Toxoplasma

Lignereux, Louis, Tong, Wen Han, Tan, Sijie, Vyas, Ajai & O’handley, Ryan, 2023, Presence of IgG antibodies is not a reliable marker of Toxoplasma gondii infection in feral mice, International Journal for Parasitology: Parasites and Wildlife 22, pp. 75-79 : 76-77

3.3. Toxoplasma B1 gene PCR

3.3.1. Mice brain samples contained DNA derived from the toxoplasma genome

The Toxoplasma genome contains B1, a gene that is specific to the parasite and which is found in multiple copies within the genome ( Burg et al., 1989). We used a PCR to amplify this gene, using genomic DNA

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isolated from the brain samples as the template (Supplementary Material 1A).

We isolated the genomic DNA from brain samples of 103 mice, of which only 1 was determined to be seropositive for anti- Toxoplasma IgG antibodies, and 102 were seronegative. Experimenters were blind to the serostatus during DNA isolation and subsequent molecular analysis. The presence of B1 was detected in brain tissue of the sole IgG positive animal. Surprisingly, an additional 52 animals were found to contain Toxoplasma B1 gene within brain tissue, indicating the presence of Toxoplasma genetic material.

The prevalence based on PCR was therefore 51.46%, 95%CI [41.93; 60.88]

The 50 animals were negative for Toxoplasma B1 (Supplementary Material 1B). PCR products from all 53 B1-positive samples were DNA sequenced and confirmed to correspond with the expected B1 sequence (Supplementary Material 1C). The target gene was successfully amplified from the laboratory-grown Toxoplasma type II Prugniaud PA7 strain, which serves as the positive control. Negative controls did not exhibit B1 amplification throughout the experiment.

3.3.2. Toxoplasma DNA detected in mice brain samples were from a type II strain

Animals with positive determination of Toxoplasma B1 were further tested for the presence of restriction enzyme sites, Sau3AI and Hhal, at 2 opposite ends of the Toxoplasma SAG 2 locus, SAG2:5′ and SAG2:3′, respectively. Presence of Sau3AI in the SAG2:5′ DNA sequence indicates the Toxoplasma to be of a type III strain. Presence of Hhal in the SAG2:3’ DNA sequence indicates the Toxoplasma to be of a type II strain. An absence of both restriction enzyme sites indicates the Toxoplasma to be of a type I strain. This process allows unambiguous determination of which Toxoplasma strain were the mice infected with. The process was carried out via a previously described nested PCR approach but with modifications to complement to our wildlife mice samples.

To test the sensitivity and accuracy of this genotyping approach, laboratory-grown Toxoplasma type II Prugniaud PA7 strain served as positive control during the nested PCR amplifications. SAG2 was readily detected from Toxoplasma type II Prugniaud PA7 and analysis by DNA Sanger sequencing revealed an absence of the Sau3Al restriction enzyme sites at the SAG2:5′ locus and a presence of Hhal in the SAG2:3’ locus DNA sequence. This indicates the Toxoplasma to be of a type II strain, confirming the accuracy of this genotyping approach. No PCR product was detected from the non-template sterile water negative control (Supplementary Material 2A and 2B). DNA Sanger sequencing was similarly carried out on laboratory-grown Toxoplasma type I RH, and there was an absence of both Sau3Al and Hhal restriction enzyme sites (Supplementary Material 2C). These analysis proved that the nested PCR approach gives consistent genotyping results.

31 Toxoplasma B1-positive mice were then taken at random to establish the Toxoplasma strains these animals were infected with. The same genotyping approach found all 31 mice to be infected with a type II Toxoplasma strain (Supplementary Material 2D). There was no detection of mixed infections. In each amplification process, the target genes were successfully amplified from the laboratory-grown Toxoplasma type II Prugniaud PA7 strain, which serves as the positive control. Negative controls did not exhibit B1 amplification throughout the experiment.