This work was supported by the Auburn University fund FOP 101002-139294-2050.

All experimental procedures were approved by IACUC of Auburn University, Auburn, Alabama.
1. Preparation of Culture Media, Dishes and Bacterial Culture
<ol>
	<li>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.</li>
	<li>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 12...

<ol>
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Rev. 32, 185-190 (2004).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+heat+stress+on+the+porcine+small+intestine:+A+morphological+and+gene+expression+study.">Effect of heat stress on the porcine small intestine: A morphological and gene expression study.</a> Comp. Biochem. Physiol. A-Mol. Integr. Physiol. 156, 119-128 (2010).</li><li>Moseley, P. L., Gisolfi, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Frontiers+in+Thermoregulation+and+Exercise.">New Frontiers in Thermoregulation and Exercise.</a> Sports Med. 16, 163-167 (1993).</li><li>Lambert, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress-induced+gastrointestinal+barrier+dysfunction+and+its+inflammatory+effects.">Stress-induced gastrointestinal barrier dysfunction and its inflammatory effects.</a> J. Anim. Sci. 87, 101-108 (2009).</li><li>Berg, A., Muller, H. M., Rathmann, S., Deibert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gastrointestinal+system+-+An+essential+target+organ+of+the+athlete's+health+and+physical+performance.">The gastrointestinal system - An essential target organ of the athlete's health and physical performance.</a> Exerc. Immunol. Rev. 5, 78-95 (1999).</li><li>Khan, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbes,+intestinal+inflammation+and+probiotics.">Microbes, intestinal inflammation and probiotics.</a> Expert Rev Gastroent. 6, 81-94 (2012).</li><li>Quigley, E. M. 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C., Bueno, A. A., de Souza, R. G. M., Mota, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+microbiota,+probiotics+and+diabetes.">Gut microbiota, probiotics and diabetes.</a> Nutr. J. 13, (2014).</li><li>Eutamene, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+Lactobacillus+paracasei+and+its+bacterial+products+to+counteract+stress-induced+gut+permeability+and+sensitivity+increase+in+rats.">Synergy between Lactobacillus paracasei and its bacterial products to counteract stress-induced gut permeability and sensitivity increase in rats.</a> J. 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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+anti-Helicobacter+pylori+activity+of+the+probiotic+strain+Bacillus+subtilis+3+is+due+to+secretion+of+antibiotics.">In vitro anti-Helicobacter pylori activity of the probiotic strain Bacillus subtilis 3 is due to secretion of antibiotics.</a> Antimicrob Agents Ch. 45, 3156-3161 (2001).</li><li>Sorokulova, I. B., Kirik, D. L., Pinchuk, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotics+against+Campylobacter+pathogens.">Probiotics against Campylobacter pathogens.</a> J. Travel Med. 4, 167-170 (1997).</li><li>Gracheva, N. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+induces+the+translocation+of+cutaneous+and+gastrointestinal+microflora+to+secondary+lymphoid+organs+of+C57BL/6+mice.">Stress induces the translocation of cutaneous and gastrointestinal microflora to secondary lymphoid organs of C57BL/6 mice.</a> J. Neuroimmunol. 171, 29-37 (2006).</li><li>Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+commensal+microflora+by+toll-like+receptors+is+required+for+intestinal+homeostasis.">Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis.</a> Cell. 118, 229-241 (2004).</li><li>Leon, L. R., Blaha, M. D., DuBose, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+cytokine,+corticosterone,+and+tissue+injury+responses+in+mice+during+heat+strain+recovery.">Time course of cytokine, corticosterone, and tissue injury responses in mice during heat strain recovery.</a> J. Appl. Physiol. 100, 1400-1409 (2006).</li><li>Ribeiro, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weight+loss+and+morphometric+study+of+intestinal+mucosa+in+rats+after+massive+intestinal+resection.+Influence+of+a+glutamine-enriched+diet.">Weight loss and morphometric study of intestinal mucosa in rats after massive intestinal resection. Influence of a glutamine-enriched diet.</a> Rev. Hosp. Cl&#236;n. Fac. Med. S. Paulo. 59, 349-356 (2004).</li><li>Vainrub, A., Pustovyy, O., Vodyanoy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+90+nm+(lambda/5)+in+an+optical+transmission+microscope+with+an+annular+condenser.">Resolution of 90 nm (lambda/5) in an optical transmission microscope with an annular condenser.</a> Opt Lett. 31, 2855-2857 (2006).</li><li>Moore, T., Globa, L., Pustovyy, O., Vodyanoy, V., Sorokulova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+administration+of+Bacillus+subtilis+strain+BSB3+can+prevent+heat+stress-related+adverse+effects+in+rats.">Oral administration of Bacillus subtilis strain BSB3 can prevent heat stress-related adverse effects in rats.</a> J Appl Microbiol. 117, 1463-1471 (2014).</li><li>Richardson, T. 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The authors declare that they have no competing financial interests.

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 p...

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.

&#160;
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich,&#160;St. Louis, MO</td>
			<td rowspan="2">P4417</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol&#160;</td>
			<td>Pharmco products Inc. Brookfield, CT, USA&#160;</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>VWR</td>
			<td>97064-334</td>
			<td></td>
		</tr>
		<tr>
			<td>MacConkey agar plates</td>
			<td>VWR</td>
			<td>470180-742</td>
			<td></td>
		</tr>
		<tr>
			<td>5% blood agar plates</td>
			<td>VWR</td>
			<td>89405-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Brucella blood with Hemin, and Vitamin K, 
			&#160;plates</td>
			<td>VWR</td>
			<td>89405-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Bouin&#8217;s Fixative</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>15990</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-10 Rat ELISA kit</td>
			<td>Invitrogen, Camarillo, CA, USA</td>
			<td>KRC0101</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-1beta Rat ELISA Kit</td>
			<td>Invitrogen, Camarillo, CA, USA</td>
			<td>KRC0011</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6 Rat ELISA Kit</td>
			<td>Invitrogen, Camarillo, CA, USA</td>
			<td>KRC0061</td>
			<td></td>
		</tr>
		<tr>
			<td>INF-gamma Rat ELISA Kit</td>
			<td>Invitrogen, Camarillo, CA, USA</td>
			<td>KRC4021</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF-alpha Rat ELISA Kit</td>
			<td>Invitrogen, Camarillo, CA, USA</td>
			<td>KRC3011</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat LPS ELISA Kit</td>
			<td>NeoBioLab, MA, USA</td>
			<td>RL0275</td>
			<td></td>
		</tr>
		<tr>
			<td>Environmental Chamber 6020-1</td>
			<td>Caron, Marietta, OH, USA</td>
			<td>6020-1</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Centrifuge&#160;</td>
			<td rowspan="2">Beckman Coulter, Indianapolis, IN, USA</td>
			<td>Optima L-90K</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Ultra Centrifuge</td>
		</tr>
		<tr>
			<td>Light microscope optical system</td>
			<td>CitoViva Technology Inc., Auburn, AL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Colony counter</td>
			<td>Fisher Scientific , Pittsburgh, PA, USA</td>
			<td>RE-3325</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td>Haier, Brooklyn, NY, USA</td>
			<td>HCMO50LA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra microplate reader</td>
			<td>Bio-Tek Instrument, Winooski, VT, USA</td>
			<td>ELx 808</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto Strip Washer</td>
			<td>Bio-Tek Instrument, Winooski, VT, USA</td>
			<td>ELx50</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Gilson, Pipetman, France</td>
			<td>P100, P200, P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>C24 Incubator Shaker</td>
			<td>New Brunswick Scientific, Enfield, CT, USA</td>
			<td>Classic C24</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking Shaker&#160;</td>
			<td>Reliable Scientific, Inc., Nesbit, MS, USA</td>
			<td>55</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>875713</td>
			<td>100mmX15mm</td>
		</tr>
		<tr>
			<td>SterilGard III Advance</td>
			<td>The Baker Company, Sanford, ME, USA</td>
			<td>SG403</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Growing Flasks</td>
			<td>Corning Incorporated, Corning, NY, USA</td>
			<td>4995</td>
			<td>PYREX 250mL Erlenmeyer flasks</td>
		</tr>
		<tr>
			<td>Optical Spectrometer Genesys 20</td>
			<td>Thermo Scientific, Waltham, MA, USA.</td>
			<td>4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sony DXC-33 Video camera</td>
			<td>Sony</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Richardson test slide</td>
			<td>Electron Microscopy Science, Hatfield, PA, USA</td>
			<td>80303</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore water purification system</td>
			<td>Millipore</td>
			<td>Direct-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Image-Pro Plus software</td>
			<td>Media Cybernetics, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triple Beam Balance</td>
			<td>OHAUS Corporation, Parsippany, NJ, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Tissue Homogenizer, Dounce</td>
			<td rowspan="3">VWR</td>
			<td rowspan="3">71000-518</td>
			<td rowspan="3"></td>
		</tr>
		<tr>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>

prevention of heat stress adverse effects in rats by bacillus subtilis strain

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.

The mean body temperature of animals before and immediately after heat stress was 36.7 &#177; 0.07 oC and 40.3 &#177; 0.17 oC, respectively (P &#60; 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.
Trans...

Watch this Scientific Journal Video about Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain at JoVE.com

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

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.

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

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

The overall goal of this experiment is to evaluate the preventative effect of the B.subtilis BSB3 strain against complications after heat stress. This method can help answer key questions in the prevention of adverse effect from heat stress. The main advantage of this technique is that it can be used to evaluate different approaches to mitigate the complications of heat stress.Demonstrating this procedure will be Ludmila Globa. And Oleg Pusovyy, research associates from our laboratory. To begin, with a single colony of Bacillus subtilis BSB3 that was grown overnight on a nutrient agar plate.Inoculate ten milliliters of nutrient broth in a flask. Grow the culture overnight at 37 degrees Celsius. Prepare 500 milliliters of sporulation medium according to the recipe shown here.And autoclave at 121 degrees Celsius for 20 minutes. Allow the medium to cool to 50 degrees Celsius before adding the following sterile solutions. In a sterile cabinet, cool the prepared medium at room temperature to 40 degrees Celsius for 20 minutes.And pour 25 milliliters into each of 20 sterile petri dishes. Allow the medium to solidify overnight. Next, spread 0.5 milliliters of the overnight Bacillus subtilis BSB3 culture onto the surface of the sporulation medium plates.Incubate the plates at 37 degrees Celsius for five days until 90%sporulation. Then use high resolution microscopy to check for phase-bright spores. To harvest the bacteria, add one milliliter of PBS to the plates, and use a sterile cell spreader to scrape the bacteria from the surface.Store the suspension at four degrees Celsius until needed. Confirm the viability of the bacteria by plating 0.1 milliliter of the appropriate dilution of a bacterial suspension on sporulation medium plates and incubate at 37 degrees Celsius for 18 to 24 hours. Use a colony counter to count bacterial colonies and calculate the titer of bacteria in the tested suspension.Then prepare a bacterial suspension in PBS at a final concentration of one times ten to the eighth CFUs per milliliter. After treating rats by oral gavage with B.subtilis BSB3 or PBS for two days, then subdividing the animals and taking their temperature, keep rats from groups one and two at room temperature for 25 minutes. Place animals of groups three and four in a climate chamber at 45 degrees Celsius and 55%relative humidity for 25 minutes.Following the measurement of the rectal temperatures of each rat again, place all animals at room temperature for four hours. Then, after euthanizing the animals, collecting the trunk blood, and isolating organs according to the text protocol, place each organ sample into pre-weighed sterile tubes and weigh. Add sterile PBS to each tube to obtain a one to ten weight per volume dilution of the sample, and use a sterile glass tissue homogenizer to generate homogeneous tissue suspensions.Next, use sterile PBS to make serial dilutions of each sample. Then plate 0.1 milliliters of all dilutions onto the surface of MacConkeys and 5%blood agar. And brucella blood agar with Hemin and Vitamin K1.After incubating the plates, use a colony counter to count the colonies on each group of plates.Express the results as the number of colony forming units, or CFUs, per gram of tissue. After preparing and staining sections of the small intestines according to the text protocol, use a high resolution microscope to measure the intestinal villi and total mucose wall thickness. Analyze at least four samples from each rat and make at least 20 measurements in each sample.To carry out high resolution light microscopy of blood samples, use the vibration isolation platform as a base for the microscope system with a video camera and computer to record live images. After calibrating test images according to the text protocol, place seven microliters of freshly drawn blood from each rat on a glass slide and add a coverslip. Then photograph and record ten image frames of 72 by 53.3 micrometers squared in each sample.Finally, using software that provides high resolution direct view optical images in real time, measure the concentration of vesicles. The mean body temperature of animals before and immediately after heat stress was 36.7 plus or minus 07 degrees Celsius and 40.3 plus or minus 0.17 degrees Celsius respectively. Exposure of rats to heat resulted in significant inhibition of villi height and total mucosal thickness.However, treatment with the BSB3 strain before stress, protected the intestine from the harmful effect of heat. For translocation of bacteria from the gut, mesenteric lymph nodes, liver, and spleen were analyzed. As illustrated here, bacteria were isolated at a concentration of 1.7 times ten to the three plus or minus 4.6 times ten to the two CFUs per gram of tissue only in samples from mesenteric lymph nodes and liver of PBS treated rats exposed to heat.On the other hand, all tested samples from control and heat stressed animals that received the BSB3 strain were sterile. No change in IL 1beta, IL-6, TNFalpha, or interferon gamma cytokine levels were registered in heat stressed rats. However, IL-10 and LPS levels were elevated in PBS treated animals before heat treatment.Therefore, B.subtilis BSB3 pre treatment prevented the rise of IL-10 and LPS in serum after heat treatment. Finally, a significant increase in the concentration of free vesicles was found in the blood of rats that received PBS before heat stress, but not in the BSB3 treated animals. It's important to remember to use a pyrogenic materials for blood collections and precision to keep serum liquids at minus 20 degrees Celsius that with repeated freezing and thawing, and to follow a sterile procedure during analysis of bacterial translocation.Following this procedure like administration of bacteria after heat stress can be performed in order to answer additional questions about complications of heat stress.

prevention-heat-stress-adverse-effects-rats-bacillus-subtilis

Research

JoVE Journal

Immunology and Infection

7.4K Görüntüleme.  Auburn University. The overall goal of this experiment is to evaluate the preventative effect of the B.subtilis BSB3 strain against complications after heat stress. This method can help answer key questions in the prevention of adverse effect from heat stress. The main advantage of this technique is that it can be used to evaluate different approaches to mitigate the complications of heat stress.Demonstrating this procedure will be Ludmila Globa. And Oleg Pusovyy, research associates from our laboratory....