Bovine bile

The trophozoites of G. intestinalis (strain WB) were maintained in axenic culture in modified Diamond’s TYI-S-33 medium [13 (link)] containing bovine bile (Sigma-Aldrich, St. Louis, MO, USA; 0.5 mg/mL) at 37 °C in 4.5 mL screw-cap flat-bottom vials or 15 mL tubes (Falcon^TM). The parasites were harvested in logarithmic phase of growth (after 72–96 h) by cooling vials or tubes for 45 min, washed three times with phosphate-buffered saline (PBS), counted in a hemocytometer and the protozoan samples were adjusted. To test the properties of LL on trophozoites, four inhibitory concentrations (ICs) were used, 50, 100, 150, and 200, based on the previously reported IC₅₀ value of 28.2 μM [11 (link)]. A total of 6000 trophozoites/mL were incubated for 48 h/37 °C in the presence of LL previously diluted in vehicle (dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA); 0.022% final concentration). As a positive control for damaged cells, albendazole (ABZ, Sigma-Aldrich, St. Louis, MO, USA) was used at 0.32 μM.

The freshly collected worms were cleaned as previously described [36 ], then washed twice with PBS, pH 7.4, and transferred to recover in a pre-warmed RPMI-1640 culture medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA) without serum and antibiotics at 37 °C under 5% CO₂, for 30 min. Five intact worms were immediately collected after recovery, washed twice with pre-warmed PBS and total RNA was extracted from each worm using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Other sets of five worms were cultured as previously described [36 ] in RPMI-1640 medium supplemented with 10% FBS with 0.5 μg/mL and 2 μg/mL of bovine bile (Sigma Aldrich, Darmstadt, Germany) at 37°C under a 5% CO₂ atmosphere for 4 and 8 h. Finally, worms were collected and proceeded to total RNA extraction.

Trophozoites of Giardia (strain ATCC^® 203333) were grown in modified TYI-S-33 medium at pH 7.0, supplemented with 10% heat-inactivated fetal calf serum and 0.5 g bovine bile (Sigma-Aldrich, St Louis, MO, USA), in 20 mL screw-cap culture vials.

Trophozoites of the G. intestinalis strain WB clone C6 (ATCC 50803) were cultured axenically at 37°C in modified Diamond's TYI-S-33 medium as previously described [38] (link). The medium was supplemented with 10% heat-inactivated bovine serum (Invitrogen) and 0.05% bovine bile (Sigma). Cultures were inoculated in 25 cm²-flasks, filled to 90% of their total volume in order to attain low-O₂ tension conditions. Trophozoites were transferred every 48 h into fresh medium when cells became confluent. For preparation of the inocula, trophozoites were detached by chilling the cultures on ice for 30 min.

We collected C2, trophozoite of G. lamblia isolates, from a patient in southwest China. We used modified trypticase yeast extract iron-serum-33 medium (TYI-S-33), which was composed of 2% casein digest, 1% yeast extract, 1% glucose, 0.2% NaCl, 0.2% l-cysteine, 0.02% ascorbic acid, 0.2% K₂HPO₄, and 0.06% KH₂PO₄. We used borosilicate glass screw-cap culture tubes to supplement this with 10% heat-inactivated bovine serum (Hangzhou Sijiqing Biological Engineering Materials Company) and 0.05% bovine bile (Sigma) at pH 6.8. Without shaking, we inoculated the culture with 4 × 10³ trophozoites per 4 mL tube at 37°C three times a week.

The analysis of resistance to different bile salts was performed comparing the two human gallbladder bile isolates IPLA60001 and IPLA60002 with the R. gauvreauii type strain DSM-19829. Comparisons were also made with R. bromii strains from the Rowett Institute (L2-63 and 5AMG), the results of which have been described previously (7 ). The analysis was carried out in triplicate by determining the MICs on M2GSC 2% agar plates supplemented with 30% CBRF and different concentrations (ranging from 0% to 12% wt/vol) of CA, TDC GC, TC, GDC, Porcine Bile Extract, Bovine Bile (purchased from Sigma-Aldrich), and Mixed Bile Salts (Oxoid, Waltham, MA). The plates were incubated at 37°C in the anaerobic cabinet for 48 h.

Krill oil with 40%, 50% and 60% phospholipid content was obtained from Luhua Biomarine Co., Ltd. (Jinan, China), and some others were lab-made. The LC-MS grade reagents, such as acetonitrile (ACN), isopropanol (IPA) and ammonium acetate (CH₃COONH₄), were purchased from Thermo Fisher Scientific (Shanghai, China). The mucin from porcine stomach, pepsin from porcine gastric mucosa (250 U/mg), gastric lipase from Rhizopus oryzae (35 U/mg), porcine lipase (200 U/mg) and bovine bile were provided by Sigma Aldrich (Shanghai, China). Porcine trypsin (250 U/mg) and whey protein isolate (WPI) were supplied by Yuanye Bio-Technology Co., Ltd. (Shanghai, China) and Sinopharm Chemical reagent Co., Ltd. (Shanghai, China), respectively. Other analytical grade chemicals were also purchased from Sinopharm Chemical reagent Co., Ltd.