The Pennsylvania State University. The Graduate School. Department of Animal Science EFFECTS OF COLOSTRUM HEAT TREATMENT ON IMMUNE

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1 The Pennsylvania State University The Graduate School Department of Animal Science EFFECTS OF COLOSTRUM HEAT TREATMENT ON IMMUNE DEVELOPMENT IN THE NEONATAL CALF A Dissertation in Animal Science by Sonia L. Gelsinger 2017 Sonia L. Gelsinger Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2017

2 The dissertation of Sonia L. Gelsinger was reviewed and approved* by the following: Arlyn J. Heinrichs Professor of Animal Science Dissertation Adviser Chair of Committee Joy L. Pate Professor of Reproductive Physiology C. Lee Rumberger and Family Chair in Agricultural Sciences Robert J. Van Saun Professor of Veterinary and Biomedical Sciences Gabriella A. Varga Distinguished Professor Emeritus of Animal Science Terry D. Etherton Distinguished Professor of Animal Nutrition Head of Department of Animal Science *Signatures are on file in the Graduate School

3 iii ABSTRACT Experiments were conducted to determine whether plasma IgG and absorption efficiency could be increased by heat treatment of colostrum with high or low initial IgG content; test the interaction of bacterial content of colostrum and heat treatment on IgG absorption in calves; and characterize absorption of IFNG, TNF, and IL1B as well as growth and immune response in calves that received heat-treated or unheated colostrum. In each experiment colostrum was pooled and divided in half to create heat-treated and unheated treatments. Heat-treated colostrum was heated to 60 o C for 30 (experiments 1 and 2) or 60 minutes (experiment 3). Colostrum in the first experiment was pooled according to IgG concentration (low, medium, or high). In experiment 2 half of the unheated and heattreated colostra were incubated to create high bacteria treatments. Calves in each experiment were randomly assigned to treatments and blood was collected 48 hours after birth to assess IgG absorption. Absorption of IgG was increased (experiment 1) or unchanged (experiments 2 and 3) by heat treatment. Plasma IgG increased with increasing colostrum IgG concentration and decreased with greater bacterial content of colostrum. To assess the effect of colostrum heat treatment on immune response, bull calves in experiment 3 received subcutaneous injections of ovalbumin at 14 and 35 days of age. Blood samples were collected 0, 4, 8, and 12 h after each injection and daily for the subsequent 10 d for cytokine and IgG analysis. Plasma IL1B concentration was reduced and calf growth pattern was altered in calves fed heat-treated colostrum. Heat treatment of colostrum alters concentrations of bioactive factors received from colostrum. Research is needed to characterize ramifications on neonatal immunity and development. Keywords: calf, colostrum, heat treatment, immunity

4 iv TABLE OF CONTENTS List of Figures... vii List of Tables... viii Acknowledgements... ix Chapter 1 Introduction... 1 References... 3 Chapter 2 A Review of Literature Describing Bovine Colostrum and Neonatal Calf Immunity... 5 Historical Overview... 5 Overview of the Immune System... 7 Immune System of the Neonatal Calf Importance of Immunoglobulin G Heat Treatment of Colostrum Cytokines in Colostrum Conclusions References Chapter 3 Heat Treatment of Colostrum Increases IgG Absorption Efficiency in High-, Medium- and Low-Quality Colostrum Abstract Introduction Materials and Methods Colostrum Treatments Calves and Sampling IgG Analysis Statistical Analysis Results and Discussion Conclusions References Chapter 4 Effect of Colostrum Heat Treatment and Bacterial Population on IgG Absorption and Health of Neonatal Calves Abstract Introduction Materials and Methods... 57

5 v Colostrum Treatments Calves and Sampling IgG and Bacterial Analysis Statistical Analysis Results and Discussion Conclusions References Chapter 5 Comparison of Immune Responses in Calves Fed Heat-treated or Unheated Colostrum Abstract Introduction Materials and Methods Colostrum Treatments Calves and Sampling Total and Ovalbumin-specific IgG Analysis Cytokine Analysis Statistical Analysis Results Discussion IgG absorption IL1B and IFNG Growth Antibody production Conclusions References Chapter 6 Conclusions Appendix A: Apparent efficiency of absorption calculation References Appendix B: Comparison of radial immunodiffusion and enzyme-linked immunosorbant assay for quantification of bovine IgG in colostrum and plasma Introduction Materials and Methods Animals and sampling Radial immunodiffusion Enzyme-linked immunosorbant assay Colostrum heat treatment Statistical analysis

6 Results and Discussion Conclusions References Appendix C: Summary of electrolyte treatments vi

7 vii LIST OF FIGURES Figure 5-1 Body temperature after subcutaneous injection with 5.0 mg/ml ovalbumin at 14 (I) and 35 (II) d of age in calves that received heat-treated (60 o C, 60 min; HT) or unheated (UH) colostrum at birth. Body temperature was not affected by colostrum treatment (P > 0.05) or calf age (P > 0.05) but changed with time after injection (P < 0.01). * indicates body temperature different from time zero Figure 5-2 Plasma IFNG and IL1B concentrations in sera after subcutaneous injection with 5.0 mg/ml ovalbumin at 14 (I and III) and 35 (II and IV) d of age in calves that received heat-treated (60 o C, 60 min; HT) or unheated (UH) colostrum at birth. Colostrum treatment did not affect plasma IFNG in calves at 14 or 35 d or IL1B at 35 d (P > 0.05). Plasma IL1B was lower in HT calves at 14 d (P = 0.01). Time after injection (P < 0.01) but not calf age (P > 0.05) affected plasma IFNG concentration. Plasma IL1B was lower in 35-dold calves (P < 0.01) and changed with time after injection (P = 0.04). * indicates concentration different from time zero. Lowercase letters indicate differences between treatments Figure 5-3 Change in ovalbumin-specific IgG concentration in sera after subcutaneous injection with 5.0 mg/ml ovalbumin at 35 d of age in calves that received heat-treated (60 o C, 60 min; HT) or unheated (UH) colostrum at birth. Ovalbumin-specific IgG concentration changed with time after injection (P < 0.01) but was not affected by colostrum treatment (P > 0.05). * indicates concentration different from time zero Figure B-1 Correlation plots of IgG concentration measured by RID and ELISA in heat-treated colostrum, unheated colostrum, and plasma Figure B-2 Histograms showing the number of repeated analyses by ELISA and RID for unheated and heat-treated (n = 58) colostrum as well as plasma (n = 104) samples Figure B-3 Correlation of IgG concentration measured by RID and ELISA methods in colostrum samples (n = 45) before and after heat-treatment

8 viii LIST OF TABLES Table 3-1 Total immunoglobulin G (mg/ml) 1 and bacterial counts (log cfu/ml) in colostrum before and after heat treatment (60 o C, 30 min) Table 3-2 Least squares means estimates for 48-h plasma IgG and apparent efficiency of absorption (AEA) of IgG in calves fed unheated or heat-treated (60 o C, 30 min) colostrum of high, medium, or low qualities Table 4-1 Mean (± SD) IgG concentration and bacterial populations in each colostrum treatment Table 4-2 Mean (± SD) birth weight, age at colostrum feedings, and blood parameters at birth for calves in each treatment Table 4-3 Plasma IgG concentration 48 hours after birth measured by RID and ELISA and efficiency of absorption in calves fed heat-treated or unheated colostrum of high or low bacterial content Table 4-4 Calf days with a health score > 2 during the first 7 d of life in calves fed heat-treated or unheated colostrum with high or low bacterial content Table 5-1 Chemical composition of calf starter mix offered to calves starting at 3 d of age Table 5-2 Summary of colostrum treatments and feeding, calf birth weight and IgG absorption as measured by radial immunodiffusion (RID) or ELISA Table 5-3 Serum cytokine concentration at birth and 24 to 48 h of age in calves fed unheated or heat-treated colostrum Table 5-4 Growth measures after ovalbumin injection at 14 and 35 d of age in calves that received heat-treated (60 o C, 60 min; HT) or unheated (UH) colostrum at birth Table B-1 Least squares means and standard errors of means for IgG concentration in plasma and colostrum measured by ELISA or RID methods and colostrum before and after heat treatment Table C-1 Total number of doses of oral electrolytes offered to each calf used in experiment 3 and reasons that electrolyte doses were offered

9 ix ACKNOWLEDGEMENTS I would like to express my sincere gratitude to all who contributed to making my PhD adventure a success. I would like begin by thanking my advisor, Dr. Jud Heinrichs for accepting me as a student. His guidance, wisdom and encouragement have been paramount to successful completion of this dissertation. I am especially grateful to him for advocating for me and challenging me to take advantage of the various opportunities available for teaching, research and extension, even when they involved travelling halfway around the world. This appreciation is also extended to others in the Animal Science Department faculty for creating an academic culture where students are challenged and encouraged to grow. I specifically thank Drs. Joy Pate, Robert Van Saun, and Gabriella Varga for donating their time and expertise as members of my dissertation committee, Drs. Troy Ott, Burt Staniar, Craig Baumrucker, Chad Dechow and Tara Felix for sharing their wisdom and experience and Mr. Dale Olver for his mentorship and encouragement in various teaching and youth extension activities. I have greatly benefitted from each of their examples and appreciate their willingness to invest in my development as a researcher, teacher, and professional. Furthermore, I would like to thank my fellow graduate students especially my labmates Felipe Pino, Alanna Kmicikewycz, and Lucas Mitchell for their constant availability when I needed them. I also extend special appreciation to Jasmine Dillon for many lunch discussions to solve the world s problems. I also recognize all of the undergraduate students and interns that helped me clean barns, feed calves, collect

10 x samples and run endless ELISAs. I could not have completed these research projects without them. Special thanks are expressed to Amanda Smith, Linda Truong, Blair Cundiff, Jason Rizo, and Chad Heffner for their time and commitment to each of my experiments. Successful completion of my PhD required cooperation from several teams and collaborators. I would like to thank teams at the Penn State Dairy Farm, Penn State Meats Lab, and Evergreen Farms for their patience with my random-hour sampling and experiment requirements. Gratitude is also expressed to the Penn State Food Science Department especially Emily Furumoto and Byron Ba for their assistance in colostrum processing. Thanks are expressed to collaborators at the University of Vermont and la Universidad de Las Palmas de Gran Canaria in Spain especially Rink Tacoma and Dr. Anastasio Arguello for broadening the scope of my research and graduate experience. This dissertation would not have been possible without much support from friends and family. I want to recognize my parents, Ernest and Linda for their patient support of my research. I also recognize the support of my friends in Penn State Christian Grads through Starbucks, on-farm meal deliveries and willingness to learn much more about cows than they ever wanted to know. Special appreciation is extended to Dr. Heather Holleman, Devon Torchiana, Sara Hoffman, Kelly Patches, and members of my Missional Communities for their prayers and mentorship throughout my program. I am also grateful to my boyfriend, Cory Arnold for his patience, commitment and undying support through the final seasons of my PhD program; and finally, to my God and Savior, Jesus Christ through whom all things are possible.

11 xi EPIGRAPH It is the glory of god to conceal a matter; to search out a matter is the glory of kings. Proverbs 25:2

12 Chapter 1 Introduction Colostrum contains high concentrations of various biologically active factors. One of these that has been the subject of much research is immunoglobulin G. Due to bovine placental structure, newborn calves are born with no circulating IgG and at high risk of disease and mortality. Calves are able to absorb whole, functional IgG molecules directly from colostrum when colostrum is ingested within 24 hours after birth (Stott et al., 1979). The current standard of the dairy industry for determining successful IgG absorption is a blood IgG concentration exceeding 10 mg/ml at 24 hours of age (Tyler et al., 1996). Calves that do not reach this concentration are described as having failure of passive transfer and experience greater risk of disease and death. One management practice shown to increase IgG absorption is heat treatment of colostrum. Studies indicate that heating colostrum to 60 o C for 30 to 60 minutes reduces bacterial count with minimal effects on viscosity and IgG concentration (Godden et al., 2006; McMartin et al., 2006); however, when fed to calves increases blood IgG concentrations by 15 to 30% (Elizondo-Salazar and Heinrichs, 2009; Kryzer et al., 2015). The mechanism for how colostrum heat treatment improves IgG absorption has not been described. The major hypothesis states that improved IgG absorption is an indirect result of a reduction in bacterial content. A conclusive test of this hypothesis is included in this dissertation.

13 2 In addition to IgG, colostrum also contains various growth factors, hormones, cytokines and other components that may have biological functions in the neonatal calf (Baumrucker and Blum, 1993; Hagiwara et al., 2000; Hammon et al., 2012). The effects of heat treatment on each of these factors and possible ramifications on neonatal development are yet to be described. The goal of this dissertation was to continue characterizing the effects of colostrum heat treatment on IgG absorption and begin exploring its effects on other aspects of calf immunity. Experiments were conducted to address the following specific aims: 1. Determine whether plasma IgG and absorption efficiency could be increased by heat treatment of colostrum with high or low initial IgG content; 2. Test the interaction of bacterial content of colostrum and heat treatment on IgG absorption in calves; and 3. characterize absorption of IFNG, TNF, and IL1B as well as growth and immune response in calves that received heat-treated or unheated colostrum.

14 3 References Baumrucker, C. R., and J. R. Blum Secretion of Insulin-Like Growth-Factors in Milk and Their Effect on the Neonate. Livest. Prod. Sci. 35: Elizondo-Salazar, J. A., and A. J. Heinrichs Feeding heat-treated colostrum or unheated colostrum with two different bacterial concentrations to neonatal dairy calves. J. Dairy Sci. 92: Godden, S., S. McMartin, J. Feirtag, J. Stabel, R. Bey, S. Goyal, L. Metzger, J. Fetrow, S. Wells, and H. Chester-Jones Heat-treatment of bovine colostrum. II: Effects of heating duration on pathogen viability and immunoglobulin. J. Dairy Sci. 89: Hagiwara, K., S. Kataoka, H. Yamanaka, R. Kirisawa, and H. Iwai Detection of cytokines in bovine colostrum. Vet. Immunol. Immunopathol. 76: Hammon, H. M., J. Steinhoff-Wagner, U. Schonhusen, C. C. Metges, and J. W. Blum Energy metabolism in the newborn farm animal with emphasis on the calf: endocrine changes and responses to milk-born and systemic hormones. Domest Anim Endocrinol. 43: Kryzer, A. A., S. M. Godden, and R. Schell Heat-treated (in single aliquot or batch) colostrum outperforms non-heat-treated colostrum in terms of quality and transfer of immunoglobulin G in neonatal Jersey calves. J. Dairy Sci. 98: McMartin, S., S. Godden, L. Metzger, J. Feirtag, R. Bey, J. Stabel, S. Goyal, J. Fetrow, S. Wells, and H. Chester-Jones Heat treatment of bovine colostrum. I: Effects

15 4 of temperature on viscosity and immunoglobulin G level. J. Dairy Sci. 89: Stott, G. H., D. B. Marx, B. E. Menefee, and G. T. Nightengale Colostral Immunoglobulin Transfer in Calves.1. Period of Absorption. J. Dairy Sci. 62: Tyler, J. W., D. D. Hancock, S. M. Parish, D. E. Rea, T. E. Besser, S. G. Sanders, and L. K. Wilson Evaluation of 3 assays for failure of passive transfer in calves. J. Vet. Intern. Med. 10:

16 Chapter 2 A Review of Literature Describing Bovine Colostrum and Neonatal Calf Immunity Historical Overview Colostrum is the first secretion of a mammary gland following a pregnancy. It has long been known that colostrum consumption is important for neonatal mammals; however, some exact functions and mechanisms are still to be elucidated. The importance of colostrum for neonatal calf survival was established in a simple experiment by Smith and Little (1922) wherein one group of calves received colostrum while the second group was deprived of colostrum. Mortality rates during the first 3 weeks of life were 0% and 75% for colostrum-fed and deprived groups, respectively. Necropsy results revealed that all of the calves that died having received no colostrum died of septicemia (Smith and Little, 1922) providing clear evidence that some components provided in colostrum played a critical role for calf immunity to bacterial infection. A flurry of experiments followed this discovery as researchers hastened to identify those components and describe their functions. A 1956 experiment reported that molecules, then described simply as agglutinins for their ability to agglutinate cells, existed in colostrum and could protect calves from infection (Ingram et al., 1956). These agglutinins also appeared to be specialized and would only cause agglutination when exposed to specific types of

17 6 bacteria (Ingram et al., 1956). Later studies were able to detail the structure of these molecules and the name immunoglobulin (Ig) was adopted linking their immunological function with their large molecular size (Butler, 1969). Researchers also realized that there were different subclasses of Ig, distinguished by different molecular structures and variable efficiency for causing cell agglutination, and the prevalence of each subclass in colostrum varied between species. The most prevalent subclass in bovine colostrum was IgG; whereas, IgA predominated in human colostrum (Butler, 1969). Subsequent experiments quickly identified IgG as a major protective component in bovine colostrum and that these large molecules could pass unaltered from the colostrum into calf circulation (Kruse, 1970; McGuire et al., 1976). This discovery spurred a series of experiments by Stott et al. (1979a, 1979b, 1979c, 1979d) to characterize the period, rate and extent of calves absorption of IgG. Others sought to discover external factors that could impact IgG absorption including feeding methods, circulating glucocorticoid concentration and prepartum energy intake by the cow (Halliday et al., 1978; Molla, 1978; Johnston and Oxender, 1979). Simple tools were developed and later enhanced to allow rapid on-farm estimation of IgG concentration based on colostrum density and refraction index (Naylor and Kronfeld, 1977; Pritchett et al., 1994; Bielmann et al., 2010). Many studies have been conducted and continue today to investigate ways to improve management of dry cows, colostrum and newborn calves to enhance IgG absorption. Heat treatment of colostrum at 60 o C for 30 to 60 minutes is one management strategy that has been shown to improve IgG absorption by calves (Johnson et al., 2007; Elizondo-Salazar and Heinrichs, 2009b). However, the mechanism by which colostrum heat treatment improves IgG is yet to be elucidated. In addition, the effects of colostrum

18 7 heat treatment on non-ig proteins and possible ramifications on neonatal development have not been well investigated. The goal of this dissertation was to continue characterizing the effects of colostrum heat treatment on IgG absorption and begin exploring its effects on other aspects of calf immunity. Overview of the Immune System The cells of the immune system can be divided into innate cells, including neutrophils and macrophages, and adaptive cells, such as B cells and T cells. Each of these groups of cells is important for recognizing and removing foreign organisms from the host. The innate cells recognize foreign antigens based on patterns found on the surface of foreign cells that are not present on host cells (Janeway and Medzhitov, 2002). Lipopolysaccharide is an example of an antigen expressed on the surface of multiple different types of bacterial cells, but not mammalian cells. Due to the general nature of these receptors, innate cells have no memory and cannot distinguish specific organisms. They simply determine whether an encountered cell is self or nonself (Janeway and Medzhitov, 2002). In contrast, adaptive immune cells produce receptors that recognize specific antigens and thus are able to distinguish and remember individual foreign species and generate different types of immune responses depending on the type of antigen (Edelman, 1973). Upon recognition of a foreign antigen, neutrophils and macrophages will initiate phagocytosis and release cytokines and reactive oxygen species into the surrounding area (Aderem and Underhill, 1999). Phagocytosed organisms will be digested within the

19 8 lysosome resulting in multiple antigenic particles which can then be presented by macrophages and dendritic cells to B and T cells to initiate cell replication and production of receptors specific for the presented antigen (Edelman, 1973). Upon initial activation, B cells begin to secrete large numbers of antigen-specific Ig molecules. The first Ig subclass produced is IgM, which has lower antigen-binding affinity compared to other Ig subclasses but can efficiently activate complement proteins to assemble and disrupt pathogenic membranes (Edelman, 1973). As the immune response continues, B cells will switch to produce other subclasses of Ig, including IgG (Li et al., 2004). CD4+ helper T cells assist in this process through receptor stimulation and cytokine production (McHeyzer-Williams et al., 2003). Similar to IgM, IgG also activates complement proteins, but is a smaller molecule than IgM (Edelman, 1973). Thus, while IgM remains mainly in circulation, IgG is readily transferred across various membranes within the body (Mostov, 1994). In addition to complement activation, IgG can also neutralize antigens and mark them for phagocytosis by macrophages and neutrophils thereby increasing the efficiency of the innate immune response (Clark, 1997; Aderem and Underhill, 1999). Cytokines are protein signaling molecules that may be secreted by multiple cell types, but immune cells are the main producers of cytokine signals; receptors exist on the surface of most cells in the body (Janeway and Medzhitov, 2002). Specific cytokines relevant to this dissertation are the proinflammatory interleukin 1β (IL1B) and tumor necrosis factor α (TNF) and the T cell secreted interferon γ (IFNG). Both IL1B and TNF are secreted by activated macrophages to induce acute inflammation including fever,

20 9 blood vessel dilation, and acute phase protein production (Janeway and Medzhitov, 2002; Dinarello, 2009). These cytokines are also important in the process of antigen presentation in macrophage-dependent T cell activation (Croft, 2009; Dinarello, 2009). Despite similar source and systemic response, IL1B and TNF differ in the kinetics of their response. Release of TNF accelerates rapidly following exposure to antigen, peaking within 2 hours and is hastily eliminated such that it is no longer detectable 8 hours after initial exposure (Oliver et al., 1993; Benjamin et al., 2016). In contrast, IL1B production occurs more slowly reaching detectable concentration approximately 4 hours after exposure and remaining high for several days (Oliver et al., 1993). While IL1B and TNF are generally associated with innate cell response, IFNG is produced by cytotoxic (CD8+) and helper 1 (CD4+) T cells and directs the ensuing adaptive response. Production of IFNG promotes a CD4+ T helper 1-type immune response, which is driven by recognition and removal of pathogen by macrophages and elimination of injured or infected host cells by CD8+ T cells (Berger, 2000; Schroder et al., 2004). This is accomplished by downregulating antibody production by B cells, activating macrophages and enhancing T cell function by increasing the expression of major histocompatibility complex (MHC) molecules on immune and non-immune cells (Schroder et al., 2004). A CD4+ T helper type-2 immune response is characterized by less systemic inflammation, reduced T cell activity and high antibody production by B cells (Mosmann and Coffman, 1989). The immune response of the newborn calf is predominantly a CD4+ T helper type-2 response (Chase et al., 2008).

21 10 Immune System of the Neonatal Calf Early in the study of immunity, researchers began investigating whether protection from disease was transferable from mother to offspring. Results from these early studies were summarized by Ratner et al. (1927). In some species when the neonate was infected immediately after birth with a disease to which the mother had recovered, the newborn was not severely affected; this mechanism did not seem to function in goats, cattle, or sheep (Ratner et al., 1927). Subsequent experiments, measuring first agglutinins and then IgG, reported that these molecules were undetectable in calf plasma at birth but increased if calves consumed colostrum (Orcutt and Howe, 1922; Stott et al., 1979a). Ratner et al. (1927) were among the first to suggest that the absence of these protective molecules in neonatal calves was due to placental structure. Due to placental inhibition of Ig transport and protection from foreign antigen in utero, calves are born with an immune system that is antigenically naïve. Thus, the adaptive immune cells in neonatal calves are incapable of recognizing foreign antigens until the foreign cells are first recognized, phagocytosed, digested, transported, and antigens are presented by innate immune cells in lymphoid tissues. The adaptive system is further handicapped by high concentrations of hormones (e. g. cortisol and prostaglandin E2) and cytokines (e. g. IL4 and IL10) produced during pregnancy and parturition, which suppress T cell function and bias the adaptive response towards a CD4+ T helper type-2 response (Fischer et al., 1981; Schroder et al., 2004; Chase et al., 2008). Unfortunately, neonatal B cells, the focal point of this type of response are present

22 11 in very low numbers at birth and not completely functional until 4 to 5 weeks of age (Nonnecke et al., 2012). In addition to low B cell numbers, CD4+ T cells are also low in newborn calves making up only 16% of the peripheral T cell population. These cells account for 30% of peripheral T cells in adult cattle (Wilson et al., 1996). In place of these adaptive cells, neonatal calves have greater numbers of circulating neutrophils and γδ+ T cells (Menge et al., 1998; Kampen et al., 2006). It is likely that, given the limitations of the adaptive immune response, neonatal calves have evolved to depend more heavily on the innate functions of their immune system. This may explain the very high proportions of peripheral γδ+ T cells, which are able to recognize antigen using receptors similar to those of innate immune cells (Bonneville et al., 2010). This allows γδ+ T cells to generate T cell type responses to foreign cells without the added step of antigen presentation. γδ+ T cells are also able to perform a wider array of functions than either CD4+ or CD8+ T cells including recruitment of innate cells, induction of cell death, and production of proinflammatory and anti-inflammatory cytokines (Bonneville et al., 2010). High numbers of γδ+ T cells in circulation grants the neonatal calf much more flexibility in immune response and may help compensate for the slower response observed by other members of the adaptive system. Importance of Immunoglobulin G Benefits of an adaptive system include a rapid, targeted, multifaceted response utilizing multiple cell types and the complement protein system to eliminate the foreign

23 12 body. Adaptive cells also produce a wider array of specific receptors, which reduce the risk of invading cells passing unnoticed by the immune system. This response is a cellmediated, specific response that can effectively eliminate foreign cells with minimal destruction to host cells (Stinchcombe and Griffiths, 2007). In contrast, the innate response is limited mainly to the neutrophilic release of reactive oxygen species and phagocytosis, which can result in excessive inflammation and damage to host tissues (Janeway and Medzhitov, 2002). Secreted Ig molecules can partially bridge the gap between the innate and adaptive systems by broadening the array of antigens recognized to alert innate cells to the presence of an invading organism and by activating the system of complement proteins to assist in eliminating the organism (Edelman, 1973; Clark, 1997). Immunoglobulin G molecules are produced by activated B cells following exposure to a specific antigen (Edelman, 1973). Upon secretion, these molecules enter general circulation via lymph fluid and may be transported from the blood into most tissues with the exception of the brain (Mostov, 1994; Clark, 1997). There are 2 regions to each IgG molecule: the variable or antigen-specific region (F ab ) and the constant or signaling region (F c ). The F ab region is specific, binding only to the antigen to which its parent B cell was activated. Regardless of the antigen recognized by the F ab region, the F c region translates an identical signal to neutrophils, macrophages, and other immune cells to initiate phagocytosis (Clark, 1997). If the F c region is not quickly recognized by a phagocytic cell, circulating complement proteins may also recognize the bound IgG molecule and initiate a cascade of proteins leading to eventual lysis of the foreign cell

24 13 (Gasque, 2004). These two regions allow IgG to combine the innate response with adaptive specificity. During the period of colostrogenesis, approximately 3 to 4 weeks prior to parturition, the bovine mammary gland begins to selectively transfer IgG from blood circulation into the forming colostrum (Baumrucker et al., 2010). There are two subclasses of bovine IgG: IgG 1 and IgG 2 (Baumrucker and Bruckmaier, 2014). Both subclasses are present in colostrum; however, IgG 1 makes up 85 to 90% of the total IgG thus the majority of this dissertation will focus on IgG 1 (Elizondo-Salazar and Heinrichs, 2009b; Verweij et al., 2014; Samarutel et al., 2016). The total concentration of IgG in bovine colostrum is highly variable ranging from 11 mg/ml to 221 mg/ml (Kehoe et al., 2007; Baumrucker et al., 2010; Kehoe et al., 2011). Factors that can affect colostrum IgG concentration include time when colostrum is collected relative to parturition, parity, dry period length and energy intake during pregnancy (Kehoe et al., 2011; Mayasari et al., 2015; Mann et al., 2016). Early researchers observed that when calves were fed colostrum within 24 after birth, the IgG molecules that were in the colostrum appeared and were functional in the blood of the calves (Kruse, 1970; Stott et al., 1979a). Since then a plethora of studies have been done investigating various means of increasing calves ability absorb IgG. Hall et al. (2014) and Kamada et al. (2007) demonstrated enhanced IgG absorption with selenium supplementation directly in colostrum or via inclusion in the diet of the prepartum cow. Others supplemented pregnant cattle with nicotinic acid, iodine,

25 14 lactoferrin and fatty acids with little to no affect on IgG absorption (Conneely et al., 2014a; Conneely et al., 2014b; Aragona et al., 2016; Connelly and Erickson, 2016). Despite extensive investigation into factors affecting IgG absorption, the exact mechanism of absorption is yet to be elucidated. Based on data from several studies showing a consistent increase in serum IgG with increasing ingestion of IgG from colostrum, a nonspecific mechanism has been assumed for IgG absorption in neonatal calves (Stott and Fellah, 1983; Chigerwe et al., 2008; Osaka et al., 2014). This assumption is reflected in the terms successful and failure of passive transfer of immunity used to describe calves that achieve serum IgG concentrations above or below 10 mg/ml, respectively (Tyler et al., 1996). Htun et al. (2016) reported increased serum IgG and IgG absorption efficiency when difructose anhydride III, a molecule that interacts with tight junctions to improve nutrient absorption, was included in the colostrum. This implies that tight junctions may be at least partially involved in absorption. Data from rodents indicate that at least 80% of IgG present in the serum of neonatal mice and rats is selectively transported in vacuoles via the neonatal Fc receptor (FcRn) present in the neonatal gut lumen (Kliwinski et al., 2013). Studies conducted using neonatal ruminants have identified FcRn on crypt cells but not enterocytic cells, implying FcRn involvement in IgG recycling into the gut lumen, rather than absorption (Mayer et al., 2002). Studies have not been done to determine presence of FcRn within the intestine of the neonatal calf; however, the inability of calves to absorb insulin-like growth factor-1 (IGF1) with the same efficiency as IgG implies that the neonatal bovine gut exhibits some degree of selectivity of absorption (Vacher et al., 1995). Further

26 research is needed to fully characterize the mechanism of absorption and factors that may manipulate it. 15 Heat Treatment of Colostrum The concept of pasteurization was developed very quickly following the discovery of microorganisms. However, due to the high concentration of proteins, the time and temperature combinations used for pasteurization of milk resulted in a thick pudding consistency when used for colostrum (Ragsdale and Brody, 1923; Elizondo- Salazar et al., 2010). The first attempts to pasteurize colostrum were done in effort to avoid transferring bovine tuberculosis to newborn calves via colostrum from infected dams. Ragsdale and Brody (1923) determined that heating colostrum to 60 o C could inactivate tuberculosis organisms in 20 minutes and maintain colostrum fluidity for up to 3 hours. Whereas heating colostrum to 62.5 o C or 72.5 o C, the temperatures used for milk pasteurization, resulted in a solid colostrum mass within 60 minutes or 75 seconds, respectively (Ragsdale and Brody, 1923). This experiment established a temperature for colostrum heat treatment based on maintaining feedability; however, lack of specific methods prevented early researchers from determining the effects of this protocol on IgG. More recent studies have provided a more thorough characterization of the effects of heat treatment on various aspects of colostrum and calf health including IgG availability. Initial experiments investigating the effects of heat on IgG heated small volumes of colostrum to temperatures ranging from 57 o C to 63 o C for up to 120 minutes to determine the time and temperature combination that could successfully reduce

27 16 bacterial count without affecting viscosity or IgG concentration or activity (McMartin et al., 2006; Elizondo-Salazar et al., 2010). McMartin et al. (2006) reported insignificant losses in IgG concentration when moderate to high quality colostrum (IgG = 76.4 ± 26.5 mg/ml) was heated to 60 o C for 120 minutes. Elizondo-Salazar et al. (2010) reported reduced IgG concentration after 30 minutes of heating at 60 o C using similar quality colostrum (IgG = 74.8 mg/ml). A subsequent study utilizing a commercial pasteurizer and a 60 o C, 60-minute heat treatment, reported greater losses in IgG when initial concentration was high (IgG 70 mg/ml) compared to lower quality colostrum (Donahue et al., 2012). Having determined that heating to 60 o C had limited effects on colostrum IgG concentration, subsequent studies sought to determine minimum time required to reduce various bacteria found in colostrum. Elizondo-Salazar et al. (2010) demonstrated a 90% reduction in total bacteria and coliform counts could be achieved within 30 and 60 minutes at 60 o C. Godden et al. (2006) later confirmed that heating colostrum to 60 o C for 30 minutes eliminated the pathogenic bacteria species Mycoplasma bovis, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enteritidis. Mycobacterium avium subsp. paratuberculosis, the causative agent of Johnes disease in cattle, required greater duration of heat treatment. This bacterium was completely eliminated in 3 of 4 replicate batches after 60 minutes. It was not detected in any sample after 90 minutes of heating (Godden et al., 2006). Specific effects of colostrum heat treatment on calves ability to absorb IgG were first investigated by researchers at the Oklahoma State University who reported greater serum IgG concentration in calves that received colostrum that had been heated to 63 o C

28 17 for 30 minutes compared to calves that received raw colostrum (Bush et al., 1981). Unfortunately, these results were never published outside of an experiment station research report. They were, however, supported by Johnson et al. (2007) who reported greater mean serum IgG concentration in calves that received heat-treated (60 o C, 60 minutes) colostrum. Elizondo-Salazar and Heinrichs (2009b) collected samples from calves every 4 hours during the first day and weekly through 8 weeks of age and observed greater serum IgG concentration in calves that received heat-treated (60 o C, 30 minutes) compared to raw colostrum from 4 hours through 5 weeks of age. This improvement in IgG absorption from heat-treated colostrum has since been confirmed in multiple field studies including both Holstein and Jersey calves (Donahue et al., 2012; Godden et al., 2012; Kryzer et al., 2015). Effects of colostrum heat treatment on non-igg colostrum proteins or other aspects of immune function in calves are limited. Johnson et al., (2007) reported no differences in leukocyte counts and function between calves that received heat-treated (60 o C, 60 minutes; n=16) or raw (n=14) colostrum. However, this conclusion is limited by the number of observations. Abd El-Fattah et al. (2014) reported reduction in lactoferrin and IGF1 concentrations after heating colostrum to 60 o C for 60 minutes. Possible ramifications on neonatal gut and immune development are yet to be characterized. Three possible hypotheses have been proposed to explain the observed increase in serum IgG concentration in calves fed heat-treated colostrum. First, the IgG molecule may be fragmented during the heating process and the methods used to quantify IgG are not able to distinguish between whole, functional molecules and molecular fragments

29 18 (Bush et al., 1981). Second, heat treatment reduces bacterial counts, thus, improved IgG absorption is an indirect result of reduced bacterial load (Johnson et al., 2007). Third, heat treatment increases colostrum viscosity implying that non-ig proteins are denatured; IgG absorption may increase as an indirect result of decreased absorption of other proteins (Elizondo-Salazar and Heinrichs, 2009b). The first of these hypotheses was investigated in two experiments using a serum neutralization assay to quantify IgG function in heat-treated colostrum and in serum from calves that received heat-treated colostrum (Godden et al., 2006; Johnson et al., 2007). Initial results indicated that IgG molecules in colostrum retained their function through the heat treatment process (McMartin et al., 2006). This conclusion was based on a small number (n=18) of colostrum samples collected before and after heating to 60 o C for 120 minutes and was limited by sample size. A subsequent experiment confirmed these results showing no difference in neutralization of bovine viral diarrhea virus by IgG in serum samples collected from calves 24 hours after consuming either heat-treated (60 o C, 60 minutes) or raw colostrum (n=100; (Johnson et al., 2007). These initial studies confirm that at least the F ab region of the IgG molecule, the region responsible for viral recognition and neutralization remains intact during heat treatment. However, neutralization assays provide no information regarding the signal transduction F c region of the molecule. Loss of the F c function would prevent the IgG molecule from communicating with immune cells severely limiting its function. A field study investigated this hypothesis indirectly by monitoring health in calves that received heattreated (n=533) or raw (n=518) colostrum (Godden et al., 2012). Results demonstrated

30 19 lower risk of diarrhea prior to weaning in calves fed heat-treated colostrum refuting the hypothesis that increased absorption is due to significant fragmentation of IgG. Researchers used two approaches to investigate the second hypothesis. The first study used an incomplete 2-by-2 factorial design in attempt to separate the confounding effects of colostral bacterial content and heat treatment on IgG absorption by calves (Elizondo-Salazar and Heinrichs, 2009a). Colostrum was pooled to create a uniform batch, which was subsequently divided in thirds. One-third remained raw. One-third remained raw and was incubated for 24 hours to allow bacteria to grow. The final third was heated to 60 o C for 30 minutes. Calves that received heat-treated colostrum had greater serum IgG concentration; bacterial count did not appear to affect IgG absorption (Elizondo-Salazar and Heinrichs, 2009a). However, lack of a heat-treated, high bacteria colostrum treatment prevented a conclusive test of the interaction between bacterial count and colostrum heat treatment. A second study used an observational approach combining data from 1,051 calves that had received heat-treated (60 o C, 60 minutes) or raw colostrum (Godden et al., 2012). Samples of individual colostrums fed to each calf were collected at the time of feeding and serum was collected once between 1 and 7 days of age. Regression analysis of the results revealed a negative correlation between serum IgG concentration and colostrum coliform count (Godden et al., 2012). These results imply that a reduction in colostrum coliform count, an inevitable result of heat treatment, would improve IgG concentration. But the confounding of the effects of heat treatment and bacterial count prevent a conclusive answer to this hypothesis. The third hypothesis is possibly the most difficult to investigate because of the relative lack of knowledge regarding protein absorption in the neonatal calf intestine. If

31 20 IgG absorption is a highly selective process as reported by rodent researchers (Kliwinski et al., 2013), then it is expected to be relatively noncompetitive and this hypothesis is most likely false. However, data suggest that IgG absorption is much less selective in neonatal ruminants (Stott and Fellah, 1983; Chigerwe et al., 2008; Osaka et al., 2014); therefore, considerable competition may exist between proteins for absorption. The precise mechanism of IgG absorption must be described to adequately address this hypothesis. Experiments to describe that mechanism are on-going. Cytokines in Colostrum Cytokines have been isolated from the colostrum of several species including cattle, pigs, and humans (Bocci et al., 1991; Hagiwara et al., 2000; Nguyen et al., 2007). Similar to IgG, cytokine concentrations are greatest in colostrum collected immediately postpartum and decrease in subsequent mammary secretions (Hagiwara et al., 2000). The source of colostrum cytokines is disputed. They could be transported into the mammary gland via transcytosis, similar to IgG (Baumrucker et al., 2010). They could also be secreted by immune cells present in colostrum (Liebler-Tenorio et al., 2002; Hagiwara et al., 2008). The role of these cytokines in the neonatal calf is not well defined at present. Data from several species suggest that neonates are able to absorb cytokines from colostrum (Goto et al., 1997; Hagiwara et al., 2001; Nguyen et al., 2007). One experiment reported greater leukocyte proliferation and increased production of reactive oxygen species by neutrophils in newborn calves that received an oral dose of IL1B (Hagiwara et al., 2001). The same group also demonstrated greater cytokine production

32 21 (IL2) and receptor expression on peripheral lymphocytes from neonatal calves following exposure to IL1B, TNF, and IFNG than when lymphocytes were exposed to antigen without prior cytokine exposure (Yamanaka et al., 2003). Lymphocytes from adult cattle exhibited the same response with and without prior cytokine exposure (Yamanaka et al., 2003). Similar results are reported from studies with human babies (Bessler et al., 1996). All of these cytokines are present in bovine colostrum and are likely absorbed during the first day postpartum (Goto et al., 1997; Hagiwara et al., 2000). Additional research is needed to determine the extent of absorption and characterize the function of colostrum cytokines in the neonate. The effect of colostrum heat treatment on cytokine absorption and function and potential ramifications on calf immune development must be investigated. Conclusions Approximately 6% of dairy farms, representing 20% of heifer calves, in the United States heat treat colostrum prior to feeding calves (NAHMS, 2016). As use of this practice continues to grow it is important to determine the mechanism by which heat treatment enhances IgG absorption. Specifically, it is necessary to determine whether situations exist when this does not occur. Additionally, research continues to reveal physiological roles for various non-ig components of colostrum. Studies are needed to determine the effects of heat treatment on these components, including potential ramifications on calf development. To address these research needs, experiments were conducted to address the following specific aims:

33 22 1. Determine whether similar increases in plasma IgG and absorption efficiency were observed when calves received heat-treated or unheated colostrum of varying IgG content; 2. Conclusively determine the combined effects of colostral bacterial content and heat treatment on IgG absorption in calves; and 3. Characterize immune responses in calves that received heat-treated or unheated colostrum in terms of growth, body temperature, and blood IFNG, IL1B, TNF and IgG concentrations; and to determine calves ability to absorb IFNG, TNF, and IL1B from heat-treated and unheated colostrum.

34 23 References Abd El-Fattah, A. M., F. H. R. Abd Rabo, S. M. El-Dieb, and H. A. Satar El-Kashef Preservation methods of buffalo and bovine colostrum as a source of bioactive components. Int. Dairy J. 39: Aderem, A., and D. M. Underhill Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17: Aragona, K. M., C. E. Chapman, A. B. Pereira, B. J. Isenberg, R. B. Standish, C. J. Maugeri, R. G. Cabral, and P. S. Erickson Prepartum supplementation of nicotinic acid: Effects on health of the dam, colostrum quality, and acquisition of immunity in the calf. J. Dairy Sci. 99: Baumrucker, C. R., and R. M. Bruckmaier Colostrogenesis: IgG(1) Transcytosis Mechanisms. J Mammary Gland Biol. 19: Baumrucker, C. R., A. M. Burkett, A. L. Magliaro-Macrina, and C. D. Dechow Colostrogenesis: Mass transfer of immunoglobulin G(1) into colostrum. J. Dairy Sci. 93: Benjamin, A. L., F. T. Korkmaz, T. H. Elsasser, and D. E. Kerr Neonatal lipopolysaccharide exposure does not diminish the innate immune response to a subsequent lipopolysaccharide challenge in Holstein bull calves. J. Dairy Sci. 99: Berger, A Th1 and Th2 responses: what are they? BMJ. 321:424. Bessler, H., R. Straussberg, J. Hart, I. Notti, and L. Sirota Human colostrum stimulates cytokine production. Biol. Neonate. 69:

35 24 Bielmann, V., J. Gillan, N. R. Perkins, A. L. Skidmore, S. Godden, and K. E. Leslie An evaluation of Brix refractometry instruments for measurement of colostrum quality in dairy cattle. J. Dairy Sci. 93: Bocci, V., K. Vonbremen, F. Corradeschi, E. Luzzi, and L. Paulesu What is the role of cytokines in human colostrum. J. Biol. Regul. Homeost. Agents. 5: Bonneville, M., R. L. O'Brien, and W. K. Born gamma delta T cell effector functions: a blend of innate programming and acquired plasticity. Nat. Rev. Immunol. 10: Bush, L. J., R. Contreras, T. E. Staley, and G. D. Adams The effect of pasteurization of colostrum on absorption of immune globulins by calves. Pages in Oklahoma Agricultural Experiment Station. Oklahoma State University, Stillwater, OK. Butler, J. E Bovine Immunoglobulins: A Review. J. Dairy Sci. 52: Chase, C. C., D. J. Hurley, and A. J. Reber Neonatal immune development in the calf and its impact on vaccine response. Vet. Clin. North Am. Food Anim. Pract. 24: Chigerwe, M., J. W. Tyler, L. G. Schultz, J. R. Middleton, B. J. Steevens, and J. N. Spain Effect of colostrum administration by use of oroesophageal intubation on serum IgG concentrations in Holstein bull calves. Am. J. Vet. Res. 69: Clark, M. R IgG effector mechanisms. Chem.Immunol. 65: