Isolation, partial purification, and immunogenicity of flagella from Tritrichomonas foetus.

Tritrichomonas foetus, the agent of bovine trichomoniasis, is a flagellate protozoan responsible for substantial economic losses to the dairy and calf industries worldwide. As yet, there is no approved treatment nor is there a sensitive diagnostic method. All these problems suggest that immunization is the best control strategy. In view of this, we isolated and partially purified flagella of the parasite by vortex homogenization followed by low-speed differential centrifugation. The resulting enriched flagellar preparation termed "crude flagellar prep" was purified further by sucrose and percoll gradients. Microscopic analysis showed that the flagellar membrane was intact. Analysis by sodium dodecyl-sulfate polyacrylamide gel electrophoresis revealed three prominent protein bands of 42, 49, and >250 kDa, and several minor bands. Immunoblotting of flagellar and whole-cell extracts revealed many flagellar antigens.

Purification and analysis of a phospholipase A2-like lytic factor of Trichomonas vaginalis.

Trichomonas vaginalis produces soluble factors that have been reported to have the ability to damage target cells in vitro, and it has been hypothesized that these factors may play a role in the pathogenesis of human trichomoniasis. A lytic factor (LF) was purified from T. vaginalis, and the molecular characteristics of LF were determined. T. vaginalis extract was subjected to hydrophobic chromatography with a 10 to 60% N-propanol gradient in 0.1 M ammonium acetate, resulting in the elution of LF from the column at 30% N-propanol. Cytotoxicity assays revealed that LF was cytotoxic to WEHI 164 cells and bovine red blood cells, and inactivation of LF by treatment with trypsin suggested that the active component of LF was a protein. Size exclusion chromatography of LF produced two fractions at 144 and 168 kDa, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LF under reducing conditions revealed two subunits of 57 and 60 kDa. Results of a fluorescence assay of LF on carboxyfluorescein-labeled liposomes composed of phosphatidylcholine-cholesterol showed that liposomes were hydrolyzed, suggesting that LF had phospholipase activity. Thin-layer chromatography analysis of BODIPY (4,4-difluoro-3a,4adiaza-s-indacene)-labeled phosphatidylcholine treated with LF demonstrated products that migrated identically to the products produced by treatment with phospholipase A(2) (PLA(2)). These results suggest that LF is a PLA(2) and may be an important virulence factor of T. vaginalis mediating the destruction of host cells and contributing to tissue damage and inflammation in trichomoniasis.