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Title: IMMUNE RESPONSE INDUCED BY N-LAUROYLSARCOSINE EXTRACTED OUTER-MEMBRANE PROTEINS OF AN ISOLATE OF EDWARDSIELLA ICTALURI IN CHANNEL CATFISH

Author
item Bader, Joel
item Shoemaker, Craig
item Klesius, Phillip

Submitted to: Fish and Shellfish Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/22/2003
Publication Date: 3/30/2004
Citation: Bader, J.A., Shoemaker, C.A., Klesius, P.H. 2004. Immune response induced by N-Lauroylsarcosine extracted outer-membrane proteins of an isolate of Edwardsiella ictaluri in channel catfish. Fish and Shellfish Immunology. 16(3):415-428.

Interpretive Summary: Edwardsiella ictaluri is the causative agent of enteric septicaemia of catfish (ESC). This disease results in the largest amount of economic lose in cultured catfish in the southeastern United States and where ever catfish are cultured. Annual loses attributed to ESC are estimated at greater than $100 million to the catfish industry. While, a live-modified, whole bacterial cell vaccine presently exists and has been shown to successfully prevent ESC, second generation vaccines are still in development. These second generation vaccines are killed vaccines and subcellular in nature. This study examined the efficacy of a lipopolysaccharide-reduced N-lauroylsarcosine outer-membrane protein (OMP) fraction as a ESC vaccine. Prior to the vaccination studies this OMP fraction was produced, immunogenicity characterized, and compared to other subcellular fractions. The OMP fraction contained less than 2% (W/V) lipopolysaccharide (LPS). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that whole cell lysates contained 27 proteins from 107 to 14.3 kDa, whereas OMP contained nine proteins from 97 to 14.3 kDa, LPS contained two proteins at 45 and 37 kDa bands and a smear of bands below 14.3 kDa, and cell wall fraction contained 21 proteins from 97 to 8 kDa. Immunogenicity was confirmed by subsequent Western blot analysis using vaccinated catfish sera. Blots showed that 97, 80, and 19 kDa proteins were immunogenic. Rapid enzyme-linked immunosorbent assays (ELISA) demonstrated that OMP produced a weak, but observable antibody response by 21-d post injection. The vaccine studies showed that OMP provided only marginal protection against ESC at the best dose of 12.5 ug/100 uL. A booster dose of 12.5 ug/100 uL OMP did not significantly enhance this protection.

Technical Abstract: A virulent isolate of Edwardsiella ictaluri (AL-93-75), the causative agent of enteric septicaemia of catfish (ESC), was used to derive a lipopolysaccharide-reduced N-lauroylsarcosine outer- membrane protein (OMP) fraction vaccine. The OMP fraction was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared to whole-cell lysate, purified lipopolysaccharide (LPS)and a crude cell-wall fraction. The OMP fraction contained less than 2% (W/V) LPS. SDS-PAGE showed that whole cell lysates contained 27 proteins from 107 to 14.3 kDa, whereas OMP contained nine proteins from 97 to 14.3 kDa, LPS contained two proteins at 45 and 37 kDa bands and a smear of bands below 14.3 kDa, and cell wall fraction contained 21 proteins from 97 to 8 kDa. Channel catfish, Ictalurus punctatus, were vaccinated with 12.5 ug/100 uL OMP and immunogenicity was confirmed by subsequent Western blots. Blots showed that 97, 80, and 19 kDa proteins were immunogenic. Rapid enzyme-linked immunosorbent assays (ELISA) demonstrated that OMP produced a weak, but observable antibody response by 21-d post injection. OMP concentrations of 3.13, 6.25, 12.5, 25, and 50 ug/100 uL total protein were tested for protective immunity. Marginal protection by relative percent survival (RPS) was only seen for fish injected with 12.5 ug/100 uL with RPSs between 55-67.5%. A booster dose of 12.5 ug/100 uL OMP did not significantly enhance protection.