Baylisascaris procyonis: In vitro culture and analysis of larval excretory - secretory antigens
Abstract
The goal of this study was to identify and characterize excretory-secretory (ES) antigens useful for immunodiagnosis of larva migrans caused by Baylisascaris procyonis. An in vitro culture technique was developed for larvae of Baylisascaris spp., and ES antigens were collected from cultures of B. procyonis, B. melis, B. columnaris, B. transfuga, Ascaris suum and Toxocara canis. ES antigens were characterized by SDS-PAGE, PAS staining, and lectin binding, and their specificity was assessed by immunoassays on western blots using polyclonal and monoclonal antibodies. Baylisascaris procyonis ES antigens consisted of a heterogeneous group of complex glycoproteins ranging from 10 kd to over 200 kd. A high degree of similarity was observed between ES antigens among the four Baylisascaris species, especially between B. procyonis and B. melis, while T. canis ES antigens presented a much more unique electrophoretic profile. Five monoclonal antibodies (Bp1-Bp5) were produced against B. procyonis ES antigens. Bp1 and Bp2 recognized common carbohydrate determinants on 14 kd ES components of B. procyonis, B. melis, and B. transfuga, while Bp4 and Bp5 recognized several protein determinants only present on ES components of B. procyonis and B. melis. Bp3 recognized protein determinants on B. procyonis and B. melis of 33-35 kd, but also cross-reacted with a high molecular weight T. canis ES component. Immunoassays utilizing B. procyonis ES antigens demonstrated extensive cross-reactivity with heterologous antisera raised in mice and rabbits. However, antibody recognition of B. procyonis ES components ranging from 33-45 kd was very useful for genus-specific immunodiagnosis. Normal human sera and T. canis positive sera cross-reacted with many B. procyonis ES antigens, including those in the 33-45 kd range. However, treatment of antigens by periodate oxidation markedly decreased cross-reactions, and allowed for the differential immunodiagnosis of human infections with B. procyonis versus T. canis. These studies demonstrated that common carbohydrate epitopes present on ES components from different ascaridoid nematodes contribute to the cross-reactions seen in immunoassays. Specific B. procyonis ES antigens, with molecular weights of 33-45 kd, may be useful for genus-specific immunodiagnosis of larva migrans caused by Baylisascaris spp.
Degree
Ph.D.
Advisors
Kazacos, Purdue University.
Subject Area
Immunology
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