Pathogen PCR amplification analysis
Conserved primers identified in the literature (see Table 1) were used to amplify the 18S subunit of the ribosomal RNA gene (18S rRNA) of Trypanosoma brucei ,Neospora caninum , Sarcocystis gigantea , Toxoplasma gondii , Babesia bovis and Theileria orientalis (Franco, Romero, Ferrari, Schnittger, & Florin-Christensen, 2018; Thompson et al., 2013; Yang et al., 2014). In the case of Plasmodium falciparum , cytochrome b primers (Schaer et al., 2013) were used. Purified genomic DNA of these seven pathogens was obtained from in vitro cultures and used to validate the 18S rRNA/cytochrome b primers and PCR amplification conditions. In this study, PCR conditions were slightly modified as indicated in Table 1. PCR amplification of the positive controls resulted in amplicons of the expected size for the seven parasites investigated (Figure 2).
The 243 genomic DNA samples were subject to PCR amplification with the four primer pairs and PCR conditions indicated in Table 1. Positive controls shown in Figure 2 were used in each PCR round. Overall, the PCR analysis did not amplify the 18S rRNA or the cytochrome b gene of the seven selected parasites in any of the 243 samples, while the same genes were successfully amplified in the positive controls within the same experiment. Despite our best efforts to obtain larger numbers of whole blood samples from Queensland (n = 4), we could only access additional serum samples of chital deer from this state (n = 105). Genomic DNA was extracted from a subset of these serum samples (n = 50) and the PCR screen for the seven parasite genera was performed (same conditions as indicated in Table 1). No amplicons were obtained for any of the 50 serum samples and these are not discussed further.