Immune Function The New Frontier in
Animal Production
Steve Puntenney, Ph.D.
OmniGen Research, LLC
Mission Statement
OmniGen Research is dedicated to
expanding the boundaries of science to
benefit animal health and productivity
through the development of natural
products for the livestock industry.
History of OmniGen Research
 1999
 May 2001
 Feb 2002
 May 2002
 Mar 2003
 Jan 2004
HBS identified as a significant issue in
OR & WA Dairies
Meetings w/ consulting veterinarians
in WA indicated a high failure rate of
autogenous vaccines & need for other
intervention identified
Prototype OmniGen placed on 6 OR &
WA Dairies
OmniGen Research, LLC formed
with NEF to discover MOA
PAP began distribution of OAF
OmniGen-AF Licensed to PAP
History of OmniGen Research
 July 2005
 June 2006
 Feb 2007
Construction on the OmniGen
Research Lab
Established OmniGen Research
Farm
Beginning of construction of dairy
and lab animal facilities
Overview of presentation
Part 1
 Acquired and innate immunity
• Macrophages
• Neutrophils
 Nutrition and immunity during
transition
 Immune function and udder
health
Part 2
 OmniGen Mastitis Studies
 Mycotic Abortions
 Mycotoxin Binding
 OmniGen Use Rates
Adaptive (antibody-mediated) immunity
Vulnerable
Janeway et al.,
2005
Impact of periparturient
immunosuppression
Increased susceptibility to diseases
-mastitis
-metritis/retained placenta
-foot infections
-respiratory infections
-gastrointestinal disorders
-lowered peak milk production
Why focus on immune function?
“A .5 kg increase in peak milk production is
equal to 108 kg of additional milk per
lactation” (Utah State University)
Innate immunity
Allows time for adaptive immunity to develop
 First line of defense
 Evolutionarily ancient
 Several components
–
–
–
–
Epithelial barriers
HCl
Digestive enzymes
Cellular component
 Macrophages
 Neutrophils
 Natural killer (NK cells)
Opsonization of Bacteria by C3b
 Opsonization is the coating of microbes with
complement components, such as C3b
(also IGg & antibodies, but later).
 Opsonized particles are more easily
phagocytosed due to the presence of
complement receptors on the plasma
membrane of phagocytic cells
Picture of macrophage surface using
scanning electron microscopy
C3b
Cytokine release
Neutrophil Trafficing
L-selectin
IL-8
Burton and Erskine, 2003
Phagocytosis by the neutrophil
Bacterium
Recognition
Killing mechanisms: Respiratory burst
via ROS
+
Phagocytosis
O2
NADP oxidase
superoxide anion (O.2-)
Bacteriocidal
hypochlorite
ion OCl-
Superoxide
dismutase
Neutrophil
myeloperoxidase
killing
killing
H2O2
Macrophages:
myeloperoxidaseIndependent mechanism
Other oxygen-derived
free radicals (OH.)
ROS: Good and Bad
 Good: kill pathogens
 Bad: tissue damage
 “Immune cells” have
high intrinsic ROS
generation and,
therefore, have high
anti-oxidant demand
Chew and Park, 2002
 “Immune cells” have
membranes high in
PUFAs which are
susceptible to ROSmediated damage
A primary mechanism by which nutrients support
immunity is via protection against ROS
+
Phagocytosis
O2
NADP oxidase
superoxide anion (O.2-)
Bacteriocidal
hypochlorite
ion OCl-
Superoxide
dismutase
Neutrophil
myeloperoxidase
Anti-oxidants (scavenge free radicals)
vitamin E, vitamin C
zinc, selenium, carotenoids, flavenoids
Cu/Zn
H2O2
Macrophages:
myeloperoxidaseindependent mechanism
Other oxygen-derived
free radicals (OH.)
Nutrition and immunology at
parturition
 Three factors conspire to affect nutrition and
immunity at parturition
– Surge in cortisol
– Decline in feed intake
 NEFA spike
 Ketone body spike
– Impaired calcium homeostasis
Impact of periparturient
immunosuppression
 Increased susceptibility to disease
-mastitis
-metritis/retained placenta
-foot infections
-respiratory infections
-gastrointestinal disorders





Parturition
Drying off
Shipping
Over-crowding
Environmental
(heat/hold)
 Excess handling
Source: Burton and Erskine, 2003
Neutrophil L-selectin
Periparturient immunosuppression
L-selectin
Cortisol brings about immunosuppression
Healthy mammary tissue
Mammary tissue in stress
Cytokines released from macrophages
signal neutrophil migration
Source: Burton and Erskine, 2003
Increased mastitis incidence due to
periparturient immunosuppression
Primiparous
Multiparous
Barkema et al., 1998
22
20
18
16
14
Control
Anionic
21
18
15
12
9
6
3
0
-3
-6
-9
-1
2
-1
5
-1
8
12
10
8
6
-2
1
DMI, kg.
Dry Matter Intake during Transition
Day Relative to Parturition
Puntenney & French 2006
Energy balance and parturition
ENE Balance (MCal/Kg)
0
-3
-6
Control
Anionic
-9
-12
-15
0
1
2
3
Week Relative to Parturition
Puntenney & French 2006
Plasma NEFA
 Depressed dry matter intake and elevated blood NEFA
concentrations during the 2 to 3 weeks prior to calving
have been associated with an increased occurrence of
ketosis (Duncan 1998, Goff 2006)
 Mobilization of adipose tissue is related to the sudden
increase in demand for energy brought on by the onset
of lactation, not parturition itself (Goff 2002)
 Elevated NEFA is related to lower DMI at 14 d
postpartum and an increased incidence of metritis and
mastitis (Goff 2006)
Impact of Plasma NEFA Concentration
on neutrophil function (dairy)
250
Oxidative burst
200
150
100
50
0
0 mM NEFA
2 mM NEFA
Scalia et al. 2006
NEFAs inhibit ROS
+
Phagocytosis
O2
NADP oxidase
superoxide anion (O.2-)
Bacteriocidal
hypochlorite
ion OCl-
X
Superoxide
dismutase
Neutrophil
myeloperoxidase
Cu/Zn
H2O2
X
Macrophages:
myeloperoxidaseindependent mechanism
Other oxygen-derived
free radicals (OH.)
β-hydroxybutyrate
 An indicator of Ketosis
 Associated with negative energy balance
 Cows with plasma BHB levels above 12 mg/dL, are
considered to be subclinically ketotic, above 15 mg/dL
clinically ketotic (LeBlanc 2005)
 Elevated BHB 14 d postcalving is associated with an
increased incidence of metritis & mastitis (Goff 2006)
βHB by Day Relative to Parturition
12
BHB, umol/L
11
10
9
8
7
6
5
4
-1
0
1
7
14
21
Day Relative to Parturition
Puntenney & French 2006
H2O2 Production
Ketones suppress leukocyte function
Hoeben et al., 1999
Ketones inhibit ROS
+
Phagocytosis
O2
NADP oxidase
superoxide anion (O.2-)
Bacteriocidal
hypochlorite
ion OCl-
X
Superoxide
dismutase
Neutrophil
myeloperoxidase
Cu/Zn
H2O2
X
Macrophages:
myeloperoxidaseindependent mechanism
Other oxygen-derived
free radicals (OH.)
Calcium signaling in PBMN cells
Hypocalcemia impairs immune cell
function
Control cows
Cows with milk fever
Kimura et al., 2006
Control
Anionic
Day Relative to Parturition
21
18
15
9
12
6
3
0
-3
-6
-9
-1
2
-1
5
-1
8
12
10
8
6
-2
1
DMI, kg.
22
20
18
16
14
What are the effects of OmniGen-AF
on neutrophil function?
 Respiratory burst
(ROS generation)
 Neutrophil NET
formation
 Phagocytosis Assay
Respiratory burst assay
Combine
-neutrophils (+/- OmniGen-AF)
-Cytochrome C
-Zymosan A (from yeast)
Incubate 30 min, 37oC
Assess Cytochrome-C reduction by color change
(655 nm)
Effects on Respiratory Burst
6
ROS generation
5
4
0 nM PMA
5 nM PMA
3
2
1
0
Control
OmniGen
Omnigen-AF effect (P<0.01)
OmniGen-AF restored phagocytosis and
ROS generation in Stress Challenge
+
Phagocytosis
O2
NADP oxidase
superoxide anion (O.2-)
Bacteriocidal
hypochlorite
ion OCl-
Superoxide Cu/Zn
dismutase
H2O2
Neutrophil
myeloperoxidase
Macrophages:
myeloperoxidaseindependent mechanism
Other oxygen-derived
free radicals (OH.)
Production of NETs by neutrophils
Inactive neutrophils
Activated neutrophils with NETs
+ interleukin-8 (IL-8)
Source: Brinkmann et al., 2004, Science
Association of neutrophil “NETs”
with bacteria
S. aureus
Gram +
Salmonella
Gram -
Shigella
Gram -
Neutrophil NET assay
Neutrophils +/- OmniGen-AF
Add to microplate with varying
Concentration of PMA (activator)
Incubate 30 minutes at 37oC
Add Sytox green (extracellular
DNA marker)
Read in fluorescence plate reader
485 excitation, 525 emmision
Change in DNA fluorescence
relative to 0 nM PMA control
OmniGen-AF enhanced NET release in
immunosuppressed sheep neutrophils
1.2
1
0.8
Control
OmniGen
0.6
0.4
0.2
0
0 nM
0.5 nM
5 nM
50 nM
Phorbol ester (PMA) concentration
OmniGen-AF as main effect (P<0.01)
Phagocytosis Assay
Sheep
E. coli Strain 487
(+/- OmniGen)
Neutrophils
Combine in ratio of 30 E. coli:1 neutrophil
Incubate 37oC, 2 hr
(allow killing of E. coli)
Add MTT
(live E. coli metabolize to formazin, purple)
Incubate 4 hr, 37oC in CO2
Stop rx, incubate 4 hr
Read at 655nm on plate reader
OmniGen-AF enhanced phagocytosis in
stress challenged sheep neutrophils
Killing of E. coli
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Control
OmniGen-AF
P<0.01
Moving beyond molecular biology
DNA
L-selectin
IL8R, ICE
IL4R
thymopoeitin
Cell function
Organ function
Whole animal
Populations
Mastitis
 Mastitis is defined as an
infection of part of the
mammary gland
 Cost – about an
average of $200/cow
annually. . . . .$2 billion
to the industry
 “A disease of man
transmitted to the cow”
 Impossible to eradicate,
but practical to control.
Effect on Milk Composition & Quality
 Rapid increase in somatic cell count
(leukocytes)
Milk volume, milk fat, SNF,
protein, lactose, casein,
calcium & shelf life
Whey protein, albumin,
sodium, chloride & pH
(bad things)
Economic Loss due to Mastitis
Anatomy of the
Mammary Gland
 All pathogens enter through
the streak canal via
-employee hands
-contaminated bedding
-equipment insults
-cow to cow transfer
Teat Sphincter
Streak canal
DeLaval, Inc. 2006
 Contagious
Pathogens
- Streptococcus agalactiae (very short life o/s the udder; responds to dc
treatment and teat dips) gram +
- Staphylococcus aureus (resistant to antibiotics; chronic) gram + (CPS)
 Environmental
- Other Strep. spp.
- Dysgalactiae (chronic mastitis) gram +
- Uberis (contaminated bedding) gram +
- Coliforms
- E. coli (hot mastitis, quick acting,
high temp & shock) gram –
- Klebsiella spp. (high incidence in
wood shavings) gram – (CPS)
-Enterobacter sp. gram –
-Aerobacter aerogenes gram –
CPS type cell walls supress complement C3b
activity & resist phagocytosis
Staph. aureus
Other Pathogens
 Coagulase negative staph (live on skin; don’t cause clinical mastitis gram +
(CPS)
 Psuedomonas aeruginosa (lives in water; responds to Cl) gram – (CPS)
 Corynebacterium pyogenes (antibiotic resistant) gram + (CPS)
 Nocardia spp. (standing water; acts like coliform) gram +
 Mycoplasma spp. (spreads easily, shuts down milk gram –
production in 24 hours, equipment related)
occasional; dirty mastitis tubes
 Proteus spp. – not common gram- (CPS)
 Serratia spp. – chronic mastitis gram – (CPS)
 Yeasts
 Fungi
usually not significant
Part 2




Mastitis Field Studies With OmniGen
Mycotic Abortions
Mycotoxin Studies
OmniGen Use Rates
Case Study No. 7 - Texas
- 3200 cow dairy, open lot design, silage based TMR
- Initiated an off/on/off protocol:
Off - Aug. 1, 2003 - Sept. 27, 2003 (45 d)
On - Sept. 28, 2003 - Dec. 30, 2003 (93 d)
Off - Jan. 1, 2004 - Mar. 3, 2004 (63 d)
Case Study No. 7 - Texas
Summary of Cull/Dead Records by Feeding Period
Off
(45 days)
109
36
On
(93 days)
163
45
Off
(63 days)
144
43
Total:
Culls
Dead
Respiratory:
Culls
Dead
10
4
6
7
6
5
Digestive:
Culls
Dead
12
14
8
15
18
16
Mastitis:
Culls
Dead
33
1
8
4
28
6
Case Study No. 7 - Texas
Summary of culls and deaths by feeding period
Item
Off
On
(108 days)
(93 days)
Total:
Culls
253
163
Deaths
79
45
----------------------------------------------------------------------------Respiratory:
Culls
16
6
Deaths
9
7
Digestive:
Culls
Deaths
30
30
8
15
Mastitis:
Culls
Deaths
61
7
8
4
Case Study #9:Effect of OmniGen-AF on a
High SCC herd (4000 cows)
OmniGen Added
Avg. SCC
600000
550000
500000
Avg. SCC
450000
400000
12/17/2006
12/3/2006
11/19/2006
11/5/2006
10/22/2006
10/8/2006
9/24/2006
9/10/2006
8/27/2006
8/13/2006
7/30/2006
350000
Dominant Pathogen: Strep uberis; Minor Pathogen: E. coli
Conditions Don’t Always Go Our
Way!!
Case Study #10: Effect of OmniGen-AF
on a High SCC herd (3000 cows)
OmniGen Added
16-Jan
17-Jan
18-Jan
19-Jan
20-Jan
21-Jan
22-Jan
23-Jan
24-Jan
25-Jan
26-Jan
1-Feb
2-Feb
3-Feb
4-Feb
5-Feb
6-Feb
7-Feb
400
380
360
340
320
300
382
367
327
358
335
333
344
334
337
266
270
251
284
251
274
255
268
278
280
260
240
1/
12
/
1/ 200
14 7
/
1/ 200
16 7
/
1/ 200
18 7
/
1/ 200
20 7
/
1/ 200
22 7
/
1/ 200
24 7
/
1/ 200
26 7
/
1/ 200
28 7
/
1/ 200
30 7
/2
2/ 007
1/
2
2/ 007
3/
2
2/ 007
5/
2
2/ 007
7/
20
07
220
Dominant Pathogen: Strep uberis
Series1
High Incidence of New Infects
PSCC | PSCC
<200 | >=200 All
SCC
280 |
315 595
>=200
12% | 13% 25%
--------+-------SCC
1558 | 277 1835
<200
64% | 11% 75%
==========================
1838
592 2430
76%
24% 100%
Reduced Incidence of NI Rate after
30 days on OmniGen
PSCC |
PSCC
<200 | >=200
SCC
212 | 291 503
>=200
8% | 11% 19%
--------+-------SCC
1702 | 335 2037
<200
67% | 13% 80%
==========================
1914
626 2540
75%
24% 100%
Monitoring Fresh Cow Mastitis:
Low SCC Herd
Cows 5 to 30 days in Milk
LACT | LACT
<2 | >=2
All
SCC
15 |
27
42
>=200
6% | 10% 16%
--------+-------SCC
82 | 145 227
<200
30% | 54% 84%
==========================
97
172
269
36%
64% 100%
196,000 SCC
Monitoring Fresh Cow Mastitis:
High SCC Herd
Cows 5 to 30 days in Milk
LACT | LACT
<2 | >=2 All
SCC
3|
11
14
>=200
5% | 20% 25%
--------+-------SCC
9|
32
41
<200
16% | 58% 74%
==========================
12
43
55
21%
78% 100%
286,000 SCC
Doing it Right!!!
YIELD & DOLLAR LOSSES
Table 3. Use of DHIA somatic cell count (SCC) score to estimate production
loss due to mastitis.
Difference in milk yield*
(lb/305 days)
SCC
SCS count (1000’s) Lactation 1
> Lactation 2
0
1
2
3
4
5
6
7
12.5
25
50
100
200
400
800
1600
——
——
——
200
400
600
800
1000
* Comparisons are with lactation yields at SCC scores of 2.
Source: Current Concepts of Bovine Mastitis. NMC, 1987.
——
——
——
400
800
1200
1600
2000
Lost Production Revenue due to
Mastitis
Economic loss in milk production due to mastitis compared to a herd
with a SCC score of 3
SCS
3
4
5
6
7
Annualized Loss
500 cow herd
1000 cow herd
—
—
$24,000
$ 48,000
$48,000
$ 96,000
$64,000
$128,000
$96,000
$192,000
cents/hd.
----16¢
32¢
48¢
64¢
Based on a milk price of $12.00/cwt.
Source: Current Concepts of Bovine Mastitis. NMC, 1987.
Economics of Reduced Mastitis
 100,000 drop in SCC = @15 per cwt. Bonus
75 lbs. of milk x $0.15 = $11.2 cents
 Clinical cases of mastitis cost $300 per cow
(Guard, et al. 1996)
10% reduction in clinical cases = $10.0 cents per
head per day
 .5 SCS reduction = $8.0 cents in production revenue/day
Abortions
 Approximately 15% of abortions are mycotic
 The value of a pregnancy is $278.00 (US)
 The cost of a pregnancy loss is $555 (US)
(DeVries 2006; JDS 89:3876 – 3885)
Result in Extended Lactations
Mycotic abortions
Mycotic placentitis observed in 131/1107 placentas obtained
Aspergillus hyphae isolated from
placental tissue (78% incidence)
Phycomycete hyphae isolated from
placental tissue (11% incidence)
67% of fetal stomachs contained Aspergillus hyphae
McCausland et al., 1997
OR/WA Abortion Study
Abortions
1000000
100000
10000
A. fumigatus
(spores/ml)
1000
100
10
Co
w
#2
0
Co
w
#6
37
Co
w
61
Co
7
w
#2
34
Fe
9
tu
s
(6
Fe
17
tu
)
s
(2
34
9)
Ca
no
la
1
Mycotic Abortions: Field Studies
20
AF added
15
New crop haylage
10
5
Dec
Nov
Oct
Sep
Aug
Jul
Jun
May
Apr
Mar
Feb
Jan
0
Case Study Summary
Incidence of HBS and Abortion
--------Number of Cases---------No OmniGen1
HBS Abortions
OmniGen
HBS
Case No.
Herd Size
Abortions
12,3
1200
18
-0
-2
300
4
-0
-4
350
-31
-15
5
4500
14
>100
0
0
-------------------------------------------------------------------------------------1number
of cases reported in the previous 6 month period
2binder fed during pre-OmniGen period
3history of vaccination program for clostridium Type A
case locations: 1 & 5; Washington, 2 & 4; Oregon
OmniGen AF - Wisconsin Case Studies
Milk Yield & DMI
No OmniGen
Nov
Dec
Jan
OmniGen
Feb Mar Apr May
Case No. 9*
Milk (lb)
78
79
84
85
90
90
91
DMI (lb)
48
49
51
51
52
52
53
Case No. 10**
Milk (lb)
58
60
62
62
62
62
63
DMI (lb)
44
45
50
50
50
50
50
------------------------------------------------------------------------------------*OmniGen fed at 2oz./h/d beginning after December weigh day,
reduced to 1oz./h/d mid- January
**OmniGen fed at 2oz./h/d beginning after December weigh day,
reduced to 1oz./h/d at end of January
In Vitro Mycotoxin Binding Studies with various
commercial flow agents
University of Missouri, Columbia
% Binding by Other Commercial Products
Mycotoxin
T-Bind
Mycotex
MTB100 UltraSorb AB20
Aflatoxin
100
99
45
100
100
Fumonisin
100
NT
NT
NT
100
Ochratoxin
90
83
81
80
87
Vomitoxin
30
0
0
13
40
Zearalenone
61
70
76
100
60
---------------------------------------------------------------------------------Duplicate samples tested with 2 ppm aflatoxin, 2 ppm fumonisin, 2 ppm
ochratoxin, 4ppm vomitoxin and 2 ppm zearalenone
NT= interference, no results
In Vitro Mycotoxin Binding Studies with OmniGen AF
University of Missouri, Columbia
% Binding __________
Mycotoxin
Sample 1
Sample 2
Aflatoxin
100
100
Fumonisin
83
nt
Ochratoxin
95
88
Vomitoxin
47
55
Zearalenone
86
91
--------------------------------------------------------------------------------------------Duplicate samples tested with 2 ppm aflatoxin, 2 ppm fumonisin, 2 ppm
ochratoxin, 4ppm vomitoxin and 2 ppm zearalenone
NT= interference, no results
OmniGen-AF
 Product category - Nutritional Supplement
 Feeding Rate – 55 grams per head
 Not sold as a mycotoxin binder …..
but recent Columbia University
studies have shown it to be highly
efficacious
Review
 All mastitis organisms enter through the streak
canal – milking technique, equipment maintenance
and hygiene are paramount to low scc
 NEFA & BHB inhibit neutrophil killing ability
 OmniGen experimentally increased phagcytosis
against e. coli, increased ROS activity, &
increased nutrophil net formation in vitro
 OmniGen-AF reduced SCC in Strep uberis herds
 New infection rate is the driver of SCC
 There may be a good return on investment for
OAF in some mastitis applications
 OmniGen may not be effective against bacteria
forming capsulated cell walls (Klebsiella and
Staph)
Questions?