Protozoal parasites Flashcards

Question

Tell me the main differentiation stages of Haemoflagellates

Answer 1

Body Borer Believed to initially been a parasite of insects (may still be and mammals a minor part of biological cycle) 3 main differentiation stages **Promastigote** **Trypomastigote -** flagellated, in mammal and transfer organism to and from insect. Extracellular **Epimastigote** – flagellated form found in insect tissue **Amstigote** Amastigote – non -flagellated form present inside mammalian cells (show binary fission). Intracellular

Answer 2

Different species have different morphological forms Trypanosomes adopt different morphological forms in different hosts Flagella appears at side in tyropmastigote and epimastigote At and in promastigote

Answer 3

**Trypomastigote**-posterior localization of mitochondrial DNA aggregate, (kinetoplast K), to the nucleus (N). An attached flagellum exits from the flagellar pocket (FP) near the cell posterior. **Epimastigote**-kinetoplast is anterior to the nucleus. While the flagellum is still attached to the cell surface, it also has a long, cell-free overhang. **Promastigote**-similar arrangement of DNA-containing organelles as epimastigote, but the flagellum is unattached after exiting the FP pocket at the cell anterior. (Seen in Leishmannia – not Trypanosomes) **Amastigote** – smaller, spherical, kinetoplast anterior to the nucleus. The flagellum very short and nonmotile, - INTRACELLULAR FORM

Answer 4

The microtubule quartet (MtQ) has **opposite polarity** to the other array microtubules- (nucleated near the basal body and grows towards the cell anterior may act as a signal for the formation of the flagella). The flagellum attachment zone **(FAZ)** filament interrupts the array and attaches the flagellum to the cell surface. **PFR**, para-flagellar rod. **FP**- flagellar pocket has unique composition

Answer 5

Different species have different morphological forms Trypanosomes adopt different morphological forms in different hosts Promastigote- Leissmania Trypomastigote-?

Answer 6

Position of flagella changes with nuclei position Plasma membrane covered in protein coat made of VSG proteins

Answer 7

**Unicellular eukaryotes** The cell membrane consists of at least **three discrete domains**: the cell membrane, the flagellar membrane, and the flagellar pocket Possess a **microtubule corset** closely opposed to the cell membrane – define the shape of the cell Have a **kinetoplast** – a mass of cytoplasmic DNA found at the base of the flagellum and continuous with the (single) mitochondrion. Possess a **paraflagellar rod** – only partially characterised with unknown function (attachment to insect host? Aids motility?)

Answer 8

Mass of mitochondrial DNA which sits where the flagellar pocket will be at the end of the mitochondria Kinetoplast splits first, then flagellar then nucleus during division The kinetoplast is a specialized region of the mitochondria of trypanosomatids that harbors the most complex and unusual mitochondrial DNA found in nature. Kinetoplast DNA (kDNA) is composed of thousands of circular molecules topologically interlocked to form a single network.

Answer 9

Possess a paraflagellar rod – only partially characterised with unknown function (attachment to insect host? Aids motility?) * Attachment to insect host? Dense hemi-desmosome- like plaques associated with proximal flagellum and unsect cells * Aids motility? * Stabilises flagellum

Answer 10

**Unique to kinetoplastids** (flagellates inc. Tryp and Leish) **Mitochondrial DNA** Mass of **circular maxi- and mini- kDNA** near flagella polar body Present at one end of the branched single continuous mitochondria 10-40 copies of the mitochondrial genome (**maxicircles**) 1000 +copies of guide DNA (**minicircles- forms gRNA**) – unique correction system of mitochondrial genes akin to CRISPR

Answer 11

* **Crithidia** * **Phytomonas**

Answer 12

**Crithidia** – mongenic insect parasites (possible direct ancestor of group) Only found in choanomastigote form e.g.**, C. bombi** parasite of bumblebees implicated in decline of wild bee populations

Answer 13

**Phytomonas (phyto= plant)** * digenic insect * plant parasites. **P. staheli** causes wilt in coconut /oil palms Latin America (\>$1Billion lost/ yr). **P. leptovasorum** coffee phloem necrosis in Liberica and Arabica ($8 billion / yr). Other ssp infect tomatoes No treatment, try to prevent spread Probably initiated as insect monogenic parasite –maybe initiator of all parasitic flagellates and used as models for human flagellate trypanosomal diseases.

Answer 14

T. ambystomae. in amphibians T. antiquus, extinct (Fossil in Miocene amber) T. avium, which causes trypanosomiasis in birds T. boissoni, in elasmobranch T. brucei, which causes sleeping sickness in humans and nagana in cattle T. cruzi, which causes Chagas disease in humans T. congolense, which causes nagana in ruminant livestock, horses and a wide range of wildlife T. equinum, in South American horses, transmitted via Tabanidae, T. equiperdum, which causes dourine or covering sickness in horses and other Equidae, it can be spread through coitus. T. evansi, which causes one form of the disease surra in certain animals T. everetti, in birds T. hosei, in amphibians T. irwini, in koalas T. lewisi, in rats T. melophagium, in sheep, transmitted via Melophagus ovinus T. paddae, in birds T. parroti, in amphibians T. percae, in the species Perca fluviatilis T. rangeli, believed to be non-pathogenic to humans T. rotatorium, in amphibians T. rugosae, in amphibians T. sergenti, in amphibians T. simiae, which causes nagana in pigs. Its main reservoirs are warthogs and bush pigs T. sinipercae, in fishes (other host Leaches) T. suis, which causes a different form of surra T. theileri, a large trypanosome infecting ruminants T. triglae, in marine teleosts T. vivax, which causes the disease nagana, mainly in West Africa, although it has spread to South America T grayi – crocodiles spread by Tsetse flies T. clandestinus crocodiles spread by Leaches

Answer 15

America- some esp. rodent ones widespread **T. lewisi**, in rats T musculi in mice…initially Americas (flea)-pathogenic to man too **T cruzi** in man – Chagas disease – infectious to wide spectrum of mammals

Answer 16

**T. vivax** **T. Congolese** **T. brucei** **T. equiperdum** **T. evansi**

Answer 17

T brucei – **5 subspecies** (flies /bats!) Cause Nagana in ruminants Sleeping sickness in man Dourine in horses Surra numerous animals including dogs/ cats ruminants (Africa/ middle east /south America

Answer 18

Ruminants- Nagana- West Africe (Tsetse fly)

Answer 19

T. Congolese, causes nagana in ruminant livestock, horses, and a wide range of wildlife

Answer 20

T. equiperdum, which causes dourine or covering sickness in horses and other Equidae (also mating)

Answer 21

T. evansi, surra in certain animals

Answer 22

Salivarian trypanosomes - passed to the recipient in saliva * insects normally tsetse flies (Glossina spp.) * also, tabanid (horse) flies, vampire bats South America * tsetse fly is an amplification step **African trypanosomiasis**

Answer 23

Stercocarian trypanosomes – passed to the recipient in faeces of insects e.g., triatomid (assassin) bugs also, tabanid flies, fleas **South American trypanosomiasis**

Answer 24

Sleeping sickness in man Nagana in cattle and horses Surra in many animals Dourne in equids

Answer 25

The trypanosome and species infected

Answer 26

**_Human Disease_** **Trypanosoma brucei rhodesiense** (2%) - more acute disease, Zoonotic **Trypanosomoa brucei gambiense** (98%). **_Animal Disease_** **Trypanosoma. brucei**. **bruceii** in cattle causes Nagana **Trypanosoma. brucei equiperdum**, which causes dourine or covering sickness in Equidae (also spread at mating) **Trypanosoma. brucei evansi**, surra in many species’ animals Last 2 cannot reproduce in insects so only use them a mechanical vector Morphologically identical – genetically different

Answer 27

overtime will end up in CSF in CNS When present in blood in high amount it goes to Stumpy form Can show sexual production as evidence for DNA transfer occurring Cattle can also be infected by T.b.rhodesiense (this is not seen with gambiense form)

Answer 28

**Procyclic form (TM)** – the dividing form of the parasite found in the insect host. Mostly binary fission but are capable of recombination (? sexual reproduction)

Answer 29

**Epimastigote** – smaller form invasive form can leave gut of fly and enter its salivary gland has different flagella attachment to TM forms

Answer 30

**Metacyclic form (TM)** – the human infective form of the parasite found in the insect host- in salvarian forms it can resist salivary enzymes and reduces their production (see below for Chagas disease)

Answer 31

**Bloodstream form (TM)** – the dividing form found in the bloodstream of the mammalian host

Answer 32

Tropical Africa (grassland and forest) Long lived obligate parasites- female to 4 months as adult At least 23 species in tropical Africa Different subspecies are infected with different trypanosomes Primary host: insect Secondary host: mammals

Answer 33

Females **feed on vertebrate blood through biting** Multiple egg broods \>6 but **only requires mating once** (may mate more often) Has a uterus and releases larva at 3rd Instar stage (9d)- hatches after 4 days, then stays in for a further 9 days as continues to develop Pr**oduces one larva at a time** released from uterus. Larvae and burrows into sand or soil to pupate ie depended on mother for nutrition till adult hence female must feed often-exclusively on blood different species have different chosen hosts can take \>100% body weight in blood Dynamic populations – drop by \>90% in dry season

Answer 34

Day feeding Attracted to smell, movement, and colour (specifically blue) – large animals Often quite host specific Very good transmission system – both Mechanical and Biological Transmission, must feed regularly (Horse flies can act as Mechanical vector for trypanosomes) The presence of these flies / their parasites is a direct cause of sub-Saharan poverty – and failure in agriculture

Answer 35

Other biting flies (e.g., Horse) – mechanical transfer only Vertical trans-placental Blood transfusion/ organ transplantation Needle (rare must be when highly infectious) Sexual contact All these are relatively rare – the fly primary host is a very efficient amplification step

Answer 36

**T. b. gambiense** is found in Central and West Africa * More Chronic - ~98% of all ASS * Limited reservoirs * Easier to control **T. b. rhodesiense** is found primarily in East Africa (Botswana, Ethiopia, Kenya, Malawi, Tanzania, Uganda, Zaire, and Zimbabwe) * More Acute - ~2% of all ASS * Wildlife and Livestock infected too * Harder to control

Answer 37

Three (? two) defined stages – **100% fatal if not treated** Acute means speed of infection, severity is whether it will kill someone or not **Stage 0/** Initial infection – localised **Trypanosomal chancre** (response to localised presence of trypanosome) at the site of the infectious fly bite ~ 2 days of infection. More common in T. b. rhodesiense infection, Parasites multiply here – pathology due both to trypanosome and type IV hypersensitivity response to fly salivary proteins **Stage 1/** Haemolymphatic stage – parasite found in the blood/ Lymph Nonspecific, generalized symptoms malaise, weakness, anaemia fatigue, pruritis, hepato-splenomegaly, weight loss and intermittent fevers in waves (Immune evasion strategies here- parasite is highly antigenic) ~ 1–3 weeks after the tsetse fly bite T. b. rhodesiense ~ weeks to months after the tsetse fly bite T. b. rhodesiense V common lymphadenopathy **Stage 2/** CNS stage (often overlaps with stage 1) T. b. rhodesiense ~ 21–60 days. T. b. gambiense ~ 300–500 days, invasion of the CNS (CSF contains trypomastigotes) causes neuropsychiatric disease, (fever occurring less frequently) sleep/wake cycle reversed, daytime somnolence, nocturnal insomnia, hence the common name **Behavioural -**hallucinations, delirium, apathy, aggression, mania **Motor** - weakness, ataxia, tremor, **Sensory** - hyperaesthesia, anaesthesia, pruritis, visual hallucinations -\> resulting in seizures, coma, DEATH (inevitable if untreated)

Answer 38

**Variant specific glycoprotein– VSG** – surface glycoprotein ~ 90% of outer coat and close to 100% external surface Many (2000 **VSAs** (Variant specific antigen) genes or parts of genes in genome –one expressed at one time) ~65kD, 500 amino acids, 3 domains. **N terminal 360 amino acids**, most superficial and seen by immune system poor sequence conservation but similar secondary/ tertiary structure **The 120 C-terminal amino acids** highly conserved but masked lying next to the plasma membrane. **The C terminal pro protein** has typical transmembrane hydrophobic sequence attaching the protein to the plasma but this cleaved and the mature protein is linked to the membrane cell surface by a phosphatidylinositol glycolipid link. This allows tighter packing and masks other more conserved membrane proteins from antibodies.

Answer 39

VSG part of 12-15nm thick outer layer on membrane As the parasitemia rises, the host raises antibodies to the coat killing most of the parasites Some of the remaining parasites switch their coat protein and therefore evade the immune response TEMs of TSs of trypanosomes

Answer 40

VSG is varied because have genetic recombination and VSG expression site switching in respect to the promotor and position relative the telomere (the most telemetric version is expressed) causes changes in VSG expression

Answer 41

**VSG recombination to novel forms** Stripping and replacement of VSG also occurs ?? role of flagellar groove **VSG shedding and attachment on to RBC causes** RBC lysis and anaemia Recruitment of complement and antibodies away from the parasite Hypocomplementemia- lower complement levels as they are being used to lyse RBC

Answer 42

The infective form is the **stumpy trypomastigotes** Transition from slender (blood) to stumpy (infectious non proliferating) forms caused by a quorum sensing mechanism, stumpy induction factor (SIF) accumulates as parasite numbers increase during parasitaemia. I.e., so non-dividing infectious form only produced when high levels (consider the opposite) Where the long form is ingested, they die in the fly

Answer 43

Clinical diagnosis initially hard – non-specific signs Haematology –parasite only observed at time of high parasitaemia in early infection Lymph node biopsy PCR on blood/ CSF CSF smears Serology not good – main antibodies against VSGs …. which one? - There are some screening Ab tests.

Answer 44

Those drugs which enter the CNS and those which don't

Answer 45

**Haemolymphatic stages** (these drugs don’t enter CNS) * **Pentamidine, for T.b gambianse** - binds AT rich regions of parasite DNA -\> crosslinking (resistance observed) – discovered 1937 Resistance to Pentanidine caused by mutations, TbAT1/P2 transporter High Affinity Pentamidine Transporter Resistant trypanosomes were selected for in the field by farmers under-dosing * **Suramin, for T.b rhodesiense –** binds trypanosomal glycolytic enzymes, inhibiting energy metabolism - discovered 1916

Answer 46

\*Nausea, vomiting, diarrhoea, headache, skin tingling, and weakness. Sore palms of the hands and soles of the feet, trouble seeing, fever, and abdominal pain may also occur \*\*headache, conjunctive injection, fever, tachycardia, hiccup, diarrhoea, vomiting, tremor, slurring of speech, usually followed by convulsions and ultimately coma, death **CNS stages-** these enter CNS, but toxicity rules out use for earlier stages **Nifurtimox** (a nitrofuran) undergoes reduction and releases oxygen radicals and H2O2 which destroys parasite DNA. Mammalian cells are protected by peroxidases and superoxide dismutase **Melarsoprol\*\*** (arsenical) irreversibly binds sulfhydryl groups on pyruvate kinase reduces energy production Limited selective toxicity (1949) **Eflornithine\*\*** (polyamine biosynthesis inhibitor) irreversibly inhibits ornithine decarboxylase which makes polyamines e.g., putrescine needed in cell division, 1970 \*\* Severe side effects - ~95%+ clearance of parasite

Answer 47

**Fexinidazole -** 2018, first drug for advanced-stage sleeping sickness in thirty years- Nitroimidazole compound Acts against both stages if not severe taken orally- taken orally and enters CNS Parasite nitro reductases reduce fexinidazole to reactive intermediates free radical and superoxide release which are capable of damaging DNA and proteins

Answer 48

WHO has targeted West African trypanosomiasis for elimination as a public health problem? Strategies for elimination of West African trypanosomiasis **: active and passive case - screening** **: treatment of the confirmed cases,** **: vector control to reduce the tsetse population.** East African trypanosomiasis far greater challenge due to reservoir infection in Wildlife and Livestock. Vector control is the primary strategy along with animal health. **There is no vaccine or prophylactic drug**

Answer 49

**_Environmental_** **Map highly effected sites –** localised sequential aerosol spaying with plant spread insecticides in severe outbreaks (Ground treatments not used – residues) **Bush Clearance** – ecological issues Pour on **systemic insecticide treatment** of animals (East African trypanosomiasis) **Baits and coloured traps** (pheromone and insecticide traps) **_Human_** **Long-sleeved shirts / pants of medium-weight material** in neutral colours **Inspect vehicles** - flies are attracted to the motion and dust from moving vehicles. **Avoid bushes.** **Minimise activity** during the hottest part of the day. Together can reduce Tsetse fly attack by \>99%

Answer 50

**Nagana- Trypanosoma brucei brucei and friends** Similar disease in cattle to sleeping sickness in humans Caused by T.b. brucei, T.b. rhodesiense (T. vivax and T. congolense) More widespread and commoner than sleeping sickness – more vectors and quicker reproduction in fly Large areas of sub-Saharan Africa killing 3 million cattle per year- can’t farm cattle here Some cattle are relatively resistant (e.g., N'Dama), European species not Devastates agriculture and lifestyles

Answer 51

**_South American trypanosomiasis- SAT_** **Trypanosoma cruzi – Chagas disease** Zoonotic disease affecting 150 species of mammals inc. humans Found as **amastigote** (intracellular), trypanomastigote (blood) and epimastigote (insect) forms Stercocarian trypanosomes – passed to the recipient in faeces of insects e.g., triatomid (assassin) bugs– also tabanid flies, fleas Tabinids – horsefly – again a mechanical vector not biological one Cf T brucei – only a few other hosts

Answer 52

Assassin bugs crawls as it can’t fly Triatomine insect vector- Triatoma, Rhodnius, and Panstrongylus (or “kissing bug”) takes a blood meal and releases trypomastigotes in its faeces at bite wound. Trypomastigotes enter host through wound or at intact mucosal membranes, eg conjunctiva. In the mammal host, the trypomastigotes invade cells at site of inoculation, and differentiate into intracellular amastigotes. These multiply by binary fission, differentiate into trypomastigotes, and release into the circulation Trypomastigotes infect cells in a variety of tissues and transform into intracellular amastigotes in new infection sites-\>Clinical disease The bloodstream trypomastigotes do not replicate (cf African trypanosomes). Replication resumes only when the parasites enter another cell or in the “kissing bug” when infected by feeding on mammals that contains circulating parasites. In insect the ingested trypomastigotes transform into epimastigotes in the vector’s midgut. The parasites multiply and differentiate in the midgut and differentiate into infective metacyclic trypomastigotes (generally taken up by bug when it takes a meal) in the hindgut. Replicated via binary fission

Answer 53

Vertical trans-placental Blood transfusion/ organ transplantation Needle stick (rare must be when highly infectious) Consumption of uncooked food that is contaminated with faeces from infected triatomine bugs ??Bed bug. Why kissing bug – crawling bug living in cracks and feeding at night biting exposed skin at night often face

Answer 54

Chagas disease is transmitted to animals and people by insect vectors that are found only in the Americas. ~ 8 million people in Mexico, Central America, and South America have Chagas disease, most inapparent infection. Columbia alone ~$1Billion/yr health costs. Estimated 300,000 infected in US Large-scale population movements from rural Latin America to other regions of the world have increased the geographic distribution and changed the epidemiology of Chagas disease. In the US and Europe Chagas disease is found but is not endemic. cf other ways of transfer Infection is lifelong and may become life threatening ~ 30% will develop serious cardiac problems and 10% will develop digestive and neurological problems Bed bug can act as an amplifying host

Answer 55

**3 phases** – some clinicians fuse the 2 chronic phases **Acute phase:** occurs immediately after infection, lasts ~ 2 months There may be initial fever or swelling at site of inoculation. High numbers of parasites circulate in blood, symptoms are mild or absent. very rarely, acute infection causes cardio myositis, or encephalitis/meningitis (easy to diagnose here) **Chronic intermediate phase:** parasites are hidden mainly in heart and digestive muscles. Due to immune response parasite number are low but rarely eliminated (diagnosis difficult). In later years can lead to heart failure and /or sudden death

Answer 56

**Chronic Chagas disease:** Many people remain asymptomatic for life. However, 20–30% of infected people develop severe life-threatening problems Heart rhythm abnormalities that can cause sudden death. A dilated cardiomyopathy and heart failure Failure in GI smooth muscle -megaoesophagus or megacolon, leading to difficulties eating or defecating

Answer 57

**Entry into chronic disease** 1. Immune suppression (AIDS, steroids, or chemotherapy), Reactivation with parasites found in the circulating blood. 2. Autoimmunity: Caused by Molecular mimicry: as pathogen bears antigens like host antigens with self-responding Abs resulting in tissue damage

Answer 58

Not easy using blood smears – only acute phase PCR blood in acute phase Serological techniques often inconclusive Xenodiagnoses - is often used. Uninfected bugs are fed on suspected sufferers and monitored over a period of 3 months to see if their faeces contain the metacyclic tyrpomastogotes.

Answer 59

**Nifurtimox** (see Sleeping Sickness) and **Fexinidazole / Benzinidazole**. Both administered orally needs long treatment courses and can’t overcome pathology ~60 clearance of parasite as in cellular niche Benznidazole - see Fexinidazole (Nitroimidazole) nb this is not a benzimidazole the microtubule inhibitor – see helminth lectures All severe side effects

Answer 60

Vector - Improved housing and spraying insecticide inside housing to eliminate the bugs, Insecticide impregnated bed nets, biting usually at night, a crawling bug plastering houses and better cleanable flooring has proved effective Triatoma insecticide resistance is starting to develop. Screening of blood donations Screening at organ transplantation Early detection and treatment of new cases, including mother-to-baby (congenital) cases,

Answer 61

Work underway with Leishmania – trials underway Less easy with T. cruzi because of variations in the parasite Impossible with T. bruceii because of VSG antigenic variation.

Answer 62

Exploit trypanosome weak spots e.g., flagellar pocket (unique membrane structure), paraflagellar rod (only in kinetoplasts), kinetoplast, Genomes have been sequenced and some biochemical pathways are different to ours but common to trypanosomes Why are some cattle resistants to T.b. brucei?

Answer 63

Zoonotic Diagnosis and recognising infection in vectors/ reservoir hosts Other hosts Drug resistance – parasite (some) and insect vectors (lots?) Vaccines… Asymptomatic stages Pregnant women

Protozoal parasites Flashcards

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