AN SCI 320 FINAL

Defining Health and Disease - 01/25/24

Health: a state of complete physical, mental, and social well-being, not merely the absence of disease or infirmity.

DIsease: condition of living animal or plant body or of one its parts that impairs normal function, and is typically manifested by distinguishing signs and symptoms.

Defining “disease” is pretty hard

• Disease is relative to what people consider “normal”

• Can be subjective: “one person's diarrhea is another person normal day

• Some believe that our use of “health” and “disease” reflect value judgments

• The definition can change with time as our knowledge evolves

Illness: a person's subjective experience of their symptoms. What the patient brings to their health care provider

Disease: underlying pathology: biologically defined: the healthcare provider’s perspectives

Sickness: social and cultural conception of this condition: cultural beliefs and reactions such as fear or rejection. These affect how the patient reacts.

Naturalism

• Most prominent philosophical approach to defining health and disease

  • Reference Class: a natural class of organisms of uniform functional design: specifically, an age group or a sex of species

  • Normal Function: part or process within members of the reference class is a statistically typical contribution by it to their individual survival and reproduction

  • Disease: type of internal state which is either an impairment of normal functional ability

  • Health: the absence of disease

• Criticisms:

  • Neglects the role values play in determining healthy or diseased

  • Provide definitions that rely exclusively on info from the biological sciences, but, lacks a basis in biological theory

Normativism

• Disease is deviancy from some alternative state of affairs which is considered more desirable

  • Suggest that we both la people and medical professionals, should use health and disease in ways that reflect our values

  • Physiological or psychological states that we desire are called healthy and those we want to avoid are labeled diseases

• Criticisms

  • Cases where we agree that a state is undesirable but we disagree over whether it is a diseased state (ex. Overweightness, PMS)

  • Values can change

• Hybrid Theories

  • A condition is a disorder if and only if (a) the condition causes some harm or deprivation of benefit to the person as judged by the standards of the person’s culture (the value criterion), and (b) the condition results in the inability of some internal mechanism to perform its natural function, wherein natural function is an effect that is part of the evolutionary explanation of the existence and structure of the mechanism (the explanatory criterion)”

  • The term disease should only apply to dis-valued states with the proper biological etiology

• Criticism

  • A state where there is no evolutionary dysfunction yet we disvalue that state

What influences the balance between health and disease?

• Is aging a disease?

Colonization vs Infection - 01.30.24

Colonization (normal flora)

• Colonizing cells = 39 trillion

Normal Flora

• Most areas of the body in contact with the outside environment harbor resident microbes

• Microorganisms that normally reside at a given site and under normal circumstances do not cause disease

• Normal flora is essential for health: (a) create an environment that may prevent infections and (b) enhance host immune defenses

• Internal organs, tissues and fluids are microbe-free (relatively)

• Transient flora

  • Occupy the body for only short periods

  • Usually picked up during daily activities (hand-shake, touch a door-knob, kissing)

  • Often eliminated easily (hand-washing)

• Resident flora

  • Are permanently established (or for long periods of time)

Types of Relationships with Microbiome

• Mutualism

  • Both the host and the microbe benefit

  • Examples: ruminants and their gut microorganisms (2) E. coli-microbe receives nutrients, but produces vitamins K and B- complex

• Commensalism

  • One partner benefits, and the other neither benefits or is harmed

• Parasitism

  • One organism benefits at the expense of the host

  • Cost to the host can vary from slight to fatal

  • An external parasite (flea, ectoparasite) is said to cause infestation

  • An internal parasite (endoparasite, tapeworm) is said to cause infection

• Pathogenic

  • Organism causes damage to the hose during infection

Distribution and Diversity of Colonization

Microbial Colonization of the skin

Initial Colonization

• Prevailing paradigm

  • Uterus and contents are normally sterile and remain so until just before birth

  • Breaking of fetal membrane exposes the infant: all subsequent handling and feeding continue to introduce what will be normal flora

  • Is this paradigm entirely correct? → currently a controversial topic

Factors that influence Initial Colonization

• Maternal factors

  • Gut microbiota

  • Vaginal health

  • Periodontal disease (other infections)

  • Genetics, diet, antibiotics

• Birth

  • Vaginal vs. Cesarean delivery

• Postnatal Factors

  • Genetics

  • Breastfeeding vs formula milk

  • Medications and antibiotics

  • Diet

  • Environment

Defining Infection

• Infectious agent: viruses, bacteria, fungi, protozoa, worms and prions

• Infection: condition in which infectious agent penetrates host defenses

• Infectious disease: an infection that causes damage or disruption to tissues and organs and/or physiological homeostasis

• Endogenous infections

  • Occurs when normal flora is introduced to a site that was previously sterile (epidermis infection of wound)

• Exogenous infections

  • Caused by organisms that are normally present in the body, but have gained entrance from the environment (influenza virus infection and respiratory tract)

Types of pathogen

• True pathogen (influenza virus): infectious agent that causes disease in virtually any susceptible host

• Opportunistic pathogen (pseudomonas, candida albicans): normally harmless: causes disease when the normal flora is disrupted (by antibiotics) or when the host is immunocompromised ( by drugs or other illnesses ).

Patterns of Infection

• Localized infection: infectious agent enter the body and remains confined to a specific issue

• Systemic infection: infection spreads to several sites and tissue fluids usually the bloodstream

• Focal infection: infectious agent breaks loose for a local infection and is carried to other tissues

• Mixed infection: several microbes grow simultaneously at the infection site (poly microbial)

• Primary infection: refers to the first time you are exposed to (and infected by) a specific pathogen (UTI)
  Secondary infection: another infection by a different microbe succeeding a primary infection

• Acute Infection: comes rapidly, with severe but short-lived effects

• Chronic (persistent) infection: progresses and persists over a long period of time

Infections that go unnoticed

• Asymptomatic (subclinical) infections: although infected, the host does not show any signs of disease

  • Inapparent infection, so the individual does not seek medical attention

    • Ex. typhoid, chlamydia, HIV-1, epstein-Barr virus, Gonorrhoea

Mary Mallon, “Typhoid Mary” (1869 – 1938)

• First person in the United States identified as an asymptomatic carrier of Salmonella Typhi (typhoid fever).

• She was presumed to have infected 51 people, three of whom died, over the course of her career as a cook.

• She was twice forcibly isolated by public health authorities and died after a total of nearly three decades in isolation

Acquisitions and Transmission of infectious agent

• Communicable infection

  • Infected host an transmit the infectious agent to another host

    • Highly communicable infection is contagious

• Non-communicable infection

  • Infection does not arise through transmission from host to host

    • Occurs primarily when a composed person is invaded by his/her own normal flora

    • Contracted organism from natural, non-living reservoir

Nosocomial Infections

• Infections acquired or developed during a hospital stay.

  • From surgical procedures, equipment, personnel, and exposure to drug-resistant microorganisms

  • 2-4 million human cases/year in the US. 90,000 deaths. Impact in veterinary medicine not well studied.

  • 

What defines a particular disease?

• Signs: (objective evidence)

  • Something that can be detected/measured by someone else (tachycardia, petechiae)

• Symptoms: (subjective evidence)

  • Something that must be described by the one suffering the disease (anxiety, pain, fatigue)

• Syndrome: the complete set of signs and symptoms associated with a specific disease (metabolic syndrome)

Introduction to Epidemiology 02.01.24

Roots of Epidemiology

• Epidemiology is a fundamental science of public health.

• Epidemiology has made major contributions to improving population health.

• Epidemiology is essential to the process of identifying and mapping emerging diseases.

• There is often a frustrating delay between acquiring epidemiological evidence and applying this evidence to health policy.

EPIDEMIOLOGY

• The study and analysis of the patterns (frequency and distribution) causes and effects of disease and health -related factors in populations

  • Epi “on, upon, befall”

  • Demo “people”

  • -ology - study of

  • Epidemiology: the study of which befalls people

  • Cornerstone of public health: shapes policy decisions and evidence-based practice

Major areas of epidemiological study include

• Disease etiology

• Transmission

• Outbreak investigation

• Disease surveillance and screening

• Forensic epidemiology and screening

• Biomonitoring

• Comparison of prevention/treatment outcomes

Epidemiologist rely on:

• Scientific disciplines to better understand disease

• Statistics for efficient use of data and draw appropriate conclusions

• Social sciences to better understand proximate and distal causes

• Other fields for exposure assessment

Host:

• Immunity

• General health

• Age

• Gender

• Genetic background

• religious/cultural practices

Agent

• Pathogen traits

• Virulence

• Dose

• Incubation period

Environment

• Heat or cold stress

• Food availability

• Hygiene

• Crowding

• Cultural practices

• Presence of vectors or reservoirs for pathogen

Pathogen Traits

• Types:

  • Viruses, bacteria, fungi, protozoa, worms and prions

  • Other information (strain, serotypes, etc, ex. Salmonella enterica serovar Typhimurium)

• Virulence

  • Ability to cause severe disease

  • Virulence factors: specific mechanisms that allow pathogen to adhere to or penetrate host cell, thwart immune defenses, damage host

• Infectious dose:

  • Minimum number of pathogens required to cause illness

• Incubation period:

  • Time it takes after first exposure for the pathogen to cause signs and symptoms: influences extent of spread

Virulence: Infectious Dose (ID)

• Minimum number of microbes required to cause infection in the host

• Smaller the ID, the greater virulence

• If ID is not reached, infection will not occur

• ID₅₀ (median infectious dose): amount of pathogenic microorganism that will produce demonstrable infection in 50% of exposed hosts

Host Traits

• Immunity to pathogen

  • Previous exposure, immunization

  • Antigenic variation of pathogen can overcome

• General Health

  • malnutrition , overcrowding, fatigue

  • Developing wor;d more susceptible: crowding, poor nutrition poor sanitation

• Age

  • Very young, elderly generally more susceptible

  • Immune system less developed in young: wanes in old

  • Elderly also less likely to update immunizations

• Genetic background

  • Natural immunity varies widely

  • Specific receptors critical for infection may differ in individuals (african swine fever: domestic pigs s warthogs)

  • Sickle cell gene and resistance to malaria

• Gender

  • Females more likely to develop urinary tract infections

    • Urethra is shorter: microbes more likely to ascend

  • Pregnant animals are at more risks

  • Pregnant animals can pass on some disease to offspring

• Religious and cultural practices

  • Breastfeeding provides protective antibodies to infants

  • Consumption of raw fish can increase exposure (ex. Freshwater fish and tapeworm Diphyllobothrium latum).

Environmental Factors

• Environmental factors may increase likelihood of disease transmission opportunities or lower the hosts resistance to infection

  • Heat or cold stress

  • Food availability

  • Hygiene

  • Crowding

  • Cultural practices

  • Presence of vectors or reservoirs for pathogen

Routes of Transmission

• Direct contact: physical contact or fine aerosol droplets

  • Some pathogens cannot survive in environment

  • Hand washing considered single most important measure for preventing spread of infectious disease

  • Horizontal vs vertical transmission

• Indirect contact: passes from infected host to intermediate conveyor and the to another host

  • Some pathogens can survive for a period outside of the host

• Fomite: inanimate object that serves a role in disease transmission (pens, cups, doorknobs, clothing, boots, etc)

• Vector: any agent (insect, animal, or microorganism) that carries a pathogen and transmits it (mechanical or biologically) to human or animal hosts

• Vehicle: typically food, water, or air (droplet nuclei, aerosols) that transmits a pathogen to the host

• Reservoir: the natural habitat (living or nonliving) in which a pathogen lives and reproduces that serves as a source of infection

  • Living reservoir may be symptomatic or asymptomatic

  • Asymptomatic:: harder to identify and control spread (up to 50% of women infected with Neiserria gonorrhoeae are asymptomatic).

  • Exclusively human reservoirs are easier to control (Smallpox)

  • Non-human reservoirs (arthropod, wild animal) challenging to control)

  • Environmental reservoir: difficult or impossible eliminate (Bacillus anthracis)

Portals of entry and exit

• Skin: nicks, abrasions, punctures, incisions

• Gastrointestinal tract: food, drink, and other ingested materials

• Respiratory tract: oral and nasal cavities

• Urogenital tract: sucal, displaces organisms

• Transplacental

• feces/urine

• semen/ vaginal secretions

• Sputum

• Saliva

• Blood

• Pus and lesion exudates

• Tears

• Vomit

Cycle of transmission

Reservoirs and Modes of Transmission

Epidemiology Part 2 - 02.06

Frequency of Cases

• Prevalence: the total number or proportion of cases or events or conditions in a given population

• Incidence: the number of new cases during a specified time period

• Morbidity rate: number of people affected with certain diseases during a given period of time

• Mortality rate: number of deaths in a population due to certain disease during a given period of time

• Case-fatality rate: the percentage of people with a specific disease that dies from that disease

• Attack rate: number of people affected by a disease divided by the number of people with a specific exposure

Disease Occurrence Patterns

• Endemic: a relatively steady frequency over a long period of time in the particular geographic locale (ex. Common cold)

• Sporadic: when occasional cases are reported at irregular intervals (ex. Rabies in the US)

• Epidemic: increasing prevalence of a disease beyond what is expected (ex. Porcine epidemic diarrhea virus (PEDV) in the US in 2013)

• Pandemic: epidemic across countries and continents (HIV/AID, COVID-19)

In an outbreak of aflatoxicosis among finishing pigs in a farm of 200 pigs: 100 pigs ate feed mixed using an old batch of corn. 20 pigs first began vomiting. A day later, 30 more pigs showed similar symptoms. Over the course of the week, 10 pigs died.

• What is the prevalence of aflatoxicosis? 25%

• What is the case fatality rate? 20%

• What is the attack rate? PB: 50%

Basic Reproductive Number (R₀)

• The average number of new infectious generated by one infection in a completely susceptible population

• Measure of the intrinsic potential of an infectious agent to spread

• R₀ = C x P x D

  • C = contact rate (contact/time)

    • The average rate of contact between susceptible and infected individuals

  • P = transmissibility (infection/contact)

    • The probability of infection given contact between a susceptible and infected individuals

  • D = duration of infectiousness (time/infection)

Effective Reproductive Number ( R )

• A population will rarely be totally susceptible to an infection in the real world. The effective reproductive rate ( R ) estimates the average number of secondary cases per infectious case in a population made up of both susceptible and non-susceptible hosts.

• R= R₀ x S

• R₀ = Basic reproductive number

• S = fraction of the host population that is susceptible

Endemic vs. Epidemic

Newyrok of Infection

• Rate of spread of an infection depends on R₀ and serial interval

Serial Interval

• The time between the same stage of illness in successive clinical cases in a chain of transmissions

  • 

SARS (Severe acute respiratory syndrome)

• Outbreak: November 2022 - July 2033

• Causes by SARS coronaviru (SARS-CoV)

• Spread across 37 countries and regions

• Infected 8,098 people (reported cases)

• Killed 774 people (killed 1 in 10 people infected)

Spatial Heterogeneity

• Rural Areas

• Urban Areas

Seasonality of Infections

• Why? What causes seasonality?

  • Climate, temperature, moisture levels, allergies

  • 

Impact of Movement and Modern Transport

Pathogenesis of Infections 02.08.24

Germ Theory

• Robert koch

• Louis pasteur

Koch’s Postulates

• The microorganism must be found in abundance in all organism suffering from the disease, but should not be found in healthy organisms

• The microorganisms must be isolated from a diseased organism and grown in pure culture

• The cultured microorganisms should cause disease when introduced into a health organism

• The microorganism must be reisolated from inoculated, diseased experimental jost and identified as being identical to the original specific causative agent

1. The suspect germ must be present in every case of the disease

2. The germ must be isolated and grown in pure culture

3. The cultured germ must cause the disease when it is inoculated into health, susceptible experimental host (animal or plant)

4. The same germ must be reisolated from the diseased experimental host

Infectious Agents

• Bacteria

• Viruses

• Protozoa

• Fungi

• Parasitic worms (helminths)

• Prions

Introduction to Bacteria

Bacterial taxonomy

Bacterial Shapes

• Coccus

  • Coccobacillus

• Bacillus

  • Cocobacillus

  • vibrio

• Spiral

  • Vibrio

Bacterial Shapes

Bacteria Cell Structure

• Flagella

  • Presence is species.strain dependent

  • For motility

  • Number and arrangement vary

• Pili/Fimbriae

  • Hair-like structures

  • Fimbriae shorter than pili

  • Adhere.attach to surfaces (key step in most infections

  • “F” or sex pilus: used for transfer of genetic material (plasmid) from one bacteria to another

  • Can provide resistance against engulfment by phagocytes

Bacterial Cell Wall Structure

Gram Staining

1. Application of crystal violet

2. Application of iodine

3. Alcohol wash

4. Application of safranin

Bacterial Reproduction

1. Cell replicate its dna

2. The cytoplasmic membrane elongates, separating DNA molecules

3. Cross wall forms completely

4. Daughter cells

Rapid Bacterial Growth

1. Cytoplasmic membrane

2. Replicated chromosome

3. Septum

4. Completed septum

5. 60 mins – 90 mins – 120 mins

Bacterial Endospore Formation

• Under stressful environments, certain gram positive bacteria are capable of forming endospores

• Endospores can survive environmental assaults that would normally kill the bacteria. These stresses include high temperature, high uv irradiation, desiccation, chemical damage and enzymatic destruction

• Endospores make them of particular importance because they are not readily killed by manu antimicrobial treatments

Types of Bacterial Pathogen

• True pathogen (enterotoxigenic escherichia coli): infectious agent that causes disease in virtually any susceptible host

• Opportunistic pathogen (pseudomonas, staphylococcus): normally harmless, causes disease when the normal flora is disrupted (by antibiotics) or when the host is immunocompromised (by drugs or other illnesses).

Bacterial Adhesion

• Necessary to avoid innate host defense mechanisms ( peristalsis in the hugy and the flushing action of mucus, saliva and urine).

• Adhesion is often an essential preliminary step to colonization and then penetration through tissues

• At the molecular level, adhesion involves surface interactions between specific receptors on host cell membrane and ligands on the bacterial surface

• Nonspecific surface properties of the bacterium. Including surface charge and hydrophobicity, also contribute to the initial stages of the adhesion process

Mechanism of Adherence to cell or tissue surfaces

• Non-specific adherence: reversible attachment to the surface (dock)

  • Hydrophobic interactions

  • Electrostatic attractions

  • Atomic and molecular vibrations resulting from fluctuating dipoles of similar frequencies

  • Brownian movement

  • Recruitment and trapping by biofilm polymers interacting with bacterial glycocalyx (capsule)

• Specific adherence: irreversible permanent attachment to the surface (anchoring)

  • Formation of many specific lock and key bonds between complementary molecules on each cell surface

  • Complementary receptor and adhesion molecules must be accessible and arranged in such a way that many bonds form over the area of contact between the two cells.

  • Once the bonds are formed, attachment under physiological conditions becomes virtually irreversible.

Specific adherence of bacteria to cell or tissues

• Tissue tropism

  • Particular bacteria are known to have apartment preference for certain tissues over others

    • Ex s mutans is on dental plaque but not on tongue surfaces

• Species specificity

  • Certain pathogenic bacteria infect only certain species of animal

• Genetic specificity within a species

  • Certain strains or races within a species are genetically immune to a pathogen

Terminology: Adherence factors in host-pathogen interactions

Adhesion

• A surface structure or macromolecule that binds a bacterium to a specific surface

Receptor

• A complementary macromolecular binding site on a (eukaryotic) surface that binds specific adhesins or ligands

Lectin

• Any protein that binds to a carbohydrate

Ligand

• A surface molecule that exhibits specific binding to a receptor molecule on another surface

Mucous

• The mucopolysaccharide layer of glycosaminoglycans covering animal cell mucosal her for the purpose of DNA transfer surfaces

Fimbriae

• Filamentous proteins on surface of bacterial cells that often have adhesins for specific adherence

Common pili

• Same as fimbriae

Sex Pilusx

• A specialized pilus that binds mating prokaryotes together for the purpose of DNA transfer

Type 1 fimbriae

• Fimbriae in enterobacteriaceae which bind specifically to mannose terminate glycoproteins on eukaryotic cell surfaces

Type 4 pili

• Pili in certain gram - and gram + bacteria. In pseudomonas, thought to play a role in adherence and biofilm formation

S-layer

• Proteins that form the outermost cell envelope component of broad spectrum of bacteria, enabling them to adhere to host cell membranes and environmental surfaces

Glycocalyx

• A layer of exopolysaccharide fibers on the surface of bacterial cells which may be involved in adherence to a surface. Sometimes a general term for capsule

Capsule

• A detectable layer of polysaccharide (rarely polypeptide) on the surface of a bacterial cell which may mediate specific or nonspecific attachment

Lipopolysaccharide (LPS)

• A distinct cell wall component of the outer membrane of gram- negative bacteria with the potential structural diversity to mediate specific adherence. Probably functions as an adhesin

Bacterial Biofilm Formation

• Aggregate of microorganisms in which cells that are frequently embedded within a self-produced matrix of extracellular polymeric substance adhere to each other and surfaces

• Form on tissue surfaces (ex teeth, skin, mucosal membrane ) and inanimate surface (ex catheters kitchen counters, orthopedic implant)

• Often impervious to detergents, antibiotics and immune system

• Can act as reservoirs for repeated infections

Know the definitions

• Planktonic cells

• biofilm -enmeshed cells

• Persisters cells

Bacterial Pathogens

• Facultative intracellular pathogen

  • Salmonella, shigella, yersinia

• Obligate intracellular pathogen

  • Ex. rickettsia, chlamydia, coxiella

• Extracellular pathogen

  • Ex. vibrio cholera, pseudomonas E. coli (ETEC)

Bacterial Pathogens

• Facultative intracellular pathogen

  • Ex. salmonella, shigella, yersinia

• Obligate intracellular pathogen

  • Ex. rickettsia, chlamydia, coxiella

• Extracellular pathogen

  • Ex. Vibrio cholera, pseudomonas, E. coli (ETEC)

Bacterial Invasion

• Penetration of host cells and tissues (beyond the skin and mucous surfaces)

• Mediated by a complex array of molecules, often described an “invasins”

• Invasins can be in the form of bacterial surfaces or secreted proteins which target host cell molecules (receptors)

Bacterial Invasins: Spreading Factors

• Descriptive term for a family of bacterial enzymes that affect the physical properties of tissue matrices and intracellular spaces, thereby promoting the spread of the pathogen

• Hyaluronidase:

  • Produced by streptococci. Staphylococci, and clostridia

  • Attacks the interstitial cement (“ground substance”) of connective tissue by depolymerizing hyaluronic acid

• Collagenase

  • Produced by clostridium histolyticum and C. perfringens

  • Breaks down collagen, the framework of muscles, which facilitates has gangrene due to these organism

• NeuraminidaseL

  • Produced by intestinal pathogens such as Vibrio cholerae and Shigella

  • Degrades neuraminic acid (also called sialic acid), an intercellular cement of the epithelial cells of the intestinal mucosa

• Streptokinase and staphylokinase

  • Produced by streptococci and staphylococcus, respectively

  • Digests fibrin and prevents clotting of the blood

  • Relative absence of fibrin in spreading bacterial lesions allows more rapid diffusion of the infectious bacteria

Bacterial Toxigenicity

• exotoxins

  • Proteins produced inside pathogenic bacteria

  • gram-positive bacteria

  • Retain high activity at very high dilutions

• Endotoxins

  • Lipid potions of LPS, outer membrane of cell wall

  • Gram-negative bacteria

Virulence Factors

• Adhesion

• Invasion

• Competition for iron and nutrients

• Resistance to host immunity

• Secretion of toxins

Bacterial Conjugation: Propagation of Virulence

Biofilms - 02.13.24

Biofilm

• Highly structured microbial communities enmeshed in a self-produced slime matrix comprised of proteins, polysaccharides, eDNA, and lipids

Biofilms are highly complex

• Stringy: only contains eDNA

• Chunky: only contains proteins

• Webby: contains everything

Biofilm Development Stages

1. colonization/adhesion

   1. Non-specific attachment

      1. Physio-chemical

      2. Pili-mediated

      3. flagella-mediate

   2. Specific attachment

      1. Receptor-mediated

      2. host-ECM components

2. Aggregation

3. microlony/monolayer formation

4. Maturation

5. Dispersal

Biofilm EPS formation, Maturation (3-5)

• Bacterial communication

  • Quorum sensing

Biofilm Dispersal (6)

• Bacterial-mediated

  • Matrix degradative enzymes

  • Dissemination

  • Persistence

Why form biofilm

• To protect from predators

• Cooperation strategy for bacteria

• Biofilms provide a nutritional advantage for the bacteria

• To establish dominance in the environment

Biogenic Habitat-Forming Organisms

• protection/safety

• Shelter

• Stress response

• Predator evasion

Polymicrobial Nature

• Biofilms are predominantly polymicrobial

  • Multiple bacterial species

• Multi-organism communities

  • Bacterial-fungal and agal biofilms

High Bacterial Diversity = high survival

• Biofilm = sponge

• High diversity

  • Enhance EPS profile

  • High nutrition absorption

• Enhance metabolic capacity

  • High ability to breakdown complex molecules

Antimicrobial/Antibiotic Resistance

• Bacteria acquire/develop mutations that render the antibiotic/toxin ineffective

How?

• Intrinsic resistance

  • Inherited physiological property

    • Cell wall (gram-, gram +)

• Acquired resistance

  • Mutations

  • Gene acquisition

    • Horizontal gene transfer (HGT)

Antimicrobial/ Antibiotic Tolerance

• Ability to survive transient exposure to a high concentration of an antibiotic, despite being susceptible

• How

  • Intrinsic tolerance

    • Inherited physiological property

      • Spore formation

      • Biofilm formation

  • Acquired tolerance

    • Physiological property

      • Join a biofilm

      • Dormancy (metabolic shutdown)

        • Persister cells

Multi-Species Biofilm = Multi-functional biofilm

Why Biofilms

• Environmental stress

• Predator evasion

• Nutrient acquisition

• Antibiotic tolerance/ resistant

• Horizontal gene transfer

Biofilm Infections

• Approx. 34% of nosocomial infections

• 65% of all microbial infections

• 80% of chronic infection

Biogilm infection chronicity

• Biofilm prolong/ prevent wound healing

  • Re-epithelialization

  • Granulation tissue formation

• Manipulation of pro-inflammatory and anti-inflammatory responses

Anti-biofilm Strategies are difficult

• Antibiotics are becoming ineffective

• Metals such as silver and zinc can be toxic

• Debridement procedures are aggressive and invasive

Preventative strategies are god but not enough

• Wash hands

• Cover coughs and sneezes

• Clean surfaces

• Adequate ventilation

Introduction to Viruses - 02.15

Virions/Viruses

• Acellular and the virion consist of

  • DNA or RNA core

    • Single-stranded positive-sense RNA

    • Single-stranded negative-sense RNA

    • Double stranded RNA

    • Double stranded DNA

  • Protein coat (capsid)

  • Lipid envelope (on some viruses) and “spikes” (glycoproteins

• Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria (“bacteriophage”) and archaea.

• Can replicate only when within living host cell;

  • General rule: DNA viruses replicate within the nucleus while RNA viruses replicate within the cytoplasm

Viruses: Structure

• Structuve

  • Viral proteins

  • Envelop from host cell

  • Capsid forming virus structure

  • Nucleic acid

  • Nucleoprotein

Viruses: Classification

Two Systems of Classification

• Hierarchical virus classification system

Four Main characteristic are used

1. Nature of nucleic acid: RNA or DNA

2. Symmetry of the capsid

3. Presence or absence of an envelope

4. Dimensions of the virion and capsid

• The Baltimore Classification

Viruses can be classified into seven (arbitrary) groups

1. Double-stranded DNA

2. Single stranded (+) sense DNA

3. Double-stranded RNA

4. Single stranded (+) sense DNA

5. Single stranded (-) sense RNA

6. Single-stranded (+) sense with RNA with DNA intermediate in life-cycle

7. Double stranded DNA with RNA intermediate

Viral Pathogenesis

Viral Attachment and Entry

• Direct penetration

  • Viral capsid or genome is inject into the host cell’s cytoplasm (poliovirus)

• Membrane Fusion

  • The cell membrane is punctured and made to further connect with the unfolding viral envelope (influenza virus)

• Endocytosis

  • The host cell engulfs and takes in the viral particle (HIV)

Viral Replication

• Uncoating

  • Stripping off the viral protein coat, releasing the viral nucleic acid or genome

• Transcription / mRNA production

  • Virus takes advantage of host cell structures to replicate itself

  • For some RNA viruses, the infection RNA produces messenger RNA (mRNA) for translation into protein (virus components).

  • For others with negative stranded RNA and DNA, transcription occurs before translation

• Synthesis of virus components:

  • Viral protein synthesis: virus mRNA is translated on host cell ribosomes into two types of viral protein

• Viron assembly

  • Newly synthesized genome (nucleic acid), and proteins are assembled to form new virus particles (virions)

  • Can occur at the cell’s nucleus, cytoplasm or plasma membrane

Viral Latency

• Ability of some viruses to remain dormant within the host dell (occult infection), sometimes for years

• Viral genome can remain latent either as an episome or integrated in the host chromosome. The latter allows for replication of viral genome during host cell division

• Latency is maintained by a few viral genes that keep viral genome silent and escape from host immune system

• Latency can stop upon viral genome reactivation, often promoted by external stimuli (host stress cellular signals)

• Viruses with the ability for latency have two options when infecting a cell:

  • Enter the latency or the lytic pathway. The decision is regulated by expression of regulatory proteins part of a genetic switch system, usually repressor(s) as well as proteins controlling the stability of the latter. The outcome depends on the ratio of these key regulators. The ratio may be determined by environmental factors such as the host cell type, its shape, or the nutrient availability.

Viral Shedding

• Via Budding:

  • Most effective for viruses that require an envelope (HIV, smallpox)

  • Before budding, the viral receptor are placed on the host cell surface

  • Depletes host cell membrane → eventual host cell death

• Via Apoptosis

  • Forced cell suicide released viral progeny.

  • As apoptotic host cells are phagocytosed by macrophages, virus has the opportunity to get into macrophages to infect or be transported to other tissues in the body

• Via Exocytosis

  • Used by non-enveloped viruses

  • Host cell is not destroyed

  • Virus progeny are enclosed in vesicles and transported to cell membrane to be released

Protozoa 02/22/24

Pathogenesis of Infection: Protozoa 02/22/24

Paramecium

• A protozoan

• Abundant in freshwater, brackish, and marine environments (very abundant in stagnant basins and ponds)

• Readily cumulative

  • Widely used in classrooms and labs to study biological processes

Dictyostelium Discoideum

• Species of soli-living amoeba: referred to as “slime mold”

• Social amoeba

Protozoan Parasites

• Eukaryotic organism – “Proto-zoa” literally means “first animals”

• About 32,000 living species (34,000 are extinct)

  • 21,000 species occur as free-living organisms

  • 11,000 species are parasitic in vertebrae and invertebrate hosts

• Most protozoa are microscopic organism

  • Few grow to be seen by the naked eye, they are <50 um in size

• Nutrition is holozoic (type of heterotrophic nutrition) as in higher animals

  • Via osmotrophy OR absorb nutrients through cell membrane

  • Via phagocytosis: engulf food particles with pseudopodia or take in food through a mouth-like aperture called a cytostome

• Classified into four groups

  • Sarcodina (ameba)

    • Ex. Entamoeba

  • Mastigophora (flagellates)

    • ex. Giardia, leishmania

  • Ciliophora (ciliates)

    • ex. Balantidium

  • Sporozoa organism whose adult stage is not motile (can't move)

    • Ex. plasmodium, cryptosporidium

Protozoan Classes

• Amoebae

  • Use pseudopodia to creep or crawl over solid substrates

  • Similar to human macrophages

• Flagellates

  • Use elongate flagella: undulate to propel the cell through liquid environments

    • Flagella: whip-like extensions of the cell membrane with an inner core of microtubules arranged in a specific 2+9 configuration (similar to human spermatozoa)

• Ciliates

  • Use numerous small cilia which undulate (move up and down) in waves allowing the ell to swim in fluids

    • Cilia: “hair-like” extensions of cell membrane similar to flagella but with interconnecting basal element facilitating synchronous movement (similar to human bronchial epithelial cells)

• Sporozoa

  • “Spore-formers” form non-motile spores as transmission stages

  • Many pre-spore stages move using tiny undulating ridges or waves in the cell membrane imparting a forward gliding motion

Protozoan Life Cycles

• Short generation times

  • Enormous reproductive potential → rapidly causes acute disease

  • Increased chances for mutation → changes in virulence, drug susceptibility and other characteris

• Asexual or sexual reproduction

  • Asexual (in intermediate hosts)

    • Budding: outgrowth of a mature cell grows and becomes a new daughter cell

    • Binary fission: one nuclear division gives rise to two daughter cells (closest to mitosis)

    • Schizogony: “multiple fission” – nucleus divides repeatedly, allowing one cell to give rise to many daughter cells

  • Sexual (in definite hosts)

    • Conjugation: cells that java undergone a reduction division fuse, exchange haploid micronuclei, and separate – each gives ride to two daughter cells

• Some protozoa have complex life cy;es requiring two different host species: others require only a single host to complete the life cycle

Protozoan Life Cycles

Protozoan Terminology

• Trophozoite (“animals that feeds”)

  • Active, feeding, multiplying stage of most protozoa

  • Stage usually associated with pathogenesis

  • May be found intracellularly (within host cells) or extracellularly (in hollow organs, body fluids or interstitial spaces between cells)

  • Not very resistant to external environmental conditions and do not survive long outside of their hosts

• Cyst (process: “encystment”)

  • Formed by only some protozoa

  • Can contain one or more infective forms

  • Multiplication can occurs in the cyst of some species, excystation releases more than one organism

  • Cyst released into the environment (stools) have a protective wall

• Oocyst

  • A hardy, thick-walled stage of the life cycle of some protozoa (sporozoan): stage results from sexual reproduction

  • Oocysts of some protozoans (sporozoan) are passed in the feces of the host.

  • Oocysts of plasmodium (causative agent of malaria) developed in the body cavity of the mosquito vector

Protozoan Excystation

• Ultrastructural morphology details of an oblong-shaped Giardia sp. Protozoan cyst, revealing the filamentous nature of the cyst wall.

• The cyst is undergoing”excystation” with a flagellated trophozoite

Mode of Transmission

• Direct Transmission

  • Of trophozoites through intimate body contact, such as sexual transmission

    • Ex. trichomonas spp. Flagellates: causes trichomoniasis → infertility in humans and cattle

    • 

  • Fecal-Oral transmission

    • Environmentally-resistant cyst stages passed in feces of one host and ingested with food/water by another

    • Ex. entamoeba histolytica, giardia duodenalis

    • 

• Vector-borne transmission

  • Trophozoites taken up by blood-sucking arthropods (insect or arachnid) and passed to new hosts when they next feed

  • Ex. Trypanosoma brucei by tsetse flies to humans (sleeping sickness), cattle, horses, and other livestock . Plasmodium spp. Haemosporidia by mosquitoes to humans (malaria)

  • 

• Predator-prey transmission

  • Ziotres encysted within the tissue of prey animal (herbivore) being eaten by a predator (carnivore) which subsequently sheds spores into the environment to be ingested by new pet animals

    • Ex. toxoplasma gondii ingested by cats

  • 

Protozoan Escape Mechanisms

• Antigenic Masking (and Mimicry?)

  • Ability of a parasite to escape immune detection by covering itself with host antigens

• Blocking of Serum Factors

  • Some parasites acquire a coating of antigen-antibody complexes or non cytotoxic antibodies that sterically blocks the binding of specific antibody or lymphocytes to a parasite surface antigens

• Intracellular Location

  • The intracellular habitat of some protozoan parasites protects them from the direct effects of the host’s immune response.

  • By concealing the parasites antigens, this strategy also delays detection by the immune system

• Antigenic Variation

  • Some protozoa parasites change their surface antigens during the course of an infection

  • Parasites carrying the new antigens escape the immune response to the original antigens

• Immunosuppression

  • Parasitic protozoan infections generally produce some degree of host immunosuppression

  • This reduced immune response may delay detection of antigenic variants

  • Can reduce the ability of the immune system to inhibit the growth of and/or to kill the parasites

Protozoan Pathogenesis

• Cellular ,Tissue and Organ Damage

  • Extracellular or intracellular parasites that destroy cells while feeding can lead to organ dysfunction and serious or life-threatening consequences

• Toxic protozoal products

  • Toxins associated with some protozoa (plasmodium) can cause fever and chills (can occur cyclically)

• Interference with host function

  • Some parasites that inhabit the small intestine can significantly interfere with digestion and absorption and affect the nutritional status of the host

• Delayed-type hypersensitivity

  • Pathology as a consequence of immune response mediated by antigen-specific effect T cells

• Immunosuppression

  • Host is susceptible to secondary infection (by other pathogens)

Toxoplasma Gondii

Fungi 02/27/24

Pathogenesis of Infection: Fungi Tuesday 02/27/24

Some Terminology

• Mycoses (singular = mycosis);

  • Diseases of warm-blooded animals caused by fungi

• Medical Mycology

  • Study of fungi as animal and human pathogens

Fungal Pathogens

• Eukaryotic organisms:

  • separate from plants and animals

  • Genetically more closely related to animals than plants

• About 1.5 million different species exist

  • Only 300 species are pathogenic

  • Many pathogenic fungi are microorganisms

• Most are multicellular (molds), but some are unicellular (yeasts)

  • Dimorphic fungi can exist as molds or yeasts

• Heterotropic

  • Incapable of producing food

• Fungi feed by extracellular digestion

  • Saprobic

    • Live of dead organic matter

    • Help to cycle nutrients (decomposition)

  • Mutualistic

    • Some are in symbiosis with plant roots

  • Parasitic

    • Live on living organism, cause disease of the host

• Mostly asecual or secual reporduction and dispersion by spores

  • In contrast: yeast reproduce by budding or binary fission

• Have rigid cell wall (glucans and chitin)

Basic Fungal Structure

Fungal Cell Wall

Yeast (Unicellular Fungi)

• Yeast genus Candida

  • Most clinically relevant fungal group

  • Has multiple species which cause disease in humans and animals

• Yeast genus Cryptococcus

  • Causes opportunistic infections (meningitis, pneumonia)

  • Has a few species which cause disease in humans and animals (most common in cats but also in dogs, cattle, horses, sheep, goats, birds and wild animals)

Molds (multicellular fungi)

• Molds are extremely diverse group of organism, the vast majority of which are non-pathogenic

• Has two major pathologic groups:

  • Genus aspergillus

  • Order mucorales

• Molds and their pores are found in soil and decaying vegetation throughout the world

• Both can cause rhino-sinustitis and various forms of pulmonary infections in immunocompromised humans and animals

Dimorphic Fungi

• Can exist as either yeast or molds

• Typically exist in environment as a mold but shen spores are inhaled they grow in the host as a yeast

• Most commonly cause subacute pulmonary infections

Bacteria which Masquerade as Fungi

• Nocardia and actinomycetes were originally believed to be fungi, but now are known to be bacteria

• Develop filamentous, branching structures

• Causes subacute and chronic infections of the lungs, CNS, and soft tissue: most commonly seen in immunocompromised humans and animals

• Can be clinically indistinguishable from frugal disease

• Streptomycin, actinomycin, and streptothricin and are all medically important antibiotics isolated from actinomycetes bacteria

Risk Factors for developing fungal infections

• Use of antibiotics

• Use of drugs that suppress the immune system

  • Cancer chemotherapy drugs

  • Corticosteroids

  • Anti-organ rejection drugs (ex. Azathioprine, methotrexate and cyclosporine)

  • Tumor necrosis factor inhibitors (treat rheumatoid arthritis and related disorder

• Disorders

  • AIDS

  • Burns, if extensive

  • Diabetes

  • Hodgkin lymphoma ar other lymphomas, leukemia

• Environmental Factors

  • Warm, moist areas of the body (mouth, vagina)

  • Sweaty clothes and shoes enhance fungus growth on the skin

  • Indwelling catheters

  • Mechanical ventilation

• Genetic predisposition

  • Not well understood

General Features of Fungal Infection

• Often seen in immunocompromised patients or animals

  • Exceptions: dimorphic fungi, dermatophytes

• Clinical presentation is typically subacute to chronic

  • Exceptions: candidemia and mucormycosis

• Person-toperson transmission doesnt usually occur

  • Exceptions: dermatophytes and mucocutaneous candida infections

• There are no specific symptoms or signs which reliably distinguish fungal infections from bacterial ones

• Typical antibiotics are ineffective

  • Exception: pneumocystis

Classifications of Fungal Infections

• Superficial and Cutaneous Mycoses

  • Involve outermost (stratum corneum) or deeper (keratinized) layers of skin and its appendages (nails or hair)

  • “Tinea Infections” or “Ringworm”

  • Usually no inflammation (superficial) allergic or inflammatory response (when deeper)

  • Primarily caused by dermatophytes

• Mucocutaneous Mycoses

  • Involved skin, eyes, sinuses, oropharynx, external ears, vagina

  • Candidal Infection

    • Manifests a superficial mucocutaneous disease to invasive disease with dissemination

      • Oral candidiasis (thrush) and esophageal candidiasis are characterized by white patches on the tongue or mucosal surfaces (can be removed by scraping).

      • Vulvovaginitis is seen in the settings or oral contraceptive use, diabetes mellitus, pregnancy , and antibiotic therapy, it manifests with vaginal discharge and vulvar edema and pruritus

• Subcutaneous Mycoses

  • Often caused by saprophytes from soil or vegetation

  • Many are confined to tropical and subtropical regions

  • Involve subcutaneous tissues, muscles and fascia

  • Traumatic inoculation

  • Localized (often chronic) infection

  • Treatment usually involves use of antifungal agents and/or surgical excision

  • Treatment of some serious subcutaneous mycoses reamines unresolved

• Systemic (Deep) Mycoses

  • Diseases that occur deep within the tissues, involving vital organs and/or the nervous system

  • Entry into the body is usually through inhalation of spores or open wounds

  • Causes by the true pathogenic fungi (usually dimorphic fungi) or opportunistic saprobes

  • Usually subclinical/ subacute presentation

  • May be fatal (can also be chronic)

  • Examples: blastomycosis, coccidioidomycosis, cryptococcosis and histoplasmosis

  • Symptoms: fever, cough, chest pain, weight loss, fatigue

Allergic Disorders

• Mold exposure can trigger several allergic disorders, including:

  • Asthma

  • Allergic bronchopulmonary aspergillosis (ABPA)

  • Allergic fungal rhinitis

  • Hypersensitivity pneumonitis

• No established safe limits for indoor mold

• Visible mold growth in a home is not reliable measure of exposure

Mycotoxins

Types of Mycotoxins (Fungus poisons)

• Mushrooms

  • 50-100 toxic species

  • Identical to non-toxic ones

  • Many variety of toxins - different clinical manifestations and severity

  • Acute liver and renal failure: life-threatening

• Aflatoxins

  • Produced by Aspergillus sp.

  • At least 14 types

  • Contaminate corn, soybeans, and peanuts

  • Acutely, can cause liver failure

  • Chronic exposure increases risk of hepatocellular carcinoma

• Ergot Alkaloids

  • Produced by genus, Claviceps

  • Affected grains: tye, wheat, barely

  • Ergot alkaloids similar in structure to neurotransmitters, also have vasoconstriction properties.

Ergot Alkaloids

• Two forms of ergotism

  • Acute: seizures, hallucination, mani, vomiting, diarrhea

  • Chronic- ischemia, and dry gangrene of extremities

Dracunculus Medinensis – 02.29.24

Dracunculus Medinensis (Guinea Worm)

• Parasite: Dracunculus Medinensis - “Little Dragon from Medina”

  • A nematode: females up to 31” in length, males: ⅙ in length

• Disease: Dracunculiasis: Guinea-worm disease (GWD)

  • One of the older diseases known to humankind

    • Mentioned in number of historical texts

      • Sanskrit greek, hebrew (15-16 BS)

      • Parasite found in egyptian mummies (3000 years old_

  • Remains endemic in 3 countries: Sudan, Mali, and Ethiopia

  • Infects humans and domestic animals

  • Transmitted by drinking water

The Life Cycle of the Guinea Worm

Rod of Asclepius (symbol of medicine)

• Greek god Asclepius deity associated with healing and medicine

• Symbol continues to be used in modern times, associated with medicine and health care

• Historians believe that the extraction of Dracunculus Medinensis (“the fiery serpent”) on a stick led to the symbolism behind the Rod of Asclepius

Caduceus (Staff of Mercury and Hermes)

• The caduceus is recognized symbol of commerce and negotiation

• Mistakenly used as the symbol of medicine in the united states

Introduction to Helminths (Parasitic Worms)

Helminths (Parasitic Worms)

• Eukaryotic organism, invertebrates

• Relatively large (>1 mm long) some are very large (>1 m long)

• Have well-developed organ systems and most are active feeder

• The body is either flattened and covered with plasma membrane (flatworms) or cylindrical and covered with cuticle (roundworms)

• In their adult form, helminths are unable to multiply in humans

• Some helminths are hermaphrodites (monoecious) other have separate sexes (dioecious)

• Worldwide distribution: infection is most common and serious in poor countries. The distributions by clime, hygiene, diet and explore to vectors

• Many infections are asymptomatic, pathogenic manifestations depend on the size, activity, and metabolism of the worms. Immune and inflammatory responses also cause pathology

• Production losses due to:

  • Competition for nutrients

  • Damage to body systems (gut, liver, can lead to death)

• Animal welfare: companion animals - food animals

• Public health: zoonotic infections

Taxonomy of Helminths

Characteristic of Helminths

Host Types

• Definitive Host

  • Host where adult stages develop

• Intermediate Host

  • Host where immature stages develop to produce usually ineffective stages of it without reaching to maturity

• Paratenic Host

  • Immature stage retained but no parasite development

Transmission Route

• Fecal-Oral

  • Eggs or larvae passed in the feces of one host and ingested with food/water by another

    • Ex. ( Ingestion of Trichuris eggs leads directly to guy infections in humans.) (Ingestion of Ascaris eggs and Strongyloides larvae leads to a pulmonary migration phase before gut infection in humans)

• Transdermal

  • Infective larvae in the soil (geo-helminths) actively penetrate the skin and migrating through the tissues to the guy where adults develop and produce eggs that are released in host feces

    • Ex. larval hookworms penetrate the skin, undergoing pulmonary migration and infecting the guy where they feed on blood causing iron-deficiency anemia in humans

• Vector-borne

  • Larval stages taken up by blood-sucking arthropods or undergoing amplification in aquatic molluscs

    • Ex. (1) Onchocerca microfilariae ingested by blackflies and injected into new human hosts, (2) Schistosoma eggs release miracidia to infect snails where they multiply and form cercariae which are released to infect new hosts. Cause of “river blindness”

• Predator-prey

  • Encysted larvae within prey animals (vertebrate or invertebrates) being eaten by predators where adult worms develop and produce eggs

    • Ex. (1) Taenia cysticerci in beef and pork being eaten by humans, (2) Echinococcus hydatid cysts in offal being eaten by dogs.

Survival Strategies

• Morphological Adaptations

  • Degeneration of organs

    • Organs of locomotion

    • Tropic organs

    • Nervous system and sense organs

  • Attainment of new organs

    • Body shape (adapted to host environment and migration)

    • Developed of protective covering (cuticle is resistant to digestive enzymes)

    • Development of adhesive organs (sucks, hooks, jaws, secretory glands, acetabulum)

• Physiological Adaptations

  • Secretion of anti-enzymes and mucous

  • Facultative anaerobic respiration

  • Osmotic pressure adaptability

  • Chemotaxis

  • Hypobiosis

    • Critical hatching conditions

    • Periparturient rise

  • Reproductive

    • Hermaphroditism

    • Development of cyst wall

    • Fecundity

    • Complexity of life cycle

• Avoidance of Host Defence

  • Acquisition of host molecules to reduce antigenicity

  • Release substance the depress immune system (ex. Depress lymphocyte function, inactive macrophages, or digest antibodies )

  • Producing anti-complement factors (to protect their outer layers from lytic attack)

  • Release large amounts of antigenic material - antigen overload ( to divert immune responses, locally exhaust immune potential)

  • Induce a form of immune tolerance (infections acquired in life- before or shortly after birth)

Pathogenesis of Helminth Infections

• Direction damage from worm activity

  • Block of internal organs

    • Ex. Gi transit, blood flow through organs or lymph flow affected by worm size, migration and granuloma formation

  • Pressure exerted by growing parasites

    • Ex. large fluid-like cyst in the liver, brain, lungs or body cavity

  • Physical Damage

    • Ex. tissue necrosis, feeding by worms, damage during migration

  • Chemical damage

    • Ex. release of excretory-secretory materials, release of enzymes and factors such as anticoagulants)

  • Nutritional impact in under-nutritioned individuals or animals

• Infect Damage from Host Response

  • Hypersensitivity-base inflammatory changes

    • Ex. contribute to lymphatic blockage associated with filarial infections)

  • Local allergic responses

    • Eosinophilia, edema, and joint pain

  • Structural changes

    • Ex. villous atrophy, mucosa permeability changes

Simple (direct) Life Cycle: Enterobius Vermicularis (pinworm)

Complex Life Cycle: Paragonium westermani (Lung Fluke)

Complex Life Cycle: Fasciola Hepatica (Liver Fluker)

Prion Disease 03/05/24

Prion Diseases (transmissible spongiform encephalopathies (TSEs))

• Humans

  • Kuru (“to shake”)

  • Creutzfeldt Jakob Disease (CJD)

  • Fatal Familial Insomnia (FF)

  • Gerstmann Straussler Syndrome (GSS)

• Cattle

  • Bovine spongiform encephalopathy (Mad Cow Disease) (BSE)

• Sheep/Goats

  • Scrapie

• Deer/ELK

  • Chronic Wasting Disease (CDW)

Characteristics of TSEs (Prions Disease)

• Spongiform (brain vacuolation

• Neuronal loss, gliosis and astocytosis

• Atrophy of brain tissue

• Accumulation of misfolded prion plaques

Different Prions affect different parts of the brain

• Cerebral cortex

  • Symptoms: loss of memory, mental acuity, visual impairment (CJD)

• Thalamus

  • Damage leads to insomnia (FFI)

• Cerebellum

  • Damage leads to body coordination movement problems and hard to walk (kuru, GSS)

• Brain stem

  • Mad cow disease (BSE) – brain stem is affected

Characteristics of Infection

• Encephalitis (inflammation of the brain)

• widespread neuronal loss

• Loss of motor control

• Dementia

• Paralysis

Kuru

• Identified by epidemiology in Papua New Guinea based on anthropological research by Robert and Louise Glasse in 1950’s

• 1% of the Fore tribe was afflicted: most women, some children, few adult males

• Symptoms: headache, joint,6-12 weeks later →difficult walking, death within 1-2 years

• 1910-1920

• Evidence lead Glasse to suggest that endocannibalism was associated with disease

• Carlton Gadjusek did research and proved Kuru killed patients

From Kuru to Scrapie

• Animals needed to study TSEs

• Scrape disease in sheep is similar to kuru with symptoms and etiology

• Scrapie can be transmitted to hamster and mice, not kuru

• Mice infected with scrape were the first good animal model

• Infectious agent purified 5000 fold:

  • Nuclease resistant

  • UV and heat resistant

  • Sensitive to protease (only at high levels) and protein denaturants

Bovine Spongiform Encephalopathy (BSE) “mad cow disease”

• 1970: hydrocarbon-solvent extraction of meat and bone meal for cattle feed was abandoned in Britain

• 1986: 7000 infected cows. BSE became reportable, epidemiology suggested a prion ideas, and meat and bond meal use was abandoned

• BSE incubation period is 5 years: 1 mill cows were infected

• 1989: human consumption of bovine CNS tissue (though to have highest prion concentration) banned based on fears of transmission to humans

• 1996: new type of CJD appeared in Britain and France on young patients and different neuropathology. Linked with consumption of BSE-contaminated beef

Introduction to Prions

• “Pree-ons”

• Shortened for “proteinaceous infectious particle”

• Causes transmissible spongiform encephalopathies (TSEs) among other disease

• No treatment available to halt progression of TSEs. Treatment in humans is aimed at alleviating symptoms and making patient comfortable

• TSEs are ALWAYS FATAL

Basic Structure

• Normal prions (PrP^C ) – common in brain cells

  • Contains 200-250 amino acids twisted into coils(helices) with tails of more amino acids

• The mutated and infectious form (PrP^SC )

  • Built from same amino acids but take different shape

  • 100x smaller than known virus

Types of Prion Diseases (TSEs)

• Sporadic

  • Occur with no prion protein mutation

  • Most TSEs are sporadic

  • Cause unknown

  • Infected 1-2 mill people late in life

• Infectious

  • Ex. Kuru,, BSE (mad cow disease), Scrapie

  • Consumption of infected material

  • Iatrogenic spread (organ transplant, esp. corneaL been operated with surgical instruments used on a CJD patient)

• Familial / Hereditary

  • Due to autosomal dominant mutation of PrP

  • inherited : 10-15% of total human TSE cases

Properties of Infectious Prions (PrP^SC )

• Resistant to degradation by proteases → leads to accumulation

• B-sheet structure of PrP^SC have high affinity for other B-sheet structures (in other proteins or other PrP^SC) → forms oligomeric, insoluble aggregates that in turn form toxin amyloid plaques → interferes with cell and tissue function and death of cells and tissues

• PrP^SC are extremely resistant to heat and chemicals

• PrP^SC are very difficult to decompose biologically

  • Survive in soil for many years

• Prions are not nucleic acids (ex. DNA or RNA0

• How do infectious prions propagate>

  • During oligomerization the prions corrupt the native form of the protein into a transmissible disease conformation

Formation of Infectious Prions PrP^SC

Intro to Immunology and Innate Immune System 03.12.24

Basic Concepts of Immunology

• Immunology is the study of the body's defense against infection. We are continually exposed to microorganisms, many of which cause disease, and yet become ill only rarely

• How does the body defend itself?

• When infection does occur, how does the body eliminate the invader and cure itself?

• Why do we develop long-lasting immunity to many infectious diseases

Components of the Immune System

• Basic defense system of the body

• Composed by specialized cell types and organized structures that coordinate defense mechanisms

• Protects from harmful pathogens and disease

• Prevent and limit infection

• When unsuccessful in curving the infection disease arise

History of Immunology

• Edward Jenner, developed the first vaccine against smallpox (18th century). Develop the first vaccine by infecting a patient with cowpox and demonstrating that the patient became immune to smallpox.

• Louis Pasteur, France, 19th century: Demonstrated that infectious diseases are caused by microorganisms (Germ theory of disease). Pasteur developed the first vaccine against rabies by attenuating the rabies virus, making it less harmful.

• Robert Koch, Germany, 19th and early 20th centuries: Koch's work on tuberculosis and anthrax led to significant breakthroughs in our understanding of these diseases and their treatment. Laid the foundation for modern microbiology and the development of antibiotics.

Components of the Mucosal Immune System

• Several immune factors function in concert to:

  • Stratify luminal microbes

  • Minimize bacterial-epithelial cell contact

  • Tolerance towards food antigens and commensal microorganism (“oral tolerance”)

  • Prevent the induction of unnecessary systemic immune responses (“compartmentalization”)

• Work in concert to:

  • Survival and respond accordingly to microbial threats

  • Maintaining a balance between tolerance and defense mechanisms

Components of Barrier Function

• Intestinal barrier are made up of numerous different cell types

  • Enterocytes (absorptive)

  • Goblet cells (mucus-producing)

  • Paneth cells (found in crypts, produce antimicrobial compounds)

• All these cell types develop from a common stem cell at the base of the intestinal crypts

• Structure:

  • One layer of epithelial cells tightly adhered to each other

  • Epithelial cell shedding

  • Unidirectional flushing

  • Inteintal barrier: surface area 25000 sq ft

• The junctional complex

  • Transcellular proteins connected through adaptor proteins to the actin cytoskeleton

  • Important in maintaining cell polarity

  • Desmosomes are localized dense plaques that are connected to keratin filaments

Components of Barrier Function: Mucus Layer

• Glycoproteins called mucin by goblet cells

• Protein core with several polysaccharide molecules attached

• 2 layer of Mucin

  • Outlet layer: colonized by microorganisms

  • Inner layer: high concentrations of antimicrobial peptides prevents microbial colonization “Killing Zone”

Microbial Sensing of Intestinal Environment

• Presence of microorganisms close to epithelial surfaces are recognized by APCs via PRRs

• Activated Dcs stimulate IL-22 secretion by innate lymphoid cells

• Stimulates epithelial cell proliferation and the secretion of antimicrobial peptides:

  • Defensins, REGIII, Lactoferricin

• AMPs act as lytic enzymes disturbing microbial cell membrane

• AMPs disrupt microbial cell membrane by forming pore

Pattern Recognition Receptors (PRRs)

• Several germline-encoded receptors used to recognize different MAMPS

• Sensing of MAMPS through the PRRs induces tissue repair and epithelial cell proliferation

Immunological Components of Barrier Function:

• Lamina Propria

  • Loose connective tissue

  • Contains several immune cells

    • Antigen-presenting cells (APCs)

    • T cells and B cells

    • Innate lymphoid cells

    • Other immune mediators (complement, chemokines, cytokines)

Microbiology Research: Pre-NGS Periods

• After Louis Pasteur discovered bacteria, medical research focused mainly on their role in causing disease.

• The bacteria that reside in and on our bodies were generally regarded as either harmless commensals, or pathogens.

• Tools available only allowed us to study these microorganisms one at a time, rather than as communities.

• Research focused on the few bacteria that could be grown in vitro. But the majority of microbes that live in our bodies are extremely hard to grow in vitro

Microbiology Research: POST-NGS

• Culture-independent techniques (NGS) allow the study of the ecological complexity of the intestinal microbiota and its impact on host physiology.

• Have revolutionized the field of microbiology and broadened the lens to explore the functional roles of microbiota on host health and disease.

• Intestinal microbiota serves an important role in protecting health, by shaping metabolic and immune function both locally and systemically

• Microbial cells in intestine surpass human cells by a factor of 10

• Microbial genome is 100 x more extensive than human genome

Intestinal Microbiota role in Health and Disease

• The intestinal microbiota have likely evolved under selective pressure to effectively degrade nondigestible plant carbohydrates enhancing the host digestive efficient

• Additionally

  • Metabolic: Vitamin B, K and SCFA

  • Structural: promote epithelial cell proliferation mucin production

  • Protective: Competitive exclusion of non-resident bacteria and pathogens

• Bidirectional communication between the host and intestinal microbiota via a wide range of metabolites (SCFA, Indoles, BA)

Niche Occupation and Competitive Exclusion

• Mucin glycans are nutrients for some bacteria

• Establishing a physical barrier (niche Occupation) excluding potential pathogens

• Reduce pH by production of SCFAs acetate and lactate

• 

Components of the Immune System

Where do the cells of the immune system come from?

• Hematopoiesis in the Bone Marrow

  • Production of blood cells

  • Myeloid

  • Lymphoid

• Maturation of Lymphocytes occurs in Central Lymphoid Organs

  • Bone marrow of animals (Bursa of Fabricius in birds) for B cells

  • Thymus, for T cells

Immune Cells

• Stem cell differentiation from bone marrow

• All lymphoid and myeloid cells are derived from hematopoietic stem cells in the bone marrow

• Lymphoid and myeloid cells in circulation are collectively referred to as leukocytes or WBCs

• Cells are differentiated by appearance and surface markers, clusters of differentiation: CD4, CD8, T cell receptor and C cell receptor

Innate Immunity Faster Response NonSpecific vs Adaptive Immunity Slow response on first exposure specific, develops memory

Innate Immune system Part 2 03.14.24

Neutrophils

• Components of the immune system innate immunity

• 50-60% of circulating immune cells

• Produce potent antimicrobial toxins, stored in vesicles

• First responders to infection

• Release mesh like structure composed of cytoskeleton and DNA that traps microorganism

• Miltibulate nucleus

• Lysosomal granules

• Activation of bactericidal mechanism

• Phagocytosis and kill

• 1st white blood cell to infection site

• Die and release their contents

• Irritate surrounding tissue/recruit other cells

• Phagocytosis is improved by opsonization with immunoglobulins

• Neutrophils enter site of injury and infiltrate

• Release factors important in immune response

Eosinophils and Basophils

• Eosinophils

  • Granules

    • 0.2-3% circulating immune cells

    • Effective in killing parasites

• Basophils

  • Granules

    • <0.5% circulating immune cells

    • Coordinate immune response to parasitic infection

Macrophages and Monocytes

• Tissue resident immune cells (sentinel)

• Constantly patrolling extracell spaces

• Engulf and phagocyte microbes

• Coordinate immune activation and promote recruitment of other immune cells

• 

Macrophages

• Large cell (10-25 uM diameter)

• Main purpose: phagocytosis/kill

• Act non-specifically

• Chemotactic capability

• Release cytokines

• Potent phagocytosis when activated by T lymphocytes

• Express antigen on surface to T/B cells

Dendritic Cell

• “Professional” antigen presenting cells

• Derived from monocytes

• Located at barrier sites and lymphoid tissues (Lymph nodes, peyer’s patches)

• Main function is to present antigens to T and B cells

• Initiate immune response

• Promote tolerance towards harmless microorganism or to self antigens

Sensing Mechanisms of Innate Immunity

• Coordination of the innate immune response relies on the information provided by many types of receptors

• Pattern recognition Receptors (PRRs)

• Evolutionarily ancient pathogen system of recognition and signaling

Toll Like Receptors

• Limited specificity compared with the antigen receptors of the adaptive immune system

• Recognize elements of most pathogenic microbes

• Are expressed by many types of cells:

  • Macrophages

    • Dendritic cells

    • B cells

    • Stromal cells

    • Certain epithelial cells

• Enabling the initiation of antimicrobial responses in many tissues

• Toll-like receptor family (10 in humans and in cattle, 12 in mice)

• Extracellular and intracellular

• Bacterial, viral, year associated molecular patterns

NF-kB pathway

• Cytokines and chemokines responsible for initiation of innate immune response

NOD Like Receptors

• Nucleotide binding oligomerization dimer (NOD)- like receptors

• Intracellular

• NOD recognizes muramyl dipeptide from peptidoglycan

• Detection of bacterial peptidoglycan by NOD receptors

• Some NLR activate NFkB to initiate the same inflammatory response as the TLRs

• Other NLRs trigger a distinct pathway that induces cell death and inflammation through formation of Inflamasome

RIG-I and MDA-5

• Intracellular pattern recognition receptors

• Involved in viral recognition

• Sentinels for intracellular viral RNA produced within the cell in contract with TLRs

• Provide frontline defense against viral infections in most tissues

• 

Cytokine Signaling

• Activated macrophages secrete a range of cytokines

  • Local effects

    • Systemic effect

Cytokines signaling molecules of the immune system

Mammalian Inflammatory response in response to bacterial infiltration through the physical barrier

• Inflammatory response

  • Sensed by the integrated mast cells; neutrophils. Macrophages and effectors cells

  • Production of cytokines and chemokines

    • Interleukin-1beta

    • Interleukin-6

    • Interkelin-12

    • TNF-alpha

  • Vasodilation

  • Immune cell recruitment

Wound Healing

1. Pathogen infiltration through wound

2. Mast cells secrete factors that mediate vasodilation and vascular constriction. Delivery of blood, plasma, platelets and cells to injured area increases

3. Platelets from blood release blood-clotting proteins at wound site

4. Neutrophils secrete factors that kills and degrade pathogens

5. Neutrophil and macrophages remove pathogens by phagocytosis

6. Macrophages secrete hormones called cytokines that attract immune system cells ot the site and activate cells involved in tissue repair

7. Inflammatory response continues until the foreign material eliminated and the wound is repaired

Map

• Inflammatory trigger → inflammation → physiological purpose → pathological consequences

Inducers of Inflammations

• Exogenous Inducers

  • Pathogen associated molecular patterns (PAMPS)

  • Virulence factors from microbial inducers

  • Commensal bacteria which can also induce inflammation (through TLRs)

  • Non-microbial origin: allergens, irritants, foreign bodies, toxic compounds

  • Foreign bodies

• Endogenous Inducers

  • Sequestration of cells or molecules kept in intact cells and tissues

  • Necrotic cell death

  • Epithelial-mesenchymal transition

  • Damage to vascular endothelium

  • Chronic inflammation

  • 

Complement System

• Nine plasma proteins (C1 to C9) that are produced by the liver and circulate in the blood in inactive form

• Sequential activation leads to assembly of large donut-shaped membrane attack complex (MAC) with the assembly of the C5-C9

• MAC inserts in microorganism to form pores

• 3 outcomes of complement activation

Complement System

• Antimicrobial response continued: complement pathway

• Cascade sequences that amplify

  • Classical:

    • binding to antibodies (adaptive)

  • Alternative:

    • directly binds to foreign invader (innate) - polysaccharides from yeast, gram negative bacteria

  • Lectin Pathway:

    • stimulated by mannose containing proteins and carbohydrates on microbes (viruses and yeast)

Complement System: Classical pathway

• Binding to antibodies(Y-shaped molecules) and attached to a particular foreign invader

• Initiates complement activation cascade

• Activated complement C1

Complement System: Alternative Pathway

• Binding directly to a foreign invader non specifically activates the complement cascade

• Activates complement C1

• Pathogenic bacterial cell

The Acute phase response

• Cytokines (TND-a, IL1B, and IL-6) act on hepatocyte toL

  • Promotes a change in the profile of proteins that they synthesize

  • Blood levels of some proteins go down (Albumin), whereas levels of others increase markedly (SAA)

  • C-reactive protein: opsonization and complement activation

  • Serum Amyloid A: chemoattractant and inflammasome activation

Role of Interferon in Viral Defense Mechanisms

• Called interferon because it interferes with viral replication

• Block spread of viral replication on neighboring cells

1. Signal neighboring cells to put up barriers

2. Signal infected cells to die

3. Recruitment of white blood cells to stimulate long lasting immunity

• Released from virus-infected cells

• Transiently interferes with viral replication

• Enhance actions of NK cells

• Slows cell division and suppresses tumor growth

SARS-COV-2 interferes with interferon signaling

• SARS-CoV-2 inhibits multiple arms of the type I IFN response: Reduce IFN-b production during infection. Inhibits recognition of the foreign viral RNA by RIG-I Inhibits phosphorylation of repressor subunit of NF-kB Reducing import into the nucleus and reducing transcription of type I IFN

Effects of SARS-CoV-2 on respiration

• Moderate damage: accumulating fluid, reduced gas exchange

• Severe damage: build up of protein-rich fluid, vert limited on gas exchange

Cytokine Storm

1. Coronavirus infects lung cells

2. Immune cells, including macrophages, identify the virus and produce cytokines

3. Cytokines attract more immune cells, such as white blood cells, which in turn produce more cytokines, creating a cycle of inflammation that damages the lung cells

4. Damage can occur through formation of fibrin

5. Weakened blood vessels allow fluid to seep in and fill the lung cavities leading to respiratory failure

Adaptive Immune System-T Cell Development and Function: 03.19.24

Stages of Adaptive Immune Response

Components of the immune system: immune cells

Innate Immune System

• Both have hematopoietic cell in bone marrow

Adaptive Immune System

• Both have hematopoietic cell in bone marrow

Components of the immune system: Immune Cells

• T cells recognize specific antigen fragments presented by other cells

• Orchestrate immune responses to eliminate pathogens, infected cells, and abnormal cells

• The vast diversity of function between different T cell subsets ensures that responses are tailored to the specific need

• 

T Cell Development in Thymus

• Selection process

  • Positive selection

  • Negative selection

• Enter circulation as

• CD4+: T Helper Cell

• CD8+: Cytotoxic T cell

• Treg: T regulatory cell

T Cell Receptor

• Recognize specific antigen via the T CELL RECEPTOR (TCR)

• TCR is a multi-subunit surface protein

  • Constant region

  • Variable region

• How million of different antigen receptors can be generated from a limited amount of coding DNA in the genome

Gene Recombination

• Diversity of TCR is accomplish by unique random genetic rearrangement of germline gene segments

• Functional receptors are created during T cell development in the thymus by randomly choosing V, D and J gene segment

• When progenitor T cell arrives in the thymus thymic factors (thymosin, thymopoietin) activate RAG genes

• RAG genes encodes for recombinase enzyme that induces recombination of germline encoded DNA

• Terminal deoxynucleotidyl transferase (TdT) binds V-D-J sections by randomly adding new bases, Junctional diversification

• 

• This gene rearrangement and junctional diversification occurs on both the alpha and beta chains of TCR

• This creates a virtually unlimited TCR repertoire allowing to recognize millions of different antigens from aMHC- limited coding region of the DNA

• However, almost ⅔ of the time junctional diversification will result in a non-functional TCR

T Cells recognize antigens in the context of MHC molecules

• MHC MOLECULES (Major Histocompatibility complex) present antigens to T Cells

• 2 classes of MHC

  • MHC-I expressed in all nucleated cells present antigens to CD8+ T cells

  • MHC-II expressed by antigen presenting cells (APCs), present antigens to CD4+ T cells

• Discovered to be responsible for tissue graft rejection in transplantation studies

• T cells only recognize “own” MHC molecules

• MHC complex is an extended genetic locus that contains several polygenic genes that are involved in antigen presentation to T cells

• In humans MHC complex is called HLA (Human Leukocyte Antigen) and is located in chromosome 6

• In Cattle MHC complex is called BoLA (Bovine Leukocyte Antigen), and is located in chromosome 23

MHC Molecules that most polymorphic gene in the mammalian genome

• polygenicL the NHC region contains multiple genetic loci encoding proteins with the same function:

• In humans (chromosome 6)

  • HLA-A (14,000 alleles), B, or C molecules can all present peptides to CD8+ cell

  • HLA-DPm DQ or DR molecules can present to CD4+ T cells

  • Polymorphic: each gene in the MHC locus has several alleles within the population

  • Co-dominant expression: Alleles from each haplotype are expressed in any one individual

  • “Polygeny”, “Polymorphism” and “Co-Dominant expression” ensure a diversity of MHC molecules providing protection of a populations from virtually unlimited diversity of microbes and therefore prevents loss of entire population from emerging infections

MHC Class I Molecules

• Consist of an alpha chain associated noncovalently with a b2-microglobulin chain

• Expressed in all nucleated cells (not expressed by red blood cells)

• Main function is to present peptides to CD8+ T-lymphocytes, which kill virus-infection cells

• Viral peptides produced within the cell are digested by proteasome

• Antigen peptide fragments are transported to endoplasmic reticulum with the help of TAP (transporter protein) and loaded into MHC-1

• When MHC-I binds to an antigen it detaches from TAP and migrates from ER to cell surface

MHC Class II Molecule

• Expressed only on “professional” antigen presenting cells such as DCs and Macrophages

• Noncovalent association of polymorphic alpha and polymorphic beta chain

• Main function is to present antigens derived from extracellular pathogens to CD4+ T cells, which once stimulated activate macrophages and B cells to generate inflammatory and antibody responses respectively

• Antigen is taken up via endocytosis

• Endosome becomes increasingly acidic which activates digestive protein called endosomes/lysosomes denaturing the protein fragment into small peptides

• MHC0II molecules resides in specialized compartment hold in placed by an invariant chain

• Once the endosome containing peptide fragments duse with the MHC-II endosome invariant chain is cleaved leaving a small fragment called CLIP

• CLIP is replaced by antigen peptide and MHC-II is loaded to cell surface

T-Cell Development in Thymus

• A gene present in the thymus allows thymic epithelial cells to express many self antigens to developing T Cells

• If a double positive developing T cell engages first with MHC-II to commits to CD4+

• If they bind with high affinity to MHC they are eliminated

Central Tolerance and Peripheral Tolerance

• A subset of self-reactive T cells are allowed to survive and develop into regulatory T cells that promote and maintain tolerance towards self antigens (Central tolerance)

• In the periphery certain signals from antigen presenting cells can induce mauve T cells to differentiate into regulatory cells (Peripheral tolerance)

T Cell Function: CD8 T cells

• Once a T cell recognize an antigen, naive T cells differentiate into several functional classes of effort T cells that are specialized for different activities

• CD* T cells recognize pathogen peptides presented by MHC class I molecules

• Naive CD8 T cells differentiate into cytotoxic effector T cells that recognize and kill infected cells

T Cell Function: CD4 T Cells

• CD4 T cells have more flexible repertoire of effector activities

• Once they recognize pathogen peptides presented by MHC class II molecules

• Naive CD4 T cells can differentiate into many different subsets with different immunological functions

• Main CD4 effector subsetsL TH1, TH2, TH17, and TFH, which activate their target cells

• Regulatory T cells or Treg cells inhibit the extent of immune activation

yS T Cells, a major lymphocyte subset in calves

• Unconventional T cells

• Major subset in calves (40-60% lymphocytes)

• Directly bind antigen

• Not MHC restricted

  • Effector yS T cells, WC 1.1+

    • Early protection against intracellular infections while alpha and beta T cells effector function is blunted

  • Regulatory yS T cells, WC 1.2+

    • Immune tolerance (IL-10, TGF-Beta)

T Cells activation by APCs

• T cell activation occurs in 3 steps

1. APCs present antigens to T Cells in context of MHC molecules

2. Level of expression of B7 on APC dictated by inflammatory signals influence T cell polarization

3. Secreted co-stimulatory signals from APCs determines CD4 T cell fate

Development of Peripheral tolerance

• With no inflammatory signals APCs don't express B7

• 

Development of effector function

• When inflammatory signals prime APC activation B7 expression increases\

• 

T Cell Activation

T Cell activation

• Antigens are transported by dendritic cells to lymph nodes

• Naive T lymphocytes recirculate through these lymph nodes

• If a T cells has a receptor that recognizes the specific antigen, it becomes activated

• Once activated it will rapidly expand (Clonal Expansion) and differentiate

• Activated T cell can remain in lymph node (T Follicular Cell) to help with B activation

• Or migrate to sites of infection

C4-B cell function and Development - 03.21.

Stages of Adaptive Immune Responses

1. Establishment of infection

2. Induction of adaptive response

3. Adaptive immune response

4. Immunological memory

B cell function and development

1. Initial exposure

2. Primary immune response

   1. Primarily lgM

3. Secondary exposure

   1. Much shorter lag phase

4. Secondary immune response

   1. Isotype antibody switch

   2. Dramatic increase in antibody production

   3. More efficient binding affinity

   4. Long lasting memory

B Cell Antibodies

• B cells can directly recognize specific antigens via B cell receptors/antibodies and does not need antigen presentation by MHC

  • BCR: membrane bound

  • Antibody: secreted

• BCR/Antibodies

  • Constant region

  • Variable region (antigen specificity)

• Antigens

  • Carbohydrates

  • Lipids

  • Protein / peptide

B Cell Development

• Fetal development of B cells

  • Fetal liver (FL)

    • FL HSC

    • Pro-B

    • Pre-B

    • Immature B

  • All into B-1B cell

• Postnatal Development of B cells

  • Bone Marrow (BM)

    • BM HSC

    • Pro-b

    • Pre-b

    • Immature B

  • Into spleen

    • Transitional B-2B cell intoL

      • Follicular B-2B Cell

      • Marginal zone B cell

B Cell Development

• B Cells receptor undergoes a similar genetic rearrangement as the T cell receptor

• For a B cell to become activated more than 1 BCR must recognize the antigen and become cross link

• This requires than antigens that have repetitive epitopes so that more than one BCR can bind at the same time

• Most self protein lack repetitive epitopes providing a mechanisms of self tolerance

B Cell Activation

• Once a fully functional B cell develops in spleen it migrates to lymph nodes (Follicular B cell) or stays in Spleen (marginal B Cell)

• In germinal center of lymph node if a B cell recognizes an antigen through if a B cell recognizes an antigen through its BCR it quill rapidly start to divide (Clonal Expansion)

• After this step it requires stimulatory signals from T follicular cells to survive

• High rate of cell division induces mutations in BCR (Somatic hypermutation)

• Some mutations will lead to better binding affinity to antigen (Affinity maturation)

• Every time B cells divides in germinal centers i needs to present antigens to Tfh cells to keep dividing

• The only sources of antigen comes from “reservoirs” from Follicular DCs

• If the hypermutated BCR has low affinity it cannot take antigen from Dcs and it is programmed for cell death

• If new BCR has high affinity it can then present antigen to Tfh cells

B Cell Activation Summary

• APCs are continuously sampling luminal antigens

• Leads to antigen-specific immune responses that promote the production of dimeric lgA

• slgA aids in antigen sampling from the intestinal lumen

  • 1. TLR ligand recognition promotes naive T cell differentiation

  • 2. Tfh cells promote B cell differentiation into lgA+ B cells inside the Peyer's patch

  • 3. lgA+ B cells become plasma cells and secrete lgA to regulate gut microbiota

Antibody Classes; lgG

• Principle Ab in serum

  • 14-18 mg/mL

  • lgG1: 11 mg/mL

  • lgG2: 7 mg/mL

  • Fixes complement

  • Late response to antigen

• lgG1

  • Selective transfer (colostrum)

  • fetal/neonatal defense

  • Toxin inactivation

  • Principal milk /colostrum lg

  • Fc portion involved in endotoxin neutralization and complement fixation

• lgG2

  • Primary opsonin for phagocytosis

  • Fc portion: Opsonization and neutrophil phagocytosis

  • In cattle lead from blood to infected mammary gland and act as key opsonin to help neutrophils target and clear mastitis causing bacteria

Antibody Classes: lgM

• Largest lg

• Serum (1-3 mg/mL)

• Fixes complement

• 1st lg produced to antigen challenge

• Fc portion is involved in blocking and complement fixation

Antibody classes: lgM

• Bind to masts cells

• Binds to basophils

• Activation

• Release of

  • Histamine

• Involved in allergy progression

Antibody Classes: lgA

• Main antibody at mucosal sites

• When secreted into intestinal lumen (secretory component)

• Activates complement system in serum, but not in milk

• Local immunity an secretions

  • Prevents bacterial adherence

  • Maternal milk: very important

  • Primary Ig in colostrum of humans

Antibody Effort Functions

• Class switching promoted by Tfh cells allows to tailor antibody responses to the specific need

Allergy Induction

• Early life dysbiosis can result in aberrant immune development

• Local intestinal conditions drive immunoglobulin class switch to lgE rather than lgA

1. Allergens enter via inhalation and bind to lgE on masts cells

2. Mast cells are activated, leading to increased mucus production, nasal irritation, and contraction of bronchial smooth muscle.

Immune System - Antibodies April 2nd

Antibodies

Cell Mediated and Humoral Immunity

Function of Antibodies

• Opsonization

  • Bacterium

  • phagocyte

• Complement system activation

  • Complement system protein

  • Bacterium

  • Opsonization by C3b

  • Inflammatory response

  • Lysis of foreign cells

• Immobilization and Prevention of Adherence

  • Bacterium

  • Flagellum

• Cross-linking

  • Bacterium

  • ex. aggregation

• Antibody-dependent cellular cytotoxicity (ADCC)

  • Natural killer cell

  • Kills cell

  • Infected “sell” cell

• Neutralization

  • Virus

  • Toxin

Antibodies (Ab)

• Antibodies are also called immunoglobulins (lg)

• Y-shaped protein

• Two copies of heavy chain and light chain

  • Two identical variable regions that bind antigen: specificity

  • Stem is constant region: immune system recognition

Antibody Classes

• Five major classes: lgM, lgG, lgA, lgE, lgD

• Have some basic monomeric (single unit) structure

• Monomer: single Y shaped molecule

• Each class has different constant region of heavy chain

• Some classes from multimers (binding of multiple units).

• Each class has distinct functions and properties

Immunoglobulin G (lg G)

• Monomer (2 binding sites)

• Most common Ab (80% of total Ab in serum): half-life: 23 days (longests protection)

• Location: blood, lymph, intestine

• Can cross placenta from mother to child (in humans, and some animals, NOT in cattle, sheep, pigs and horses)

• Can activate complement (classical pathway)

• Fc region binds phagocytes

• Function:

  • Neutralization, agglutination, complement activation, opsonization and antibody-dependent cell-mediated cytotoxicity.

  • First and most abundant during secondary response

Immunoglobulin M (lg M)

• Pentamer (10 binding sites)

• 5-10% of total antibody in serumL half-life: 5 days

• Location: blood lymph, B-cell surface (monomer)

• Cannot cross placenta

• Can activate complement ( classical pathway)

• Function

  • First antibodies produced in response to first exposure to antigen

  • Neutralization, agglutination, and complement activation

  • Large size percents crossing from bloodstream to tissues. Primary role therefore in bloodstream infections

  • Monomeric form serves as B-cell receptor

Immunoglobulin A (lg A)

• Dimer with secretory component (4 binding sites)

• 10-15% of total antibody in serum; half-life: 6 days

• Location: secretion (ex. Tears, saliva, mucus, intestine, respiratory tract, milk): monomer: blood, lymph

• Cannot cross placenta

• Cannot activate complement

• Function

  • Protection of mucosal surfaces: neutralization and trapping of pathogens in mucus, prevents attachment of pathogens to epithelial cells.

  • Protects breast-fed infants against intestinal pathogens. Not absorbed by a gut after gut closes, protects the gut barrier from within the lumen

Immunoglobulin D (lg D)

• Monomer (2 binding sites)

• 0.2% of total antibody in serum: half-life: 3 days

• Location: blood, lymph and B-cell surface

• Cannot cross placenta

• Cannot activate complement

• Function

  • Serves as B-cell receptor: initiation of acquired immune response

  • Function in serum is largely unknown

Immunoglobulin E (lg E)

• Monomer (2 binding sites)

• 0.002% of total antibody in serum: half life: 2 days

• Location: bound to mast and basophils through body and blood (Fc regions binds mast cells and basophils)

• Cannot cross placenta

• Cannot activate complement

• Function:

  • Activation of basophils and mast cells against parasites and allergens

  • When bound to antigens, triggers release of histamine (and other compounds) from mast cells and basophils that contribute to inflammation and some allergic responses

  • 

Class Switching

• Plasma cells normally secret lgM when first activate

• T helper cells can induce some activated B cells to become plasma cells that secrete other antibody classes

• B cells in lymph nodes usually switch to lgG

• B cells in mucosal tissues often switch to lgA

Primary vs. Secondary Response

• First response to antigen is the primary response.

  • Adaptive immune system “remembers” specific antigen

  • If encountered again, a stronger secondary response results

• Secondary response to antigen is strong and faster because the body can remember the first encounter and fight back

  • Combination of rapid proliferation of memory cells and on-going affinity maturation on subsequent exposures

Maternal Transfer of Immunity and Vaccination 04/04

Modes of Adaptation Immunity

• Maternal transfer of antibodies to infant via placenta or breast milk

• Administration of pre-formed substances to provide immediate but short-term protection (immunoglobulin, antitoxin)

• Passive Immunity

  • Give rapid protection within 48 hours

  • Protection is temporary and wanes with time (usually few months)

Natural Passive Immunity: Maternal Transfer

• Maternal transfer of antibodies to offspring via:

  • Placenta (lgG)

  • Colostrum (primarily via lgG, short (24h) window of opportunity)

  • Breast milk (primarily secretory lgA, mostly protects against enteric pathogens)

• No memory: protection is lost once antibodies degrade.

• No evidence that some maternal lymphocytes may be transferred via breast milk: these lymphocytes may take residence in neonates' peyer's patches (role in immunity is not well-understood.)

• Not all animals are capable of trans-placental transfer of antibodies (lgG)

  • These animals are critically reliant on colostrum for passive immunity.

Artificial Passive Immunity: Immunoglobulin Therapy

• Provided by administering immunoglobulins (lg) for post-exposure prophylaxis

  • Ex.

    • human normal immunoglobulin (HNlG). Collected from pooled human donations – contains antibodies to infectious agents common in the community

    • Hepatitis B immunoglobulin (HBIG)

    • Varicella Zoster immunoglobulin (VZIG)

    • Rabies immunoglobulin

• Also used to treat some immunodeficiencies/ immune disorders

  • Lupus, multiple sclerosis, after bone marrow transplant)

• Involves use of blood-derived products

  • Safety concerns, possible side effects such as serum sickness

Vaccination: Artificial Active Immunity

Variolation

• Variolation or inoculation: first method used to immunize an individual against smallpox (Variola)

• Edward jenner noticed milkmaids who recovered from cowpox rarely got smallpox

• In 1796, Jenner exposed a boy (James Phipps) to material from cowpox lesion: then six weeks later exposed him to smallpox

  • Phipps did not catch smallpox. He was immune

  • Jenner and others worked to spread variolation: the use of less dangerous cowpox material to protect against smallpox.

• Louis Pastuer honored Jenner by using the term vaccination (vacca is latin for cow) to describe artificial induction of a community against any infectious disease.

Vaccination

• A vaccine is a preparation of pathogen or its products that elicits an immune response

• Vaccines establish immune memory without the pathogenic events that are typical of first encounter with virulent pathogen

• One of the most effective strategy for controlling infectious disease

  • Responsible for dramatic declines in childhood diseases

  • Diseases sometimes reappear and spread as a result of failure to vaccinate children

• Primary and Secondary Antibody response

• 

Vaccination: Boosting

• A booster dose is an additional vaccine dose administered after the priming dose

  • For some vaccines, only receive one or a few more boosters

  • For others, receive boosters every X years or when the antibodies titer in blood is below acceptable level

• Given to increase circulating antibodies and capacity for strong immune response

• Often necessary because priming dose insufficient to elicit protective immunity. Immune response to vaccine may be weak, particularly to certain types of vaccines

Goals of Vaccination: Individual

• Decrease severity of clinical signs after exposure

  • Most vaccines do not entirely prevent infection

  • Elicit humoral.cellular immunity that limits pathogen replication/spread and lessens clinical signs

  • Prevent mortality by reducing severity of pathogenesis

• Increase total number of pathogens (infectious dose) needed to cause illness in a vaccinated individual

• Decrease shedding of pathogen

  • Vaccinated individuals may still shed virus when vaccinated, but do not become clinically ill

  • Reduce spread by reducing shedding (concentration or duration of shedding)

Goals of Vaccination: Population Level

• Not all individuals can be vaccinated

  • Population sometimes to large: sometimes diffuse to reach all

  • Allergies adverse response to vaccination

  • Immunocompromised (young, old, or ill)

• Herd immunity develops when critical portion of population is immune to disease – protects unvaccinated individuals

  • Infectiou agent unable to spread due to insufficient susceptible hosts

  • In a freely, randomly mixing population, this threshold is ~70% but can be lower or higher depending on population and pathogen traits

Herd Immunity:

Requirements of a “perfect” vaccine

• Must be safe: must not cause disease, minimal side effects

• Give long lasting protection, preferably with a single dose

• Protect against both illness and death

• Prevent shedding (reduces spread)

• Provide rapid protection after administration

• Ideally low cost, stable, easy to administer (oral vs. needle)

• Can differentiate infected from vaccinated (i.e DIVA: no impact on surveillance efforts)

Types of Vaccines

• Attenuated whole-agent vaccines (Modified live (MLV) )

• Inactivated whole-agent vaccines (“Killed”)

• Subunit (proteins or carbohydrates)

  • Recombinant: antigen inserted into another virus vector

  • Conjugated: carbohydrate antigen attached to a protein to help immune system “see” the poorly antigenic component

• Toxoid: inactivated toxin used as a vaccine

• 

Attenuated Whole-Agent Vaccines

• Attenuated: weakened form of pathogen

  • Grow under conditions resulting in mutations, or genetically manipulated to replace or delete pathogenicity genes

  • Replicates in recipientL disease undetectable or mild

  • Often stimulates both humoral and cellular immunity

• Advantages: single dose usually induces long-lasting immunity

  • Can also inadvertently immunize others by shedding of live vaccine

• Disadvantages: can sometimes cause illness in vaccine recipient

  • Can occasionally revert or mutate, become pathogenic

  • Cause cause overt symptoms in immunocompromised individuals

  • Generally not recommended for pregnant women

  • Usually require refrigeration to remain potent – live organism

Inactivated Whole-Agent Vaccines

• Inactivated: unable to replicate “killed”

  • Treated with formalin or other chemical that does not significantly change surface epitopes

  • Primarily stimulate humoral immunity (antibodies)

• Advantages: cannot cause infection or revert to pathogenic forms

  • Generally safer and fewer adverse events or side effects

  • Are sometimes more heat stable than inactivated

• Disadvantages: no replication, so no amplification in vivo: less robust immune responses

  • Cannot spread vaccine by shedding (only vaccinate recipient)

  • Several booster doses usually needed for adequate immunity

  • Often contain adjuvant to enhance immune response

Subunit Vaccines

• Break virus into components, immunize with purified components

• Clone appropriate viral gene, express in bacteria, yeast, insect cells, cell culture, purify protein

• Antigen usually a capsid or membrane protein

• Advantages:

  • Proteins produced by recombinant DNA technology

  • Contain no viral genomes that can replicate or escape

  • No contamination with infectious virus or foreign proteins

• Disadvantages:

  • Expensive

  • Poor antigenicity (low, level, short duration response).

  • Usually stimulate production of antibody, not cytotoxic T cells

  • Lack of good delivery system (injections are best, not well liked)

Adjuvants

• Adjuvants are non-specific stimulators of the immune system that are added to improve vaccine response

• Mechanism of action

  • Depot effects: localization of antigen to the site of inoculation. Slow release and clearance of antigen: prolong immune response (ex. Mineral oil)

  • Surface Effects: through presentation of antigen as particles: activate and hence activity of APCs (ex. Particulate adjuvant: aluminum hydroxide)

  • Inflammation effects: direct stimulation of the immune response (ex. Monophosphoryl lipid A – non-toxic component of LPS)

• Essential for efficacy of inactivated vaccines

• Less commonly required for attenuated vaccines

  • Active replication extends period of immune system exposure allowing for adequate response

Comparison of Attenuated and Inactivated Vaccines

Routes of Administration

• Vaccines are most commonly injected into the skin of muscle tissues

  • Needle and syringe (parenteral)

• Needle-less administration easier and often cheaper

  • Oral

  • Intranasal

  • Intradermal

  • Mass delivery – aerosol or water

  • In ovo delivery to poultry – chicken hatches with immunity

Safety of Vaccines

• Therapeutic index: risk-versus-benefit of receiving vaccine

• All vaccines have side effects

  • Some are mild: fatigue, swelling and pain at site

  • Relatively common – counsel clients on these side effects

  • Some are severe: severe allergic responses, high fevers, seizures, vaccine reversion to wild-type, death

  • Exceedingly rare for most vaccines

• Risks can outweigh benefits for some vaccines

Vaccine Failure

• Bad vaccine - product problems

• Incorrect delivery

  • Wrong vaccine, wrong route, wrong dose

  • Inactivated vaccine (inappropriate storage conditions)

• Wrong animal/timing

  • Immunocompromised or sick animal

  • Maternal antibody interference (too young)

  • Given too proximate to exposure to achieve protection

    • At least 5-10 days to have sufficient response to protect

  • Poor responder: some animals just don't respond well to vaccines

    • Protect these animals by herd immunity

• Too few boosters: one response may be insufficient for complete protection

Immune Disorders and Chronic DIsease - 04/09

Immune Hypersensitivity Reactions (tolerance)

• An overreaction of the immune system: hypersensitivity reactions require a pre-sensitized (immune) state of the host

• Includes allergies and autoimmunity

• Reactions may be uncomfortable to damaging to occasionally fatal

• Four classifications

  • Type 1: Allergy, Anaphylaxis and atopy (immediate)

  • Type II ; cytotoxic, antiBody-dependent

  • Type III: immune complex disease

  • Type IV: Delayed-type hypersensitivity

Immune System and Sterile Inflammation

• Tissue injury but no infectious agent present

• Mechanical trauma

• Ischemia-reperfusion injury (IRI)

  • Injury to the tissues results from the initial hypoxia but also from the restoration of blood flow and re-oxygenation, which exacerbates inflammation

  • Acute myocardial infarctions, cerebral infarctions, acute kidney injury and solid organ transplantation

  • Gastric dilatation and volvulus (GDV) - commonly seen in larg, deep-chested dogs

• Crystal-induced inflammation: crystal deposition within joints leads to gouty arthritis and elicit the classical signs of inflammation including redness, pain, heat, swelling and loss of function

• Particle-induced lung disease

  • Asbestosis and silicosis

• Toxin exposure

• Innate recognition of tissue damage.

  • Involves damage-associated molecular patterns (DAMPs)

DAMP-Sensing and Sterile Inflammation

Immune System and Chronic Inflammation

• Chronic inflammation

  • If antigen persist, antigen-reactive T cells can drive continued

  • Recurrent activation of immune processes (ex. inflammation)

  • Causes tissue damage (autoimmune diseases and inflammatory diseases), which then causes immune activation. Cyclical process

  • Adiposity is a major contributor to sub-clinical chronic inflammation

• Important role of inflammation in pathogenesis of chronic disease: atherosclerosis, type 2 diabetes, Alzheimer's disease, cancer, etc.

Adipose and Chronic Inflammation

Chronic Disease in Animals

• Some common examples

  • Arthritis and other orthopedic conditions

  • Chronic kidney disease

  • Hepatitis and other liver diseases

  • Skin allergies (atopy)

  • Diabetes mellitus

  • Cushing’s and addison's disease

  • Inflammatory bowel disease

  • Hyperthyroidism (cats) and hypothyroidism (dogs)

  • Alzheimer's disease

Antimicrobials and Antibiotics 04.16.24

Definitions

Sterilization

• Complete elimination or destruction of all organisms and viruses by chemical or physical means

Sanitization

• The process of reducing microbial contamination to an acceptable “safe” level

Decontamination

• A treatment of an inanimate object/surface to kill microorganisms or remove contamination is safe to handle. No quantitative implication

Disinfectant

• A chemical agent that is applied to inanimate objects to kill most pathogenic microbes, but not all microbes. They may not be effective against bacterial spores. Usually not safe on host tissues

Antiseptic

• A chemical agent that is applied to living tissue to kill most pathogenic microbes, but not all microbes. Usually safe on host tissue

Antimicrobials

• Inhibits growth of microorganisms

Antibacterial

• Inhibits growth of bacteria

Antibiotic

• Inhibits growth of microorganism

• Made by other microorganism

• Usually extended to include synthetic drugs

Bacteriostatic vs. Bactericidal

Bacteriostatic

• Reversible inhibition of bacterial growth

• When the antibiotic is removed, the bacteria can replicate

• Some bacteriostatic drugs may be bactericidal at high concentrations

• Examples: sulfonamides, tetracyclines, chloramphenicol

Bactericidal

• Kills bacteria

• When the antibiotic is removed, almost none of the bacteria can replicate

• Examples: penicillin, vancomycin, rifampin

Time before “Modern” Antibiotics

First Treatments:

• 1500 BC

  • Egyptians used honey, lard and lint for dressing wounds

  • Enzymatic production of hydrogen peroxide in most honeys

  • High sugar content (high osmolarity)

  • Presence of other bioactive factors

• Over 2,000 years ago

  • Moly bread was used in china, greece, serbia, egypt and other ancient civilizations as treatment for infected wounds

Metal Solutions

• Arsenic

  • Used since antiquity

  • One of the first antimicrobial compounds and was effective against syphilis

  • Arsenic is very toxic to the patient

• Mercury

  • Very effective antimicrobial agent

  • Used to sterilize surfaces and kill microbes

  • Still used as preservatives in vaccines

• Bacteria can develop really high levels of resistance to metals (5-10mM)

Discovery of Penicillin

• September 3, 1928: Alexander Fleming (Professor of Bacteriology) discovers that growth of Staphylococcus (cause boils, sore throats and abscesses) was inhibited by growth of mold (Penicillium notatum).

• June 1929: findings published in the British Journal of Experimental Pathology, with only a passing reference to penicillin's potential therapeutic benefits.

• Howard Florey, Ernst Chain and colleagues at Oxford University turned penicillin from a laboratory curiosity into a life-saving drug.

• 1940:

  • Florey showed that penicillin could protect mice against infection from deadly Streptococci.

• 1941:

  • a 43-year old policeman, Albert Alexander, became the first recipient of the Oxford penicillin.

  • Developed a life-threatening infection with huge abscesses affecting his eyes, face, and lungs.

  • Penicillin was injected and within days he made a remarkable recovery.

  • But supplies of the drug ran out and he died a few days later.

  • Plans to make penicillin available for British troops on the battlefield led to large scale production of the antibiotic.

Penicillin: Mechanisms of Action

1. A bacterial cell wall is composed of macromolecule of peptidoglycan composed of NAG-NAM chains that are cross-linked by peptide bridges between the NAM subunits

2. New NAG and NAM subunits are inserted into the wall by enzymes allowing the cell to grow. Normally, oher enzymes link new NAM subunits to old NAM subunits with peptide crosslinks

3. Penicillin interferes with the linking enzymes, and NAM subunits remain unattached to their neighbors. However the cell continues to grow as it adds more NAG and NAM subunits

4. The cell bursts from osmotic pressure because the integrity of peptidoglycan is not maintained

Antibiotics: Timeline of Discovery

Antibiotics: Mechanisms of Action

Spectrum of Activity

• Broad Spectrum

  • Active against both gram-positive and gram-negative organisms

  • Examples: tetracyclines, phenicols, fluoroquinolones, “third-generation” and “fourth-generation” cephalosporins

• Narrow Spectrum

  • Have limited activity and are primarily only useful against particular species of microorganisms

  • Example:

    • Glycopeptides and bacitracin are only effective against Gram-positive bacteria, whereas polymyxins are usually only effective against gram negative bacteria

    • Aminoglycosides and sulfonamides are only effective against aerobic organisms, while nitroimidazoles are generally only effective for anaerobes

General Characteristics of Antimicrobial Drugs

• Selective Toxicity

  • Ability of drug to kill or inhibit pathogen while damaging host as little as possible

• Therapeutic Dose

  • Drug level required for clinical treatment

• Toxic Dose

  • Drug level at which drug becomes too toxic for patient (i.e produces side effects)

• Therapeutic Index

  • Ratio of toxic dose to therapeutic dose

Level of Antimicrobial Activity

• Effectiveness of antimicrobial are expressed in two waysL

  • Minimal inhibitory concentration (MIC)

    • Lowest concentration of drug that inhibits growth of pathogen

  • Minimal lethal concentration (MLC)

    • Lowest concentration of drug that kills pathogen

• Two techniques are routinely used to determine MIC and MLC

Dilution Susceptibility Tests

• Inoculate media containing different concentrations of drug

• Monitor growth by plate counts or optical density (OD) at 600 nm

• Plot the OD 60 nm vs concentration

• MIC:

  • The lowest concentration showing no growth

• MLC:

  • Tubes showing no growth are subcultured into drug-free medium

  • Lowest corresponding original concentration from which microbe cannot be recovered is MLC

Kirby-Bauer Disk Diffusion Tests

• Disks impregnated with specific drugs are placed on agar plates inoculated with test microbe

• Drug diffuses from disk into agar, establishing concentration gradients

• Observe clear zones (no growth) around disks

• Diameters of zone used to quantitate susceptibility or resistance

Synergism of Combination Antibiotic Therapy

Antibiotic Combination Therapy

• Indication for combination therapy may include:

  • Infections caused by multiple microorganism (abdominal and pelvic infections)

  • Nosocomial infections, which may be caused by many different organisms

  • Serious infections in which a combination is synergistic (ex. An aminoglycoside and an antipseudomonal penicillin for pseudomonas infections)

  • Likely emergence of drug-resistant organism if a single drug is used (ex. tuberculosis )

  • Fever or other signs of infection in immunosuppressed patients. Combinations of antibacterial plus antiviral and/or antifungal drugs may be needed

Effectiveness of Antimicrobial Drugs Therapy

• Factors that influence effectiveness:

  • Susceptibility of pathogen to drug

  • Single vs combination therapies

  • Ability of drug to reach site of infection

  • Ability of drug to reach concentrations in body that exceeds MIC of pathogen

Factors Influencing the MIC in the body during treatment

• Amount Administered

• Route of administration

• Duration of Therapy

  • Varies from single dose to year.

  • For most acute infections, ~7-10 days until the patient has been asymptomatic for 48 to 72 hours

• Other Pharmacokinetics (fate of a substance in the body)

  • Rate of uptake

  • Route (kidney, liver) of clearance (elimination) from body

  • Half-life, which is affected by diseases (liver or kidney disease) and other drugs

  • Interactions with other drugs

  • Dosing schedule, particularly compliance

  • Side effects and idiosyncratic (“Type B reactions”) responses

• Immune Status of Patient

• Antibiotic Drug Resistance

Antimicrobials and Antibiotics (continued) 04.18

Antibiotic Use in Agricultural Animals

• Therapeutic vs non-therapeutic uses

  • Antibiotics are given in sub-therapeutic concentrations (below MIC) in feed to promote better feed conversion and growth

• Estimated use in agriculture in United States

  • ~16 million pound/year for non-therapeutic use in animals (institute of medicine, 1989)

  • 24.6 million pounds/year for non-therapeutic purposes in chickens, cattle, and swine. Compared to just 3..0 million pounds for human medicine. (union of Concerned Scientists, 1989)

  • 17.8 million pounds of antimicrobials used for animals, only 3.1 million pounds are used non-therapeutically. (Animal Health Institute: pharmaceutical industry-sponsored)

• Many antibiotics used in animals are ones used in human medicine

  • Examples: tetracyclines, penicillins, and sulfonamides

  • Examples of exceptions: ionophores (used routinely in beef calves in feedlots and some dairy heifers)

Antibiotic Use in Agricultural Animals

Misuse of Antimicrobial Drug Therapy

• Alternation in microflora

  • Susceptibility to secondary infections

    • (children given antibiotics for routine upper respiratory infections often get diarrhea (ex. Susceptible to aggressive antibiotic-resistant C. difficile).

  • Long-term health effects

    • Obesity, allergy, atopic and autoimmune disease, infectious disease

• Drug-resistant microflora

Chronic Impacts of Early Antibiotic Use

• Antibiotics

  • Early in life

  • Multiple courses of broad spectrum antibiotics

• Dysbiosis

  • Imbalance in gut microbiota

  • Loss of keystone species

  • Loss of microbial diversity

  • Alterations in metabolic capacity

  • Blooms of pathogens

• Disease

  • Later in life

  • Obesity

  • Allergy and atopic disorders

  • Autoimmune disease

  • Infectious disease

Chronic Impact of Early Antibiotic Use

Differential Impacts of Antibiotics on Microflora

Antibiotics Drug Resistance

Terminology

• Multiple drug resistance (MDR)

  • Organism is resistant to more than one drug in three or more antimicrobial categories

• Extensively drug resistant (XDR)

  • Organism is resistant to at least one agent in all but two or fewer antimicrobial categories

• Pan Drug-resistant (PDR)

  • Greek prefix “pan”, meaning “all”

  • Organism is resistant to all agents in all antimicrobial categories

Emergence and Transmission of Antimicrobial-Resistance

• Introduction of a resistant organism

  • Presence of a patient or animal with a resistant microorganism

• Emergence of a new resistant organism

  • Selective pressure from antimicrobial use

• Clonal Dissemination

  • Inadequate hand hygiene

  • Insufficient use of barrier isolation

  • Inattention to environmental reservoirs or vectors

How antibiotic resistance occurs

1. Lots of germs, a few are drug resistant

2. Antibiotics kill bacteria causing the illness, as well as good bacteria protecting the body from infection

3. The drug-resistant bacteria are now allowed to grow and take over

4. Some bacteria give their drug-resistance to other bacteria, causing more problems

Role of Poor Compliance in Antibiotic Resistance

Strength of Biofilms

• Predator evasion

• Antibiotic tolerance/ resistance

• Horizontal gene transfer

Biofilms Promote Antibiotic Tolerance and Resistance

• Reversible phase

  • Motility

  • Adhesion

• Irreversible phases

  • Mutration

  • Dispersion

  • Propagation

Mechanism of Antibiotic Resistance

Dynamics of Drug Resistance

• People or animals who receive an antibiotic are more likely to harbor bacteria resistant to that antibiotic and biochemically unrelated antibiotics

• People or animals who frequent environments in which antibiotics are used are more likely to harbor drug-resistant bacteria, even if they have not received antibiotics, this applies to patients as well as to staff

• The probability of harboring drug-resistant bacteria returns to normal within a few weeks after antibiotic therapy is discontinued or after absence from the antibiotic-rich environments. But not entirely eliminated

• The prevalence of drug-resistant bacteria in the community is increasing due to increasing use of antibiotics in the environment ‘

• Antibiotics, use them and lose them

Cost of Antibiotic Resistance

• Every year in the United States, more than two million people get antibiotic resistant (ABR) infections. At least 23,00 people die as a result

• In the united states, antibiotic resistance add $20 billion in direct healthcare costs, with additional societal cost of ~$35 billion a year in lost productivity

• Reducing ABR infections by just 20% would save $3.2- $5.2 billion in health care costs each year and cut up to $11.3 million additional in-hospital days for patients with ABR infections

• What is the impact of ABR to animals and veterinary care?

  • Not well-understood

State of New Antibiotic Drug Development

New Antibiotics: They're not profitable to make. Who pays?

Retarding Emergence of Resistance

• Maintenance of therapeutic levels

  • Ensure patient compliance

  • Avoid the use of drugs when the MIC is at or only slightly below the attainable level

  • Prevent biofilms and treat them aggressively

• Use combinations of antibiotics when indicated (but not otherwise)

• Avoid over and ill-advised use of antibiotics

  • Prescriptions for infections that won't respond

  • Tendency to use hot new drugs

  • Self medication

Spread of Antibiotic Resistance

Biosecurity 04.23.24

Defining Biosecurity

• Preventive measures designed to reduce risk of

  • Infectious diseases

  • Exposure to toxin and other contaminants

  • Bioterrorism

  • Zoonotic transfer

Importance of Biosecurity

• Health animals for healthy people

• To ensure safe food for the population

• Protect the livelihood of individuals and families. Mant family owned farms and production

• Protect a vital industry in the country

• Meet policy requirements for competitive global trade

• Mitigate economic consequences of a disease outbreak

• Beef Cattle Production

  • Largest single segment of US agriculture

  • Great than 1 million businesses, farms, and ranches, 98% of US farms

  • $88.25 billion to US economy

  • 26.4 billion pounds of beef

  • 92.0 million head (all cattle and calves)

• Pork Production

  • 60,000 pork producers : 550,000 jobs

  • More than 110 million hogs/year

  • $23.4 billion to US economy

  • Exports over 2.2 million metric tons annually of pork and pork related products (26% of US production)

• Poultry Industry

  • World's largest producer and second largest exporter of poultry meat and a major egg producer

  • Broiler meat: $45 billion and 36.9 billion pounds annually

  • Eggs: $13.5 billion and 96.4 billion eggs annually

Some Factors that influence Biosecurity

• Globalization

  • Increasing travel and movement of people across borders

  • Increased trade in food and agricultural products

  • New agricultural production and food processing technologies

  • High dependence of some countries on food imports

  • Shift from country independence to county interdependence for effective biosecurity

• Advances in communications and global access to biosecurity information

• Great public attention to biodiversity, the environment and the impact of agriculture on both

• Scarcity of technical and operational resources

• Legal obligations: local, national and international laws and restrictions

Increased Risk of Exposure

• Farm density

  • Other production facilities within a few miles

• Animal movement

  • Especially if animals leave, then return to the premises

• Traffic on and off the premises

  • Vehicles (feed, milk, garbage, rendering) and drivers

• Human activity

  • Employees, service personnel, visitors

• Equipment sharing

  • Between facilities, oro between animal groups within the facility

• Access by wildlife

  • Such as insects, birds, rodents, feral animals

• Housing difficult to clean

  • Well-thought out construction and facility lay out is important

• Mortality disposal near animal housing

Some element of biosecurity planning

• Biosecurity coordinators on premises identified and recorded

• Organized training program with records of training

• Lines of separation on buildings with required sanitation

• Perimeter buffer areas defined: defining which areas of the facility are “hot” and “cold”

• PPE on premise for employees

• Vector control for multiple species of pests

• Equipment control for multiple species of pests

• A mortality management plan

• Manure and other waste management

• Replacement / new animals

• Water management

• Feed and new material management

A Biosecurity Plan

• Embodies multiple components

  • Risk perception

  • Risk assessment

  • Risk management

  • Risk communication

• Designed to improve disease control

  • Provide foreign and domestic diseases

  • Provide tools to minimize risk

• Disease risk cannot be totally eliminated

  • Decrease exposure to existing disease agents

• No one-size fits-all answer

Risk Perception

• Different perceptions of risk

  • First identify what is viewed as a threat

• Factors influencing perception

  • Previous experience

  • Media

  • Environment

• Common beliefs to overcome

  • We have always done it this way

  • I've had most everything on this farm

  • It's too expensive

• New beliefs to embrace

  • Disease outbreak can and do happen

  • Prevention is less costly than treatment

  • Too much is financially invested to lose

  • Prevention through awareness and management

Risk Assessment

• Objective evaluation is critical

  • Possible vs. probable

  • Perform site specific hazard analysis (geographical, indoors versus outdoor raises animals)

  • Identify sources of potential infection

  • Consider the health status and species of animals

• Identify strengths and weaknesses of existing infrastructure / practice

  • Identify areas needing protection

  • Ascertain site-specific pathways for potential disease movement

  • Be aware of the change over time. Regular re-assessment is important

• Disease prediction is complicated

  • Depends on interaction of many different factors

• Fundamental knowledge of disease is important

  • Route of transmission

  • Understand the epidemiologic triad of each disease

Risk Management

• Evaluate facility / operation

  • Preemptively identify challenges

• Develop systematic plan

  • Establish well-defined line of separation

  • Dirty (contaminated) / clean (protected)

• Prioritization of biosecurity measures

  • Focus on highest risk first

  • Consider probability of occurrence

  • Consider ease/ cost of implementation vs. consequences

  • Easy to implement often means greater compliance

  • Inexpensive yet yield rewards

• Tailored management plan: no common formula

  • Immediate challenges

  • Short term goals

  • Long term goals

• Seek several expert advice, remain open to suggestions

Risk Communication

• Communication is key

• Plan must be understood and supported to be effective

• Reporting and reporting requirements

Legislative requirements for animal health

• The legal requirement to notify the local animal health divisional office at the first sign or suspicion of notifiable disease

• Awareness of the legal requirements during a disease outbreak, such as foot-and-mouth disease (restriction of movement measures)

• Familiarity with the legal requirements relevant to farm businesses

• Knowledge of current legislation concerning high profile issues ex. Fallen stock, animal identification and traceability

• An understanding of the food production regulations

Levels of a Biosecurity Plan

• Conceptual

  • Location, geospatial sitting, orientation of the facility

• Structural

  • Capital investment, construction, to prevent disease spread

• Operational

  • Processes, management practices, standard operating procedure to exclude or contain diseases

Conceptual Biosecurity

• Evaluate existing facility

  • Facility location, geospatial sitting, orientation, scope , size

• Risk level

  • High risk: greater farm density, close to wildlife area, large groups managed as one population

• Best practices

  • Separation, isolation with enhanced distance to neighboring livestock/ livestock facilities

  • Manage smaller groups of animals as biosecure units

  • Distance to wildlife habitats and roads

• Mitigate / compensate for vulnerabilities

  • Eliminate (make less attractive) wildlife and pest habitat

  • Reroute traffic away from animal areas

  • Create smaller biosecure groups

Structural Biosecurity

• Construction and capital investment

  • Physical design and maintenance

• Paved parking away from barns

• Fences, barriers leading to entrances to conduct biosecurity protocols

• Locations for cleaning / disinfecting

• On-site laundry for outerwear

• Specialized anteroom at entry

Structural Biosecurity: Danish Entry system

• Visible line of separation

• Site-specific biosecurity attire

• Appropriate biosecurity protocols for entering and exiting

Operational Biosecurity

• Processes, management practices, standard operating procedures to exclude or contain disease

  • On-farm movements and managements

  • People, animals, supplies, equipment, vehicles, and other items

  • Understand effectiveness of your mitigation looks (disinfectants)

• Should be based on general specific risk assessments

  • Mitigation of conceptual and structural vulnerabilities, and know disease

• Apply strategic actions at critical control points

  • Focus on inputs and outputs

  • Entrance and exits

  • Work paths

  • Processes

• Clearly identify separation of clean and dirty.

Development of a Biosecurity Plan

• Step 1: prioritize the disease agent

  • Consider species/ susceptibility, housing, management, wildlife exposure

• Step 2: conduct a facility assessment

  • Identify pathways/movements

• Step 3: Implement processes to minimize impact of disease

  • Prevent movements (and practices) that carry disease

Assessment of Animal Health

• Physical conditions

  • Condition and smell of hair coat and skin, body scoring

• Sleeping habits

  • Time of day, time spent standing or lying

• Attitude

• Posture

  • Movement or weight shifting from one foot or side

• Eating and drinking habits

  • Time of day, amounts, time spent eating and drinking water