Biological Resource Center for <i>Toxoplasma</i>

Camila Rocaboy; Marie-Laure Dardé; Dominique Aubert; Aurélien Mercier; Naïma Ortis; Karine Passebosc-Faure; Homayoun Riahi; Sandie Escotte; Martine Gatet; Isabelle Villena

doi:10.5334/ojb.61

(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 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 (8×10⁶/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.

The BRC Toxoplasma belongs to the following networks: Biobanques (INSERM), BBMRI-ERIC, FBRCMi and Club 3C-R.

Classification (1)

Microorganism

Parasite, protozoan

Species

Toxoplasma gondii

Classification (2)

Biological samples and associated data.

Keywords

Toxoplasma gondii, strains, parasite, biological resource center.

Context

Spatial coverage

France, Reims: 49° 15’ 46” North/4° 02’ 05” East

France, Limoges: 45° 51’ 00” North/1° 15’ 00” East

Temporal coverage

Since 2002.

Temporal coverage for accessibility

Since 2010.

(2) Methods

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).

Figure 1

Toxoplasma gondii strains are stored in liquid nitrogen.

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 long-term preservation

Liquid nitrogen

Storage temperature

–80°C; –196°C

Shipping temperature from patient/source to preservation or research use

NA

Shipping temperature from storage to research use

Dry Ice (–80°C)

Quality assurance measures

Parasite revival in cell culture or in mouse after defrosting.

Storage location verification.

Source of associated data

National Reference Center for Toxoplasmosis

Original research works

Ethics Statement

Animals were housed and fed according to the animal care facilities guidelines, and procedures were approved by ethic committees on animal use.

Number of authorization for Reims’ site delivered by the ethic committee for animal experimentation: APAFIS#11913-2017102316076105 v2.

Number of authorization for Limoges’ site delivered by the ethic committee for animal experimentation: APAFIS #15267-2018052909366202 v2.

Constraints

Animals were housed and fed according to the animal care facilities guidelines, and procedures were approved by ethic committees on animal use.

(3) Bioresource description

Object name

Toxoplasma gondii

Bioresource name

Biological Resources Center of Toxoplasma

Bioresource acronym: BRC Toxoplasma

Bioresource location

BRC Toxoplasma

Pôle de Biologie Médicale et Pathologie du CHU de Reims

Laboratoire de Parasitologie

rue Général Koenig

FR 51092 Reims Cedex FRANCE

BRC Toxoplasma

CHU DUPUYTREN - CBRS

Laboratoire de Parasitologie

2 avenue Martin Luther King

FR 87042 Limoges FRANCE

Bioresource contact

crb-reims@wanadoo.fr

Bioresource URL

www.toxocrb.com

Identifier used

(if available: DOI, etc.)

Bioresource type

Tachyzoites or bradyzoites

Type of sampling

Each cryovial contains 8×10⁶ tachyzoites in 1 ml of cryopreserving media, or a precise number of cysts in 1.5 ml, or DNA extract from 8×10⁶ tachyzoites in 1 ml tubes.

Anatomical site

NA

Disease status of patients/source

– Human strains:
- Congenital toxoplasmosis
- Toxoplasmosis in immunodeficient patients (AIDS or transplant)
- Acquired toxoplasmosis in immunocompetent patients
– Animal strains
- Chronic infection
- Abortion

Clinical characteristics of patients/source

NA

Size of the bioresource

The biobank stores around 1500 strains from different hosts (humans or animals): 1000 human strains and 500 from different animal species.

BRC Toxoplasma includes 30–60 new strains per year.

Vital state of patients/source

NA

Clinical diagnosis of patients/source

NA

Pathology diagnosis

NA

Control samples

Cysts and tachyzoites number per cryovial.

Mycoplasma detection.

Microsatellite genotyping before and after cell culture.

Biospecimen type

Microorganism class B and DNA

Release date

Since 2010

Access criteria

The catalogue of BRC Toxoplasma is built on several criteria validated by the scientific council. General conditions are available on the website. All requests are recorded.

(4) Reuse potential

NA

[B1] Ajzenberg D, Collinet F, Mercier A, Vignoles P, Dardé, ML. Genotyping of Toxoplasma gondii isolates with 15 microsatellite markers in a single multiplex PCR assay. J. Clin. Microbiol. 2010; 48: 4641–4645. DOI: https://doi.org/10.1128/JCM.01152-10

[B2] Aubert D, Ajzenberg D, Richomme C, Gilot-Fromont E, Terrier ME, De Gevigney C, Game Y, Maillard D, Gibert P, Dardé ML, Villena I. Molecular and bio¬logical characteristics of Toxoplasma gondii isolates from wildlife in France. Veterinary parasitology. 2010; 171(3–4): 346–349. DOI: https://doi.org/10.1016/j.vetpar.2010.03.033

[B3] Demay S, Begaud E, Raynal-Malchy E, Talignani L, Casaregola S, Fave lA, Briand M, Valence-Bertel F, Dardé ML, Villena I, Guyonneaud R, Pressigout M, Bizet C. BRC-LIMS: software for the management of biological resource Centres of microorganism. London: ESBB, 2015, 29 sept–2th Oct.

[B4] Desmonts G, Couvreur J. L’isolement du parasite dans la toxoplasmose congénitale: intérêt pratique et théorique. Arch. Fr. Pédiatr. 1974; 31: 157–166.

[B5] Lorenzi H, Khan A, Behnke MS, Namasivayam S, Swapna LS, Hadjithomas M, Karamycheva S, Pinney D, Brunk BP, Ajioka JW, Ajzenberg D, Boothroyd JC, Boyle JP, Dardé ML, Diaz-Miranda MA, Dubey JP, Fritz HM, Gennari SM, Gregory BD, Kim K, Saeij JP, Su C, White MW, Zhu XQ, Howe DK, Rosenthal BM, Grigg ME, Parkinson J, Liu L, Kissinger JC, Roos DS, Sibley LD. Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Tox¬oplasma gondii genomes. Nature communications. 2016; 7: 10147. DOI: https://doi.org/10.1038/ncomms10147

[B6] Mzabi A, Escotte-Binet S, Le Naour R, Ortis N, Audonnet S, Dardé ML, Aubert D, Villena I. Optimi¬zation of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry. Cryobiology. 2015; 71(3): 459–463. DOI: https://doi.org/10.1016/j.cryobiol.2015.09.002

[B7] Richomme C, Aubert D, Gilot-Fromont E, Ajzenberg D, Mercier A, Ducrot C, Ferté H, Delorme D, Villena I. Genetic characterization of Toxoplasma gondii from wild boar (Sus scrofa) in France. Veterinary Parasitol¬ogy. 2009; 164(2–4): 296–300. DOI: https://doi.org/10.1016/j.vetpar.2009.06.014

Reading: Biological Resource Center for Toxoplasma

Bioresource Papers

Biological Resource Center for Toxoplasma

Authors:

Camila Rocaboy ,

CHU de Reims, FR

Marie-Laure Dardé,

CHU de Limoges; Université de Limoges, FR

Dominique Aubert,

CHU de Reims; Université de Reims Champagne-Ardenne, EA 7510 ESCAPE, FR

Aurélien Mercier,

Université de Limoges, FR

Naïma Ortis,