measuring infectivity( week 5)

When is human volunteer research considered suitable?

When the disease is:

• Not life-threatening

• Easily treatable

• Preventable (e.g., with a vaccine)

• Study does not cause harm to participants

What was the Tuskegee Syphilis Study?

: An unethical study where Black men with syphilis were left untreated to observe disease progression.

What did the Belmont Report establish?

A: Ethical principles and federal guidelines for human subject research.

What are the three main principles of the Belmont Report?

• Respect for persons

• Beneficence

• Justice

What does “Respect for persons” mean?

Individuals must give informed consent and have autonomy.

what is beneficence

maximize benegits and minimize harmwha

what is justice

fair distribution of risks and benefits

t or f human trials are acceptable if they produce valuable scientific knowledge

false, ethical standards must still be met regardless of benefit.

: True or False: It is ethical to study a disease in humans if it is not life-threatening but has no treatment.

false, lack of treatment increases risk and raises ethical concerns

what is the biggest ethical issue in the tuskegee syphils study

lac of infomred consent and wthholdng treatment

t or f participants can be exposed to harm if they consent

false consent does not justfy unnecessary harm

: Why is the Tuskegee Syphilis Study considered an example of injustice?

: It targeted a vulnerable racial group and denied them treatment.

Why must diseases in human trials be treatable or preventable?

To ensure participants are not exposed to irreversible or serious harm.

What type of research is MOST ethically acceptable in humans?

Research with:

• Minimal risk

• High benefit

• Available treatment if needed

What changed after the Belmont Report?

: Strict federal regulations and oversight (IRBs) for human research.

What are the 3 main principles of the Belmont Report?

1. Respect for persons → informed consent

2. Beneficence → maximize benefit, minimize harm

3. Justice → fair distribution of risks/benefits

What does “informed consent” require?

articipants must:

• Understand risks/benefits

• Participate voluntarily

• Be able to withdraw

who overssess human research

Institutional review board Irb

what does an rb do

approves monitors, and reviews all biomeidcal and behavioral research involving humans

t or f if a study mnmzes harm it automatically satisfies all ethical principles

false, it must also ensure justice and iinformed consent

what is a prospectve study

a clncal study that follows subjects forward n tem often, involving infection or treatment

what is a retrosepective study

a study that looks back at events that already occured (past outbreaks)

t or f retrospective stduies involved expermental manpulation

false they only analyze past data

what makes an ideal animal model

mimics human disease symptoms

same route of infecton

smlar diease progression

what are key considereations when choosng an animal model

• cost and ease of maintenance

• relevance to human diease

• abilty for genetc manipulation

• differences in physiology behavior

• use of germ frem (gnotobitoci) animals

why is coprophagy in mice mportant experimnetally

it affects infecton ad microbiome results (Behaviorla differences vs humans)

t or f if an animal gets the disease it is automatically a good model

flase, route symptomes and progression must match humans

who regulates anmal research

IACUC ((Institutional Animal Care and Use Committee)

what must be demonstrated before usiing aiamsl

• No alternative model exists

• Strong scientific rationale

• Hypothesis-driven research

what are key ethical equirements n aniimal experments

• Minimize number of animals

• Reduce pain and discomfort

• Use anesthesia

• Proper care before, during, after

• Euthanize if animals become moribund

how often are anmal protocols reviewed

annually

t or f aniamsl can be used if its convenents for researchs

false, must be necessary wth no alternativest

t or f using more aniimals increases accuracy and is preferred

false must use the minmum number needed

why are invertebrate models often used in research

• no iacuc approval required

• cheap and easy to maintain

• good for hgh troughput studies

t or f all animal research equires iacuc approval

false, iinvertebrates are generally not regulated

what is a major limiation of all inverterbrate models

lack of adaptive immune system

what is caenohabditis elegans(worms) commonly used to study

Bacterial pathogenesis (e.g., gut infection models)

How is infection studied in C. elegans?

Replace normal food with pathogenic bacteria and observe gut colonization.

What happens during infection in C. elegans?

• pathogen accumulates in gut

• Intestinal distension

• Death of worm

What is “slow-killing” in C. elegans?

death accumulation of live bacteria in the gutwh

what is fast killing

rapd death due to toxins produced by bacteriat

why is c elegans a good model organism

• fully sequenced genome

• many genetic tools

• rapid life cycel

• cheap and easy to grow

  • good for high troughput screening

what are limitaons of c elegans

• no adaptive mmunty

• cannot grow at 37

• different anatomty

  • doesnt naturally encounter all human pathogens

t or f c.. elegans can model all human nfecton

false, temperatrure and biolgy limit thiswh

why is inabiloity to grow at 37 important

many human pathgoens grow at that temp

What is Galleria mellonella (moth) used for?

A: Studying bacterial infection and toxin effects

What is “melanization”?

Darkening response indicating immune activation in larvae.

Why is Galleria useful compared to C. elegans?

A: Can survive at 37°C (closer to human conditions)

what are limitations of galleria mellonella

• genome not fully sequences

• limted gentc tools

• ahrder to generagte mutatns

t or f gallera s genetcally better studed then c elegans

false galleraia is not fully sequenced

What is Drosophila melanogaster used for? (FRUT FLY)

A: Studying bacterial, viral, and parasitic infections

: Why is Drosophila considered the most versatile invertebrate model?

• Can model bacteria, viruses, AND parasites

• Fully sequenced genome

• Extensive mutant libraries

What immune pathways in Drosophila are similar to humans?

A: Innate immune pathways (e.g., TLR-like pathways, IL-1 signaling)

What is a key experimental limitation in Drosophila infection studies?

A: Often requires high bacterial doses.

True or False: Drosophila has adaptive immunity.

❌ False — only innate immunity.

Q: Which invertebrate model can study viruses and parasites?

A: Drosophila melanogaster

Which model…mathc them

• Has best genetics? → C. elegans / Drosophila

• Can survive at 37°C? → Galleria mellonella

• Can study viruses/parasites? → Drosophila

• Is NOT regulated by IACUC? → All invertebrates

What happens to Galleria mellonella during infection?

Melanization (darkening) → progresses from active → sluggish → dead

True or False: Galleria mellonella can be easily genetically mutated.

A: ❌ False — lacks well-developed genetic tools

Why is Galleria useful despite poor genetics?

• Can survive at 37°C

• Shows clear infection phenotypes (melanization)

What makes Drosophila unique compared to other invertebrate models?

A: Can be used to study bacteria, viruses, AND parasites

What is special about the immune system of Drosophila melanogaster?

Innate immune pathways are similar to mammals (e.g., TLR-like signaling)

What are key advantages of Drosophila?

• Fully sequenced genome

• Extensive mutant libraries

• Strong genetic tools

t or f all nverebrate models can study viral nfectons

false only flys

What is Acanthamoeba castellanii used to study? amoeba

intacellular pathogens that infect phagocytic cells

why is amoeba a good model for macrophages

infection processes are smlar both engluf pathgoenswh

what does axenic growth mean

growht free of other orgnassms

what are major limatons of amoeba

• polypod genomes (complex)

  • diffcult to genetcally manpulate 9poor transfecton succes

what are CFUS used for

measruing number of viable bacteria (bacterial load)

what is surviival curve

tracks surval of infected organsm over timewha

what do LD50 and D50 measure

• LD does that kills 50%

  • ID dose that infects 50

what is biophotonc imagne

uses light often luminescent bacteria to track nfection n live animals

What is a competition assay?

A: Compares fitness/virulence of two bacterial strains in the same host

: True or False: CFUs measure total bacteria (alive + dead).

A: ❌ False — only live bacteria

Why are time points important in infection studies?

A: Infection dynamics change over time (early vs late stages)

True or False: A higher CFU always means higher virulence.

❌ False — virulence also depends on host damage and toxins

What is typically compared in survival curves?

wild vs mutantwh

: True or False: Survival curves measure bacterial load directly.

A: ❌ False — they measure survival, not CFUs

: If a mutant has longer survival time than wild-type, what does that mean?

A: Mutant is less virulent

How does it work? imagine

Bacteria are engineered to express luciferase (lux operon from Vibrio species), producing light detected by a sensitive camera

What does the light indicate?

location and intesnity of infection

why is biophotonic imaging usefull

• no need to sacrifice animals at each time point

• can track infection in the same animal over time

  • shows spatiial distributioon where infection is

t or f biophotonic imaging replaces CFU measuremnets entirely

flase it complements but does not fully replace CFUwhy

: What is a key advantage over CFU plating?

A: Multiple time points without killing animals

True or False: LD₅₀ can compare virulence between different species (e.g., cholera vs dysentery).

A: ❌ False — only valid within the SAME disease/model system

True or False: Lower LD₅₀ means lower virulence.

A: ❌ False — lower LD₅₀ = MORE virulent

What is a competition assay?

A: Infection with a mixture of wild-type and mutant bacteria

hat is the “input ratio”?

Ratio of mutant to wild-type bacteria BEFORE infection

what is the output ratio

ratio after infection

why must strains be trackable

to be able to distringuish them

what is the formula for CII

ci= output/inputwha

what does a CI=1 mean

no difference in virulencewhat

what does CI> 1 mean

mutant is more virulent (outcompletes wild typewha

what does CI <1 mean

mutant is less virulent

Q: True or False: If a mutant grows slower, it may appear less virulent in a competition assay.

true fitness affects resultswhat

what are trans effects

wild type bacteria may complement mutant defects masking phenotypet

t or f CI only reflects virulence

false, also reflects overall fitness

HIGH-YIELD CONNECTIONS (EXAM GOLD)

• Survival curve → time to death

• LD₅₀ → dose to kill

• ID₅₀ → dose to infect

• CFU → # of live bacteria

• Biophotonic imaging → where infection is over time

• Competition assay → relative fitness (WT vs mutant

What is a CFU?

A colony-forming unit = one viable bacterium (or group) that can grow into a visible colony on a plate

How do you measure CFUs?

• Take sample (tissue, culture, etc.)

• Serially dilute

• Plate on agar

• Count colonies

• Calculate back to original concentration