Module 3: Techniques

Question 1

Questions when making an infection model

Answer 1

• virulence factors
• contribution of host
• transmission
• susceptibility to antibiotics
• Vaccination

Question 2

Why are infetion models important

Answer 2

• identity virulence factros and steps involved in infection and development of diseases
• hypothesis-driven research

Question 3

What does a good infection model consist of

Answer 3

1. is suspeptible to infection
2. mirrors all disease symtpoms that occur during infection
3. easy to work with an inexpensive

Question 4

What is the best model to study human diseases

Answer 4

human

Question 5

What are ethical principles to abide to

Answer 5

1. respect for individuals with consent
2. beneficence by maximising enefits and miniising harm
3. justice by fair treatment and risk distrubutions
4. approval by ethical board

Question 6

Compare prospective vs retroactive study

Answer 6

• P: experiments with human volunteers like vaccines
• R: events have allready happen like with an outbreak

Question 7

Name 6 non human animal models

Answer 7

1. rodents
2. zebra fish
3. cat
4. dog
5. pig
6. rabbit
7. primates

Question 8

What are 6 criteria for animal model?

Answer 8

1. Cost and ease to maintain and manipulate the animal model.
2. Similarity to the disease (symptoms, location of the bacteria, transmission route, outcome).
3. Are the results consistent with observations of the human disease?
4. Ethical consideration: intensity of pain, death is not an ethical experimental endpoint.
5. Genetic uniformity of the animal model (inbred mice, less noisy).
6. Genetic modification of the animal model (transgenic: knockout, knock-in

Question 9

What is the problem of using animals for human disease, with examples

Answer 9

some human disease have different course in animals

1. Salmonella typhi, typhimirum wild type salmonella present different in mice and people
2. Model host-pathogen interaction: Citrobacter rodentium in mouse mimics S. typhimurium infection of human

Question 10

Discuss Caenorhabditis elegans- worm

Answer 10

PRO:

• model genetic organism, simple body plan, transparent, small size, generation time

CON

• does not replicate symptoms

Question 11

What is the humanised mouse modell

Answer 11

• zap immune system, put human version of immune system in mouse using injected bone marrow cells
• hard to do
• mouse is still mouse = not that helpful

Question 12

What are 4 characteristic of Gnotobiotic animals

Answer 12

• Germ free animals, no bacteria in or on them
• PRO
  
  • no partial imunitiy or cross creation (when u have normal microbiotic u produce antibidoeies agasint urself, but immune system has learned it knows, sometimes those antibiodtises sometimes are helpful for ur own microbiotic but all react against pathogen
  • by change
  • noise free animal model
• CON:
  
  • underveleopled mucosa-associated lympoid tissues
  • dont know what bacteria is, immune system doesnt know what to do

Question 13

What is the pipeline for animal model asics

Answer 13

1. grow bacteria in axenic medium or host and standarsise dose
2. innoculate dose into animal
3. Monitor the growth of bacteria in particular site, such as spleen, liver, lung, gastro-intestinal tract, by the colony forming unit method (CFU).
4. Monitor the survival of animal. However, death is not an ethical experimental outcome, so the animal needs to be sacrificed before death occurs from the experiment (based on symptoms).

Question 14

What is biophotonic imaging

Answer 14

see where infection is happening

Question 15

What is LD50 and ID50, and benefits, CONS

Answer 15

• LD50: dose to kill 50% of animals (Lethal Dose 50%)
• ID50: dose to infect 50% of animals (Infection Dose 50%)
  
  • have to determine what a positive infection is , for eg CFU 10^8 in tissue C, appearance of symtpms, size of abcess
• PRO:
  
  • both reflect cumaltive effect of colonisation, production of symptom and host damage
• CON
  
  • only mutation of essential virulance factors will have an effect
  • many animals
  • might be varance during infection. when infected to infection there are not clone strains might be variable
  watch the pro and con section

Question 16

What does the compeition assay asses

Answer 16

• Which one of the mutant or the wild type is more fit for the host
• which one outcompetes the other
  
  • the two bacteria will fight each other, the better one does better
    (output (test)/input(control) divided by input(test) / input (control)

Question 17

What is avirulenc egene

Answer 17

gene that cools reaction, may…

Question 18

trans competition

Answer 18

• wild type supplies missing fdactpors in such quantities that mutangt doesnt have to do
• will grow along with it

Question 19

What does a tissue culture model entail, PRO, CON, EG

Answer 19

• in animal model many variable cant be controlled
• PRO:
  
  • culturd mammalian cells used to provde simplier system with fewer variable that can be controlled
  • cultured cells are more permissive
  • more ethical
  • straighfoward intrepereation (can look at one specieifc thing, like ability to invade cell)
• CON: lacks complexity of the animal
• USE: primary cells, immortalised tumor cells, co-culture

watch image explaination and the last secyion yoo

Question 20

What is the step by step for tissue infection

Answer 20

1. innoculate at specific dose from bacterial patogen
2. dilution serires
3. CFU count
4. growth curve

Question 21

Name manners of studing disease

Answer 21

• Gnotobiotic animals
• surivial curve
• biophotonic imaging
• LD50 and ID 50
• Competition Assay
• Tissue Culture Model
• Gentamicin protection assay
• invasion success curve
• fluorescence microscopy
• huamised cells

Question 22

What are virulence factors, how come

Answer 22

• produce involve in disease caused bypathogen
  1. product is linked to one gene or a group of gene in the genome of bacterial pathogen
  2. direct product of group of genes (like protein)
  3. product of metaolic pathway meaning enzymes involved in pathway are encoded by genes
  4. one VF requires other to be effected (eg: secretion viruelnce factor)

Question 23

What is the molecular verision of Koch’s postulates

Answer 23

1. The phenotype or property under investigation should be associated with pathogenic members of a genus or pathogenic strains of a species.
2. The gene(s) associated with the supposed virulence trait should be isolated (identified) by molecular methods. Specific inactivation of the gene(s) associated with the suspected virulence trait should lead to a measurable loss in pathogenicity or virulence.
3. Reversion or allelic replacement of the mutated gene should lead to restoration of pathogenicity (complementation). Restoration of pathogenicity should accompany the reintroduction of the wild-type gene(s)

Question 24

Go through example of molecular koch’s postuate

Answer 24

1. all Salmonella typhimurium can invade epithelail cells
2. all strains posses group of genes SPI-1, if mutates no invasion
3. introduction of plasmid with inA restores orginal phenotype (complementation)

Question 25

What is the polar effect

Answer 25

mutation of upstream gene affect expression of downstream genes

Question 26

What are limitation of molecular Koch’s postualtes

Answer 26

1. non pathogenic strains sometimes dont exists meaning they are strict intracellular pathogen
2. Identification of the genes that could be involved in a phenotype is not trivial.
3. Genetic tools are not available for all bacterial species.
4. Sometimes, many genes perform the same function: redundancy of virulence factors

Question 27

Give example of limitation of molecular Koch’s postulate

Answer 27

The gene involved in a phenotype in one strain could be also present in another strain causing a different disease. Ex: both S. typhi and S. typhimurium possess SPI-1, but S. typhi causes a systemic infection, whereas S. typhimurium causes a local infection. Howevr, both invade intestinal cells