Unit 1 Stress Response Notes 2025 Jan 20 PDF

Unit 1. Response to Physical, Chemical Can we produce animals that are more and Biological Stressors: resilient to stress to support animal health? Why is animal health so important? This course will review and discuss (Slide 2)...

Unit 1. Response to Physical, Chemical Can we produce animals that are more and Biological Stressors: resilient to stress to support animal health? Why is animal health so important? This course will review and discuss (Slide 2) management practices, genetic selection strategies, and immunological approaches Animal health is of paramount that can be used to maintain or improve importance to today’s livestock and animal health, and many of these approaches aquaculture producers. When animals are are focused on minimizing stress. excessively stressed, their health can be negatively impacted, and this can manifest We need a basic understanding of as reduced production and product quality, neurobiology and immunology, and to be and increased risk of disease. aware of the concept of biomarkers for monitoring animal health before we move Today’s consumers demand high- forward with these topics. quality and reasonably priced products from animals that are raised ethically with Response to stress (Slide 3): minimal stress. Domesticated animals are routinely Many livestock pathogens are also subjected to different stressors; these zoonotic; therefore, maintaining animal include psychological, physical, chemical, health is a good strategy to reduce risk of and biological stressors (i.e. microbes and human disease - “One Health” approach. their toxins). Since the 1950s, producers have Stressor: Something that disrupts become depended on antimicrobials (i.e. homeostasis in an organism, and in doing antibiotics, anthelmintics) to prevent and so, elicits a stress response. treat disease. However, we now know that the wide-spread use of antimicrobials in Stress response: Is an evolutionary the livestock and aquaculture industries conserved response that involves activation has contributed to the development of of multiple physiological systems during antimicrobial resistant (AMR) pathogens. perceived danger; these include the cardiovascular, metabolic, musculoskeletal, Therefore, we need to develop alternate neuroendocrine and immune systems. strategies to maintain or improve animal health. The stress response manifests as “fight”, “flight” or “freeze” (fainting goat example) For the past 30-40 years, animal responses. breeders have focused on improving production traits; however, genetic These goats have congenital myotonia, selection for performance is inversely which is an inherited disorder. The causative correlated with animal health and fertility; gene CLCN1 codes for a chloride channel perhaps, we need to re-evaluate our protein that contains a missense mutation; selection priorities? the amino acid alanine is replaced with a proline residue, making the protein Climate change is expected to adversely dysfunctional. affect the health of livestock and aquaculture species. Perceived fear by these goats causes muscle contraction during the flight response, and muscle locking occurs Resilience to stress (Slide 4): because chloride levels cannot be quickly restored to non-stress levels. These goats A stress resilient animal can resist, cope were selectively bred for in the Southern with (adapt or habituate) and completely USA supposedly because they were recover from (restore homeostasis) unable to escape easily. Maybe not the perceived danger. (i.e. Bamboo bends in a best breeding program for species severe storm but bounces back to its original survival! form after the storm has passed.) Short-term (acute) activation of the Genetics, environment, and epigenetic stress response (i.e. minutes to hours) is mechanisms also determine resilience to designed to enhance survival of an stress. organism by restoring physiological homeostasis. Hence, a little stress can be a An animal that has low stress resilience is good thing! easily overcome by a stressor, or may become sensitized to a stressor, which can Excessive or chronic activation of the lead to hyper-responsiveness to the same stress response however (i.e. several hours stressor, or other stressors, during per day for weeks or months), can lead to subsequent exposure. Either scenario can a variety of psychological, metabolic and lead to disease. reproductive disorders, as well as immunological disorders that increase All animals have the potential to be susceptibility to infectious and overwhelmed by a stressor or combination autoimmune diseases and cancer. of stressors. For a stress response to occur, an Examples of physical, chemical, biological, organism must first sense the stressor; this and psychological stressors (Slide 5): is facilitated through touch, taste, smell, hearing and sight sensory input, the Here is a list of potential physiological, recognition of “non-self” and “danger chemical, biological and psychological signals” by the immune system, and by stressors that may elicit a stress response in communication between the host and gut domestic and companion animals. These microbiome. stressors are likely to vary within and across species and may interact (i.e. environmental This sensory input is relayed to the brain interactions) to elicit the stress response. via neural transmitters, hormones, and cytokines, where it can be perceived as Example 1: Predicted climate-driven danger, which elicits the stress response. changes in spatio-temporal distribution of Haemonchus contortus in European sheep Variation in the stress response is (Slides 6-8): determined by genetics, environmental factors, and by gene-by-environment Slide 7: Q0 is an estimate of potential interactions that are regulated by infection pressure - the average number of epigenetic mechanisms. Variation occurs second-generation mature adult worms at the level of sensory input, stress produced by a single adult worm during its perception, as well as the stress response. lifetime. Note: Spatial and temporal changes in Q0 occur throughout the year in Europe. 2 Slide 7: Representative concentration lived in an apartment, which breed of dog might be pathways (RCP) are different greenhouse most appropriate? gas concentration (GHG) trajectories, where GHG emissions are expected to 1) Border collie, 2) Cavalier King Charles spaniel, peak between 2010-2020 (RCP2.6), 3) Jack Russell terrier, 4) German Shorthaired around 2040 (RCP4.5), around 2080 pointer. (RCP6), or continue to rise throughout the 21st century (RCP8.5). 2. Increasing dairy production in Brazil (Slide 11): Note: Model predictions suggest that Q0 is expected to increase along with increased Q. Which breed of cattle would be most appropriate GHG emissions in Northern Europe. for increasing milk production in Brazil? 1) Guzera, 2) Holstein, 3) Holstein/Gir cross, 4) Gir. Example 2: Risk of viral infection is 10 X greater in the morning (Slide 9): 3. Increasing helminth resistance in Canadian sheep (Slide 12): -Time of day regulates herpes and influenza A virus progression in mice Q. If you were faced with the challenge of increasing resistance of sheep to gastrointestinal -Viral infection worsens when circadian parasites, which genetics could be introgressed into rhythm is abolished by disruption of a key Canadian flocks? 1) Merino, 2) Rambouillet, 3) circadian clock gene encoding the Gulf Coast, 4) Suffolk. transcription factor BMAL1- this has implications for “jet lag” during travel, Q. How could this be achieved? and for adjusting light: dark cycles. -Cross breeding/gene editing -Human BMAL1 levels in blood have 4. Choosing a fish species for an aquaponics seasonal variation and are lowest in winter company (Slides 13-14): months- implications for increased risk of infection during winter. Q. If you were looking for a fish species that will grow well in warm greenhouse water, which -Some viruses can differentially target species might be most appropriate? 1) Largemouth components of the molecular circadian bass, 2) Arctic Char, 3) Rainbow trout, 4) Tilapia. clockwork for their own gain. Q. Are our livestock and aquaculture species Matching animals to their environment matched appropriately to current farming (Slides 10-17): practices? (Slide 15): When it comes to animal production and Example: The changing environment of laying management, it is critical that an animal hens (Slide 16): be appropriately matched both genetically and epigenetically with its environment, Chickens have gone from free-range to battery because a miss-match can lead to stress cage housing, and now to either “enriched” avery that may increase risk of disease. housing systems, or free-range organic systems during the past 100 years! 1. Choosing a pet for your apartment (Slide 10): Q. How has this impacted them? Q. If you were looking for a pet to help cope with -bone fractures, parasitic infections another lockdown for your young family, and you 3 Q. Are exotic species such as water buffalo, 4. Specific or not? ostrich, and new aquaculture species matched to 5. Systemic versus local? current intensive farming systems? (Slide 17: 6. Repeatable 7. Potential for high-throughput analysis Q. Are endangered species that have been re- 8. Economical introduced into the wild from zoos/aquariums 9. Assay developed appropriately matched to the wild for optimal 10. Real-time precision monitoring survival? (Slide 17): Physiology of the neuroendocrine stress response: In addition to making sure that animals are appropriately matched to their environment, there Conventional thinking is that the stress response are many management practices that can be used starts at the level of the brain, where two different to minimize animal stress, and maximize stress stress axes, the sympathetic-adrenal-medullary resilience, and we will focus on some of these (SAM) axis and hypothalamic-pituitary-adrenal- throughout the course. cortex (HPA) axis, are activated as part of the physiological response to the perceived danger. Before we proceed, it is imperative that we understand the physiology of the stress response. The SAM axis is immediately activated This understanding will help us to identify (seconds) during stress and the peak response biological markers (biomarkers) of stress, which occurs within minutes, whereas activation of the are needed to monitor stress levels in animals. HPA axis is delayed (minutes) and peaks within hours. Q. How do we know that an animal is stressed? (Slide 18): Note: In terms of research, we often investigate the SAM and HPA axes independently during the -Increased flock/herd mortality stress response; however, there is bidirectional -Weight loss communication between these axes during -Reduced fertility activation and inactivation of the stress response, -Clinical disease as they both share the adrenals. -Behavioral changes (i.e. anorexic, recumbent, aggressive, self-destructive, For example, immediate activation of the SAM learned helplessness) axis during stress contributes to adrenal cortex glucocorticoid (GC) secretion, and stress-induced These endpoints are either non-specific, GCs can also regulate the secretion of or difficult to measure, may not be catecholamines from the adrenal medulla. sensitive enough to provide early detection, or detection technology is not Sympathetic-adrenal-medullary (SAM) axis yet reliable or developed. (Slide 21): Q. Is there a biomarker of stress? (Slide 19): The SAM axis initiates the “fight, flight and freeze” responses to stress, which involve changes Some potential criteria for biomarkers of in neuroendocrine and immune function that can stress (Slide 20): affect animal behavior, metabolism, cardiovascular and musculoskeletal functions, 1. Easy to obtain tissues (i.e. blood, urine, respiration, and disease resistance. saliva, milk, feces, tissue biopsy) 2. Sampling should cause minimal This is achieved by the immediate release of discomfort to animal acetylcholine (Ach) from sympathetic nerve fibers 3. Sensitive that innervate the adrenal medulla (splanchnic 4 nerve). Ligation of Ach to adrenal chromaffin -Bioactivity is short-lived (T1/2= minutes), in part, cell nicotinic Ach receptors (NiAchR) triggers because enzymes (i.e. catechol-O- the secretion of vesicles into the circulation that methyltransferases and monoamine oxidases) contain the catecholamines epinephrine (EPI) quickly inactivate catecholamines. and some norepinephrine (NEPI). The adrenals are the primary source of basal and stress- -Tissue-specific expression of α and β adrenergic induced circulating EPI, whereas, circulating receptors can also affect catecholamine NEPI primarily comes from other tissues. The bioactivity in target tissues. contribution of EPI versus NEPI to physiological and behavioral changes during the Hypothalamic-pituitary-adrenal axis (Slide 23): stress response varies across species and tissues. Activation of the HPA axis begins with the Catecholamines mediate their physiological secretion of corticotrophin releasing hormone responses via tissue- and cell-specific (CRH) and other hormones with similar and/or expression of α and β adrenergic receptors. synergistic functions (i.e. arginine vasopressin (AVP) and urocortin) from neurons within the Basal concentrations of circulating paraventricular nucleus (PVN) of the catecholamines vary throughout the day and hypothalamus. These neuropeptides are season because they are under the influence of transported to the pituitary via neurons that project hormones such as melatonin (pineal gland), into the posterior pituitary, and by the portal adrenocorticotropin hormone (ACTH, anterior blood. Within the anterior pituitary, CRH can bind pituitary), GC (adrenal cortex), and gonadal to high affinity CRH receptor (CRH-R1) on steroids. corticotroph cells. Synthesis of Epinephrine (Slide 22): CRH binding to CRH-R1 initiates the production of adrenocorticotropin hormone (ACTH) from NEPI and EPI are synthesized from L-tyrosine proopiomelanocortin (POMC), as well as POMC by adrenal chromaffin cells through an gene transcription; an enzyme called pro-hormone enzymatic pathway, which ends with the convertase 1 (PC1) is responsible for cleavage of conversion of NEPI to EPI via the enzyme the pro-hormone POMC into bioactive ACTH; phenylethanolamine N-methyltransferase ACTH is then secreted into the circulation, and (PNMT). subsequently binds to melanocortin 2 receptors (MC2R) on adrenal cells within the adrenal Since catecholamines are so physiologically cortex. influential, circulating concentrations are tightly regulated by the following mechanisms: Steroidogenesis of glucocorticoids (Slide 24): -Rate of catecholamine synthesis is regulated by Ligation of ACTH with MC2R triggers adrenal enzyme activity and expression levels (i.e. steroidogenesis, which involves the conversion of PNMT: various types of stressors can also free cholesterol by cytochrome P450 (CYP) differentially affect PNMT mRNA and protein isozymes and 3β-hydroxysteroid dehydrogenase expression, as well as PNMT activity. (3β-HSD) into GC; corticosterone in birds and rodents, and cortisol in fish and domesticated and -Catecholamine bioavailability is regulated by companion mammals. different plasma binding proteins. Only 50% is bioavailable in human serum. GC such as cortisol elicit a range of physiological responses, including altering metabolism (i.e. inducing hepatic gluconeogenesis, deposition/metabolism of 5 lipids, muscle protein catabolism), altering behavior, and regulating the innate and -Under normal circumstances, circulating acquired immune responses to help deal ACTH and GC concentrations are regulated with the stressor and restore homeostasis. throughout the day and season by circadian clock genes that are expressed within the GC mediate their physiological effects suprachiasmatic nuclei (SCN) of the by binding to high affinity hypothalamus- located just above the optic mineralocorticoid receptors (MR) and chiasma. These central clock genes are low affinity glucocorticoid receptors (GR) responsive to environmental cues such as that can act as nuclear transcription light and feeding cycles, and the balance of factors for unique sets of genes. stimulatory and inhibitory clock gene expression has been shown to regulate CRH Expression levels of MR and GR, and secretion from the PVN. In diurnal species, their ratio (MR:GR), influence GC this allows for high early morning and low bioactivity within different tissues. evening GC concentrations; the opposite occurs in nocturnal species. This increase in Since GC can affect many physiological GC helps the body meet fluctuating energy processes, GC concentration must also be requirements throughout the day/night and tightly regulated: season. Role of MR and GR in maintaining GC Other signals influencing adrenal GC functions (Slide 25): secretion: -Circulating hepatic binding proteins, -Sympathetic signaling from the SCN via including cortisol-binding globulin the splanchnic nerve has been shown to (CBG) as well as albumin (ALB), help provide rapid light/dark signals to the regulate the bioavailability of circulating adrenals, which leads to GC secretion GC. These binding proteins sequester 95% independent of ACTH. of GC during non-stress states, which means that only 5% of GC is normally -Circadian cycling of catecholamine bioactive. During stress, stored CBG is secretion, which is under the influence of released to help buffer increasing GC peripheral circadian clock genes expressed levels; however, when CBG becomes within the adrenal medulla, likely also saturated with GC, circulating influences adrenal GC cycling, since cycling concentrations of bioactive GC are is retained ex vivo during cell culture. dramatically increased. Circadian cycling of GC and disease: Hypothalamic-pituitary-adrenal axis continued (Slide 26): Since circadian cycling of adrenal GC subsequently contributes to the cycling of -Tissue GC concentrations are also locally gene products in other tissues (i.e. liver, regulated by the expression of isozymes lymphoid tissues, brain), it is not surprising 11β-HSD2 (abundant in placenta, testis, that disrupted adrenal circadian signalling kidney and brain) and 11β-HSD1 has been associated with many human (abundant in the liver, adipose tissue and psychological disorders (post-traumatic brain); these isozymes respectively stress disorder, bipolar disorder, major catalyze the conversion of bioactive depression, schizophrenia, seasonal cortisol into inactive cortisone, and vise- affective disorder), metabolic (obesity, Type versa. 2 diabetes) and immune disorders (lupus, 6 rheumatoid arthritis, multiple sclerosis, Proposed biomarkers of the stress response (Slide autoimmune thyroiditis, asthma), and 28): cancer (breast, lung, ovary and kidney). Since sensory input and stress perception Negative feedback signaling within the are difficult to quantify, emphasis has been HPA axis during circadian cycling and placed on identifying direct or indirect stress (Slide 27): biomarkers of the stress response. Since MR are the high affinity GC Direct Biomarkers: receptors, their contribution to circadian cycling in HPA tissues is greater than GR. 1. Catecholamines: During acute stress however, when MRs -Most often measured in blood, and to a become saturated with GC, GC-mediated lesser extent in urine. physiological responses are primarily the result of GC ligation with GRs. -In-dwelling catheters are required for real- time blood sampling due to their rapid Circadian and stress-induced release during stress, their very short T1/2 in concentrations of GC are also regulated by circulation, and sensitivity to animal negative feedback signals within the HPA handling (acute stress biomarker). axis. -Levels in blood likely don’t represent regional MR participate in negative feedback tissue differences in sympathetic activity. signaling during day/night circadian cycling, and MR expression levels -As of yet, there is no universally accepted determine sensitivity to stress. MR are methodology and assay sensitivity varies: High highly expressed within the hippocampus Performance Liquid Chromatography (HPLC) and decreased hippocampal MR versus tandem mass spectrometry (MS/MS) with expression has been associated with liquid chromatography (LC), versus Enzyme- elevated levels of plasma ACTH and GC. Linked Immunosorbent Assay (ELISA). GR participate in negative feedback 2. Glucocorticoids: signaling in response to stress. These -Most widely used biomarker of the stress negative feedback signals occur at the response- most stable and easy to get a tissue level of the hypothalamus, pituitary, and sample. hippocampus, where GR expression is high. -Influenced by day/night/season, so a sampling protocol is essential. During chronic stress, MR and GR expression levels become attenuated. This -Decreases with age (i.e. for pigs, cortisol stabilizes can adversely affect HPA axis negative around 20 weeks of age and is about 40% lower feedback signaling and is associated with than at 12 weeks of age). increased basal GC concentrations and sustained increases in GC concentration -Gender differences (barrows 15% > gilts). during stress. -Is elevated at parturition in some species (i.e. sheep) -Total cortisol (blood- acute stress biomarker) versus free bioactive cortisol (saliva- acute stress 7 biomarker, feces and hair -chronic stress biomarker). -Is not affected by age, gender, or day/night/ season. -Circulating GC concentrations may not represent GC concentrations in regional tissues and vice -Is being used as an acute stress biomarker in pigs. versa. -ELISA versus RIA -Non-adrenal sources of GC (i.e. skin, intestines, placenta, adipose tissues, leukocytes etc.) 5. Blood pressure: -Invasive measurement of arterial blood pressure -Temporal response requires multiple requires surgical catheter implantation. sampling….$. -Blood pressure cuff can be placed over metacarpal -ELISA versus Radio Immune Assay (RIA- artery, but this procedure requires anesthetic, and is greatest sensitivity) not as reliable as the invasive method described above. -Different sampling protocols are required for acute versus chronic stress assessment. 6. Heart Rate (Slide 29): -Heart rate monitors can now be used in precision 3. ACTH: agriculture to measure sympathetic tone of -Influenced by day/night/season. livestock in real-time. Their reliability is currently under review, although the harness apparatus is -Changes with age? problematic for free ranging livestock. -Gender differences? -Acute stress monitor? -Circulating concentrations likely don’t represent Physiology of the immune response to microbial tissue ACTH concentrations. stressors: -Acute stress biomarker (blood). Body defense (Slide 30): -Chronic stress biomarker? For an animal to be able to maintain physiological homeostasis in an -Non-pituitary sources of ACTH (skin, environment rich with pathogenic bacteria, leukocytes) fungi, parasites, and viruses (microbial stressors), it must be able to do the -ELISA versus RIA following: Indirect Biomarkers: 1. Prevent pathogen entry into the body using physical (i.e. skin, mucous) and 4. Chromogranin A: physiological barriers (i.e. pH, mucous, -Is secreted by adrenal medulla chromaffin cells temperature). along with EPI and NEPI, but also by anterior pituitary corticotrophs. 2. Recognize the invading pathogen as non- self if barriers are breached (sensory -Is more stable than EPI and NEPI, and in perception of danger). humans, saliva chromogranin A is considered a reliable indicator of increased sympathetic tone - correlates well with NEPI concentration. 8 3. Restore homeostasis by responding Immunoregulation and disease (Slide 31): appropriately to eliminate the pathogen (stress response). The immune system is comprised of two arms: the innate and acquired (adaptive) If these are achieved, the animal will immune system. The innate immune system remain productive and develop enhanced is ancient, highly conserved across species, long-term protection against the pathogen. and is the first to be activated in response to microbial stressors. If the animal is not successfully able to eliminate a pathogen, disease will occur. Immunity: Is the protection provided by the This is a welfare issue, it can affect immune system to resist microbial infection. productivity and possibly lead to death, Humoral proteins and cells making up the and may be a human health issue. innate and acquired immune system are the effectors providing this protection. Disease: An illness or condition that prevents the body or mind from working Just as the SAM and HPA axes are tightly normally. (Merrian-Webster defn.) regulated, the innate and acquired immune responses are tightly self-regulated and are Animals also host commensal microbial also regulated by signals from the populations that reside on the epithelium neuroendocrine system (i.e. SAM, PNS, and of the skin and at mucosal surfaces of the HPA axes) to ensure that these immune gastrointestinal, respirator, and urogenital responses can effectively restore tracts. Under normal conditions, the host homeostasis. learns to tolerate these microbes because they have an important symbiotic If the innate and acquired immune relationship with the host: responses are attenuated, an animal will not be able to effectively eliminate pathogens or 1. The host provides nutrients and an tumor cells. environment that supports microbe survival. In contrast, excessive and prolonged innate or acquired immune responses can result in 2. The microbes make certain nutrients extensive host tissue damage and can lead to available to the host and help protect a variety of disorders including: against invading pathogenic microbes. 1. Acute or chronic inflammatory disease. If tolerance to these microbes does not develop normally at an early age, or 2. The development and proliferation of tolerance is broken during the animal’s tumor cells. life, this can also lead to disease. 3. Sensitization to environmental antigens The ability of animals to recognize and that results in atopic disease (i.e. atopic respond to pathogens as non-self and dermatitis, asthma, food allergy, hay fever). tumor cells as modified-self and to tolerate non-self commensal microbes is 4. Sensitization to self-antigens that may facilitated by the immune system. lead to autoimmune disease. 9  Three major layers of host defense (Slide th The immune system- our 6 sensory 32): system: Innate immunity The immune system constitutes our sixth sensory system that alerts the central The innate immune response manifests as nervous system (CNS) to microbial the host inflammatory response, and the danger. During microbial invasion for secretion of the pro-inflammatory cytokines example, surveillance cells of the immune TNFα, IL-1 and IL-6 is triggered by the system release signaling molecules called recognition of danger signals by soluble and cytokines (i.e. TNFα, IL-1 and IL-6) as cell membrane pattern-recognition receptors well as various neuropeptides that bind to (PRRs). their respective receptors on neural cells within the gut, skin, liver/spleen, These danger signals include highly respiratory and urogenital tracts and brain. conserved microbial-associated molecular These neural cells become activated and patterns (MAMPs) making up microbial subsequently alert the CNS to danger. membranes, and host alarm signals (alarmins) that are released by damaged or These danger signals are perceived activated host cells. within the brain, and the CNS responds with neural (i.e. SAM, PNS) and While the innate immune response can be endocrine (i.e. HPA) signals that help highly effective at controlling and regulate the immune response to ensure its eliminating pathogens, it has limited effective, but not excessive. Bidirectional specificity, and does not provide long-term communication among these systems enhanced protection against pathogens - occurs within what is collectively referred referred to as immunological memory. to as the neuroendocrine-immune system. The host inflammatory response is The release of pro-inflammatory typically localized to site(s) of infection so cytokines (TNFα, IL-1 and IL-6) during that tissue damage is minimized. However, immune system activation affects many during a severe infection with a highly different tissues, and the systemic virulent pathogen, localized immunological response is referred to as the acute-phase defenses may not be sufficient to contain the response. infection. Dissemination of the pathogen into the circulation to other tissues can In the hypothalamus for example, these trigger a potentially damaging condition cytokines elicit a fever response, and IL-1 referred to as Systemic Inflammatory induces sickness behaviour (i.e. sleepiness Response Syndrome (SIRS). In extreme and anorexia). cases, SIRS can lead to sepsis, organ failure and death. If an animal survives SIRS, it is In the muscle, these cytokines cause typically immunocompromised and is at protein catabolism, which mobilizes high risk to secondary infections for amino acids that are used to make host months-years. defense and tissue repair proteins. Acquired immunity Many of these proteins are synthesized by the liver and are referred to as hepatic For long-term immunity to occur, the host acute-phase proteins (APP). must mount an acquired immune response that is highly specific and has capacity for 10 immunological memory and increased 1. Phagocytosis and killing of the microbe efficiency. This acquired immune response is triggered by the recognition of 2. Processing microbial proteins into microbial antigens (usually proteins) by: peptides 1. Immunoglobulins; which include 3. Migration from the site(s) of infection to membrane B cell receptors (BCR) on B- secondary lymphoid tissues (i.e. lymph lymphocytes (B cells) and secreted nodes, Peyer’s patches, spleen etc.) antibodies that are produced by terminally differentiated B cells referred 4. Up-regulated expression of membrane to as plasma cells. bound antigen receptors called major histocompatibility complex molecules 2. T cell receptors (TCR) on T- (MHC) that are loaded with the processed lymphocytes (T cells). microbial peptide antigens. The acquired immune response can 5. Presentation of microbial antigens to manifest as a humoral antibody response antigen-specific T cells via MHC-TCR (AbMIR) that targets extracellular interactions; this is referred to as antigen pathogens and their toxins, or a cell- presentation. mediated immune response (CMIR) that targets intracellular pathogens. Unique These antigen-specific T cells become cytokines drive these polarized immune activated to proliferate and terminally responses. For example, IL-4 and IL-13 differentiate into the effector T cells of the steers AbMIR, whereas IFNg steers acquired immune system (i.e. cytotoxic T CMIR. cells). Essential features of the acquired Antigen-specific B cells may also immune response (Slide 33): recognize the microbial antigens, and with the help of an antigen-specific T helper cell, Although the acquired immune response these B cells may become activated to is more effective than the innate immune proliferate and terminally differentiate into response for controlling and/or the effector B cells (antibody-secreting eliminating pathogens, it takes longer for plasma cells). the host to mount (day-to-weeks as opposed to minutes-to-hours) because a Memory B and T cells are also generated higher level of immune system during the acquired immune response, and orchestration is required to elicit an these facilitate long-term immunity. acquired immune response, which also involves the innate inflammatory The innate immune response provides an response. immediate level of protection that buys time for the more effective acquired immune For an acquired immune response to response to occur and is also required for an occur for example, there must be effective acquired immune response to sufficient inflammation to activate occur. surveillance cells of the innate immune system to carry out the following activities: 11 Immunological biomarkers of the stress Examples: response to microbial stressors (Slide 34): -Bovine IgG1 (AbMIR) versus IgG2 (CMIR) in blood; IgG1 is the predominant immunoglobulin Hundreds of immune-related proteins are isotype found in bovine milk. produced during the innate and acquired immune responses to microbial stressors and may serve as -Bovine mucosal IgA and IgM biomarkers of microbial stress. -Bovine IgE (very short half-life in circulation) Biomarkers of acute microbial stress: Q. Should we measure basal or inducible 1. Cytokines: Innate pro-inflammatory cytokines antibodies? (TNFα, IL-1 and IL-6). -Basal antibody levels may be around the assay 2. Hepatic APPs: Many immune-related APPs are limit of detection (i.e. ELISA). produced during the acute-phase response to microbial infection (i.e. serum amyloid A, C- -AbMIR can be induced using a novel antigen or reactive protein, haptoglobin, and certain commercial vaccine to assess the capacity to mount complement proteins). These APPs, however, an immune response. lack microbial specificity and are also induced by GC in response other types of stressors. Q. Should we measure total or antigen-specific antibody levels? 3. Temperature: Febrile responses are easy to monitor but are not always elicited in response to Antibody response to antigen challenge (Slide 36): microbial stress. The kinetics of the host antibody response varies Biomarkers of chronic microbial stress: depending on antigen concentration, antigen half- life, number of antigen exposures and antigen exposure duration. 1. Cytokines: Cytokines driving AbMIR (IL-4 and IL-13), or CMIR (IFNg). Factors confounding antibody production (Slide 37): 2. Antibodies: Antibodies have microbial specificity, are highly sensitive and potentially Antibody production may be confounded by long-lasting indicators of microbial exposure, immune health status. which also makes them ideal for diagnosis of specific diseases. Examples: Q. Which antibody type (isotype) would be most -Acute exposure to different types of stressors can appropriate to measure as a biomarker of stimulate immune cell trafficking to support the microbial stress? (Slide 35) host immune response in the skin and at mucosal surfaces - this could enhance vaccine-induced -There are different antibody isotypes and sub- antibody production. isotypes (i.e. bovine IgM, IgG1/2, IgA). -Chronic exposure to different types of stressors -Antibody isotypes are species- and tissue- causes immunosuppression® attenuated antibody specific production. 12 -Some microbes (i.e. viruses) can modulate the -Biological -acidosis, sleep deprivation, intense host immune system to evade detection (i.e. exercise, pathogen challenge suppress AbMIR or CMIR); this could affect When the stressor is of microbial origin, a panel disease diagnosis as well as vaccine efficacy. of immune system biomarkers is typically used to assess the innate and acquired (AbMIR and Stages of Bovine Johne’s disease (JD) caused by CMIR) immune responses. Antibody levels are Mycobacterium avium subsp. paratuberculosis most commonly measured due to ease of (MAP) (Slide 38): sampling, and their specificity makes them extremely useful for diagnostic testing of specific Seroconversion takes time, which means early microbial infections. diagnosis of JD is difficult. Also, the host immune response may change depending on the Genetics and epigenetics of stress (Slide 3): stage of infection, which means a biomarker panel may be necessary for diagnosis of JD. Variation in gene activity is controlled at the level of the genome and epigenome: Summary of biomarkers of stress response: Genetic variants predetermine gene There are many potential biomarkers of the activity and are permanent and inherited by stress response. There are pros and cons to each offspring (i.e. single nucleotide biomarker, and there is no single biomarker that polymorphisms (SNP). can be used to assess responsiveness to all types of stressors. Therefore, a panel of direct and/or Epigenetic variants are established by indirect biomarkers, reflecting the SAM and environmental quos during mitosis and HPA responses, should be used to assess acute provide an adaptive mechanism for stress response. individual phenotypic change. Since epigenetic modifications may be inherited, In the case of chronic stress, it may be they can also contribute to phenotypic necessary to administer a stressor to assess an variation within populations. animal’s resilience to stress. This could be achieved by subjecting an animal to a highly Genetics of stress controlled stress regime involving one or more stressors. Once the stressor is administered, the As discussed previously, genetics likely SAM, HPA or immune responses can be contributes to variation in sensory input, monitored over time to assess stress resistance, stress perception, as well as the stress habituation, recovery or sensitization. response. Since the stress response is easiest to assess, it is the preferred phenotype for genetic selection. Examples of highly controlled stress regime: Blood cortisol level for example, is a -Psychological -isolation, re-grouping phenotype of moderate-to-high heritability. Heritability estimates (H2) have been found -Physical -heat, cold, transport, noise, electrical, to vary across species and are influenced by restraint time of day and type of stressor. -Chemical -fungal mycotoxins or bacterial Examples: toxins (MAMPs-lipopolysaccharide), pro- inflammatory cytokines, neuropeptides (i.e. -Human twin study estimates of H2 for CRF, AVP, ACTH) cortisol at awakening (0.28), 30 min after awakening (0.60), and in the evening (0.08). 13 Gustafsson PA et al. Twin Res Hum package DNA as chromatin (DNA Genet 2011: 14: 553. wrapping) within the nucleus. Histone proteins allow for chromatin -Porcine cortisol response 60 min post unwinding/winding, which allows/restricts ACTH challenge (0.68). Larzul C et al. transcription factor accessibility to genes. 2015: 9: 1929. -Histone tails are susceptible to -Barn swallow basal corticosterone (0.15) modifications (i.e. methylation, acetylation and handling stress-induced corticosterone and phosphorylation) by various enzymes. (0.34). Jenkins BR et al. 2014 Proc Bio These modifications alter the 3-dimensional Sci 2014: 281. configuration of chromatin, which subsequently affects accessibility of -Rainbow trout cortisol response (0.41) to transcription factors and transcriptional 3-hr confinement. Overi O et al. 2005 machinery to the DNA. Integr Comp Biol 45: 465. 3. RNA-based mechanisms such as -Sheep 4-hr cortisol response (0.3) to noncoding RNAs (ncRNA). bacterial endotoxin lipopolysaccharide (LPS) challenge; this MAMP was used to -ncRNA are regulatory gene products that simulate an acute bacterial infection. You do not become translated into proteins. Q et al. 2008 Toxicol Appl Pharmacol 230: 1; You Q et al. 2008 Vet Dermatol -MicroRNA (miRNA) is a single-stranded 2008: 19: 174; Pant SD et al. 2016 ncRNA (< 22 nucleotides) that binds to Livestock Sci 2016: 187: 40. complementary nucleotides (seed region) found within the 3’ untranslated region These moderate-to-high heritability (UTR) of target mRNA. estimates indicate that selective breeding -miRNA binding to mRNA usually results could be performed to improve livestock in either mRNA degradation, or repressed resilience to different stressors. mRNA translation. Ovine HPA response to bacterial LPS All three of these mechanisms greatly challenge (Slide 39): contribute to regulation of genes within the neuroendocrine-immune system, and they Epigenetics of stress (Slide 40): are subject to influence by environmental quos (i.e. stressors). This raises the There are three known mechanisms of possibility that they may be targeted to epigenetic modification that influence improve livestock stress resilience. gene activity: Example heat stress and zebra finch song 1. Methylation of cytosine nucleotides by (Slide 41): DNA methyltransferases -DNA methylation is typically associated -The zebra finch embryo is responsive to changes with transcriptional silencing. in maternal song at higher ambient temperatures® altered reproductive success and 2. Modification of histone proteins thermal preference. -Affects chromatin accessibility by transcription factors. Q. Is this a mechanism that helps prepare the newborn for its new environment? Histones are scaffold proteins that help 14  Normal peak GC levels are detected in the Examples of neuroendocrine-immune genes morning, but they fail to decrease during the subject to epigenetic modifications: evening. 1. A response element, for the transcription -In humans, symptoms include accumulation of factor nerve growth factor-induced protein A, central fat, muscle weakness, skin lesions, bone found within the promoter region of the GR fractures, increased susceptibility to infection gene is subject to DNA methylation. Stress- caused by impaired immune function, induced methylation of this response element hypertension, diabetes, cardiovascular failure and influences negative feedback regulation of the stroke. HPA axis. -In humans, polymorphisms in the ubiquitin- 2. GR levels are also down-regulated by specific protease 8 (USP8) gene, which indirectly miRNA-18 and miRNA-124a binding to the GR contributes to increased ACTH production, are a mRNA 3’ untranslated region (UTR). risk factor for Cushing’s disease. 3. IFN-γ is a key cytokine that promotes a -Also, a mutation inactivating armadillo repeat CMIR against intracellular pathogens. GCs containing 5 (ARMC5), a tumor suppressor gene, induce histone deacetylase, which reduces can cause adrenal hyperplasia associated with accessibility of transcription factors to the Cushing’s disease. promoter region of IFN-γ ® reduced IFN-γ gene expression. Addison’s disease: Is most caused by an autoimmune response against adrenal cells that Responses to stress: the good, bad and ugly: results in atrophy of the adrenal cortex ® underproduction of GC. In most cases this causes The physiological response to stress is a highly loss of negative feedback at the pituitary ® high complex process that is predetermined by POMC production and subsequent ACTH and genetics and is also subject to epigenetic melanocyte-stimulating hormone (MSH) regulation by environmental quos (i.e. production. stressors). -In humans, symptoms include excessive melanin While the stress response is designed to pigmentation, low blood pressure, muscle enhance survival of an organism by restoring weakness, weight loss, fever, anxiety, nausea and physiological homeostasis, excessive or chronic vomiting, muscle and joint pain and mood change. activation of the stress response can lead to a variety of disorders that increase susceptibility -In humans, polymorphisms in genes coding to infectious and autoimmune diseases and immune-related molecules (MHC and CTLA4) and cancer. Risk of these disorders is predetermined genes involved in steroidogenesis (CYP21A2, by genetics and may be influenced by epigenetic CYP17A1, CYP11B1, 3β-HSD2) are risk factors modifications. for Addison’s disease. Genetic variants in neuoendocrine-immune Epigenetic modifications of neuoendocrine- genes contribute to inherited risk of disease: immune genes contribute to life-long risk of disease: Examples: (Slides 42-43) If an animal is not able to recover from stress, Cushing’ disease: Over production of GC, in habituate to stressor, or becomes sensitized to a most cases caused by a pituitary gland tumor stressor because of epigenetic modifications, risk that results in over production of ACTH. 15 of disease is likely to increase. The neuroendocrine-immune system is especially sensitive to epigenetic modifications during late fetal and neonatal development. It is hypothesized that these modifications allow the developing neuroendocrine-immune system to adapt to the novel postnatal environment, which includes developing immunological tolerance to the newly colonized skin and mucosal microbiota. When the epigenome is miss- matched with the animal’s environment, risk of disease is increased. Developmental origins of health and disease (DOHaD) hypothesis (Side 44): David Barker discovered that maternal undernutrition contributes to low birth weight, which is a risk for the development of adult ischemic heart disease, obesity, and diabetes (Thrifty phenotype). The DOHaD hypothesis has expanded to include other stressors such as maternal over nutrition, psychological disorders, changes in the microbiome, and exposure to pathogens and xenobiotics. 16