Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

All experimental procedures were approved by IACUC of Auburn University, Auburn, Alabama.

1. Preparation of Culture Media, Dishes and Bacterial Culture

1. Inoculate 10 ml of nutrient broth in a flask with a single colony of Bacillus subtilis BSB3 culture, grown overnight on a nutrient agar plate.
2. Make 500 ml of sporulation medium¹⁹ (per liter: Bacto Nutrient Broth, 8 g; 10% (w/v) KCl, 10 ml; 1.2% (w/v) MgSO₄ x 7H₂O, 10 ml; agar, 15 g) and autoclave at 121 ^oC for 20 min. Allow to cool to 50 ^oC and add the following sterile solutions:1 M Ca(NO₃)₂, 1 ml; 0.01 M MnCl₂, 1 ml; 1 mM FeSO_4, 1 ml.
3. Cool prepared medium to 40 ^oC for 20 min at room temperature and pour 25 ml into each of 20 sterile Petri dishes in the sterile cabinet. Let the medium solidify overnight.
4. Spread 0.5 ml of overnight culture of Bacillus subtilis BSB3 onto the surface of the medium in the Petri dishes. Incubate culture at 37 ^oC for 5 days until 90% sporulation. Reveal phase-bright spores by high resolution microscopy.
5. Harvest bacteria by adding 1 ml of sterile phosphate buffered saline (PBS) to the plate and scraping bacteria from a surface using a sterile cell spreader. Store the suspension in the refrigerator until needed.
6. Confirm viability of bacteria by plating 0.1 ml of the appropriate dilution (10^-5 to 10^-6) of a bacterial suspension on sporulation medium plates followed by incubation for 18 – 24 hr at 37 ^oC.
7. Count bacterial colonies on the plates using a colony counter and calculate the titer of bacteria in the tested suspension. Prepare bacterial suspension with the final concentration 1 x 10⁸ CFU/ml in PBS.

2. Animals

1. Use 32 male Sprague–Dawley rats (250 – 300 g). Maintain animals under specific pathogen-free conditions with free access to standard pellet chow and water. Establish a mean temperature (21 ± 1 ^oC), humidity (50 ± 5%) and a 12 hr light-dark cycle.

3. Experimental Design

1. Start treatment of animals by oral gavage¹¹, after 2 days of acclimatization. Use syringe with the oral gavage needle. Treat 16 rats with B. subtilis BSB3 suspension (1 ml per rat) twice a day for two days (B. subtilis group). Treat 16 rats with PBS (1 ml per rat) twice a day for two days (PBS group) (Figure 1).
2. After treatment, subdivide each group (8 rats per group): Group 1- control (PBS/no stress), Group 2- B. subtilis (B. subtilis /no stress), Group 3- stress (PBS/heat stress) and Group 4- B. subtilis + stress (B. subtilis/heat stress).
3. Measure the rectal temperature of each rat using an electronic digital thermometer. Keep rats of groups 1 and 2 at room temperature for 25 min. Place animals of groups 3 and 4 in a climate chamber at 45 ^oC and relative humidity 55% for 25 min.
4. Afterwards, measure the rectal temperature of each rat in all groups using an electronic digital thermometer. Place all animals at room temperature for 4 hr.
5. Anesthetize rats with isoflurane (2 – 4%) in a sealable anesthesia jar and observe until the rats are deeply anesthetized (absence of response to toe pinch). Euthanize rats by rapid decapitation with a guillotine.
6. Collect trunk blood from each rat²⁰ (15 – 16 ml) with the apyrogenic pipettes into apyrogenic tubes to obtain serum and immediately take samples of blood (7 µl from each rat) with a pipette for microscopic examination.
7. Put tubes with the blood in a refrigerator for at least 30 min to allow clotting. Centrifuge tubes at 20 ^oC, 7,000 x g for 10 min and quickly remove serum with a pipette. Aliquot serum obtained from each rat into 50 µl volumes and store at -20 ^oC until assay.

4. Surgical Procedure

1. Cut/trim all fur from the abdomen and thorax of the underside of the carcass with electric shears. Place the carcass in a sterile cabinet and treat the shaved surface of the carcass with 70% alcohol.
2. Use a sterile scalpel to create a midline incision of the abdomen. Remove liver, mesenteric lymph nodes, spleen and small intestine from each rat using sterile tweezers.

5. Analysis of Bacterial Translocation 

1. Place each sample of liver, mesenteric lymph nodes and spleen into pre-weighed sterile tubes and weigh. Calculate the weight of the sample as a difference between the weight of a tube with a sample and the weight of an empty tube.
2. Add sterile PBS to the tube to obtain a 1:10 dilution (w/v) of the sample and homogenize each sample with a sterile glass tissue homogenizer to obtain a homogeneous suspension²¹.
3. Make serial dilutions (10^-1 to 10^-4) of each sample in sterile PBS. Plate 0.1 ml of all dilutions of each tissue onto surface of MacConkey's and 5% blood agar and Brucella blood agar with hemin (0.005 g/L) and vitamin K1 (0.01 g/L) plates.
4. Incubate MacConkey's and 5% blood agar plates aerobically and Brucella blood agar plates under anaerobic conditions at 37 ^oC²².
5. Count colonies of aerobic bacteria after 24 hr, and colonies of anaerobic bacteria after 48 hr of incubation using a colony counter. Express the results as the number of colony-forming units (CFU) in a gram of tissue.

6. Histological Analysis 

1. Remove small intestine samples from each rat by the surgical procedure (steps 4.1 – 4.2). Fix samples in Bouin’s Fixative, embed in paraffin, prepare 6 µm sections and stain sections with hematoxylin and eosin using standard procedures²³.
2. Use a high-resolution microscope system for measuring height of intestinal villi and total mucosal thickness in each sample (magnification 100X)²⁴. Analyze 4 samples from each rat and make at least twenty measurements in each sample. Express results in micrometers.

7. Cytokine Assay

1. Use commercial rat ELISA kits for IL-1β; IL-6; TNF-α; INF-γ, and IL-10 to measure levels of cytokines in serum according to manufacturer’s protocol.
2. Generate standard curves for each set of samples assayed using standards for the relevant kit. Define the level of cytokines in each sample by comparison of experimental data with the appropriate standard curve.

8. Lipopolysaccharide Assay

1. Analyze level of LPS in serum using a rat LPS ELISA kit according to manufacturer’s protocol. Estimate concentration of LPS in samples by comparison of the obtained values with the values of the standard curve.

9. High Resolution Light Microscopy of Blood

1. Use the microscope system described previously^25,26. Use a vibration isolation platform as a base for the microscope system and video camera and computer²⁵ to record the live images. Calibrate test images using a Richardson slide as previously described²⁷.
2. Place 7 µl of freshly drawn blood from each rat on a glass slide, coverslip, photograph and record ten image frames of 72 × 53.3 µm² in each sample (magnification 1,700X).
3. Measure the concentration of vesicles using software which provides high-resolution direct-view optical images in real time. Examine a minimum of 20 image frames for each experimental condition²⁸.

10. Statistics

1. Express all values as mean and standard deviation. Perform statistical analysis and graph plotting. Analyze the results with the two sample t-test, set the significance level at 0.05 to define statistical significance.