title: Antibodies that recognize alpha- and beta-tubulin inhibit in vitro growth of the fish parasite Trypanosoma danilewskyi, Laveran and Mesnil, 1904.
abstract: We have previously demonstrated that immunization of goldfish with excretory/secretory (ES) products of Trypanosoma danilewskyi in conjunction with Freund's complete adjuvant protected naïve goldfish from challenge with live parasites. In the present study, we report that T. danilewskyi alpha- and beta-tubulin, which were identified in ES products, induce an immunological response in cold-blooded hosts such as fish. The cDNA sequences of both the T. danilewskyi alpha- and beta-tubulin subunits were determined and subsequently cloned into a prokaryotic expression vector for production of recombinant proteins in Escherichia coli. The coding sequences for the trypanosome alpha- and beta-tubulin were 1353 and 1326bp, respectively, resulting in production of recombinant proteins of M(r) 55-60kDa. An affinity-purified rabbit IgG raised against the recombinant alpha-tubulin recognized recombinant alpha- and beta-tubulin, and native T. danilewskyi tubulin. Incubation of cultured parasites with greater than 40microg of purified rabbit-anti-recombinant alpha-tubulin IgG in vitro caused a dramatic inhibition of parasite growth after as little as 24h of cultivation. Specific anti-tubulin antibodies were responsible for the inhibition of parasites in culture since incubation with similar concentrations of an isotype control had no effect on parasite growth in vitro after 7 days of cultivation. Our results indicate that T. danilewskyi tubulin was antigenic to goldfish, and since antibodies to trypanosome tubulin have a marked effect on trypanosome growth in vitro, that tubulin may be one of the target molecules for control of trypanosomiasis in fish.
authors.affiliation: Department of Biological Sciences, University of Alberta, CW-405 Biological Sciences Building, Edmonton, Alta. T6G 2E9, Canada.
authors.forename: Debbie A
authors.initials: DA
authors.lastname: Plouffe
authors.forename: Miodrag
authors.initials: M
authors.lastname: Belosevic
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology*; D000953:Antigens, Protozoan / Q000737:chemistry / Q000276:immunology; D003001:Cloning, Molecular; D019084:Fluorescent Antibody Technique, Indirect; D006054:Goldfish / Q000276:immunology* / Q000469:parasitology*; D007114:Immunization; D007074:Immunoglobulin G / Q000276:immunology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D016415:Sequence Alignment; D014345:Trypanosoma / Q000235:genetics / Q000254:growth & development / Q000276:immunology*; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D014404:Tubulin