Probiotic Approach for Prevention of Heat Stress-Related Complications Iryna Sorokulova, Ludmila Globa, Oleg Pustovyy, Vitaly Vodyanoy Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849 RESULTS Body temperature - Exposure of rats to high temperature resulted in significant increase of body temperature. The mean body temperature of rats before and immediately after heat stress were 36.7±0.07oC and 40.3 ±0.17oC, respectively (P<0.05). Histology - Morphometric study of intestine showed that heat stress significantly inhibited villi height and total mucosal thickness in rats (Figures 2A,B; 3). INTRODUCTION Temperature is one of the most challenging stressors, affecting human and animal health. Extreme heat stress can damage the gastrointestinal mucosa, which protects the internal environment of the body from bacteria and bacterial endotoxins (lipopolysaccharides). Dysfunction of this protective barrier results in increased intestinal permeability and diffusion of toxic bacterial components from the gut lumen to the blood. Gut microbiota play key role in keeping mucosal barrier function. By modulating the intestinal microbiota one might offer a novel and non-invasive therapeutic approach for promoting host well-being by prevention and treatment of the adverse effects of stress in the gut. . Figure 3. Histological images of intestinal mucosa A- PBS/25oC; B-PBS/45oC; C- Probiotic/25oC; D-Probiotic/45oC. OBJECTIVES The main goal of this study was to determine the efficacy of probiotic bacteria B. subtilis BSB3 in prevention of heat stress-related complications. Figure 2.. Rats were pre-treated with PBS ( ) or probiotic ( ) METHODS Ethics Statement - All animal procedures were approved by the Auburn University Institutional Animal Care and Use. The study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Bacillus probiotic strain - B. subtilis BSB3 with promising probiotic activity was used in this study. Animals - Adult male Sprague–Dawley rats (Harlan Laboratories, USA) weighing 250–300 g were used. The animals were housed two in a cage under specific pathogen free conditions and were acclimatized for 2 days at temperature (21 ± 1o C), humidity (50 ± 5%) and lighting (12-h day/12-h night) with free access to water and standard food. Experimental design. Rats were treated by oral gavage with B. subtilis probiotic or PBS twice a day for two days. Half of the rats of each group were exposed to heat stress (45o C, relative humidity 55% for 25 min) while the remaining rats were placed at identical conditions but at 25oC (Figure 1). Bacterial translocation - Mesenteric lymph nodes, liver, and spleen were removed under sterile conditions and homogenates were plated onto 5% blood and MacConkey’s agar plates for recovery of aerobic bacteria and Brucella blood agar plates supplemented with vitamin K1 and hemin for anaerobic bacteria. Histological analysis – Ileum samples were removed from each rat, fixed in Bouin’s Fixative , embedded in paraffin, sectioned at 6 micrometers, slide mounted, hematoxylin and eosin (H&E) stained, and cover-slipped. Intestinal villi height and total mucosal thickness were measured for each sample using a high resolution microscope system. Translocation of bacteria - All samples were sterile in control animals (groups 1 and 2) and in heat-stressed animals, pre-treated with probiotic (group 4). From MLN and liver of heat-stressed animals (group 3) bacterial cultures were isolated (Fig. 4). Figure 4. Protective effect of probiotic against bacterial translocation. Rats were pre-treated with PBS ( ) or probiotic ( ) Figure 5. Serum IL-10 concentration in animals of different groups. Rats were pre-treated with PBS ( ) or probiotic ( ) Cytokines- Heat stress did not result in change of IL-1β; IL-6, TNF-α, INF-γ cytokines level. Significant increase of IL-10 level was found in rats, exposed to heat and pre-treated with PBS (group 3- PBS/Stress) – Figure 5. No change of IL-10 concentration was determined in animals treated with probiotic before heat stress (group 4 – Probiotic/Stress). Serum LPS concentration - Level of LPS significantly increased in serum of heat-stressed animals, which received PBS before stress exposure (Figure 6). Concentration of LPS in serum of rats, pre-treated with probiotic before heat stress, was not significantly changed in comparison with non-stressed animals (4.3±0.03 ng ml-1 and 2.1±0.02 ng ml-1 correspondingly) (P>0.05). Vesiculation of rat erythrocytes after exposure to heat - The concentration of free vesicles increased after exposure to heat stress from (1.4±0.2)×106 to (3.8±0.3)×106 vesicles μl-1 in rats, pre-treated with PBS (Figure 7). In probiotic treated animals exposed to high temperature no significant change in concentration of free vesicles was found (Figure 7). Cytokines assay - Serum levels of cytokines were measured using commercial rat cytokine-specific sandwich enzyme-linked immunosorbent assay (ELISA) kits for IL-1β; IL-6; TNF-α; INF-γ, and IL-10 (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer’s instructions. The minimum detectable doses of these kits were: IL-1β (<3 pg/mL); IL-6 (<5 pg/mL); TNF-α (<4 pg/mL); INF-γ (<13 pg/mL), IL-10 (<5 pg/mL). Serum concentration of LPS was analyzed by rat LPS ELISA kit (NeoBioLab, Cambridge, MA, USA), which is a quantitative sandwich immunoassay. High resolution light microscopy of blood were performed using the microscope system built in our laboratory. Statistics - All results were presented as mean and standard deviation. Statistical calculations and graph plotting were carried out using Microcal™ Origin version 6.0 (Northhampton, MA) and 2010 Microsoft Excel. Figure 1. Experimental design Figure 6. Serum LPS concentration in animals of different groups. Rats were pre-treated with PBS ( ) or probiotic ( ) Figure 7. Vesicles concentration in blood of rats. Rats were pre-treated with PBS ( ) or probiotic ( ) CONCLUSIONS: B. subtilis BS3B strain was effective in the prevention of complications related to heat stress in rats. ACKNOWLEDGMENTS – This work was supported by Auburn University Funds #10100021392942050.