Purification and characterization of three larval excretory-secretory proteins of Dirofilaria immitis

Two proteins were previously described in the excretory-secretory products (ES) collected from Dirofilaria immitis during the molt from the third stage to the fourth stage in vitro. The two proteins were purified using cation exchange and reverse phase HPLC. During the purification of these two proteins, a third protein was identified that co-migrated with one of the others during previous gel analysis. All three had molecular masses of 20-23 kDa as determined by Tris-glycine SDS-PAGE and have been designated 20, 22L and 22U kDa proteins. The three proteins were digested with trypsin. Amino acid sequences were subsequently determined for four peptides and the N-terminus of the 20 kDa protein, five peptides of the 22L kDa protein and three peptides of the 22U kDa protein. The 20 and 22L kDa proteins were quite similar based on sequence and purification characteristics. The 22U kDa protein, but not the 20 and 22L kDa proteins, was also identified in adult worms using tryptic mapping and amino acid sequencing techniques. Immunoblot analysis demonstrated that the 20 and 22L kDa proteins were specifically recognized by sera from dogs immune to infection by D. immitis but not by sera from infected non-immune dogs. The 22U kDa protein was weakly recognized by the same immune sera but not by the infected non-immune dog sera. Since the 20 and 22L kDa proteins appear to be larval specific, associated in time with the molt from L3 to L4 and are specifically recognized by immune dog sera, they are good vaccine candidates.