FOR RUMINANTS. kemin.com/guthealth

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1 FOR RUMINANTS kemin.com/guthealth

2 What is CLOSTAT? CLOSTAT contains a proprietary, patented strain of Bacillus subtilis PB6. PB6 is a unique, naturally occurring, spore-forming microorganism. Kemin has identified and selected PB6 as an active substance that helps maintain the balance of microflora in the intestinal tract of livestock (Figure 1). Figure 1. Bacillus subtilis PB6 in the intestine. Features Contains PB6, a unique, patented strain of Bacillus subtilis Stable during processing and packaging Stable when blended with other feed ingredients Stable under normal commercial pelleting conditions Compatible with antibiotics, coccidiostats and organic acids commonly used in the animal feed industry Proven efficacy in research Stability Mineral premix concentrate (oxides, sulfates) for three weeks Long term - temperatures ranging from 5 C (41 F) to 65 C (149 F) for one year Short term F (90 C) for 10 minutes Resistant to acid conditions of ph 2.0 for 90 minutes Stable during processing and packaging, when blended with other feed ingredients, and under normal commercial pelleting conditions

3 Mode of Action Bacillus subtilis PB6 secretes active substances that are inhibitory toward various strains of pathogens including Clostridium, Salmonella and E.coli spp 1 (Figure 2). Figure 2. Pathogen cell wall disruption. Zone of Inhibition and Bacillus subtilis PB6 A zone of inhibition test examines bacteria sensitivity and resistance to certain compounds. The size of the zone surrounding the compound disk on the plate is an indication of microbial susceptibility to the compound (Figure 3). 2 Enteric Disease Pathogens There are many different pathogens causing intestinal health issues in ruminants. The most notable are E.coli, Salmonella spp, Clostridial spp, Cryptosporidium, Rotavirus and Coronavirus (Table 1). Of particular interest are E.coli, Salmonella and Clostridial bacteria. Each of these types of bacteria, while different in many aspects, can similarly target the intestinal tract, cause death by the systemic effects of toxins and show up clinically as bloody diarrhea. 4 Table 1. Enteric disease pathogens. Figure 3. Bacillus subtilis PB6 and Clostridium perfringens inhibition. 3 Pathogen Epidemiology E. Coli Attaches to intestinal wall and secretes toxins that disrupt normal gut function. 5 Salmonella spp. Clostridia spp. Invades the mucosa of the small and large intestine and produces toxins. Invasion of epithelial cells stimulates the release of pro-inflammatory cytokines, which induce an inflammatory reaction. The acute inflammatory response causes diarrhea and may lead to ulceration and destruction of the mucosa. 6 Secretes toxins that destroy intestinal lining and increase permeability of blood vessels. Overgrows in environments rich in protein, readily available energy. 7 Cryptosporidium parvum Causes changes in intestinal mucosa, including partial villous atrophy, crypt lengthening and inflammation. 8 Rotavirus and Coronavirus Infection of the small intestinal mucosa leads to loss of surface area and malabsorption of nutrients by intestinal villi. 9

4 Bacillus subtilis PB6 Research Research conducted with Bacillus subtilis PB6 has demonstrated antagonistic activity against a wide range of pathogens (Table 2) (Figure 4). 10 Table 2. Characteristics of Bacillus subtilis PB6. 10 Bacterial Strains Inhibition Zone Diameter (mm) Clostridium perfringens ATCC ± 0.44 Escherichia coli CVCC ± 0.14 Salmonella typhimurium ATCC ± 0.11 Staphylococcus aureus CVCC ± 0.26 Listeria monocytogenes CMCC ± 0.76 Vibrio alginnolyficus ± 0.43 It is important to note Salmonella is rarely considered part of the normal flora of the intestinal tract. 7 Asymptomatic cattle can shed Salmonella for prolonged periods after infection and, in the case of Salmonella Dublin infection, some cattle may be intermittent shedders for life. 7 Clostridial organisms are normally found in the digestive tract of cattle and only become problematic with dietary stress, injury, management changes, parasitism or other stress related events. 11 Rapid growth of C. perfringens results in the production of toxins, which can lead to intestinal tract disorders both in young and mature ruminants. A B C Figure 4. Characteristics of Bacillus subtilis PB6 on A) Clostridium perfringens ATCC13124, B) Escherichia CVCC1555, C) Salmonella typhimurium ATCC References 1. Yeow-Lim Teo, Alex., Tan, Hai-Meng. Applied and Environmental Microbiology, Aug. 2005, p Todar, K., Bacterial Resistance to Antibiotics. Todar s Online Textbook of Bacteriology. Accessed Kemin Internal Image IMG-KANA McGuirk, Sheila M., Dealing with Salmonella and Clostridium Problems on our Dairies. University of Wisconsin, School of Veterinary Medicine. 5. Enterohemorrhagic E. coli (EHEC) pathogenesis. Front Cell Infect Microbiol. 2012; 2: 90. Published online 2012 Jul 12. Prepublished online 2012 May 30. doi: /fcimb Y Nguyen1 and Vanessa Sperandio. 6. Ralph A. Giannella. Chapter 21 Salmonella. Medical Microbiology, 4th edition McGuirk, Sheila M., Managing Clostridial Diseases in Cattle. University of Wisconsin, School of Veterinary Medicine. 8. Ernest A. Meyer. Chapter 80 Other Intestinal Protozoa and Trichomonas Vaginalis. Medical Microbiology, 4th edition Rotaviral and coronaviral diarrhea. Vet Clin North Am Food Anim Pract Nov;1(3): Torres-Medina A, Schlafer DH, Mebus CA. 10. Kemin Internal Document, Songer, J. Glenn, Clostridial Enteric Diseases of Domestic Animals., Clinical Microbiology Reviews, Apr. 1996, p Kemin Internal Image used with Permission. 13. P. R. Broadway, CLOSTAT Reduces the Negative Impacts of a Salmonella Challenge in Weaned Holstein Steers. J. Anim. Sci. Volume 95, Supplement 1, This material is based upon work supported by the U.S. Department of Agriculture, Bioscience Research Lab, Agricultural Research Service, Lubbock, TX under agreement No. KANA Any opinions, findings, conclusion, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 14. Kluger, M.J. and B.A. Rothenburg Fever and reduced iron: their interaction as a host defense response to bacterial infection. Science 203(4378): Patent U.S.7,247,299.

5 Reducing the Negative Impact of Pathogenic Salmonella Typhimurium in Weaned Holstein Steers A research trial conducted with the United States Department of Agriculture at the Livestock Issues Research Unit in Lubbock, TX evaluated the potential for CLOSTAT, a patented Bacillus subtilis active microbial, to reduce the severity of salmonellosis in weaned Holstein steers challenged with Salmonella Typhimurium. 13 Calves were fed either control diets (no CLOSTAT) or 13 g/h/d CLOSTAT in a starter ration for 35 days. Calves were then assigned to one of four treatments, consisting of CLOSTAT or no CLOSTAT and Salmonella (1.6 x 10 6 Salmonella Typhimurium) or no Salmonella. The CLOSTAT calves displayed decreased rectal temperatures (P< 0.001) after the study compared to the control calves challenged with Salmonella. Mounting an immune response to a pathogen challenge requires a significant amount of energy. It has been estimated that an increase in core body temperature by 1 o C requires an increase of percent in an animal s metabolic rate. 14 Mediating this change in body temperature would potentially spare glucose, allowing energy to be put towards other productive functions. NoSal Control NoSal CLOSTAT Sal Control Sal CLOSTAT Rectal Temperature, C Summary Time Relative to Salmonella Challege, h Figure 5. Effect of CLOSTAT on rectal temperatures of weaned Holstein steers challenged with Salmonella Typhimurium. CLOSTAT is an active microbial containing a proprietary, patented strain of Bacillus subtilis PB6. Over 200 internal R&D documents referencing PB6 research trial work. North America China Europe India SE Asia In vitro analysis evaluating PB6 efficacy against multiple strains within bacterial organisms. 15 Commercial animal research trials demonstrating consistent response. Gut health is a highly complex system including the structural integrity of the intestine, the balance of microflora and the status of the immune system. Kemin offers a comprehensive platform of leading solutions impacting key areas of gut health.

6 Comprehensive approach to gut health by strengthening intestinal integrity, microbial balance and immune function. Integrity: Maintaining the strength of the intestinal barrier Protection: Supporting immune function Balance: Diversifying the population of microbes Gut health is a highly complex system including the structural integrity of the intestine, the balance of microflora and the status of the immune system. Kemin offers a comprehensive platform of industry leading solutions for livestock, poultry and equine. Kemin offers a range of nutritional solutions for raising healthy animals. We understand your need to raise healthy livestock that gives consumers the nutritional and health benefits they are looking for, while also returning a profit. Our products and services help you with: Nutrition Feed Quality Gut Health Pathogen Control KEMIN.COM/GUTHEALTH Kemin Industries, Inc. and its group of companies All rights reserved. Trademarks of Kemin Industries, Inc., U.S.A. BR