Institute of Bacteriology and Mycology University of Leipzig Microbiological and immunological...

Institute of Bacteriology and MycologyInstitute of Bacteriology and MycologyUniversity of LeipzigUniversity of Leipzig

Microbiological and Microbiological and immunological results of immunological results of dairy cows fed one year dairy cows fed one year

with Effective with Effective Microorganisms (EM)Microorganisms (EM)

Monika Krueger, Karen Thaens, W. Monika Krueger, Karen Thaens, W. SchroedlSchroedl

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 immunologicalresults of dairy cows fed one yearwith Effective Microorganisms (EM)

The GIT is the focus of interaction of The GIT is the focus of interaction of the outside to the inside. Food the outside to the inside. Food modulates microorganisms and their modulates microorganisms and their products – metabolites, hormones, products – metabolites, hormones, toxins, etc. supporting or disturbing toxins, etc. supporting or disturbing numerous metabolic, immune and numerous metabolic, immune and nervous functions of the GIT.nervous functions of the GIT.


High-yielding dairy cows are influenced by High-yielding dairy cows are influenced by diets rich of fat, proteins and carbohydrates. diets rich of fat, proteins and carbohydrates. These diets promote lipolytic, proteolytic and These diets promote lipolytic, proteolytic and saccharolytic microorganisms.saccharolytic microorganisms.

They overgrow and damage the They overgrow and damage the gastrointestinal barrier.gastrointestinal barrier.

Microorganisms and their toxins translocate. Microorganisms and their toxins translocate. They damage the immune system and they They damage the immune system and they are the cause of milk losses and diseases.are the cause of milk losses and diseases.


Objective IObjective I

In a German dairy farm, cows and their In a German dairy farm, cows and their calves were infected by calves were infected by Salmonella Salmonella Zerro, a relatively unknown serotype, Zerro, a relatively unknown serotype, over a period of several months.over a period of several months.

At the beginning of the infection some At the beginning of the infection some calves and cows diseased severely. At calves and cows diseased severely. At present, salmonellosis is eradicated.present, salmonellosis is eradicated.


Objective IIObjective II Measures:Measures:

Effective Microorganisms (EM) were given Effective Microorganisms (EM) were given in feed fromin feed from

27. 03.03 - 25.02.04,27. 03.03 - 25.02.04,

November 03: Lactulose application inNovember 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.


Material and MethodsMaterial and Methods

A dairy farm of about 1400 cows and A dairy farm of about 1400 cows and their calves in Saxony was involved in their calves in Saxony was involved in these investigations. these investigations.

Faeces and blood plasmas of 20 Faeces and blood plasmas of 20 randomly selected cows of the highest randomly selected cows of the highest yielding group, 15 cows of the medium yielding group, 15 cows of the medium yielding group, 15 heifers, 15 dried off yielding group, 15 heifers, 15 dried off cows and 15 cows immediately after birth cows and 15 cows immediately after birth were investigated.were investigated.



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

Material and Material and methodsmethods

NNumber of investigated animalsumber of investigated animals

27.03.27.03. 14.05.14.05. 24.09.24.09. 25.02.25.02.

number ofnumber of

all all animalsanimals

8080 7272 5656 4747

group Igroup I 2020 2020 1919 1818

group IIgroup II 1515 1414 1010 66

group IIIgroup III 1515 1313 99 99

group IVgroup IV 1515 1212 88 66

group Vgroup V 1515 1313 1010 88


The following parameters were used to evaluate the effect of 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

Lactobacillus spp.Lactobacillus spp.

Bifidobacterium spp.Bifidobacterium spp.

YeastsYeasts

Salmonella serotypesSalmonella serotypes

Bdellovibrio spp.Bdellovibrio spp.

StaphylococciStaphylococci

Bacteriodes spp. Bacteriodes spp.

MRS-agar (OXOID)MRS-agar (OXOID)

MRS-agar (OXOID)MRS-agar (OXOID)

Sabouraud-agar (SIFIN)Sabouraud-agar (SIFIN)

Rappoport enrichment medium, Rappoport enrichment medium, Kauffmann-agar (SIFIN)Kauffmann-agar (SIFIN)

Two component-agar acc. to Two component-agar acc. to Stolp 1963Stolp 1963

Staphylococcus-Medium No. Staphylococcus-Medium No. 110 (OXOID110 (OXOID

Neomycine, Desoxycholat Neomycine, Desoxycholat Nutritional-agarNutritional-agar


MethodParameter

Material and methods

BloodBloodParametersParameters CRPCRP HaptoglobinHaptoglobin LPS binding LPS binding

ProteinProtein IgG and IgM anti-IgG and IgM anti-

LPS antibodies E. LPS antibodies E. coli J5coli J5

IgG and IgM anti-IgG and IgM anti- SalmonellaSalmonella Cerro Cerro

–antibodies–antibodies

MethodMethodELISAELISAELISAELISAELISAELISA ELISAELISA

ELISAELISA


Material and methods


Quantitative results of Quantitative results of microbiologicalmicrobiological

investigationsinvestigations

of faeces from cows of faeces from cows treated treated

with EMa with EMa

Effects of EMa on total aerobic bacterial Effects of EMa on total aerobic bacterial counts in faeces of cowscounts in faeces of cows

0

1

2

3

4

5

6

7

8

9

H1 H2 H3 H4

*

*

H1H1 H2H2 H3H3 H4H4

medianmedian 4,79 x 104,79 x 1055 4,17 x 104,17 x 1066 1,07 x 101,07 x 1066 2,45 x 102,45 x 1066

* p < 0,05 to H1

* *

lg/g

Effects of EMa on anaerobic total Effects of EMa on anaerobic total bacterial counts in faeces of cowsbacterial counts in faeces of cows

0

1

2

3

4

5

6

7

8

9

H1 H2 H3 H4

* *

H1H1 H2H2 H3H3 H4H4

medianmedian 2,00 x 102,00 x 1066 1,51 x 101,51 x 1077 9,12 x 109,12 x 1066 1,82 x 101,82 x 1077* *

* p < 0,05 to H1

lg/g

Effects of EMa on counts of lactobacilli Effects of EMa on counts of lactobacilli in faeces of cowsin faeces of cows

0

1

2

3

4

5

6

7

8

H1 H2 H3 H4

**

H1H1 H2H2 H3H3 H4H4

medianmedian 1,32 x 101,32 x 1055 1,05 x 101,05 x 1066 3,89 x 103,89 x 1055 1,07 x 101,07 x 1055*

* p < 0,05 to H1

*

lg/g

Effects of EMa on bifidobacteria countsEffects of EMa on bifidobacteria countsin faeces of cowsin faeces of cows

0

1

2

3

4

5

6

7

8

9

H1 H2 H3 H4

**

*

H1H1 H2H2 H3H3 H4H4

medianmedian 4,57 x 104,57 x 1055 1,41 x 101,41 x 1066 7,94 x 107,94 x 1066 1,78 x 101,78 x 1077* * *

* p < 0,05 to H1 H3, H4: p < 0,05 to H2

lg/g

Effects of EMa on aerobic Gram-Effects of EMa on aerobic Gram-negative bacterial counts in faeces of negative bacterial counts in faeces of

cowscows

0

1

2

3

4

5

6

7

H1 H2 H3 H4

*

H1H1 H2H2 H3H3 H4H4


* p < 0,001 to H1, H3 und H4

*

lg/g

Effects of EMa on yeast counts in Effects of EMa on yeast counts in faeces of faeces of

cowscows

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

H1 H2 H3 H4

*

H1H1 H2H2 H3H3 H4H4

medianmedian 3,02 x 103,02 x 1033 5,89 x 105,89 x 1033 4,27 x 104,27 x 1033 3,98 x 103,98 x 1033*

* p < 0,05 to H1, H3 und H4

lg/g

Effects of EMa on Bacteroides counts in Effects of EMa on Bacteroides counts in faeces of cowsfaeces of cows

0

1

2

3

4

5

6

H1 H2 H3 H4

H1H1 H2H2 H3H3 H4H4

medianmedian 1,17 x 101,17 x 1044 1,66x 101,66x 1044 5,37 x 105,37 x 1033 2,75 x 102,75 x 1033

*

* p < 0,05 to H2

*

lg/g

Effects of EMa on staphylococcal counts Effects of EMa on staphylococcal counts in faeces of cows in faeces of cows

0

1

2

3

4

5

6

7

H1 H2 H3 H4

H1H1 H2H2 H3H3 H4H4


lg/g

Effects of EMa on Clostridium perfringens Effects of EMa on Clostridium perfringens countscounts

in faeces of cowsin faeces of cows

0

1

2

3

4

5

6

H1 H2 H3 H4

H1H1 H2H2 H3H3 H4H4


lg/g

Effects of EMa on alkalic Effects of EMa on alkalic phosphatase in faeces of phosphatase in faeces of

cowscows

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

H1 H2 H3 H4

U/g

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.

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.

Results of bacteriological Results of bacteriological investigations of group I and Vinvestigations of group I and V

SalmonelSalmonellala

BdellovibBdellovibriorio

ProtozoaProtozoa

H1H1 00 00 0*0*

H2H2 00 00 0*0*

H3H3 77 00 15*15*

H4H4 00 00 1111

Quantitative results of Quantitative results of volatilevolatile

fatty acidsfatty acids

of faeces from cows of faeces from cows treated treated

with EMa with EMa


Effects of EMa on the amount of Effects of EMa on the amount of volatile fatty acids in faeces of volatile fatty acids in faeces of

cows (µmol/g)cows (µmol/g)

0

5

10

15

20

25

30

35

40

45

H1 H2 H3 H4

C2

C3

C4

C5

* * *

* * **

**

*

* p < 0,05 to H1

Effects of EMa on the amount of Effects of EMa on the amount of volatile fatty acids in faeces of volatile fatty acids in faeces of

cows (mol%)cows (mol%)

0

10

20

30

40

50

60

70

80

90

H1 H2 H3 H4

C2

C3

C4

sonst

* *

** *

* p < 0,05 to H1

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.

Quantitative results of Quantitative results of immunological parameters immunological parameters

of blood plasma of blood plasma

of cows treated with EMa of cows treated with EMa


C-reactive ProteinC-reactive Protein

CRP

Probennahme

1 2 3 4 5

CR

P

(µg/m

l)

0

200

400

600

800

1000

*

*

* p < 0,05 to H2

Beginning of the vaccination

HaptoglobinHaptoglobin

Haptoglobin

Probennahme

1 2 3 4 5

Ha

pto

glo

bin

(µ

g/m

l)

0

1000

2000

3000

4000

5000

6000

7000

*

*

* p < 0,05 to H2


LPS-binding ProteinLPS-binding Protein

LBP

Probennahme

1 2 3 4 5

LB

P (

µg/m

l)

0

10

20

30

40

50

60

*

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

IgG-anti-LPS E.coli,J5IgG-anti-LPS E.coli,J5

IgG-anti-LPS E.coli,J5

Probennahme

0 1 2 3 4 5

IgG

-an

ti-L

PS

(R

E/m

l)

0

200

400

600

800

1000

1200

1400


IgG-anti-IgG-anti-S.S.CerroCerro

IgG-anti-S.Cerro

Probennahme

0 1 2 3 4 5

IgG

-an

ti-S

.Ce

rro

(R

EE

/ml)

0

500

1000

1500

2000

2500

3000

*

*

* p < 0,05 to H1 H4: p < 0,05 to H2 und H3

Beginning of vaccination

IgM-anti-S.CerroIgM-anti-S.Cerro

IgM-anti-S.Cerro

Probennahme

0 1 2 3 4 5

IgM

-an

ti-S

.Ce

rro

(R

EE

/ml)

0

200

400

600

800

1000

1200

* *

*

* p < 0,05 to H1 Beginning of

vaccination

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.

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.

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.

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.

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.

Institute of Bacteriology and Mycology University of Leipzig Microbiological and immunological...

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