Research | coolp

Overview

Coccidian parasites (Eimeria, Toxoplasma) and their relatives (Cryptosporidium) rely on their oocyst form to infect numerous host species including humans (Dumètre et al. 2012; Shapiro et al. 2019) We try to better understand how oocysts remain infectious when exposed to chemical and physical stress outside and then inside the host, by probing the structural, mechanical and adhesion properties of the oocyst wall using quantitative imaging and biophysical approaches.

We hope our research will help identify new intervention strategies to reduce environmental contamination by these parasites.

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Imaging the oocyst

The size of oocysts, ranging from 4 to 40 µm for most species, and their refringence make them easy to visualize under simple microscopes. This can allow to observe oocyst distorsion and large openings in their walls. To gain further into potential changes in parasite morphology and wall structure following exposition to chemical and physical stress, we exploit the natural blue fluorescence of the coccidian oocyst walls and/or use fluorescent markers such as antibodies or lectins as proxies for the wall ultrastructure (Freppel et al. 2019). In particular, a decrease in wall autofluorescence intensity is linked to the loss of the outer wall layer in Eimeria and Toxoplasma oocysts as revealed by transmission electron microscopy (Dumètre et al. 2013; 2021). By applying AI-based image analyses tools such as Stardist and Celldetective, we can quantify the structure of oocysts based on their apparent size, area and autofluorescence. This greatly helps us to screen chemical and physical factors that can visibly affect the overall parasite morphology.

Multiscale characterization of the oocyst mechanics and adhesion

We hypothesize that oocysts can resist harsh conditions due to their capacity to retain their integrity. To address this hypothesis, we adapt single-cell force measurement techniques, e.g. atomic force microscopy (AFM) and microindentation, coupled to imaging to provide a quantitative characterization of the oocyst mechanics and the forces required for deforming even breaking at the wall layer scale (~100 nm) and whole oocyst scale. With these tools, we have started to characterize the resistance of the parasites to chlorinated disinfectants and demonstrated that the inner oocyst wall layer is the key wall structure that sustains the integrity and potential infectivity of oocysts facing adverse conditions.

In addition to mechanics, our previous AFM experiments revealed that environmental factors can modify adhesion of oocysts, e.g. household bleach making them stickier (Dumètre et al. 2013). We hypothesize that higher adhesion of the parasites allows them to be retained and even concentrated in food, water sediments and soils in conjunction with the physicochemical parameters of these environmental matrices (Dumètre et al. 2012; Kinsey et al. 2023).

We are now looking at similar or divergent mechanical and adhesion behaviors in oocysts exposed to thermal treatment or other chemical disinfectants such as ozone.

Mechanical indentation and rupture of the walls of a T. gondii oocyst by using a glass microneedle. Scale bar = 5 µm. From Freppel et al. (2019).

Phagocytosis of Toxoplasma gondii oocysts by RAW 264.7 macrophages assessed by:

(A) micromanipulation using glass micropipettes under aspiration. Bar = 5 µm

(B) fluorescence imaging showing in blue, DAPI macrophage nucleus and autofluorescent oocysts (arrow), and in red phalloidin-stained actin filaments of the macrophages). Bar = 10 µm

(C,D) transmission electron microscopy showing (C) sporozoites (Sp), an unsporulated oocyst (N), and openings in the oocyst and sporocyst walls (arrows), and (D) tachyzoites developing within the macrophage. Bar = 1 µm. From Freppel et al. (2016)

Implications for preventing the waterborne and foodborne transmission of oocysts

We study how T. gondii and C. parvum oocysts survive environmental conditions and common household and industrial inactivation treatments including chlorination and ozone. We explore the effects of such treatments on the structure, permeability, and mechanics of the parasites, and in collaboration with parasitologists and 'omics' experts, we investigate whether treatments modify the oocyst transcriptome, proteome, and infectivity. To this aim, we contribute to develop alternative approaches to assess the infectivity of native vs. treated parasites by using cell culture coupled to qPCR and surrogate models. In particular, we aim at determining whether oocysts of the chicken E. acervulina and mouse E. papillata, which are produced more easily than T. gondii oocysts, can be used as non pathogenic surrogate models of T. gondii to assess the efficacy of physical and chemical decontamination treatments.

Implications for host infection by oocysts

Following ingestion with water or food, oocysts travel rapidly in the host digestive tract down to the small intestine where the oocyst wall has to open to release the infective forms (sporozoites) it contains. Depending on the coccidian species, this process has been recognized to likely involve physical (mechanical) stimuli (of unknown nature) and/or the action of digestive factors from the host (enzymes and biliary salts) or the parasite (sporozoite proteases). However, in certain coccidian species such as Toxoplasma, parenteral inoculation of oocysts can lead to the same burden of infection as the digestive route, suggesting that the digestive microenvironment is dispensable for a successful infection (Freppel et al., 2016), and that host cells such as phagocytes may contribute to initiating infection.

As a complementary approach, we are interested in the contribution of phagocytic cells such as macrophages and neutrophils in processing the oocyst wall and hosting parasite development by studying, using micropipettes and optical tweezer techniques, the dynamics of oocyst internalization by phagocytes and the ability of parasites to overcome degradation to replicate and/or use these cells as Trojan horses (Freppel et al., 2016; Ndao et al., 2020).

Selected publications (click on the title to get full text)

Dynamics of Toxoplasma gondii oocyst phagocytosis by macrophages. Ndao O, Puech PH, Bérard C, Limozin L, Rabhi S, Azas N, Dubey JP, Dumètre A. Frontiers in Cellular and Infection Microbiology 2020; 10: 1-9
Structure, composition, and roles of Toxoplasma gondii oocyst and sporocyst walls. Freppel W, Ferguson DJP, Shapiro K, Dubey JP, Puech PH, Dumètre A. The Cell Surface 2019; 5:100016.
Macrophages facilitate the excystation and differentiation of Toxoplasma gondii sporozoites into tachyzoites following oocyst internalisation. Freppel W, Puech PH, Ferguson DJP, Azas N, Dubey JP, Dumètre A. Scientific Reports 2016; 6:33654.

Selected publications (click on the title to get full text)

Surrogates of foodborne and waterborne protozoan parasites: a review. Augendre L, Costa D, Escoote-Binet S, Aubert D, Villena I, Dumètre A, La Carbona S. Food and Waterborne Parasitology 2023; 33:e00212.
Effect of household bleach on the structure of the sporocyst wall of Toxoplasma gondii. Dumètre A, Dubey JP, Ferguson DJP. Parasite 2021 28: 68
Toxoplasma gondii oocyst infectivity assessed using a sporocyst-based cell culture assay combined with quantitative PCR for environmental applications. Rousseau A, Escotte-Binet S, La Carbona S, Dumètre A, Chagneau S, Favennec L, Kubina S, Dubey JP, Majou D, Bigot-Clivot A, Villena I, Aubert D. Applied and Environmental Microbiology 2019; 85(20):e01189-19.
Evaluation of propidium monoazide-based qPCR to detect viable oocysts of Toxoplasma gondii. Rousseau A, Villena I, Dumètre A, Escotte-Binet S, Favennec L, Dubey JP, La Carbona S. Parasitology Research 2019.
Assessing viability and infectivity of foodborne and waterborne stages (cysts/oocysts) of Giardia duodenalis, Cryptosporidium spp., and Toxoplasma gondii: a review of methods. Rousseau A, La Carbona S, Dumètre A, Robertson LJ, Gargala G, Escotte-Binet S, Favennec L, Villena I, Gérard C, Aubert D. Parasite 2018; 25:14.
Simultaneous detection of the protozoan parasites Toxoplasma, Cryptosporidium and Giardia in food matrices and their persistence on basil leaves. Hohweyer J, Cazeaux C, Travaillé E, Languet E, Dumètre A, Aubert D, Terryn C, Dubey JP, Azas N, Houssin M, Favennec L, Villena I, La Carbona S. Food Microbiology 2016; 57:36-44.
Development of a qRT-PCR method to assess the viability of Giardia intestinalis cysts, Cryptosporidium spp., and Toxoplasma gondii oocysts. Travaillé E, La Carbona S, Gargala G, Aubert D, Guyot K, Dumètre A, Villena I, Houssin M. Food Control 2016; 59:359-65.
Effects of ozone and ultraviolet radiation treatments on the infectivity of Toxoplasma gondii oocysts. Dumètre A, Le Bras C, Baffet M, Meneceur P, Dubey JP, Derouin F, Duguet JP, Joyeux M, Moulin L. Veterinary Parasitology 2008; 153(3-4):209-13.

Transport dynamics of T. gondii oocysts in soils

We study the fate and transport of oocysts in soils as a function of soil physicochemical properties and soil water chemistry properties. For this, we apply oocysts onto columns, which contain with sand, natural loamy sand soils or sandy loam soils, and subject them to artificial rainfall in absence or presence of surfactants, monovalent and divalent cations, and humic substances at different concentrations. Quantitative polymerase chain reaction (qPCR) is used to detect and numerate oocysts in soil leachates to evaluate their breakthrough and in soil matrices to examine their spatial distribution. We are interested in differences in the rate and extent of transport of oocysts as a function of physical and chemical parameters tested.

Selected publications (click on the title to get full text)

Detection, fate, and transport of the biohazardous agent Toxoplasma gondii in soil water systems: Influence of soil physicochemical properties, water chemistry, and surfactant. Kinsey E, Korte C, Gouasmia S, L'Ollivier C, Dubey JP, Dumètre A, Darnault CJG. Environmental Microbiology Reports 2023; 15:596-613.
Environmental transmission of Toxoplasma gondii: oocysts in water, soil, and food. Shapiro K, Bahia-Oliveira L, Dixon B, Dumètre A, de Wit LA, VanWormer E, Villena I. Food and Waterborne Parasitology 2019; 12:e00049.
Interaction forces drive the environmental transmission of pathogenic protozoa. Dumètre A, Aubert D, Puech PH, Hohweyer J, Azas N, Villena I. Applied and Environmental Microbiology 2012; 78(4):905-12.
Quantitative estimation of the viability of Toxoplasma gondii oocysts in soil. Lélu M, Villena I, Dardé ML, Aubert D, Geers R, Dupuis E, Marnef F, Poulle ML, Gotteland C, Dumètre A, Gilot-Fromont E. Applied and Environmental Microbiology 2012; 78(15):5127-32.
Development of a sensitive method for Toxoplasma gondii oocyst extraction in soil. Lélu M, Gilot-Fromont E, Aubert D, Richaume A, Afonso E, Dupuis E, Gotteland C, Marnef F, Poulle ML, Dumètre A, Thulliez P, Dardé ML, Villena I. Veterinary Parasitology 2011; 183(1-2):59-67.
Detection of Toxoplasma gondii oocysts in environmental soil samples using molecular methods. Lass A, Pietkiewicz H, Modzelewska E, Dumètre A, Szostakowska B, Myjak P. European Journal of Clinical Microbiology and Infectious Diseases 2009; 28(6):599-605.

Collaborations & Fundings

Nothing possible without these following people and labs:

Pierre-Henri Puech, Laurent Limozin @ LAI Marseille, Julien Husson@ Polytechnique Institute Paris, Mattie Pawlowic @ Dundee: Imaging and mechanics of oocysts
David Ferguson @ Oxford Univ and Artemis Kosta @ IMM Marseille: Electron microscopy
Isabelle Villena @ Reims Univ, Anne Silvestre@ INRAE Tours, Stéphanie La Carbona @ Actalia Company: 0ocyst production, treatments, and infectivity assays in different matrices
Christophe Darnault @ Clemson Univ: Oocyst transport in soils

... and thanks to funders: ANR, Région PACA, AMU, CNRS, INSERM, ...