Identification and Characterization of Differentially Expressed IgM Transcripts of Channel Catfish Vaccinated with Antigens of Virulent Aeromonas hydrophila

Abstract

Channel catfish (Ictalurus punctatus) is the top species produced in US aquaculture and motile Aeromonas septicemia, caused by virulent Aeromonas hydrophila (vAh), is one of the most severe diseases that afflict catfish farms. Previously, vaccination of fish with extracellular proteins (ECP) of vAh was shown to produce a robust antibody-mediated immune response against vAh infection. In this study, we analyzed IgM transcripts that were differentially expressed in the head kidney and liver of ECP-immunized and mock-immunized (control) fish with emphasis on a variable domain of heavy chain. Quantitative PCR analysis indicated that immunized fish produced significantly more IgM transcripts than control fish. Full-length IgM heavy chain cDNA was cloned, which encoded typical IgM peptide, including signal peptide, variable domain (VH), constant domain (CH), and carboxyl terminal peptide. Great sequence diversity was revealed in a VH segment, with the third complementarity diversity region (CDR3) being most variable. Using germline VH gene grouping method, variants (clones) of VH characterized in this study belonged to nine VH families. The most unique variants (approximately 49%) were found in the VH2 family. Vaccinated fish apparently had more unique variants than in the control fish. There were 62% and 79% of unique variants in the head kidney and liver of vaccinated fish, respectively, while 44% and 27% unique variants in the head kidney and liver of control fish, respectively. Among the unique variants in VH2 family, approximately 87% of them were found in vaccinated fish. Two-dimensional gel electrophoresis of semi-purified IgM protein confirmed that matured IgM protein was as variable as IgM transcripts identified in this study, with isoelectric points crossing from 6 to 10. Results of this study provided insight into the molecular and genetic basis of antibody diversity and enriched our knowledge of the complex interplay between antigens and antibodies in Ictalurid catfish.