# Eimeria acervulina is a promising surrogate for Toxoplasma gondii oocysts exposed to chemical and physical treatments

Laure Augendre, Sandie Escotte-Binet, Dominique Aubert, Isabelle Villena, Jean-Michel Répérant, Stéphanie La Carbona, Aurélien Dumètre

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## To cite this version:

Laure Augendre, Sandie Escotte-Binet, Dominique Aubert, Isabelle Villena, Jean-Michel Répérant, et al.. Eimeria acervulina is a promising surrogate for Toxoplasma gondii oocysts exposed to chemical and physical treatments. Experimental Parasitology, 2025, 278, pp.109049. 10.1016/j.exppara.2025.109049. anses-05387569

HAL Id: anses-05387569

https://anses.hal.science/anses-05387569v1

Submitted on 23 Jan 2026

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# *Eimeria acervulina* is a promising surrogate for *Toxoplasma gondii* oocysts exposed to chemical and physical treatments

Laure Augendre<sup>a,b</sup>, Sandie Escotte-Binet<sup>a</sup>, Dominique Aubert<sup>a</sup>, Isabelle Villena<sup>a</sup>, Jean-Michel Répérant<sup>c</sup>, Stéphanie La Carbona<sup>b</sup>, Aurélien Dumètre<sup>d,*</sup>

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## ARTICLE INFO

**Keywords:**

*Eimeria acervulina*

*Toxoplasma gondii*

Coccidian oocysts

Surrogates

Food decontamination processes

Inactivation

## ABSTRACT

Oocysts of the coccidian parasite *Toxoplasma gondii* withstand a wide range of chemical and physical factors, contributing to their food- and water-borne transmission to humans worldwide. Assessing the efficacy of processes to remove or inactivate oocysts at a pilot or industrial scale encounters major ethical, economic, and methodological constraints. The coccidian parasite *Eimeria acervulina* has been proposed as a non-human pathogenic alternative of *T. gondii* to assess food decontamination, however it is not known whether the two parasites exposed to chemical and thermal treatments parallel in terms of oocyst structure and infectivity. Using bioassays and lectin-based assays combined with flow cytometry and fluorescence microscopy analyses, this study shows that *E. acervulina* and *T. gondii* oocysts display similar response to heating and/or freezing and bleach or NaOH treatments, as in maintaining infectivity, with *E. acervulina* oocysts retaining their size and structure better than *T. gondii*. Collectively, our results suggest that *E. acervulina* is a reliable model for studying the response of *T. gondii* oocysts to certain chemical and physical agents. It could therefore serve as an affordable, non-pathogenic substitute for *T. gondii* when evaluating food decontamination processes, particularly in industrial settings.

## 1. Introduction

*Toxoplasma gondii* is a coccidian parasite that causes toxoplasmosis in humans and other warm-blooded animals (Dubey, 2010). It is one of the most widespread parasitic infections worldwide, with a seroprevalence in humans of approximately 30% (Robert-Gangneux and Dardé, 2012). Infection occurs mainly by eating undercooked meat containing the cysts of the parasite, or through the consumption of water and raw fruits and vegetables contaminated with oocysts (Shapiro et al., 2019). Oocysts are the environmentally resistant forms of the parasite that are excreted exclusively by domestic cats and wild felids. Though the contribution of each route to human contamination remains largely unknown, several epidemiological studies have highlighted that both endemic and epidemic food- and water-borne oocyst-related infections might be more frequent than previously thought, notably in South America (reviewed by (Álvarez García et al., 2021; Pinto-Ferreira et al.,

2019)). Irrespective of the route of contamination, *T. gondii* infections are generally asymptomatic in immunocompetent people, whereas they can cause severe neurological and ocular diseases in immunocompromised individuals and congenitally infected foetuses (Robert-Gangneux and Dardé, 2012).

While *T. gondii* cysts can be relatively easily killed by cooking meat properly, oocysts are much more difficult to inactivate (Mirza Alizadeh et al., 2018). They can survive for weeks, even years, under most environmental conditions encountered in soils, fresh and marine waters, foodstuffs and fresh produce (Shapiro et al., 2019). In addition, they withstand common chemical inactivation agents used for surface, food and water decontamination (Gérard et al., 2019; Jones and Dubey, 2010; Mirza Alizadeh et al., 2018). The resistance of oocysts to adverse conditions is attributed to the bilayered structure, mechanics, and molecules of their oocyst and sporocyst walls (Dumètre et al., 2013, 2021) and to Late Embryogenesis Abundant (LEA) proteins the oocysts

<footnote><sup>a</sup> EA 7510 ESCAPE Epidemiosurveillance and Circulation of Parasites in the Environment, University of Reims Champagne-Ardenne, Faculty of Medicine, 51 Rue Cognacq Jay, 51096, Reims, France</footnote>

<footnote><sup>b</sup> ACTALIA Food Safety, 310 Rue Popielujko, Saint-Lô, 50000, France</footnote>

<footnote><sup>c</sup> ANSES, Virology, Immunology, Avian and Rabbit Parasitology Unit, Ploufragan-Plouzané-Niort Laboratory, BP 53 Ploufragan, 22440, France</footnote>

<footnote><sup>d</sup> Aix Marseille University, CNRS, INSERM, Laboratory Adhesion and Inflammation (LAI), 13009, Marseille, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: aurelien.dumetre@univ-amu.fr (A. Dumètre).</footnote>
express under certain environmental stress conditions (Arranz-Solís et al., 2023).

To minimize human exposure to foodborne pathogens, the food industry implements control measures, i.e. chemical and/or physical treatments, to remove or inactivate them to guarantee safe final produces. However, assessing the efficacy of such processes on *T. gondii* oocysts from laboratory to industrial scales is hampered by various ethical, economic, and methodological constraints. Indeed, (i) large quantities of oocysts are required, which can only be obtained by infecting laboratory cats (Martorelli Di Genova et al., 2019), (ii) handling oocysts is biohazardous due to their low infectious dose, estimated between 1 and 10 oocysts in pig and rodent models (Dubey et al., 1996, 1997), and (iii) there are no reliable, accessible, and faster alternatives to mouse bioassay as the gold standard to assess oocyst infectivity (Rousseau et al., 2018), which is in practice impossible to implement in routine laboratories for parasite infectivity assessment in food samples. In this context, researchers are increasingly seeking safe and reliable alternatives to reduce the use of *T. gondii* oocysts for research and development purposes in the food domain (Augendre et al., 2023).

The coccidian *Eimeria* parasites have emerged as potential surrogates to improve methods to detect oocysts of *T. gondii* and its closely relative *Cyclospora cayetanensis* in food samples (Lalonde and Gajadhar, 2016a, 2016b) and to study their persistence and infectivity following storage and decontamination procedures (Kniel et al., 2007; Lee and Lee, 2001, 2003). *Eimeria* spp. can infect a wide range of domestic and wild animals but not humans (Martorelli Di Genova and Knoll, 2020). *Eimeria acervulina*, *E. maxima*, and *E. tenella*, which cause coccidiosis in chicken, are the best characterized species and can be relatively easily produced in chickens raised in experimental facilities. *E. acervulina* and *T. gondii* oocysts share similar size (18 × 14 µm and 13 × 11 µm, respectively), structure and molecular composition, in particular of their oocyst wall (Freppel et al., 2019; Mai et al., 2009; Samuelson et al., 2013). However, the two parasites notably differ in the organization of the sporozoites, the oocyst and sporocyst walls surround: *E. acervulina* contains four sporocysts with two sporozoites each versus two sporocysts with four sporozoites each in *T. gondii*. *Eimeria* oocysts appear to resist the same range of environmental conditions and physical and chemical inactivation procedures as *T. gondii* oocysts, thanks to their oocyst wall (Bellı et al., 2006; Daugschies et al., 2013; Long, 1982; Shearer et al., 2007).

With the perspective of proposing *E. acervulina* oocysts as suitable substitutes for *T. gondii* to assess the efficacy of decontamination treatments applied to food matrices, the objective of this study was to characterize and compare the integrity of the wall and the infectivity of *E. acervulina* and *T. gondii* oocysts exposed to various chemical and thermal treatment conditions. To this end, we applied treatments that were previously shown to affect the wall properties and/or the infectivity of *T. gondii* oocysts (Dubey et al., 1970; Dumètre et al., 2013, 2021).

## 2. Materials and methods

All methods were performed in accordance with the relevant guidelines and regulations.

### 2.1. Parasites

*Eimeria acervulina* oocysts of P3A strain (originally isolated and purified in 2000 in ANSES Ploufragan, France, and since maintained in this laboratory) (Répérant et al., 2021) were produced following an experimental procedure approved by the French Ministry of Agriculture under the agreement number #34204-2021120116495122V3. Briefly, 23-day-old ISA Brown chickens (ISA Hendrix, Mûr-de-Bretagne, France) were orally inoculated with 5000 oocysts in 1 ml saline solution. Four to six days post infection (dpi), droppings were collected and oocysts were purified by flotation on a saturated NaCl solution (specific gravity 1.18)

(Répérant et al., 2021). The sporulation process was carried out by depositing the oocysts in a 2 % potassium dichromate ($K_2Cr_2O_7$) solution in a thin layer on sterile Petri dishes containing agar-agar that were subsequently incubated in an oven at 28 °C and 65 % humidity for 2–3 days (Tong and Yang, 1990). Drying out of the agar plates was prevented by daily addition of a 2 % $K_2Cr_2O_7$ solution. The progress of sporulation was monitored microscopically every day by observing the contents of the plates, until ~95 % oocysts were sporulated. Then, oocysts were carefully aspirated from the plate surface, and the plates were washed several times with 2 % $K_2Cr_2O_7$ solution to ensure complete recovery of the oocysts. After that, oocysts were counted on a McMaster cell counting chamber using an Olympus CX33 microscope at x400 magnification under bright field. The oocyst batch was stored in the 2 % $K_2Cr_2O_7$ solution at 4 °C and used for all the experiments within 6 months of production.

The ME49 genotype II strain (purchased as frozen cysts from the Biological Resources Centre for Toxoplasma, Reims, France) was used to produce *T. gondii* oocysts. For this, a 3-month-old *T. gondii* free cat (purchased from Isoquimen company, Barcelona, Spain) was infected following an experimental procedure approved by the ethical committee of University Reims Champagne-Ardenne under the agreement number B51-454-3 and the APAFIS project #35705-2022030311362368v3. After a one-week acclimation period, the cat was food withheld for 8 h before being orally infected with approximately 3200 cysts in a brain homogenate from 3 infected laboratory mice (purchased from Charles River Laboratory, Neuilly-sur-Seine, France). Cat faeces were then collected daily between 2 and 16 dpi and were processed for oocyst purification using the sucrose flotation technique (specific gravity 1.2) (Villena et al., 2004). Purified oocysts were then allowed to sporulate in a 2 % sulfuric acid ($H_2SO_4$) aqueous solution at room temperature (RT) for at least 7 days under constant agitation and aeration. The sporulation rate was about 40 %. Both unsporulated and sporulated oocysts were enumerated microscopically on a Kova slide using a Zeiss Axioskop 40 microscope at x400 magnification under bright field. The oocyst batch was stored in a 2 % $H_2SO_4$ aqueous solution at 4 °C and used for all experiments within 6 months after production.

### 2.2. Oocyst treatments

Prior to any treatments, oocysts were washed three times in sterile distilled water ($dH_2O$) and once in PBS at 5000 g for 5 min to remove $K_2Cr_2O_7$ (*E. acervulina*) or $H_2SO_4$ (*T. gondii*). Then, oocysts were enumerated microscopically on a Kova slide under bright field and epifluorescence under UV excitation, using a Zeiss Axioskop 40 microscope at x400 magnification equipped with suitable epifluorescence filters ($\lambda_{exc}$ 330–380 nm/$\lambda_{em}$ 420 nm) for detecting their blue autofluorescence (Bellı et al., 2006).

For chemical treatments, the parasites were incubated with 1 ml of household bleach solution (Lacroix Javel, 131181) containing 3.6 % active chlorine for 30 min at 4 °C, or 1 ml of NaOH 20 % for 1 h at RT. Oocysts were then washed three times in PBS at 5000 g for 5 min to remove bleach or NaOH and stored at 4 °C in 500 µL PBS before further experiments. For thermal treatments, oocysts in 50 µL PBS were either (i) heated in a dry block heater for 5 min at 95 °C, and then cooled at RT for 20 min, (ii) incubated for 48 h at –80 °C, and then thawed at RT for 1 h, or (iii) submitted to six successive thermal shocks (5 min, –80 °C/4 min, 99 °C) prior to be cooled for 1 h at RT. Irrespective of the treatments, oocysts were then stored at 4 °C until further analyses.

### 2.3. Lectin-binding assay

The integrity of the walls of untreated and treated oocysts of *E. acervulina* and *T. gondii* was characterized by incubating the parasites with FITC-conjugated Wheat germ agglutinin (WGA), Maclura pomifera (MPL) or *Bauhinia purpurea* (BP) lectins (CliniSciences, France) that have been shown to differentially bind to the oocyst, the sporocyst, and/or the
sporozoite respectively (Bushkin et al., 2012; Dumètre et al., 2021; Fabian et al., 2021; Fuller and McDougald, 2002; Harito et al., 2016). We assumed that lectin labelling was only possible if the structures the lectins recognize were accessible after stripping of the oocyst surface and/or infiltration through the wall due to loss of integrity. Lectins were used at a final concentration of 25 µg/ml (MPL) and 50 µg/ml (WGA and BP) in PBS according to the manufacturer recommendations. After a 1 h incubation at 37 °C, oocysts were washed three times in PBS at 5000 g for 5 min and examined for FITC fluorescence and oocyst wall autofluorescence by microscopy and flow cytometry (FCM). Data presented are representative of five independent experiments of treatments performed, for each parasite species, on the same batch of oocysts.

## 2.4. Fluorescence microscopy

Parasites were observed for FITC-lectin labelling as wet mounts using a Zeiss Axio observer microscope at x400 magnification equipped with a plan-Apochromat 63x/1.4 Oil DIC M27 objective and piloted by Meta-Morph™ (Gataca Systems) for fluorescence acquisition. Samples were examined for oocyst autofluorescence and FITC fluorescence using the appropriate filter sets ($\lambda_{exc}$ 335 nm/$\lambda_{em}$ 450 nm and $\lambda_{exc}$ 450 nm/$\lambda_{em}$ 490 nm, respectively).

## 2.5. Flow cytometry

Oocysts were transferred in FACS tubes in a final volume of 500 µl and analysed using an Accuri™ C6 flow cytometer (Becton Dickinson) equipped with UV 375 nm- (on FL2 and FL4 channels) and blue 473 nm-lasers (on FL1 and FL3 channels) for oocyst autofluorescence and FITC-lectin detection respectively. At least 10⁴ events were acquired per sample and treatment condition. The oocyst population was gated based on Forward Scatter-Area (FSC-A) for oocyst size and Side Scatter-Area (SSC-A) for oocyst internal complexity. Because the *T. gondii* oocyst suspension contained 60 % unsporulated/40 % sporulated oocysts, we gated separately each oocyst subset based on their respective FSC/SSC and autofluorescence intensity, sporulated oocysts being more complex and autofluorescent due to the additional presence of sporocysts compared to unsporulated oocysts. This was not done for *E. acervulina* because the oocyst suspension contained ~95 % sporulated oocysts and their sporocyst wall is weakly autofluorescent, which makes it difficult to separate sporulated and non-sporulated oocysts by FCM. All FCM data were analysed using the BDAccuri™ C6 software (v1.0.264.21). A control sample (without lectin labelling and without treatment) was included in each run with acquisition and analysis gates set accordingly. Lectin intensity data were expressed as fold change in lectin fluorescence intensity, calculated as follows: fold change = mean fluorescence intensity of oocysts incubated with lectin/mean fluorescence intensity of oocysts incubated in absence of lectin.

## 2.6. Bioassay of *E. acervulina* oocysts in chickens

The procedure was approved by the Ethics Committee Anses/ENVA/UPEC in accordance with the recommendations of the National Committee for Reflection on Animal Experimentation N°16 (permission dated December 13, 2022, no. 06). One hundred one-day-old coccidial-free female ISA Brown chicks were purchased from a local poultry-breeding centre (ISA Hendrix, Mûr-de-Bretagne, France) and were raised in an insulated room at a temperature of 30-37 °C. The chickens were provided *ad libitum* access to water and a commercial starter feed without coccidiostatic feed additives. At the age of 16 days, all chickens were weighed, and eighty birds were randomly allocated to four groups (20 chickens per group), considering the weight to obtain groups with similar average weight. Chickens were housed in wire-floored cages equipped with a tray placed under the wire floor. One day later, chickens were infected with either 10⁵ untreated (positive control) or frozen (negative control), or bleach- or NaOH-treated oocysts of *E. acervulina* in

1 ml sterile saline by oral gavage. To limit the number of chickens for the experiments, we chose to evaluate only freezing as a treatment known to inactivate the parasites. The cages with birds infected with frozen oocysts were placed on top of the cage with birds infected with untreated oocysts to avoid accidental contamination of the birds in the frozen group. No bird was excluded during the assay and for the subsequent analyses.

The growth status, presence of diarrhoea was recorded from 4 to 7 dpi for each group of chickens. Faeces were collected 4-6 days after infection, and oocysts were enumerated using the McMaster counting method. Oocyst excretion in each treatment group was expressed relative to oocyst expression in the untreated group. At 7 dpi, all birds were weighed and euthanized using electronarcosis followed by bleeding. Their duodenum and jejunum were examined for lesion scoring according to the Johnson and Reid scale (Johnson and Reid, 1970), which ranges from 0 (no lesions) to 4 (extended severe lesions). The weight gain value of animals inoculated with treated oocysts was expressed relative to the weight gain value (arbitrarily set at 100 %) of animals inoculated with untreated oocysts. Blind measurements were ensured by recording only the wingband number of the birds, without information of the group to which they belonged.

## 2.7. Bioassay of *T. gondii* oocysts in mice

Mouse bioassay was approved by the local ethical committee of University Reims Champagne-Ardenne under the agreement number B-51-454-4 and the APAFIS project #11913-2017102316076105v2. Eighty 8-week-old Swiss albino mice (purchased from Charles River Laboratory, Neuilly-sur-Seine, France) were maintained under specific-pathogen-free conditions with free access to diet and water. After a one-week acclimation period, mice were randomly divided into sixteen groups of 5 mice each. Mice were injected intraperitoneally with 1, 10, 100, or 1000 untreated (positive control) or frozen (negative control), or bleach-, or NaOH-treated sporulated *T. gondii* oocysts suspended in 200 µl sterile saline. To limit the number of mice for the experiments, we chose to evaluate only freezing as a treatment known to inactivate the parasites. To minimize potential confounders, each experimental group was housed in a single cage, and the cages were placed on shelves with a maximum of four cages per shelf. This arrangement helped to control for potential environmental variables, such as cage location, while ensuring that each group was kept in similar conditions. To assess the effect of treatments on the infectivity of *T. gondii* oocysts, blood samples (~20 µl per mouse) were collected from the tail 30 days after infection, centrifuged and sera were analysed for *T. gondii* antibodies by the modified agglutination test (Dubey and Desmonts, 1987). No animals were excluded during the assay and for the subsequent analyses. At the end of the bioassay period, all mice were euthanized by CO₂ asphyxiation, then verified dead by waiting for rigor mortis to set in after 1 h, in accordance with the relevant guidelines and regulations.

## 2.8. Statistical analysis

GraphPad Prism v5.03 (GraphPad software, San Diego, CA, USA) was used for the statistical analysis. Data are presented as mean ± standard deviation (SD) unless stated. The Shapiro-Wilk test was used to assess the normality of the data distribution. Data with normal distribution (Weight gain variable) were analysed using a parametric Student t-test using the untreated group as a control. Data with non-normal distribution were analysed using the non-parametric Kruskal-Wallis test followed by the Dunn's post-hoc test (AF and lectin intensity variables) or Mann-Whitney test (Lesion score variable). Differences with a p value < 0.05 were considered statistically significant. No animal, experimental unit or data point was excluded from the analysis, whatever the experimental group.
## 3. Results

### 3.1. Treatments can affect differentially the overall structure of sporulated *T. gondii* and *E. acervulina* oocysts

Flow cytometry (FCM) analyses showed that sporulated *T. gondii* oocysts were more heterogeneous in size and complexity following NaOH treatment and heating since only 14.7 % and 49.9 % of oocysts were in the control untreated oocyst gate respectively (Fig. 1A). Interestingly, NaOH also affected somewhat the overall structure of unsporulated *T. gondii* oocysts with only 66.0 % oocysts in the untreated control gate (Supplementary Fig. 1A). Apart from NaOH, and to a lesser extent heating, other treatments had no obvious effect on the structure and organization of *T. gondii* oocysts (Fig. 1A and Supplementary Fig. 1A and B). *E. acervulina* oocysts appeared more resistant than sporulated *T. gondii* oocysts. They were not structurally affected overall by the different chemical and thermal treatments (Fig. 1B).

Then, FCM was used to investigate potential modification of the global oocyst autofluorescence (AF) intensity as an indirect indicator of damages to the structure of their oocyst and sporocyst walls. Untreated and treated sporulated *T. gondii* oocysts exhibited a higher AF intensity than unsporulated ones (Supplementary Fig. 1C) and *E. acervulina* oocysts (Fig. 1C), of ~0.6–0.7 log₁₀ values. This was presumably due to the highest AF intensity of their sporocyst wall (see AF panels in

Supplementary Fig. 2B vs. 2A for comparison between species). Compared to untreated parasites, AF intensity was slightly increased in *E. acervulina* oocysts after NaOH treatment, whereas it did not markedly vary in *T. gondii* oocysts under various treatments whatever the sporulation stage (Fig. 1C and Supplementary Fig. 1C).

### 3.2. Most of treatments did not modify the permeability of *E. acervulina* and *T. gondii* oocysts to fluorescent lectins

FITC-conjugated WGA, MPL, and BP lectins were used to evaluate modifications in permeability of the oocyst substructures. These lectins have been documented by fluorescence microscopy to differentially bind to the coccidian oocyst and sporocyst walls and the sporozoite respectively (Bushkin et al., 2012; Dumètre et al., 2021; Fabian et al., 2021; Fuller and McDougald, 2002; Harito et al., 2016). The Supplementary Fig. 2A and B shows that WGA bound to the oocyst wall of both species, sometimes at focal points, and MPL and BP to the wall and/or the inner content of their oocysts and/or sporocysts. FCM analyses showed that fold changes in WGA, MPL or BP lectin fluorescence intensity did not differ in both unsporulated and sporulated *T. gondii* oocysts exposed to various treatments compared to untreated parasites (Fig. 2 and Supplementary Fig. 3). A similar trend was observed in treated *E. acervulina* oocysts compared to untreated ones, except that freezing/heating cycles increased BP intensity (p < 0.05) (Fig. 2).

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Flow cytometry analyses of the size-complexity and autofluorescence intensity of sporulated *Toxoplasma gondii* and *Eimeria acervulina* oocysts under various treatments. Size-complexity cytograms of sporulated *T. gondii* (A) and *E. acervulina* (B) oocysts. Red gates correspond to untreated oocysts as controls. The percents indicate the number of events that are included in these gates relative to the total number of acquired events. (C) Distribution of the autofluorescence (AF) intensity of sporulated *T. gondii* and *E. acervulina* oocysts. Each symbol represents the mean AF intensity values of ~10⁴ events acquired per sample by flow cytometry. The line is the median of the distribution of data from five independent experiments. Data distributions were analysed using the non-parametric Kruskal-Wallis test followed by Dunn's post-hoc test for multiple comparisons between treatment conditions per parasite species. Differences between conditions for each parasite species were plotted as a p values map and were considered significant when p < 0.05.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Distribution of FITC-lectin intensities in sporulated *Toxoplasma gondii* and *Eimeria acervulina* oocysts. Data are represented as fold change in lectin fluorescence intensity as calculated as follows: fold change = mean fluorescence intensity of oocysts incubated with lectin/mean fluorescence intensity of oocysts incubated in absence of lectin. Each symbol represents the ratio of the mean intensity values of ~10⁴ events acquired per sample by flow cytometry. The line is the median of the distribution of data from five independent experiments. Data distributions were analysed using the non-parametric Kruskal-Wallis test followed by Dunn's post-hoc test for multiple comparisons between treatment conditions per sporulation stage. Differences between conditions for each parasite species were plotted as a p values map and were considered significant when p < 0.05. (A) WGA, wheat germ agglutinin; (B) MPL, *Maclura pomifera* lectin; (C) BP, *Bauhinia purpurea*.</figcaption></figure>

### 3.3. Treatments impact similarly *E. acervulina* and *T. gondii* oocyst infectivity

Table 1 shows that, except for the freezing group, chickens orally infected by untreated or treated *E. acervulina* parasites excreted oocysts in their faeces from 4 to 6 dpi. However, oocyst excretion tended to be lower in chickens from the bleach and NaOH groups than in the untreated group. There were no significant variations in the weight gain of chickens in treated groups compared with the untreated group, except

again for chickens inoculated with frozen oocysts, which exhibited a higher weight gain (p < 0.05, Table 1). An intestinal lesion score of 3.0 ± 0.3 was reported in chickens inoculated with untreated oocysts (Table 1). Lesion score was not different in animals from the bleach group (2.9 ± 0.3, p > 0.05), however it was significantly lower in animals from the NaOH (score = 2.5 ± 0.6, p < 0.05) and the freezing groups (score = 0, p < 0.001). Collectively, these results showed that freezing was able to completely inactivate *E. acervulina* oocysts, while NaOH, but not bleach, appeared to partially affect their infectivity.
<table><caption>**Table 1**
Effect of treatments on the pathogenicity of *E. acervulina* oocysts in chickens. For each treatment condition, weight gain and lesion score data were compared to the untreated group by using a Student t-test (*, p < 0.05), or a Mann-Whitney test (*, p < 0.05; ***, p < 0.001), respectively.</caption><thead><tr><th>Inoculum</th><th>Treatment</th><th>Nb. of chickens</th><th>Mean weight gain (%)<sup>a</sup></th><th>Mean lesion score ± SD</th><th>Total of oocysts shed (4–6 dpi)<sup>b</sup></th></tr></thead><tbody><tr><td>100 000</td><td>Untreated</td><td>20</td><td>100</td><td>3.0 ± 0.3</td><td>2.0 x 10<sup>7</sup></td></tr><tr><td>100 000</td><td>Freezing</td><td>20</td><td>105.3*</td><td>0.0 ± 0.0***</td><td>0</td></tr><tr><td>100 000</td><td>Bleach</td><td>20</td><td>96.7</td><td>2.9 ± 0.3</td><td>1.3 x 10<sup>7</sup></td></tr><tr><td>100 000</td><td>NaOH</td><td>20</td><td>98.9</td><td>2.5 ± 0.6*</td><td>0.710<sup>7</sup></td></tr></tbody></table>

a the mean weight gain value was set arbitrarily at 100 % for chickens inoculated with untreated oocysts. For other treatments, values were calculated relative to the untreated group.

b dpi, day post-infection.

The infectivity of *T. gondii* oocysts was assessed by mouse bioassay (Table 2). No mortality was recorded in the different groups of mice inoculated with the different oocyst doses. The infectious dose of untreated oocysts was established between 100 and 1000. Oocysts were inactivated by freezing, but not by NaOH as 4 out of 5 mice inoculated with 1000 oocysts developed *T. gondii* antibodies. However, while mice inoculated with 100 untreated oocysts were infected (2/5), no mouse infection was observed with the same dose of oocysts treated with NaOH, indicating somewhat loss of infectivity for treated oocysts. Unexpectedly, all mice inoculated with bleach-treated oocysts remained negative. We retrospectively quantified the oocysts in each inoculum by qPCR and found that the mice in the bleach group had received very few oocysts compared to the other groups (Supplementary Table 1). As observed in *E. acervulina*, freezing seems to completely inactivate *T. gondii* oocysts, while NaOH can partially reduce their infectivity.

## 4. Discussion

The effects of various chemical and physical treatments were evaluated on the integrity and infectivity of *E. acervulina* and *T. gondii* oocysts with the perspective to propose *E. acervulina* as surrogate of *T. gondii* to assess the efficacy of food decontamination treatments. FCM was used to investigate modifications of the size-structure of oocysts of both species. Our results show that *E. acervulina* oocysts retain their size and structure better than *T. gondii* ones suggesting that, regarding the overall structure, *E. acervulina* oocysts are less affected by treatments. One prerequisite for an organism to be a good surrogate to assess process efficacy is to be as resistant as or more resistant than its target. Hence, from the structure point of view, *E. acervulina* oocysts seemed to be promising as a surrogate of *T. gondii* oocysts. In *T. gondii*, NaOH and to a lesser extent heating were the most deleterious treatments and led to a heterogeneous distribution of the parasites according to their size and structure. These observations suggest that these two treatments could distort even open *T. gondii* oocysts and cause the release of the sporocysts.

<table><caption>**Table 2**
Effects of treatments on the infectivity of sporulated *T. gondii* oocysts to mice. Mice were injected intraperitoneally with different numbers of sporulated oocysts (5 mice/oocyst dose/treatment) and, 30 days after, analysed for *T. gondii* antibodies by using a modified agglutination test.</caption><thead><tr><th rowspan="2">Treatment</th><th colspan="4">Number of sporulated oocysts inoculated per mouse</th></tr><tr><th>1000</th><th>100</th><th>10</th><th>1</th></tr></thead><tbody><tr><td>Untreated</td><td>4/5<sup>a</sup></td><td>2/5</td><td>0/5</td><td>0/5</td></tr><tr><td>Freezing</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td></tr><tr><td>Bleach</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td></tr><tr><td>NaOH</td><td>4/5</td><td>0/5</td><td>0/5</td><td>0/5</td></tr></tbody></table>

a number of *T. gondii* seropositive mice/number of inoculated mice.

We then investigated whether the oocyst walls retained their structure following treatments by characterizing their autofluorescence (AF) under UV. Oocyst AF is thought to be due to dityrosine bonds occurring in the inner oocyst wall layer and the sporocyst wall in chicken *Eimeria* (Belli et al., 2003, 2009; Walker et al., 2016), and the outer layer of the oocyst and sporocyst walls in *T. gondii* (Dumètre et al., 2013, 2021). First, we observed that untreated sporulated *T. gondii* oocysts were more autofluorescent than *E. acervulina*, presumably due to their sporocyst wall. Second, we compared AF of treated vs. untreated sporulated oocysts to highlight any significant changes in the bilayered structure of their walls (e.g. loss of one of the layers) (Dumètre et al., 2013, 2021). By using FCM, we did not observe any decrease in the global AF intensity of oocysts of both species. However, we cannot exclude that some treatments such as bleach had attacked the oocyst surface, and even stripped the outermost oocyst wall layer, as previously reported using imaging techniques more resolutive than FCM (Dumètre et al., 2013, 2021; Mai et al., 2009).

We then gained further information on the permeability of the oocyst walls following treatments by using lectins that were reported to differentially bind to the oocyst and sporocyst walls, and the sporozoites. WGA has been shown to label the outer layer wall of aged (10 years-old) or pepsin-treated *T. gondii* oocysts (Harito et al., 2016), MPL the inner side of the inner oocyst wall layer and the outer sporocyst wall layer in *T. gondii* and *E. tenella* (Bushkin et al., 2012; Dumètre et al., 2021; Fabian et al., 2021), and BP the surface and the apical part of *E. tenella* sporozoites (Fuller and McDougald, 2002). These lectins likely bind to yet unidentified wall glycoproteins bearing GlcNAc (WGA), α-GalNAc (MPL), and galactose (BP) moieties. Here, we confirmed such binding patterns at the wall level, but also observed that WGA could bind to relatively fresh (less than 6 months-old) *T. gondii* and *E. acervulina* oocysts respectively, in contrast to what Harito et al. observed previously (Harito et al., 2016). Differences between studies may be due to different parasite purification and storage protocols, which could result in some exposure of WGA-reactive glycoproteins on the oocyst surface. Another interesting result from our study is that BP can be used to highlight the oocyst content of *T. gondii* and *E. acervulina* as described in *E. tenella* (Fuller and McDougald, 2002).

However, the use of these different lectins did not allow us to show significant modifications in the wall permeability of treated oocysts of *T. gondii*. In *E. acervulina*, only fold change in BP fluorescence intensity was increased following repeated freezing/heating (–80 °C/+99 °C) suggesting that this treatment affected the oocyst integrity, probably leading to rupture of their walls and the subsequent infiltration of BP down to the sporocyst/sporozoite level. The absence of significant differences in the intensity changes of the other lectins and/or other treatment conditions could result from the modification or elimination of the glycoproteins targeted by the lectins due to the different treatments, in particular chemical treatments, or the inability of lectins to access their targets. These findings question the use of fluorescent lectins as permeability markers of the oocyst walls. Other molecules such as fluorophores (FITC) or dextrans of different sizes, though less specific, could be more suitable to investigate modifications in the oocyst permeability following treatments.

Freezing (–80 °C, 48 h) had no significant effects on the overall structure and integrity of *E. acervulina* and *T. gondii* oocysts, but as expected, frozen parasites were no longer infective to chickens and mice respectively. Bleach did not inactivate *E. acervulina* oocysts as previously described (Daugschies et al., 2013). The same might be true for *T. gondii*, as indicated by previous *in vitro* and *in vivo* studies (Dubey et al., 1970; Villegas et al., 2010; Wainwright et al., 2007). Indeed, in this study, qPCR analyses highlighted experimental issues with bleached oocyst inoculum hence questioning *in vivo* results obtained for this treatment. As observed in this study, household bleach appeared to have little effect on the overall oocyst morphology and inner content. The coccidian oocyst wall is known to resist bleach treatments (Dumètre et al., 2013, 2021; Mai et al., 2009), which is mainly due to its inner layer, however
the sporocyst wall could provide a second protective barrier to sporozoites in case of oocyst wall permeabilization or rupture, allowing them to maintain their infectivity (Dumètre et al., 2021).

Results showed a partial effect of NaOH on the oocyst infectivity of both species, accordingly with Dubey et al. (1970). The effects of NaOH at concentrations found in cleaning products on the coccidian oocysts are poorly documented. It is known that oocysts are less resistant to basic solutions than acid ones used as preservatives (Daugschies et al., 2013; Dubey et al., 1970), however the structural and molecular basis of this phenomenon remains to investigate. In addition to the oocyst and sporocyst walls, the sporozoite metabolism could contribute to resistance to NaOH treatment.

## 5. Conclusion

This study shows that bleach and NaOH treatments respectively do not or partially inactivate *E. acervulina* and *T. gondii* oocysts compared to thermal treatments. FCM analyses and lectin binding assays suggest that the walls of oocysts and sporocysts, even if altered by chemical treatments, may still protect to some extent the sporozoites allowing them to maintain their infectivity. Collectively, our results indicate that *E. acervulina* may be a reliable model of *T. gondii* oocysts exposed to chemical treatments and thermal treatments, and perhaps to other physical treatments as shown following exposure to γ irradiation (Lee and Lee, 2001). This parasite could therefore serve as an affordable, non-pathogenic substitute for *T. gondii* when evaluating food decontamination processes in food matrices, particularly in industrial environments. In this context, further studies are required to provide reliable and cost-effective techniques for screening *E. acervulina* oocyst infectivity without the use of chicken bioassay.

## CRediT authorship contribution statement

**Laure Augendre:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Sandie Escotte-Binet:** Writing – review & editing, Validation, Methodology, Investigation, Formal analysis, Data curation. **Dominique Aubert:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Formal analysis, Data curation, Conceptualization. **Isabelle Villena:** Writing – review & editing, Resources, Project administration, Funding acquisition. **Jean-Michel Répérant:** Writing – review & editing, Writing – original draft, Validation, Resources, Methodology, Investigation, Formal analysis. **Stéphanie La Carbona:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Aurélien Dumètre:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

We gratefully thank Sandra Audonnet (cytometry platform URCA-Cyt) and Pierre-Henri Puech (LAI, Marseille) for helpful discussions on interpretation of flow cytometry data. This work was supported by the UMT ACTIA PROTORISK 2 and the University of Reims Champagne-Ardenne. Laure Augendre PhD thesis was funded by ACTALIA and the French Research National Agency (ANR) grant BreakingTheWall (ANR-22-CE35-0008).

# Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.exppara.2025.109049.

## Data availability

Data will be made available on request.

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