Development of a Molecular Snail Xenomonitoring Assay to Detect Schistosoma haematobium and Schistosoma bovis Infections in their Bulinus Snail Hosts
<p>Singleplex (<b>A</b>); ETTS2 + ETTS1) and multiplex (<b>B</b>); multiplex ETTS2 + ETTS1 + ITS2_Schisto_F, (<b>C</b>); ETTS2 + ETTS1 + ITS2_Schisto_R) PCRs on laboratory-bred <span class="html-italic">Bulinus wrighti</span> (<span class="html-italic">B.w.</span>) and <span class="html-italic">Schistosoma haematobium</span> (<span class="html-italic">S.h.</span>) gDNA separately (1; <span class="html-italic">B.w.</span>, 2; <span class="html-italic">S.h.</span>) and combined (3; <span class="html-italic">B.w.</span> + <span class="html-italic">S.h.</span>). When <span class="html-italic">B.w.</span> and <span class="html-italic">S.h.</span> 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 <span class="html-italic">Schistosoma</span> amplicon (T) (~1000), with the additional <span class="html-italic">Schistosoma-</span>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 <span class="html-italic">Schistosoma</span> gDNA. L1 = HyperLadder I (Bioline, London, UK). -ve = negative, no template control. ITS = internal transcribed spacer.</p> "> Figure 2
<p>Multiplex ITS xenomonitoring assay trial at 55 °C (<b>A</b>) and 60 °C (<b>B</b>). Includes gDNA of <span class="html-italic">Bulinus wrighti</span> of both BioSprint (Lane 1 and 5) and DNeasy extractions (Lane 2 and 6) and gDNA of <span class="html-italic">Schistosoma haematobium</span> (Lane 3 and 5) and <span class="html-italic">S. bovis</span> (Lane 4 and 6). Combinations of <span class="html-italic">B. wrighti</span> and <span class="html-italic">S. haematobium</span> (Lane 5) or <span class="html-italic">S. bovis</span> (Lane 6) gDNA shown. Sn = snail amplicon, T = trematode amplicon, S = <span class="html-italic">Schistosoma</span> amplicon, and A = non-specific amplicon or artifact. L1 = HyperLadder I. L2 = HyperLadder IV (Bioline, London, UK). -ve = negative, no template control.</p> "> Figure 3
<p>Gel showing the secondary singleplex ITS xenomonitoring (SIX) PCR for 1) <span class="html-italic">Schistosoma haematobium</span> gDNA; 2) <span class="html-italic">S. bovis</span> gDNA; 3) <span class="html-italic">S. haematobium</span> + <span class="html-italic">B. wrighti</span> gDNA; 4) <span class="html-italic">S. bovis</span> + <span class="html-italic">B. wrighti</span> gDNA. -ve = non-template negative control. L1 = HyperLadder I (Bioline, London, UK).</p> "> Figure 4
<p>Sensitivity tests of ITS1-2-F PCR performed with serial dilutions of <span class="html-italic">Schistosoma haematobium</span> and <span class="html-italic">S. bovis</span> gDNA in the presence of <span class="html-italic">Bulinus wrighti</span> gDNA. L1 = HyperLadder I. L2 = HyperLadder IV (Bioline, London, UK).</p> "> Figure 5
<p>Experimental infections of <span class="html-italic">Bulinus truncatus</span> with <span class="html-italic">Schistosoma haematobium</span> (1–24), field-collected <span class="html-italic">B. globosus</span> infected with <span class="html-italic">Euclinostomum</span> sp. (<span class="html-italic">Euc</span>.) and field-collected <span class="html-italic">B. nasutus</span> shedding <span class="html-italic">Echinostoma</span> sp. cercariae (<span class="html-italic">Ech</span>.). The <span class="html-italic">S. haematobium</span> 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 = <span class="html-italic">B. truncatus</span> sample that was shedding <span class="html-italic">S. haematobium</span> cercariae 11 weeks after exposure. The positive control (+ve) is a mix of <span class="html-italic">B. wrighti</span> and <span class="html-italic">S. haematobium</span> control gDNA. L1: HyperLadder I, L2: HyperLadder IV (Bioline, London, UK).</p> "> Figure 6
<p>Gel images for the multiplex ITS xenomonitoring (MIX) PCR amplicons for 94 non-patent <span class="html-italic">Bulinus globosus</span> collected from Wambaa, Pemba, United Republic of Tanzania. The text under each amplicon denotes the outcome of the <span class="html-italic">Schistosoma</span> sp. targeted sequencing where relevant (i.e., presence of 538 bp amplicon), which resulted in either <span class="html-italic">S. haematobium</span> (<span class="html-italic">S.h</span>.) or sequencing failure (F). The presence of a trematode band without the presence of the <span class="html-italic">Schistosoma</span> band indicated a non-<span class="html-italic">Schistosoma</span> 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 (<span class="html-italic">n</span> = 8). <span class="html-italic">B. globosus</span> with a patent <span class="html-italic">S. haematobium</span> infection (Cham10.1 see [<a href="#B6-molecules-25-04011" class="html-bibr">6</a>]) was run as a positive control (+ve) and also represented the amplicon profile obtained for the seven patent <span class="html-italic">B. globosus</span> snails (five and two with <span class="html-italic">S. bovis</span> and <span class="html-italic">S. haematobium</span> infections, respectively (see <a href="#sec2dot4-molecules-25-04011" class="html-sec">Section 2.4</a>). -ve = the non-template negative control. L1—HyperLadder IV (Bioline, London, UK).</p> "> Figure 7
<p>Primer annealing positions flanking and internal to the ITS1 + 2 rDNA targets. Primer positions were mapped to <span class="html-italic">Schistosoma haematobium</span> and <span class="html-italic">S. bovis</span> ITS1 + 2 reference data [<a href="#B59-molecules-25-04011" class="html-bibr">59</a>] and to a <span class="html-italic">Bulinus globosus</span> and <span class="html-italic">B. nasutus</span> DNA reference (Pennance et al., unpublished data). For <span class="html-italic">Schistosoma</span> 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 <span class="html-italic">Bulinus</span> DNA, the primer combinations produced one fragment, ranging in size between 1232 and 1263 due to interspecies variation. For <span class="html-italic">Schistosoma</span> species identification, four SNPs were present at bp positions 90, 145, 195, and 265 in the ITS2 rDNA region, allowing differentiation of <span class="html-italic">S. haematobium</span> and <span class="html-italic">S. bovis</span> following ITS2 sequencing.</p> ">
Abstract
:1. Introduction
2. Results
2.1. In Silico and In Vitro Primer Evaluation
2.2. Analytical Sensitivity
2.3. Experimental Snail Infections
2.4. Specificity Testing
2.5. Testing on Field Samples
2.6. Schistosoma spp. cox1 RD-PCR
3. Discussion
3.1. The Sensitivity of MIX PCR Assay
3.2. Benefits of an Updated Molecular Xenomonitoring Protocol for Schistosomiasis Surveillance
3.3. Limitations of Molecular Xenomonitoring Approaches for Schistosomiasis Surveillance
4. Materials and Methods
4.1. Primer Selection and In Silico Evaluation
4.2. Bulinus and Schistosoma Genomic DNA Extractions
4.3. PCR Conditions, Amplicon Visualization, and Sequencing
4.4. In Vitro Primer Testing and Assay Validation
4.5. Sensitivity Testing
4.6. Specificity Testing and Validation on Field Samples
4.7. Testing the Schistosoma cox1 Rapid-Diagnostic PCR (RD-PCR) for Secondary Species Identification
4.8. Ethical Statement
Author Contributions
Funding
Conflicts of Interest
References
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Sample Availability: Samples (DNA extracts of snails and parasites) are available from the authors upon appropriate request. |
Schistosoma Species | ITS 2 Schistosome Species-Specific SNP Positions (bp) | |||
---|---|---|---|---|
SNP1 (90) | SNP2 (145) | SNP3 (195) | SNP4 (265) | |
S. haematobium | S. h (G) | S. h (C) | S. h (G) | S. h (C) |
S. bovis | S. b (A) | S. b (T) | S. b (A) | S. b (T) |
Primer (Direction) | Primer Sequence (5′-3′) | Primer Position | State | Reference |
---|---|---|---|---|
ETTS1 (Reverse) | TGCTTAAGTTCAGCGGG | 28S 5′ end (ITS2 3′ flanking region) | U | Kane et al. (1994) [31] |
ETTS2 (Forward) | TAACAAGGTTTCCGTAGGTGA | 18S 3′ region (ITS1 5′ flanking region) | U | Kane et al. (1994) [31] |
ITS2_Schisto_F (Forward) | GGAAACCAATGTATGGGATTATTG | ITS1 3′ end (5.8S 5′ flanking region) | S | Schols et al. (2019) [27] |
ITS2_Schisto_R (Reverse) | ATTAAGCCACGACTCGAGCA | ITS2 (middle) | S | Schols et al. (2019) [27] |
Infection Group | No. of B.t. Exposed | No. of S.h. Miracidia Used | No. of B.t. Preserved at 24 h | No. of B.t. Checked for Patency at 11 wks and Preserved (no. Shedding + ve) |
---|---|---|---|---|
1 | 45 | 1 | 22 | 22 1 (0) |
2 | 43 | 2 | 21 | 19 1 (2) |
3 | 45 | ~7 | 23 | 21 1 (7) |
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Pennance, T.; Archer, J.; Lugli, E.B.; Rostron, P.; Llanwarne, F.; Ali, S.M.; Amour, A.K.; Suleiman, K.R.; Li, S.; Rollinson, D.; et al. Development of a Molecular Snail Xenomonitoring Assay to Detect Schistosoma haematobium and Schistosoma bovis Infections in their Bulinus Snail Hosts. Molecules 2020, 25, 4011. https://doi.org/10.3390/molecules25174011
Pennance T, Archer J, Lugli EB, Rostron P, Llanwarne F, Ali SM, Amour AK, Suleiman KR, Li S, Rollinson D, et al. Development of a Molecular Snail Xenomonitoring Assay to Detect Schistosoma haematobium and Schistosoma bovis Infections in their Bulinus Snail Hosts. Molecules. 2020; 25(17):4011. https://doi.org/10.3390/molecules25174011
Chicago/Turabian StylePennance, Tom, John Archer, Elena Birgitta Lugli, Penny Rostron, Felix Llanwarne, Said Mohammed Ali, Amour Khamis Amour, Khamis Rashid Suleiman, Sarah Li, David Rollinson, and et al. 2020. "Development of a Molecular Snail Xenomonitoring Assay to Detect Schistosoma haematobium and Schistosoma bovis Infections in their Bulinus Snail Hosts" Molecules 25, no. 17: 4011. https://doi.org/10.3390/molecules25174011