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.