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STRATEGIES TO MITIGATE THE ADVERSE EFFECTS OF ENDOTOXINS IN TRANSITION DAIRY COWS

Italian Society of Buiatria, Parma 23-24 Nov, 2017Nov 24, 2017

Burim N. Ametaj, DVM, PhD

University of AlbertaCanada

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Outline of the presentation

¨ Endotoxin-related diseases

¨ What are the sources of endotoxins in a dairy cow?

¨ Does endotoxin translocate into the systemic circulation?

¨ How can we prevent health implications of endotoxins?

¤Grain processing technology

¤Oral vaccination (metritis and mastitis)

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Endotoxin-related Diseases3

START HERE

Eckel & Ametaj, 2016

What are the sources of endotoxins in a dairy cow?

¨ There are three sources of endotoxins in dairy cows

¤ Rumen

¤ Postpartum uterus

¤ Udder

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Proof of endotoxin elevation in the rumen during high grain diets

16-fold increase in endotoxin at 45% barley grain TMR

5

Emanuel et al., 2008

0

2,000

4,000

6,000

8,000

10,000

12,000

1 3 5 7 10

LP

S n

g/m

L

Experimental day

Ruminal fluid LPS in cows fed different amounts of grain

0% Barley grain

15% Barley grain

30% Barley grain

45% Barley grain

Does endotoxin translocate through GI tract: Ussing chamber study

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Endotoxin translocation through rumen increases: in

vitro study7

Emanuel et al., 2007

0

100

200

300

400

500

600

4.5 5.5 6.5

LP

S (µ

g/c

m2/h

)

pH value of perfusate on the mucosal side of rumen tissue

Translocation of LPS from E. coli 055:B5 across rumen

tissue from steers

Trt: P < 0.01

Endotoxin translocation through colon8

Emanuel et al., 2007

0

50

100

150

200

250

5.5 6.5 7.4

LP

S (µ

g/c

m2/h

)

pH value of perfusateon mucosal side of colon tissue

Translocation of LPS from E. coli 055:B5 Across Colon

Tissue of Steers

Trt: P < 0.01

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Rumen pH related to rumen LPS9

Ametaj et al., 2010

Infected mammary as source of endotoxin

¨ Gangrenous mastitis was associated with a plasma

LPS concentration of 85.2 pg/mL, than control cows

at 4.7 pg/mL (18-fold higher) – Hakogi et al.,

(1989)

¨ Intramammary administration of LPS in the udder

enhanced plasma LPS (5-13-fold) – (Dosogne et al.,

2002)

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Mastitis and endotoxin translocation

Hakogi et al., 1989

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Fate of endotoxin in systemic circulation and host response

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Eckel & Ametaj, 2016

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Fate of endotoxin in blood – Macrophage Pathway

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Fate of endotoxin in blood – Lipoprotein Pathway

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q Barley Grain Technology

q Oral Vaccine

New Technologies to mitigate ERD15

Grain and inflammation16

¨ Feeding high amounts of grain triggers

inflammation in dairy cows

¨ High amounts of grain are associated with

increased levels of endotoxin in the rumen fluid

¨ High grain diets is associated with elevated blood

SAA, CRP, and LBP

Emmanuel et al., 2008

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Elevated ruminal fluid LPS with high grain diets

17

0

2,000

4,000

6,000

8,000

10,000

12,000

1 3 5 7 10

LP

S n

g/m

L

Experimental day

Ruminal fluid LPS in cows fed different amounts of grain

0% Barley grain

15% Barley grain

30% Barley grain

45% Barley grain

Emmanuel et al., 2008

Feeding grain is associated with systemic inflammation – Elevated Blood SAA

18

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

40,000

45,000

50,000

1 3 5 7 10

SA

A (

ng/m

L)

Experimental day

PLasma SAA in cows fed different amounts of grain

15% Concentrate mix

30% Concentrate mix

45% Concentrate mix

60% Concentrate mix

Emmanuel et al., 2008

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Feeding grain is associated with systemic inflammation – Elevated blood CRP

19

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

1 3 5 7 10

CR

P (

ng/m

L)

Experimental day

Plasma CRP in cows fed different amounts of grain

15% Concentrate mix

30% Concentrate mix

45% Concentrate mix

60% Concentrate mix

Emmanuel et al., 2008

Feeding grain is associated with systemic inflammation – Elevated blood LBP

20

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

18,000

1 3 5 7 10

LB

P (

ng/m

L)

Experimental day

Plasma LBP in cows fed different amounts of grain

15% Concentrate mix

30% Concentrate mix

45% Concentrate mix

60% Concentrate mix

Emmanuel et al., 2008

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Barley grain treated with LA prevent elevation of LPS in rumen fluid

21

0

100

200

300

400

500

600

700

1 5 11 14 16 19 21

LP

S (

ng/m

L)

Experimental day

Ruminal LPS in cows fed LA-(0.5) or water-treated barley

LA-Treated Barley

Water-Treated Barley

Adaptation period Measurement period

Iqbal et al., 2012

New barley grain technology lowers the amount of endotoxin in the rumen fluid

¨ Increases resistant starch by 24%

¨ Lowers the amount of endotoxin in the rumen fluid

¨ Lowers the acute phase response as evidenced by lower haptoglobinand serum amyloid A

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0

200

400

600

800

1000

1200

1400

1 5 11

Ru

men

en

do

tox

in (n

g/m

L)

Measurement days (d)

Trt: P < 0.001

Day:P < 0.001Day x Trt: P = 0.019

n = 8SEM = 52.920

400

450

500

550

600

650

700

750

800

850

900

1 5 11

Pla

sma

ha

pto

glo

bin

(µ

g/d

L)

Measurements day

Trt: P = 0.37

Day:P = 0.82Day x Trt: P = 0.94

n = 8SEM = 79.012

a

b

Iqbal et al., 2012

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Development of a vaccine against Endotoxin-related diseases

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Oral vaccination24

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Rationale for the vaccine25

Evaluate health and immunology responses of

repeated oral immunization or intravenous

administration of increasing doses of LPS from

Escherichia coli 0111: B4, two weeks before

and one week after calving

Study 1- Objectives 26

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Materials and methods27

¨ 32 clinically healthy nearing term heifers and

multiparous Holstein cows assigned to four groups

of eight cows each

¨ Two groups of n=8 cows each treated either withoral LPS or oral saline (control) (Experiment 1)

¨ Two groups of n=8 cows each treated withintravenous LPS or saline (control) (Experiment 2)

¨ LPS was administered at increasing doses of

0.01µg, 0.05µg, and 0.1µg/kg BW on day 1 and 3

of each week

Materials and methods28

¨ Blood samples were collected twice per week

during 2 wk and 1 wk before and 1 wk after

parturition as well as once per week during wk 2, 3,

and 4 after parturition.

¨ Measurements

¤ Plasma acute phase proteins: SAA, LBP, haptoglobin

by ELISA using commercially available kits.

¤ Plasma immunoglobulins: IgA, IgG, and IgM by

ELISA kit EndoCab specific to endotoxin

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Blood analyses29

¨ Measurements:

¤ Plasma metabolites: Cortisol, NEFA, BHBA, glucose,lactate, insulin, and cholesterol using commerciallyavailable kits

¤ Plasma minerals: Calcium, iron, zinc, and copper weremeasured by atomic absorption spectrometry

Results: Oral LPS – Plasma SAA30

0

10,000

20,000

30,000

40,000

50,000

60,000

W1D1 W2D1 W3D1 W4 W5 W6

SA

A (

ng/m

L)

Time around parturition (wk)

Plasma SAA in dairy cows around parturition

Oral LPS

Oral saline

P

a

r

t

u

r

i

t

i

o

n

Trt P > 0.05

Week P > 0.05

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Results: Intravenous LPS – Plasma SAA31

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

40,000

45,000

50,000

W1D1 W2D1 W3D1 W4 W5 W6

SA

A (

ng/m

L)

Time around parturition (wk)

Plasma SAA in dairy cows around parturition

IV Saline IV LPSP

a

r

t

u

r

i

t

i

o

n

Results: Oral LPS – Plasma LBP32

0

10,000

20,000

30,000

40,000

50,000

60,000

70,000

80,000

W1D1 W2D1 W3D1 W4 W5 W6

LB

P (

ng/m

L)

Time around parturition (wk)

Plasma LBP in dairy cows around parturition

Oral LPS

Oral salineP

a

r

t

u

r

i

t

i

o

n

Trt P < 0.05

Week P < 0.05

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Results: Intravenous LPS – Plasma LBP33

0

10,000

20,000

30,000

40,000

50,000

60,000

70,000

80,000

90,000

100,000

W1D1 W2D1 W3D1 W4 W5 W6

LB

P (

ng/m

L)

Time around parturitionj (wk)

Plasma LBP in dairy 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 Hp34

0

200

400

600

800

1,000

1,200

W1D1 W2D1 W3D1 W4 W5 W6

Hap

toglo

bin

(µ

g/m

L)

Time around parturition (wk)

Plasma haptoglobin in dairy cows around parturition

Oral LPS

Oral saline

P

a

r

t

u

r

i

t

i

o

n

Trt P > 0.05

Week P < 0.05

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Results: Intravenous LPS – Plasma Hp35

0

200

400

600

800

1,000

1,200

1,400

1,600

W1D1 W2D1 W3D1 W4 W5 W6

Hap

toglo

bin

(µg/m

L)

Time around parturition (wk)

Plasma haptoglobin in cows around parturition

IV Saline

IV LPSP

a

r

t

u

r

i

t

i

o

n

TRT P < 0.01

Results: Oral LPS – Plasma NEFA36

0

200

400

600

800

1,000

1,200

1,400

W1D1 W1D2 W2D1 W2D2 W3D1 W3d2 W4 W5 W6

NE

FA

(m

g/m

L)

Time around parturition (wk)

Plasma NEFA in cows around parturition

Oral LPS

Oral Saline

P

a

r

t

u

r

i

t

i

o

n

TRT P < 0.01

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Results: Intravenous LPS – Plasma NEFA37

0

200

400

600

800

1,000

1,200

1,400

W1D1 W1D2 W2D1 W2D2 W3D1 W3d2 W4 W5 W6

NE

FA

(m

g/m

L)

Time around parturition (wk)

Plasma NEFA 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 BHBA38

0

500

1,000

1,500

2,000

2,500

W1D1 W1D2 W2D1 W2D2 W3D1 W3D2 W4 W5 W6

BH

BA

(m

g/m

L)

Time around parturition (wk)

Plasma BHBA in cows around parturition

Oral LPS

Oral Saline

P

a

r

t

u

r

i

t

i

o

n

TRT P < 0.01

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Results: Intravenous LPS – Plasma BHBA39

0

200

400

600

800

1,000

1,200

1,400

1,600

1,800

W1D1 W1D2 W2D1 W2D2 W3D1 W3D2 W4 W5 W6

BH

BA

(m

g/m

L)

Time around parturition (wk)

Plasma BHBA in cows around parturition

IV Saline

IV LPS

P

a

r

t

u

r

i

t

i

o

n

Results: Oral LPS – Plasma anti-LPS IgA

antibodies40

0.03

0.035

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

Oral LPS

Oral Saline

P

a

r

t

u

r

i

t

i

o

n

Trt effect P = 0.25