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.
Highlights
Immunoglobulins (Ig) in teleosts are highly specialized glycoproteins that are developed to recognize various antigens, especially those from harmful microbes, and recruit other cells and molecules to destroy invading pathogens, playing an important role in adaptive immunity [1,2,3]
Knowledge of the expression of specific IgM genes in response to specific immunogens is limited and those antigen-driven IgM genes that are expressed from the catfish repertoire upon exposure to virulent Aeromonas hydrophila (vAh) antigens is of interest
Experimental fish were immunized with extracellular proteins (ECP) or mock-immunized with phosphate-buffered saline (PBS); 3 weeks after immunization, all fish were challenged with fatal dose of virulent A. hydrophila
Summary
Immunoglobulins (Ig) in teleosts are highly specialized glycoproteins (antibodies) that are developed to recognize various antigens, especially those from harmful microbes, and recruit other cells and molecules to destroy invading pathogens, playing an important role in adaptive immunity [1,2,3]. Three isotypes of Ig, IgM, IgD, and IgT/Z, have been identified from the genomic sequences of different teleost fish species [4,5]. IgM and IgD have been found in the genome and IgM appears to be the major isotype that responds to pathogens [6,7,8]. Upon exposure to an antigen, germline-coded individual V, D, and J genes are selected from the repertoire; they undergo alternation within B cells via somatic recombination, a process that deletes some randomly distributed number of nucleotides on their boundaries joins them together with random non-templated nucleotides to form a specific IgM transcript [10,12,13]. Knowledge of the expression of specific IgM genes in response to specific immunogens is limited and those antigen-driven IgM genes that are expressed from the catfish repertoire upon exposure to vAh antigens is of interest
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