Biological Resource Center for Toxoplasma

(1) Bioresource Overview Project description The Biological Resource Center (BRC) for Toxoplasma gondii is a certified structure (NF S96-900 standard) that manages the storage of Toxoplasma gondii strains (protozoan parasite) and strain derivatives from human or animal toxoplasmosis to make them available to the scientific community. The BRC Toxoplasma works through a network of correspondents who send isolates to the parasitology departments of Reims and Limoges University hospitals (France). The managers are Pr Isabelle VILLENA (Reims) and Pr Marie-Laure DARDE (Limoges). The biological resources are stored in cryobanks located in both sites. Toxoplasma gondii strains which integrate the BRC Toxoplasma collection come from:

The Biological Resource Center (BRC) for Toxoplasma gondii is a certified structure (NF S96-900 standard) that manages the storage of Toxoplasma gondii strains (protozoan parasite) and strain derivatives from human or animal toxoplasmosis to make them available to the scientific community.
The BRC Toxoplasma works through a network of correspondents who send isolates to the parasitology departments of Reims and Limoges University hospitals (France). The managers are Pr Isabelle VILLENA (Reims) and Pr Marie-Laure DARDE (Limoges). The biological resources are stored in cryobanks located in both sites.
Toxoplasma gondii strains which integrate the BRC Toxoplasma collection come from: -The French ToxoBs network -National and international research collaborations -Regular depositors Development of BRC Toxoplasma has been also consolidated by the creation of a National Reference Center of Toxoplasmosis coordinated by Pr I.VILLENA, with an associated laboratory dedicated to Toxoplasma strains in Limoges (France).
Each site possesses its own specificity: cell culture multiplication, storage of DNA extracts from catalog's strains and distribution are managed by Reims's team, microsatellite genotyping and epidemiological data by Limoges's team.
The objectives of the BRC Toxoplasma are: -The heritage conservation of strains and strains derivatives. -The support for research projects thanks to the characterization of strains. -Diversification of the geographical and host species origin of strains by collecting isolates from research teams at a national and international level.
The biobank stores around 1500 strains from different hosts (humans or animals) and from different countries around the world. Strains of human origin are accompanied by clinical data (congenital toxoplasmosis, immunodeficient or immunocompetent patient, severity of clinical form). The online catalog currently offers 37 strains: reference strains (RH, Me49, PRU, VEG) and 33 strains selected for their belonging to the 16 haplogroups currently described for Toxoplasma gondii including 6 highly divergent strains from the Amazonian forest.
For 21 of them, the complete genome is available via ToxoDB and genomic analysis has been published in Lorenzi et al. [5]. The strain geographical origins are diversified: Europe, North and South America, Africa, Caribbean islands.
Toxoplasma gondii strains are available as tachyzoites suspensions obtained in cell culture (8×10 6 /cryovials) and as cysts in mouse's brain suspensions.
Toxoplasma gondii strains and their derivatives (DNA) are available to the research community or industrialists. The order is made via the website of the BRC where a catalog of well-characterized strains is available: www.toxocrb. com.

Classification (2)
Biological samples and associated data.

Temporal coverage for accessibility
Since 2010.

Steps
To obtain Toxoplasma gondii strains, Swiss mice are inoculated with biological samples like amniotic fluid, placenta, blood or broncho-alveolar liquid for strains from human origin, or muscles or brains for strains from animal origin [2,4,7]. Then parasites are isolated from mouse's brain or ascitic fluid and genotyped by 15 microsatellite markers [1]. The isolation protocol has been approved and accepted by the relevant national and regional ethics committees.
According to acceptance criteria set by the BRC (number of cysts, genotyping results and clinical data), isolates are added to the collection.
Bradyzoites are purified by a Percoll gradient in order to initiate cell culture on VERO cells for tachyzoite multiplication.
Strains are stored in liquid nitrogen (-196°C) in 1 mL cryovials [6] (Figure 1) and DNA extracts are stored at -80°C. Each cryovial contains a defined number of parasites (suspensions of tachyzoites or cysts).
The security of the resources is ensured by the permanent temperature monitoring of the storage enclosures: freezers are temperature monitored by a software and the liquid nitrogen level in tanks is regularly monitored by the BRC staff. Moreover, the collection is duplicate in Reims and Limoges. The following data are recorded on BRCLims software [3] and are available for each strain: genotype (microsatellite typing, and for some of them haplogroup membership), mouse virulence, host and tissue origin, date of isolation, geographical origin and disease.
To order a strain, a request must be submitted at the BRC Toxoplasma scientific council (crb.toxobs@unilim. fr) by filling an order form available on the website. After approval by the scientific council, a Material Transfer Agreement is signed and the resources are sent at controlled temperature by a qualified carrier.
The BRC valuation is ensured by the publications that must quote the bank when research is carried out with BRC Toxoplasma strains or derivatives.
To deposit a strain in the BRC Toxoplasma, a request must be submitted by filling a deposit agreement form available on the website. After approval by the direction of the BRC, the biological resources are sent at controlled temperature by a qualified carrier.

Stabilization/preservation
Strains are stored in liquid nitrogen. DNA is stored at -80°C.

Type of sampling
Each cryovial contains 8×10 6 tachyzoites in 1 ml of cryopreserving media, or a precise number of cysts in 1.5 ml, or DNA extract from 8×10 6 tachyzoites in 1 ml tubes.