Rapid identification of virulent type I strains of the protozoan pathogen Toxoplasma gondii by PCR-restriction fragment length polymorphism analysis at the B1 gene

Abstract

Sequence analysis at the 35-fold-repetitive B1 locus identified three restriction sites capable of discriminating type I (mouse-virulent) from type II or III (mouse-avirulent) strains of Toxoplasma gondii. B1 PCR-restriction fragment length polymorphism analysis of 8 type I, 17 type II, and 8 type III strains confirms the specificity of the assay. It should now be possible to ask whether strain genotype affects the severity and type of clinical disease in humans.

FIG. 1

Sequence analysis at B1. (A) Polymorphic sites that exist between the three major lineages are represented. B1 sequences were obtained from three archetypal type I, II, and III strains (RH, Prugniaud, and CEP, respectively). The numerical positions annotated refer to the numbered sites in the published sequence (GenBank accession no. AF179871). Sites demarcated by an asterisk indicate polymorphic sites at which two nucleotides are observed at significant levels (the upper letter represents the more abundant nucleotide) in the PCR-amplified material. Nucleotide positions 366, 504, and 1586 fall within potential restriction sites for XhoI (C/TCGAG), PmlI (CAC/GTG), and XcmI (CCAN₅/N₄TGG), respectively, in the type II and III sequences. PCR primers used for amplification and sequencing were as follows: S1, 5′-CGACAGAAAGGGAGCAAGAG (positions 10 to 29); S2, 5′-CCGGGCAAGAAAATGAGAT (positions 1005 to 1023); AS1, 5′-ACGCTGTGTCTCCTCTAGGC (positions 1043 to 1024); and AS2, 5′-CATGGTTTGCACTTTTGTGG (positions 1991 to 1972). Parenthetical numbers indicate GenBank nucleotide positions. (B) DNA sequence electropherogram of the B1 PCR-amplified population for RH (type I), Prugniaud (type II), and CEP (type III). Dye peaks shown bracket the polymorphic XhoI and PmlI restriction endonuclease sites. At positions 366 and 504, denoted by the asterisks, two nucleotide peaks are clearly present only in the sequences from the avirulent strains.

FIG. 2

B1 PCR-RFLP analysis distinguishes virulent from avirulent strains. (A) XhoI, PmlI, and XcmI digestion of B1 PCR-amplified DNA from RH (I), PDS (II), and CEP (III). (B) PmlI digestion of B1 PCR-amplified product from parasite genomic DNA preparations of seven type I, seven type II, and six type III strains was carried out. For more detailed information on strains amplified, see references and .