Marcadores moleculares para famílias de Trematódeos transmitidos por moluscos do gênero Biomphalaria Preston, 1910

Abstract

The Trematoda class encompasses helminths whose life cycle is complex passing through different hosts during its development. Because Biomphalaria snails act as intermediate hosts, they are very important for the maintenance of the biological cycle of several trematodes. It is important to properly recognize and identify trematode parasites that infect Biomphalaria, since different groups may have an impact on human and animal health, besides economical and ecological impacts. The taxonomy based on morphological characters of the larval form of trematodes is methodologically complicated, highlighting the need for new auxiliary techniques capable of identifying these parasites at any evolutionary stage of their cycle. Therefore, molecular tools appear as an attractive alternative. This work aims at identifying molecular markers capable of distinguishing four important families of trematodes, Clinostomidae, Echinostomatidae, Schistosomatidae and Strigeidae, all transmitted by molluscs of the genus Biomphalaria in the neotropical region. Using the online tool TipMT, developed by our group, we have designed trematode family-specific primers targeting the ribosomal DNA region optimized to be used in multiplex PCR, a technique that presents high specificity, low cost and simple execution. gDNA samples of the trematode larvae shed by molluscs of the genus Biomphalaria and from parasitized molluscs from the Medical Malacology Collection (Fiocruz-CMM) were used as a template for the PCR reactions. The panel of primers identified in this study was effective in identifying and distinguishing the four trematode families at the same PCR condition. The specificity of the primers was confirmed using gDNA obtained from other trematodes families, nematode and Biomphalaria species present in the brazilian territory. The primers were sensitive in the range of 0.1 ng to 1 ag of DNA of the parasite. This methodology was also effective for the detection of coinfections. Through a simple, fast and accurate methodology, it is possible to accurately identify and distinguish the trematode families included in this study in biological samples in a single PCR reaction. A family level identification provides important information about probable hosts and impacts generated in the affected region, thus allowing the designing of better strategies to control.