11/29/17

21

Results: Intravenous LPS – Plasma anti-LPS

IgA antibodies41

0.04

0.045

0.05

0.055

0.06

0.065

0.07

0.075

W1D1 W2D1 W3D1 W4 W5 W6

IgA

(M

U/m

L)

Time around parturition (wk)

Plasma IgA in cows around parturition

IV Saline

IV LPS

P

a

r

t

u

r

i

t

i

o

n

Trt P > 0.05

Results: Oral LPS – Plasma anti-LPS IgM

antibodies42

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

W1D1 W2D1 W3D1 W4 W5 W6

IgM

(M

U/m

L)

Time around parturition (wk)

Plasma IgM in cows around parturition

Oral LPS

Oral Saline

P

a

r

t

u

r

i

t

i

o

n

Trt effect P < 0.01

11/29/17

22

Results: Intravenous LPS – Plasma anti-LPS

IgM antibodies43

0

0.005

0.01

0.015

0.02

0.025

W1D1 W2D1 W3D1 W4 W5 W6

IgM

(M

U/m

L)

Time around parturition (wk)

Plasma IgM in cows around parturition

IV Saline

IV LPS

P

a

r

t

u

r

i

t

i

o

n

Trt P < 0.01

Results: Oral LPS – Plasma anti-LPS IgG

antibodies44

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

W1D1 W2D1 W3D1 W4 W5 W6

IgG

(M

U/m

L)

Time around parturition (wk)

Plasma IgG in cows around parturition

Oral LPS

Oral Saline

P

a

r

t

u

r

i

t

i

o

n

Trt effect P < 0.001Week P < 0.05

11/29/17

23

Results: Intravenous LPS – Plasma anti-LPS

IgG antibodies45

0

0.02

0.04

0.06

0.08

0.1

0.12

W1D1 W2D1 W3D1 W4 W5 W6

IgG

(M

U/m

L)

Time around parturition (wk)

Plasma IgG in cows around parturition

IV Saline

IV LPS

P

a

r

t

u

r

i

t

i

o

n

Trt P < 0.01

TRT P < 0.01

Results: Oral LPS – Plasma calcium46

90

95

100

105

110

115

W1D1 W1D2 W2D1 W2D2 W3D1 W3D2 W4 W5 W6

Ca

lciu

m (

mg

/L)

Time around parturition (wk)

Plasma Ca in cows around parturition

Oral Saline

Oral LPS

P

a

r

t

u

r

i

t

i

o

n

11/29/17

24

Results: Intravenous LPS – Plasma calcium

47

90

95

100

105

110

115

W1D1 W1D2 W2D1 W2D2 W3D1 W3D2 W4 W5 W6

Ca

lciu

m (

mg

/L)

Time around parturition (wk)

Plasma Ca in cows around parturition

IV Saline

IV LPS

P

a

r

t

u

r

i

t

i

o

n

Results: Oral LPS – Plasma insulin48

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

W1D1 W2D1 W3D1 W4 W5 W6

Insu

lin

(m

g/m

L)

Time around parturition (wk)

Plasma insulin in dairy cows around parturition

Oral LPS

Oral Saline

P

a

r

t

u

r

i

t

i

o

n

Trt effect P < 0.001

11/29/17

25

Results: intravenous LPS – Plasma insulin49

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

W1D1 W2D1 W3D1 W4 W5 W6

Insu

lin

(m

g/m

L)

Time around parturition (wk)

Plasma insulin in cows around parturition

IV Saline

IV LPS

P

a

r

t

u

r

i

t

i

o

n

TRT P < 0.01

Milk yield in oral LPS-treated cows50

Milk yield oral group

5

10

15

20

25

30

35

40

45

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Days in milk

Mil

k i

n k

gs

Oral saline

Oral LPS

11/29/17

26

Milk yield in intravenous-LPS treated cows51

Milk yield I/V group

5

10

15

20

25

30

35

40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Days in milk

Mil

k y

ield

in

kg

s

I/V saline

I/V LPS

52

Table 3.1 Incidence of postpartal metabolic disorders in the two experimental groups sprayed

orally with saline (control) or saline with lipopolysaccharide from Escherichia coli 0111:B4

(treatment) twice per wk during wk -2, and -1 before calving and 1 wk after calving.

Control Group

Cow number

Clinical disease

Treatment

group Cow number

Clinical disease

2360

2113

2013

2163

2412

2168

2268

2371

307

Off feed 5 day,

Temperature 40oC

Calf dead, Retained

Placenta, Metritis

Twice Mastitis, Off

feed, Temperature 39oC

Off feed 20 d post parturition, Fatty liver

Ketosis 3 d pp

Normal

Normal

Off feed, Treated with

antibiotics

Downer cow, culled

2349

2369

2370

2310

2406

2417

2277

2423

Normal

Normal

Lameness 31 d pp

Mastitis 56 d pp

Normal

Normal

Normal

Normal

Normal

Oral LPS

11/29/17

27

Intravenous LPS – Displaced abomasum and Retained placenta

53

¨ Intermittent iv

administration of LPS

caused high incidence

of LDA and RP

Zebeli and Ametaj, (2011)

Conclusion of Study 1 (Oral LPS)54

¨ Experiment 1

¨ No clinical signs of metabolic diseases during first

month after parturition in cows administered orally

with LPS

¨ More than 50% of cows in control groups had one

or more metabolic diseases

¨ Oral administration of LPS did not affect

concentration of SAA, Hp, anti-LPS IgA, and

insulin; whereas LBP decreased

11/29/17

28

Conclusion of Study 1 (Oral LPS)55

¨ Plasma anti-LPS IgG was lower and IgM higher in

cows treated with oral LPS (primary immune

response triggered)

¨ NEFA and BHBA were lower in cows treated with

oral LPS

¨ Milk production was lower in the first 2 wks after

calving

Conclusion of Study 1 (iv LPS)56

¨ Experiment 2 (iv LPS)

¨ Induced chronic endotoxemia (iv LPS) associated

with greater incidence of Displaced abomasum and

Retained placenta

¨ Intravenous LPS had no effect on SAA, BHBA,

and IgA concentrations

¨ Glucose, lactate, anti-LPS IgM, Ca2+, and insulin in

the plasma decreased

¨ Plasma Hp, LBP, anti-LPS IgG increased

11/29/17

29

Conclusion of Study 1 (Experiment 2)57

¨ Milk production was suppressed in iv LPS treated

cows (Prolactin suppressive effects of LPS)

¨ DMI decreased in iv LPS treated cows

1) To test whether oronasal administration of

LPS prepartum affects health status and milk

production of dairy cows

2) To evaluate whether oral LPS and LTA affect

immunity, metabolism, health status, and

milk yield

Study 2 - Objectives58

11/29/17

30

Mat&Methods: Study 2 – Oronasal LPS

59

Experiment 1 - Oronasal LPS

Animals

q TRT group n = 50 dry off cows

q CTR group n= 50 dry off cows

Treatments

q Dose 1 = 0.01 µg/kg body wt; (d -28)

q Dose 2 = 0.05 µg/kg body wt; (d -25 and -21)

q Dose 3 = 0.1 µg/kg body wt ; (d -18 and -14)

q Doses dissolved in 3 mL of NaCl solution

Controls

q 3 mL of NaCl solution

Route of administration

q Nasal 1/3 and oral 2/3 of the dose

Mat&Methods: Study 2- Oral LPS+LTA60

Animals

� TRT group n = 15 dry off cows

� CTR group n = 15 dry off cows

Treatments

LPS doses

� Dose 1 = 0.01 µg/kg body wt (6.5 µg/cow); (d -28)

� Dose 2 = 0.05 µg/kg body wt (32.5 µg/cow); (d -25 and -21)

� Dose 3 = 0.1 µg/kg body wt (65 µg/cow); (d -18 and -14)

LTA dose� Flat dose (120 µg/animalDoses dissolved in 2 mL of (0.85%) NaCl solution

Controls

� 2 mL of NaCl solution

Route of administration

� Oral

11/29/17

31

Results: Oronasal LPS61

Saliva and vaginal mucus sIgA in oronasal-LPS treated cows

62

11/29/17

32

Milk SCC in cows treated oronasally with LPS

63

0

50

100

150

200

250

300

350

400

450

500

1 2 3 4

SC

C,

10

3cell

s/m

L

Week after parturition

Trt P < 0.10Week P = 0.51

Trt þ week P = 0.91

Results: Plasma Acute phase proteins64

11/29/17

33

Results: Clinical observations65

Clinical Observations of cows

Treatment Lameness Retained

placenta

Cows >1

disease

P = 0.24 P = 0.12 P = 0.09

LPS 9 6 4

CTR 15 13 11

Study 2 – Experiment 2/LPS-LTA66

¨ Objective

¤ To test whether oral administration of LPS and LTA before parturition will affect immunity, metabolism,

health status and milk production of dairy cows

11/29/17

34

sIgA in the vaginal mucus of cows treated

with oral LPS and LTA67

40.00

60.00

80.00

100.00

120.00

140.00

160.00

180.00

200.00

220.00

240.00

-28 -7 7 28

Va

gin

al m

ucu

s im

mu

no

glu

bu

lin

A (n

g/m

L)

Time relative to calving (days)

TrtP = 0.01

Time P = 0.02

Trt x time P = 0.13

Saliva anti LPS sIgA in cows treated orally

with LPS and LTA68

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

-4 -1 +1 +4

Sa

liv

a I

gA

(n

g/m

L)

Week around parturition

TrtP < 0.01

Week P = 0.04

Trt x week P < 0.01

11/29/17

35

69

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

-4 -1 +1 +4

Imm

un

ogl

ub

uli

n A

(AM

U/m

L)

Week around parturition

TrtP < 0.01

Week P = 0.22

Trt x week P = 0.74

a

0.000

0.005

0.010

0.015

0.020

0.025

-4 -1 +1 +4

Imm

un

olg

ub

uli

n G

(G

MU

/mL

)

Week around parturition

TrtP < 0.01

Week P = 0.13

Trt x week P = 0.75

0.010

0.011

0.012

0.013

0.014

0.015

0.016

-4 -1 +1 +4

Imm

un

ogl

ub

uli

n M

(M

MU

/mL

)

Week around parturition

TrtP < 0.01

Week P = 0.19

Trt x week P = 0.94

b

c

Plasma TNF in cows treated orally with LPS

and LTA70

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

-4 -1 +1 +4

TN

F-ɑ

(n

g/m

L)

Week around parturition

TrtP = 0.02

Week P = 0.87

Trt x week P = 0.89

11/29/17

36

Plasma lactate in cows treated orally with

LPS and LTA71

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

-28 -25 -21 -14 -7 7 14 21 28

La

cta

te(m

mo

l/L

)

Time relative to calving (days)

Trt: P < 0.01

Time: P = 0.11

Trt × time: P = 0.36

Plasma NEFA in cows treated orally with LPS and LTA

72

0

50

100

150

200

250

300

350

400

-28 -25 -21 -14 -7 7 14 21 28

NE

FA

(йE

q/L

)

Time relative to calving (days)

Trt: P = 0.36Time: P < 0.01

Trt þ time: P = 0.78

11/29/17

37

73

Milk composition and yield in cows treated

orally with LPS and LTA74

11/29/17

38

Conclusions75

¨ Oronasal LPS lowered concentrations of glucose in

the plasma and SCC in milk

¨ Increased concentrations of salivary and vaginal

mucus sIgA

¨ Had no effects on plasma concentrations of SAA

and LBP

¨ Tended to lower lameness and RP as well as the

number of cows with more then 1 disease

Conclusions76

¨ Oral LPS+LTA increased salivary and vaginal mucus sIgA

¨ It lowered plasma anti-LPS IgG and IgM as well as TNF-alpha and lactate

¨ It had no effect on plasma anti-LPS IgA and NEFA

¨ Treatment lowered the incidence of RP, lameness and metritis

¨ It lowered the number of days cows were lame and number of diseased days in cows with more than 1 disease

11/29/17

39

Objectives

To evaluate immune responses, health status, and productivity to repeated oral or oro-parenteral exposure to LPS and LTA

Study 3 – Best route and dose of LPS

and LTA77

Materials and methods78

¨ Animals and treatments

• 400 cows randomly assigned to 4 groups (n=50) treated with different doses of LPS and LTA

¨ Cow sample collection• Blood: -4wk to +4wk, once/wk

• Milk: +1wk to +4wk, once/wk

¨ Data collection

• Feed intake: -4wk to +4wk

• Milk yield: 0 to 100 DIM

• Milk composition: CP, fat, lactose, SCC, MUN, and TS

¨ Clinical observation

• Signs of disease and treatment records for cows and calves

• Calf growth: +1wk to +4wk

11/29/17

40

Results - Insulin79

Results - NEFA80

11/29/17

41

Acute phase proteins - LBP81

0

5

10

15

20

25

-4 -1 1 4

LB

P C

on

entr

ati

on

(µ

g/m

L)

Week Relative to Calving

CTR

TRT1

TRT2

TRT3

Trt: P = 0.82

Wk: P < 0.01

Trt x wk: P = 0.01

Acute phase proteins - Haptoglobin82

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

-4 -1 1 4

Hp

Co

nen

tra

tio

n (

mg

/mL

)

Week Relative to Calving

CTR

TRT1

TRT2

TRT3

Trt: P = 0.27

Wk: P < 0.01

Trt x wk: P = 0.21

11/29/17

42

Serum Immunoglobulin G - IgG83

0

100

200

300

400

500

600

-4 -1 1 4

Seru

m I

gG

Co

ncen

tra

tio

n (

ng

/mL

)

Week Relative to Calving

CTR

TRT1

TRT2

TRT3

Trt: P = 0.25

Wk: P < 0.01

Trt x wk: P = 0.58

Serum Immunoglobulin M - IgM84

0

50

100

150

200

250

-4 -1 1 4

Seru

m I

gM

co

ncv

en

tra

tio

n (

ng

/mL

)

Week relative to calving

CTR

TRT1

TRT2

TRT3

Trt: P = 0.40

Wk: P = 0.02

Trt x wk: P = 0.62

11/29/17

43

Serum Interleukin-185

400

450

500

550

600

650

700

-4 -1 1 4

Seu

m I

L-1

co

ncen

tra

tio

n (

pg

/mL

)

Week Relative to Calving

CTR

TRT1

TRT2

TRT3

Trt: P = 0.50

Wk: P = 0.14

Trt x wk: P = 0.27

Conclusions86

¨ Treatments did not trigger systemic inflammation or

influence the normal host metabolism

¨ Treatments decreased the incidence rate of several

periparturient diseases of dairy cows

¨ Two treatments numerically lowered SCC (Somatic

cell count) on the 3rd week of lactation

q Results support the potential utilization of this new

vaccine in dairy cows during the dry off period

11/29/17

44

QUESTIONS

THANK YOU87