Virulent aeromonas vaccines and methods

Abstract

Aeromonas hydrophila is a reemerging pathogen of channel catfish ( Ictalurus punctatus ); 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 of A. hydrophila isolates 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 virulence A. hydrophila isolates revealed four fimbrial proteins unique to strain ML09-119. In this work, we expressed and purified four A. hydrophila fimbrial proteins (FimA, Fim, MrfG, and FimOM) and assessed their ability to protect and stimulate protective immunity in channel catfish fingerlings against A. hydrophila ML09-119 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 with A. hydrophila strain ML09-119. Bacterial concentrations in liver, spleen, and anterior kidney were significantly (p < 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 < 0.05) at 21 days post-vaccination. Altogether, Fim and FimMrfG recombinant proteins have potential for vaccine development against virulent A. hydrophila infection. Genomic subtraction revealed three outer membrane proteins present in strain ML09-119 but not in the low virulence reference A. hydrophila strain; 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 from A. hydrophila ML09-119 and were expressed into Escherichia coli . 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 subsequent A. hydrophila ML09-119 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 against A. hydrophila epidemic strain infection. However, TbpA protein failed to provide such strong protection. Recombinant ATPase from A. hydrophila also showed promise as a vaccine antigen. A live attenuated vaccine was prepared that combined the advantages of a live attenuated vaccine (ESC-NDKL1 (ΔgcvPΔsdhCΔƒtdA) mutant of Edwardsiella ictaluri ) against enteric septicemia of catfish (ESC) and three immunogenic recombinant proteins (Fim, FimMrfg, and ATPase) against A. hydrophila infection. 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 the A. hydrophila ML09-119, 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 < 0.05) lower mortalities than sham-vaccinated group.

We claim: 
     
         1 . A composition for providing immunological protection from disease caused by  Aeromonas hydrophila , said composition comprising:
 a strain of  Aeromonas hydrophila  comprising a recombinant fimbrial protein in the group consisting of Fim and FimMrfG.   
     
     
         2 . The composition of  claim 1 , wherein the recombinant fimbrial protein is FimA. 
     
     
         3 . The composition of  claim 1 , wherein the recombinant fimbrial protein is FimOM. 
     
     
         4 . The composition of  claim 1 , wherein the disease caused by  Aeromonas hydrophila  is  Aeromonas hydrophila  strain infection. 
     
     
         5 . The composition of  claim 1 , wherein the disease caused by  Aeromonas hydrophila  is virulent  Aeromonas hydrophila  infection. 
     
     
         6 . The composition of  claim 1 , wherein the composition further comprises anadjuvant. 
     
     
         7 . The adjuvant of  claim 6 , wherein said adjuvant is a non-mineral oil adjuvant. 
     
     
         8 . The adjuvant of  claim 7 , wherein said adjuvant is Montanide ISA 763 AVG. 
     
     
         9 . A method of providing immunological protection to an animal from a disease caused by a pathogenic bacterial strain of  Aeromonas hydrophila  in the animal comprising:
 providing to the animal an effective amount of a bacterial strain of  Aeromonas hydrophila  comprising a recombinant fimbrial protein in the group consisting of Fim and FimMrfG. 
 
     
     
         10 . The method of  claim 9 , wherein the recombinant fimbrial protein is FimA. 
     
     
         11 . The method of  claim 9 , wherein the recombinant fimbrial protein is FimOM. 
     
     
         12 . The method of  claim 9 , wherein the fimbrial protein of the bacterial strain of  Aeromonas hydrophila  is expressed in a live attenuated strain of another catfish pathogen. 
     
     
         13 . The pathogen of  claim 12 , wherein the pathogen is  Edwardsiella ictaluri . 
     
     
         14 . The method of  claim 9 , wherein the fimbrial protein of the bacterial strain of  Aeromonas hydrophila  is expressed in a virus.