Virulentvaccines and methods

Abstract

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<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 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 (ΔgcvPΔsdhCΔfrdA) 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<0.05) lower mortalities than sham-vaccinated group.