We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.
This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.
Different stressors affect human and animal health. Temperature is one of the most stressful factors, causing chronic, acute and even lethal illnesses1. Changes in intestinal morphology and a loss of gut barrier integrity after heat stress were documented in many cases2,3. This protective barrier is responsible for defense against translocation of gut bacteria and their toxins, in particular lipopolysaccharides (LPS), from the lumen to the internal circulation4,5. Stability of the gut microbiota significantly influences intestinal barrier function, the immune response of the host, and tolerance to stress conditions6. Thus, modulation of the intestinal microbiota can provide a novel approach for prevention of stress-related adverse effects.
Beneficial bacteria have been used as a promising strategy to modify the gut microbiota for successful management of various clinical conditions, such as diarrhea, inflammatory bowel disease, atopic dermatitis, metabolic disorders7-10. Probiotic effects of beneficial bacteria include production of essential metabolites to support intestinal health, stimulation of the immune system, promotion of lactose tolerance, restoration of epithelial dysfunction. Probiotics were effective in prevention of the stress-related complications in vitro and in animal models11,12.
Researchers have given more attention to Bacillus bacteria as probiotics because these bacteria support intestinal homeostasis13 and have beneficial effects on the host14. Our previous data demonstrated high efficacy of Bacillus probiotics against pathogens in vitro15,16 and in clinical trials17,18. Here, we aim to evaluate the preventive effect of B. subtilis BSB3 strain against complications after heat stress.
All experimental procedures were approved by IACUC of Auburn University, Auburn, Alabama.
1. Preparation of Culture Media, Dishes and Bacterial Culture
2. Animals
3. Experimental Design
4. Surgical Procedure
5. Analysis of Bacterial Translocation
6. Histological Analysis
7. Cytokine Assay
8. Lipopolysaccharide Assay
9. High Resolution Light Microscopy of Blood
10. Statistics
The mean body temperature of animals before and immediately after heat stress was 36.7 ± 0.07 oC and 40.3 ± 0.17 oC, respectively (P < 0.05). Exposure of rats to heat (group 3) resulted in significant inhibition of villi height and total mucosal thickness (Figures 2, 3). Oral administration of BSB3 strain before stress protected intestine from the harmful effect of heat.
Translocation of bacteria from the gut was determined by bacteriological analysis of mesenteric lymph nodes (MLN), liver and spleen of each rat. Bacterial cultures in a concentration of 1.7 x 103 ± 4.6 x 102 CFU/g tissue (Figure 4) were isolated only from MLN and liver of rats pre-treated with PBS and exposed to heat (group 3). All tested samples from control rats (groups 1, 2) and from heat-stressed animals received a BSB3 strain (group 4) were sterile. No bacteria were isolated from spleen samples.
No change of IL-1β; IL-6, TNF-α, INF-Γ cytokines levels were registered in heat-stressed rats. In the serum of animals treated with PBS before heat exposure significant elevation of IL-10 and LPS levels was found (group 3) – (Figures 5, 6). Pre-treatment with B. subtilis BSB3 (group 4) prevented the rise of IL-10 and LPS in serum of heat-stressed animals.
A significant increase of free vesicles concentration from (1.4 ± 0.2) × 106 to (3.8 ± 0.3) × 106 vesicles µl-1 (p < 0.001) was found in the blood of rats that received PBS before heat stress (Figures 7, 8). In a group of animals pretreated with BSB3 no change in the number of free vesicles was found after heat exposure (Figure 7).
Figure 1. Study Design. Two groups of rats (16 rats in each group) were treated by oral gavage with B. subtilis BSB3 (B. subtilis group) or with PBS (PBS group) twice a day. On day 3 each group was subdivided by 8 rats in each group. Rats from group 3 and 4 were placed at 45 oC for 25 min. Control animals (groups 1 and 2) were kept at room temperature. Please click here to view a larger version of this figure.
Figure 2. Measurement of Intestinal Villi Height (A) and Mucosal Thickness (B) in Rats. Rats pre-treated with PBS or B. subtilis BSB3 were exposed to 45 oC (Stress) or to room temperature (Control). Each group consisted of 8 rats. Twenty measurements of each parameter in each sample were taken. Values are presented as the mean and the standard deviation. The results were analyzed with the two sample t-test at significance level 0.05 to define statistical significance. Please click here to view a larger version of this figure.
Figure 3. Histological Images of Intestinal Mucosa Stained with Hematoxylin and Eosin. Rats were pre-treated with PBS or B. subtilis BSB3 and exposed to 45 oC (Stress) or to room temperature (Control). A. PBS/no stress; B. PBS/heat stress; C. B. subtilis/no stress; D. B. subtilis/heat stress. Villi height is indicated by arrows. Bar 200 µm. Please click here to view a larger version of this figure.
Figure 4. Total Number of Bacteria in Tissues of Rats. Rats pre-treated with PBS or B. subtilis BSB3 were exposed to 45 oC (Stress) or to room temperature (Control). Each group consisted of 8 rats. Mesenteric lymph nodes, liver, and spleen were aseptically removed from each rat, homogenized and plated onto agar media to recover aerobic and anaerobic bacteria. Total count of translocated bacteria (aerobic and anaerobic) is presented as colony-forming units (CFU) per gram of tissue. Values are presented as the mean and the standard deviation. Please click here to view a larger version of this figure.
Figure 5. Level of IL-10 in Serum of Rats. Rats pre-treated with PBS or B. subtilis BSB3 were exposed to 45 oC (Stress) or to room temperature (Control). Each group consisted of 8 rats. Blood serum was obtained from each rat and analyzed with a commercial rat ELISA kit. Values are presented as the mean and the standard deviation. The results were analyzed with the two sample t-test at significance level 0.05 to define statistical significance. Please click here to view a larger version of this figure.
Figure 6. LPS Concentration in Serum of Rats. Rats pre-treated with PBS or B. subtilis BSB3 were exposed to 45 oC (Stress) or to room temperature (Control). Each group consisted of 8 rats. Blood serum was obtained from each rat and analyzed with a commercial rat ELISA kit. Values are presented as the mean and the standard deviation. The results were analyzed with the two sample t-test at significance level 0.05 to define statistical significance. Please click here to view a larger version of this figure.
Figure 7. Number of Vesicles in Blood of Rats. Rats pre-treated with PBS or B. subtilis BSB3 were exposed to 45 oC (Stress) or to room temperature (Control). A small droplet (7 µl) of freshly drawn blood was placed on a glass slide, coverslipped, photographed and recorded ten image frames in each sample (magnification 1,700X). Concentration of vesicles was measured using software which provides high-resolution direct-view optical images in real time. Twenty image frames were examined for each experimental condition. Values are presented as the mean and the standard deviation. The results were analyzed with the two sample t-test at significance level 0.05 to define statistical significance. Please click here to view a larger version of this figure.
Figure 8. Increase of the Vesicles Concentration in Blood After Heat Stress. Video image of blood of rats pretreated with PBS before exposure to room temperature (A) or to 45 oC (B). A small droplet (7 µl) of freshly drawn blood was placed on a glass slide, coverslipped, photographed and recorded ten image frames in each sample (magnification 1,700X). The vesicle is indicated as V. Bar 6 µm. Please click here to view a larger version of this figure.
Exposure to high temperature results in serious health conditions29. Prevention and early detection of complications related to heat stress are of vital importance30. The presented protocol can be used to evaluate the efficacy of various approaches for the prevention of heat stress adverse effects.
Critical steps within this protocol include: the heat treatment procedure (45 oC, 25 min); the usage of apyrogenic materials during collection and processing of blood to prevent contamination with enterotoxins; keeping serum aliquots at -20 oC to avoid repeated freezing and thawing; and following sterile procedure during analysis of bacterial translocation. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress side effects. For other routes of administration and other compounds, this protocol can be modified. For example, the time of treatment before heat stress can be extended. Oral administration can be changed to rectal administration. Bacillus subtilis bacteria were used for prevention of heat stress complications by oral treatment of animals before exposure to elevated temperature. In order to cure complications of heat stress, this protocol can be modified by the use of bacteria for the treatment of animals after exposure to heat.
It was shown that the elevation of body temperature caused an increase of erythrocyte vesiculation, and this was completely prevented by pre-treatment with B. subtilis bacteria. Thus, the increase of the vesicles concentration in the blood during exposure to heat may indicate the level of heat stress and provides diagnostic evidence for the stability of erythrocytes and their resistance to heat. It is also a very useful and rapid test for the evaluation the efficacy of preventive treatment against heat stress complications. The induction of heat shock proteins as a marker of heat stress was previously discussed30, but this approach is time-consuming and cannot be used for real-time diagnosis of response to heat stress.
The presented approach for prevention of heat stress adverse effects is simple, cost effective and does not require additional equipment or special facilities. Future application of this work will help to understand the mechanisms of protection against heat stress complications and characterize the new procedures for this protection. This method is of significance for road workers, military personnel, athletes – groups of people that are at risk for heat stroke during intense outdoor physical activity29. The proposed approach allows one to examine the level of heat stress and to evaluate various methods to protect the health of people working in extreme conditions.
The authors have nothing to disclose.
This work was supported by the Auburn University fund FOP 101002-139294-2050.
Phosphate buffered saline (PBS) | Sigma-Aldrich, St. Louis, MO | P4417 | |
Ethyl Alcohol | Pharmco products Inc. Brookfield, CT, USA | 64-17-5 | |
Agar | VWR | 97064-334 | |
MacConkey agar plates | VWR | 470180-742 | |
5% blood agar plates | VWR | 89405-024 | |
Brucella blood with Hemin, and Vitamin K, plates |
VWR | 89405-032 | |
Bouin’s Fixative | Electron Microscopy Sciences, Hatfield, PA, USA | 15990 | |
IL-10 Rat ELISA kit | Invitrogen, Camarillo, CA, USA | KRC0101 | |
IL-1beta Rat ELISA Kit | Invitrogen, Camarillo, CA, USA | KRC0011 | |
IL-6 Rat ELISA Kit | Invitrogen, Camarillo, CA, USA | KRC0061 | |
INF-gamma Rat ELISA Kit | Invitrogen, Camarillo, CA, USA | KRC4021 | |
TNF-alpha Rat ELISA Kit | Invitrogen, Camarillo, CA, USA | KRC3011 | |
Rat LPS ELISA Kit | NeoBioLab, MA, USA | RL0275 | |
Environmental Chamber 6020-1 | Caron, Marietta, OH, USA | 6020-1 | |
Centrifuge | Beckman Coulter, Indianapolis, IN, USA | Optima L-90K | |
Ultra Centrifuge | |||
Light microscope optical system | CitoViva Technology Inc., Auburn, AL | ||
Colony counter | Fisher Scientific , Pittsburgh, PA, USA | RE-3325 | |
Freezer | Haier, Brooklyn, NY, USA | HCMO50LA | |
Ultra microplate reader | Bio-Tek Instrument, Winooski, VT, USA | ELx 808 | |
Auto Strip Washer | Bio-Tek Instrument, Winooski, VT, USA | ELx50 | |
Pipettes | Gilson, Pipetman, France | P100, P200, P1000 | |
C24 Incubator Shaker | New Brunswick Scientific, Enfield, CT, USA | Classic C24 | |
Rocking Shaker | Reliable Scientific, Inc., Nesbit, MS, USA | 55 | |
Petri dishes | Fisher Scientific, Pittsburgh, PA, USA | 875713 | 100mmX15mm |
SterilGard III Advance | The Baker Company, Sanford, ME, USA | SG403 | |
Culture Growing Flasks | Corning Incorporated, Corning, NY, USA | 4995 | PYREX 250mL Erlenmeyer flasks |
Optical Spectrometer Genesys 20 | Thermo Scientific, Waltham, MA, USA. | 4001 | |
Sony DXC-33 Video camera | Sony | ||
Richardson test slide | Electron Microscopy Science, Hatfield, PA, USA | 80303 | |
Millipore water purification system | Millipore | Direct-Q | |
Image-Pro Plus software | Media Cybernetics, MD, USA | ||
Triple Beam Balance | OHAUS Corporation, Parsippany, NJ, USA | ||
Tissue Homogenizer, Dounce | VWR | 71000-518 | |