Morphological and MALDI-TOF MS identification of freshwater snails, including intermediate hosts of schistosomes in Gabon.

Abstract

A thorough knowledge of intermediate snail hosts and their geographical distribution is essential to understanding the transmission of schistosomiasis and to inform interventions in Gabon. Accurate identification of both snails species and Schistosoma species is therefore essential. The aim of this study was to identify the intermediate hosts of Schistosoma spp. (snails) using MALDI-TOF MS. MethodsSnails were collected from various sites across Gabon and were morphologically identified to species or genus level. Snail species were randomly selected, dissected and subjected to MALDI-TOF MS and molecular analysis. Parasites were detected by cercarial emission, followed by qPCR analysis and sequencing. ResultsA total of 3,722 snails were collected and morphologically identified as Bulinus truncatus (n=1845), Bulinus forskalii (n=381), Gyraulus costulatus (n=680), Bellamya sp. (n=67) and Melania sp. (n=749). Of the 3,722 snails collected, 21.2% (n = 791) were selected for further analysis, including Bu. truncatus (n = 301), Bu. forskalii (n = 158), and Gy. costulatus (n=147). A subset of 65 snails was subjected to molecular identification to confirm morphological classification, using COI, ITS, and 18S gene. BLAST analysis of the resulting sequences showed 97-100% identity with reference sequences of Bu. truncatus, Bu. forskalii, and Gy. costulatus available in GenBank. Analysis of the MS spectra from the 791 snails revealed that 78.2% (n = 618) were of high quality, with signal intensities exceeding 3,000 arbitrary units (a.u.). The MALDI-TOF MS reference database was subsequently updated with 12 high-quality spectra, comprising four representative spectra for each snail species. A blind test of 606 remaining spectra against the database. A blind test of the remaining 606 spectra, using the updated MALDI-TOF MS reference database, demonstrated reliable identification of 100% (Bulinus forskalii, 147/147; Gyraulus costulatus, 158/158) and 96% (Bulinus truncatus, 289/301) of the specimens. These results were consistent with morphological identification, with log score values (LSVs) ranging from 1.7 to 2.8. Principal component analysis (PCA) showed that Bulinus species clustered according to their geographical origin. Overall, 17,66% of the snails analysed were found to be positive for Schistosoma sp. Further analysis, including qPCR and sequencing, revealed that the snails were infected with Schistosoma haematobium. ConclusionWe report the first data on the identification of snails by MALDI-TOF MS in Gabon and show that Bu. truncatus and Bu. forskalii are potential intermediate hosts of S. haematobium. We also show a heterogeneous spatiotemporal distribution of snails in south-eastern Gabon. The role of G. costulatus in the transmission of schistosome remains unclear. Author SummarySchistosomiasis is a disease caused by trematodes of the genus Schistosoma. Freshwater snails of the genera Biomphalaria, Bulinus and Oncomelania release infectious cercariae, which are transmitted to the final host (human or animal). It is the second most widespread parasitic disease after malaria, and among the most important neglected tropical diseases in the world. Snail identification is extremely important for monitoring snail populations and schistosomiasis. Identification has long relied on morphological criteria and molecular biology, both of which have several drawbacks. However, numerous studies have reported the performance of MALDI-TOF MS, a technology that allows species to be identified based on their proteins, as a reliable, rapid and easy-to-use tool in many fields. Recently, it has been described as an effective tool for snail identification and even for tracing the origin of the intermediate hosts of schistosomiasis. The objective of this study was to accurately identify freshwater snails, including the intermediate hosts of schistosomiasis in Gabon.