Toxoplasma gondii, (Nicolle & Manceaux, 1908)

Blaga, Radu, Aubert, Dominique, Thébault, Anne, Perret, Catherine, Geers, Régine, Thomas, Myriam, Alliot, Annie, Djokic, Vitomir, Ortis, Naïma, Halos, Lénaïg, Durand, Benoît, Mercier, Aurélien, Villena, Isabelle & Boireau, Pascal, 2019, Toxoplasma gondii in beef consumed in France: regional variation in seroprevalence and parasite isolation, Parasite (Paris, France) 26 (77), pp. 1-14 : 4

Bioassay for T. gondii in mice

The in vivo experiments were approved by the local Animal Research Ethics Committee (ComEth Anses, EnvA, UPE) of Maisons-Alfort. In order to limit suffering and distress, mice were acclimated for 7 days after their arrival. Cages were filled with paper strips. Animal health and behaviour were monitored daily. Mice were observed based on the following criteria:

– external physical appearance (disheveled or spiked hairs, watering eyes, bent back, tremors),

– behaviour (exploratory behaviour decrease, unusual posture, prostration),

– behavioural response to external stimuli (no response).

If any of these criteria were critically altered, mice were subsequently euthanised and examined post-mortem to investigate the parasitic load. Euthanasia consisted in CO 2 asphyxiation, followed by cervical dislocation.

After approval from the local Animal Research Ethics Committee, between 5 and 11 heart samples, randomly chosen from among those with the highest titers of agglutinating antibodies, were bioassayed weekly in three outbred female Swiss Webster mice (Charles River Laboratory, France). Additionally, nine seronegative hearts, randomly selected from the total number of samples, were bioassayed in mice. Briefly, each whole heart was mixed and incubated at 37 °C for 1.5 h with trypsin (final concentration 0.25%). The suspension was then filtered, pelleted by centrifugation, washed in saline, and resuspended in a saline solution containing penicillin G, streptomycin and amoxicillin to limit bacterial proliferation. This homogenate was inoculated intraperitoneally into three mice [ 1, 59]. Mice were monitored twice daily with food and water supplied ad libitum. In case of acute toxoplasmosis, the mice were culled by CO 2 asphyxiation, followed by cervical dislocation, and samples of brain were taken for analysis. Mice were bled 4 weeks post-inoculation and their serum was tested at 1:25 dilution for T. gondii antibodies with the MAT. Later on, mice were culled 60 days post-inoculation by cervical dislocation, and their brains were examined for tissue cysts.

Genotyping of T. gondii isolates

Brain cysts from seropositive mice were isolated by percoll gradient centrifugation [ 15]. DNA was extracted using a QIAamp DNA MiniKit (Qiagen, Courtaboeuf, France), and genotyping analysis of T. gondii DNA was performed with 15 microsatellite markers in a single multiplex PCR assay, as described elsewhere [ 3]. All strains isolated were cell cultivated and banked in the Toxoplasma Biological Resource Center ( BRC, Reims).