Biomphalaria molecular xenomonitoring

Biomphalaria molecular xenomonitoring

All 589 Biomphalaria snails were screened for S. mansoni and other Trematoda species infections using molecular xenomonitoring. A total of 14 (2.4%) samples failed to amplify the Biomphalaria internal control ITS locus, and so the amplifications were repeated using the same protocol, with all 14 successfully amplifying the Biomphalaria ITS locus during the repeat molecular xenomonitoring PCR. All three target loci were amplified when using DNA extracted from the one B. pfeifferi snail shedding S. mansoni cercariae, as well when using DNA isolated from 19 additional non-shedding Biomphalaria .

Te S. mansoni ND 5 locus was successfully amplified during the secondary S. mansoni ND 5 singleplex PCR in all 20 Biomphalaria samples that had amplified all three target loci during the initial molecular xenomonitoring PCR. All 20 ND5 amplicons were confirmed as S. mansoni through ND5 analysis, confirming infection with S. mansoni . Te prevalence of S. mansoni infection in these 589 Biomphalaria was therefore increased from 0.17% based on cercarial shedding to 3.4% based on molecular xenomonitoring. Biomphalaria snails infected with S. mansoni were identified at four of the 41 (9.6%) malacological survey sites ( Fig. 5 View Fig ). At site 13, three of 175 collected Biomphalaria (1.7%) were infected with S. mansoni ; at site 18, two of 66 collected Biomphalaria (3%) were infected with S. mansoni ; and at site 27, five of 151 collected Biomphalaria (4%) were infected with S. mansoni . At site 21, however, nine of 80 Biomphalaria (11.25%) were infected with S. mansoni , the highest prevalence of Biomphalaria infections across all malacological surveillance sites. Te number of malacological surveillance sites where intestinal schistosomiasis transmission was identified was therefore increased from just one using cercarial shedding to four using molecular xenomonitoring.

Te S. mansoni ND 5 TCS haplotype network is shown in Fig. 6 View Fig . Two distinct clusters were formed, each comprising three unique haplotypes. One unique haplotype consisting of two S. mansoni ND 5 sequences was identified at site 13; no other haplotypes were found at this site. One unique haplotype comprising one S. mansoni ND 5 sequence was identified at site 21. Te remaining four haplotypes were present at more than one single site, with two being present at three sites (sites 18, 21 and 27). All 20 S. mansoni ND 5 sequences were uploaded to the GenBank repository (Accession numbers: PP889740-PP889759).

Only the Biomphalaria ITS and Trematoda ITS loci were amplified in 12 Biomphalaria DNA samples, indicating infection with non- S. mansoni trematodes. Te Trematoda ITS gel band was successfully excised and genotyped in all 12 of these samples. Of these 12 samples, six were identified as Uvulifer spp. , which were all present at site 13, and the remaining six were identified as Petasiger spp. , which were present at sites 18, 20, 21 and 27 (Additional file 1: Table S2), based on ITS analysis. No genetic variation was found between any ITS sequence data for Uvulifer spp. or between any ITS sequence data for Petasiger spp. All six Uvulifer spp. ITS sequences and all six Petasiger spp. ITS sequences were uploaded to the GenBank repository (Accession numbers: PP510464- PP510469 and PP510476-PP501481, respectively). An example agarose gel image of the high-throughput molecular xenomonitoring PCR assay [ 24] is shown in Additional file 4: Figure S1 View Fig . Additional data generated during Biomphalaria molecular xenomonitoring can be found in Additional file 5: Dataset S1, Biomphalaria molecular xenomonitoring data.