Dirofilaria immitis: Immunohistochemical Localization of Acid Proteinase in the Adult Worm

Abstract

The distribution of Dirofilaria immitis acid proteinase in adult worm tissue was examined biochemically and immunohistochemically. About 45% of the total proteinase activity of 700g supernatant, which was obtained from the 0.25 M sucrose homogenate of live adult worms, was found in the 100,000g supernatant by subcellular centrifugation analysis. The distribution pattern of the proteinase activity observed by Percoll density gradient centrifugation coincided with that of glucose-6-phosphatase, a marker cytosolic enzyme, suggesting that the acid proteinase was present in vivo in a membrane-free form, possibly in the cytosol or secretory fluid. Immunostaining for the proteinase in the parasite tissue using the IgG1 (κ-type) monoclonal antibody, H-1, revealed immunoreactive enzyme primarily inside the cell as small grains, but not on the cell surface, and immunoreactivity was distributed widely in worm tissues such as lateral cords, dorsal and ventral median cords, the anterior end of the parasite, and intestinal epithelial cells in granular form. In the male reproductive system, the testicular wall and germ cells were labeled with the antibody, and in the female, uterine walls, fertilized eggs, and developing eggs as well as microfilaria were labeled. In conclusion, D. immitis acid proteinase is widely distributed in the adult parasite tissue, possibly functioning not only in nutrition metabolism but also in production of sperm and microfilariae, etc.