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. 2020 Sep 2;25(17):4011.
doi: 10.3390/molecules25174011.

Development of a Molecular Snail Xenomonitoring Assay to Detect Schistosoma haematobium and Schistosoma bovis Infections in their Bulinus Snail Hosts

Affiliations

Development of a Molecular Snail Xenomonitoring Assay to Detect Schistosoma haematobium and Schistosoma bovis Infections in their Bulinus Snail Hosts

Tom Pennance et al. Molecules. .

Abstract

Schistosomiasis, a neglected tropical disease of medical and veterinary importance, transmitted through specific freshwater snail intermediate hosts, is targeted for elimination in several endemic regions in sub-Saharan Africa. Multi-disciplinary methods are required for both human and environmental diagnostics to certify schistosomiasis elimination when eventually reached. Molecular xenomonitoring protocols, a DNA-based detection method for screening disease vectors, have been developed and trialed for parasites transmitted by hematophagous insects, such as filarial worms and trypanosomes, yet few have been extensively trialed or proven reliable for the intermediate host snails transmitting schistosomes. Here, previously published universal and Schistosoma-specific internal transcribed spacer (ITS) rDNA primers were adapted into a triplex PCR primer assay that allowed for simple, robust, and rapid detection of Schistosoma haematobium and Schistosoma bovis in Bulinus snails. We showed this two-step protocol could sensitively detect DNA of a single larval schistosome from experimentally infected snails and demonstrate its functionality for detecting S. haematobium infections in wild-caught snails from Zanzibar. Such surveillance tools are a necessity for succeeding in and certifying the 2030 control and elimination goals set by the World Health Organization.

Keywords: bovine; control; disease; elimination; parasite; schistosomiasis; surveillance; urogenital.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Singleplex (A); ETTS2 + ETTS1) and multiplex (B); multiplex ETTS2 + ETTS1 + ITS2_Schisto_F, (C); ETTS2 + ETTS1 + ITS2_Schisto_R) PCRs on laboratory-bred Bulinus wrighti (B.w.) and Schistosoma haematobium (S.h.) gDNA separately (1; B.w., 2; S.h.) and combined (3; B.w. + S.h.). When B.w. and S.h. DNA was combined (A3, B3, C3), two amplicons were produced by the ETTS1 + ETTS2 primers, a larger snail amplicon (Sn) (~1200 bp) and a smaller Schistosoma amplicon (T) (~1000), with the additional Schistosoma-specific primers producing either a 538 bp (B3; ITS2_Schisto_F) or 835 bp (C3; ITS2_Schisto_R) amplicon (S). A larger amplicon (A) (~1400–1600 bp) was also observed to be amplified in some reactions, and this was thought to be a PCR artifact or additional primer targets in the Schistosoma gDNA. L1 = HyperLadder I (Bioline, London, UK). -ve = negative, no template control. ITS = internal transcribed spacer.
Figure 2
Figure 2
Multiplex ITS xenomonitoring assay trial at 55 °C (A) and 60 °C (B). Includes gDNA of Bulinus wrighti of both BioSprint (Lane 1 and 5) and DNeasy extractions (Lane 2 and 6) and gDNA of Schistosoma haematobium (Lane 3 and 5) and S. bovis (Lane 4 and 6). Combinations of B. wrighti and S. haematobium (Lane 5) or S. bovis (Lane 6) gDNA shown. Sn = snail amplicon, T = trematode amplicon, S = Schistosoma amplicon, and A = non-specific amplicon or artifact. L1 = HyperLadder I. L2 = HyperLadder IV (Bioline, London, UK). -ve = negative, no template control.
Figure 3
Figure 3
Gel showing the secondary singleplex ITS xenomonitoring (SIX) PCR for 1) Schistosoma haematobium gDNA; 2) S. bovis gDNA; 3) S. haematobium + B. wrighti gDNA; 4) S. bovis + B. wrighti gDNA. -ve = non-template negative control. L1 = HyperLadder I (Bioline, London, UK).
Figure 4
Figure 4
Sensitivity tests of ITS1-2-F PCR performed with serial dilutions of Schistosoma haematobium and S. bovis gDNA in the presence of Bulinus wrighti gDNA. L1 = HyperLadder I. L2 = HyperLadder IV (Bioline, London, UK).
Figure 5
Figure 5
Experimental infections of Bulinus truncatus with Schistosoma haematobium (1–24), field-collected B. globosus infected with Euclinostomum sp. (Euc.) and field-collected B. nasutus shedding Echinostoma sp. cercariae (Ech.). The S. haematobium DNA amplicon was present (+ve) in 13 of the 23 non-patent snails (11 at 24 h post-exposure, and two at 11 weeks post-exposure), highlighted by the arrow. Lane 24 = B. truncatus sample that was shedding S. haematobium cercariae 11 weeks after exposure. The positive control (+ve) is a mix of B. wrighti and S. haematobium control gDNA. L1: HyperLadder I, L2: HyperLadder IV (Bioline, London, UK).
Figure 6
Figure 6
Gel images for the multiplex ITS xenomonitoring (MIX) PCR amplicons for 94 non-patent Bulinus globosus collected from Wambaa, Pemba, United Republic of Tanzania. The text under each amplicon denotes the outcome of the Schistosoma sp. targeted sequencing where relevant (i.e., presence of 538 bp amplicon), which resulted in either S. haematobium (S.h.) or sequencing failure (F). The presence of a trematode band without the presence of the Schistosoma band indicated a non-Schistosoma trematode infection (Tr.). Other non-specific bands, in this case, larger bands (NA), were also observed in these snail populations, which did not amplify with the secondary SIX PCR. x = sample failure with no control amplicon. Arrows highlight the presence of the ~1000 bp trematode band when present (n = 8). B. globosus with a patent S. haematobium infection (Cham10.1 see [6]) was run as a positive control (+ve) and also represented the amplicon profile obtained for the seven patent B. globosus snails (five and two with S. bovis and S. haematobium infections, respectively (see Section 2.4). -ve = the non-template negative control. L1—HyperLadder IV (Bioline, London, UK).
Figure 7
Figure 7
Primer annealing positions flanking and internal to the ITS1 + 2 rDNA targets. Primer positions were mapped to Schistosoma haematobium and S. bovis ITS1 + 2 reference data [59] and to a Bulinus globosus and B. nasutus DNA reference (Pennance et al., unpublished data). For Schistosoma DNA, the primer combinations produced two fragments; 1) ETTS2–ETTS1 (1005 bp) and either 2) ITS2_Schisto_F-ETTS1 (538 bp) or 3) ITS2_Schisto_R-ETTS2 (835 bp). For Bulinus DNA, the primer combinations produced one fragment, ranging in size between 1232 and 1263 due to interspecies variation. For Schistosoma species identification, four SNPs were present at bp positions 90, 145, 195, and 265 in the ITS2 rDNA region, allowing differentiation of S. haematobium and S. bovis following ITS2 sequencing.

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