Whole mitochondrial genome alignments of the O. volvulus (NC_001861; [20]) and O. ochengi (obtained from genomic sequence available at http://www.nematodes.org/genomes/onchocerca_ochengi/; Blaxter Lab, University of Edinburgh) sequences was performed to identify a PCR compatible region that contained (i) a restriction site that was unique to one species, and (ii) additional polymorphism(s) that would result in a difference in the melting temperature between amplicons generated for each of the 2 species. A 79-bp region within the nad1 gene spanning 8,655- to 8,733-bp of the O. volvulus mitochondrial genome was chosen that contained 2 synonymous and 1 non-synonymous nucleotide C > T transitions (C8683T [Ala > Ala, C8693T [Leu > Phe], & C8714T [Leu > Leu]) between the mtDNA alignment of the 2 species, 1 of which (C8693T; Leu > Phe) was found in an ApaI restriction site that is present in O. volvulus but absent in O. ochengi (Fig. 1a). A fourth discriminating variant was identified in this 79 bp region (G8659A; Leu > Leu); however, it was not ultimately used as it was located at the 5’ end of the optimal forward primer binding site. Melt curve analysis demonstrated a shift in Tm between the 2 sequences, with a mean Tm of 79.8 °C and 78.6 °C for the O. volvulus- and O. ochengi-derived sequences, respectively (Fig. 1b). HRM species determination of the unknown larval samples was performed by automated clustering of the unknown larval melt curves to curves derived from the control adult O. volvulus (Fig. 1b, c; green) and O. ochengi (Fig. 1b, c; red) samples. This translated into a significant and consistent deviation in melt curves between O. volvulus and O. ochengi samples as depicted in the fluorescence difference plots (Fig. 1c); these plots accentuate the difference in melt profiles throughout the temperature range (Fig. 1b) relative to a reference curve (set to O. ochengi adult control), and in turn, emphasizes differences between groups of sequences. To confirm that the difference between the 2 groups of melt curves were consistent with the prediction that the ApaI restriction site was present in the O. volvulus sequences but not in the O. ochengi sequences, 42 larvae-derived, 4 adult-derived and 2 cloned PCR products were analysed by restriction digest, of which a representative gel is shown in Fig. 1d. The melt curve and restriction digest data were 100 % concordant in the samples analysed, demonstrating that both approaches were equally predictive of the species in question. Finally, cloned amplicons derived from both the adult control samples and 10 larvae (5 from each melt curve group) were analysed by Sanger sequencing, confirming the discriminating variants and concordance with the reference whole mitochondrial genome sequences for the 2 species. This result does not, however, exclude the possibility that additional genetic variation may exist within the HRM-amplicon in either or both species of Onchocerca examined (or other potential Onchocerca species endemic to onchocerciasis regions [21–23]) that could result in melt curves that deviate from the O. volvulus and O. ochengi control sequences described here. For example, an analysis of the mitochondrial sequence of the O. ochengi Siisa variant [21] revealed a T,T,C haplotype that differed from the T,T,T haplotype of the adult O. ochengi presented (Dr Adrian Streit, personal communication). The melt temperature (Tm) of this O. ochengi Siisa variant is predicted to be 79.4 °C, compared to 79.7 °C for O. volvulus and 79.1 °C for O. ochengi (Tm’s were simulated using IDTDNA OligoAnalyser [https://www.idtdna.com/calc/analyzer] using the following parameters: 0.5 uM oligonucleotide, 50 mM NaCl, and 3 mM MgCl concentrations), which if present would have generated a melt curve and Tm shift intermediate to the O. volvulus and O. ochengi control sequences used. Although a single base change is unlikely to confound the interpretation between the melt profiles of the O. volvulus and O. ochengi control samples described here, this assay does offer further opportunity beyond conventional O150 assays to explore genetic variation among and within species. HRM requires that any comparison of unknown samples be made against known positive control samples; therefore, to explore potential variation beyond the assay described here, amplicons that result in melt curves that do not cluster with controls must be sequenced to identify and confirm potential novel variation present.
Of the flies dissected, 166 contained Onchocerca larvae (Additional file 1: Table S1); only a single larva was recovered from each of 123 flies, whereas 43 flies contained multiple larvae (Additional file 1: Table S2; 25.90 % of total; range: 2–6). Of the flies from which larvae were processed by HRM (129 flies in total), 4 contained mixed species infections (Additional file 1: Table S2; 14.29 % of flies with multiple larva, 3.10 % of all flies processed), i.e. they contained both O. volvulus and O. ochengi larvae, which must represent larval uptake by the blackfly from 2 blood meals from different hosts at different times. This is not surprising, given the prevalence of cattle and human cohabitation, and that blackflies will seek a blood meal from either host [24, 25]. The overall proportion of each larval stage was similar between both species (Additional file 1: Table S3; χ
2 = 3.397, df = 2, P = 0.091), however, the spatial distribution of each stage within the fly was not equal; although a greater proportion of L3 were found in the head relative to the thorax in both species, the difference between both head and thorax was greatest in O. volvulus with a higher proportion of L3 collected from the head (Additional file 2: Figure S1, Additional file 1: Table S3; χ
2 = 6.822, df = 2, P = 0.016). This finding suggests that while the rate of development within the fly may be similar between the species, O. volvulus may migrate towards the head of the fly earlier that O. ochengi. These observations (co-infection and likely equivalent transmission potential [based on prevalence of L3]), do suggest that the human and bovine hosts are constantly exposed to both parasites which, in turn, raises an interesting question in regard to the number of times host-switching may have occurred between human and cattle (a speciation hypothesis whereby the most recent common ancestor of O. ochengi and O. volvulus was a cattle parasite that established in the human host [26]).
The distribution of both species throughout the sampling region was not uniform (Fig. 2). Although no temporal differences between sampling years for each species was seen (Additional file 1: Table S2: χ
2 = 1.075, df = 2, P = 0.292), a significant spatial trend was observed that suggested that the western communities sampled had a lower prevalence of O. volvulus (22.96 %, n = 135; Black Volta, Tain and Tombe river basins), a roughly equal prevalence of O. volvulus and O. ochengi in the central Pru river basin (47.22 % O. volvulus; n = 36), and a higher prevalence of O. volvulus (92.86 %, n = 14) in the eastern-most Daka river basin (Additional file 1: Table S4; χ
2 = 32.145, df = 2, P = 5.234e-8). These results are not necessarily in contrast to a recent investigation of persistent O. volvulus with negligible O. ochengi transmission in Ghana [9]. Much of that study was focused on 2 southern Ghanaian communities, and very few or no larvae were found in the 2 communities that were shared with this study (Asubende and Agborlekame, respectively); moreover, 3 of the 7 communities used by Lamberton et al. were east of the Black Volta Lake, which would be consistent with high O. volvulus prevalence observed in the eastern-most community (Wiae) sampled here. We speculate that the high infection rate reported and difference in prevalence of O. ochengi in a number of study regions presented here is correlated with the high numbers of cattle in the north-western river basins, particularly during the dry season (December to April). However, given that the sample size of larvae for many communities was low (median = 6.5, range = 1–55 larvae/community; limited by the number of larvae present in the blackfly populations and therefore by the number of blackflies screened), further sampling is required to support these findings, particularly in the Daka river basin where Onchocerca larvae were obtained from only a single community out of the five communities in which flies were sampled.