br Materials and Methods br Results Overall of the TNP

Materials and Methods
Results Overall, 17·0% of the TNP and 23·5% of the LMNP fly specimens were positive for T. pallidum DNA (Table 1). Not all samples were positive or were tested for all three loci (Tables 1 and 2, and Table S2). The polA PCR reaction was positive less frequently than the detection rates obtained from the PCRs targeting tp0548 or tp0574. In 17 fly specimens, DNA extracts from both the body and the corresponding transport fluid were tested (Table S2). Two of the specimens were positive for tp0548 in the body extract and transport fluid and seven specimens became positive only when the transport fluid was PCR analyzed. Sequences derived from both extracts of the same fly specimen were identical (fly# 105, Table S2). For the tp0547 gene extracts from eight fly bodies were positive, of which seven were also positive in the transport fluid. The seven polA and two tp0574 sequences were identical to published orthologs from other T. pallidum the later elementary years (reference sequences in GenBank: U57757 [polA], M88769 [tp0574]). There was no variation at these two loci in all tested fly specimens. In contrast, in the tp0548 gene (nt 40 to 347, GenBank: CP000805; Fig. S3) variation was found among various T. pallidum subspecies and strains, which were included as reference, as well as among sequence data obtained from the fly samples. Among the 308 investigated sites, 66 were variable, of which 24 were parsimony-informative (including gaps; Fig. S3). Phylogenetic tree reconstructions based on tp0548 sequence data (Fig. 1) show that the T. pallidum sequences obtained from fly specimens were diverse. This does not change even when a neighbor-joining tree is constructed (Fig. S4). Most sequences obtained from flies cluster with human reference TPE strains (str. Gauthier and str. CDC2), as well as str. F-B, and the sequences derived from T. pallidum-infected baboons at LMNP (n=28, sample material originates from a study published elsewhere (Knauf et al., 2012)). Interestingly, 19 of the fly specimens (TNP n=9, LMNP n=10) exhibit a tp0548 sequence that is identical to that obtained from samples of infected baboons as well as the type “j” sequence that was obtained from a human patient with genital ulceration and infected with a strain closely related to T. pallidum subsp. endemicum (Grange et al., 2013) (unpublished data; Smajs et al.). Four additional sequences (three haplotypes) obtained from fly specimens, all from LMNP, also cluster within the TPE-containing clade, each differing from the LMNP baboon strain sequence in just a single nucleotide. tp0548 sequence data were also evaluated on the basis of the enhanced typing system (Marra et al., 2010), which uses only the sequence region between nt 130 and 215 (with reference to GenBank: CP000805) (Marra et al., 2010). As a result and with regard to the currently described subtypes (Read et al., 2016), the subtype distribution within the fly samples encompasses 23 sequences of dominating type “j”, two sequences of subtype “a” and a yet undescribed subtype “o” (n=1; Fig. S5, Table S2). Fly species varied between the national parks (Fig. 2). Two fly species, Chrysomya putoria and Musca sorbens, seem to be disproportionally involved in the acquisition of T. pallidum. A full dataset of all fly specimens is presented in the Supplementary material.
Discussion Our results indicate that T. pallidum DNA can be frequently detected on wild caught flies, at least in areas of high prevalence of T. pallidum infection in primates. The molecular confirmation of T. pallidum DNA isolated from flies in this study, together with the recent detection of treponemal DNA on fly specimens from a yaws-endemic area of Papua New Guinea (unpublished data; Mitja et al.), supports the hypothesis of vector carriage of treponematoses, including yaws. Variation in frequency of loci tested positive in fly specimens (Table 2) is most likely associated with a low copy number of treponemes in fly specimens as well as differences of sensitivity of the different PCR assays. However, it must be noted that also the amount of template DNA might have influenced the results since we have used 2 instead of 4μl template DNA in the polA PCR. All PCRs applied in this study have been published elsewhere and are frequently used for the detection of T. pallidum in humans. The identity of the tp0548 sequence isolated from olive baboons and the recovery of the same sequence from multiple fly specimens from Tanzania strongly suggests that flies often come into contact with the spirochete on NHPs. Our data are limited in that we did not attempt to isolate viable T. pallidum from the flies, which would be necessary to investigate the contagiousness of insects that carry the bacterium or the organism\'s ubiquity in the environment. T. pallidum is still not cultivable by standard microbiological techniques. Furthermore, based on the three loci that were amplified, it is not possible to identify the sources of contact with the bacteria, although it is most likely that, within the national park, baboon lesions are the major target for necrophagous flies near baboon groups. Our findings are consistent with earlier studies that demonstrated the presence (Kumm et al., 1935; Kumm, 1935b), and fly-associated transmission of treponemes (Lamborn, 1936; Castellani, 1907; Kumm and Turner, 1936; Satchell and Harrison, 1953; Thomson and Lamborn, 1934).