation in gcvP.
- the glycine cleavage system functions in providing 5,10- methylenetetrahydrofolate as a source for 1C moieties.
- Our plan is to construct a mutant containing deletions in gcvP (our current mutant) and in another enzyme that serves to provide 5,10-methylenetetrahydrofolate through an alternative pathway. Knocking out both pathways should cause improved attenuation.
- Mutation of genes encoding TCA cycle enzymes exemplified by EiAKMut05 and EiAKMut12, shows great potential as a strategy for an effective live attenuated E. ictaluri vaccine.
- TCA cycle enzymes were recently discovered as an effective strategy for vaccine development in Salmonella (which is closely related to Edwardsiella). We have found that knocking out a single TCA cycle gene does not always cause complete attenuation, but knocking out two genes can cause complete attenuation. Specifically, a Salmonella sdhCDA-frdABCD double mutant was fully avirulent and effective as a vaccine, while a Salmonella sdhCDA mutant was not fully attenuated.
- a combination mutant can be constructed that has deletions in sdhC (the gene mutated in EiAKMut05) and mdh (the gene mutated in EiAKMut12), as well as a second sdhC combination mutant that has a knockout in another enzyme that encodes a related TCA cycle enzyme.
- sdhC the gene mutated in EiAKMut05
- mdh the gene mutated in EiAKMut12
- USE OF OTHER BACTERIAL SPECIES AS LIVE ATTENUATED VACCINES FOR VARIOUS HOSTS [0059]
- the method and compositions disclosed herein are not limited to Edwardsiella ictaluri, but can be used in other bacteria as well.
- Salmonella enterica is closely related to Edwardsiella ictaluri and is in the same bacterial family (Enterobacteriaceae).
- the pathogenesis of salmonellosis in mammals is also similar to the pathogenesis of enteric septicemia of catfish caused by E. ictaluri.
- the mutation of these genes in Salmonella will result in development of an effective live attenuated vaccine for prevention of salmonellosis in various animal hosts.
- the genus Yersinia is also in the same family as Edwardsiella and Salmonella, and the disease pathogenesis of Yersinia is similar to enteric septicemia of catfish. Therefore, the mutation of these genes will be effective for development of live attenuated vaccines for Yersinia pestis, which causes bubonic plague in humans, Y. enterocolitica and Y. pseudotuberculosis, which cause gastrointestinal disease in humans and other mammals, and Y. ruckeri, which causes enteric redmouth disease in salmonid fish.
- the mutation of these genes may be an effective strategy for development of live attenuated vaccines for pathogenic Escherichia coli, Shigella flexneri, and Shigella dysenterieae, which are also closely related to E. ictaluri.
- the mutation of these genes can also be used for development of live attenuated vaccines against Francisella tularensis, which causes tularemia in humans, because the disease pathogenesis is similar to enteric septicemia of catfish.
- bacterial pathogens that we anticipate mutation of these genes may be effective for development of live attenuated vaccines include Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, Haemophilus influenzae, Haemophilus ducreyi, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Actinobacillus actinomycetemcomitans, Avibacterium paragallinarum, Moraxella catarrhalis, Moraxella bovis, Pseudomonas aeruginosa, Coxiella burnetii, Bordetella bronchiseptica, Bordetella pertussis, Bordetella parapertussis, Bordetella avium, Burkholderia mallei, Burkholderia pseudomallei, Neisseria meningitidis, Neisseria gonorrhoeae, Brucella abortus,
- BLMS may be an effective tool for identification of new gene targets for development of live attenuated vaccines.
- ictaluri harboring multiple gene disrupting (non-functional) mutations of the identified gene targets provided in the list of glycine cleavage system (gcvP), serine hydroxymethyltransferase, succinate dehydrogenase, malate dehydrogenase, 2-oxoglutarate dehydrogenase, negative regulator of sigma E activity (rseB), hypothetical protein pEI1_p1, electron transport complex protein RnfB, Fimbrial chaperon protein, Putative RNA one modulator protein pEI1_p4, UDP-glucose 6- dehydrogenase, fumarate reductase (frdA), and other genes encoding enzymes in the tri- carboxylic acid (TCA) cycle.
- gcvP glycine cleavage system
- rseB hypothetical protein pEI1_p1
- RnfB electron transport complex protein
- Fimbrial chaperon protein Putative RNA one modul
- gcvP glycine cleavage system
- sdhC succinate dehydrogenase
- mdh malate dehydrogenase
- frdA fumarate reductase
- triple mutant ESC-NDKL1 ( ⁇ gcvP ⁇ sdhC ⁇ frdA) strain was superior to the triple mutant ESC-NDKL2 ( ⁇ gcvP ⁇ sdhC ⁇ mdh) strain because our previous work showed that individual gene disrupting mutants in the gcvP and mdh genes showed superior results compared to other single mutants. Also, the individual gene disrupting mutants in the sdhC gene showed no mortality in immunized fish.
- TCA cycle The primary role of the TCA cycle is to provide NADH, which is used by bacterial cells for ATP synthesis via the electron transport chain for the complete catabolism of non-preferred carbon sources and the subsequent generation of reducing potential and biosynthetic intermediates. Also, several enzymes of TCA cycle require iron, for example, aconitase, succinate dehydrogenase complex, and fumarase.
- epidermidis perceives environmental changes through alterations in TCA cycle activity, leading to changes in the intracellular levels of biosynthetic intermediates, ATP, or the redox status of the cell (Vuong et al., 2005). These changes in the metabolic status of the bacteria result in attenuation.
- Serrovar Typhimurium Salmonella enterica
- mutant stains with a deletion of genes encoding TCA cycle enzymes ⁇ mdh, ⁇ sucCD, and ⁇ sdhCDAB replicated to higher levels than the wild-type in resting and activated macrophages, which suggests an enhanced ability to survive under antimicrobial conditions (Bowden et al., 2010).
- enterica ⁇ frdABCD ⁇ sdhCDA double mutants with complete TCA cycles may exhibit to be effective live vaccine strains for animal and human like ⁇ frdABCD ⁇ sdhCDA double mutants of other intracellular bacterial pathogens (Mercado-Lubo et al., 2008).
- fumarate reductase was found to be essential for colonization of mouse gastric mucosa (Ge et al., 2000).
- Fumarate reductase (frd) and succinate dehydrogenase (sdh) are physiologically reversible isoenzymes in the TCA cycle that are induced under anaerobic and aerobic respiratory chain, which can replace each other with functionally related membrane-bound enzyme complexes (Guest, 1981; Maklashina et al., 1998; supra).
- Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron-sulfur subunit, which contains three distinct iron-sulfur clusters, and the catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site for interaction with quinones (Cecchini et al., 2002).
- Glycine cleavage system serves as a one carbon donor (C1 unit); serine hydroxymethyltransferase is another one carbon donor source (supra).
- glycine cleave enzyme system is induced by high concentrations of glycine, and a gcv mutant was unable to use glycine as a C1 source and excrete glycine (Meedel and Pizer, 1974; Plamann et al., 1983; Stauffer et al., 1994), and we have shown that E. ictaluri gcvP is required for virulence (Karsi et al., 2009).
- the laboratory challenge was followed by a field trial mimicking commercial catfish production to evaluate the efficacy of the ESC-NDKL1 ( ⁇ gcvP ⁇ sdhC ⁇ frdA) in earthen pond conditions, the best vaccination regime (immersion, oral, and immersion-oral combination), and also, compare the efficacy of the ESC-NDKL1 to the commercially available vaccine, AQUAVAC-ESC.
- the fry were vaccinated with an ESC-NDKL1 by immersion (19 days post-hatch) in the early summer, and the oral booster was included in late summer (80 days post-hatch).
- This vaccination schedule time was chosen to provide protection during the high incidence ESC season that occurs in the late spring and early fall when water temperatures are in the 18-28° C range, the optimal temperature range for ESC development (Plumb, 1988; Thune et al., 1994).
- ESC development During the 95 days of the pond study, there was no difference between the five earthen ponds considering the water quality parameters. Overall means of DO and temperature are within what is expected for catfish fingerling production under commercial conditions (Tucker, 1990). The results from field study depend on the survival of fingerlings in each pond at the end of the study. The probability of survival depends on the initial stocking number (300 fish/pen) and remaining number at the harvest. The daily dead fish cannot be gathered in this study.
- the immersion vaccination with ESC-NDKL1 strain provided significant protection against ESC even after four months from exposure under field trials, this is a great advantage over the currently available vaccine.
- the mean individual fish weight of thirty fish was observed to be higher in the three ESC-NDKL1 ( ⁇ gcvP ⁇ sdhC ⁇ frdA) vaccinated ponds (immersion, oral, immersion-oral) and AQUAVAC-ESC than the sham-vaccinated group.
- the individual fish lengths obtained from the ESC-NDKL1 vaccinated ponds were higher than a sham-vaccinated pond, although statistically analysis showed no significant differences.
- a critical factor not evaluated in this study is the claim that the delay in fry stocking and use of a primary nursery phase can reduce early fry mortality and reduce unaccounted fish losses (Morrison et al., 1995).
- a typical production cycle for channel catfish industry involves the stocking of 7 to 10 days old fry directly into earthen ponds and growing them for 5 to 10 months. However, this method has resulted in high levels of mortality during the first 30 days (Carpenter, 2001).
- AQUAVAC-ESC was included in this study for comparison.
- the field study is, however, somewhat difficult to directly compare with other similar studies because there are different aspects that vary from one study to another such as stocking density, the length of growing period, quality of feed utilized, management techniques, and water parameters.
- Past studies have shown that AQUAVAC-ESC (RE-33) provides protection in fish against virulent E. ictaluri isolates when vaccinated 7 to 72 days post-hatch based on laboratory findings and limited field studies (Shoemaker et al., 1999; Wise et al., 2000).
- ESC-NDKL1 is a strong candidate for use as a live attenuated vaccine for catfish fry and fingerling in commercial hatcheries and fish farms.
- the following description more particularly discloses the steps used in practicing the claimed triple mutant E. ictaluri live attenuated vaccines.
- Materials and Methods [0077] Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this study are listed in TABLE 3. E. ictaluri was grown at 30 o C using brain heart infusion (BHI) broth and agar (Difco, Sparks, MD).
- Escherichia coli were grown at 37 o C using Luria-Bertani (LB) broth and agar (Difco).
- E. coli CC118 ⁇ pir and SM10 ⁇ pir/S17-1 ⁇ pir were used for cloning mutated fragments into the pMEG-375 plasmid and conjugal transfer of pMEG-375 suicide plasmid into E. ictaluri.
- Ampicillin was used at 100 ⁇ g/ml to maintain pMEG-375, and colistin was used at 12.5 ⁇ g/ml for counter selection against E. coli SM10 ⁇ pir following conjugation.
- pEi ⁇ frdA and pEi ⁇ mdh were mobilized into double mutant Ei ⁇ gcvP ⁇ sdhC by conjugation (for methods, see Karsi and Lawrence, 2007; incorporated herein by reference for all that it teaches that is not contradictory to the present disclosure).
- the recipient bacteria were spread on BHI agar containing colistin and ampicillin for selecting E. ictaluri with integrated vector, and positive colonies were followed by streaking on BHI agar with 5% sucrose and 0.35% mannitol for allelic exchange and loss of pMEG-375 with the sacB gene. Deleted regions were amplified from the resulting ampicillin sensitive colonies and confirmed by sequencing.
- Vaccine safety in specific pathogen free (SPF) catfish fry (3.17 ⁇ 0.05 cm, 335.92 ⁇ 20.02 mg) and fingerlings (7.75 ⁇ 0.08 cm, 4,500 ⁇ 14.07 mg) was determined for the two triple mutants and commercial live attenuated vaccine (Klesius and Shoemaker, 1999).
- One treatment group was used as a sham control. 14-day old catfish fry were stocked into 20 tanks at a rate of 40 fish/tank, and three- month-old catfish fingerlings were stocked into 20 tanks at a rate of 25 fish/tank.
- Fry experiment included four replicates and fingerling experiment included three replicates per group. Experiments were conducted in 40-L tanks supplied with flow-through water and fed two times per day.
- Vaccination doses were 6.0 x10 6 CFU/ml water and 4.5 x10 7 CFU/ml water for catfish fry and fingering, respectively. Mortalities were recorded daily for 21 days, and swab samples from the dead fish were plated on BHI agar for confirmation of the causative pathogen. At 21 days post-vaccination, vaccinated and sham control groups were immersion exposed to Wt E. ictaluri 93-146 containing 3.8 x10 7 CFU/ml water, and fish mortalities were monitored daily for 21 days.
- RPS Relative percent survival
- Dissolved oxygen (DO) and temperature were monitored twice daily in the morning and afternoon using a with a portable dissolved oxygen meter (YSI model 550A, YSI Inc., San Diego, California) on the pond bank. Water was added to the ponds periodically to replace that which was lost through evaporation and seepage.
- Stocking of the fry and vaccination schedule on June 25, 2015, approximately 6,000 17 day-old specific pathogen free (SPF) catfish fry were stocked into five tanks (1200/tank) supplied with flow-through dechlorinated water. Water temperature was maintained at 25-26° C throughout indoor conditions. The five tanks corresponded to five treatment groups (immersion, oral, immersion-oral combination, commercial vaccine, and sham-vaccinated).
- catfish fry (19 days post-hatch) in three treatment groups were immersion vaccinated indoors (3.93 x10 7 CFU/ml of water for 1 h).
- Fry in the sham-vaccinated group were exposed to an equivalent volume of brain heart infusion (BHI) broth indoors.
- BHI brain heart infusion
- the tanks were observed daily for mortalities.
- Fry in all treatments were moved to the ponds on July 27, 2015.
- the fry were transferred in aerated containers and stocked into ponds at a rate of 1200 fry/pond (300 fry/pen).
- the pens were covered with a lid to prevent birds and other animals from preying on the fish.
- Feeding fish were fed twice a day by hand, once in the morning and afternoon, with a commercial catfish feed. Changing to a larger feed pellet was determined according to the behavior and size development of the fry in each pond. Fish were observed after feeding, and the activity of feeding was documented.
- Vaccine preparation and oral vaccination to prepare oral vaccination, an overnight culture of ESC-NDKL1 ( ⁇ gcvP ⁇ sdhC ⁇ frdA) containing 3.52x10 9 CFU/ml was mixed with commercial feed pellets at a rate of 20% (weight to volume). The vaccine-feed was mixed by a hand mixer until all liquid was absorbed. The average amount of feed consumed one week before vaccination was used to estimate the amount of feed to use on vaccination days.
- ictaluri containing 2.71x10 9 CFU/ml was mixed with commercial feed at a rate of 20% (weight to volume), and each pond was fed for five consecutive days (average feed 600 g/pond for five days) followed by a five-day break, then another five days of exposure. Following vaccination, fish were fed regular feed without adding the vaccine to feed for 21 days. [0086] Harvesting the ponds and measuring procedures: the study was terminated on November 1, 2015 (35 days after wild-type infection) when the water temperature was less than l8°C. Fingerling fish were collected after three months of growing in net pens in earthen ponds. At the end of the trial, fish were harvested and euthanized in water containing 300 mg/L MS-222, and fish numbers and body measurements were collected.
- the number of live fish in a replication at the end of the trial was the outcome assessed using an events/trials syntax.
- Treatment was the fixed effect evaluated in the model. Replication within a treatment group was included as a random effect in the model.
- the BHI (sham) and AQUAVAC-ESC treatment groups were the referents for comparisons of the effect of the other treatments using an LSMestimate statement.
- the results of the analysis were presented as odds ratios for survival and probability of survival. [0089]
- the effect of the different treatments on the total weight of fish within a replication at the end of the trial was assessed by analysis of variance using PROC GLIMMIX in SAS for Windows 9.4. The results of the analysis were presented as least squares means and their standard errors.
- the BHI (sham) and AQUAVAC-ESC treatment groups were the referents for comparisons of the effect of the other treatments using an LSMestimate statement, adjusting the p-values for multiple comparisons with the simulate option.
- the effects of the different treatments on the weight and length of 30 fish within a replication at the end of the trial were assessed in separate mixed model analyzes using PROC GLIMMIX in SAS for Windows 9.4. Treatment was the fixed effect assessed in each model while replication within a treatment group was included as a random effect. The results of the analysis were presented as least squares means and their standard errors.
- the BHI (sham) and AQUAVAC-ESC treatment groups were the referents for comparisons of the effect of the other treatments using an LSMestimate statement, adjusting the p-values for multiple comparisons with the simulate option.
- the distribution of the conditional residuals was evaluated for each model to determine the appropriateness of the statistical model for the data. A significance level of 0.05 was used for all analyses.
- the mortality rate in wild-type challenged fingerlings was over 81% (see FIG. 6A).
- the vaccine efficacy of the mutants was determined by challenging vaccinated fingerling with wild-type E. ictaluri by immersion three weeks after vaccination.
- ESC-NDKL1 mutant protected fingerlings significantly (p ⁇ 0.01) compared to ESC-NDKL2 and AQUAVAC-ESC.
- the ESC-NDKL1 vaccinated group showed 18.95% mortality while the ESC-NDKL2 and AQUAVAC-ESC showed 79.47% and 54.49% mortalities, respectively (FIG.6B).
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- Cell Biology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Live attenuated bacteria vaccines against enteric septicemia of fish, especially catfish, and methods related to the same. Mutant strains of the bacteria Edwardsiella ictaluri (a pathogenic bacterial strain of Enterobacteriaceae) are provided. The mutant Edwardsiella ictaluri bacteria (or other pathogenic bacterial strain of Enterobacteriaceae) contain one or more gene deletions or disruptions that result in less virulent bacterial strains as live attenuated vaccine compositions against virulent wild-type Edwardsiella ictaluri bacteria (or other pathogenic bacterial strain of Enterobacteriaceae). The mutant strains showing the best immunological protection and safety as a vaccine are the triple mutants ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) strain and ESC-NDKL2 (ΔgcvPΔsdhCΔmdh) strain, with the ESC- NDKL1 strain providing the greatest safety and efficacy of these two triple mutants.
Description
LIVE ATTENUATED CATFISH VACCINE AND METHOD OF MAKING
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001 ] This application is an International Patent Application claiming priority to U.S. Application No. 15/171,367 to Mark L. Lawrence and Attila Karsi filed on June 2, 2016, now issued as U.S. Patent No. , the contents of which are incorporated herein by reference in their entirety.
FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under 2004-35204- 14211, 2009-65119-05671 and 2014-70007-22359 awarded by the National Institute of Food and Agriculture, USDA. The government has certain rights in the invention.
FIELD OF THE INVENTION
[0003] The present invention is generally directed toward a live attenuated vaccine for fish, and more particularly, it is directed toward mutant strains of Edwardsiella ictaluri as a live attenuated vaccine against enteric septicemia of catfish and methods related to the same.
BACKGROUND OF THE INVENTION
[0004] Channel catfish farming is the largest contributor to U.S. aquacultural production. In 2015, catfish growers in the US produced more than $361 million worth of catfish, of which $201 million (55.7%) was from Mississippi (www.nass.usda.gov). Catfish farmers reported 37% mortality rate (U.S. Department of Agriculture, 2011) due to enteric septicemia of catfish (ESC). Edwardsiella ictaluri (also referred to herein as E. ictaluri) is
the causative agent of enteric septicemia of catfish. Like some other species in the Enterobacteriaceae, E. ictaluri has the ability to resist killing by professional phagocytes. In particular, E. ictaluri is resistant to channel catfish neutrophils, which is an important aspect of pathogenesis because (1) neutrophils are the predominant cell type in channel catfish intestinal tract immune cells, and (2) the intestine is an important site of entry for E. ictaluri. E. ictaluri is also resistant to killing by the alternative complement pathway in channel catfish. Although a commercial live attenuated vaccine is now available and antibiotics are still used against E. ictaluri and ESC, ESC is still a major threat to the catfish industry (Klesius and Shoemaker, 1999). Live attenuated vaccines often offer the best prospect for vaccines by providing good protection against diseases through stimulating cellular immune responses (Id.), but the currently available commercial vaccine leaves much to be desired in performance and safety. [0005] The tri-carboxylic acid (TCA) cycle supplies intermediates and ATP for bacterial biosynthesis, thus transcription of genes encoding the enzymes of the TCA cycle is regulated by the availability of amino acids (Somerville et al., 2003). The TCA cycle exerts a metabolic regulation over the synthesis of capsular polysaccharide by gluconeogenesis (Sadykov et al., 2010). Previous research into the role of the metabolic pathway in pathogenic bacteria, such as in Escherichia coli, has demonstrated that succinate dehydrogenase and fumarate reductase as part of the TCA cycle are related membrane-bound enzyme complexes (Cecchini et al., 2002). It has been shown that Salmonella enterica serovar Typhimurium requires glycolysis and glucose for intracellular replication in macrophage, and the complete TCA cycle is needed for full virulence in the murine infection model (Yimga et al., 2006). [0006] In E. ictaluri, some live attenuated vaccine candidates have been developed, including auxotrophic (Lawrence et al., 1997; Thune et al., 1999) and iron-
siderophore uptake (Abdelhamed et al., 2013) mutants. Also, we reported that TCA and one- carbon (C1) metabolism pathways contribute to E. ictaluri pathogenesis (Dahal et al., 2014; Dahal et al., 2013). In particular, some TCA and C1 metabolism mutants provided good protection against wild-type E. ictaluri infections in fingerling, which was not the case when tested in catfish fry (14-day old). Thus, there is still a need for a live attenuated bacterial vaccine against ESC in fish, especially catfish, which has improved safety and protection levels of the TCA and C1 metabolism mutants. SUMMARY OF THE INVENTION [0007] Now disclosed are compositions for use as live attenuated vaccines and methods related to the same that overcome the stated deficiencies in the prior art, as well as other benefits. The compositions comprise mutant strains of the bacteria Edwardsiella ictaluri or another pathogenic bacterial strain of Enterobacteriaceae. Preferably, the mutant strains are the triple mutants ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) and ESC-NDKL2 (ΔgcvPΔsdhCΔmdh) in the bacteria Edwardsiella ictaluri or another pathogenic bacterial strain of Enterobacteriaceae. More preferably, the mutant strain is the ESC-NDKL1 strain in the bacteria Edwardsiella ictaluri. BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Further advantages of the invention will become apparent by reference to the detailed description of preferred embodiments when considered in conjunction with the drawings, which are not to scale, wherein like reference characters designate like or similar elements to the several drawings as follows: [0009] FIG. 1 depicts an overview of mutant screening procedures using BLMS method.
[0010] FIG.2 depicts the results of a vaccine efficacy trial and shows the percent mortalities resulting from vaccination using mutants developed through the BLMS method. Percent mortalities are the mean of four replicate tanks per treatment. PBS is saline control, Wt (“wild-type”) is parent strain 93-146, and AQUAVAC-ESC is a commercially available live attenuated vaccine. Capital letters above each bar indicate statistical groupings. Groups marked with the same capital letter do not show statistically significant differences. (P < 0.05) [0011] FIG. 3 depicts the results of percent mortalities resulting from challenge with parent strain 93-146 twenty one days post-vaccination using mutants developed through the BLMS method. As in FIG. 2, percent mortalities are the mean of four replicate tanks per treatment. PBS is saline control, Wt is parent strain 93-146, and AQUAVAC-ESC is a commercially available live attenuated vaccine. Capital letters above each bar indicate statistical groupings. Groups marked with the same capital letter do not show statistically significant differences. (P < 0.05) [0012] FIG. 4 is a photograph of an electrophoresis gel confirming the ESC- NKDL1 and ESC-NKDL2 in-frame deletion constructs using PCR. The first lane is 1Kb Plus marker. [0013] FIGS.5A and 5B are bar graphs showing percent mortality of catfish fry. FIG. 5A is a bar graph showing percent mortalities of catfish fry immunized with ESC- NKDL1, ESC-NKDL2, AQUAVAC-ESC, and sham control. FIG. 5B is a bar graph showing percent mortalities of catfish fry challenged with wild-type E. ictaluri at 21 days post-immunization. (*p < 0.05; **p < 0.01; ***p < 0.005) [0014] FIGS. 6A and 6B are bar graphs showing percent mortality of catfish fingerlings. FIG. 6A is a bar graph showing percent mortalities of catfish fingerlings
immunized with ESC-NKDL1, ESC-NKDL2, AQUAVAC-ESC, and sham control. FIG.6B is a bar graph showing percent mortalities of efficacy for catfish fingerlings challenged with wild-type E. ictaluri at 21 days post-immunization. (*p < 0.05; **p < 0.01; ***p < 0.005) [0015] FIG. 7A is a bar graph showing the mean number of fish remaining in each pen at harvest. This data represents the mean of four replicate pens in each earthen pond. FIG.7B is a bar graph showing the probability of survival of each treatment. The data represents the mean of four replicate pens in each earthen pond. [0016] FIG. 8 is a bar graph showing the mean total weight of fish per pen at harvest. This data represents the mean of four replicate pens in each pond. [0017] FIG. 9 is a bar graph showing the mean individual fish weight for thirty fish from each pen. This data represents the mean of four replicate pens in each pond. [0018] FIG. 10 is a bar graph showing the mean individual fish length for thirty fish from each pen. The data represents the mean of four replicate pens in each pond. DETAILED DESCRIPTION
[0019] The BLMS method described herein was successfully used in the development of a vaccine for channel catfish against E. ictaluri. Although the following embodiment describes the methods as applied in vaccine development against E. ictaluri, the BLMS method is widely applicable to the screening of any bacterial species. Other methods were employed below to prepare mutant E. ictaluri for development of live attenuated vaccines for channel catfish against E. ictaluri. These other methods can be used to create the equivalent mutants in other bacterial species for live attenuated vaccines in channel catfish and in other fish and/or catfish species.
[0020] Technological developments in functional genomics allow detection of molecular phenotypes that evade detection at the physiological or morphological levels. We disclose a new high-throughput functional genomics tool that we call bioluminescence mutant screening (BLMS) that translates molecular genotypes (gene mutations) to physiological phenotypes (light production) in bacteria and allows application of forward genetics. [0021] BLMS involves random transposon mutation of a bacterial strain expressing bacterial luciferase operon (luxCDABE) in a stable plasmid. Following this approach, we produced a random E. ictaluri mutant library that conditionally expresses luxCDABE genes from a stable plasmid, pAKgfplux2, which allows the tracking of mutants in a pool under different experimental conditions. [0022] In an alternative embodiment, luxCDABE genes could also be incorporated into bacterial chromosomes for a similar BLMS purpose. Chromosomal insertion of luxCDABE operon may require more sensitive instrumentation to alleviate the reduced amount of bioluminescence produced from a single copy lux operon. Our BLMS procedure requires use of IPTG because our mutant library expresses lux operon conditionally from a lacZ promoter on pAKgfplux2, which also carries a lacIq suppressor gene. To eliminate use of IPTG in bacterial strains without the presence of lacIq gene in their genome use of a mutant library constitutively expressing lux operon from a stable plasmid, such as pAKlux2 and pAKgfplux1, would be preferred. [0023] Through screening 2,256 mutants from E. ictaluri mutant library, we were able to identify 14 attenuated mutants at the end of in vitro BLMS and in vivo fish screening. Eight mutants were common to neutrophil and serum screening while only four and only two mutants were identified as neutrophil and serum mutants, respectively. The fourteen
identified E. ictaluri mutants were first characterized in terms of their virulence and vaccine potential and later mutated genes in each mutant were determined by transposon insertion sites identification. Finally, selected mutants were compared to a commercial vaccine (AQUAVAC-ESC®) to determine their commercial value. Findings from our research suggest that BLMS is a very powerful screening method for development of live attenuated vaccines. Fourteen mutants identified by utilizing BLMS showed reduced virulence as compared to wild-type E. ictaluri and provided increased protection from ESC compared to non-vaccinated controls. Among the 14 mutants, we observed redundant mutations in two genes. Three different mutants harbored transposon insertion at different locations in gcvP, which encodes glycine cleavage system protein P. Two different mutants harbored transposon insertion at the same location in rseB, which encodes a negative regulator of sigma E activity. This provides confirmation that BLMS procedure is a sensitive method detecting true targets. [0024] BLMS can detect novel virulence relevant genes located on native plasmids or show importance of native plasmids in bacterial virulence if random mutation occurs in the origin of replication of native plasmids. While applying BLMS to E. ictaluri, we observed that at least one of the two native plasmids of E. ictaluri (pEI1) may be important in E. ictaluri virulence because two different locations were targeted on this plasmid in two different mutants (EiAKMut04 and EiAKMut10). [0025] Production of Edwardsiella ictaluri mutant library [0026] A library of random transposon insertion mutations in conditionally bioluminescent E. ictaluri strain 93-146 carrying pAKgfplux2 was generated. The library containing approximately 15,000 mutants was created by using a derivative of the mariner transposon Himar1 carried on pMAR2xT7 plasmid. The library consisted of mutants arrayed
in 39 384-well plates. A duplicate of the whole library was also prepared. The produced mutant library is compatible with genetic footprinting of the mutants with transposon-site hybridization (TraSH) analysis. [0027] Identification of serum and neutrophil susceptible Edwardsiella ictaluri mutants [0028] We used the high throughput bioluminescence mutant screening (BLMS) procedure to identify virulence relevant genes of gram negative bacteria in vitro. We screened 2,256 mutants against both serum and neutrophils using BLMS and identified 180 mutants exhibiting light reduction during incubation with these host factors. A second round screening of these 180 mutants in quadruplicate samples allowed us to identify 35 serum, 39 neutrophil, and 26 both serum and neutrophil susceptible mutants for in vivo studies (100 total mutants for continued study). A general outline of the integrated procedures including in vitro BLMS and in vivo fish screening applied can be seen in FIG. 1. Injection of catfish with 100 BLMS-selected mutants resulted in identification of 14 attenuated mutants including 8 mutants susceptible to both serum and neutrophils, 4 susceptible to neutrophils, and 2 susceptible to serum, which were further characterized in vivo. [0029] Characterization of virulence and vaccine potentials of Edwardsiella ictaluri mutants [0030] In vitro BLMS allowed us to reduce the number of target mutants to an amenable size for in vivo characterization. Fish were infected with the 14 mutants, and their attenuation and vaccine potentials were determined (see TABLE 1). Virulence of EiAKMut07 and EiAKMut09 appeared to be higher than other mutants while E. ictaluri wild-type was the most virulent in immersion immunization. EiAKMut02, EiAKMut07, EiAKMut10, and EiAKMut12 provided more protection than other mutants. Similarly,
EiAKMut07 and EiAKMut09 were also the most virulent strains in the injection immunization though they were attenuated as compared to wild-type strain. Virulence of the second category of mutants in injection immunization ranged from 1.25% to 11.67%, while the third category of mutants including EiAKMut02, EiAKMut03, EiAKMut04, and EiAKMut06 seemed to be non-virulent. After immersion infection, efficacy of EiAKMut02, EiAKMut08, and EiAKMut12 were statistically superior to others. TABLE 1. Summary of in vivo mutant characterization results
[0031] Identification of MAR2xT7 insertions in Edwardsiella ictaluri genome
[0032] MAR2XT7 insertion locations were determined using single primer PCR amplification of transposon ends and nested primer sequencing. We determined the insertion locations of MAR2XT7 and identified disrupted (non-functional) genes from all fourteen mutants (TABLE 2). Gene identification using database searches indicated that glycine cleavage system protein P (gcvP) was disrupted in three of the mutants (EiAKMut02, EiAKMut03, and EiAKMut08) but at different locations within the genetic sequence. Similarly, negative regulator of sigma E activity (rseB) gene was mutated at the same location in EiAKMut01 and EiAKMut07. Interestingly, two genes located on one of the native plasmids of E. ictaluri (pEI1) were also mutated. One of these genes was a putative RNA one modulator protein while the other was a hypothetical protein. TABLE 2. Summary of insertion identification results
N, neutrophil sensitive; S, serum sensitive; NS, neutrophil and serum sensitive; MAR2XT7, mariner transposon; ^, insertion point; TA, two base TA duplication; lowercase letters, 7 bp flanking unique gene sequences of E. ictaluri. [0033] Attenuation and efficacy of Edwardsiella ictaluri mutants and AQUAVAC-ESC [0034] We compared our attenuated E. ictaluri mutants with a commercial live attenuated vaccine to determine whether our mutants provide reduced virulence and improved protection against the wild-type E. ictaluri infections. As can be seen from FIG.2 and FIG. 3, attenuation and efficacy experiments indicated that some of our mutants performed better than AQUAVAC-ESC while others did not. Immersion immunization indicated that AQUAVAC-ESC, EiAKMut02, EiAKMut05, EiAKMut08, and EiAKMut13 were completely attenuated while others showed increased attenuation as compared to wild- type E. ictaluri. Infection of immunized fish indicated that EiAKMut05 provided the best protection with no mortality in the immunized fish. Six other mutants indicated improved protection as compared to AQUAVAC-ESC. EiAKMut13 performed inferior to commercial vaccine in terms of protection, but EiAKMut13 provided slightly greater protection against wild-type infection as compared to the sham vaccinated fish.
[0035] EXAMPLE: LIVE ATTENUATED VACCINE FOR USE IN CATFISH [0036] The following description more particularly discloses the steps used in practicing the BLMS method as applied to the BLMS E. ictaluri live attenuated vaccine. [0037] Bacterial strains, plasmids, and growth conditions. Escherichia coli (E. coli) SM10 λpir was used as the donor strain in conjugations for transfer of pAKgfplux2 and pMAR2XT7 into Edwardsiella ictaluri strain 93-146. E. ictaluri and E. coli DH5α carrying pAKgfplux2 were used as negative and positive controls in neutrophil and serum screening experiments. E. coli strains were grown using Luria-Bertani (LB) broth and agar plates at 37o C and E. ictaluri was grown using brain heart infusion (BHI) broth and agar plates at 30o C. Antibiotics were added to the following final concentrations: ampicillin (100 μg/ml), colistin (12.5 μg/ml), gentamicin (12.5 μg/ml). 2 mM Isopropyl-ß-D-thiogalactopyranoside (IPTG) was used in growth medium and screening assays to induce expression of bacterial luciferase operon (luxCDABE) from the lacZ promoter in pAKgfplux2. E. ictaluri minimal medium was used to eliminate auxotrophic mutants. [0038] Construction of Edwardsiella ictaluri mutant library. MAR2xT7 insertions were generated by introducing pMAR2xT7 from E. coli SM10 λpir into E. ictaluri carrying pAKgfplux2 in conjugal mating as described in Karsi, A. & Lawrence, M.L. Broad host range fluorescence and bioluminescence expression vectors for Gram-negative bacteria. Plasmid 57, 286-295 (2007), herein incorporated by reference. Transposants were selected on 20 X 20 cm LB agar plates containing 12.5 μg/ml colistin and 12.5 μg/ml gentamicin. Putative transposants were picked robotically using a Flexsys Colony Picker (GENOMIC SOLUTIONS, Ann Arbor, MI) into 60 μl of LB broth containing colistin and gentamicin in 384-well microtiter plates and grown overnight in HIGRO shaker (GENOMIC SOLUTIONS)
at 30o C. A duplicate library was prepared by the Flexsys Colony Picker before sterile glycerol was robotically added to the cultures at a final concentration of 20%. Plates were sealed with aluminum foil to prevent cross contamination, lids were taped to prevent freeze drying, and libraries were frozen at -80° C. [0039] Catfish serum and neutrophil preparation. Specific pathogen free (SPF) fish facility at the College of Veterinary Medicine, Mississippi State University maintains SPF channel catfish. For serum preparation, 1-2 kg SPF catfish were anesthetized in water containing 200 mg/l tricaine methanesulfonate (MS-222) and blood was collected at 1% of body weight. A recovery period of one month was given for subsequent blood collections. Serum was obtained and stored at -80° C as single use aliquots. Neutrophils were isolated from the single cell suspensions of anterior kidney cells of SPF catfish (38.63±0.68 cm, 424.20±23.34 g) using discontinuous percoll gradient centrifugation procedure described previously. Purity of neutrophils collected from the 1.060-1.080 interface was determined using flow cytometry. [0040] In vitro mutant screening using catfish serum and neutrophils. 384- well plates containing the frozen mutants were taken out from the -80° C and aluminum cover is removed immediately. Plates were centrifuged briefly and returned to 4° C until the culture thawed completely. Four 96-well plates containing 195 µl of BHI medium with colistin and gentamicin antibiotics and 2 mM IPTG were prepared. Five microliters of mutant bacteria were inoculated in each well and were grown at 30° C by shaking at 250 RPM for 16-18 hours. The next day, 10 µl of mutant culture containing approximately 106 CFU was mixed with 90 µl of catfish serum containing 2 mM IPTG and plates were returned to the imaging chamber of an IVIS Imaging System 100 Series (XENOGEN CORP., Alameda, CA).
[0041] Initial bioluminescence of the serum was detected after five minute pre- incubation of samples in the imaging chamber adjusted to 30o C to eliminate temperature effect on light production. After initial imaging, subsequent images were captured from the same plates at every 15 minute intervals during the 90 minute data collection. Images were analyzed and photons emitted from each well were quantified using Living Image Software v2.50 (XENOGEN CORP.). Percent light change between the first and last measurement times was determined for each mutant and compared to serum resistant (E. ictaluri 93-146 pAKgfplux2) and serum sensitive (DH5α pAKgfplux2) controls included in each plate. [0042] Neutrophil screening was accomplished by setting up phagocytosis assays including freshly isolated neutrophils with 75% or higher purity, 15% SPF catfish serum, 2 mM IPTG, and mutant bacteria. Neutrophil to bacteria numbers were adjusted to give a ratio between 1:40 and 1:80. Bioluminescence imaging was conducted as described above in the serum screening procedure. Percent bioluminescence change in 2,256 mutants was calculated and compared to those of positive and negative controls. [0043] One hundred and eighty mutants with reduced bioluminescence were re- screened against serum and neutrophils in quadruplicate samples and data were analyzed using General Linear Model procedure of SAS v 9.1 (SAS Institute Inc., Cary NC). 100 mutants were selected for in vivo screening studies. [0044] In vivo mutant screening. SPF channel catfish (5.20±0.18 cm) were transferred from the SPF fish facility to 40 L flow-through tanks with dechlorinated municipal water. Fish were maintained in well-aerated tanks with a water temperature of 28o C throughout the experiments. After one weak of acclimation, fish were anesthetized in water containing 100 mg/l MS-222 and each mutant was injected into 15 catfish at a concentration of approximately 1 x 107 CFU in 100 μl phosphate-buffered saline (PBS).
Wild-type and PBS injected fish were also included in the experiment as positive and negative controls. Fish were monitored daily and dead fish were removed from the tanks. Percent mortality rates indicated attenuation state of serum, neutrophil, and both serum and neutrophil mutants. Fourteen mutants with the highest attenuation rates were further characterized. [0045] Determination of virulence and vaccine potentials. Virulence and efficacy of the 14 mutants were characterized by infecting catfish by both intraperitoneal injection and immersion. Each 40 liter flow-through tank contained twenty fish, and four tanks were used for each mutant. Fish were allowed to acclimate for one week. Quadruplicate wild-type and PBS controls were also included in all experiments. Bacteria numbers were adjusted to be equal by determining OD600 readings and adjusting volumes accordingly. In the first study, fish (13.80±0.26 cm, 25.83±1.49 g) were infected by immersion in water containing 1 x 106 CFU /ml for one hour. After 21 days, immunized fish were infected with wild-type E. ictaluri by immersion in water with 1x 107 CFU/ml for one hour. Fish were monitored and dead fish were removed daily. In the second study, fish (14.61±0.33 cm, 32.70±2.36 g) were anesthetized and infected by injecting 1 x 105 CFU in 100 μl PBS. After 21 days, fish were infected by immersion as described above. Virulence and efficacy of each mutants and controls were calculated from the fish mortality rates. [0046] Identification of transposon insertion sites. Transposon insertion sites were identified by using a single primer PCR protocol. Mutants were grown for 18 hours and genomic DNA was prepared using WIZARD Genomic DNA Purification Kit (PROMEGA). In the first round of PCR reaction, the transposon specific template was amplified linearly for 40 cycles. A second round produced specific and non-specific amplicons due to low annealing temperature at 30o C. The final round further amplified the amplicons. The 25 µl PCR reaction contained 0.2 mM dNTPs, 0.2 µM transposon specific primer, 1.5 mM MgCl2,
buffer, and 1.25 units of Taq polymerase (PROMEGA). The five µl single primer PCR reaction was cleaned with 2 µl of EXOSAP-IT enzyme mix (USB CORP.) according to the manufacturer’s instructions. Twenty micoliters of BIGDYE v3.1 sequencing reaction contained 2 µl of EXOSAP-IT enzyme mix treated template and 10 µM nested transposon specific primer. Transposon specific sequences were trimmed and remaining bacterial sequences were searched against nucleotide and protein databases using BLAST program. [0047] Vaccination studies. Virulence and efficacy of mutants were compared to a commercial vaccine (AQUAVAC-ESC). Experiment contained 10 mutants, a mixed group containing Mut02, Mut04, Mut05, and Mut06, a commercial live attenuated vaccine, and wild-type and sham controls. Two of the mutants (Mut02 and Mut08) harbored transposon insertions in the same gene but at different locations and therefore served as an internal control in the experiments. Each 40 liter flow-through tank contained 25 fish and four tanks were assigned to each group. Fish were allowed to acclimate for two weeks before bacterial challenges. Bacteria numbers were adjusted to be equal by determining OD600 readings and adjusting volumes accordingly. For vaccination, fish (11.62±0.16 cm, 15.36±0.65 g) were infected by immersion in water containing 2 x 107 CFU/ml for one hour. After 21 days, immunized fish were infected with wild-type E. ictaluri by immersion in water with 1 x 107 CFU/ml for one hour. Fish were monitored and dead fish were removed daily. Mean percent mortalities for each group were calculated, arcsine-transformed, and analyzed using PROC GLM procedure of SAS 9.1 (SAS Institute Inc., Cary, NC). [0048] Analysis of the Mutants [0049] Succinate-ubiquinone oxidoreductase (SQR) encoded by the sdhCDAB gene cluster and menaquinol-fumarate oxidoreductase (QFR) encoded by the frdABCD gene cluster are part of the tri-carboxylic acid (TCA) cycle and are structurally and functionally
related membrane-bound enzyme complexes. EiAKMut05 has an insertion in the sdhC gene, which encodes one of four subunits of the succinate dehydrogenase complex. SdhC is one of the two subunits that anchor the complex in the cytoplasmic membrane. Succinate dehydrogenase is part of the aerobic respiratory chain and the Krebs cycle, oxidizing succinate to fumarate while reducing ubiquinone to ubiquinol. It is closely related to fumarate reductase, which catalyzes the reverse reaction. Succinate dehydrogenase and fumarate reductase can replace each other at different relative rates and with different apparent substrate affinities. Because of fumarate reductase’s ability to convert succinate to fumarate, sdhCDA mutant of Salmonella enterica serovar Typhimurium were slightly attenuated and complete attenuation was achieved by succinate dehydrogenase/ fumarate reductase double mutation. In E. ictaluri, sdhC is the first gene in a polycistronic operon that encodes the four components of succinate dehydrogenase; therefore, it is possible that the mutation in sdhC has a polar effect on expression of downstream genes. Our results indicate that attenuation of E. ictaluri was achieved with sdhC mutation without a need for generating double mutants in frd genes. An explanation for this could be that fumarate reductase’s ability to convert succinate to fumarate in E. ictaluri is not as efficient as compared to Salmonella and E. coli or E. ictaluri sdhC mutant is cleared from the fish before bacteria can activate fumarate reductase, or an anaerobic condition triggering use of fumarate reductase does not occur during fish infection. Our recent analysis of E. ictaluri proteome showed that many proteins involved in the tri-carboxylic acid (TCA) pathway including the fumarate reductase complex present and TCA pathway significantly represented in E. ictaluri (unpublished data). In E. coli sdhC mutants, SdhC activity is located in the cytoplasm, and it utilizes artificial electron acceptors; in contrast, wild-type E. coli has membrane-associated SdhC activity with ubiquinone as the electron acceptor. In E. coli, fumarate reductase is expressed under anaerobic conditions with glucose as a carbon source. Although SdhC has similar function, hydrophobicity, and protein size to the membrane-binding subunit from
fumarate reductase (FrdC), SdhC and FrdC do not share significant sequence identity. In Helicobacter pylori, fumarate reductase was found to be essential for colonization of mouse gastric mucosa. In E. coli and Salmonella, succinate dehydrogenase is known to contribute to pathogenicity. The organic acids formate and succinate have a protective effect in stationary phase cells against killing effects of antimicrobial peptide BPI, which appears to disrupt the bacterial respiratory chain. Maintenance of protective levels of formate and succinate requires the activity of formate dehydrogenase and succinate dehydrogenase, respectively. E. ictaluri also encodes the formate dehydrogenase complex in its genome. [0050] Mutants 2, 3, and 8 all had insertions in gcvP, which encodes a protein that is part of the glycine cleavage system. The glycine cleavage system is a loosely associated four subunit enzyme complex that catalyzes the reversible oxidation of glycine to form 5, 10- methylenetetrahydrofolate, which serves as a one carbon (“1C”) donor. It is one of two sources of 1C units with serine hydroxymethyltransferase being the other (and is considered the more important source). Expression of the glycine cleavage enzyme system is induced by glycine, and gcv mutants are unable to use glycine as a 1C source and excrete glycine. The glycine cleavage system is also part of the formyltetrahydrofolate biosynthesis system. GcvP is a 104 kDa protein that catalyzes the decarboxylation of glycine. In E. ictaluri, gcvP is the third gene in a three gene operon; it is located downstream of gcvH and gcvT, which encode subunits of the glycine cleavage system. E. ictaluri also has a gene that encodes serine hydroxymethyltransferase. The glycine cleavage system has not been linked with virulence previously, and our disclosed composition and method are the first to employ it. [0051] Mutant 1 had an insertion in rseB, which encodes one of two negative regulators of sigmaE. RseA is considered the major regulator of sigmaE. SigmaE is expressed in response to heat shock and other stresses on membrane and periplasmic proteins, including misfolding of outer membrane proteins, hyperosmotic stress, metal ion exposure,
changes in LPS structure, and starvation signal ppGpp. SigmaE is required for heat-induced transcription of rpoH, which encodes heat shock factor sigma32 and other heat shock proteins. RseB is a periplasmic protein that interacts with RseA. RseB stimulates binding of RseA to sigmaE, thereby assisting RseA in tethering sigmaE to the cytoplasmic membrane. Degradation of RseA releases sigmaE and allows it to interact with the core enzyme of RNA polymerase to initiate transcription. Although mutations in rseA cause increased sigmaE activity, an rseB mutant shows wild-type sigmaE activity under inducing conditions and exhibits a small increase in sigmaE activity under non-inducing conditions. In E. ictaluri, rseB is the third gene in a polycistronic operon. It is downstream of rpoE, which encodes sigmaE, and rseA, and it is upstream of rseC, which encodes a positive regulator of sigmaE. SigmaE is required for Salmonella virulence and mediates Salmonella resistance to oxidative stress and antimicrobial peptides. SigmaE is also required for Salmonella to survive intracellularly. We disclose the first report of RseB being associated with virulence. [0052] Mutant 6 has an insertion in rsxB, which encodes one of six proteins that form a SoxR reducing system in E. coli. SoxR is a regulatory protein that senses superoxide and nitric oxide and induces expression of an oxidative stress response. When SoxR is activated by oxidation of its [2Fe-2S] cluster, it induces expression of SoxS, which is a transcriptional regulator that induces expression of superoxide dismutase and other oxidative response proteins. The SoxR reducing system inactivates SoxR, thereby turning off the oxidative stress response. In E. coli, when any of the six rsx genes are mutated, SoxS is constitutively expressed, leading to induction of oxidative stress response. In Salmonella, SoxS is not essential for virulence, but SoxS was found to contribute to virulence in an E. coli mouse pyelonephritis model. In E. ictaluri, rsxB is the second in the six gene rsx operon. [0053] Mutant 4 has an insertion in a gene encoding a hypothetical protein located on one of the two E. ictaluri constitutive plasmids, pEI1. The protein has >50% identity with
Salmonella effector proteins with leucine rich repeats that are secreted through a type III secretion system. The 618 amino acid protein appears to be in a monocistronic operon. [0054] EXAMPLE: IDENTIFYING MUTANTS THAT FAIL TO ATTACH TO THE HOST EPITHELIUM. [0055] The BLMS method can also be used to identify bacterial mutants that fail to attach to the host molecules, cells, or surfaces. Attachment and colonization of the host epithelium is an indispensable first step to any bacterial infection and can be achieved through a variety of diverse mechanisms. To investigate these attachment mechanisms in Edwardsiella ictaluri, we used random insertion of the pMar2xT7 transposon to generate a library of 1728 mutants. Each mutant expressed bioluminescence constitutively from the plasmid pAKlux1. This library was then screened in a high throughput fashion using an IVIS Living Image System (XENOGEN) in a series of nested in vivo challenges using a skin abrasion model we developed. Twenty mutants that displayed a decrease in their ability to colonize the channel catfish epithelium were identified. Results from this study will delineate mechanisms of E. ictaluri attachment to channel catfish skin and could lead to improved methods for prevention of enteric septicemia of catfish. [0056] Combinations of mutations. Combinations of mutations can be constructed using the pathways we have disclosed. Specifically, in-frame deletions in TCA cycle enzymes and glycine cleavage system protein can be constructed to create greater attenuation while retaining antigenicity. Mutation of the glycine cleavage system as a vaccine strategy is a new strategy that has never been previously reported. EiAKMut2 has a mutation in gcvP. The glycine cleavage system functions in providing 5,10- methylenetetrahydrofolate as a source for 1C moieties. Our plan is to construct a mutant containing deletions in gcvP (our current mutant) and in another enzyme that serves to
provide 5,10-methylenetetrahydrofolate through an alternative pathway. Knocking out both pathways should cause improved attenuation. [0057] Mutation of genes encoding TCA cycle enzymes, exemplified by EiAKMut05 and EiAKMut12, shows great potential as a strategy for an effective live attenuated E. ictaluri vaccine. Knockout of genes encoding TCA cycle enzymes was recently discovered as an effective strategy for vaccine development in Salmonella (which is closely related to Edwardsiella). We have found that knocking out a single TCA cycle gene does not always cause complete attenuation, but knocking out two genes can cause complete attenuation. Specifically, a Salmonella sdhCDA-frdABCD double mutant was fully avirulent and effective as a vaccine, while a Salmonella sdhCDA mutant was not fully attenuated. A combination mutant can be constructed that has deletions in sdhC (the gene mutated in EiAKMut05) and mdh (the gene mutated in EiAKMut12), as well as a second sdhC combination mutant that has a knockout in another enzyme that encodes a related TCA cycle enzyme. [0058] USE OF OTHER BACTERIAL SPECIES AS LIVE ATTENUATED VACCINES FOR VARIOUS HOSTS [0059] The method and compositions disclosed herein are not limited to Edwardsiella ictaluri, but can be used in other bacteria as well. Because the genes discovered in this research project are well conserved in bacteria, the mutation of these genes in other bacterial pathogens can be utilized for development of effective live attenuated vaccines to prevent other diseases. For example, Salmonella enterica is closely related to Edwardsiella ictaluri and is in the same bacterial family (Enterobacteriaceae). The pathogenesis of salmonellosis in mammals is also similar to the pathogenesis of enteric septicemia of catfish caused by E. ictaluri. The mutation of these genes in Salmonella will
result in development of an effective live attenuated vaccine for prevention of salmonellosis in various animal hosts. Similarly, the genus Yersinia is also in the same family as Edwardsiella and Salmonella, and the disease pathogenesis of Yersinia is similar to enteric septicemia of catfish. Therefore, the mutation of these genes will be effective for development of live attenuated vaccines for Yersinia pestis, which causes bubonic plague in humans, Y. enterocolitica and Y. pseudotuberculosis, which cause gastrointestinal disease in humans and other mammals, and Y. ruckeri, which causes enteric redmouth disease in salmonid fish. [0060] The mutation of these genes may be an effective strategy for development of live attenuated vaccines for pathogenic Escherichia coli, Shigella flexneri, and Shigella dysenterieae, which are also closely related to E. ictaluri. The mutation of these genes can also be used for development of live attenuated vaccines against Francisella tularensis, which causes tularemia in humans, because the disease pathogenesis is similar to enteric septicemia of catfish. Other bacterial pathogens that we anticipate mutation of these genes may be effective for development of live attenuated vaccines include Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, Haemophilus influenzae, Haemophilus ducreyi, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Actinobacillus actinomycetemcomitans, Avibacterium paragallinarum, Moraxella catarrhalis, Moraxella bovis, Pseudomonas aeruginosa, Coxiella burnetii, Bordetella bronchiseptica, Bordetella pertussis, Bordetella parapertussis, Bordetella avium, Burkholderia mallei, Burkholderia pseudomallei, Neisseria meningitidis, Neisseria gonorrhoeae, Brucella abortus, Legionella pneumophila, Helicobacteri pylori, and Campylobacter jejuni. Mutation of these genes may also be effective for development of live attenuated vaccines for gram-positive pathogens such as Listeria monocytogenes. In addition, BLMS may be an effective tool for identification of new gene targets for development of live attenuated vaccines.
[0061] Building on the BLMS method studies, we created bacterial mutant strains of the pathogenic bacterial strain of Enterobacteriaceae, E. ictaluri, harboring multiple gene disrupting (non-functional) mutations of the identified gene targets provided in the list of glycine cleavage system (gcvP), serine hydroxymethyltransferase, succinate dehydrogenase, malate dehydrogenase, 2-oxoglutarate dehydrogenase, negative regulator of sigma E activity (rseB), hypothetical protein pEI1_p1, electron transport complex protein RnfB, Fimbrial chaperon protein, Putative RNA one modulator protein pEI1_p4, UDP-glucose 6- dehydrogenase, fumarate reductase (frdA), and other genes encoding enzymes in the tri- carboxylic acid (TCA) cycle. Specifically, the gene coding for glycine cleavage system (gcvP) and three genes related to the TCA cycle, including succinate dehydrogenase (sdhC), malate dehydrogenase (mdh), and fumarate reductase (frdA), were targeted by in frame gene disrupting mutations to test live attenuated vaccines of E. ictaluri in farm-raised channel catfish. We constructed and tested double mutants of EiΔfrdAΔsdhC and EiΔgcvPΔsdhC, but these did not show the level of safety and efficacy as compared with the respective individual deletion mutants. By adding a third gene disrupting (non-functional) mutation, we synergistically increased safety and efficacy in fry and fingerling catfish to a level that was not additive of the individual mutants or the double mutants plus third mutants. This was unexpected, especially given the prior work in S. enterica showing complete attenuation with deletion mutations in succinate dehydrogenase and fumarate reductase. Even more surprising was that the triple mutant ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) strain was superior to the triple mutant ESC-NDKL2 (ΔgcvPΔsdhCΔmdh) strain because our previous work showed that individual gene disrupting mutants in the gcvP and mdh genes showed superior results compared to other single mutants. Also, the individual gene disrupting mutants in the sdhC gene showed no mortality in immunized fish. Thus, it would have been expected to have superior results for the ESC-NDKL2 (ΔgcvPΔsdhCΔmdh) strain, but the complex interplay
of these pathways appears to have resulted in synergistic rather than mere additive effects on safety and efficacy in live attenuated vaccine trials. [0062] The primary role of the TCA cycle is to provide NADH, which is used by bacterial cells for ATP synthesis via the electron transport chain for the complete catabolism of non-preferred carbon sources and the subsequent generation of reducing potential and biosynthetic intermediates. Also, several enzymes of TCA cycle require iron, for example, aconitase, succinate dehydrogenase complex, and fumarase. Thus, during growth under conditions of low iron availability, the TCA cycle activity was dramatically reduced (Somerville et al., 1999; Varghese et al., 2003). In a previous study, culturing Staphylococcus epidermidis with an increasing TCA cycle stimulator fluorocitrate dramatically decreased polysaccharide intercellular adhesion (PIA) synthesis and biofilm production without impairing glucose catabolism, the growth rate, or the growth yields (Zhu et al., 2009), which lead to speculation that S. epidermidis perceives environmental changes through alterations in TCA cycle activity, leading to changes in the intracellular levels of biosynthetic intermediates, ATP, or the redox status of the cell (Vuong et al., 2005). These changes in the metabolic status of the bacteria result in attenuation. Recent studies using serovar Typhimurium (Salmonella enterica) described that mutant stains with a deletion of genes encoding TCA cycle enzymes Δmdh, ΔsucCD, and ΔsdhCDAB replicated to higher levels than the wild-type in resting and activated macrophages, which suggests an enhanced ability to survive under antimicrobial conditions (Bowden et al., 2010). S. enterica ΔfrdABCDΔsdhCDA double mutants with complete TCA cycles may exhibit to be effective live vaccine strains for animal and human like ΔfrdABCDΔsdhCDA double mutants of other intracellular bacterial pathogens (Mercado-Lubo et al., 2008). In Helicobacter pylori, fumarate reductase was found to be essential for colonization of mouse gastric mucosa (Ge et
al., 2000). These data suggest that the conversion of succinate to fumarate plays a key role in bacterial virulence. [0063] Previously, we conducted immersion trials in catfish fingerlings by using EiΔsdhC, EiΔfrdA, EiΔfrdAΔsdhC, and EiΔgcvPΔsdhC mutants as part of or continuing research of the BLMS method findings, which provided significant protection against wild- type E. ictaluri (Dahal et al., 2014; Dahal et al., 2013). These bacterial mutants retained the ability to penetrate catfish mucosa and persist and colonize posterior kidney similar to wild- type E. ictaluri, suggesting that attenuation is not due to mutants’ inability to invade the host. In our previous report, similar results were observed with transposon insertion mutants (Karsi et al., 2009). However, our previous study showed that catfish fry (<15 days post hatch) were more sensitive to E. ictaluri than fingerlings, due to immaturity of the acquired immune organs as lymphoid populations in the anterior renal haematopoietic tissue, and the first appearance of splenic red and white pulp compartmentalisation in fry (Patrie-Hanson and Jerald Ainsworth, 1999). Of the combination mutants, only EiΔgcvPΔsdhC showed safety in catfish fry over 30% mortality and provided protection against E. ictaluri infection post- immunization. This was unexpected; therefore, the current work aimed to improve the safety and efficacy of EiΔgcvPΔsdhC by constructing a combination of triple mutants associated with TCA cycle and C1 metabolism protein derivated from EiΔgcvPΔsdhC against wild-type E. ictaluri infection in catfish fry and fingerling. [0064] The vaccine trials conducted under laboratory conditions indicated that immersion immunization of channel catfish fry and fingerling with ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) provided significant safety and protection. Fumarate reductase (frd) and succinate dehydrogenase (sdh) are physiologically reversible isoenzymes in the TCA cycle that are induced under anaerobic and aerobic respiratory chain, which can replace each other with functionally related membrane-bound enzyme complexes (Guest, 1981;
Maklashina et al., 1998; supra). Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron-sulfur subunit, which contains three distinct iron-sulfur clusters, and the catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site for interaction with quinones (Cecchini et al., 2002). Glycine cleavage system (gcv) serves as a one carbon donor (C1 unit); serine hydroxymethyltransferase is another one carbon donor source (supra). Expression of the glycine cleave enzyme system is induced by high concentrations of glycine, and a gcv mutant was unable to use glycine as a C1 source and excrete glycine (Meedel and Pizer, 1974; Plamann et al., 1983; Stauffer et al., 1994), and we have shown that E. ictaluri gcvP is required for virulence (Karsi et al., 2009). [0065] The laboratory challenge was followed by a field trial mimicking commercial catfish production to evaluate the efficacy of the ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) in earthen pond conditions, the best vaccination regime (immersion, oral, and immersion-oral combination), and also, compare the efficacy of the ESC-NDKL1 to the commercially available vaccine, AQUAVAC-ESC. Under field conditions, the fry were vaccinated with an ESC-NDKL1 by immersion (19 days post-hatch) in the early summer, and the oral booster was included in late summer (80 days post-hatch). This vaccination schedule time was chosen to provide protection during the high incidence ESC season that occurs in the late spring and early fall when water temperatures are in the 18-28° C range, the optimal temperature range for ESC development (Plumb, 1988; Thune et al., 1994). [0066] During the 95 days of the pond study, there was no difference between the five earthen ponds considering the water quality parameters. Overall means of DO and temperature are within what is expected for catfish fingerling production under commercial conditions (Tucker, 1990). The results from field study depend on the survival of fingerlings
in each pond at the end of the study. The probability of survival depends on the initial stocking number (300 fish/pen) and remaining number at the harvest. The daily dead fish cannot be gathered in this study. Other production parameters such as the mean of total weight, individual fish weight, and individual fish length were also taken into consideration as an indication of vaccination benefits. [0067] The field study indicated the ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) vaccination improved survival in the pond by approximately 5.96, 8.76, and 26.67 times the oral, immersion, and immersion-oral combination, respectively. The three ESC-NDKL1 vaccinated ponds were significantly protected compared to sham-vaccinated, and the AQUAVAC-ESC vaccinated ponds. Further supporting the survival results, the mean total weight for fish ponds vaccinated with the ESC-NDKL1 strain (immersion, oral, and immersion-oral) were higher than the sham-vaccinated, and the AQUAVAC-ESC vaccinated fish ponds, which indicated that vaccinated populations grew better than the non-vaccinated fish and commercially available vaccine. [0068] Comparing the survival and total weight of the three ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) vaccination strategies showed that better survival of immersion-oral combination (95.22% survival) vaccination over oral vaccination (81.67% survival) and immersion vaccination (86.74% survival). Although statistical differences were not observed, especially when comparing small numbers of fish, economic benefits may exist. This observation is consistent with the study using an E. ictaluri killed bacteria vaccine indicated that immersion vaccination followed by an oral booster administered through the feed resulted in lower mortality and higher agglutinating antibody titers compared with an immersion-only or non-vaccinated fish (Plumb, 1993).
[0069] The catfish pond vaccinated by ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) through immersion showed better survival than an oral vaccinated pond. This could be due to the vaccination by immersion ensures that all fish are exposed to the vaccine, but feeding vaccination does not ensure that all fish receive the vaccine strain. Indeed, it is important to note that the immersion vaccination with ESC-NDKL1 strain provided significant protection against ESC even after four months from exposure under field trials, this is a great advantage over the currently available vaccine. [0070] In this study, the mean individual fish weight of thirty fish was observed to be higher in the three ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) vaccinated ponds (immersion, oral, immersion-oral) and AQUAVAC-ESC than the sham-vaccinated group. Similarly, the individual fish lengths obtained from the ESC-NDKL1 vaccinated ponds were higher than a sham-vaccinated pond, although statistically analysis showed no significant differences. [0071] A critical factor not evaluated in this study is the claim that the delay in fry stocking and use of a primary nursery phase can reduce early fry mortality and reduce unaccounted fish losses (Morrison et al., 1995). A typical production cycle for channel catfish industry involves the stocking of 7 to 10 days old fry directly into earthen ponds and growing them for 5 to 10 months. However, this method has resulted in high levels of mortality during the first 30 days (Carpenter, 2001). In the present study, we stocked the fry in the earthen pond at 50 days post-hatch. Using oral-immersion as an example, 4.39% of fry that died from the stocking until the time of harvesting from the ponds. This shows that the use of the nursery phase results in less fry mortality during the first 30 days than stocking fry directly into ponds (Morrison et al., 1995). [0072] AQUAVAC-ESC was included in this study for comparison. The field study is, however, somewhat difficult to directly compare with other similar studies because
there are different aspects that vary from one study to another such as stocking density, the length of growing period, quality of feed utilized, management techniques, and water parameters. Past studies have shown that AQUAVAC-ESC (RE-33) provides protection in fish against virulent E. ictaluri isolates when vaccinated 7 to 72 days post-hatch based on laboratory findings and limited field studies (Shoemaker et al., 1999; Wise et al., 2000). For example, a laboratory study reported that 12-day old fry vaccinated by AQUAVAC-ESC via immersion with a dosage between 5 x 105 and 1 x 106 CFU/mL resulted in lower mortality (33.3 %) than that of a non-vaccinated group (78.7 %) (Shoemaker et al., 1999). In another study, 72-day old fry vaccinated by AQUAVAC-ESC at a dose of 1 x 106 reported a lower percent mortality (58.5%) for the vaccinated group as compared to the non-vaccinated group (77.5 %) in the laboratory. In a second part of this same study under field conditions, 21-day old, vaccinated fry resulted in no protection, as shown by similar percent mortalities after exposure to an E. ictaluri epizootic occurring in a commercial catfish pond (Wise et al., 2000). [0073] In conclusion, our laboratory and field studies showed that ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) strain with mutations in combined TCA cycle enzymes and C1 metabolism protein was significantly attenuated and provided vaccine efficacy against ESC. The field study also showed that immersion vaccination followed by oral booster increased survival of catfish when they were exposed to the pathogenic bacterium. ESC-NDKL1 is a strong candidate for use as a live attenuated vaccine for catfish fry and fingerling in commercial hatcheries and fish farms. [0074] EXAMPLE: LIVE ATTENUATED TRIPLE MUTANT VACCINE FOR USE IN CATFISH
[0075] The following description more particularly discloses the steps used in practicing the claimed triple mutant E. ictaluri live attenuated vaccines. [0076] Materials and Methods [0077] Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this study are listed in TABLE 3. E. ictaluri was grown at 30o C using brain heart infusion (BHI) broth and agar (Difco, Sparks, MD). Escherichia coli were grown at 37o C using Luria-Bertani (LB) broth and agar (Difco). E. coli CC118 λpir and SM10 λpir/S17-1 λpir were used for cloning mutated fragments into the pMEG-375 plasmid and conjugal transfer of pMEG-375 suicide plasmid into E. ictaluri. Ampicillin was used at 100 μg/ml to maintain pMEG-375, and colistin was used at 12.5 μg/ml for counter selection against E. coli SM10 λpir following conjugation. TABLE 3. Bacterial strains and plasmids.
[0078] Construction of in-frame deletion mutants. Overlap extension PCR (Horton et al., 1990) was used to generate in-frame deletions of E. ictaluri genes. EiΔgcvPΔsdhC mutant and plasmids with ΔfrdA and Δmdh fragments (pEiΔfrdA and pEiΔmdh) were reported previously (Dahal et al., 2013; incorporated herein by reference for all that it teaches that is not contradictory to the present disclosure). pEiΔfrdA and pEiΔmdh were mobilized into double mutant EiΔgcvPΔsdhC by conjugation (for methods, see Karsi and Lawrence, 2007; incorporated herein by reference for all that it teaches that is not contradictory to the present disclosure). The recipient bacteria were spread on BHI agar containing colistin and ampicillin for selecting E. ictaluri with integrated vector, and positive colonies were followed by streaking on BHI agar with 5% sucrose and 0.35% mannitol for
allelic exchange and loss of pMEG-375 with the sacB gene. Deleted regions were amplified from the resulting ampicillin sensitive colonies and confirmed by sequencing. The two triple mutants evaluated in this study were named as ESC-NDKL1 (EiΔgcvPΔsdhCΔfrdA) and ESC-NDKL2 (EiΔgcvPΔsdhCΔmdh). [0079] Safety and efficacy of the ESC-NDKL1 (EiΔgcvPΔsdhCΔfrdA) and ESC- NDKL2 (EiΔgcvPΔsdhCΔmdh) in catfish in laboratory trials. Vaccine safety in specific pathogen free (SPF) catfish fry (3.17 ± 0.05 cm, 335.92 ± 20.02 mg) and fingerlings (7.75 ± 0.08 cm, 4,500 ± 14.07 mg) was determined for the two triple mutants and commercial live attenuated vaccine (Klesius and Shoemaker, 1999). One treatment group was used as a sham control. 14-day old catfish fry were stocked into 20 tanks at a rate of 40 fish/tank, and three- month-old catfish fingerlings were stocked into 20 tanks at a rate of 25 fish/tank. Fry experiment included four replicates and fingerling experiment included three replicates per group. Experiments were conducted in 40-L tanks supplied with flow-through water and fed two times per day. Water temperature was adjusted to 25° C throughout the experiments. Vaccination doses were 6.0 x106 CFU/ml water and 4.5 x107 CFU/ml water for catfish fry and fingering, respectively. Mortalities were recorded daily for 21 days, and swab samples from the dead fish were plated on BHI agar for confirmation of the causative pathogen. At 21 days post-vaccination, vaccinated and sham control groups were immersion exposed to Wt E. ictaluri 93-146 containing 3.8 x107 CFU/ml water, and fish mortalities were monitored daily for 21 days. Relative percent survival (RPS) was calculated according to the following formula: RPS = [1-(% mortality of vaccinated fish/% mortality of non-vaccinated fish)] x 100. [0080] Determine efficacy of the ESC-NDKL1 (EiΔgcvPΔsdhCΔfrdA) in catfish fry under commercial pond culture conditions. Pond preparation: five identical earthen ponds (0.12 acre each, with an average depth of 1.5 m) located at the South Farm
Aquaculture Research Facility at Mississippi State University were used to grow catfish fry into fingerlings. The designated ponds (A13, B3, B11, B12, and B13) were drained and dried four weeks before stocking. The ponds were supplied with groundwater. Three weeks before stocking the ponds were fertilized with Perfect Pond Plus Fertilizer (Alabama, USA) and dissolved oxygen was measured daily before stocking. Supplemental aeration was provided to each pond by Air-O-Lator 24 h and seven days week. We placed four square pens (4 x 4 x 4 feet) in each pond representing four replicates for each treatment for statistical comparisons. The four pens in each pond were located in a square pattern around the Air-O- Lator to enhance the aeration in the pens and remove dissolved oxygen as a factor in mortality counts. The pens were covered with a lid to prevent birds and other animals from preying on the fish. [0081] Water quality management: throughout the experiment, the ponds were managed according to industry practices. Dissolved oxygen (DO) and temperature were monitored twice daily in the morning and afternoon using a with a portable dissolved oxygen meter (YSI model 550A, YSI Inc., San Diego, California) on the pond bank. Water was added to the ponds periodically to replace that which was lost through evaporation and seepage. [0082] Stocking of the fry and vaccination schedule: on June 25, 2015, approximately 6,000 17 day-old specific pathogen free (SPF) catfish fry were stocked into five tanks (1200/tank) supplied with flow-through dechlorinated water. Water temperature was maintained at 25-26° C throughout indoor conditions. The five tanks corresponded to five treatment groups (immersion, oral, immersion-oral combination, commercial vaccine, and sham-vaccinated). On June 27, 2015, catfish fry (19 days post-hatch) in three treatment groups (immersion, immersion-oral, and commercial) were immersion vaccinated indoors (3.93 x107 CFU/ml of water for 1 h). Fry in the sham-vaccinated group were exposed to an
equivalent volume of brain heart infusion (BHI) broth indoors. The tanks were observed daily for mortalities. Fry in all treatments were moved to the ponds on July 27, 2015. The fry were transferred in aerated containers and stocked into ponds at a rate of 1200 fry/pond (300 fry/pen). The pens were covered with a lid to prevent birds and other animals from preying on the fish. [0083] Feeding: fish were fed twice a day by hand, once in the morning and afternoon, with a commercial catfish feed. Changing to a larger feed pellet was determined according to the behavior and size development of the fry in each pond. Fish were observed after feeding, and the activity of feeding was documented. [0084] Vaccine preparation and oral vaccination: to prepare oral vaccination, an overnight culture of ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) containing 3.52x109 CFU/ml was mixed with commercial feed pellets at a rate of 20% (weight to volume). The vaccine-feed was mixed by a hand mixer until all liquid was absorbed. The average amount of feed consumed one week before vaccination was used to estimate the amount of feed to use on vaccination days. On August 21, 2015, oral vaccination was conducted for two groups (oral, and immersion-oral) by feeding vaccine-feed daily for five days (average feed 500 g/pond for five days), followed by five days feeding with no vaccine, and followed by five days feeding with vaccine. The other ponds were fed similarly but without adding the vaccine to feed. [0085] ESC Challenge: on September 26, 2015 (three months after immersion vaccination, and 35 days following the initial oral-vaccination), when water temperatures were conducive for E. ictaluri infection (22-24°C), fish were challenged with
E. ictaluri strain 93-146 in the feed. Overnight culture of wild-type E. ictaluri containing 2.71x109 CFU/ml was mixed with commercial feed at a rate of 20% (weight to volume), and each pond was fed for five consecutive days (average feed 600 g/pond for five days) followed
by a five-day break, then another five days of exposure. Following vaccination, fish were fed regular feed without adding the vaccine to feed for 21 days. [0086] Harvesting the ponds and measuring procedures: the study was terminated on November 1, 2015 (35 days after wild-type infection) when the water temperature was less than l8°C. Fingerling fish were collected after three months of growing in net pens in earthen ponds. At the end of the trial, fish were harvested and euthanized in water containing 300 mg/L MS-222, and fish numbers and body measurements were collected. Thirty individual fish, representing 10% of the initial stocking population, from each pen, were selected randomly to determine the average individual weight and length. Body weight was determined to the nearest 0.1 g, and length was measured to the nearest 1 mm. The mortality rate for each pen was determined based on initial stocking numbers and numbers of remaining fish in each pen at the end of the study. [0087] Statistical analysis. In the laboratory experiment, the effect of treatment on time to death was analyzed using survival analysis. Separate models were developed for fry safety, fry efficacy, fingerling safety, and fingerling efficacy. The Kaplan-Meier estimator was used to estimate the survivor functions using PROC LIFETEST, SAS for Windows 9.4 (SAS Institute, Inc., Cary, NC, USA). The data for all outcomes was right censored at 21 days post-vaccination or post-challenge. The log-rank test and Wilcoxon test statistics were used to assess the effect of treatment. When treatment was found to have a significant effect, Dunnett’s adjustment for multiple comparisons was used to compare each of the treatments to wild-type. The survivor functions were graphically displayed as Kaplan- Meier plots using PROC SGPLOT. An alpha level of 0.05 was used to determine statistical significance for all analyses.
[0088] In the field study, the effect of the different treatments on the survival of fish was assessed with mixed model logistic regression using PROC GLIMMIX in SAS for Windows 9.4 (SAS Institute, Inc., Cary, NC, USA). The number of live fish in a replication at the end of the trial was the outcome assessed using an events/trials syntax. Treatment was the fixed effect evaluated in the model. Replication within a treatment group was included as a random effect in the model. The BHI (sham) and AQUAVAC-ESC treatment groups were the referents for comparisons of the effect of the other treatments using an LSMestimate statement. The results of the analysis were presented as odds ratios for survival and probability of survival. [0089] The effect of the different treatments on the total weight of fish within a replication at the end of the trial was assessed by analysis of variance using PROC GLIMMIX in SAS for Windows 9.4. The results of the analysis were presented as least squares means and their standard errors. The BHI (sham) and AQUAVAC-ESC treatment groups were the referents for comparisons of the effect of the other treatments using an LSMestimate statement, adjusting the p-values for multiple comparisons with the simulate option. [0090] The effects of the different treatments on the weight and length of 30 fish within a replication at the end of the trial were assessed in separate mixed model analyzes using PROC GLIMMIX in SAS for Windows 9.4. Treatment was the fixed effect assessed in each model while replication within a treatment group was included as a random effect. The results of the analysis were presented as least squares means and their standard errors. The BHI (sham) and AQUAVAC-ESC treatment groups were the referents for comparisons of the effect of the other treatments using an LSMestimate statement, adjusting the p-values for multiple comparisons with the simulate option.
[0091] The distribution of the conditional residuals was evaluated for each model to determine the appropriateness of the statistical model for the data. A significance level of 0.05 was used for all analyses. [0092] Construction of the E. ictaluri ESC-NDKL1 and ESC-NDKL2 strains. Two triple mutants ESC-NDKL1 (EiΔgcvPΔsdhCΔfrdA) and ESC-NDKL2 (EiΔgcvPΔsdhCΔmdh) were constructed successfully (see FIG. 4). A large majority of the frdA (95.44%) and mdh genes (89.74%) were confirmed to be deleted in-frame (see TABLE 4). TABLE 4. Properties of selected E. ictaluri TCA cycle and C1 metabolism genes and percentage of gene deleted.
[0093] Safety and efficacy of ESC-NDKL1 and ESC-NDKL2 in catfish. The ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) and AQUAVAC-ESC showed no mortality in catfish fry vaccination while very low mortalities in ESC-NDKL2 (ΔgcvPΔsdhCΔmdh) (0.6%) and sham control (0.5%) were observed. These results indicated that ESC-NDKL1 and ESC- NDKL2 were safe in catfish fry (see FIG. 5A). To determine vaccine efficacy of ESC- NDKL1 and ESC-NDKL2, fry were challenged with wild-type E. ictaluri by immersion exposure three weeks after vaccination. Fry were protected significantly (p < 0.005) by ESC- NDKL1 vaccination with 6.08 % mortality while the mortality in the sham vaccinated group was 94.44%. The ESC-NDKL2 and AQUAVAC-ESC mutants showed lower but similar protection rates at 72.60% and 79.96%, respectively (FIG.5B). [0094] Fingerlings also showed similar results: ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) vaccination showed no mortality, while ESC-NDKL2 (ΔgcvPΔsdhCΔmdh), AQUAVAC-ESC, and sham vaccination showed small percent mortalities (1.45%, 1.33%, and 1.67%, respectively). The mortality rate in wild-type challenged fingerlings was over 81% (see FIG. 6A). The vaccine efficacy of the mutants was determined by challenging vaccinated fingerling with wild-type E. ictaluri by immersion three weeks after vaccination. ESC-NDKL1 mutant protected fingerlings significantly (p < 0.01) compared to ESC-NDKL2 and AQUAVAC-ESC. The ESC-NDKL1 vaccinated group showed 18.95% mortality while the ESC-NDKL2 and AQUAVAC-ESC showed 79.47% and 54.49% mortalities, respectively (FIG.6B). [0095] The efficacy ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) vaccine in catfish in earthen ponds. When the study was terminated and ponds harvested, an average of 254 fish/pen (86.74% probability of survival) remained in the ESC-NDKL1 immersion vaccinated pond, 242 fish/pen (81.67% probability of survival) remained in the ESC-NDKL1 oral vaccinated pond, and 285 fish/pen (95.22% probability of survival) remained in the ESC-
NDKL1 immersion-oral combination vaccinated pond. This was significantly higher from both the average of 135 fish/pen (42.75% probability of survival) remained in the sham- vaccinated pond (p < 0.05) and 184 fish/pen (61.51% probability of survival) remained in the AQUAVAC-ESC vaccinated pond (p < 0.05). Conversely, there was no significant difference in the probability of survival between the AQUAVAC-ESC vaccinated pond and sham-vaccinated pond (p <0.1092) (see FIGS.7A & 7B). [0096] The mean total weight for each pen for fish vaccinated with the ESC- NDKL1 (ΔgcvPΔsdhCΔfrdA) by immersion (2791.25 g), oral (2511.75 g), and immersion- oral combination (2931.6 g) were significantly higher (p < 0.0002, 0.0016, and 0.0002, respectively) than sham-vaccinated pond (1186.5 g). While no significant difference was found in the total weight observed between three ESC-NDKL1 vaccinated ponds (oral, immersion, and immersion-oral combination) compared with each other, there was a significant difference between the ESC-NDKL1 vaccinated ponds (oral, immersion, and immersion-oral combination) ponds and the AQUAVAC-ESC vaccinated pond (1861.75 g) (p < 0.2559, 0.0414, and 0.0267, respectively) (see FIG.8). [0097] The mean individual fish weights for 30 fish were 10.73, 11.92, 9.50, 10.06, and 7.48 g for immersion, oral, immersion-oral, AQUAVAC-ESC, and sham- vaccinated groups, respectively (see FIG. 9). Significantly higher individual fish weights were observed in the fish vaccinated with ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) by oral and immersion methods than the sham-vaccinated pond (p<0.0042 and 0.0513, respectively). Whereas, no significant differences were noted between ESC-NDKL1 immersion-oral vaccinated fish and AQUAVAC-ESC compared with sham-vaccinated pond. [0098] Mean individual fish lengths for 30 fish were 10.41, 10.84, 10.18, 10.91, and 9.45 cm for immersion, oral, immersion-oral, AQUAVAC-ESC, and sham-vaccinated
groups, respectively. The differences in individual fish lengths were not significant between the vaccinated fish (ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) and AQUAVAC-ESC), and sham- vaccinated fish (see FIG.10). [0099] The terms "comprising," "including," and "having," as used in the claims and specification herein, shall be considered as indicating an open group that may include other elements not specified. The terms "a," "an," and the singular forms of words shall be taken to include the plural form of the same words, such that the terms mean that one or more of something is provided. The term "one" or "single" may be used to indicate that one and only one of something is intended. Similarly, other specific integer values, such as "two," may be used when a specific number of things is intended. The terms "preferably," "preferred," "prefer," "optionally," "may," and similar terms are used to indicate that an item, condition or step being referred to is an optional (not required) feature of the invention. [00100] The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention. It will be apparent to one of ordinary skill in the art that methods, devices, device elements, materials, procedures and techniques other than those specifically described herein can be applied to the practice of the invention as broadly disclosed herein without resort to undue experimentation. All art-known functional equivalents of methods, devices, device elements, materials, procedures and techniques described herein are intended to be encompassed by this invention. Whenever a range is disclosed, all subranges and individual values are intended to be encompassed. This invention is not to be limited by the embodiments disclosed, including any shown in the drawings or exemplified in the specification, which are given by way of example and not of limitation.
[00101] While the invention has been described with respect to a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the invention as disclosed herein. Accordingly, the scope of the invention should be limited only by the attached claims. [00102] All references throughout this application, for example patent documents including issued or granted patents or equivalents, patent application publications, and non- patent literature documents or other source material, are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference (as some are done so), to the extent each reference is at least partially not inconsistent with the disclosure in the present application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).
Claims
1. A composition for providing immunological protection from an enteric septicemia caused by Edwardsiella ictaluri, said composition comprises a live attenuated strain of Edwardsiella ictaluri comprising gene disrupting mutations in genes coding for three proteins in the group consisting of glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), malate dehydrogenase (mdh), and fumarate reductase (frdA).
2. The composition of claim 1, wherein the three proteins having the gene disrupting mutations are glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), and fumarate reductase (frdA).
3. The composition of claim 1, wherein the three proteins having the gene disrupting mutations are glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), and malate dehydrogenase (mdh).
4. The composition of claim 1, wherein the gene disrupting mutations are in frame gene disrupting mutations.
5. A mutant bacterial strain of Edwardsiella ictaluri comprising gene disrupting mutations in genes coding for three proteins in the group consisting of glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), malate dehydrogenase (mdh), and fumarate reductase (frdA).
6. The mutant bacterial strain of claim 5, wherein the three proteins having the gene disrupting mutations are glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), and malate dehydrogenase (mdh).
7. The mutant bacterial strain of claim 5, wherein the three proteins having the gene disrupting mutations are glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), and fumarate reductase (frdA).
8. The mutant bacterial strain of claim 5, wherein the gene disrupting mutations are in frame gene disrupting mutations.
9. A composition for providing immunological protection from an enteric disease caused by a pathogenic bacterial strain of Enterobacteriaceae comprising a live attenuated
strain of the pathogenic bacterial strain of Enterobacteriaceae comprising gene disrupting mutations in genes coding for three proteins in the group consisting of glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), malate dehydrogenase (mdh), and fumarate reductase (frdA).
10. The composition of claim 9, wherein the three proteins having the gene disrupting mutations are glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), and malate dehydrogenase (mdh).
11. The composition of claim 9, wherein the three proteins having the gene disrupting mutations are glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), and fumarate reductase (frdA).
12. The composition of claim 9, wherein the gene disrupting mutations are in frame gene disrupting mutations.
13. A method of providing immunological protection to an animal from an enteric disease caused by a pathogenic bacterial strain of Enterobacteriaceae in the animal comprising providing to the animal an effective amount of a live attenuated strain of the pathogenic bacterial strain of Enterobacteriaceae comprising gene disrupting mutations in genes coding for three proteins in the group consisting of glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), malate dehydrogenase (mdh), and fumarate reductase (frdA).
14. The method of claim 13, wherein the three proteins having the gene disrupting mutations are glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), and malate dehydrogenase (mdh).
15. The method of claim 13, wherein the three proteins having the gene disrupting mutations are glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), and fumarate reductase (frdA).
16. The method of claim 13, wherein the gene disrupting mutations are in frame gene disrupting mutations.
17. A method of providing immunological protection to an animal from an enteric disease caused by a pathogenic bacterial strain of Edwardsiella ictaluri in the animal
comprising providing to the animal an effective amount of a live attenuated strain of the pathogenic bacterial strain of Edwardsiella ictaluri comprising gene disrupting mutations in genes coding for three proteins in the group consisting of glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), malate dehydrogenase (mdh), and fumarate reductase (frdA).
18. The method of claim 17, wherein the three proteins having the gene disrupting mutations are glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), and malate dehydrogenase (mdh).
19. The method of claim 17, wherein the three proteins having the gene disrupting mutations are glycine cleavage system (gcvP), succinate dehydrogenase (sdhC), and fumarate reductase (frdA).
20. The method of claim 17, wherein the gene disrupting mutations are in frame gene disrupting mutations.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15/171,367 | 2016-06-02 | ||
US15/171,367 US9700611B2 (en) | 2008-06-23 | 2016-06-02 | Live attenuated catfish vaccine and method of making |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2017209917A1 true WO2017209917A1 (en) | 2017-12-07 |
Family
ID=60477782
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2017/032345 WO2017209917A1 (en) | 2016-06-02 | 2017-05-12 | Live attenuated catfish vaccine and method of making |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2017209917A1 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9375467B2 (en) * | 2008-06-23 | 2016-06-28 | Mississippi State University | Live attenuated catfish vaccine and method of making |
-
2017
- 2017-05-12 WO PCT/US2017/032345 patent/WO2017209917A1/en active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9375467B2 (en) * | 2008-06-23 | 2016-06-28 | Mississippi State University | Live attenuated catfish vaccine and method of making |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Vetter et al. | Biofilm formation is not required for early-phase transmission of Yersinia pestis | |
Le Roux et al. | Genome sequence of Vibrio splendidus: an abundant planctonic marine species with a large genotypic diversity | |
HU219535B (en) | Vaccines comprising attenuated bacteria expressing recombinant proteins | |
US9492521B2 (en) | Vaccines for control of epidemic Aeromonas hydrophila generated by markerless gene deletion | |
Wang et al. | Construction of a Streptococcus iniae sortase A mutant and evaluation of its potential as an attenuated modified live vaccine in Nile tilapia (Oreochromis niloticus) | |
Choi et al. | Generation of two auxotrophic genes knock-out Edwardsiella tarda and assessment of its potential as a combined vaccine in olive flounder (Paralichthys olivaceus) | |
Han et al. | Deletion of luxS further attenuates the virulence of the avian pathogenic Escherichia coli aroA mutant | |
Nho et al. | Improving safety of a live attenuated Edwardsiella ictaluri vaccine against enteric septicemia of catfish and evaluation of efficacy | |
Triet et al. | Development and potential use of an Edwardsiella ictaluri wzz mutant as a live attenuated vaccine against enteric septicemia in Pangasius hypophthalmus (Tra catfish) | |
Edrees et al. | Construction and evaluation of type III secretion system mutants of the catfish pathogen Edwardsiella piscicida | |
US9375467B2 (en) | Live attenuated catfish vaccine and method of making | |
CN102140430A (en) | Mouse-typhus salmonella gene-deletion mutant strain without containing resistance marks, vaccine and application thereof | |
Valle et al. | Construction and characterization of a nonproliferative El Tor cholera vaccine candidate derived from strain 638 | |
US10232027B2 (en) | Live attenuated Edwardsiella ictaluri vaccine and method of using the same | |
US9700611B2 (en) | Live attenuated catfish vaccine and method of making | |
Mo et al. | Phenotypic characterization, virulence, and immunogenicity of Edwardsiella tarda LSE40 aroA mutant | |
WO2017209917A1 (en) | Live attenuated catfish vaccine and method of making | |
Holden et al. | Avian pathogenic Escherichia coli ΔtonB mutants are safe and protective live-attenuated vaccine candidates | |
Shah et al. | Effect of metC mutation on Salmonella Gallinarum virulence and invasiveness in 1-day-old White Leghorn chickens | |
US11219678B2 (en) | Live attenuated Edwardsiella ictaluri vaccine and method of using the same | |
Edrees et al. | An Edwardsiella piscicida esaS mutant reveals contribution to virulence and vaccine potential | |
CN106661544B (en) | Vaccine for livestock production system | |
WO2010084350A1 (en) | Mutant pathogenic bacterial and live attenuated vaccine compositions | |
WO2013185337A1 (en) | Edwardsiella tarda mutant strain and application thereof | |
Porras | Intraspecific Variability of Edwardsiella piscicida and Cross-protective Efficacy of a Live-attenuated Edwardsiella ictaluri Vaccine in Channel and Channel× Blue Hybrid Catfish |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 17807196 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 17807196 Country of ref document: EP Kind code of ref document: A1 |