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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.
Microbiological and immunologicalresults of dairy cows fed one yearwith Effective Microorganisms (EM)
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.
Microbiological and immunologicalresults of dairy cows fed one yearwith Effective Microorganisms (EM)
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.
Microbiological and immunologicalresults of dairy cows fed one yearwith Effective Microorganisms (EM)
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.
Microbiological and immunologicalresults of dairy cows fed one yearwith Effective Microorganisms (EM)
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.
Microbiological and immunologicalresults of dairy cows fed one yearwith Effective Microorganisms (EM)
Microbiological and immunologicalresults of dairy cows fed one yearwith 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
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
Microbiological and immunologicalresults of dairy cows fed one yearwith Effective Microorganisms (EM)
The following parameters were used to evaluate the effect of EM:
Microbiological and immunologicalresults of dairy cows fed one yearwith 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
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
Microbiological and immunologicalresults of dairy cows fed one yearwith Effective Microorganisms (EM)
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
Microbiological and immunologicalresults of dairy cows fed one yearwith Effective Microorganisms (EM)
Material and methods
Microbiological and immunologicalresults of dairy cows fed one yearwith Effective Microorganisms (EM)
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
medianmedian 1,78 x 101,78 x 1044 3,09 x 103,09 x 1055 3,89 x 103,89 x 1044 3,98 x 103,98 x 1044
* 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
medianmedian 9,12 x 109,12 x 1044 1,07 x 101,07 x 1055 1,15 x 101,15 x 1044 1,17 x 101,17 x 1055
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
medianmedian 1,66 x 101,66 x 1044 5,50 x 105,50 x 1033 7,08 x 107,08 x 1033 6,03 x 106,03 x 1033
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
Microbiological and immunologicalresults of dairy cows fed one yearwith Effective Microorganisms (EM)
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
Microbiological and immunologicalresults of dairy cows fed one yearwith Effective Microorganisms (EM)
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
Beginning of the vaccination
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
Beginning of the vaccination
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.