Mississippi State University

Mark L Lawrence

Attila Karsi

Hossam Abdelhamed

Frank Austin

Daniel B Paulsen

Daniel W Scruggs

Dongyou Liu

A Jerald Ainsworth

is a reemerging pathogen of channel catfish (); recent outbreaks from 2009 to 2014 have caused the loss of more than 12 million pounds of market size catfish in Alabama and Mississippi. Genome sequencing revealed a clonal group ofisolates with unique genetic and phenotypic features that is highly pathogenic in channel catfish. Comparison of the genome sequence of a representative catfish isolate (ML09-119) from this virulent clonal group with lower virulenceisolates revealed four fimbrial proteins unique to strain ML09-119. In this work, we expressed and purified fourfimbrial proteins (FimA, Fim, MrfG, and FimOM) and assessed their ability to protect and stimulate protective immunity in channel catfish fingerlings againstML09-119 infection for vaccine development. Our results showed catfish immunized with FimA, Fim, FimMrfG, and FimOM exhibited 59.83%, 95.41%, 85.72%, and 75.01% relative percent survival, respectively, after challenge withstrain ML09-119. Bacterial concentrations in liver, spleen, and anterior kidney were significantly (p&#x3c;0.05) lower in vaccinated fish compared to the non-vaccinated sham groups at 48 h post-infection. However, only the Fim immunized group showed a significantly higher antibody titer in comparison to the non-vaccinated treatment group (p&#x3c;0.05) at 21 days post-vaccination. Altogether, Fim and FimMrfG recombinant proteins have potential for vaccine development against virulentinfection. Genomic subtraction revealed three outer membrane proteins present in strain ML09-119 but not in the low virulence referencestrain; the major outer membrane protein OmpAI (OmpA1), TonB-dependent receptor (TonB-DR), and transferrin-binding protein A (TbpA). Here, the genes encoding OmpAI, tonB-DR, and tbpA were cloned fromML09-119 and were expressed into. The purified recombinant OmpA, TonB-DR, and TbpA proteins had estimated molecular weights of 37.26, 78.55, and 41.67 kDa, respectively. Catfish fingerlings vaccinated with OmpA1, TonB-DR, and TbpA emulsified with non-mineral oil adjuvant were protected against the subsequentML09-119 infection with 98.59%, 95.59%, and 47.89% relative percent survival (RPS), respectively. Furthermore, the mean liver, spleen, and anterior kidney bacterial loads were significantly lower in catfish vaccinated with the OmpA1 and TonB-DR than the non-vaccinated control group. ELISA demonstrated that catfish immunized with OmpA1, TonB-DR, and TbpA produce significant antibody response by 21 days post-immunization. Therefore, data generated during the study suggest that OmpAI and TonB-DR proteins could be used as potential candidates for vaccine development againstepidemic strain infection. However, TbpA protein failed to provide such strong protection. Recombinant ATPase fromalso showed promise as a vaccine antigen. A live attenuated vaccine was prepared that combined the advantages of a live attenuated vaccine (ESC-NDKL1 (&#x394;gcvP&#x394;sdhC&#x394;frdA) mutant of) against enteric septicemia of catfish (ESC) and three immunogenic recombinant proteins (Fim, FimMrfg, and ATPase) againstinfection. Our results showed channel catfish fingerlings immersion-vaccinated with ESC-NDKL1::pETfim, ESC-NDKL1::pETmrfG, and ESC-NDKL1::pETATPase exhibited 100%, 91.67%, and 100% percent survival after challenge with theML09-119, which was significantly less than non-vaccinated group (88.89% mortality). In a second study, Catfish immunized with NDKL1::pETfim, ESC-NDKL1::pETmrfG, ESC-NDKL1::pETATPase had significantly (p&#x3c;0.05) lower mortalities than sham-vaccinated group.

Virulentvaccines and methods

A live attenuatedbacterium lacking a viable gene encoding a functional evpB protein and a method of using the same to protect fish against infection from virulent. The methods and compositions for protecting fish against infection from virulentcomprising administering to a fish a therapeutically effective amount of an attenuatedbacterium lacking a viable gene encoding a functional protein selected from the group consisting of evpB, fur and hfq. The bacterium may include an insertion and/or deletion mutation in the gene. The fish include catfish, preferably catfish fingerling or a catfish fry. The composition may be delivered via immersion delivery, an injection delivery, an oral delivery, or combinations thereof.

Live attenuatedvaccine and method of using the same

A live attenuatedbacterium lacking a viable gene encoding a functional evpB protein and a method of using the same to protect fish against infection from virulent. The methods and compositions for protecting fish against infection from virulentcomprising administering to a fish a therapeutically effective amount of an attenuatedbacterium lacking a viable gene encoding a functional EvpB protein. The bacterium may include an insertion and/or deletion mutation in the evpB gene. The fish include catfish, preferably catfish fingerling or a catfish fry. The composition may be delivered via immersion delivery, an injection delivery, an oral delivery, or combinations thereof.

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Live attenuated bacteria vaccines against enteric septicemia of fish, especially catfish, and methods related to the same. Mutant strains of the bacteria(a pathogenic bacterial strain of) are provided. The mutantbacteria (or other pathogenic bacterial strain of) contain one or more gene deletions or disruptions that result in less virulent bacterial strains as live attenuated vaccine compositions against virulent wild-typebacteria (or other pathogenic bacterial strain of). The mutant strains showing the best immunological protection and safety as a vaccine are the triple mutants ESC-NDKL1 (&#x394;gcvP&#x394;sdhC&#x394;frdA) strain and ESC-NDKL2 (&#x394;gcvP&#x394;sdhC&#x394;mdh) strain, with the ESC-NDKL1 strain providing the greatest safety and efficacy of these two triple mutants.

Live attenuated catfish vaccine and method of making

A high throughput bioluminescence mutant screening procedure is disclosed. This procedure utilizes robotics, and bacterial luciferase to allow real-time monitoring of mutant viability. The procedure was used to decelop a live attenuated vaccine for a catfish against, which is further claimed herein. Additionally, genes from other bacterial species are disclosed which may also be used to create vaccines.

A high throughput bioluminescence mutant screening procedure is disclosed. This procedure utilizes robotics, and bacterial luciferase to allow real-time monitoring of mutant viability. The procedure was used to develop a live attenuated vaccine for a catfish against, which is further claimed herein. Additionally, genes from other bacterial species are disclosed which may also be used to create vaccines.

Live attenuated bacteria vaccines are provided. Also provided are methods by which such vaccines can be obtained, including: a method by which a copy of the dam gene from a pathogenic bacteria is cloned into a plasmid capable of replication in the same bacteria species such that it is overexpressed from either a lac promoter, tac promoter, araBAD promoter, trc promoter, trp promoter, T7, SP6, or T5 bacteriophage promoters, a native promoter from that species, or other appropriate promoter. The plasmid containing the dam gene is then transferred into the pathogens to cause increased expression of Dam resulting in the formation of a live attenuated bacterial vaccines. Alternative methods for producing the vaccine are also provided including altering or replacing the chromosomal promoter for the native dam gene so as to alter Dam expression or mutating or replacing the native dam gene so as to alter the expression of Dam in a pathogenic bacteria.

Methods of preparation of live attenuated bacterial vaccine by alteration of DNA adenine methylase (Dam) activity in those bacteria

A method for identifying virulent strains ofthat includes the use of primers or probes in a PCR assay or hybridization technique that employs primers or probes, which are specific for virulence-specific genes of. Also provided is a method of control ofstrains that have been identified using the method of the present invention.

Growing community of inventors

Starkville, MS, United States of America

Mark L Lawrence