*2.2. Use of the Gut-Loop Model for Investigating Immune Responses and Infection with C. parvum* 2.2.1. Intestinal Stimulations

Intestinal responses to immunostimulants were evaluated with isolated loops in lambs and ex vivo with explants. Just after the end-to-end anastomosis, 150 µL of stimulants were injected into loops with an 8-mm 30G needle at the following concentrations: bacterial lipopolysaccharide (LPS) (10 µg/mL, LPS-EB, Invivogen, San Diego, CA, USA), R848 (10 µg/mL, Resiquimod, Invivogen, San Diego, CA, USA) and yeast cell wall fraction 1 (YCW1) or 2 (YCW2) (5 mg/mL, Phileo by Lesaffre, Marcq-en-Barœul, France). The two YCW were obtained from a *Saccharomyces cerevisiae* (*S. cerevisiae*) strain and differed in their polysaccharide composition. YWC1 is composed of an equilibrated content between β-glucans and mannoproteins (at minima 20% of each sugar compound), whereas YCW2 is enriched in β-glucans (50%). Twenty-four hours after surgery, animals were euthanized and intestinal tissues were collected either from the loops or from the ileum reconnected to the intestinal transit, and explants were generated with biopsy punches of 8 mm diameter. Tissue samples were incubated in culture medium (RPMI; 10% FBS; 100 U/mL Penicillin; 100 µg/mL Streptomycin; 100 µg/mL Normocin; 250 ng/mL Amphotericin B) alone or with LPS at a concentration of 10 µg/mL, for 4 h at +37 ◦C and 5% CO2. Three explants per condition were used, referred as triplicates.

### 2.2.2. RNA Extraction and RT-qPCR

RNA extractions from explants or pieces of loop were performed after homogenization in TRIzol (Invitrogen, Waltham, MA, USA) with an Ultra-turrax and processed according to manufacturer's recommendations. Total RNA was reverse transcribed with the iScript RT SuperMix kit (Biorad, Hercules, CA, USA) and amplified by quantitative real-time PCR (RT-qPCR) in presence of EVA® Green using CFX96 Touch Real-Time PCR Detection System (Biorad, Hercules, CA, USA). Results were expressed as 2e−∆Ct, following normalization with three stable reference genes (*hprt*, *gapdh* and *actb*). mRNA was quantified for *il1α*, *il1β*,

*mx1*, *cxcl1*, *cxcl2*, *cxcl8* and *tnfα* genes known to be upregulated by YCW extracts and/or TLR-agonists used in this study, LPS and R848 (see Table S1 for primer sequences).

#### 2.2.3. *C. parvum* Infection

Intestinal loops were injected with 150 µL of physiological serum (0.9% NaCl) or with <sup>150</sup> <sup>µ</sup>L of parasite solution containing 1.5 <sup>×</sup> <sup>10</sup><sup>4</sup> or 1.5 <sup>×</sup> <sup>10</sup><sup>5</sup> oocysts of *C. parvum* in presence or not of yeast cell wall fractions. The *nluc*-INRAE transgenic strain of *C. parvum* [16], which expresses the nanoluciferase enzyme was used in the study to quantify parasite load. All intestinal samples were collected after 24 h of infection with *C. parvum*.

#### 2.2.4. Parasite Burden

The level of infection in the intestinal loops was determined by two complementary methods: The assessment of the luciferase activity of the *C. parvum nluc*-INRAE transgenic strain and the quantification of the *C. parvum 18S* (*Cp18S*) gene expression by RT-qPCR. For relative light unit (RLU) quantification, 500 µL of lysis buffer (50 mM Tris HCl; 2 mM DTT; 2 mM EDTA; 10% glycerol; 1% Triton) were added to biopsy punch, followed by a 30 min incubation at +4 ◦C. Samples were then vortexed during 1 min and centrifuged at 12,000<sup>×</sup> *<sup>g</sup>* for 1 min. Twenty-five <sup>µ</sup>L of supernatant were mixed with 25 <sup>µ</sup>L of Nano-Glo® substrate (1/50, Promega, Madison, WI, USA) and luminescence activity measured with a GloMax® plate reader (Promega, Madison, WI, USA). The values were expressed in RLU per cm<sup>2</sup> of intestinal tissue. *Cp18S* gene mRNA was quantified using the forward 5 0 -TAGAGATTGGAGGTTGTTCCT-30 and reverse 50 -CTCCACCAACTAAGAACGGCC-30 primers and results are expressed as 2e−∆Ct, after normalization with the expression of three reference genes (*hprt*, *gapdh* and *actb*).

#### 2.2.5. Histology and Immunofluorescence

Following dissection and PBS wash, intestinal samples were immediately incubated in a 4% paraformaldehyde PBS solution during 24 h at +4 ◦C for tissue fixation. After 2 washes of 4 h at +4 ◦C in PBS, samples were incubated in a 30% sucrose PBS solution before inclusion in OCT (Cellpath, Newtown, UK) and conserved at −20 ◦C until histological analyses. Seven µm-thick histological sections were realized with the 3050S Leica cryotome (Leica, Wetzlar, Germany).

Intestinal sections were incubated with a rat polyclonal serum against oocyst antigens (as previously used [17]), overnight in the dark at +4 ◦C and then washed twice with PBS. The secondary antibody coupled with Alexa 594 (Invitrogen, Waltham, MA, USA) was added for 1 h in the dark and washed twice with PBS. Finally, cell nuclei were stained with Hoechst (Invitrogen, Waltham, USA) during 2 min and then washed twice with PBS. Immunofluorescent staining of *C. parvum* were analyzed by microscopy with the Eclipse 80i Nikon Microscope (Nikon, Tokyo, Japan).

#### 2.2.6. Statistics

Statistical analyses were performed using the GraphPad Prism v6 software (GraphPad Software, San Diego, CA, USA). The Kruskal–Wallis non-parametric test and the Dunn's multiple comparison test were used to determine the significance of difference for parasite load determination by luminescence measure and RT-qPCR between the different conditions. *p*-values of less than 0.05 were considered statistically significant. The linear regression analysis was used to establish the correlation curve between parasite load evaluated by luminescence measure and *Cp18S* gene expression.

#### **3. Results**
