The acquisition of nucleotide sequence data of Toxoplasma gondii is hard to achieve due to the difficulty of isolating the parasite from asymptomatic hosts; thus, researchers must attempt genotyping from clinical samples where the DNA of the parasite is heavily diluted within the host’s DNA [5, 13]. To deepen the analysis of the mixed infections found in the SAG3 locus, we cloned and sequenced DNA from clinical samples that showed a triple infection pattern by PCR-RFLP.
Mixed infections due to T. gondii are demonstrated by PCR-RFLP when more than one restriction pattern is visualized and by sequencing when two peaks overlap due to different nucleotides at the same position [11, 14, 15]. However, one disadvantage of PCR-RFLP is that a maximum of three coinfections can be suspected when the restriction patterns of the three classic alleles are visualized for a given locus, just as we reported for the SAG3 marker in clinical samples from feral cats in Quintana Roo .
By cloning the PCR amplification products, we confirmed the mixed infections of TgCatMxQR6 and TgCatMxQR3, demonstrating the presence of five and three different sequences, respectively. From the isolated clones, we obtained classical alleles (I, II and III) as well as others that harboured unique SNPs. This is an unprecedented result, since, as far as we know, there is no prior evidence of more than three different T. gondii genotypes in a single blood sample . The result is so surprising that we thought it might be due to cross contamination in the laboratory, mistakes introduced by the polymerase or chimaeras produced during the PCR procedures. However, during the amplification and cloning assays, we included appropriate controls to assure there was no contamination, and in every PCR assay, hot-start high-fidelity AmpliTaq Gold™ polymerase was used, which has a barely noticeable error rate (2.6 × 105) . To reduce the probability of chimaera production, we also implemented several technical measures, such as an increased elongation time (1.5–2.0 min per cycle), low quantity of DNA as a template (ratio of the host:parasite DNA 1 × 10−5), a reconditioning step (dilution 1:2 of the amplification products of the multiplex PCR before the nested PCR) and the use of DMSO as a reaction stabilizer [17,18,19]. In addition, DNA from only one of the reference strains was used, and most of the sequences differed from the control strain (RH).
This phenomenon has also been reported in Malawi and Cambodia in patients infected with the related apicomplexan Plasmodium, where the use of nPCR allowed the identification of up to four genetic variants, but when they applied high-resolution molecular techniques (massively parallel pyrosequencing -MPP-), they were able to identify up to 16 genetic variants in samples that had previously yielded three genotypes only . These results have implications for the prognosis of the disease and drug therapy because it has been reported that when patients are infected with several strains of Plasmodium, drug treatment can “help” some variants to perform better after removal of other competitor strains . This could also occur in mixed T. gondii infections in hyperendemic regions, where some strains may be resistant to some chemotherapeutics and are the cases that do not respond to the current treatment against the parasite. In addition, the two sampled cats were feral, and these animals obtained their whole diet from hunting small mammals and birds; if the cats consume prey infected with different genotypes within several days, it is possible that more than three genotypes could be found circulating in their blood because tachyzoites can be found up to 10 days after oral infection [22,23,24].
Two unique polymorphisms were found in the sequences TgCatMxQR6d and TgCatMxQR6f. The first of them (A/G, 159 position) is a type II strain SNP, while the second (A/G, 110 position) has been reported in sequences isolated from China (GenBank® KU599378, KU599384, KU599385, and KU599386); however, both sequences are new. The finding of sequences that carry unique SNPs in Mexico has also been reported in pigs from Yucatan: Cubas-Atienzar et al.  found 18 different Alt. SAG2 sequences in addition to the I and III classic alleles in blood and muscle samples of 40 swine. One of the three sequences found here (TgCatMxQR3b) that did not have 100% identity with those reported elsewhere is an intragenic recombinant between type I × II. This phenomenon has also been reported in one dog from Chiapas, where a recombinant sequence I × II was reported and it produced an atypical SAG3 allele . Cubas-Atienzar et al.  also reported I × II intragenic recombinants at Alt. SAG2 and SAG3 markers and one I × II × III at the GRA6 locus. Therefore, these results suggest that there are more intragenic recombination events than previously thought, and they are frequent in hyperendemic regions.
Four out of ten sequences of SAG3 reported here, including one from a dog from Chiapas, were located in unique nodes of the cladogram, in some cases up to five mutational steps far away from the classic I, II, and III alleles, which confirms our hypothesis that there are endemic T. gondii strains in Mexico. The other six sequences were grouped inside the nodes of the classic alleles, with sequences obtained mainly from the USA and Europe (France, Portugal and Turkey), where genetic diversity of the parasite is reduced. This may be due to the presence of few native species of felids in these regions (other than feral domestic cats); in the USA and Canada, only cougar (Puma concolor) and red lynx (Lynx rufus) are naturally distributed; and in Europe, there are only three species: European wild cat (Felis silvestris silvestris), northern lynx (Lynx lynx) and Iberian lynx (Lynx pardinus), so there would be a lower probability of genetic recombination events . In the smallest nodes, isolates from tropical regions of South America and the Caribbean were found, where there are up to nine species of felines besides the domestic cat, and therefore greater genetic diversity of the parasite .
Jiang et al.  analysed the frequency of genotypes isolated in the USA from domestic and wild animals and concluded that in domestic/urban transmission cycles, new genotypes are rarely found, while in the sylvatic transmission cycle, there is high genetic diversity (up to 10-fold) with a greater frequency of new and atypical strains. The cats included in the present study lived in a sylvatic environment where they coexisted with five wild feline species .