SEPARATION AND CRYOPRESERVATION OF NEOSPORA-CANINUM TISSUE CYSTS FROMMURINE BRAIN

A protocol was developed for the separation, concentration, enumeratio n, and cryopreservation of Neospora caninum tissue cysts from mouse br ains. Brains from chronically infected mice were homogenized and tissu e cysts counted in 10-mu l aliquots. Tissue cysts were separated from brain homogenates by centrifugation at 4,400 g on 35% (v/v) Percoll/ph osphate-buffered saline (PBS) continuous-density gradients. After remo val of the brain layer, the separated tissue cysts were concentrated b y diluting the remaining solution with PBS and centrifuging at 500 g. The pellet was resuspended in PBS and tissue cysts were enumerated. Fi fty percent of tissue cysts were recovered from brains centrifuged onc e and 64% from brains centrifuged twice. Tissue cysts were preserved w ith 7.5% dimethyl sulfoxide in horse serum at -60 C. After thawing, br adyzoites were digested in an acid/pepsin solution and placed onto Ver o cell cultures. Neospora caninum tachyzoites were recovered from cell cultures, indicating that bradyzoites retained viability after concen tration and cryopreservation. Separated tissue cysts ranged in diamete r from 107 mu m to 15 mu m (average = 31 mu m), and the average bradyz oite dimensions were 2 x 7.5 mu m. These methods make it possible to s tore viable N. caninum tissue cysts for oral-infectivity trials and ot her studies.