Blue Buffalobuffalo Clinicalclinical Reportreport

BLUEBLUE BUFFALOBUFFALO CLINICALCLINICAL REPORTREPORT

VOLUME NO. 1 Hydrolyzed for Food Clinical Evidence for: Intolerance KEY POINTS HF

1 ACTIVATED MAST CELL RESTING MAST CELL

BLUE Natural Veterinary Diet HF Hydrolyzed for Intact Protein features Food Intolerance IgE salmon hydrolysate, a Hydrolyzed protein novel protein with a mean particles molecular weight of 2 kDa to help reduce the risk of adverse reactions to food.

Inﬂammatory 2 mediator release

Multiple research study findings support that Hydrolyzed proteins Intact protein initiating too small to trigger BLUE Natural Veterinary type 1 immune response immune response Diet HF provides an ideal approach for nutritionally managing pets with Figure 1. Hydrolyzed proteins help avoid immune reactions. adverse food reactions:

•Novel protein plus low molecular weight BLUE Natural Veterinary Diet HF Hydrolyzed for Food Intolerance •No animal protein antigen contamination Often times the ingredient exclusions in a reaction to food due to a non-immunological diet are just as important as the inclusions. abnormal physiological response. Digestive •High digestibility This is essential in cases of true food enzyme deficiencies, garbage ingestion, •Preferred palatability allergies as well as food intolerance. Food vasoactive amines, contaminants such as hypersensitivity (allergy) is the term used to bacteria, metabolic, toxic, idiosyncratic •Ingredients preferred describe the clinical disease induced by food or pharmacological effects of foods or by clients ingestion in which there is an immunological food additives all can contribute to food reaction. This response is typically due intolerance. 1, 4, 5 to IgE-mediated type I hypersensitivity; In a clinical setting, food allergy and food however, types III and IV also are highly intolerance are rarely differentiated and 1 suspected. The immunological reaction is frequently respond to a similar dietary usually attributed to dietary water-soluble approach. Because the precise immunologic glycoproteins that have molecular weights processes of most adverse food reactions are 2, 3 ranging from 10 to 70 kDa. usually not known,4, 6, 7 on a practical level, Food intolerance (also known as food the phrase adverse food reactions (AFR) is sensitivity) is the term used for an adverse used to reference both conditions. NUTRITIONAL APPROACH TO CHART 1. HELP MANAGE PETS WITH ADVERSE HF MOLECULAR WEIGHT COMPARED WITH STUDY: FINISHED PRODUCT FOOD REACTIONS: COMMON ALLERGENS ELISA-TEKTM ANTIGEN TESTING

1) PROTEIN HYDROLYSATES PURPOSE Once diagnosed, patients with adverse Ensure that BLUE Natural Veterinary Diet food reactions must be carefully managed HF finished product does not contain other to minimize the potential for allergen common protein sources that might elicit exposure and triggering of immune an immune response. responses. One clinically proven option is the use of hydrolyzed protein diets, in STUDY DESIGN which the protein source is hydrolyzed Samples of BLUE Natural Veterinary to small molecular weights that are not Diet HF Hydrolyzed for Food Intolerance 8 allergenic (Figure 1).Studies have shown finished product from every production that 50% to 80% of dogs allergic to intact 2) AVOIDING COMMON FOOD ALLERGENS run are subjected to protein contaminant proteins have clinically improved when Since allergies are inappropriate or testing by commercially available 3, 9, 10 fed foods with protein hydrolysates. abnormal reactions of the immune system enzyme-linked immunosorbent assay, against a normal protein, allergies can ELISA-TEKTM.13 This highly sensitive form to any protein. The most common testing is designed to detect the presence food allergens are proteins with a of common food protein sources STUDY: PROTEIN POLYPEPTIDE molecular weight between 10 kDa and including beef, poultry, egg and soy. Test HYDROLYSATES MOLECULAR WEIGHT 70 kDa. Smaller proteins are normally samples are also collected throughout the ANALYSIS too little to elicit an immune reaction, production run to verify each run prior to while larger proteins cannot normally release and to validate the effectiveness access the body across the GI mucosa. The of steps taken in the manufacturing PURPOSE most commonly identified food allergens process, such as equipment cleanout and To show that BLUE Natural Veterinary in dogs and cats are listed in Table 1.6 burnout (a high-heat process to sterilize Diet HF Hydrolyzed for Food Intolerance Reactions to carbohydrate sources, such as the equipment), before and after each is formulated to reduce the risk of immune corn, rice and potato, have been reported manufacturing run. responses, with highly hydrolyzed salmon but appear to be much less common. 10 protein as the sole animal protein source. RESULTS12 TABLE 1. This testing continues to show results that STUDY DESIGN MOST COMMONLY IDENTIFIED FOOD meet our strict standards for evidence of Salmon hydrolysate intended for use ALLERGENS IN DOGS AND CATS6 contaminating proteins prior to release in BLUE Natural Veterinary Diet HF of the finished product and validates our Hydrolyzed for Food Intolerance formulas cleanout procedures. is regularly subjected to molecular Dogs (n=198) Cats (n=89) weight analysis by conventional Beef (36%) Beef (20%) In summary, by utilizing salmon as the testing methodology at AGROBIO Dairy (28%) Dairy (14.6%) protein source for hydrolysis, BLUE Laboratories.11 Fish (13%) Natural Veterinary Diet HF uniquely Wheat (15%) (e.g. sardines, tuna) 12 addresses both nutritional approaches RESULTS Egg (10%) Lamb (6.7%) for AFR: protein hydrolysis AND The majority of the protein hydrolysis Chicken (9.6%) Poultry (4.5%) avoidance of common food allergens. products from the salmon hydrolysate Lamb/Mutton (6.6%) Barley/Wheat (4.5%) This combined benefit of a novel protein in BLUE Natural Veterinary Diet HF Soy (6%) Diverse (11%) source and a low molecular weight helps Hydrolyzed for Food Intolerance have a reduce the risk of immune responses or mean molecular weight of 2 kDa or less. Additionally, to ensure that the finished other adverse reactions to food. • 78.2% of the water-soluble peptides in product does not contain other common the salmon hydrolysate ingredient sample protein sources that might elicit an 3) ENHANCED DIGESTIVE EFFICIENCY had a molecular weight of 2 kDa or less immune response, tests were conducted The use of highly digestible proteins has to determine for any evidence of common long been recommended for managing • 97.3% of the water-soluble peptides in the animal protein contaminants. food allergies. Hydrolysis of a protein salmon hydrolysate ingredient sample had enhances digestive efficiency and studies a molecular weight of 10 kDa or less show that BLUE Natural Veterinary Diet HF is highly digestible as well as results in ideal stool quality.12

BLUE BUFFALO CLINICAL REPORT species-appropriate housing and managed CHART 2. HIGH DIGESTIBILITY RESULTS consistently during the study, including STUDY: NUTRIENT ANALYSIS providing access to activity/exercise. Food AND DIGESTIBILITY consumption was monitored daily and body weights were recorded prior to study initiation and on study days 1, 3 and 5. PURPOSE Stool quality observations were made at least twice daily and scores were Prove that BLUE Natural Veterinary Diet recorded. The scoring scale ranged from HF Hydrolyzed for Food Intolerance is a 1 for diarrhea to 5 for hard, dry crumbly highly digestible pet food. feces and was aided by photographs of STUDY DESIGN examples. In this study, a stool score between 3 and 4 is considered to represent Three groups of adult dogs (n=6 each ideal fecal consistency for dogs and cats. for Canine Digestibility Studies 1, 2 and 3) and 3 groups of adult cats (n=7 RESULTS12 each for Feline Digestibility Studies 1, Overall, feeding BLUE Natural Veterinary 2 and 3) from a commercial research Diet HF Hydrolyzed for Food Intolerance facility were enrolled in the studies. in both dog and cat studies resulted All animals selected were clinically primarily in moist, formed (score of 3) or healthy. Animals were individually fed well-formed, sticky (score of 3.5) stools. the species-appropriate BLUE Natural Veterinary Diet HF Hydrolyzed for Food FIGURE 2. STOOL QUALITY SCORING Intolerance diet once daily as their sole source of nutrition for 10 days. Animals were maintained individually in standard, Mean Dry Matter Mean Protein species-appropriate housing and managed Digestibility Digestibility consistently during the study, including CANINE STUDIES FELINE STUDIES providing access to activity/exercise. Food consumption was monitored daily STUDY: and body weights were recorded on DETERMINING STOOL QUALITY days 1 through 6 and on day 10. On the last day of the study, a fecal sample from each animal as well as a sample PURPOSE of BLUE Natural Veterinary Diet HF Hydrolyzed for Food Intolerance diet These 5 studies were conducted to show was sent to a commercial laboratory that feeding BLUE Natural Veterinary for nutrient analysis. The results of Diet HF Hydrolyzed for Food Intolerance these analyses were used to calculate could result in ideal stool quality (fecal digestibility values, including dry matter consistency) in healthy dogs and cats. digestibility. Digestibility analysis was STUDY DESIGN performed according to the recommended protocol for use in the determination Three groups of adult dogs and 2 groups of metabolizable energy of pet food as of adult cats (n=10 each for Canine 4) HIGH PALATABILITY defined by AAFCO.14 Stool Quality Studies 1, 2 and 3 and for Feline Stool Quality Studies 1 and 2) Because of its impact on compliance and acceptability, high palatability is an RESULTS12 were enrolled in the studies. All animals important component of the nutritional Mean results from three studies in each selected were clinically healthy. Animals approach to adverse food reactions. species showed that BLUE Natural were individually fed the species- Studies show dogs and cats prefer BLUE Veterinary Diet HF is highly digestible. appropriate BLUE Natural Veterinary Diet HF Hydrolyzed for Food Intolerance diet Natural Veterinary Diet HF Hydrolyzed once daily as their sole source of nutrition for Food Intolerance over the leading for 7 days. For cats, diet was made hydrolyzed therapeutic pet food.15 available over a 4-hour period. Animals were maintained individually in standard,

Hydrolyzed for Food BLUE BUFFALO CLINICAL REPORT HF Intolerance REFERENCES STUDY: PALATABILITY TESTING UTILIZING TWO-PAN STANDARD 1. Scott, D.W., Miller, W.H., Griffin, C.E. Skin immune ASSAY TECHNIQUES system and allergic skin diseases. in: Muller and Kirk’s small animal dermatology. 6th ed. WB Saunders, PURPOSE 5) CLIENT INGREDIENT PREFERENCE Philadelphia; 543–666, 2001. Compare responses of dogs and cats 2. Biourge VC, Fontaine J, Vroom MW. Diagnosis of to BLUE Natural Veterinary Diet PET OWNER INSIGHTS18 adverse reactions to food in dogs: efficacy of a soy- HF Hydrolyzed for Food Intolerance In a survey of 300 pet owners, owners isolate hydrolyzate-based diet. J Nutr 134:2062-2064, palatability versus the leading hydrolyzed report that they prefer the top ingredients 2004. therapeutic pet food.15 in BLUE Natural Veterinary Diet HF 3. Puigdemont A, Brazis P, Montserrat S, et al. 5 to 1 over the ingredients in the leading Immunological responses against hydrolyzed soy STUDY DESIGN16, 17 hydrolyzed protein therapeutic diet. protein in dogs with experimentally induced soy Thirty adult dogs and 30 adult cats were Meeting client needs and preferences hypersensitivity. Am J Vet Res, 67:484-488, 2006. enrolled in the palatability studies. All is key to encouraging increased client 4. Kennis RA. Use of atopic dogs to investigate adverse animals selected were from calibrated compliance. reactions to food. JAVMA 221(5):638-640, 2002. palatability panels and were clinically healthy. Animals were maintained CLINICAL IMPACT 5. Leistra MHG, Markwell PJ, Willemse T. Evaluation of individually in standard, species- The studies discussed in this Clinical selected-protein-source diets for management of dogs appropriate housing and managed Report provide evidence supporting the with adverse reactions to foods. J Am Vet Med Assoc consistently during the study, including molecular weight analysis, antigen testing, 219:1411-1414, 2001. providing access to activity/exercise. digestibility, palatability and consumer 6. Verlinden A, Hesta M, Millet S, et al. Food allergy insights for BLUE Natural Veterinary Diet in dogs and cats: a review. Crit Rev Food Sci Nutr, Per standard protocol, each animal was HF. These findings support that BLUE 46:259-273, 2006. individually offered 2 stainless steel Natural Veterinary Diet HF provides an 7. Bryan J, Frank L. Food allergy in the cat: a diagnosis bowls, one containing BLUE Natural ideal approach to nutritionally manage by elimination. J Fel Med Surg, 12:861-866, 2010. Veterinary Diet HF Hydrolyzed for Food pets with adverse food reactions while 8. Roudebush P, Guilford WG, Jackson HA. Adverse Intolerance and the other the leading satisfying pet owner preferences for reactions to food. In Hand MS, Thatcher CD, Remillard hydrolyzed therapeutic pet food (leading quality, natural ingredients. RL, et al. (eds): Small Animal Clinical Nutrition, 5th ed, diet) once daily for 2 days. Dogs were offered 400 g of each diet and cats For more information about Blue Buffalo Topeka, Kan, 2010, Mark Morris Institute, p 609. were offered 100 g of each diet. Bowl Quality Assurance Testing and Clinical 9. Jackson HA. Food allergy in dogs—clinical signs and placement was reversed daily and both Research please visit TrueBLUEVets.com diagnosis. Eur J Comp Anim Pract. 19: 230-233, 2009. bowls were presented for 30 minutes for or call 1-888-323-BLUE. 10. Gaschen FP, Merchant SR. Adverse food reactions dogs and for 4 hours for cats. If one diet in dogs and cats. Vet Clin North Am Small Anim Pract was completely consumed prior to the 41:361-379, 2011. end of the test period, both bowls were 11. GROUPE Qualtech, Agrobio Laboratories analysis removed. Food consumption was recorded report, January 2016. for each animal and each diet on each day. 12. Blue Buffalo Co., Ltd., data on file, 2015. 12 13. ELISA-TEKTM Sigma-Aldrich® Elisa Technologies, Inc. RESULTS Gainesville, FL. 14. AAFCO (2015) Official Publication of the Association of American Feed Control Officials Inc., Champaign, IL. 15. GfK scanned data, 2015. 16. Smith J.C., Rashotte M.E., Austin T., Griffin R.W. Fine-grained measures of dogs’ eating behavior in single-pan and two-pan tests. Neurosci. Biobehav. Rev (8)243–251, 1984. 17. Aldrich GC, Koppel K. Pet Food Palatability Evaluation: A Review of Standard Assay Techniques Dogs and cats consumed significantly and Interpretation of Results with a Primary Focus on (P<0.001) more HF Hydrolyzed for Food Limitations. Animals. 5(1):43-55, 2015. Intolerance than the leading diet. 18. Blue Buffalo Co., Ltd., Consumer Study, July 2015.

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