I&I A2

Parasite

an organism that resides on or within another living organism in order to find the environment and nutrients it requires for growth and reproduction

What are the 2 most prevalent human parasites?

helminths: worms

Protozoans: unicellular

TRUE OR FALSE: Helminths are more pathogenic than protozoans

FALSE

What are other terms for the helminths a) tape worm, b) flukes, c) roundworm?

a) cestode

b) trematode

c) nematode

What is the a) residence, b) pathology and c) symptoms of Schistosome /blood fluke?

a) blood

b) acute or chronic

c) abdominal pain, diarrhea, bloody urine or poo, anaemia, enlarged liver and spleen

What is the a) residence, b) symptoms and c) pathology of tape worm?

a) gut

b) abdominal pain, diarrhea, weight loss

c) chronic

What is the a) residence, b) symptoms and c) pathology of stronglioides (round worm)?

a) penetrate skin→ to the lungs→ gastrointestinal tract

b) abdominal pain, diarrhea, wheezing, itching

c) acute and chronic

What is the a) residence, b) symptoms and c) pathology of guinea worm (round worm)?

a) subcutaneous

b) blister formation with burning sensation

c) chronic, sort of

Giardia symptoms/how can it manifest? (7)

Asymptomatic

acute short-lasting diarrhea (1-3 weeks)

Nutritional disorders

weight loss

allergies

lower cognitive function

cancer

Giardia transmission routes

• person-person

• Water/food borne.

• Zoonotic.

TRUE OR FALSE: There are 8 known Giardia genotypes, with different ones most associated with human to human and animal transmission

FALSE: Genotypes A and B are most human infecting AND zoonotically transmitted

Giardia has a binucleated genome. What are the functions of each of the two nuclei?

BOTH transcriptionally active and replicate at the same time (same functions)

What organelles does Giardia have and not have?:

• ER

• feeding organelle

• Golgi

• Mitochondrion

• nucleus

HAS:

• ER

• feeding organelle

• nucleus

DOESN’T HAVE:

• Golgi

• Mitochondrion

What are the challenges in treating giardia? (4)

• Negative impact of main drug (5-Nitroimidazoles) on microbiome

• Short term & long-term side effects of drugs

• drug resistance is common in some areas.

• difficult to culture

Blood Fluke life cycle (7 steps)

1. eggs shed from excrement

2. miracidia released from eggs

3. miracidia picked up by snails

4. miracidia→ sporocysts

5. sporocysts→ cercariae; released (into water)

6. cercariae burrow under human skin

7. cercariae→ schistosomulae; enter bloodstream and migrate to target organs

TRUE OR FALSE: Parasites are sexless

FALSE: often they are, but some, for instance schistosomes have both male and female

Tapeworm lifecycle (7 steps)

1. eggs released into environment

2. ingestion by animal host

3. oncospheres hatch and move through intenstinal wall to muscle

4. oncospheres→ cysticerci

5. humans ingest not-fully-cooked, infected meat

6. attachment to intestine

7. shedding of egg containing segments from end of tail

Guinea worm life cycle (5)

1. larvae consumed by copepod

2. Human or animal consumes infected copepods through food or water

3. larva penetrates stomach an intestine→ migrates to underneath the skin

4. fertilized worm penetrates skin, causing a blister

5. larva released from submerged blister by mature worm

Leishmania lifecycle

1. sand-fly sucks blood→ a or b

1. injects promastigote

2. a) promastigote phagocytosed by macrophage

3. promastigote→ amastigote

4. multiplication and cell lysis in macrophage and other cells

5. b) ingests infected cells

6. amastigote→ promastigote (in gut)

7. division and migration to proboscis (sucker)

What are the a) cutaneous/mucocutaneous and b) visceral manifestations of Leishmaniasis?

a) ulcer/scar tissue, destruction of mucus membrane

b) Fever, enlargement of Spleen & Liver, Post-kala-azar dermal Leishmaniasis

What are the species of human malaria? (5)

Plasmodium…

falciparum

vivax

malariae

ovale

knowlesi

Malaria lifecycle (6 steps)

1. gametocytes circulate in mosquito blood

2. male and female gametocytes fuse in gut→ motile stage

3. oocyst formation and multiplication

4. sporozoite formation and travel to salivary gland

5. transmission to humans→ travel to liver and multiplication

6. merozoites enter blood and enter RBC→ replicate→ lyse RBC

a) What are the main symptoms of malaria and b) what percentage of cases show all three?

a) inflammation from cytoadherence to vasculature, acidosis, anaemia

b) 35%

What are the top three symptomatic configurations associated with malaria cases?

1. cytoadherence + acidosis + anaemia (35%)

2. cytoadherence + acidosis (28%)

3. acidosis (24%)

To what demographic is malaria the biggest threat and why?

pregnant women in 1st and 2nd pregnancies; placenta is a site of cytoadherence for dominant placental binding antigen PfEMP1 VAR2CSA which is relatively conserved, immunity develops by 3rd pregnancy, causes low birth weight and impaired development

How much more likely are those affected by cerebral malaria to a) develop neurological disability and b) die from respiratory and diarrheal diseases?

a) 28x

b) 4x

What social and economic factors affect and are affected by malaria? (6)

• Political stability

• Economic stability.

• Changes in climate

• Pandemics

• Education

• GDP

What is available to prevent or combat malaria and what are the limitations? (3)

bug netting→ needs reapplication of insecticide, can result in insecticide resistance

clinical drugs→ drug resistance

vaccine→ limited efficacy

TRUE OR FALSE: Immunity to malaria is impossible

TRUE

Is the infectious dose of malaria high or low?

low

What are the impacts of RBC malaria infection on the immune response/body/cells? (5)

• increased size of RBC

• increased rigidity of RBC

• reduced oxygen flow (hypoxia)

• RBC express surface proteins to stick to microvasculature

• Inflammation

a) What is cytoadherence and b) antibody recognition of malaria mediated by?

a +b) P. falciparum erythrocyte membrane protein 1 (PfEMP1)

b) multiple PfEMP1 proteins; can be switched between to evade immune response

How do mosquitos adapt to develop resistance for malarial control insecticides? (2)

Mutated sodium channel

Upregulation of detoxifying enzymes

What causes artemisinin activation?

parasites digest Haemoglobin causing build up of Haem and Fe²⁺

What chronic diseases does T. gondii cause to which groups? (3)

congenital toxoplasmosis: vertically transmitted to fetus

Cerebral toxoplasmosis: in the immunocompromised

Ocular toxoplasmosis: 4.5% of infections

T. gondii Life Cycle: just the general gist tbh… (3 steps)

1. shed parasites ingested via water or food

2. interconvert between asexually reproducing bradyzoites (persistence, slow replication, chronic) and tachyzoites (mobile, fast replication, acute)

3. In definitive host: bradyzoites→ merozoites; undergo sexual replication and shedding

TRUE OR FALSE: T. gondii can be transmitted alongside blood and organ donation

TRUE

What is the difference in manifestation of T. gondii infection in a) immunocompetent and b) immunocompromised patients?

a) Asymptomatic/ flu-like symptoms, swollen lymph nodes, tissue damage

b) encephalitis, pulmonary and cardiac disease

What other conditions is chronic T. gondii infection associated with?

Neurological, behavioural and neuropsychiatric disorders

Heart disease

What is the a) total transmission of T. gondii during pregnancy and what is it specifically in b) first, c) second, d) third trimester.

a) 20-30%

b) 5-10%

c) 15-30%

d) 30-72%; 75% subclinical

Why are felines the definitive host for T. gondii?

Lack intestinal Δ-6-desaturase needed for linoleic acid metabolism, causing linoleic acid build up which promotes parasite gametogenesis

a) What are the apical organelles and b) what do they collectively mediate? (5)

a) see image

b) Egress, attachment, entry, modification, downregulating immune responses

T. gondii has expanded ligand families. What are they and what are they for?

• SRS (surface proteins): attachment

• MIC (microneme): adhesion and invasion

• RON/ROP (rhoptry)

• GRA (dense granule): modification

T. gondii Egress Triggers (5)

• Fas:FasL ligand binding→ caspase activation→ ↑ [Ca²⁺]

• Perforin insertion into host membrane→ ↓ [K⁺]→ ↑ [Ca²⁺]

• abscisic acid increases above threshold

• acidification in the parasitophorous vacuole→ Ca²⁺-dependent secretion of micronemes→ ↓ suppression by K⁺

• NTPases deplete host cell ATP→ ion pumps run dry→ ↓ [K⁺]

TRUE OR FALSE: T. gondii is hard to fight against because it can change expression to inhibit intrinsic and/or extrinsic apoptotic pathways

FALSE: Can just inhibit both

How can T. gondii overcome host Cell Apoptosis? (3)

• inhibit Bax from oligomerizing with Bak, which would release cytochrome C

• Upregulates anti-apoptotic molecules

• interference with caspase-8 inhibits Fas

intracellular tissue cysts of T. gondii are (reactivated/quiescent) in immunocompromised and (reactivated/quiescent) in immunocompetent, causing (acute/chronic) infection.

intracellular tissue cysts of T. gondii are (reactivated/quiescent) in immunocompromised and (reactivated/quiescent) in immunocompetent, causing (acute/chronic) infection.

like aight bro…

Symptoms of cryptosporidiosis

• Gastroenteritis

• Diarrhoea

• Dehydration

• Malnutrition

(__) of cows are exposed to cryptosporidium by 2 weeks old

95%

TRUE OR FALSE: Cryptosporidium has multiple definitive hosts

TRUE: Does sexual replication in every host (hosts= mammals)

The Cryptosporidium lifecycle (at least 6 steps)

1. parasite-containing oocyst enters host

2. oocyst activated in intestine by bile salts

3. sporozoite released from oocyte

4. sporozoite infects intestinal epithelial cell

5. three cycles of asexual replication forming (from 1 meront) 8 merozoites each

6. sexual determination into male (female= non-replicating): forms gamont which releases 16 gametes

7. fertilization→ diploid zygote→ meiosis→ sporulation

Cryptosporidium transmission routes

faecal-oral

food/water borne

How is water treated for cryptosporidium and why?

irradiation, ozone, chlorine dioxide needed because oocytes are ethanol and bleach resistant

Cryptosporidium oocytes can survive for (__) after being shed

months!

What is outside/on top of an intestinal epithelial cell?

How does Cryptosporidium trigger host recognition? (2)

most importantly, is recognised by STAT1 and IFNγ (as well as other IFNs)

How does Cryptosporidium limit immune response? (4)

• Downregulation of STAT1 & IFNγ

• Inhibition of apoptosis

• >150 exported proteins; likely immune modulators

• MEDLE2 triggers ER stress; limits inflammatory response

What organelle does Crypto lack?

functional mitochondria

What features make replication of Crypto unusual?

• mitosis with uncondensed chromosomes

• Nuclear envelope doesn’t break down during mitosis

• very rapid meiosis; produces 8 daughter parasites in <1hr

Challenges in studying Cryptosporidium (5)

• Understanding of this biology usually poor

• Lack of continuous in vitro culture

• No long-term storage

• Genetic modification difficult

• Small size of parasite

a) American and b) African trypanosomiasis (scientific name)

a) Trypanosoma cruzi

b) Trypanosoma brucei

a) American and b) African trypanosomiasis: what disease does it cause?

a) chagas disease

b) sleeping sickness

What is the pathogenesis of Chagas disease? (2 phases)

acute phase 2 months post infection with mild symptoms

over time 30% develop cardiac complications and 10% develop digestive, neurological or mixed alterations

a) what are the treatments for Chagas disease and b) why are they not so effective (3)?

a) benznidazole or nifurtimox

b) long treatment period, early symptoms hard to catch, causes serious side effects in 40%

Trypanosoma cruzi lifecycle (7 steps)

1. multiplying epimastigotes in midgut (of triatomine)

2. metacyclic trypomastigotes in hindgut

3. triatomine bug takes blood meal

4. trypomastigotes penetrate cells at wound site

5. trypomastigotes→ amastigotes and undergo binary fission

6. amastigotes→ trypomastigotes→ burst from cells

Trypanosoma brucei (HAT) life cycle (6 steps)

1. bloodstream trypomastigotes→ procyclic trypomastigotes in midgut

2. divide by binary fission

3. leave midgut→ epimastigotes; multiplication in salivary gland

4. epimastigotes→ metacyclic trypomastigotes

5. tsetse fly takes blood meal

6. metacyclic trypomastigotes→ bloodstream trypomastigotes; carried around body

TRUE OR FALSE: Trypanosoma brucei is fully extracellular

TRUE

What outcomes does Trypanosoma brucei result in in patients? (2)

• Sequestration in the adipose tissue results in wasting

• Neurological symptoms from parasites in the cerebrospinal fluid

What treatments are available for a) early, b) non-severe late, c) late T.b. gambiense from HAT?

a) Pentamidine

b) Fexinidazole

c) Nifurtimox –Eflornithine combination therapy

What treatments are available for a) early, b) late T.b. rhodensiense from HAT?

a) Suramin

b) Melarsoprol; up to 10% fatal! 😱

kinetoplast: what is it/where is it found and what does it do?

Dense mitochondrial DNA network in trypanosome

Needed for survival in arthropods, loss associated with drug resistance

The trypanosome genome encodes (__) surface proteins and expresses (__) at one time, anchored to the surface membrane with (__)

The trypanosome genome encodes very many🤷‍♀️ surface proteins and expresses one at one time, anchored to the surface membrane with glycosyl inositol phosphate

Do out of (+)ssRNA , (-)ssRNA and dsRNA viruses, which have the following features: a) RdRp, b) nucleocapsid

a) dsRNA

b) (-)ssRNA, dsRNA

name the following structures on linear RNA

The structure shown is…

a pseudonot

Viral RNA can replicate with (high/low) fidelity polymerase

Viral RNA can replicate with (high/low) fidelity polymerase

What are the two modes of RNA transcription initation and what are the primers required?

de novo; no primer

primer-dependent; terminal or 5’-Cap proteins

TRUE OR FALSE: Virus (+)ssRNA genome is not modified before translation

FALSE

TRUE OR FALSE: The (+)ssRNA genome resembles cellular mRNA

TRUE

How do alphaviruses differ from (+)ssRNA viruses?

make subgenomic RNA in addition to full length complementary genomic RNA

What are some examples of alphaviruses and what disease do they cause?

Togaviridae; Rubella, Sindbis, Ross River Fever

Caliciviridae; Norovirus

(+)ssRNA replication during infection (7 steps)

1. binding to receptor

2. entry into an endosome

3. genome and VPg primer protein is released into cytoplasm

4. translation in polypeptide

5. cleavage of polypeptide

6. replication→ accumulating (+) strands

7. packaging and release

What structures does polio have that assist it during replication? (3)

• cloverleaf at 5’ end

• Cre transcription element

• pseudoknot at the 3’ end

What is the function/the process of this function in action of Cre? (4 steps)

1. Two 3CD protein complexes join to the stem of Cre

2. an additional 3D protein joins to the Cre loop

3. VPg binds with primarily A-U nucleotide pairing to loop

4. Two Uracils synthesized on VPg giving primer function

Poliovirus transcription (6 steps)

1. viral 3AB protein joins to host membrane→ recruits PCBp

2. host PCBP2 and the viral 3CD bind to the 5′ cloverleaf-like structure of +RNA; host PABP bound to the 3′ poly(A) tail

3. interaction between PCBP2 and PABP→ ends of the RNA genome come together

4. cleavage of 3CD and the release of the RdRp

5. synthesis of (-)RNA utilising Vpg-UU as a primer

6. dsRNA is then partly unwound by the chaperone activity of the host hnRNPC and the binding of PCBP2 and 3CD to the 5′ end of the (+)RNA

Where is the Cre element in the genome?

can be anywhere

What is the product of subgenomic RNA?

structural proteins

To transcribe subgenomic RNA, alphaviruses have (__) in/on (__)

To transcribe subgenomic RNA, alphaviruses have subgenomic promoter on the (-)RNA strand

TRUE OR FALSE: Sub-genomic RNAs are not not packaged within virions

TRUE

How does polio initiate translation?

Via IRES element/structure on poliovirus mRNA that directly binds to cellular translation factors and shifts all the translational machinery to Poliovirus translation

Is function of IRES CAP-dependent or independent?

independent

What virus other that polio a) recruits ribosome in a similar manner and b) what is the difference?

a) hepatitis C

b) HCV does not destroy the cellular protein translation machinery thus does not directly kill hepatocytes

What kind of RNA genome does Coronavirus have?

(+)ss

Does coronavirus have subgenomic RNA?

yes

SARSCoV-2 Discovery process (9 steps)

1. Bronchoalveolar lavage fluid (BALF) collected from sick patients.

2. Bronchoalveolar Lavage fluid used to directly inoculate Vero (Monkey Kidney) cells

3. Total RNA isolated

4. Screened by RT-PCR for other known respiratory pathogens.

5. RNA present is reverse transcribed to cDNA

6. DNA Sequencing then carried out using Illumina and Nanopore Platforms.

7. de novo sequencing to construct genome from overlap between reads

8. RT-PCR primers specific for SARSCoV-2 designed

9. Full genome sequencing methods developed to monitor viral evolution

What type of dye is used for the RT-PCR of covid samples and why is it problematic?

SYBR Green intercalating dye binds any double-stranded DNA which causes false positive fluroscence if your sample is contaminated or non-specific primers are used

What do melt curves in RT-PCR show?

multiple peaks indicate multiple products

How can the specificity of fluroscence generators in RT-PCR be improved?

TRUE OR FALSE: The main limitation of probe based RT-PCR is that only one gene target can be made to produce fluorescence.

FALSE: probes for different targets can be made to emit at different wavelengths.

The main downside would probably be that no melt curve can be generated