Toxoplasma gondii

Yang, Yurong, Dong, Hui, Su, Ruijing, Li, Tongyi, Jiang, Nan, Su, Chunlei & Zhang, Longxian, 2019, Evidence of red panda as an intermediate host of Toxoplasma gondii and Sarcocystis species, International Journal for Parasitology: Parasites and Wildlife 8, pp. 188-191 : 189

2.4. Isolation of viable T. gondii from red panda tissues by bioassay in mice

Fifty gram tissue samples (heart, tongue, diaphragm, and leg muscle) of eight red pandas were bioassayed in mice respectively. Tissues from each red panda were pooled, homogenized and digested in pepsin. The homogenates were then inoculated into BALB/c mice (n = 5) and/or gamma interferon (γ- IFN) knockout mice (n = 2) subcutaneously as previously described (Dubey, 2010). Tissues (lung, brain or mesenteric lymph nodes) smears of dead mice were examined for T. gondii tachyzoites or cysts individually. Survivors were bled on day 60 post-inoculation (DPI) and a 1:25 and 1:200 dilution of serum from each mouse was tested for T. gondii antibodies by MAT. If tissue cysts or tachyzoites were not found in mouse tissues, homogenized lung, brain and heart tissues were subpassaged into new groups of mice subcutaneously.

2.5. DNA isolation and polymerase chain reaction (PCR) identification of T. gondii , N. caninum and Sarcocystis neurona

The DNA was extracted from digestion striated muscle using a commercial DNA extraction kit (Tiangen Biotec Company, DP304). The DNA isolated from T. gondii (CT1 strain) or N. caninum (NC1 strain) was used as a reference for PCR, they were kindly provided by Dr. JP Dubey (ARS, USDA) and Dr. Jing Liu ( China Agricultural University, China). PCR assays for T. gondii , N. caninum and S. neurona were performed using the specific primer pairs TOX5/TOX8, NP6/NP21 and JNB33// JNB54, the products were expected to be 450 bp, 328 bp, and 1100 bp, respectively ( Schares et al., 2008; Yamage et al., 1996; Dubey et al., 2001).