1. Parasitology: Contents
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Malaria Toxoplasmosis Leishmaniasis
Complement Activation Pathway
African Trypanosomiasis (Sleeping Sickness)
South American Trypanosomiasis (Chagas Disease)
Schistosomiasis Immunity to Filariasis
Hookworms
Human Intestinal Nematodes
Are Worms Good for Us?
Cestodes (Tapeworms)
Parasite GenomesParasite Vaccines
2. Parasitology Lecture 2
Malarial Parasites ‐ Plasmodium Invasion
Anopheles is a genus of mosquito. There are
approximately 460 recognised species: while over 100 can
transmit human malaria, only 30‐40 commonly transmit
parasites of the genus Plasmodium that cause malaria
which affects humans in endemic areas.
This photo shows a female mosquito. Only females feed
off humans, since the blood they collect is a nutrition‐rich
medium that is ideal for them to develop their eggs in.
Their gender is also easily classified as males have
feathery antennae.
There are over 1000 species of plasmodium that cause malaria in a variety of animals, but only four of these
are transmitted in humans:
P. falciparum ‐ Most widespread and responsible for most deaths
Found in Tropical Africa, Asia, Latin America
P. vivax ‐ Less virulent than P. falciparum and rarely fatal
Found worldwide in tropic regions,
P. ovale ‐ Rare compared to the above species, and substantially less dangerous than P. falciparum
Found in Tropical West Africa
P. malariae ‐ "Benign malaria", not nearly as dangerous as that produced by P. falciparum or P. vivax
Found worldwide, but patchy distribution
Malaria transmission requires an average temperature of >15°C, and the vector cannot survive above 3000m
The life cycle of malaria essentially follows a sequence of 4 phases; the first of which is sexual and only
occurs in anopheline mosquitoes (multiplication of the parasite does not occur here). This is followed by
three asexual phases, which involve multiplication of the parasite in the human body.
The spleen fights off infections on the blood, and so splenomegaly (an enlarged spleen) is a primary
symptom of malaria in children. Artwork has been discovered from centuries back depicting this, such as that
of Hippocrates palpating a child’s abdomen. A cartoon from WWII depicting the symptoms of malaria is
shown below. In modern warfare, statistics show that more soldiers die from malaria than enemy fire in
areas where malaria is endemic.
3. Stages of infection of red blood cells:
Individual parasites enter the RBCs, and can be seen on microscope images as little black dots
“Signet ring” phase (seen due to a large food vacuole and the peripherally situated nucleus)
Multiplication of the parasites inside the RBCs
Percentage parasitaemia refers to the percentage of red blood cells infected with parasites; from the ratio of
infected: non‐infected RBCs, a patient’s prognosis can be determined
The Life Cycle of Plasmodium
Sporozoites are injected into the bloodstream of a human host as a female mosquito feeds (A), they remain
in the bloodstream for 20 to 30 minutes, then head to hepatocytes in the liver (B), where they form
merozoites (daughter cells of protozoan parasites). After 30 to 40 minutes, the merozoites burst from the
hepatocytes, infect red blood cells (C) and rapidly reproduce asexually. Every 2 to 3 days, they burst from the
red blood cell hosts and go on to infect more RBCs (D). Antimalarial drugs can reduce these infections but
there is as yet no complete cure for malaria.
Some parasites go on to form male and female gametocytes (E), which can be picked up by the next
mosquito that feeds on the human host (F). Inside the gut of the mosquito the gametes fuse to form a zygote
(G), that penetrates, and forms a cyst in, the gut wall. The zygote eventually ruptures and releases many
sporozoites that head for the salivary glands of the mosquito (H), where they await injection into another
human host.
The sexual and first asexual phases of the plasmodium life cycle only occur in anopheline mosquitoes, the
second asexual phase is in the liver, and the third in the blood, which is repeated many times. Every asexual
phase begins with feeding and growth, and every phase ends when new invasive parasites appear.
4. The Sexual Phase of Plasmodium (Inside Anopheline mosquitoes)
Ingested gametocytes swell and discharge osmophillic bodies (that can
tolerate high sugar concentrations) into the red blood cells. This disrupts
the RBC membrane, releasing gametocytes. During development of the
male gametocyte.
During development of the male gametocyte, the DNA is replicated
three times, so the nucleus of the activated gamete has 8 complete sets
of DNA. Eight kinetosomes are then formed in a microtubule organising
centre. Each kinetosome is the base and growing point for a flagellum
(axoneme). Eight flagella are formed in total, and the gamete explodes
releasing flagellum. This process is called exflagellation and occurs within
a few minutes after ingestion of the infected blood by the mosquito.
Each of the eight flagellum is a spermatozoa cell (sperm)! They actively
swim to the female gametocyte, fertilisation leads to the formation of a
zygote. In the next 5 to 10 hours, this develops into an ookinete (the first
invasive stage of the parasite’s life cycle), in which major changes occur
and apical complexes form, which allow the parasite to penetrate cells.
The ookinete is capable of moving spontaneously, it glides through the blood meal and penetrates the
stomach of mosquitos to form a thick‐walled structure known as an oocyst under the mosquito's outer gut
lining, where the next phase begins.
mosquito
midgut
Many oocysts on the inner mosquito midgut A single oocyst
5. Ookinete
Sporozoite
The First Asexual Phase of Plasmodium (Inside Anopheline mosquitoes)
In this stage, the ookinete becomes a sporozoite.
First it develops from an ookinete to an oocyst
The phase lasts for 8 to 35 days, and the ookinete grows rapidly to 80μm in diameter. The oocyst projects
into the haemocoel of the insect and feeds on the haemoglobin of the insect’s blood meal, in this time, DNA
replication occurs
A haemocoel is a cavity or series of spaces between the organs of organisms with open circulatory systems. A
combination of blood, lymph, and interstitial fluid called haemolymph circulates through the haemocoel.
Each oocyst contains at least 1,000 sporozoites that burst out and migrate to the salivary glands of the
mosquito
Merozoite
The apical complexes
(marked by blue circles) are
located in the same place in
these structurally different
morphologies of the
plasmodium parasite
6. apical
ring microtubules
Second Asexual Phase of Plasmodium (Inside human hosts)
Sporozoites are injected into the human host’s bloodstream (1) by a feeding mosquito,
within 30 to 40 minutes they leave the blood and enter Kupffer cells (2); macrophage‐like cells that line
the liver capillaries. They then leave these cells and enter the nutrient‐rich hepatocytes (3), where rapid
growth occurs (40μm in 48 hours) and lose their morphology, rounding up to become trophozoites called
merozoites (4). (Trophozoites are protozoan parasites in the activated, feeding stage in their life cycle).
After this growth period, one sporozoite divides into 10,000 to
30,000 merozoites, this process of division is called schizogony
(or merogony), and the many dividing bodies are referred to as
shizonts.
NB: Because the sporozoite enters the hepatocytes, forming its own vacuole (3 & 4), it does not enter the
cytoplasm of the cell, and therefore an infected person shows no symptoms in this stage of infection,
however, in P. vivax, some trophozoites become a latent stage called hypnozoites, that cause relapses years
later.
collar
rhoptry
3‐layered pellicle
Don’t need to know these names, just understand the
basic structure of the apical complex
7. Third Asexual Phase of Plasmodium (Inside human hosts)
The merozoites burst out of the hepatocytes and invade red blood cells. Once inside they ingest the
haemoglobin and first become erythrocytic schizonts, which divide to form between 16 and 18 merozoites.
This process takes 2 to 3 days overall and suffers experience malaise as their red blood cells synchronously
burst, releasing the merozoites and the substances contained inside the RBC. After several blood cycles
(about 4 days), some trophozoites differentiate into gametocytes, which remain dormant in the RBCs until a
person is bitten by a mosquito.
In order to invade the red blood cells, the merozoites must recognise and bind to RBC receptors, they then
deform the erythrocyte in order to enter the cell. NB: Erythrocytes are quite structurally tough due to their
sturdy cytoskeleton structure!
The major surface proteins on a human erythrocyte (RBC) are band 3 anion transporters, and glycophorins (a
family of sialic acid‐rich glycoproteins A, B, C and D), Glycophorins A & C, and band 3 anion transporters span
the RBC membrane. The invasion by a RBC depends on the glycophorins, especially the N‐terminal O‐linked
tetrasaccharide.
Other species require different molecules. P. vivax requires duffy blood group antigens in order to invade
erythrocytes, and in tropical areas where this parasite is endemic, there is a natural selection pressure for
individuals without these specific antigens.
The merozoite initially attaches anywhere on the erythrocyte surface (1) by the fibrils of its MZ surface coat.
A tight junction is formed, followed by subsequent invagination and formation of a parasitophorous vacuole
(2). The thick MZ surface coat is sloughed off as the merozoite enters the RBC (3). Entry follows the
alignment of its apical end, and as in the second stage, the merozoite remains in its own vacuole rather than
the erythrocyte itself.
The formation of a parasitophorous vacuole involves a dramatic re‐organisation of the RBC cytoskeleton,
which occurs as organelles in the merozoite called rhoptries and micronemes secrete invasion molecules,
such as RESA (ring erythrocyte surface antigen molecule) that enhance the fluidity of the RBC membrane.
The RBC cytoskeleton is very tough, because of its highly ordered network which is very difficult to disrupt.
The most prominent component of this cytoskeleton is a fibrous polypeptide called spectrin. The spectrin
forms tetramers, that are organized into a meshwork fixed to the membrane by the protein ankyrin. Ankyrin
is itself connected to a transmembrane protein called 'band 3' or anion exchanger protein. Spectrin is also
linked to a transmembrane protein called glycophorin C by the protein known as 'band 4.1.' Thus the
meshwork is anchored to the membrane at multiple sites. Band 4.1 stabilizes the association of spectrin with
actin, as does the protein adducing.
1 2 3 4
8. After entry of the RBC, the trophozoites feed by producing enzymes which degrade haemoglobin, the most
dramatic changes involve the shape and deformability of the RBC, they are identified by their irregular shape
and the presence of membrane knobs. After parasite invasion, the RBCs become sticky and attach to blood
vessel walls, decreasing the velocity of blood flow. This is an effective strategy employed by the parasite that
ensures it is kept away from the spleen for as long as possible. (Remember, it is the spleen that destroys
infected blood cells (this is why splenomegaly is a primary sign of malaria; it is caused by dilation of the
splenic sinuses due to an increased lysis of red and white blood cells.
The image below shows a false colour scanning electron micrograph of an infected red blood cell, merozoites
have burst from the cell and have started to invade the neighbouring cell.
9. The knobs found on the surfaces of erythrocytes are parasite derived and parasite induced. They are
important in the adhesion of parasitized RBCs to deep seated capillary endothelium, which slows blood flow
and allows the parasites to avoid destruction in the spleen for as long as possible.
This can cause cerebral malaria, especially with P. falciparum. Cerebral malaria can be fatal, a person can slip
into coma in very short periods of time due to the short multiplication period of parasites after the
sequestration of red blood cells.
The image below is of an affected brain; the red spots are sites of petechial haemorrhage. A petechia is a
small (1‐2mm) red or purple spot on the body, caused by a minor haemorrhage due to burst capillary vessels
10. A seropositivity incidence for different
countries can be established by analysing
sera (blood plasma) for antibodies :
Parasitology Lecture 10
Toxoplasma gondii
Toxoplasma gondii is a member of the
apicomplexan phylum, which consists of
members who have an apical complex that is
of great importance for penetrating host
cells. This phylum includes the Plasmodium
parasites that cause malaria. T. gondii is an
obligate intracellular protozoan parasite that
infects almost any warm-blooded mammal or
bird and is the most successful on the planet;
affecting 12-80% of the human population.
Since Toxoplasma is easier to manipulate
than Plasmodium, lots of research into this
parasite has helped to understand malaria.
Human cells infected with T. gondii. Parasites (yellow-
green) rupture out of a dying host cell (blue); in the
background is an intact host cell containing a large
parasite-filled vacuole
Ctenodactylus gundi
In 1908 T. gondii was discovered in the gundi (above), whilst looking for the Leishmania
parasite in Africa. (In retrospect the correct name for the parasite should have been
Toxoplasma gundii since the scientists that discovered it incorrectly identified the host’s
name.) A series of suggestions were then made about the parasite’s life cycle:
1939 - identified as a congenital infection
1960 - suggested it was transmitted through ingestion of uncooked meat (carnivorism)
1965 - found to appear in populations of carnivorism (such as in Paris*)
1970 - the parasite was linked to cat faeces and the life cycle was finally understood
*In a survey in Paris (where raw meat is routinely eaten), Desmonts et al. (1965) found over 80% of the adult population
sampled had antibodies to T. gondii.
11. William McPhee Hutchison (1924-1998) was born in Glasgow and studied Zoology at
Glasgow University. While working at the University of Strathclyde in Glasgow, in the 1960’s,
he demonstrated that Toxoplasma gondii was a parasite of cats which shed oocysts in
faeces. Hutchison’s work was rewarded with the Robert Koch Medal and Prize in 1970.
T. gondii life cycle
The definitive host of T. gondii is the cat. Felines ingest either oocysts (containing
sporozoites, or tissues that are infected with bradyzoite cysts. The parasites then burst out
of the cysts and invade the intestinal epithelium; both forms can differentiate into male
and female gametes and after fertilisation they become oocysts containing sporozoites.
Any warm-blooded animal can ingest these oocysts, which releases sporozoites into the
intestine that invade the tissue. They differentiate into the tachyzoite stage and
disseminate throughout the body, invading cells and becoming bradyzoite cysts. These
unsporulated oocysts are passed in the cat’s faeces and, outside the host, turn into
sporulated oocysts (containing sporozoites), which feed on soil and water .
If another warm-blooded animal eats infected tissue (through uncooked mead or
contaminated water), the bradyzoites are released into the intestine, differentiate back
into tachyzoites and then disseminate round the body where they eventually become
bradyzoite cysts again. If a bradyzoite cyst is ingested by a cat, bradyzoites invade the
intestinal epithelium and differentiate into male and female gametes and then become
oocysts again. Tachyzoite transmission through the placenta can infect a foetus and cause
developmental problems.
12. Parasite movement
Parasite movement does not involve projection of pseudopod-like extensions, they rely on
an intricate linear motor system that is sandwiched between the parasite’s plasma
membrane and a pair of membranes known as the inner membrane complex. Actin and
myosin, together with special ‘gliding associated proteins” are involved’. The actin is linked
to the trans-membrane adhesive proteins (see next page).
The inner membrane complex rides against the plasma membrane. This movement takes
place at the ‘moving junction’ of the host cell as the parasite enters, and helps it move into
the cell after pre-digestion of the membrane with (step 5, bottom diagram on next page)
For more information see the attached article (not recommended by lecturer):
Formin’ an invasion machine: actin polymerization in invading apicomplexans
(Holder, 2008)
Sporozoites (“seed animals”) infect new hosts. The oocysts
form in the feline intestinal epithelium, unsporulated oocysts
are shed in faeces for 3-18 days and sporulate for a period of
three weeks outside the cat’s body. They contaminate water,
soil, fruits and vegetables and are very stable, especially in
warm and humid environments. Although the cats only shed
these organisms for a short period of time, re-infection can
raise the number of organisms present at a given time.
Tachyzoites (“fast animals”) are in an asexual stage of rapid
growth. They inhabit intermediate hosts and accumulate
inside almost any nucleated cell. The parasites are secreted
into the bloodstream, causing an acute disease known as
parasitaemia. This is limited by the immune response,
which induces the change of the tachyzoites into cyst-
forming bradyzoites.
bradyzoites (“slow animals”) are sessile (slow-growing) and
inhabit intermediate hosts. In chronic toxoplasmosis, the
bradyzoite presents as irregular crescent-shaped clusters
(pseudocysts) in infected neural and muscular tissues (brain and
skeletal and cardiac muscle). These cysts persist to cause chronic
disease and if an individual becomes immunocompromised ,
they can cause acute encephalitis.
13. Roprtries and micronemes are specialised secretory organelles that contain numerous enzymes
released during the penetration process
14. Clinical manifestations of Toxoplasma
infection are usually asymptomatic (and thus
undiagnosed). They may occur in outbreaks,
as with Vancouver, 1995, or in isolated cases.
The incubation period is usually about 4 to 21
days and infection can result in focal
lymphoadenopathy, where the lymph glands
swell at the infection site, and flu-like
symptoms, such as fever, sore throats and
headaches. There are also claims of T. gondii
inducing an altered behaviour.
The parasite was introduced to test rats,
which subsequently became mildly attracted
to cat urine, the parasite is believed to be
more likely to invade the amygdala of the
brain, which is involved in a variety of fear-
related behaviours.
Remember, that if a rat is attracted to cat urine, there is a greater possibility that the
animal will be consumed, allowing the parasite to continue its life cycle inside a feline host.
In immuno-compromised patients, reactivation of a latent disease can cause fatal
pneumonia, ocular problems, and in 25% of HIV patients it causes encephalitis, leading to
coma and death. Diagnosis of encephalitis in HIV patients in France alone was 800 in 1992,
and 200 in 2002, due to anti-virals that slow the destruction of the immune system.
Vertical transmission to infants can also occur and can lead to problems such as diminished
vision or blindness in 14% of infants, hydrocephalus and intracranial calcifications, leading
Prevalence of T. gondii in livestock
1 pig feeds 300 to 400 people, and
remember that more than one pig
may be used in any one product!
Health risks for T. gondii
DALY = YLL + YLD
(DALY: disability adjusted life year, YLL: years lost to
mortality, YLD: number of years lived with a disability).
In comparison to common infections, T. gondii is very
successful and it is therefore a significant health issue
15. Conditions occurring after several months or years include visual impairment, mental and
cognitive abnormalities and seizures or learning disabilities, but the early diagnosis and
treatment of these conditions reduces the risk of complications.
Immune response to T. gondii
It is probable that a rise in the immune response to Toxoplasma causes them to
differentiate into the bradyzoite stages; this is a Th1 response with IFN-γ. The immune
response keeps the infection in check and dormant, but Toxoplasma also secretes a
molecule that enhances the Th1 response. It does this by enhancing the production of the
cytokine IL-12, which promotes IFN-γ production by T cells.
The molecule cyclophilin 18 (made by the parasite) binds to the chemokine receptor ‘CCR5’
on antigen-presenting dendritic cells to induce the formation of IL-12, which can then act
on T cells, as mentioned above, to promote IFN-γ formation.
It does this to prevent intermediate host mortality and allow them to distribute infection.
The incidence of congenital Toxoplasmosis from different
countries. The rate is especially high in France
Clinical manifestations are usually
asymptomatic at birth (70 to 90% of
cases), but can appear as rash,
lymphoadenopathy, hepatosplenomegaly,
hyperbilirubinaemia (jaundice), anaemia
and thrombocytopaenia (few platelets). In
<10% of cases the classic triad of
chorioretinitis (inflammation of the
choroid), intracranial calcifications and
hydrocephalus (an accumulation of
cerebrospinal fluid in the ventricles, or
cavities, of the brain) can result.
to CNS abnormalities in 11.4% of infants, miscarriage or still births in 5% of cases (this is
also common in livestock). It can also cause premature birth and intra-uterine growth
retardation.
The inverse relationship between the incidence of
foetal infection and the severity of the foetal damage;
infection is most severe if contracted in the first
trimester of pregnancy than if it were contracted in the
third trimester (Remington et al., 1995)
Hydrocephalus as a result of congenital Toxoplasmosis
16. Toxoplasma is believed to affect the behaviour of humans by slowing reaction times and
making one more likely to take risks, and people with Toxoplasma infections are twice as
likely to be involved in a car accident, although scientists are unsure which of these factors,
contributes to this result. In rats it has been observed that the most basic of instincts; a fear
of cats, can be overridden, the rat becomes attracted to cat urine, meaning it is more likely
to be eaten and continue the parasite’s life cycle.
For more information, watch the Horizon video at:
http://193.60.156.105/wmv/lifesciences/bodysnatchers/body2.wmv
Does Toxoplasma influence sex ratios?
Woman that are seropositive for Toxoplasia are seen to have more sons than uninfected
women and mice infected with Toxoplasma produce more males in their litters during the
early stages of the disease. This could be because males are known to ‘roam’ more than
females; they leave their habitats more often and are more likely to act more
spontaneously and adventurously and take bigger risks.
Treatment
Treatment of Toxoplasma includes sulphonamides, pyrimethamine and other anti-
malarials. Spiramycin is used to reduce the risk of congenital infection transmission and a
live vaccine of the cystless strain of T. gondii is available for immunisation of sheep. There is
currently no human vaccine and no drugs can currently target encysted bradyzoites.
T. gondii has molecules on its surface such as profilin, that acts as ligands for the toll-like
receptors (TLRs); single membrane-spanning proteins that activate the immune cell
response on recognition of these molecules. TLR ligands are structurally conserved in
pathogens, and distinguishable from host molecules. Activation of this receptor leads to
activation of MYD88; a universal adaptor protein, that drives the production of protein
complexes that regulate cell activities and, in turn, produce IL-12.
17. Parasitology Lecture 3
Leishmania
Leishmaniasis is a disease caused by protozoan parasites of the
genus Leishmania. and is transmitted by certain species of
sandfly; by Phlebotomous in the “old world” (Asia and Europe),
and by Lutzomyia in the “new world” (South America). Many of
these species infect humans, but they are primarily classified as a
zoonosis (an infectious disease that is transmitted from
vertebrate animals to humans). Visceral Leishmaniasis is a severe
form of the disease in which the parasites have migrated to the
vital organs.
Leishmania is a very complex group of infectious agents, containing the following species:
L. tropica ‐ L. tropica
L. major ‐ L. major
L. aethiopica ‐ L. aethiopica
L. mexicana ‐ L. mexicana, L. amazonensis, L. garnhami, L. pifanoi, L. venezuelensis
L. braziliensis ‐ L. braziliensis, L. guyanensis, L. panamensis, L. peruviana
L. donovani ‐ L. donovani, L. infantum, L. chagasi
The incidence rate of infection is about 400,000 per year, there are about 12 million cases in the
world, and it can be fatal, leading to over 20,000 deaths a year in India alone.
Leishmania is an obligate intracellular parasite that live in cells of the macrophage lineage in the
immune system. Although the parasitic forms infecting macrophages are morphologically identical,
clinical manifestations are very diverse depending on which types of macrophages are affected; they
can all act as hosts for different Leishmania
Top right: Leishmania parasite entering a macrophage cell
18. An infected female sandfly introduces flagellated promastigotes into the host skin, where they are
taken up by macrophages. Here they lose their flagella and become amastigotes, they then multiply
and burst out of the cell to infect more macrophages. If the infected macrophages are ingested by a
sandfly, they develop into infectious metacyclic promastigotes that are introduced into another
mammal as the sandfly next feeds.
the morphological differences between
promastigotes (flagellated) and amastigotes
Cultured macrophages
show parasite infection
Leishmania skin lesion
Leishmania is a disfiguring disease that can lead
to destruction of body parts, such as the ear or
nose (see right), due to cartilage destruction, and
secondary bacterial infections can often result.
Blemishes that appear on the skin are caused by
uncontrolled lesion growth due to parasitic
infection.
19. Leishmania is primarily a zoonosis; a disease that is transmitted between an animal and a human.
Transmission of a disease from humans to animals is typically referred to as “reverse zoonosis”. The
Leishmania parasite exhibits its effects in a wide variety of hosts, including wild rodents, sloths and
dogs. There is an enormous reservoir of infection for this disease.
What is the aim of a parasite on entering a mammalian host and how does the host respond?
The parasite aims to enter the macrophage cells of the reticuloendothelial system, the host wants to
protect itself and as the first line of defence, employs the innate (or non specific) immune system,
which consists of cells which respond to pathogens in a generic way without conveying long‐lasting
immunity to the host. Paradoxically the main type of cells involved in the host defence are the
macrophages themselves.
The infective stage of the parasite is a flagellated promastigotes (in its metacyclic form), that is
about 20‐25μm long including the flagellum. When a parasite interacts with a host, the parasite/
host surface interface is very important. The Leishmania promastigotes have three types of
molecule of their surface; Lipophosphoglycan (LPG), glycoprotein GP63 and glycoinositol
phospholipids (GIPLs)
Lipophosphoglycan is composed of an inositol lipid anchor and many repeating saccharide units.
LPG is a component of the glycocalyx; a network of polysaccharides that project from cellular
surfaces and allow attachment to various surfaces, as well as providing protection for the cell.
Glycosylphosphatidylinositol (GPL) anchored proteins such as GP63 (a protease of molecular weight
63 KDa) increase in levels during transformation of non‐infective promastigote forms to infective
metacyclic forms.
LPG
5 x 106 molecules per
cell
GIPL
1 x 107 molecules
per cell
GP63
0.5 x 106 molecules
per cell
20. The Complement System
The complement system is a biochemical cascade that is activated when foreign bodies enter the
cells and helps clear pathogens from an organism. Inactive precursors (or zymogens) from the liver,
circulate in the blood until activated, and on activation they are cleaved into two or more
fragments. The major fragment has two biologically active sites; one to bind the target and the
other to act as the enzymatic site.
The complement functions to aid the process of phagocytosis through ‘opsonisation’ by sticking to
the foreign body and allowing it to be targeted, it also promotes cell lysis through the insertion of
channels in the foreign body that lead to cell lysis due to a change in osmotic gradient. Complement
also aid chemotaxis and inflammation due to the existence of smaller proteins
Complement Activation & Membrane Attack Complex (MAC) Formation
NB: This is in much greater depth than is needed, use as reference only!
Antibodies bind to antigens on the bacterium surface. A C1 complex composed of 1 molecule of C1q
and 2 molecules of both C1r and C1s bind to the aggregated antibody molecules and cross‐
phosphorylation of the C1r and C1s complexes take place (1). They then cleave the complement
protein C4, into a large C4b fragment, which binds to the bacterial membrane, and a small C4a
fragment, which acts as an anaphylatoxin. Anaphylatoxins trigger degranulation of endothelial cells,
mast cells and phagocytes, producing a local immune response
The C4b complex is activated by the enzyme C2b, and after the attachment of C3b, this
C4b/C2b/C3b complex leads to the production of many more C3b proteins, which bind to the
bacterial membrane and induce its phagocytosis (2). C5 then attaches to the C4b/C2b/C3b complex,
and is cleaved to form C5a; a potent anaphylatoxin and important chemoattractant (a substance
that promotes chemotaxis). C5b then dissociates from the C4b/C2b/C3b complex, and acts to
initiate the formation of the membrane attack complex.
C5b associates with C6 and C7, and C7 allows this complex to insert itself into the bacterial
membrane. C8 then binds to this complex and inserts itself into the cell membrane. This newly
formed C5b/C6/C7/C8 complex then catalyses the addition of many C9 molecules, which arrange
themselves in a cylindrical manner across the membrane surface, creating a pore (3) that disrupts
the ionic and osmotic balance across the membrane, thus killing the bacterial cell.
Infiltration of Macrophages by Leishmania
Leishmania binds with GP63 and LPG molecules to CR1 (complement receptor 1), CR3 and mannose
1 2 3
21. fructose receptors on macrophage (Mφ) surfaces. These attachments to the CR1 and CR3 molecules
occur either directly, or indirectly via complement components.
The parasite is not destroyed as it is engulfed because the LPG molecules increase in thickness when
progressing from the non‐infective to infective stages. This process of elongation increases the
thickness of the glycocalyx from 7 nm to 17 nm, and also stops the MAC complex from forming,
GP63 is also important, because it proteolytically cleaves the complement component C3b into its
inactive form, and so prevents lysis. LPG and GP63 also interact with complement receptor sites on
the macrophage that do not activate the macrophage cell.
Finally when a macrophage engulfs a foreign body, they usually enter an endosomal compartment
that fuses with lysozymes and destroys the pathogen. It survives this by creating a parasitophorous
vacuole, LPG molecules with a pH of between 4.2 and 5.2 inhibits lysosomal enzymes (this technique
is employed in many parasites that establish themselves in living host cells. LPG also inhibits Protein
Kinase C, that is involved in the generation of toxic macrophage metabolites, and GP63 inactivates a
host’s proteases, especially around a pH of 4.0.
Amastigotes (cells with no flagella) also make scavenger enzymes such as superoxide dismutase and
glutathione peroxidase
22. Parasitology Lecture 8
Leishmania Prophylaxis
Before reading this lecture understand lecture 3 ‘Leishmania’, most importantly, remember
it is an obligate intracellular parasite that lives in macrophage cells.
Luckily for researchers, Leishmania is a zoonosis and mice are natural hosts of the parasite.;
we can therefore use mice as hosts for research purposes. Like in humans, there is a
spectrum of disease in mice, some are resistant, others susceptible, and others are affected
in different ways. This resistance or susceptibility is inherited.
The infection is controlled in two ways: initially by macrophages in the reticuloendothelial
system, followed by the induction of adaptive immunity, allowing specific pathogens to be
targeted and antibodies produced against them. Research suggests that the basis for the
difference in the resistance or susceptibility of mice relies on a single major genetic locus,
and to investigate, a congenic mouse strain was produced by conventional genetic
breeding. A congenic mouse strain is when one mouse is genetically identical to another,
only differing in the single gene under investigation.
Concept
In order to create a congenic mouse strain, cross a resistant strain with a susceptible one
and backcross the F1 progeny that are still susceptible with the resistant strain.
This process is repeated, and after several backcrosses the resulting mice all have the
resistant strain’s genes, except the one determining the mouse’s susceptibility (so the
mouse remains susceptible).
The response to a Leishmania infection of these susceptible mice is then compared to
that of the original resistant strain
Analogy
Think of how to make a dry martini, starting with 50% gin and 50%
vermouth. The olive represents the gene you want to conserve (the one
that makes the mouse susceptible to Leishmania).
To make a martini more dry you spill out half of the drink and add more
gin, but keep the olive, if you do this for long enough the drink will
eventually contain all gin, but still contains the original olive.
Eventually, the single gene controlling Leishmania susceptibility was identified and named
Lsh. Scientists working round the world also identified single genes that confer resistance to
a specific infective agent; mycobacteria causes tuberculosis and is controlled by the Bcg
locus, and salmonella is controlled by the Ity locus.
23. The genes were eventually cloned and called Nramp (natural resistance associated
macrophage protein). Mouse strains which varied in their initial response to Leishmania
show mutations in this gene and database searches showed that the gene encoded a
divalent ion transporter molecule, although its function is not entirely understood.
Nitric Oxide Pathway
Nitric Oxide (NO) is a short‐lived but very toxic molecule that is produced by macrophages.
When a Leishmania parasite enters a macrophage it creates a parasitophorous vacuole in
order to evade the immune system, but NO can readily diffuse across lipid membranes;
meaning it can easily get into the parasitophorous vacuole and destroy the parasite.
Oxygen Citrulline
L‐Arginine Nitric Oxide
NO Synthase
Tetrahydrobiopterin
NADPH
Tetrahydrobiopterin and NADPH are two essential cofactors for the above process. NO is
also spontaneously oxidised to form NO2‐ and NO3‐ ions, but NO2‐ can be reduced back to
NO in low pH conditions. Note that the parasitophorous vacuole has a low pH...
Nramp‐1 (Natural resistance associated macrophage protein 1 )
This is an integral membrane protein expressed exclusively in cells of the immune system in
mice, which is recruited to the membrane of a phagosome (vacuole formed around a
particle) upon phagocytosis. It has a hydrophobic core of ten transmembrane domains and
scientists believe it could be involved in transporting NO2‐ ions into the parasitophorous
vacuole. In theory the concentrated NO2‐ ions would then be reconverted back to NO inside
the vacuole, due to its low pH, and would kill the parasite.
Schematic representation of the structure of the Nramp protein and its orientation in the cell membrane
24. However, more recent work has modified the above view. It has been suggested that
Nramp‐1 is a divalent cation transporter and , for example, extrudes Mn2+ from the
parasite. Other cations such as Fe2+ and Zn2+ may be involved and the process depends on
the pH ‐ the more acidic the solution, the faster the transport of these ions. Manganese
(Mn2+) ions are essential co‐factors for the production of an enzyme called superoxide
dismutase (SOD). With less Mn2+, the parasites cannot produce as much SOD and are
therefore more susceptible to the superoxide produced by macrophages.
Superoxide (O2‐)
Superoxide is a biologically toxic molecule that is deployed by the immune system to kill
invading microorganisms. In phagocytes, superoxide is produced in large quantities by the
enzyme NADPH oxidase for use in oxygen‐dependent killing mechanisms of invading
pathogens. Because superoxide is toxic, nearly all organisms living in the presence of
oxygen contain isoforms of the enzyme superoxide dismutase. SOD detoxifies reactive
superoxide radicals produced by activated macrophages and is therefore a major
determinant of intracellular survival of Leishmania.
Adaptive Immunity
When the innate immune system fails, the adaptive
immune system kicks in (T and B lymphocytes), enabling
mouse strains that are initially susceptible to infection to be
able to control it. We need to understand why this
happens:
Macrophages are activated by cytokines such as IFN‐γ
(interferon gamma), which are produced by CD4+ T cells. To
see if these CD4+ T cells are important, an adaptive transfer
of immunity is performed, where lymphocytes from one
mouse species are transformed to another. These adaptive
transfer studies must be performed in inbred strains of
mice, this ensures that the donor and recipient are
genetically identical so the transplanted tissue is not
rejected by the recipient.
A mouse was infected with Leishmania, its CD4+ T cells were then removed and
transplanted into another mouse of the inbred strain. The ability to control infection of
mice that did get the CD4+ T cells , and those that did not were compared.:
The inbred mouse strain C3H healed faster after receiving
the CD4+ T cells.
Another inbred mouse strain BALB/c suffered a worse
manifestation of the disease.
Therefore it was determined that CD4+ T cells have two different effects in
different strains of mice!
26. Parasitology Lecture 6
African Trypanosomiasis (Sleeping Sickness)
The trypanosome parasite is a single‐celled
kinetoplasmic, extracellular protozoan; it contains
kinetoplasts (disk‐shaped masses of circular DNAs
inside a large mitochondrion that contains many
copies of the mitochondrial genome), lives in the
host’s bloodstream and can be a zoonosis, many of
these characteristics are rather like that of
Leishmania. The parasites are transmitted to humans
through the bite of a tsetse fly of the genus
Glossinna and never exist outside a host.
Currently over 66 million people are exposed to the disease in 36 countries, there are
300,000 new cases a year, many with advanced stages of the disease and in the 1960s, the
prevalence o sleeping sickness had been reduced to less than 100 cases per 100,000 people
per annum, but following the independence of many countries and outbreaks of civil war,
the changes in health policies have led to a significant rise in the number of cases of
sleeping sickness, and this is worryingly on the increase.
There are two forms of the disease that are caused by two different species:
Trypanosoma gambiense ‐ named after the Gambia in West Africa, causes a chronic
infection lasting years, affects countries of Western and Central Africa.
Trypanosoma brucei rhodesiense ‐ named after Rhodesia, now called Zimbabwe,
causes acute illness lasting several weeks, affects Eastern and Southern Africa.
Gambia
Zimbabwe
Due to the large number of livestock affected, cattle farming in these areas is poor, which also affects the economy
27. The initial clinical signs of trypanosomiasis are fever, weakness,
headache, swollen lymph nodes and joint pains, followed by
anaemia , heart problems and oedema. In advanced stages the
parasite invades the CNS. People can no longer concentrate and
exhibit mood changes, lethargy and increasing torpor (temporary
hibernation ‐ a state of decreased physiological activity, usually
characterized by a reduced body temperature and rate of
metabolism). Eventually this disease leads to coma and death.
Trypanosoma predominantly lives as a free‐living organism in the bloodstream, until it
enters the CNS, where it causes the most serious symptoms. If untreated, the infection is
fatal and there is currently only one drug, discovered in 1932, called melarsopral, that is an
arsenic‐based, and therefore very toxic, drug with serious side effects. Of the small number
of people who get this treatment, approximately 1,000 die every year from arsenic
encephalopathy, which may manifest as seizures, mental status changes, and coma.
Like most protozoa, Trypanosoma exists in a
number of different forms; the main forms being
slender and stumpy, although a variety are
illustrated by the diagram to the left. They are found
in the blood and are transmitted when an infected
tsetse bites a human host. Only the stumpy
morphologies survive in the tsetse midgut, then
undergo a series of multiplication stages which end
with the parasite relocating to the salivary glands.
The infective stages are known as metacyclics (the
same terminology as with Leishmania).
Tsetse flies are quite big, their
bites are very painful and
cause large sores to form.
The Trypanosoma
flagellum looks
like a fin, sitting
on top of its body
Note the characteristic arrangement
of the microtubules in the flagella of
the Trypanosoma parasite and also
the kinetoplast at the anterior end of
the organism
28.
29. Once in the mammalian host, the Trypanosoma change into the long slender forms and
multiply by binary fission with a doubling time of about 6 hours; some change into the
stumpy forms that will go on to infect flies. A characteristic feature of chronic infection in
man and animals is the occurrence of regular fluctuations in the numbers of parasites
present in the blood, this observation was known for many years and 1910 an Italian
scientist called Massaglia stated
“trypanolytic crises are due to the formation of anti‐bodies in the blood. A few
parasites escape destruction because they become used or habituated to the action of
these antibodies. These are the parasites which cause the relapses”
This was a very prophetic statement, in the sense that the structure of antibodies was not
to be discovered until about 50 years later in the 1960s. He relied on development of lab
models and techniques for purifying tryptases (the most abundant secretory granule‐
derived serine proteases contained in mast cells that have recently been used as a marker
for mast cell activation) and electron microscopy.
In his investigations he Identified a thick surface coat, and
analysis of different clones of the parasites (obtained by
the organisms dividing by binary fission) showed that they
had “biochemically” different clones from one another.
Each coat was different enough to be encode by a
different gene and each type was known as a variant
antigen type. The surface molecule is known as variable
surface glycoprotein (VSG), and has a molecular weight of
61kDa, they exist as a very tightly packed monolayer
above the parasite’s lipid bilayer, and cover the entire
organism’s surface.
Massaglia looked at the peptide sequences of four different clones and saw that the amino
acid sequence was different at the beginning of the VSG, confirming that each VSG was
encoded by a different gene; about 10% of the biomass of the organism consists of the VSG
alone! At any one time in the blood of a mammal, the majority of blood stream forms are
slender and express only one type of VSG, but there are also a few stumpy non‐dividing
forms that co‐exist with them.
The immune system of the host recognises the dominant VSG in the population of
Trypanosoma, over the next few days they then make the specific antibody and kill 99% of
the pathogens; the ones that express the dominant VSG, by complement fixation and
opsonisation. However, some pathogens in the population have a different VSG type that
the antibody is not designed to recognise, so these survive, grow up and reproduce by
binary fission to increase the population size. This process constantly repeats itself and is
responsible for the fluctuating parasite levels found in a host suffering from chronic
infection (see diagram on following page).
NB: Antibodies are very specific, they can distinguish between proteins with a single
different amino acid, or even a single enantiomer of an amino acid
30. glycoproteins are recognised produces the
fluctuating levels of parasites in the blood.
A new VSG type is recognised by B lymphocytes,
which engulf the parasite and display its antigens on
their outer membrane. They then act as antigen‐
presenting cells in the lymph nodes and stimulate Th
cells to produce specific lymphokines (types of
cytokine) that enable B cell clonal expansion, where
the B cells mature into plasma cells that produce
antibodies.
Activated B cells subsequently produce antibodies
which help to inhibit pathogens until phagocytes or
the complement system, for example, clears the
host of the pathogens.
The VSG switch still occurs in animals that cannot make antibodies, and if you move the
parasites between animal hosts faster than they can make new antibodies, the parasite still
switches its VSGs regardless. This process of VSG switching even occurs in vitro! The
number of VSGs the trypanosome can make is probably in the region of 1,000!
The question that remains is how the trypanosomes manage this VSG switching...
A tell‐tale symptom of chronic Trypanosoma infection is a fluctuating
concentration of parasites in the host’s blood stream
The Immune Response
One in about 104 to 105 parasites express a heterotype VSG, but the rest express a single
homotype which is recognised by the immune system, causing an immune response to take
place. The antigens allow the parasites to be lysed via the complement system (see lecture
3 ‐ Leishmania), and also stimulates agglutination and phagocytosis.
Effectively the immune system allows for the
selection of the heterotypes, which then go on to
reproduce, and the repetition of this process as new
32. Since about half the VSG genes that have been studied are telomere linked, and there are
hundreds of genes to encode for hundreds of different VSGs, there must be hundreds of
chromosomes present in their genome.
But, since the trypanosomes have a normal amount of DNA, they must have an array of
different sized chromosomes.
Mini chromosomes ~ 105 nucleotides
Small chromosomes ~ 2‐7x105 nucleotides
Middle chromosomes ~ 2x106 nucleotides
Large chromosomes ~ >2x106 nucleotides
So, we accept that VSG genes are found on chromosomes of all sizes, but since a gene on a
large chromosome can also be expressed as a copy on a medium sized chromosome, it
suggests that translocation can take place between chromosomes. A further degree of
variation can also arise from recombination, where the VSG gene is generated by the
“joining” of segments of two different telomere linked genes which each code for part of
the resultant VSG.
This may help explain why telomeres are the sites of expression; they have highly repetitive
stretches of DNA such as short tandem repeats, and since such areas are highly likely to
undergo recombination, more variation can be generated.
Finally, a further new piece of information adds to reasons why only one VSG is expressed
at any one time. There is a special place within the trypanosome nucleus where this
process occurs, and where all the necessary molecules required for expression are
sequestered (separated and stored). This site is called the expression site body, and is
found in the nucleolus.
This was discovered by Miguel Navarro and Keith Gull in the University of Manchester a few
years ago. They also identified the enzyme which did the transcribing, and to their surprise
it was RNA polymerase I (Pol I) which is not usually employed for protein transcription!
35. Although the early stage disease is not usually severe, chronic symptoms may develop after
10 to 30 or 40 years, which includes cardiac problems such as enlarged hearts and cardiac
arrest. South American footballers have dropped dead on the pitch to an apparently
unknown cause, later found to be due to trypanosomiasis. Other chronic symptoms include
dilation of the digestive tract (megacolon and megaesophagus), accompanied by severe
weight loss. Swallowing difficulties may be the first symptom of digestive disturbances and
may lead to malnutrition.
A C
B
D
A: Many of trypanosome parasites are found in the tissues of infected individuals. B: an enlarged heart, a
light probe here shows just how thin the apex becomes on enlargement. C: A dilated colon, all muscle tone
has been lost and extensive swelling is present. D: a dilated colon has been cut open to expose the inside.
T.cruzi has a remarkable capacity to invade every nucleated cell it encounters, so it can
survive and replicate in many cells. As with many protozoans it lives (at least for part of its
life cycle) in a parasitophorus vacuole, but unlike other protozoans such as Leishmania,
engulfment is not an actin mediated phagocytic event and the membrane of the
parasitophorus vacuole is not derived form the plasma membrane of the host cell.
Instead, the parasitophorous vacuole is derived from the membranes of lysosomes.
Lysosomes are organelles that contain digestive enzymes (acid hydrolases). They digest
excess or worn‐out organelles, food particles, and engulfed pathogens by fusing with
vacuoles and dispensing their enzymes, digesting the vacuole’s contents.
36. 1) The parasite secretes oligopeptidase B molecules that bind G protein coupled
receptors on the host cell surface. This activates phospholipase C, which then
induces an increase in cellular calcium.
2) The parasite secretes Cruzipain, which cleaves kininogen into kinins;
inflammatory mediators that bind to cell surface kinin receptors, eventually
activate PLC and induce a rise in intracellular calcium.
Following accumulation of lysosomes under the parasite attachment site, lysosome
membranes fuse and create a membrane of the parasitophorus vacuole. It is believed that
the parasite enters the cell by a combination of its own movement and the ‘pulling and
recovery’ of lysosomal membranes along microtubules that ‘drag’ the it in.
Most parasites do not want to allow lysosomal fusion, but T. cruzi actually requires it!
The function of a lysosome in an animal cell
The parasite attaches to varied molecules (matrix proteins or integrins) on a cell surface.
Both Transforming growth factor beta receptor II (TGFβRII) and Receptor Tyrosine Kinases
(RTKs) can act in this way, and they both these expressed on many cell types
This complementary binding triggers a rise intracellular calcium in host cell, causing an
accumulation of lysosomes under the site where the parasite attaches. T. cruzi effectively
‘fools’ the host cell into thinking there is some damage to the cell membrane and triggers a
cellular wound repair process. The intracellular Ca2+ concentration is raised, and lysosomes
are then transported to the ‘wounded’ membrane along microtubules.
The parasite is thought to increase cellular calcium by a variety of processes, including:
39. Parasitology Lecture 4
Invasion by Helminths (Parasitic Worms)
Platyhelminthes and Nematoda
Platyhelminthes (Flatworms) include tapeworms and planarians (non‐parasitic flatworms)
Trematoda is a class within the above phylum, containing blood flukes and schistosomes
Nematoda (Roundworms)
Caenorhabditis and Filariae are genera of the above phylum
Geohelminths are a group of soil‐transmitted parasites, that include hookworms such as
Ancylostoma duodenale
Necator americanus
In this lecture, we will deal with two different species that have very different morphologies
Schistosomiasis (or bilharzia)
A diseased caused by schistosomes that affects over 250 million people and is most commonly
found in Asia, Africa, and South America, especially in areas where the water contains numerous
freshwater snails, which may carry the parasite.
3 major species infect man: 2 major species infect animals:
Schistoma mansoni
Schistoma haematobium
Schistoma bovis (cattle)
Schistoma mattei (sheep)
Schistoma japonicum
It is a chronic debilitating disease and many infections are asymptomatic, with mild anaemia and
malnutrition being common in endemic areas. Acute schistosomiasis (Katayama's fever) may occur
weeks after the initial infection, especially by S. mansoni and S. Japonicum. Manifestations of the
disease include: abdominal pain, cough, diarrhoea, eosinophilia (extremely high eosinophil count),
fever, fatigue and hepatosplenomegaly (enlargement of both the liver and the spleen). Occasionally
central nervous system lesions occur: cerebral granulomatous disease may be caused by ectopic S.
japonicum eggs in the brain
S. haematobium
Terminal spine at the posterior end
Usually associated with urinary schistosomiasis.
Adults are usually found in venous plexuses
around the urinary bladder, they release eggs
that traverse the bladder wall and can cause
haematurea and fibrosis of the bladder
S. mansoni
Lateral spine at the side
S. japonicum
No spine present
Adults are usually found in the mesenteric or rectal veins and release
eggs which move to the lumen of the intestine and pass out in faeces.
The rest of the eggs are filtered at the periportal tracts of the liver,
and can sometimes cause liver fibrosis
(fibrosis is formation of excess fibrous connective tissue).
41.
their glycocalyx is shed (via microvilli), and a new double unit membrane is formed from
membranous vesicles. The trigger for this change is a stepwise transfer in saline concentration. A
larval schistosome that has shed its tail becomes known as a schistosomulum.
“Swimmer’s itch” can be caused by some schistosomes from another species (e.g. birds). They
attempt to penetrate a human’s skin, but do not secrete the right enzymes to aid penetration and
so they die in a host’s skin, causing an itchy rash (above images).
Cercariae are 125μm long and 25μm
in diameter, with a 200μm long tail.
They are covered by a continuous
syncitial tegument (multinucleated
dermal cells), that is about 0.5μm
thick. The exterior surface consists
of a glycocalyx, and trilaminar
plasma and basement membranes. They have a primitive nervous system and primordial digestive
and reproductive systems (the earliest systems of that kind).
Scanning electron micrograph of an
early skin‐stage schistosomulum
Scanning electron micrograph of
a lung‐stage worm
Scanning electron micrograph of a late
skin‐stage schistosomulum
42. Schistosomes have oral and ventral suckers. They
may lie in the mesenteric veins for months on
end, and both males and exist in copulation from
this point in their life cycle until death.
♀
♂
You can estimate how heavily someone is infected by counting the number of parasite eggs in their
urine or faeces. Urine from heavily infected people is cloudy, and usually bloody. This disease was
identified in ancient Egypt, as “the aaa‐disease” and the identifying symptom was the discharging
phallus. In its later stages, schistosomiasis can cause massive liver fibrosis, leading to an expanded
abdomen, the prognosis for late liver fibrosis is not good.
Miracidium
The intermediate host is a freshwater snail, the
schistosome eggs hatch on contact with
freshwater, releasing Miracidium (200μm x 40μm).
Their surface is covered by anucleate epithelial
plates that are covered in cili, and allow the
organism to swim very fast. They can swim about
2mm/sec, and to turn it angles its body in a
“rudder‐like” manner.
Salinity changes tell the organism when to slow
down as it approaches a snail and following
penetration of the snail through either its mouth
or foot, the epithelial plates are shed. Inside the
digestive gland of the snail the organisms
differentiate into sporocysts and reproduce
asexually. Inside the sporocysts cercariae form, this process takes about 3 weeks in S. mansoni.
These cercariae can remain viable for up to 48 hours outside a host in freshwater bodies.
These cercariae penetrate an unsuspecting human host, and the cycle begins again.
Schistosomes are flatworms; they are Trematodes.
Next we deal with Hookworms, which are Nematodes.
Refer back to lecture 1 for the differences between these,
but you should really know them by now!
43. vector. Vaccination is therefore
an attractive possible solution,
A: A freshwater snail is surrounded by miracidium. B: Bodies of fresh
water in less developed countries such as Africa, carry schistosomes
Parasitology Lecture 5
Schistosomiasis Vaccination
Schistosomiasis is very difficult A B
to control in areas that are
poorly developed on political
and social levels. Because their
infrastructures are not well
developed it is difficult to
introduce medication to treat
the infected, or to introduce
molluscicides to kill off the
but it is still difficult to introduce the vaccine into these areas ‐ smallpox has been
eradicated in many countries, but still affects those where aid is less easy to acquire.
In order to find a vaccine for this disease, it is necessary to understand immunity. The
question that needs addressing is how can schistosomes live in the blood streams for
extended periods of time without being attacked by the host immune system...
A B C
Recapping the schistosome life cycle: Cercariae (A) penetrate a human host, migrate to the portal blood system and
mature to schistosomes (B), where they stay in the venous blood supply. The male forms a gynecophoric canal that the
female lies in, and they produce many eggs a day that hatch outside the body into miracidium (C) (here: S. mansoni)
How do the schistosome parasites avoid the host’s immune response?
Various laboratory models can be used to investigate infection in order to see how the
animal and the parasite interact: mice, rats, baboons, rhesus monkeys and cattle. When
infected with a strain of S. mansoni, these animals do show evidence of immunity to
various extents:
Rhesus monkeys show a strong immunity
Baboons have less resistance than rhesus monkeys
Rats manage to terminate the primary infection
Mice show a limited response and chronic infection ensues (results)
NB: S. mansoni does not usually affect animals such as wild rodents, and so this may have
an effect on the results shown in the above investigation.
44. Smithers & Terry worked with Rhesus monkeys infected with S. mansoni in the late ‘60s
and found that they were able to destroy cercariae worms from a second schistosomiasis
infection, but could not destroy the adults that had already established in the first
infection. Immunity could either be stimulated with large numbers of irradiated cercariae
(so they would die before they became adults), or with transplanted adults from another
monkey. The conclusions drawn were that Immunity appeared to operate against the larval
stages and not the adult stages, but could be stimulated by both. This is known as
concomitant immunity. In the same way, some people have been known to develop a
resistance to a secondary cancer, but this defence has no effect on the established primary
tumour.
In 1977, it was discovered that all investigated blood‐dwelling organisms contain an outer
surface covering (tegument) that is unique in nature, consisting of fused cells surrounding
the worm with a single continuous double‐bilayer membrane. On penetration of the
cercariae into the host skin, the outer glycocalyx is shed from their syncytial membrane,
and within 30 minutes the 2nd lipid bilayer is laid down. Membranous bodies that have a
double lipid bilayer are made in the perikarya in the sub‐tegumental cells; they fuse with
the cell surface to create the new membrane structures (microvilli with bulbous tips form
on the schistosome membrane due to this process).
The homogenates of adult worms were found to contain many of the molecules that were
found in the host species; antigens are shared, decreasing the antigenicity of the parasite
(its capacity to induce an immune response). This is due to acquisition of the host
molecules, rather than mimicry of them since when schistosome adults are incubated in a
red blood cell medium containing larvae, they acquire RBC antigens (A and B blood group
antigens) into their tegument via adhesion to their surfaces, allowing them to disguise
themselves as “self” and thus avoid detection by the immune system.
45. Experiment to show the acquisition of host blood group molecules
Schistosomes were taken at the ages of 1) three hours and 2) four days and incubated with
both anti‐schisto‐antibodies from an infected animal and anti‐host RBC antibodies to give
four samples. The antibodies produced on the cell membranes were labelled with gold
particles so they would show up on an electron microgram. The type of protein that is
present on the schistosomulum membrane can therefore be identified. (NB: A
schistomulum is the immature form of a parasitic schistosome after it has entered the
blood vessels of its host.)
Young 3 hour schistosomulum + anti‐schisto
‐antibody. The schistosome antibody is
displayed on the membrane
4 day old schistosomulum + anti‐RBC‐antibody.
The test now confirms the presence of RBC
antibodies on the membrane
Young 3 hour schistosomulum + anti‐RBC‐
antibody. There are no RBC antibodies
detected on the cell membrane
4 day old schistosomulum + anti‐RBC‐
antibody. There are now no schisto
antibodies detected on the cell membrane
Where the young schistosomulum once showed the presence of schisto proteins on its cell
membrane, the more mature organism displays the host RBC proteins, and is therefore
disguised as “self” rather than “non‐self”
Concept
If you were to infect a monkey with schistosomes, the schistosomes would coat
themselves with monkey antigens
If you were to infect a mouse with schistosomes, the schistosomes would coat
themselves with monkey antigens
If you then take a monkey and immunise it with mouse proteins, the monkey’s immune
system sees them as foreign bodies and makes antibodies that can be used in the case of
future exposure. This monkey can now make antibodies to mouse proteins and is called
an “anti‐mouse” monkey.
46. Eosinophils attacking a schistosome cell 1: a schistosome cell, 2: a schistosome after exposure to
cytotoxic granules, 3: a necrotic mass
Experiment used to determine the presence of host antigens on the tegument of adult
Schistosoma mansoni
Since the monkey in experiment 4 had already been immunised, it was ready to mount an
attack on any mouse proteins that were deemed foreign. In all other cases, the parasite is
initially recognised as foreign, allowing long‐term immunity to be achieved, but then the
parasite acquires a “self” protein coat, allowing the adults to evade the immune system.
This led to the idea that the schistosomula could be artificially transformed to convey anti‐
larval immunity. In vitro killing assays were then carried out using the antibodies IgG and
IgE, and cells like eosinophils and macrophages. If the cells were incubated with an IgE that
was specific for the parasite surface, eosinophils would selectively attack them with
cytotoxic granules. This is known as antibody dependent cell mediated cytotoxicity.
1 2 3
47. The prevalence of infection with Schistosoma haematobium in a population living in an
endemic area.
This graph shows a peak of infection intensity
of children roughly aged 6 to 15, but this is
followed by a decrease as a person
progresses into adulthood.
This could be due to adults spending less time
in the water as they age, since children are
more likely to play in the water etc., or it
could be that children are susceptible to
infection in early life, but over time immunity
builds up and renders the adults immune to
infection.
To investigate the resistance theory, re‐infection studies must be carried out
Praziquantel is a drug that is not licensed for use in the UK for human consumption, but is
available as a vetinary anthelmintic (drugs used to expel parasitic worms), or for certain
humans on a named‐patient basis. It clears up infection, making sure human subjects are
infection‐free before the re‐infection trials, to eliminate the variation in egg numbers
counted in faeces and urine.
The re‐infection study was carried out in Gambia using S. haematobium. Control for
exposure was done through controlling the subjects’ exposure to water, and before the
study was carried out, the field staff moved into the village a year in advance so they could
get to know every individual in the village and their personal histories. Every person in the
village was then monitored during the study and the following factors were recorded:
Activity and time in water
Densities of snails (infected and uninfected) in the water at the time
Densities of cercariae in the water at the time
A massive statistical analysis was then carries out, which took all the data into account.
The study took place along the Gambia
River in the small villages of Madina,
Samaco and Njarinjufa. The Gambia is
the smallest country in Africa
48. The adults in the study were found to be immune, and their immune parameters were thus
measured: parasite specific antibodies, eosinophils and other granulocytes.
A strong association was found to exist
between the types of antibody a person had
and their resistance to re‐infection. The
population was divided into quintiles (5
groups) on the basis of their IgG4 or IgE
response; those in the highest quintile for
IgE were found to be 10 times less likely to
be re‐infected than the lower quintile, and
those in the highest quintile for IgG4 were
found to be 10 times more likely to be re‐
infected than the lowest quintile.
The eggs of the parasite are the main pathogenic stage, the move to the outside
environment is associated with the release of enzymes and metabolic products which
stimulate very strong eosinophil and macrophage rich granuloma (which are T‐cell
dependent). This is particularly severe when the eggs are washed back into the liver
because lots of leucocytes flood into the liver to try and contain the infection, but this leads
to granuloma formation and a reduced blood flow. I
In S. mansoni infections, this results in alterations in blood flow leading to
hepatosplenomegaly as more RBCs are made in response to the reduced blood flow,
leading to a high blood pressure, oesophageal varices, and eventually death if untreated.
In S. haematobium, damage to the bladder and urethra can result, leading to renal failure
January &
February
Pre‐study
chemotherapy
Exposure to
Re‐infection
Low
Medium
High
April & May
Check efficacy
of treatment
Rains arrive
2‐9 Year Olds
Some
Some ++
High
August to
November
Transmission ‐
water conduct
observations
10‐14 Year Olds
None
Some
Some/ ‐
April & May
Re‐infection
intensity
Adults
None
None
None
Specific IgE against WWH shows a significant inverse
correlation (p<0.05) with re‐infection
49. ← Oesophageal varices caused
by hypertension can
haemorrhage spontaneously
and can therefore be fatal if
not treated
A large infiltrate of leucocytes
to the liver can cause
autoimmune liver fibrosis as
they try to attack the foreign
bodies (schistosome eggs)
trapped there↓
← Hepatosplenomegaly due to schistosome infection
The miracidia inside schistosome eggs release histolytic secretions when trapped in the
host tissues, Th cells recognise the egg antigens and release lymphokines (lymphocyte
cytokines) that cause inflammatory cells to aggregate, forming a granuloma, and leading to
hypersensitivity over long periods of time. The body eventually realises it’s doing more
harm than good through the immune response and so switches off the hyperactive
immunity, reducing the granuloma size and allowing the eggs to exit without too much
reaction (see below)
50. Lymphatic filariasis can be transmitted by
two main species of filarial nematodes:
Wuchereria bancrofti and Brugia malayi. It
causes inflammatory damage to, and the
dysfunction of the lymphatic system,
causing fluid accumulation which leads to
conditions such as elephantiasis (swollen
limbs), and hydrocoele. Filariasis also causes
filarial fever, lymphangitis and dermato‐
lymphangio‐adenitis (inflamed skin and
lymph nodes). These parasites live in the
lymphatic system and they are either so
large, or cause so much damage, that the
fluid can’t drain.
Onchocerciasis (river blindness) is caused by
the microfilaria of the parasite Onchocerca
volvulus. Here, the adult parasites live in
lymphatic nodules and lay microfalariae,
which migrate to the dermis and cause
itching and ageing of the skin. When they
migrate to the cornea, they can also cause
river blindness. In areas where this is
endemic, the older generation often rely on
the guidance of children who can still see.
The thread‐like filarial parasites live in the
blood (C), and inside lymphatic vessels (D).
They live for 4‐6 years, producing millions of
microfilariae, which circulate in the blood.
A B
Hydrocoele testis (B) is common in regions where the
parasites are endemic, and can on rare occasions affect the
breast tissue in women (A)
Parasitology Lecture 9
Immunity to Filariasis
The parasite is transmitted by the blackfly (Simulium), or a mosquito (Anopheles)
A B
C D
52. Pathology is mainly due to the adult worm living in the lymphatics or sinuses of the lymph
node. Because the worms are very active, they thrash around a lot and cause dysfunction
of, and inflammatory damage to the lymph system. The thichening of vessel walls from this
damage causes incompetent lymph valves, and ultimately leads to the blockage of the
lymphatics, especially upon the death of the worm (from senility ‐ old age)
Elephantiasis is due to fluid accumulation, typically in the legs, but it
can also affect the arms. Due to secondary infections, the skin
eventually becomes cracked where bacteria and fungi flourish, a
severe fever is also a common symptom. Treatment for this involves
scrubbing the affected area to get rid of the bacteria and fungi that
cause the exacerbation.
Hydrocoele is the most common clinical manifestation. In endemic
areas 40 to 60% of adult males are affected by this. It is caused
when adult worms localise in scrotal lymphatics and cause fluid
accumulation.
Chyluria is often due to hidden internal damage to the
kidneys and lymphatic system, resulting in an intermittent
discharge of lymph into the renal pelvis and subsequently
into the urine. Chyle is a milky white bodily fluid that
consists of lymph and emulsified fats or free fatty acids.
This gives urine of an affected individual the characteristic
appearance as seen to the left.
The glass on this chyluria sufferer’s bedside table does not
contain milk; it is a fresh urine sample!
Treatment
There is no vaccine available for this disease, the only ways to stop it are to interrupt its
transmission or control the morbidity. To interrupt its transmission one must eliminate the
microfilariae from the bloodstream with a single dose of a 2 drug regimen (albendazole
with diethylcarbamazine or ivermectin) once a year for four to six years. This, however, is
difficult to keep up for such a long period. Controlling its morbidity involves assisting the
lymph slow and preventing secondary infections by introducing forms of basic hygiene.
Morbidity refers to a diseased state, disability, or poor health due to any cause.
Onchocerciasis (river blindness)
The Onchocerca volvulus adults are 30 to 80 cm long and live in
nodules in the human skin. They live for about 12 years and
produce many microfilariae (about 0.3μm long), which circulate in
the skin and cause pathology. Transmission is via the bite of an
infected black fly (Simulium), which breed in areas of fast‐flowing
water. Across Africa they are distributed across some areas of
Venezuela, Brazil, Columbia and Equador.
53. The nodules lie in the subcutaneous tissue, about 1 to 5 cm in diameter. An infected person
may have several hundred at various locations of their bodies, such as the skull, ribs,
elbows, hips, thighs and knees. Just one tiny nodule contains many adult parasites, but
remember it is not the adults that cause the real problems!
Palpation of a nodule (A), and an idea of the number of parasites contained within (B)
A B
Eye manifestations are caused when the microfilariae migrate to the eye, where they die
causing a profound inflammatory response and scarring where the cornea is left very
opaque (see p.1). Onchocerciasis is the second leading cause of blindness of infectious
origin, leaving 18 million people infected, 800,000 visually impaired and 270,000 blind.
Skin manifestations
These are less widely reported than river blindness,
involving chronic dermatitis and intense itching due to
the dying microfilariae that cause a subcutaneous
inflammatory response causing the skin to itch and
become swollen and chronically thickened, known as
“lizard skin”. The skin also becomes lax due to the
destruction of elastic fibres, and may lose pigmentation,
commonly known as “leopard skin”.
Often people have to resort to scratching themselves
with rocks to alleviate this maddening itch.
Control of the parasites is through the drug ivermectin to kill the worms, and through the
spraying of the black fly breeding grounds with larvicides to break the transmission cycle.
Immunology
For further information on immunology, see back sheet entitled “Basic Immunology”
There is a fascinating host‐parasite interaction in this case; the infected human is exposed
to multiple life cycle stages, each interacting with different parts of the immune system.
The following data focuses on lymphatic filariasis, but onchocerciasis also shares common
immunological features...
54. Filariasis is a spectral disease. Multiple infection from a mosquito vector can lead to
immunity, tolerance or an inappropriate immune response and an immunology is
important in the understanding of these groups:
Immunity ‐ no symptoms and no microfilaraemia infection
Tolerance ‐ no symptoms, but an present microfilaraemia infection
The category which most individuals fall under
Inappropriate immune response ‐ symptoms include adenolymphangitis
(inflammation of the lymph nodes and vessels) and periodic fever. Chronic disease
includes oedema and chyluria, and elephantiasis may also result.
In order to understand filarial immunity, comparisons must be drawn between the three
main groups: endemic normals (EN), microfilaraemics (MF) and those suffering from
chronic pathology (CP). Humoral responses include antibody isotopes and antigenic
epitopes, while cellular responses include a proliferative response with CD4+ T cells:
Th1 cells (type 1) secrete IFN‐γ (interferon gamma)
Th2 cells (type 2) secrete IL‐4, 5, 9 and 13
T‐reg (regulatory) secrete IL‐10 and TGF‐β (transforming growth factor beta)
The immune response cabn be determined by the secreted cytokine (Immunoglobulin)
levels, as seen in the previous lecture (Leishmania Prophylaxis)
IgG1 IgG2 IgG3 IgG4 IgE
Endemic Normal 13 2 4 25 12
Microfilaraemic 32 3 6 761 9
Elephantiasis 75 40 36 222 43
Low levels of all Igs
Large IgG4 amounts
Large amounts of all
Human filariasis: Levels of antigen specific isotypes of IgG (μm/ml) and of IgE (ng/ml) antibodies
measured against adult B. malayi somatic extract
From the above table it is clear that endemic normals tend to have higher lower antibody
responses, while the elephantiasis patients have the highest. Those that asymptomatic
have high IgG4 and lower IgE levels. Endemic normals and sufferers of elephantiasis have a
lower IgG4:IgE ratio. IgG4 has been proposed as a blocking antibody, therefore the IgG4:IgE
ratio may be important when considering immunity.
High IgG4 levels may reduce some pathology, but this is undesirable if the effector
mechanism is via some form of IgE‐mediated ADCC (Antibody‐Dependent Cell‐mediated
Cytotoxicity) where the effector mechanism is not known.
55. Hyporesponsiveness is specific to the filarial
antigen; it can be seen that in individuals with
microfilariae infection (Mf+), the
responsiveness of the T cells to the filarial
antigen is greatly reduced, whereas if an
irrelevant antigen, such as that of
streptococcus) is introduced into an individual
with microfilariae infection, there is no
change in the T cell response.
Here, human PBMCs (peripheral blood
mononuclear cells) in Mf+ individuals have
reduced IFN‐γ levels, but intact IL‐4 levels.
This shows that the Th1 arm of the immune
response is turned off in the case of
microfilaraemics, while the Th2 response
remains in tact. This is hyporesponsiveness of
the Th1 cells, but drug treatment with
diethylcarbamazine (DEC) partially restores
the responsiveness of the human PMBCs, as
shown in the following graphs:
Summary of data so far:
The largest population are asymptomatic microfilaraemics with reduced T cell responses
but with high IL‐4, IL‐10, TGF‐β and low IFN‐γ levels. diethylcarbamazine treatment
restores both T‐cell responsiveness and IFN‐γ production. Endemic normals and those
suffering from elephantiasis tend to have low worm numbers and greater IFN‐γ levels.
