Toxoplasma gondii subsp. isolation

Gamble, Amandine, Ramos, Raül, Parra-Torres, Yaiza, Mercier, Aurélien, Galal, Lokman, Pearce-Duvet, Jessica, Villena, Isabelle, Montalvo, Tomás, González-Solís, Jacob, Hammouda, Abdessalem, Oro, Daniel, Selmi, Slaheddine & Boulinier, Thierry, 2019, Exposure of yellow-legged gulls to Toxoplasma gondii along the Western Mediterranean coasts: Tales from a sentinel, International Journal for Parasitology: Parasites and Wildlife 8, pp. 221-228 : 224

2.5. T. gondii isolation and genotyping

Tissues for parasite isolation were collected from the necropsied gulls that tested positive for anti- T. gondii antibodies. Brains and cardiac muscles were prepared and inoculated intraperitoneally to three female Swiss mice (Mus musculus) following the procedure described in Mercier et al. (2010). Given the low parasitic burden found in the tissues of most T. gondii hosts, mouse bioassay was necessary for an upstream amplification of parasitic burden to reach the sensitivity thresholds of most genotyping techniques. Considering the risk of bacterial proliferation due to the sampling conditions and the decomposition state of the gull samples, those were treated with an antibiotic solution (1000 U/ml penicillin and 100 μg streptomycin/ml in saline solution) before inoculation. A 200 μl aliquot of the gull brain and heart preparations was also used for DNA extraction using the QIAamp ª DNA MiniKit (Qiagen, France) and quantification of T. gondii by real-time PCR targeting the T. gondii 529 bp repetitive element as previously described (Homan et al., 2000). The mice were monitored daily and all surviving mice were euthanized at four weeks post-inoculation. Brain from the MAT-positive mice were screened for cysts by microscopic examination. Live parasites were cryopreserved until analysed. DNA from 200 μl of mouse brain tissue was extracted and T. gondii isolates were genotyped using the length polymorphism of 15 multilocus microsatellite markers located on 11 different chromosomes in a multiplex PCR assays as detailed in Ajzenberg et al. (2010). All experimental procedures in mice were conducted according to European guidelines for animal care (‘‘Journal Officiel des Communautés Européennes’’, L358, December 18, 1986) and approved by the Regional Ethics Committee Limousin (Registration Number: CREEAL 3-07-2012).