Unit 1: Stress Response to Physical, Chemical and Biological Stressors

Why is animal health so important

When animals are excessively stressed, their health can be negatively impacted, and this can manifest as reduced production and product quality, and increased risk of disease

Many livestock pathogens are also zoonotic; therefore, maintaining animal health is a good strategy to reduce risk of human disease - “One Health” approach

What are AMR pathogens

However, we now know that the widespread use of antimicrobials in the livestock and aquaculture industries have contributed to the development of antimicrobial resistant (AMR) pathogens.

Therefore, we need to develop alternate strategies to maintain or improve animal health

What is the relationship between genetic selection for performance and animal health and fertility?

For the past 30-40 years, animal breeders have focused on improving production traits; however, genetic selection for performance is inversely correlated with animal health and fertility

How is climate change expected to affect the health of livestock and aquaculture species?

Climate change is expected to adversely affect the health of livestock and aquaculture species. introduction of micro-toxins and weather born diseases

What are biomarkers?

A biomarker (short for biological marker) is an objective measure that captures what is happening in a cell or an organism at a given moment.

What are the different types of stressors?

include psychological, physical, chemical, and biological stressors (i.e. microbes and their toxins)

What is a stressor?

Something that disrupts homeostasis in an organism, and in doing
so, elicits a stress response.

What are some physical stressors?

Heat

cold

climate change

shipping

air quality

water quality

light intensity

injury

stray voltage

UV radiation

processing

noise

restraint/confinement

surgical

predation

What are some chemical stressors?

Mycotoxins (are naturally occurring toxins produced by certain moulds (fungi) and can be found in food)

Heavy metals

organochlorines

poisonous plants

What are some biological stressors?

Nutrient excess

feed restriction

parturition

negative energy balance during lactation

acidosis

inadequate sleep

over training

dehydration

infectious (bacteria, virus, fungi, parasite)

What are some psychological stressors?

Mixing

weaning

herding

showing

What is a stress response?

Is an evolutionary conserved response that involves activation
of multiple physiological systems during perceived danger; these include the cardiovascular, metabolic, musculoskeletal, neuroendocrine and immune systems.
The stress response manifests as “fight”, “flight” or “freeze” (fainting goat example) responses.

Why do some goats faint?

These goats have congenital myotonia, which is an inherited disorder. The causative gene CLCN1 codes for a chloride channel protein that contains a missense mutation; the amino acid alanine is replaced with a proline residue, making the protein dysfunctional. Perceived fear by these goats causes muscle contraction during the flight response, and muscle locking occurs because chloride levels cannot be quickly restored to non-stress levels. These goats were selectively bred for in the Southern USA supposedly because they were unable to escape easily. Maybe not the best breeding program for species survival

What is short term stress otherwise known as?

Short-term (acute) activation of the stress response (i.e. minutes to hours) is
designed to enhance survival of an organism by restoring physiological
homeostasis. Hence, a little stress can be a good thing

What is excessive or long term stress

Excessive or chronic activation of the stress response however (i.e. several hours
per day for weeks or months), can lead to a variety of psychological, metabolic and reproductive disorders, as well as immunological disorders that increase
susceptibility to infectious and autoimmune diseases and cancer.

What happens when a stress response occurs?

For a stress response to occur, an organism must first sense the stressor; this
is facilitated through touch, taste, smell, hearing and sight sensory input, the
recognition of “non-self” and “danger signals” by the immune system, and by
communication between the host and gut microbiome. This sensory input is relayed to the brain via neural transmitters, hormones, and cytokines, where it can be perceived as danger, which elicits the stress response. Variation in the stress response is determined by genetics, environmental factors, and by gene-by-environment interactions that are regulated by epigenetic mechanisms. Variation occurs at the level of sensory input, stress perception, as well as the stress response.

What is resilience to stress?

A stress-resilient animal can resist, cope with (adapt or habituate) and completely recover from (restore homeostasis) perceived danger. (i.e. Bamboo bends in a severe storm but bounces back to its original form after the storm has passed.) Genetics, environment, and epigenetic mechanisms also determine resilience to stress. An animal that has low-stress resilience is easily overcome by a stressor or may become sensitized to a stressor, which can lead to hyper-responsiveness to the same stressor, or other stressors, during subsequent exposure. Either scenario can lead to disease. All animals have the potential to be overwhelmed by a stressor or combination of stressors

What is Q0 (Q naught) ?

Q0 is an estimate of potential infection pressure - the average number of second-generation mature adult worms produced by a single adult worm during its lifetime Note: Model predictions suggest that Q 0 is expected to increase along with increased GHG emissions in Northern Europe

True or false risk of infection is 10X greater in the morning than night time?

True: Time of day regulates herpes and influenza A virus progression in mice. Viral infection worsens when circadian rhythm is abolished by disruption of a key circadian clock gene encoding the transcription factor BMAL1- this has implications for “jet lag” during travel, and for adjusting light: dark cycles. Human BMAL1 levels in blood have seasonal variation and are lowest in winter months- implications for increased risk of infection during winter. Some viruses can differentially target components of the molecular circadian clockwork for their own gain

How do we know that an animal is stressed?

Increased flock/herd mortality
-Weight loss
-Reduced fertility
-Clinical disease
-Behavioral changes (i.e. anorexic, recumbent, aggressive, self-destructive,
learned helplessness)
These endpoints are either non-specific, or difficult to measure, may not be sensitive enough to provide early detection, or detection technology is not yet reliable or developed

What are some of the potential criteria for biomarkers of stress?

1. Easy to obtain tissues (i.e. blood, urine,
saliva, milk, feces, tissue biopsy)
2. Sampling should cause minimal discomfort to the animal
3. Sensitive 4. Specific or not?
5. Systemic versus local?
6. Repeatable
7. Potential for high-throughput analysis
8. Economical
9. Assay developed
10. Real-time precision monitoring

What are the two stress axes?

The sympathetic-adrenal-medullary

(SAM) axis and hypothalamic-pituitary-adrenal-cortex (HPA) axis, are activated as part of the physiological response to the perceived danger.

When does the SAM axis activate?

The SAM axis is immediately activated (seconds) during stress and the peak response occurs within minutes

When does the HPA axis activate?

activation of the HPA axis is delayed (minutes) and peaks within hours.

true or false; there is bidirectional communication between the axes

true, In terms of research, we often investigate the SAM and HPA axes independently during the stress response; however, there is bidirectional communication between these axes during activation and inactivation of the stress response, as they both share the adrenals. For example, immediate activation of the SAM axis during stress contributes to adrenal cortex glucocorticoid (GC) secretion, and stress-induced GCs can also regulate the secretion of catecholamines from the adrenal medulla.

What does the SAM axis initiate?

The SAM axis initiates the “fight, flight and freeze” responses to stress, which involve changes in neuroendocrine and immune function that can affect animal behavior, metabolism, cardiovascular and musculoskeletal functions, respiration, and disease resistance.

How is the fight or flight process achieved?

This is achieved by the immediate release of
acetylcholine (Ach) from sympathetic nerve fibers that innervate the adrenal medulla (splanchnic nerve).

Ligation of Ach to adrenal chromaffin cell nicotinic Ach receptors (NiAchR) triggers the secretion of vesicles into the circulation that contain the catecholamines epinephrine (EPI) and some norepinephrine (NEPI).

The adrenals are the primary source of basal and stress- induced circulating EPI, whereas, circulating NEPI primarily comes from other tissues.

The contribution of EPI versus NEPI to physiological and behavioural changes during the stress response vary across species and tissues

How do catecholamines mediate their physiological responses?

Catecholamines mediate their physiological responses via tissue- and cell-specific expression of α and β adrenergic receptors. Basal concentrations of circulating catecholamines vary throughout the day and
season because they are under the influence of hormones such as melatonin (pineal gland), adrenocorticotropin hormone (ACTH, anterior pituitary), GC (adrenal cortex), and gonadal steroids

Where are NEPI and EPI synthesized from and what by?

NEPI and EPI are synthesized from L-tyrosine by adrenal chromaffin cells through an enzymatic pathway,

How does NEPI convert to EPI?

The conversion of NEPI to EPI via the enzyme phenylethanolamine N-methyltransferase (PNMT)

How is the rate of catecholamine synthesis regulated?

is regulated by enzyme activity and expression levels (i.e. PNMT: various types of stressors can also differentially affect PNMT mRNA and protein expression, as well as PNMT activity

What is catecholamine bioavailability regulated by?

Catecholamine bioavailability is regulated by

different plasma binding proteins. Only 50% is bioavailable in human serum

Why is catecholamine bioactivity short-lived?

Bioactivity is short-lived (T 1/2 = minutes), in part, because enzymes (i.e. catechol-O methyltransferases and monoamine oxidases) quickly inactivate catecholamines. -Tissue-specific expression of α and β adrenergic receptors can also affect catecholamine bioactivity in target tissues

Describe the activation process of the HPA axis

Activation of the HPA axis begins with the
secretion of corticotrophin releasing hormone (CRH) and other hormones with similar and/or synergistic functions (i.e. arginine vasopressin (AVP) and urocortin) from neurons within the paraventricular nucleus (PVN) of the hypothalamus. These neuropeptides are transported to the pituitary via neurons that project into the posterior pituitary, and by the portal blood. Within the anterior pituitary, CRH can bind
to high affinity CRH receptor (CRH-R1) on
corticotroph cells. CRH binding to CRH-R1 initiates the production of adrenocorticotropin hormone (ACTH) from
proopiomelanocortin (POMC), as well as POMC gene transcription; an enzyme called pro-hormone convertase 1 (PC1) is responsible for cleavage of the pro-hormone POMC into bioactive ACTH;
ACTH is then secreted into the circulation, and subsequently binds to melanocortin 2 receptors (MC2R) on adrenal cells within the adrenal cortex

Describe the steroidogenesis of glucocorticoids

Ligation of ACTH with MC2R triggers adrenal steroidogenesis, which involves the conversion of free cholesterol by cytochrome P450 (CYP)
isozymes and 3β-hydroxysteroid dehydrogenase 3β-HSD) into GC; corticosterone in birds and rodents, and cortisol in fish and domesticated and
companion mammals

What are the physiological responses of GC? (CORTISOL)

GC such as cortisol elicit a range of physiological responses, including altering metabolism (i.e. inducing hepatic gluconeogenesis, deposition/metabolism of lipids, muscle protein catabolism), altering behavior, and regulating the innate and acquired immune responses to help deal with the stressor and restore homeostasis

How do GC mediate their physiological effects?

GC mediate their physiological effects by binding to high affinity

mineralocorticoid receptors (MR) and low affinity glucocorticoid receptors (GR)

that can act as nuclear transcription factors for unique sets of genes.

Expression levels of MR and GR, and their ratio (MR:GR), influence GC

bioactivity within different tissues. Since GC can affect many physiological

processes, GC concentration must also be tightly regulated

Whats the Role of MR and GR in maintaining GC functions

Circulating hepatic binding proteins, including cortisol-binding globulin

(CBG) as well as albumin (ALB), help regulate the bioavailability of circulating

GC. These binding proteins sequester 95% of GC during non-stress states, which

means that only 5% of GC is normally bioactive. During stress, stored CBG is

released to help buffer increasing GC levels; however, when CBG becomes

saturated with GC, circulating concentrations of bioactive GC are dramatically increased.

Tissue GC concentrations are locally regulated by…

Tissue GC concentrations are also locally

regulated by the expression of isozymes

11β-HSD2 (abundant in placenta, testis,

kidney and brain) and 11β-HSD1

(abundant in the liver, adipose tissue and

brain); these isozymes respectively

catalyze the conversion of bioactive

cortisol into inactive cortisone, and vise-

versa.

Under normal circumstances, circulating ACTH and GC concentrations are regulated throughout the day and season by

circadian clock genes that are expressed within the

suprachiasmatic nuclei (SCN) of the

hypothalamus- located just above the optic

chiasma. These central clock genes are

responsive to environmental cues such as

light and feeding cycles, and the balance of

stimulatory and inhibitory clock gene

expression has been shown to regulate CRH

secretion from the PVN. In diurnal species,

this allows for high early morning and low

evening GC concentrations; the opposite

occurs in nocturnal species. This increase in

GC helps the body meet fluctuating energy

requirements throughout the day/night and

season

what are the Other signals influencing adrenal GC secretion

Sympathetic signaling from the SCN via

the splanchnic nerve has been shown to

provide rapid light/dark signals to the

adrenals, which leads to GC secretion

independent of ACTH.

-Circadian cycling of catecholamine

secretion, which is under the influence of

peripheral circadian clock genes expressed

within the adrenal medulla, likely also

influences adrenal GC cycling, since cycling

is retained ex vivo during cell culture.

explain the Circadian cycling of GC and disease

Since circadian cycling of adrenal GC

subsequently contributes to the cycling of

gene products in other tissues (i.e. liver,

lymphoid tissues, brain), it is not surprising

that disrupted adrenal circadian signalling

has been associated with many human

psychological disorders (post-traumatic

stress disorder, bipolar disorder, major

depression, schizophrenia, seasonal

affective disorder), metabolic (obesity, Type

2 diabetes) and immune disorders (lupus,

rheumatoid arthritis, multiple sclerosis,

autoimmune thyroiditis, asthma), and

cancer (breast, lung, ovary and kidney)

Negative feedback signaling within the

HPA axis during circadian cycling and

stress

Since MR are the high affinity GC

receptors, their contribution to circadian

cycling in HPA tissues is greater than GR.

During acute stress however, when MRs

become saturated with GC, GC-mediated

physiological responses are primarily the

result of GC ligation with GRs.

Circadian and stress-induced

concentrations of GC are also regulated by

negative feedback signals within the HPA

axis.

MR participate in negative feedback

signaling during

day/night circadian

cycling, and MR expression levels

determine sensitivity to stress. MR are

highly expressed within the hippocampus

and decreased hippocampal MR

expression has been associated with

elevated levels of plasma ACTH and GC

GR participate in negative feedback

signaling in response to…

stress. These

negative feedback signals occur at the

level of the hypothalamus, pituitary, and

hippocampus, where GR expression is

high.

During chronic stress, MR and GR

expression levels become…

attenuated. This

can adversely affect HPA axis negative

feedback signaling and is associated with

increased basal GC concentrations and

sustained increases in GC concentration

during stress.

Describe the important elements of catecholamines as a direct biomarker

1. Catecholamines:

-Most often measured in blood, and to a

lesser extent in urine.

-In-dwelling catheters are required for real-

time blood sampling due to their rapid

release during stress, their very short T1/2 in

circulation, and sensitivity to animal

handling (acute stress biomarker).

-Levels in blood likely don’t represent regional

tissue differences in sympathetic activity.

-As of yet, there is no universally accepted

methodology and assay sensitivity varies: High

Performance Liquid Chromatography (HPLC)

versus tandem mass spectrometry (MS/MS) with

liquid chromatography (LC), versus Enzyme-

Linked Immunosorbent Assay (ELISA)

Describe the important elements of glucocorticoids as a direct biomarker

Most widely used biomarker of the stress

response- most stable and easy to get a tissue

sample.

-Influenced by day/night/season, so a sampling

protocol is essential.

-Decreases with age (i.e. for pigs, cortisol stabilizes

around 20 weeks of age and is about 40% lower

than at 12 weeks of age).

-Gender differences (barrows 15% > gilts).

-Is elevated at parturition in some species (i.e.

sheep)

-Total cortisol (blood- acute stress biomarker)

versus free bioactive cortisol (saliva- acute stress

biomarker, feces and hair -chronic stress

biomarker).

-Circulating GC concentrations may not represent

GC concentrations in regional tissues and vice

versa.

-Non-adrenal sources of GC (i.e. skin, intestines,

placenta, adipose tissues, leukocytes etc.)

-Temporal response requires multiple

sampling....$.

-ELISA versus Radio Immune Assay (RIA-

greatest sensitivity)

-Different sampling protocols are required for

acute versus chronic stress assessment

Describe the important elements of ACTH as a direct biomarker

Influenced by day/night/season.

-Changes with age?

-Gender differences?

-Circulating concentrations likely don’t represent

tissue ACTH concentrations.

-Acute stress biomarker (blood).

-Chronic stress biomarker?

-Non-pituitary sources of ACTH (skin,

leukocytes)

-ELISA versus RIA

Describe the important elements of Chromogranin A as an indirect biomarker

4. Chromogranin A:

-Is secreted by adrenal medulla chromaffin cells

along with EPI and NEPI, but also by anterior

pituitary corticotrophs.

-Is more stable than EPI and NEPI, and in

humans, saliva chromogranin A is considered a

reliable indicator of increased sympathetic tone -

correlates well with NEPI concentration Is not affected by age, gender, or day/night/

season.

-Is being used as an acute stress biomarker in pigs.

-ELISA versus RIA

Describe the important elements of Blood presure as an indirect biomarker

5. Blood pressure:

-Invasive measurement of arterial blood pressure

requires surgical catheter implantation.

-Blood pressure cuff can be placed over metacarpal

artery, but this procedure requires anesthetic, and is

not as reliable as the invasive method described

above.

Describe the important elements of heart rate as an indirect biomarker

Heart rate monitors can now be used in precision

agriculture to measure sympathetic tone of

livestock in real-time. Their reliability is currently

under review, although the harness apparatus is

problematic for free ranging livestock.

-Acute stress monitor?

What are the 3 things an animal should do for it to maintain physiological homestasis?

For an animal to be able to maintain

physiological homeostasis in an

environment rich with pathogenic bacteria,

fungi, parasites, and viruses (microbial

stressors), it must be able to do the

following:

1. Prevent pathogen entry into the body

using physical (i.e. skin, mucous) and

physiological barriers (i.e. pH, mucous,

temperature).

2. Recognize the invading pathogen as non-

self if barriers are breached (sensory

perception of danger).

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3. Restore homeostasis by responding

appropriately to eliminate the pathogen

(stress response)

if the 3 things they do to maintain homestasis are achieved what happens?

If these are achieved, the animal will

remain productive and develop enhanced

long-term protection against the pathogen

if they cant elimate a pathogen what happens?

If the animal is not successfully able to

eliminate a pathogen, disease will occur.

This is a welfare issue, it can affect

productivity and possibly lead to death,

and may be a human health issue

what is a disease?

Disease: An illness or condition that

prevents the body or mind from working

normally. (Merrian-Webster defn.)

Animals also host commensal microbial populations that reside on the…

epithelium

of the skin and at mucosal surfaces of the

gastrointestinal, respirator, and urogenital

tracts. Under normal conditions, the host

learns to tolerate these microbes because

they have an important symbiotic

relationship with the host:

1. The host provides nutrients and an

environment that supports microbe

survival.

2. The microbes make certain nutrients

available to the host and help protect

against invading pathogenic microbes

what happens if tolerance to microbes doesnt occur

If tolerance to these microbes does not

develop normally at an early age, or

tolerance is broken during the animal’s

life, this can also lead to disease.

The ability of animals to recognize and

respond to pathogens as non-self and

tumor cells as modified-self and to

tolerate non-self commensal microbes is

facilitated by the immune system

what are the two arms of the immune system comprised of?

The immune system is comprised of two

arms: the innate and acquired (adaptive)

immune system. The innate immune system

is ancient, highly conserved across species,

and is the first to be activated in response to

microbial stressors

what is immunity?

Immunity: Is the protection provided by the

immune system to resist microbial infection.

Humoral proteins and cells making up the

innate and acquired immune system are the

effectors providing this protection

how is it ensured the immue responses can restore homeostasis

just as the SAM and HPA axes are tightly

regulated, the innate and acquired immune

responses are tightly self-regulated and are

also regulated by signals from the

neuroendocrine system (i.e. SAM, PNS, and

HPA axes) to ensure that these immune

responses can effectively restore

homeostasis

If the innate and acquired immune

responses are attenuated

an animal will not

be able to effectively eliminate pathogens or

tumor cells

In contrast, excessive and prolonged innate or acquired immune responses can result in

extensive host tissue damage and can lead to a variety of disorders including:

1. Acute or chronic inflammatory disease.

2. The development and proliferation of tumor cells.

3. Sensitization to environmental antigens

that results in atopic disease (i.e. atopic

dermatitis, asthma, food allergy, hay fever).

4. Sensitization to self-antigens that may

lead to autoimmune disease.

What happens during microbial invasion?

The immune system constitutes our sixth

sensory system that alerts the central

nervous system (CNS) to microbial

danger. During microbial invasion for

example, surveillance cells of the immune

system release signaling molecules called

cytokines (i.e. TNFα, IL-1 and IL-6) as

well as various neuropeptides that bind to

their respective receptors on neural cells

within the gut, skin, liver/spleen,

respiratory and urogenital tracts and brain.

These neural cells become activated and

subsequently alert the CNS to danger

what is done to ensure the immune response is effective and not excessive?

These danger signals are perceived

within the brain, and the CNS responds

with neural (i.e. SAM, PNS) and

endocrine (i.e. HPA) signals that help

regulate the immune response to ensure its

effective, but not excessive. Bidirectional

communication among these systems

occurs within what is collectively referred

to as the neuroendocrine-immune system.

what is the acute-phase response?

The release of pro-inflammatory

cytokines (TNFα, IL-1 and IL-6) during

immune system activation affects many

different tissues, and the systemic

response is referred to as the acute-phase

response

what do the cytokines do in the hypothalamus?

In the hypothalamus for example, these

cytokines elicit a fever response, and IL-1

induces sickness behaviour (i.e. sleepiness

and anorexia).

what do the cytokines cause in the muscle?

In the muscle, these cytokines cause

protein catabolism, which mobilizes

amino acids that are used to make host

defense and tissue repair proteins

tissue repair proteins are synthesized by the liver and are referred to as

hepaticacute-phase proteins (APP)

The innate immune response manifests as the host inflammatory response…

and the

secretion of the pro-inflammatory cytokines

TNFα, IL-1 and IL-6 is triggered by the

recognition of danger signals by soluble and

cell membrane pattern-recognition receptors

(PRRs)

danger signals include what?

These danger signals include highly

conserved microbial-associated molecular

patterns (MAMPs) making up microbial

membranes, and host alarm signals

(alarmins) that are released by damaged or

activated host cells

While the innate immune response can be highly effective at controlling and

eliminating pathogens, it has limited specificity, and does not provide long-term

enhanced protection against pathogens - referred to

immunological memory

The host inflammatory response is typically localized to site(s) of infection SO THAT….

tissue damage is minimized. However,

during a severe infection with a highly

virulent pathogen, localized immunological

defenses may not be sufficient to contain the

infection. Dissemination of the pathogen

into the circulation to other tissues can

trigger a potentially damaging condition

referred to as Systemic Inflammatory

Response Syndrome (SIRS). In extreme

cases, SIRS can lead to sepsis, organ failure

and death. If an animal survives SIRS, it is

typically immunocompromised and is at

high risk to secondary infections for

months-years.

What must the host do for loing term immunity?

For long-term immunity to occur, the host

must mount an acquired immune response

that is highly specific and has capacity for

immunological memory and increased

efficiency. This acquired immune

response is triggered by the recognition of

microbial antigens (usually proteins)

what recognizes the microbial antigens?

1. Immunoglobulins; which include

membrane B cell receptors (BCR) on B-

lymphocytes (B cells) and secreted

antibodies that are produced by

terminally differentiated B cells referred

to as plasma cells.

2. T cell receptors (TCR) on T-

lymphocytes (T cells).

What are the 2 responss that the aquired immine response can manifest as?

The acquired immune response can

manifest as a humoral antibody response

(AbMIR) that targets extracellular

pathogens and their toxins, or a cell-

mediated immune response (CMIR) that

targets intracellular pathogens. Unique

cytokines drive these polarized immune

responses. For example, IL-4 and IL-13

steers AbMIR, whereas IFNg steers

CMIR

Although the acquired immune response is more effective than the innate immune

response for controlling and/or eliminating pathogens, it takes longer for

the host to mount (day-to-weeks as opposed to minutes-to-hours) because?

because a

higher level of immune system

orchestration is required to elicit an

acquired immune response, which also

involves the innate inflammatory

response

For an acquired immune response to occur for example, there must be sufficient inflammation to activate surveillance cells of the innate immune system to carry out the following activities what are the 5 activities?

1. Phagocytosis and killing of the microbe

2. Processing microbial proteins into peptides

3. Migration from the site(s) of infection to secondary lymphoid tissues (i.e. lymph nodes, Peyer’s patches, spleen etc.)

4. Up-regulated expression of membrane bound antigen receptors called major

histocompatibility complex molecules (MHC) that are loaded with the processed microbial peptide antigens.

5. Presentation of microbial antigens to antigen-specific T cells via MHC-TCR

interactions; this is referred to as antigen presentation.

when antigen specif t cells get activated to proliferate and terminall differntiae where do they do that?

These antigen-specific T cells become

activated to proliferate and terminally

differentiate into the effector T cells of the

acquired immune system (i.e. cytotoxic T

cells).

what also helps recognize microbial antigens?

Antigen-specific B cells may also

recognize the microbial antigens, and with

the help of an antigen-specific T helper cell,

these B cells may become activated to

proliferate and terminally differentiate into

the effector B cells (antibody-secreting

plasma cells).

what cells are also genrated during hte acquire immune response?

Memory B and T cells are also generated

during the acquired immune response, and

these facilitate long-term immunity

The innate immune response provides an

immediate level of protection that buys time

for the more effective acquired immune

response to occur and is also required for an

effective acquired immune response to

occur.

Hundreds of immune-related proteins are

produced during the innate and acquired immune

responses to microbial stressors and may serve as

biomarkers of microbial stress

What are the 3 biomarkers of acute microbial stress?

1. Cytokines: Innate pro-inflammatory cytokines

(TNFα, IL-1 and IL-6).

2. Hepatic APPs: Many immune-related APPs are

produced during the acute-phase response to

microbial infection (i.e. serum amyloid A, C-

reactive protein, haptoglobin, and certain

complement proteins). These APPs, however,

lack microbial specificity and are also induced by

GC in response other types of stressors.

3. Temperature: Febrile responses are easy to

monitor but are not always elicited in response to

microbial stress

What are the 2 biomarkers of chronic microbial stress?

1. Cytokines: Cytokines driving AbMIR

(IL-4 and IL-13), or CMIR (IFNg).

2. Antibodies: Antibodies have microbial

specificity, are highly sensitive and potentially

long-lasting indicators of microbial exposure,

which also makes them ideal for diagnosis of

specific diseases

Which antibody type (isotype) would be most appropriate to measure as a biomarker of

microbial stress?

There are different antibody isotypes and sub-

isotypes (i.e. bovine IgM, IgG1/2, IgA).

-Antibody isotypes are species- and tissue-

specific

Examples:

-Bovine IgG1 (AbMIR) versus IgG2 (CMIR) in

blood; IgG1 is the predominant immunoglobulin

isotype found in bovine milk.

-Bovine mucosal IgA and IgM

-Bovine IgE (very short half-life in circulation)

Should we measure basal or inducible antibodies?

Basal antibody levels may be around the assay

limit of detection (i.e. ELISA).

-AbMIR can be induced using a novel antigen or

commercial vaccine to assess the capacity to mount

an immune response

Your professor's question is asking whether we should measure basal (resting) antibody levels or inducible antibody levels when studying immune responses.

Here’s what their answer means:

Basal antibodies are the ones already present in the body without any new infection or vaccination. However, their levels might be too low to detect using common lab tests like ELISA (a test that measures antibody levels in a sample).

Inducible antibodies are produced after the immune system is stimulated by a new antigen (a substance that triggers an immune response) or a vaccine. Measuring these helps us see how well the immune system can respond to a challenge.

So, your professor is suggesting that instead of just measuring the low basal levels, it might be better to use a vaccine or antigen to trigger an immune response and then measure how many antibodies the body can actually produce—which gives a clearer picture of immune function.

Should we measure total or antigen-specific antibody levels?

Antibody response to antigen challenge (Slide 36): The kinetics of the host antibody response varies

depending on antigen concentration, antigen half- life, number of antigen exposures and antigen

exposure duration

Your professor is asking whether we should measure total antibodies (all antibodies in the body) or antigen-specific antibodies (antibodies that target a particular pathogen or vaccine).

Their answer explains that the host's antibody response (how the body produces antibodies after encountering an antigen) depends on several factors:

Antigen concentration – If there's more antigen (virus, bacteria, or vaccine component), the immune system may produce more antibodies.

Antigen half-life – Some antigens stay in the body longer, allowing a stronger or more prolonged immune response. Others break down quickly, leading to a shorter response.

Number of antigen exposures – The immune system responds more strongly if it has seen the antigen before. More exposures (like booster shots) generally lead to higher antibody levels.

Antigen exposure duration – If the immune system is exposed to an antigen for a longer time, it may produce a more sustained antibody response.

In short, your professor is highlighting that antigen-specific antibody levels provide more useful information than total antibody levels because they directly show how well the immune system responds to a particular challenge.

what are some factors confounding antibody production

Antibody production may be confounded by immune health status.

Examples:

-Acute exposure to different types of stressors can

stimulate immune cell trafficking to support the

host immune response in the skin and at mucosal

surfaces - this could enhance vaccine-induced

antibody production.

-Chronic exposure to different types of stressors

causes immunosuppression® attenuated antibody

production

Some microbes (i.e. viruses) can modulate the

host immune system to evade detection (i.e.

suppress AbMIR or CMIR); this could affect

disease diagnosis as well as vaccine efficacy

Stages of Bovine Johne’s disease (JD) caused by

Mycobacterium avium subsp. paratuberculosis

(MAP)

Seroconversion takes time, which means early

diagnosis of JD is difficult. Also, the host

immune response may change depending on the

stage of infection, which means a biomarker

panel may be necessary for diagnosis of JD.

Summary of biomarkers of stress response accute stress

There are many potential biomarkers of the

stress response. There are pros and cons to each

biomarker, and there is no single biomarker that

can be used to assess responsiveness to all types

of stressors. Therefore, a panel of direct and/or

indirect biomarkers, reflecting the SAM and

HPA responses, should be used to assess acute

stress response

Summary of biomarkers of stress response accute stress

In the case of chronic stress, it may be

necessary to administer a stressor to assess an

animal’s resilience to stress. This could be

achieved by subjecting an animal to a highly

controlled stress regime involving one or more

stressors. Once the stressor is administered, the

SAM, HPA or immune responses can be

monitored over time to assess stress resistance,

habituation, recovery or sensitization

example of highly controlled stress regime

-Psychological -isolation, re-grouping

-Physical -heat, cold, transport, noise, electrical,

restraint

-Chemical -fungal mycotoxins or bacterial

toxins (MAMPs-lipopolysaccharide), pro-

inflammatory cytokines, neuropeptides (i.e.

CRF, AVP, ACTH) -Biological -acidosis, sleep deprivation, intense

exercise, pathogen challenge

When the stressor is of microbial origin, a panel

of immune system biomarkers is typically used to

assess the innate and acquired (AbMIR and

CMIR) immune responses. Antibody levels are

most commonly measured due to ease of

sampling, and their specificity makes them

extremely useful for diagnostic testing of specific

microbial infections

Genetics and epigenetics of stress (

Variation in gene activity is controlled at

the level of the genome and epigenome:

Genetic variants predetermine gene

activity and are permanent and inherited by

offspring (i.e. single nucleotide

polymorphisms (SNP).

Epigenetic variants are established by

environmental quos during mitosis and

provide an adaptive mechanism for

individual phenotypic change. Since

epigenetic modifications may be inherited,

they can also contribute to phenotypic

variation within populations

Genetics of stress

As discussed previously, genetics likely

contributes to variation in sensory input,

stress perception, as well as the stress

response. Since the stress response is easiest

to assess, it is the preferred phenotype for

genetic selection.

Blood cortisol level for example, is a

phenotype of moderate-to-high heritability.

Heritability estimates (H 2 ) have been found

to vary across species and are influenced by

time of day and type of stressor.

Examples of blood cortisol?

Human twin study (Gustafsson et al. 2011)

Cortisol heritability at awakening (0.28): Low genetic influence, meaning environmental factors play a bigger role.

30 min after awakening (0.60): Higher genetic influence, suggesting genes significantly affect cortisol levels after waking up.

Evening cortisol (0.08): Very low heritability, meaning evening cortisol levels are mostly shaped by daily experiences, not genetics.

Pigs (Larzul et al. 2015)

Cortisol response after ACTH challenge (0.68): ACTH is a hormone that stimulates cortisol release. A high heritability (0.68) means genetic factors strongly influence how pigs respond to stress, which could help in breeding stress-resistant pigs.

Barn swallows (Jenkins et al. 2014)

Basal corticosterone (0.15): Low heritability, meaning environmental factors mainly determine resting hormone levels.

Stress-induced corticosterone (0.34): Moderate heritability, so genes play a bigger role in how barn swallows respond to stress.

Rainbow trout (Overi et al. 2005)

Cortisol response (0.41) to 3-hour confinement: Moderate heritability suggests that genes influence how trout react to being confined, but the environment also plays a role.

Sheep (You Q et al. 2008, Pant SD et al. 2016)

4-hour cortisol response (0.3) to bacterial endotoxin (LPS) challenge: LPS mimics bacterial infection to test immune-stress response. A heritability of 0.3 means genetic factors contribute to cortisol response, but environment is still important.

Conclusion:

Since many of these cortisol responses have moderate-to-high heritability, it suggests that selective breeding could be used in livestock (like pigs and sheep) to develop animals that are more resilient to stress. This means choosing animals with desirable stress responses to improve welfare and productivity in future generations.

There are three known mechanisms of epigenetic modification that influence gene activity: what are they?

1. Methylation of Cytosine Nucleotides by DNA Methyltransferases

DNA methylation is typically associated with transcriptional silencing.

2. Modification of Histone Proteins

Affects chromatin accessibility by transcription factors.

Histones are scaffold proteins that help package DNA as chromatin (DNA wrapping) within the nucleus. Histone proteins allow for chromatin unwinding/winding, which allows/restricts transcription factor accessibility to genes.

Histone tails are susceptible to modifications (i.e. methylation, acetylation, and phosphorylation) by various enzymes.

These modifications alter the 3-dimensional configuration of chromatin, which subsequently affects accessibility of transcription factors and transcriptional machinery to the DNA.

3. RNA-Based Mechanisms Such as Noncoding RNAs (ncRNA)

ncRNA are regulatory gene products that do not become translated into proteins.

MicroRNA (miRNA) is a single-stranded ncRNA (< 22 nucleotides) that binds to complementary nucleotides (seed region) found within the 3’ untranslated region (UTR) of target mRNA.

miRNA binding to mRNA usually results in either mRNA degradation or repressed mRNA translation.

All three of these mechanisms greatly contribute to regulation of genes within the neuroendocrine-immune system, and they are subject to influence by environmental quos (i.e. stressors). This raises the possibility that they may be targeted to improve livestock stress resilience.

Examples of neuroendocrine-immune genes subject to epigenetic modifications:

1. A response element, for the transcription

factor nerve growth factor-induced protein A,

found within the promoter region of the GR

gene is subject to DNA methylation. Stress-

induced methylation of this response element

influences negative feedback regulation of the

HPA axis.

2. GR levels are also down-regulated by

miRNA-18 and miRNA-124a binding to the GR

mRNA 3’ untranslated region (UTR).

3. IFN-γ is a key cytokine that promotes a

CMIR against intracellular pathogens. GCs

induce histone deacetylase, which reduces

accessibility of transcription factors to the

promoter region of IFN-γ ® reduced IFN-γ

gene expression

Responses to stress: the good, bad and ugly

The physiological response to stress is a highly

complex process that is predetermined by

genetics and is also subject to epigenetic

regulation by environmental quos (i.e.

stressors).

While the stress response is designed to

enhance survival of an organism by restoring

physiological homeostasis, excessive or chronic

activation of the stress response can lead to a

variety of disorders that increase susceptibility

to infectious and autoimmune diseases and

cancer. Risk of these disorders is predetermined

by genetics and may be influenced by epigenetic

modifications

cushing’ disease (important)

Overproduction of Glucocorticoids (GC)

In most cases, overproduction of GC is caused by a pituitary gland tumor that leads to excessive ACTH production.

Normal peak GC levels are detected in the morning, but they fail to decrease in the evening.

Symptoms in Humans:

Accumulation of central fat

Muscle weakness

Skin lesions

Bone fractures

Increased susceptibility to infection due to impaired immune function

Hypertension, diabetes, cardiovascular failure, and stroke

Genetic Risk Factors for Cushing’s Disease:

Polymorphisms in the USP8 gene (ubiquitin-specific protease 8) indirectly contribute to increased ACTH production and are a risk factor for Cushing’s disease.

Mutations in the ARMC5 gene (armadillo repeat-containing 5), a tumor suppressor gene, can cause adrenal hyperplasia associated with Cushing’s disease.