1. Institute of Bacteriology and Mycology University of Leipzig Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM) Monika Krueger, Karen Thaens, W. Schroedl

2. Introduction Microorganisms of the gastrointestinal tract (GIT) influence health and performance of animals and human beings. They live in a close relationship to their hosts. Quality and quantity of the microorganisms influence digestion, metabolism and immuno- logical reactivity of the host. Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM)

3. The GIT is the focus of interaction of the outside to the inside. Food modulates microorganisms and their products – metabolites, hormones, toxins, etc. supporting or disturbing numerous metabolic, immune and nervous functions of the GIT. The GIT is the focus of interaction of the outside to the inside. Food modulates microorganisms and their products – metabolites, hormones, toxins, etc. supporting or disturbing numerous metabolic, immune and nervous functions of the GIT. Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM)

4. High-yielding dairy cows are influenced by diets rich of fat, proteins and carbohydrates. These diets promote lipolytic, proteolytic and saccharolytic microorganisms. High-yielding dairy cows are influenced by diets rich of fat, proteins and carbohydrates. These diets promote lipolytic, proteolytic and saccharolytic microorganisms. They overgrow and damage the gastrointestinal barrier. They overgrow and damage the gastrointestinal barrier. Microorganisms and their toxins translocate. They damage the immune system and they are the cause of milk losses and diseases. Microorganisms and their toxins translocate. They damage the immune system and they are the cause of milk losses and diseases. Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM)

5. Objective I In a German dairy farm, cows and their calves were infected by Salmonella Zerro, a relatively unknown serotype, over a period of several months. At the beginning of the infection some calves and cows diseased severely. At present, salmonellosis is eradicated. Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM)

6. Objective II Measures: Measures: Effective Microorganisms (EM) were given in feed from Effective Microorganisms (EM) were given in feed from 27. 03.03 - 25.02.04, 27. 03.03 - 25.02.04, November 03: Lactulose application in November 03: Lactulose application in feed over 6 weeks, feed over 6 weeks, November 03: vaccination against sal- November 03: vaccination against sal- monellosis by farm specific vaccine. monellosis by farm specific vaccine. Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM)

7. Material and Methods Material and Methods A dairy farm of about 1400 cows and their calves in Saxony was involved in these investigations. A dairy farm of about 1400 cows and their calves in Saxony was involved in these investigations. Faeces and blood plasmas of 20 randomly selected cows of the highest yielding group, 15 cows of the medium yielding group, 15 heifers, 15 dried off cows and 15 cows immediately after birth were investigated. Faeces and blood plasmas of 20 randomly selected cows of the highest yielding group, 15 cows of the medium yielding group, 15 heifers, 15 dried off cows and 15 cows immediately after birth were investigated. Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM)

8. Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM) Material and Methods Schedule of investigation: - before EM application - 6 weeks after permanent EM application - 6 months after permanent EM application - 12 months after permanent EM application

9. Material and methods N umber of investigated animals Material and methods N umber of investigated animals 27.03.14.05.24.09.25.02. number of all animals all animals80725647 group I group I20201918 group II group II1514106 group III group III151399 group IV group IV151286 group V group V1513108 Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM)

10. The following parameters were used to evaluate the effect of EM: Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM) Material and Methods Faeces: Parameter Total aerobic bacterial count Gram-negative bacterial count Total anaerobic bacterial count Clostridium perfringens Method Nutritional-agar (SIFIN) McConkey-agar (SIFIN) Columbia-agar (OXOID) Blood-agar with Neomycine and Polymyxine B

11. Lactobacillus spp. Lactobacillus spp. Bifidobacterium spp. Bifidobacterium spp. Yeasts Yeasts Salmonella serotypes Salmonella serotypes Bdellovibrio spp. Bdellovibrio spp. Staphylococci Staphylococci Bacteriodes spp. Bacteriodes spp. MRS-agar (OXOID) Sabouraud-agar (SIFIN) Rappoport enrichment medium, Kauffmann-agar (SIFIN) Two component-agar acc. to Stolp 1963 Staphylococcus-Medium No. 110 (OXOID Neomycine, Desoxycholat Nutritional-agar Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM) Method Parameter Material and methods

12. iesiesiesies BloodParameters  CRP  Haptoglobin  LPS binding Protein  IgG and IgM anti- LPS antibodies E. coli J5  IgG and IgM anti-  Salmonella Cerro –antibodies Method ELISA ELISA Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM) Material and methods

13. Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM) Quantitative results of microbiological investigations of faeces from cows treated with EMa

14. Effects of EMa on total aerobic bacterial counts in faeces of cows H1H2H3H4median 4,79 x 10 5 4,17 x 10 6 1,07 x 10 6 2,45 x 10 6 * p < 0,05 to H1 ** lg/g

15. Effects of EMa on anaerobic total bacterial counts in faeces of cows H1H2H3H4median 2,00 x 10 6 1,51 x 10 7 9,12 x 10 6 1,82 x 10 7 ** * p < 0,05 to H1 lg/g

16. Effects of EMa on counts of lactobacilli in faeces of cows H1H2H3H4median 1,32 x 10 5 1,05 x 10 6 3,89 x 10 5 1,07 x 10 5 * * p < 0,05 to H1 * lg/g

17. Effects of EMa on bifidobacteria counts in faeces of cows H1H2H3H4median 4,57 x 10 5 1,41 x 10 6 7,94 x 10 6 1,78 x 10 7 *** * p < 0,05 to H1H3, H4: p < 0,05 to H2 lg/g

18. Effects of EMa on aerobic Gram-negative bacterial counts in faeces of cows H1H2H3H4median 1,78 x 10 4 3,09 x 10 5 3,89 x 10 4 3,98 x 10 4 * p < 0,001 to H1, H3 und H4 * lg/g

19. Effects of EMa on yeast counts in faeces of cows H1H2H3H4median 3,02 x 10 3 5,89 x 10 3 4,27 x 10 3 3,98 x 10 3 * * p < 0,05 to H1, H3 und H4 lg/g

20. Effects of EMa on Bacteroides counts in faeces of cows H1H2H3H4median 1,17 x 10 4 1,66x 10 4 5,37 x 10 3 2,75 x 10 3 * * p < 0,05 to H2 * lg/g

21. Effects of EMa on staphylococcal counts in faeces of cows H1H2H3H4median 9,12 x 10 4 1,07 x 10 5 1,15 x 10 4 1,17 x 10 5 lg/g

22. Effects of EMa on Clostridium perfringens counts in faeces of cows H1H2H3H4median 1,66 x 10 4 5,50 x 10 3 7,08 x 10 3 6,03 x 10 3 lg/g

23. Effects of EMa on alkalic phosphatase in faeces of cows U/g

24. Results EMa in feed of cows significantly increased the total aerobic and anaerobic bacterial counts in faeces of cows at 6 weeks and 12 months of application. Significant increases of lactobacilli and bifidobacteria were seen in faeces 6 weeks and 12 months ( only bifidobacteria) of application.

25. Results Gram-negative aerobic bacteria and yeasts were significantly increased at 6 weeks of application. Bacteroides, staphylococci and Clostridium perfringens were significantly reduced at 12 months of application only in case of bacteroides but the isolation number of C.perfringens (35% to 22%) and staphylococci (20% to 12%) were reduced from the beginning to the end of our investigations.

26. Results of bacteriological investigations of group I and V SalmonellaBdellovibrioProtozoa H1000* H2000* H37015* H40011

27. Quantitative results of volatile fatty acids of faeces from cows treated with EMa Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM)

28. Effects of EMa on the amount of volatile fatty acids in faeces of cows (µmol/g) * * * *** * * * * * p < 0,05 to H1

29. Effects of EMa on the amount of volatile fatty acids in faeces of cows (mol%) * * * ** * p < 0,05 to H1

30. Results EMa were effective on the amount of volatile fatty acids in faeces of our cows.The amounts of acetic acid, propionic acid and butyric acid increased during the whole time of investigation. The relationship of acetic to butyric acid was reduced. This means more butyric acid was provided for the colonic enterocytes.

31. Quantitative results of immunological parameters of blood plasma of cows treated with EMa Microbiological and immunological results of dairy cows fed one year with Effective Microorganisms (EM)

32. C-reactive Protein * * * p < 0,05 to H2   Beginning of the vaccination

33. Haptoglobin * * * p < 0,05 to H2  Beginning of the vaccination 

34. LPS-binding Protein * * p < 0,05 to H2 Beginning of the vaccination  

35. IgG-anti-LPS E.coli,J5   Beginning of the vaccination

36. IgG-anti-S.Cerro * * * p < 0,05 to H1 H4: p < 0,05 to H2 und H3   Beginning of vaccination

37. IgM-anti-S.Cerro ** * * p < 0,05 to H1 Beginning of vaccination  

38. Results The acute phase proteins C-reactive protein and haptoglobin increased at 6 and 12 months of EMa application in comparison to 6 weeks. LPS binding protein decreased at 12 months of EMa application. This means that towards the end of our investigations the cows were more able to stabilize their innate unspecific immune system.

39. Results In comparison to this, the specific immunological parameters - IgG-anti- LPS-antibodies and IgM-anti-S. Cerro- antibodies were reduced and IgG-anti-S. Cerro-antibodies were increased.

40. Conclusions I The application of EMa by feed to high yielding cows for about one year was suitable to increase healthful bacteria as lacto-bacilli and bifidobacteria. The increase of healthful bacteria was accompanied by increasing amounts of volatile fatty acids.

41. Conclusions II EMa were able to induce more propionic and butyric acids in relationship to acetic acid. During application of EMa the acute phase proteins C-reactive protein and haptoglobin increased and LBP decreased, the specific antibodies against Salmonella Cerro increase (IgM, 6 weeks and 6 months) or decrease (IgG, 12 months) because of growing defense capacity of the animals.

42. Conclusions III Altogether the combined measurements of EMa, Lactulose and vaccination against Salmonella Cerro were able to defense salmonellosis and to stabilize the health status of the investigated animals.