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
Microbiological and immunological
results of dairy cows fed one year
with Effective Microorganisms (EM)
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 immunological reactivity of the host.
Microbiological and immunological
results of dairy cows fed one year
with Effective Microorganisms (EM)

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)

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.

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)
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)
Objective II
Measures:
Effective Microorganisms (EM) were given
in feed from
27. 03.03 - 25.02.04,
November 03: Lactulose application in
feed over 6 weeks,
November 03: vaccination against salmonellosis by farm specific vaccine.
Microbiological and immunological
results of dairy cows fed one year
with Effective Microorganisms (EM)
Material and Methods


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.
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
Microbiological and immunological
results of dairy cows fed one year
with Effective Microorganisms (EM)
Material and methods
Number of investigated animals
27.03.
14.05.
24.09.
25.02.
number of
all
animals
80
72
56
47
group I
20
20
19
18
group II
15
14
10
6
group III
15
13
9
9
group IV
15
12
8
6
group V
15
13
10
8
Microbiological and immunological
results of dairy cows fed one year
with Effective Microorganisms (EM)
Material and Methods
The following parameters were used to
evaluate the effect of EM:

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
Microbiological and immunological
results of dairy cows fed one year
with Effective Microorganisms (EM)
Parameter

Lactobacillus spp.

Bifidobacterium spp.

Yeasts

Salmonella serotypes

Bdellovibrio spp.

Staphylococci

Bacteriodes spp.
Material and methods
Method
MRS-agar (OXOID)
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)
Material and methods
Blood
Parameters
 CRP
 Haptoglobin
 LPS binding
Protein
 IgG and IgM antiLPS antibodies E.
coli J5
 IgG and IgM anti
Salmonella Cerro
–antibodies
Method
ELISA
ELISA
ELISA
ELISA
ELISA
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
Effects of EMa on total aerobic bacterial
counts in faeces of cows
median
H1
H2
4,79 x 105
4,17 x 106
*
H3
H4
1,07 x 106
2,45 x 106
lg/g
9
8
*
7
*
6
5
4
3
2
1
0
H1
* p < 0,05 to H1
H2
H3
H4
*
Effects of EMa on anaerobic total bacterial
counts in faeces of cows
median
H1
H2
2,00 x 106
1,51 x 107
H3
6
* 9,12 x 10
H4
1,82 x 107
lg/g
9
*
8
*
7
6
5
4
3
2
1
0
H1
* p < 0,05 to H1
H2
H3
H4
*
Effects of EMa on counts of lactobacilli in
faeces of cows
median
H1
H2
1,32 x 105
1,05 x 106
H3
5
3,89
x
10
*
H4
5
1,07
x
10
*
lg/g
8
7
*
*
6
5
4
3
2
1
0
H1
* p < 0,05 to H1
H2
H3
H4
Effects of EMa on bifidobacteria counts
in faeces of cows
median
H1
H2
4,57 x 105
1,41 x 106
H3
H4
* 7,94 x 106
* 1,78 x 107
lg/g
9
8
*
*
*
7
6
5
4
3
2
1
0
H1
* p < 0,05 to H1
H2
H3
H4
H3, H4: p < 0,05 to H2
*
Effects of EMa on aerobic Gram-negative
bacterial counts in faeces of cows
median
H1
H2
H3
H4
1,78 x 104
3,09 x 105
3,89 x 104
3,98 x 104
*
lg/g
7
*
6
5
4
3
2
1
0
H1
* p < 0,001 to H1, H3 und H4
H2
H3
H4
Effects of EMa on yeast counts in faeces of
cows
median
H1
H2
3,02 x 103
5,89 x 103
H3
* 4,27 x 103
H4
3,98 x 103
lg/g
4,5
*
4
3,5
3
2,5
2
1,5
1
0,5
0
H1
* p < 0,05 to H1, H3 und H4
H2
H3
H4
Effects of EMa on Bacteroides counts in
faeces of cows
median
H1
H2
H3
H4
1,17 x 104
1,66x 104
5,37 x 103
2,75 x 103
lg/g
6
5
*
4
3
2
1
0
H1
* p < 0,05 to H2
H2
H3
H4
*
Effects of EMa on staphylococcal counts in
faeces of cows
median
H1
H2
H3
H4
9,12 x 104
1,07 x 105
1,15 x 104
1,17 x 105
lg/g
7
6
5
4
3
2
1
0
H1
H2
H3
H4
Effects of EMa on Clostridium perfringens counts
in faeces of cows
median
H1
H2
H3
H4
1,66 x 104
5,50 x 103
7,08 x 103
6,03 x 103
lg/g
6
5
4
3
2
1
0
H1
H2
H3
H4
Effects of EMa on alkalic
phosphatase in faeces of cows
U/g
0,35
0,3
0,25
0,2
0,15
0,1
0,05
0
H1
H2
H3
H4
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
investigations of group I and V
Salmonella
Bdellovibrio Protozoa
H1
0
0
0*
H2
0
0
0*
H3
7
0
15*
H4
0
0
11
Microbiological and immunological
results of dairy cows fed one year
with Effective Microorganisms (EM)
Quantitative results of volatile
fatty acids
of faeces from cows treated
with EMa
Effects of EMa on the amount of volatile fatty
acids in faeces of cows (µmol/g)
*
45
*
*
40
35
30
C2
C3
C4
C5
25
20
15
10
*
5
*
*
*
*
*
*
0
H1
* p < 0,05 to H1
H2
H3
H4
Effects of EMa on the amount of volatile fatty
acids in faeces of cows (mol%)
90
*
80
*
70
60
C2
C3
C4
sonst
50
40
30
20
*
*
10
*
0
H1
* p < 0,05 to H1
H2
H3
H4
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.
Microbiological and immunological
results of dairy cows fed one year
with Effective Microorganisms (EM)
Quantitative results of
immunological parameters
of blood plasma
of cows treated with EMa
C-reactive Protein
CRP
1000
*
CRP (µg/ml)
800
600
400
*
200
0
1
2
3

4
Probennahme
* p < 0,05 to H2
Beginning of the vaccination
5
Haptoglobin
Haptoglobin
7000
*
6000
Haptoglobin (µg/ml)
5000
4000
3000
2000
1000
*
0
1
2
3

4
Probennahme
* p < 0,05 to H2
 Beginning of the vaccination
5
LPS-binding Protein
LBP
60
50
LBP (µg/ml)
40
*
30
20
10
0
1
2
3

4
Probennahme
* p < 0,05 to H2
 Beginning of the vaccination
5
IgG-anti-LPS E.coli,J5
IgG-anti-LPS E.coli,J5
1400
1200
IgG-anti-LPS (RE/ml)
1000
800
600
400
200
0
0
1
2
3
Probennahme
 Beginning of the vaccination

4
5
IgG-anti-S.Cerro
IgG-anti-S.Cerro
3000
*
IgG-anti-S.Cerro (REE/ml)
2500
2000
*
1500
1000
500
0
0
1
2
3

4
5
Probennahme
* p < 0,05 to H1
H4: p < 0,05 to H2 und H3
 Beginning of vaccination
IgM-anti-S.Cerro
IgM-anti-S.Cerro
1200
*
IgM-anti-S.Cerro (REE/ml)
1000
*
800
600
*
400
200
0
0
1
2
3
Probennahme
* p < 0,05 to H1
Beginning of vaccination

4
5
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-antiLPS-antibodies and IgM-anti-S. Cerroantibodies 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.