Epidemiology of toxoplasmosis: role of the tick Haemaphysalis longicornis

Survival and infectivity of T. gondii in a tick’s body

The transgenic RH/GFP strain of T. gondii (kindly provided by Dr. Xuan, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan) was maintained in human foreskin fibroblasts cells cultured in Dulbecco’s Modified Eagle Medium (Gibco® DMEM), with 10 % of fetal bovine serum(FBS) at 37 °C in a 5 % CO₂ incubator. Purification of tachyzoites was performed, as described previously [18]. Parasites and cultured cell debris were washed several times in cold phosphate-buffered saline (PBS), and the resulting pellets were resuspended in cold PBS and passed through a 27-gauge needle and a 5.0 ?m pore filter (Millipore Corp., Billerica, MA, USA). The H. longicornis ticks were maintained in our laboratory. A single engorged female was used to establish the tick colony. A colony of parthenogenesis H. longicornis ticks was initiated from one engorged female collected from a deer at the Shanghai Wildlife Park, China. Ticks were reared in a dark incubator at 25 °C, with 92 % relative humidity and fed on a New Zealand white rabbit. After three generations under laboratory conditions, the tick colony was established at the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China [15].

Parasite injection was performed as previously described with some modifications [19]. The injections were done using 10 ?L microcapillaries (Drummond Scientific, Broomall, PA, USA) drawn to fine-point needles using a micropipette puller (Narishige, Tokyo, Japan). The needles were then loaded onto a microinjector (Narishige, Tokyo, Japan). T. gondii (10³) in 0.5 ?l of PBS and 0.5 ?l of the buffer alone were microinjected from fourth coxae into the haemocoel of unfed adult ticks fixed on a glass slide with adhesive tape in experimental and control groups, respectively. Injected ticks were incubated for three, five, seven, 10, 15, 20, and 30 days at 25 °C, and then DNA was purified from pools (10 ticks) of injected ticks for qPCR detection. The 529-bp DNA fragment in T. gondii can be used as a quantitative target for parasite numbers [16, 17]; the relative amount of the target of the 529-bp element of T. gondii compared to the tick actin gene (tick house-keeping gene) is regarded as the marker of the number of parasites in ticks. After microinjection, the tick’s body lyses were also observed for live parasites using a fluorescence microscope.

Infectivity of T. gondii in ticks was confirmed as follows: Ticks, previously injected with T. gondii, were homogenized (groups of 10 adults) in 1.5 ml of normal saline (0.9 % NaCl) containing penicillin and streptomycin. Ticks, previously injected with buffer PBS, were homogenized as controls. Mice (BALB/c, eight weeks old) were injected (0.5 ml/mouse, three mice) intraperitoneally with the tick homogenate in antibiotic solution. Injected mice were observed daily. When clinical symptoms occurred in mice that were killed, such as apathy, depression, and abdominal swelling, appeared, peritoneal dropsy was performed to observe for live parasites using a fluorescence microscope.