A total of 10 genotyped T. gondii strains were utilized as shown in Table 1, and the genomic DNA was prepared as described previously [17].

The SOD gene was amplified by PCR from genomic DNA of T. gondii with 1 pair of primes: 5´-ATGGTATTCACTTTGCCCCCGCT-3´ (forward prime) and 5´-TCATTTCAAGGCATTCTCCAAG-3´ (reverse primer). The design of primers was based on SOD gene of T. gondii RH isolate available in GenBank database (AF029915). The amplification reaction was performed in a volume of 20 μl containing 2 μl template DNA, 10 μl 2×1 Step buffer (0.5 U Taq polymerase), 1 μl of each primer, and 6 μl RNase-free dH₂O. The target DNA was amplified under the following conditions: 94˚C for 30 sec, 63.6˚C for 1 min, and 72˚C for 1 min. The PCR amplification products were confirmed by electrophoresis in a 1.5% agarose gels and staining with ethidium bromide followed by visualization under UV.

The SOD PCR amplification products from representative T. gondii strains were digested with restriction enzymes Xba I and EcoR I, respectively, and incubated at 37˚C for 3 hr. The restriction fragments were separated by electrophoresis in 1.5% agarose gel and staining with ethidium bromide, followed by visualization under UV.

The SOD PCR products were purified with the DNA purification kit (TransGen Biotech, Beijing, China) and ligated with the pEASY-T1 vector (TransGen Biotech) according to the manufacturer's instructions, and then transformed into Escherichia coli DH5α competent cells. The transformed cells carrying the insert were successively selected by blue-white screening, PCR, and restriction enzyme digestion. The positive colonies were sequenced by Beijing Genomics Institute Company (Beijing, China). The obtained SOD gene sequences from different T. gondii strains were aligned using Multiple Sequence Alignment Program Clustal ×1.83. Phylogenetic reconstructions based on the sequences of SOD gene among different T. gondii strains were performed using 3 methodologies, namely maximum parsimony (MP), Bayesian inference (BI), and maximum likelihood (ML) [18,19].

The amplification of the SOD gene resulted in a single product of approximate 1,700 bp in length on agarose gel for all tested T. gondii strains (Fig. 1). There are no obvious differences in all tested T. gondii strains after digestion of the amplified SOD products with EcoR I and Xba I, revealing that subtypes I, II, and III could not be differentiated in this condition (Fig. 2). Then, the amplicons of all isolates were sequenced. The sequencing results showed that the SOD gene was 1,712 bp in length for the TgCatBr64, 1,709 bp in length for PTG, 1,706 bp in length for RH, 1,702 bp in length for TgCgCa1, and 1,707 bp for the remaining 6 strains. Besides, the A + T contents of these sequences ranged from 50.1% to 51.1% among all isolates. The alignment of all 10 sequences revealed nucleotide polymorphisms at 43 positions, with an intraspecific variation of 0-1.0% (Table 2). Of these variable nucleotide positions, there were 29 transitions (C↔T, T↔G, A↔C, and A↔G), 7 transversions (A↔T and C↔G), and 7 deletions in all the sequences. These results showed 97.5% sequence similarity of SOD gene among all the tested isolates, suggesting considerable sequence diversity. Amino acid polymorphisms at 24 positions were detected among 10 T. gondii strains, including 20 amino acid position substitutions and 4 amino acid position deletions (Table 3).

Phylogenetic analysis of the 10 examined T. gondii isolates based on the SOD gene sequences demonstrated 2 major clusters (Fig. 3). TgCatBr5, TgCatBr64, and TgCgCa1 strains were clustered in 1 clade, and PRU, MAS, GT1, CTG, TgTgucan, PTG, and RH were clustered in the other clade. Overall, the topologies of all trees based on nucleotide sequences inferred by 2 different methods were similar, with only small differences of bootstrap values.