Immune Function - The New Frontier in Animal Production

1. Immune Function - The New Frontier in Animal Production Steve Puntenney, Ph.D. OmniGen Research, LLC OmniGen

2. 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.

3. History of OmniGen Research • 1999 HBS identified as a significant issue in OR & WA Dairies • May 2001 Meetings w/ consulting veterinarians in WA indicated a high failure rate of autogenous vaccines & need for other intervention identified • Feb 2002 Prototype OmniGen placed on 6 OR & WA Dairies • May 2002 OmniGen Research, LLC formed with NEF to discover MOA • Mar 2003 PAP began distribution of OAF • Jan 2004 OmniGen-AF Licensed to PAP

4. History of OmniGen Research • July 2005 Construction on the OmniGen Research Lab • June 2006 Established OmniGen Research Farm • Feb 2007 Beginning of construction of dairy and lab animal facilities

5. 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

6. Adaptive (antibody-mediated) immunity Vulnerable Janeway et al., 2005

7. Impact of periparturient immunosuppression Increased susceptibility to diseases -mastitis -metritis/retained placenta -foot infections -respiratory infections -gastrointestinal disorders -lowered peak milk production

8. 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)

9. First line of defense Evolutionarily ancient Several components Epithelial barriers HCl Digestive enzymes Cellular component Macrophages Neutrophils Natural killer (NK cells) Innate immunity Allows time for adaptive immunity to develop

10. 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

11. Neutrophil Trafficing L-selectin IL-8 Burton and Erskine, 2003

12. Phagocytosis by the neutrophil Bacterium Recognition

13. Killing mechanisms: Respiratory burst via ROS O2 + Phagocytosis NADP oxidase superoxide anion (O.2-) H2O2 Superoxide dismutase Bacteriocidal hypochlorite ion OCl- Neutrophil myeloperoxidase Macrophages: myeloperoxidase- Independent mechanism killing killing Other oxygen-derived free radicals (OH.)

14. Good: kill pathogens Bad: tissue damage “Immune cells” have high intrinsic ROS generation and, therefore, have high anti-oxidant demand “Immune cells” have membranes high in PUFAs which are susceptible to ROS-mediated damage ROS: Good and Bad Chew and Park, 2002

15. O2 + Phagocytosis NADP oxidase superoxide anion (O.2-) H2O2 Superoxide dismutase Bacteriocidal hypochlorite ion OCl- Neutrophil myeloperoxidase Macrophages: myeloperoxidase- independent mechanism Other oxygen-derived free radicals (OH.) A primary mechanism by which nutrients support immunity is via protection against ROS Cu/Zn Anti-oxidants (scavenge free radicals) vitamin E, vitamin C zinc, selenium, carotenoids, flavenoids

16. 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

17. Impact of periparturient immunosuppression • Increased susceptibility to disease -mastitis -metritis/retained placenta -foot infections -respiratory infections -gastrointestinal disorders

18. Periparturient immunosuppression • Parturition • Drying off • Shipping • Over-crowding • Environmental (heat/hold) • Excess handling L-selectin Neutrophil L-selectin Source: Burton and Erskine, 2003

19. Cortisol brings about immunosuppression Healthy mammary tissue Mammary tissue in stress Cytokines released from macrophages signal neutrophil migration Source: Burton and Erskine, 2003

20. Increased mastitis incidence due to periparturient immunosuppression Primiparous Multiparous Barkema et al., 1998

21. Dry Matter Intake during Transition Puntenney & French 2006

22. Energy balance and parturition Puntenney & French 2006

23. 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)

24. Impact of Plasma NEFA Concentration on neutrophil function (dairy) Oxidative burst Scalia et al. 2006

25. O2 + Phagocytosis NADP oxidase superoxide anion (O.2-) H2O2 Superoxide dismutase Bacteriocidal hypochlorite ion OCl- Neutrophil myeloperoxidase Macrophages: myeloperoxidase- independent mechanism Other oxygen-derived free radicals (OH.) NEFAs inhibit ROS Cu/Zn X X

26. 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)

27. βHB by Day Relative to Parturition Puntenney & French 2006

28. Ketones suppress leukocyte function H2O2 Production Hoeben et al., 1999

29. O2 + Phagocytosis NADP oxidase superoxide anion (O.2-) H2O2 Superoxide dismutase Bacteriocidal hypochlorite ion OCl- Neutrophil myeloperoxidase Macrophages: myeloperoxidase- independent mechanism Other oxygen-derived free radicals (OH.) Ketones inhibit ROS Cu/Zn X X

30. Hypocalcemia impairs immune cell function Control cows Calcium signaling in PBMN cells Cows with milk fever Kimura et al., 2006

32. Respiratory burst (ROS generation) Neutrophil NET formation Phagocytosis Assay What are the effects of OmniGen-AF on neutrophil function?

33. 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)

34. Effects on Respiratory Burst ROS generation Omnigen-AF effect (P<0.01)

35. OmniGen-AF restored phagocytosis and ROS generation in Stress Challenge O2 + Phagocytosis NADP oxidase superoxide anion (O.2-) H2O2 Superoxide dismutase Bacteriocidal hypochlorite ion OCl- Neutrophil myeloperoxidase Macrophages: myeloperoxidase- independent mechanism Other oxygen-derived free radicals (OH.) Cu/Zn

36. Production of NETs by neutrophils Inactive neutrophils Activated neutrophils with NETs Source: Brinkmann et al., 2004, Science + interleukin-8 (IL-8)

37. Association of neutrophil “NETs” with bacteria S. aureus Salmonella Shigella Gram + Gram - Gram -

38. 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

39. OmniGen-AF enhanced NET release in immunosuppressed sheep neutrophils Change in DNA fluorescence relative to 0 nM PMA control Phorbol ester (PMA) concentration OmniGen-AF as main effect (P<0.01)

40. Phagocytosis Assay Sheep E. coli Strain 487 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)

41. OmniGen-AF enhanced phagocytosis in stress challenged sheep neutrophils Killing of E. coli P<0.01

42. Moving beyond molecular biology DNA Cell function Organ function Whole animal Populations L-selectin IL8R, ICE IL4R thymopoeitin

43. 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.

44. 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)

45. Economic Loss due to Mastitis

46. 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

47. Pathogens • Contagious - 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

48. 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

49. Part 2 • Mastitis Field Studies With OmniGen • Mycotic Abortions • Mycotoxin Studies • OmniGen Use Rates

50. 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)