Development of multi-species probiotics against calf diarrhea inducing pathogen and its validation of the effect on growth performance and intestinal microflora composition in Holstein Calves

학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2014. 2. 최윤재.Calf diarrhea is the most common and severe disease in young calves in that major economic losses to dairy farms. During a pre-weaning period, young calves are susceptible to many infectious pathogens, especially E.coli K99 causing diarrhea and dysentery with blood and mucus in the feces. The use of antibiotics helps to alleviate diarrhea, lowers the calf mortality and decreases the protein requirement in the young calves. The abuse of antibiotics to animal, however, may lead antibiotics resistance to potential human pathogens. Because of these circumstances, feeding antibiotics have been prohibited since 2011 in Korea. Therefore, development of antibiotic alternatives is required for sustainable livestock production. Probiotics are live microorganisms, suggesting that it may have beneficial effects on a host gut ecosystem. I have chosen lactic acid bacteria (LAB) since LAB are the most common type of microbes producing lactic acid, bacteriocins and other metabolic products which protect pathogen colonization and modulate immune responses. Previously, two strains of lactic acid bacteria such as Lactobacillus plantarum genome shuffling 1 (LP-GS1) and Pediococcus acidilactici genome shuffling 4 (PA-GS4) with improved antimicrobial activity against E.coli K99 and E.coli O157 were achieved, respectively, by genome shuffling method. In this study, the multi-species probiotics contained LP-GS1 and PA-GS4 were treated in Holstein calves. This newly developed multi-species probiotics include Bacillus subtilis T4 as a digestive enzyme source and Saccharomyces boulardii as an intestinal regulator and supplier of protein and mineral. Total of 40 holstein male calves (age 5-18 days) were randomly assigned to four diet groupsNegative control (NC, no treatment), Positive control (PC, antibiotics treatment), Wild type LAB added probiotics mixture (WPM, mixture of Pediococcus acidilactia PA175, Lactobacillus plantarum LP177, Saccharomyces boulardii SB, and Bacillus subtilis T4), and Genome shuffled LAB added probiotics mixture (GPM, mixture of Genome shuffled Pediococcus acidilactia PA-GS4, Genome shuffled Lactobacillus plantarum LP-GS1, SB, and T4). Test and control groups were fed using a milk replacer and a calf starter with probiotics mixture (109 cfu each strain/d/head), with antibiotics (neomycin sulfate) or no treatment for 8 weeks. Growth effects of multi-species probiotics were tested in Holstein calves. GPM group showed same average daily gain as PC group. And there was 50% mortality in NC and WPM group while 90% of calves were survived in PC and GPM group. Furthermore, GPM showed good modulation effect of intestinal microflora. Potential pathogens such as E.coli and Clostridium perfringens were lower in GPM group than in NC group. In addition beneficial bacteria such as Pediococcus acidilactici, Bacillus subtilis and Lactobacillus spp were higher in GPM group than in NC group. These results suggest that newly developed multi-species probiotics could use as promising antibiotic alternatives for making environment-friendly livestock products.Contents Summary I Contents III List of Tables and Figures VI List of Abbreviations VIII I. Introduction 1 II. Review of Literature 3 1. Neonatal calf diarrhea 3 2. Probiotics 4 1) Concept of probiotics 4 2) Intestinal regulatory effect of probioitcs 6 3. Properties of probiotics 7 1) Lactic acid bacteria 7 (1) Pediococcus acidilactici 9 (2) Lactobacillus plantarum 9 2) Bacillus subtilis 10 3) Saccharomyces boulardii 11 III. Materials and Methods 12 1. Preparation of multi-species probiotics 12 1) Microbial strains 12 2) Lactic acid bacteria strain 13 (1) Anti-microbial activity test 13 (2) Physiological test 14 ① pH curve 14 ② Growth curve 15 (3) Acid tolerance test 15 (4) Bile resistance test 15 3) Sample preparation of multi-species probiotics 15 4) Probiotics survivability in low temperature storage 18 2. Holstein calves feeding test 19 3. Growth performance 21 4. Fecal microflora analysis 21 1) Quantitative real-time polymerase chain reaction 21 2) MCK agar assay 22 5. Blood analysis 23 6. Statistical analysis 24 IV. Results and Discussion 25 1. Characterization of probiotics 25 1) Anti-microbial activity test 25 2) Physiological test 28 3) Acid tolerance test 29 4) Bile resistance test 29 5) Survivability in low temperature 30 2. Probiotics preparation 33 3. Growth performance in Holstein calves 35 4. Intestinal microflora composition 37 1) Quantitative real-time polymerase chain reaction 37 2) MCK agar assay 40 5. Blood analysis 41 1) Serum immunoglobulin 41 2) Blood cell counting 45 6. Conclusion 47 V. Literature Cited 49 VI. Summary in Korean 58 VII. Acknowledgement 60Maste