Toxoplasma gondii

Ferreira, Flávia Batista, Macêdo-Júnior, Arlindo Gomes de, Lopes, Carolina Salomão, Silva, Murilo Vieira, Ramos, Eliézer Lucas Pires, Júnior, Álvaro Ferreira, Vitaliano, Sérgio Netto, Santiago, Fernanda Maria, Santos, André Luis Quagliatto, Mineo, José Roberto & Mineo, Tiago Wilson Patriarca, 2020, Serological evidence of Toxoplasma gondii infection in Melanosuchus niger (Spix, 1825) and Caimam crocodilus (Linnaeus, 1758), International Journal for Parasitology: Parasites and Wildlife 12, pp. 42-45 : 43

2.3. Attempts to directly detect T. gondii in muscle samples from an alligator

We attempted direct detection of parasite DNA and isolation of the parasite in a single alligator with positive serology results that we had access to fragments of muscles from its tail, in accordance with protocols previously described ( Dubey, 1998; Lopes et al., 2016). Briefly, dissociated tissue fragments were inoculated in mice by the intraperitoneal route, which were observed during 30 days for morbidity and survival. Blood samples were collected in the end of the experiment and evaluated for seroconversion by ELISA. Also, approximately 500?l of the processed tissue samples was added to a confluent monolayer of HeLa cells, to observe whether parasite multiplication would be detected during a 30-day period. A quantitative real time polymerase chain reaction (qPCR) using SYBR green detection system (Promega Co, Madison, WI, USA) was used for the attempts to detect parasite DNA in those tissue fragments, through the amplification of T. gondii 's Tg529 region (Forward: 5′-GCTCCTCCAGCCGTCTTG-3’; Reverse: 5′-CCTCAC CCTCGCCTTCAT-3′). The assays were performed in a real-time PCR thermal cycler (StepOnePlus, Thermo Scientific, EUA), along with a standard curve of known amounts of T. gondii DNA extracted from culture derived tachyzoites.