Gallbladder

Litters of two-day old New Zealand White infant rabbits with the lactating doe were acquired from a commercial breeder (Milbrook Farm, Amherst, MA). The following day, the infant rabbits were administered cimetidine (50 mg kg⁻¹ via intraperitoneal injection; Hospira, IL) 3 hr prior to oro-gastric inoculation with either 1×10⁹ cfu wild type V. parahaemolyticus, or one of the isogenic mutants, or sodium bicarbonate solution (2.5 g in 100 mL; pH 9) using a size 4 French catheter (Arrow International, Reading, PA). To prepare the inocula, cultures of bacteria grown for ∼18 hr at 30°C were harvested by centrifugation (5 mins 6000 g), and the cell pellet resuspended in sodium bicarbonate solution (pH 9) to a final concentration of 2×10⁹ cfu mL⁻¹. Following inoculation, the infant rabbits were monitored frequently for clinical signs of illness. Disease was scored at euthanasia as follows: no gross disease (no adherent fecal material on fur and intestines appear normal), intestinal fluid (no adherent fecal material on fur but intestines appeared red, swollen and contained fluid), diarrhea (liquid fecal material stains or adheres to fur, and intestines appeared red, swollen and contained fluid). In most experiments, rabbits were euthanized at fixed times after infection (i.e. 12, 18, 28 or 38 hr PI), but rabbits were euthanized prior to these time points if they appeared moribund (categorized as ‘dead’ in Table 1 and Figure 1A). At necropsy, the intestinal tract from the duodenum to the rectum was removed and processed for microbiological, microscopic and histologic analyses. For some rabbits, the internal organs including the gall bladder, spleen and liver were also collected, homogenized and plated on selective media to check for systemic spread of V. parahaemolyticus.
To determine fluid accumulation ratios (FARs), an approx. 5 cm length of the distal small intestine was isolated from the rest of the intestine using silk ligatures. The intestinal section was weighed and then cut every 0.5 cm to release any luminal fluid, and the tissue pieces reweighed. The FAR was calculated as the weight of fluid divided by the weight of the drained tissue. The electrolyte and protein concentrations in serum and diarrheal fluid collected from the ceca of infected rabbits were measured on an Olympus Analyzer (AU-2700) at the Brigham and Woman's Hospital clinical laboratory.
The number of V. parahaemolyticus cfu in tissue samples taken from the small and large intestine, cecum and stool were determined after homogenization, serial dilution and plating on LB media containing 50 µg mL⁻¹ carbenicillin as described previously [24] (link). For unknown reasons, rabbits were occasionally not colonized by the pathogen i.e., no V. parahaemolyticus cfu were detected in any tissue sample. These rabbits (less than 10%, regardless of strain tested) were excluded from all further analyses. However, any rabbits that contained detectable numbers of V. parahaemolyticus cfu in at least one tissue sample were included; for these rabbits, the lower limit of detection was reported for sections where no colonies were detected at the lowest dilution plated, and this value was used in calculation of mean cfu.
For routine histological analyses, tissue segments were fixed in 10% neutral buffered formalin, processed for paraffin embedding and stained with hematoxylin and eosin (H&E). The slides were semi-quantitatively assessed for infiltration of inflammatory cells (heterophils), cell proliferation, and tissue damage by a pathologist blinded to the origin of the tissue. Each histological parameter was evaluated on a 0–4 scale as follows: 0 (normal), 1 (mild), 2 (moderate), 3 (severe) and 4 (severe and extensive).

Bile acids were extracted from liver, whole gallbladder bile, whole small intestine with content, and dried feces in 90% ethanol. Briefly, solid tissues and feces were homogenized in 90% ethanol and incubated at 50°C overnight. After centrifugation, the clear supernatant was used for total bile acid measurement with a bile acid assay kit. To collect fecal samples, an individual mouse was placed in a jar briefly and fresh feces were collected. Bile acid pool was calculated as the total bile acids in liver, gallbladder, and small intestine. For LC-MS analysis of bile acid composition, ethanol extracts were dried and then resuspended in injection buffer containing 0.1% formic acid in 1:1 water: mixture of acetonitrile and methanol (1:1) and subsequently analyzed on a Thermo Fisher Scientific UltiMate 3000 UHPLC with a Waters Cortecs C18 column (Waters Acquity UPLC HSS T3 1.8 um, 2.1x150 mm, part No. 186003540) and a TSQ Quantis triple quadrupole mass spectrometer. The running condition is as follow: Solvent A: 0.1% formic acid; Solvent B: 0.1% formic acid in 1:1 methanol: acetonitrile. Flow rate: 0.3 ml/min. Gradient: 52%–90% B in 18 min, 90% to 52%B in 0.1 min, hold for 4 min. Run time: 22 min. TSQ Quantis triple quadrupole mass spectrometer Ion Mode: Ion Source Type: H-ESI; Spray Voltage: Static; Negative Ion (V): 2500; Sheath Gas (Arb): 50; Aux Gas (Arb): 10; Sweep Gas (Arb): 1; Ion Transfer Tube Temp (°C): 325; Vaporizer Temp (°C): 350; Polarity: Negative; Cycle Time (sec): 0.8. Other LC-MS parameters (Retention time, ion monitoring) are listed in supplemental Table S1. Standard curves for bile acids and internal standard glyco-CDCA-d4 (G-CDCA-d4) were generated with purified compounds and relative area under the curve was calculated. To measure T-CDCA-d4 metabolism in fecal slurry mixtures, fresh fecal sample was resuspended in a reaction buffer consisting of 10% PBS (pH=7.4), and 90% 3 mM sodium acetate (pH = 5.2) to a final suspension of 4 mg fecal sample/ml. T-CDCA-d4 was added to a final concentration of 20 μg/ml and the mixture was incubated at 37°C for 6 h. An equal amount of methanol was added and the mixture was incubated on ice for 1 h to precipitate protein. After centrifugation, an aliquot of the supernatant was vacuum dried and resuspended in injection buffer. LC-MS measurement of bile acids was performed as described above.