Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis

The utility of sequence polymorphisms in the dense granule antigen GRA6 gen e as typing markers for Toxoplasma gondii was investigated. The coding regi on of GRA6 was amplified, sequenced and compared for 30 Toxoplasma strains from eight different zymodemes (Z1-Z8). Sequence alignment identified nucle otide polymorphisms at 24 positions out of 690 bp, which correlated with mu rine-virulence. Types I, II, and III could be distinguished from each other on the basis of three; 10, and six variable positions, respectively. Two d eletions of 15 bp and 3 bp existed in the avirulent (type II) strains. With one exception, all polymorphic positions resulted in amino acid substituti ons, and the two gaps of 15 bp and 3 bp caused the deletion of six amino ac ids in type II strains. Intra-specific polymorphisms were also found in the virulent group. A high degree of sequence polymorphism correlating with th e phenotypes of T. gondii strains points to the GRA6 gene being a good mark er for strain characterisation and typing of the isolates of this apicomple xan. The large variety of amino acid changes supports the view that the GRA 6 protein plays an important role in the antigenicity and pathogenicity of T. gondii. The existence of polymorphic restriction sites for endonuclease MseI was used to develop a PCR-RFLP method which could simply differentiate the three different groups (types I, III III) of T. gondii. (C) 2000 Austr alian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.