Bivalve mollusks as sentinels: Molecular detection of Toxoplasma gondii on Maranhão Island in northeastern Brazil

Bivalve mollusks belong to a class of invertebrates that are cosmopolitan and globally traded. Therefore, this study aimed to investigate the occurrence and genetic assessment of Toxoplasma gondii, based on the SAG1 gene in bivalve mollusks from natural growing areas on Maranhão Island in northeastern Brazil. Oysters ( Crassostrea sp.), mussels ( Mytella sp.) and clams ( Anomalocardia sp.) were collected from natural mangrove áreas in São Luís, Paço do Lumiar, Raposa and São José de Ribamar on Maranhão Island during the period of January to December 2022 (rainy and dry seasons). The samples were organized into pools of gills from 3 animals, their DNA was extracted and subjected to detection of T. gondii (SAG-1 gene), and the positive samples subjected to additional nested PCR for the markers APICO, BTUB, SAG3, 3’ SAG2, 5’ SAG2, Alt.SAG2 and GRA6 for genetic characterization. Sequences obtained were analyzed for phylogenetic reconstruction using the MEGA X program. Three mussel samples tested positive (3/60; 1.8%), all collected in the rainy season from Raposa and São José de Ribamar. These samples were subjected to nested-PCR for other T. gondii markers; however, there was no amplification. BLASTn analysis confirmed 98 to 100% genetic similarity with T. gondii sequences. Although based on a single gene, which limits robust genotype inference, phylogenetic analysis of the SAG1 gene indicated clustering of the detected sequences with reference sequences related to classical genotypes. The current study provides an update on the molecular occurrence of the zoonotic protozoan T. gondii in an estuarine area of northeastern Brazil, and highlights the importance of research involving monitoring of shellfish harvesting areas, given their role in public health and food safety.