Sodium taurocholate

Fecal samples were collected daily from mice infected with wildtype C. parvum. Oocysts were purified from fecal material using sucrose and cesium chloride gradient centrifugation [28 (link)].
To obtain sporozoites, oocysts were resuspended in 600 μL of PBS and treated with 200 μL of bleach (Clorox, California, USA) on ice for 10 min. After three washes with PBS by centrifugation, sodium taurocholate (Sigma, New Jersey, USA) was added to the oocyst suspension to a final concentration of 0.75%. The oocyst suspension was incubated at 37°C for 60 min, and the sporozoites released were harvested by centrifugation at 16,000 g for 3 min and resuspended at the concentration of 5 x 10⁷ sporozoites/80 μL of SF buffer (Lonza, Basel, Switzerland).
For the transfection of sporozoites, donor and CRISPR/Cas9 plasmids (50 μg in 100 μL each) were added to the sporozoite suspension in a 100-μL electroporation cuvette (Lonza). The sporozoites were electroporated using the EH100 program on an AMAXA 4D Nucleofector System (Lonza).

The experimental protocol was approved by the Institute Animal Care and Use Committee of Wenzhou Medical University (WYYY-AEC-2023-108), and all experimental procedures described were performed in accordance with established institutional guidelines and approved protocols. Rats were housed in a cage (four rats per cage) in a temperature-controlled room with a 12-h light-dark cycle. Food and water were available ad libitum.
Forty male Sprague–Dawley rats were obtained from the Animal Laboratory Center of the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, CN). The rats were starved for 24 h before surgery but were allowed to drink water freely during this period. They were then injected with 2% pentobarbital (0.3 mL/100 g, subcutaneously) in the abdomen and restrained in the supine position.
The ANP rat model was established using a modified Aho method, involving the injection of 3.5% sodium taurocholate (86339 Sigma, America) into the bile duct at a constant rate (Fig. 1A).
For the acute necrotizing pancreatitis-associated intestinal injury model (ANP-IR model) group, we injected necrotic pancreas and PAAF, which were collected from the ANP group, into the triangular region formed by the left renal artery and ureter at a constant rate. The injection volume used was 0.1 mL/100 g (Fig. 1B). Supplemental Fig. S1 presents photographs taken during the fabrication of the ANP-IR model.

For testing the production of hydrolytic enzymes, a protocol adapted from Corbu and Csutak [31 (link)], Zajc et al. [32 (link)], and Avram et al. [33 (link)] was used. An inoculum with 1McFarland density was obtained, starting from a fresh 24 h culture on YPGA medium. The suspension (10 µL) was spotted on the surface of culturing media specific for determining the ability to secrete specific hydrolytic enzymes: hemolysins (on Sabouraud Dextrose Agar medium (SDA) (Oxoid) (Hampshire, UK) with 50 mL/L ram blood); amylases (on YP culture media containing 0.5% yeast extract (Carl Roth, Karlsruhe, Germany), 1% bacteriological peptone (Sigma Aldrich, Burlington, MA, USA) and 1% NaCl (Carl Roth) supplemented with 10% starch); caseinases (on YP medium with 10% casein from milk (Carl Roth)); extracellular deoxyribonucleases (DN-ases) (on DNase Test Agar medium—Difco, East Rutherford, NJ, USA, supplemented with 0.01% toluidine blue (Sigma Aldrich, Burlington, MA, USA)); phospholipases (on SDA culture media with 100 mL/vL egg yolk, 1.17 g/L NaCl (Carl Roth, Karlsruhe, Germany), and 0.001% CaCl₂∙2H₂O (Carl Roth)); gelatinase-type proteolytic enzymes (on a specific culturing media containing 0.1% yeast extract; 0.5% sodium taurocholate (Sigma Aldrich, Burlington, MA, USA); 1% bacteriological peptone; 0.1% NaCl; 3% gelatin (Sigma Aldrich) and 1.5% agar-agar). After inoculation, the plates were incubated at 37 °C for 72 h and monitored daily to observe a positive reaction according to the reference protocols (the appearance of a clear area around the culturing spot for the hemolysins production; the appearance of a clear zone after staining with Lugol solution (Carl Roth) for amylase production; the presence of clear halos or a white precipitate around the colonies for the production of caseinases, gelatinases and phospholipases; or a light halo/ pale pink halo for the DN-ase production test). The results were recorded as arbitrary units (“−” negative result; “+” positive result, registered after the estimated time, “D”—delayed positive results; the positive result was registered after the optimal time of determination) at the end of the incubation time. Since the yeast strains were isolated from the surface of different plants, the production of the siderophore-like compounds was also determined using a specific culturing media (1% bacteriological peptone, 0.1% ammonium ferric citrate (Sigma Aldrich, Burlington, MA, USA), 2% agar) supplemented with 0.1% esculin (Sigma Aldrich, Burlington, MA, USA). The positive result in this case is indicated by the appearance of a brown to black colored zone around the yeast spot after at least 24 h of incubation at 37 °C.

Before infection, purified C. parvum oocysts were treated with a 40% bleach solution (commercial laundry bleach containing 8.25% sodium hypochlorite) diluted in Dulbecco’s Phosphate Buffered Saline (DPBS; Corning Cellgro) for 10 min on ice. Oocysts were then washed 4 times in DPBS containing 1% (wt/vol) bovine serum albumin (BSA; Sigma) before resuspending to a final concentration of 1x10⁸ oocyst/ml in DPBS with 1% BSA. For some experiments, oocysts were excysted prior to infection by incubating the oocysts with 0.75% sodium taurocholate (w/v; Sigma) in DPBS at 37°C for 60 min. As indicated, excysted oocysts were filtered through a membrane with 1 μm pore size (Whatman, VWR International) to remove unexcysted oocysts from sporozoites. Sporozoites were spun down at 1,250 x g for 3 min and then resuspended in 50% CM prior to adding to ALI monolayers. Oocysts or filtered sporozoites were added to monolayers in 30 μl of 50% CM three days post top medium removal. After 3 hr, monolayers were washed twice with DPBS to remove extracellular parasites and re-establish the air-liquid interface.