Variation in primary sequence and tandem repeat copy number among i-antigens of Ichthyophthirius multifiliis

Abstract

The immobilization antigens (i-antigens) of Ichthyophthirius multifiliis are potential vaccine candidates for the prevention of ‘white spot’ disease in freshwater fish. These antigens vary with respect to antigenicity and molecular mass, and at least five i-antigen serotypes have been identified among parasite isolates thus far. In previous studies, the gene and corresponding cDNA encoding a ∼48 kDa i-antigen from parasite isolate G1 (serotype A), had been cloned and sequenced. We now report on the isolation of two new genes, designated IAG52A[ G5] and IAG52B[ G5], encoding ∼52/55 kDa i-antigens from a parasite isolate representing a different serotype, namely, D. Based on their deduced sequences, the ∼52/55 kDa gene products have the same structural features as the 48 kDa protein including hydrophobic N- and C-termini, periodic cysteine residues with the potential for metal binding, and tandemly repetitive amino acid sequence domains that span their length. Nevertheless, the products of these genes vary in their tandem repeat copy number, and share only ∼50% homology overall. When expressed in heterologous systems, the products of the newly described genes react strongly with monospecific polyclonal antisera against the i-antigens of serotype D and are clearly i-antigens. It would nevertheless appear that mRNA transcripts from the two genes are present at widely different levels within parasites themselves. Analysis at the protein level using 2-D SDS-PAGE would further suggest that multiple i-antigens are expressed within the same serotype at any given time.