Differentiation of Toxoplasma gondii from closely related coccidia by riboprint analysis and a surface antigen gene polymerase chain reaction

Abstract

The tachyzoite of the human pathogen Toxoplasma gondii is morphologically indistinguishable from the proliferative stages of some other zoonotic coccidia, including Sarcocystis. To determine the identity of such coccidia obtained from human tissues and other sources, we compared riboprints (through restriction enzyme analysis of the polymerase chain reaction [PCR]-amplified small subunit rRNA gene) of the following protozoa: the RH and ts-4 strains of T. gondii, lines OH3 and S11, which are two recently isolated T. gondii-like parasites from Brazil, Neospora caninum, Sarcocystis species, and the malarial parasite Plasmodium berghei. In addition, the protozoan genomes were examined by PCR for homologs of surface antigen genes of T. gondii, and by Southern hybridization to the heterologous rRNA gene probe pSM 389. Strains OH3, S11, ts-4, and RH shared identical riboprints, and OH3, S11, and ts-4 have p22 and p30 surface antigen gene structures similar to RH. In contrast, riboprints for N. caninum and T. gondii differ with respect to Dde 1 sites, and moreover, their genomes vary significantly from one another at both the p22 and p30 gene loci. The riboprints of Sarcocystis and P. berghei differ markedly from T. gondii and N. caninum and from each other. Bam HI pSM 389 restriction fragment length polymorphisms differentiate ts-4 from RH, OH 3, and S11. Our results confirm that OH3 and S11 are indeed T. gondii, but that N. caninum and T. gondii are likely to be separate species, thereby resolving previous uncertainties concerning the identity of these parasites. Together, the variation in riboprints and surface antigen gene structure reflects the phylogenetic diversity among these coccidia, and in addition, confirms the value of riboprinting in the identification of apicomplexan parasites such as T gondii.