Lab 3. Parasite diversity I. Microparasites
The microparasites include the protozoans, bacteria, fungi and viruses. They are
characterized by their small size (almost always microscopic and often only 1-2
micrometers in size), their modes of reproduction, and their ability to illicit strong and
specific host immunity. They are fascinating to study, partly because they include the
majority of the major parasitic diseases of humans, but also because they demonstrate
brilliant adaptations for the parasitic way of life. Unfortunately, their small size makes
them exceptionally difficult to study. This difficulty leads to the fact we know so little
about the general biology of even our most pathogenic microparasites. In this lab, living
material is very difficult to acquire. We will focus on the protozoan parasites via demo
material, slide material and projector slides.
Protozoa
The study of protozoans requires considerable patience and skill as a microscopist. In
order to find the diagnostic characters on your slides, you will probably require the use of
oil immersion techniques (if you are shaky on the use of this technique see me for a
refresher!).
The one-celled parasitic protozoans are an extremely diverse assemblage of species.
Your slide box contains only a very small sample of this diversity. As you go through the
protozoans, pay particular attention to the many morphological differences between
species, as well as the diversity of life-cycles. You will see that there are direct and
indirect modes of transmission, sexual and asexual multiplicative phases and various
levels of pathological response by the host.
Phylum Mastigophora
Trypanosoma rhodiense and T. brucei
T. rhodesiense is the causative agent of the dangerous human disease, African sleeping
sickness. Over 10,000 cases are diagnosed annually; 50% of these die and the other 50%
suffer permanent damage. T. brucei is responsible for the immensely important disease
of livestock known as nagana. Both use tsetse flies as vectors. African sleeping sickness
has plagued humans ever since they first encroached on the domain of the tsetse fly. The
disease has kept over 4 million sq miles of grazing land in Africa out of agricultural
production. Consider the political, ecological and socioeconomic implications if a
vaccine for sleeping sickness were ever found (note: a third species, T. cruzi, is known as
American trypanosomiasis and infects over 10 million people; it uses a reduvid bug as
vector).
This group is characterized by a spindle-shaped body containing a central nucleus (or
kinetosome). The flagellum arises from this structure. A kinetoplast is also situated near
the base of the flagellum. It is a mass of DNA within a single mitochondrion and is
easily seen with the light microscope. Your slide is a smear from an infected human’s
blood. You will see numerous trypomastigotes. This stage is the final developmental
stage of the trypansomes (see diagram). Under oil immersion, you should be able to see
the undulating membrane, flagellum, nucleus and kinetoplast.
Trypomastigotes undergo asexual reproduction (binary fission) in the vertebrate host
(human, domestical mammal, native ungulates). The fact that the parasite can survive in
so many reservior hosts is a major factor in the epidemiology of the disease. During a
blood meal, the tsetse flies ingest infective trypomastigotes. Once ingested, these
become procylic trypomastigotes and these multiply by binary fission and then
metamorphose to an epimastigote form in the insects forgut. These migrate to the
salivary gland and transform into metacyclic typomastigotes, which can then be
inoculated into another mammalian host during a blood meal. This species is fascinating
for its ability to evade the host’s immune system by continuously changing the antigenic
structure of their external surface (the phenominon of antigenic variation).
Trichomonas vaginalis
This is a cosmopolitan species, found in the urogenital tracts of men and women.
Members of this group are easily recognized by having an anterior tuft of flagella, a stout
median rod (the axostyle) and an undulating membrane. It is an immense family,
members of which form the span of free-living, commensal (one group is found only in
the gut of termites; another is found between the teeth, and on the tonsils, of humans) and
parasitic lifestyles. Refer back to your notes on the relationships between Rhizobium and
Agrobacterium, and between the pathogenic vs. mycorrhizal fungi. Can you imagine any
parallels ? Can you imagine the selection pressures occurring on the Trichomonads that
may have led to their different ways of exploiting hosts ?
The parasitic species are unusual in that they do not use vectors (e.g. tryps) or cysts
(Giardia) for transmission. Really, the only option left is to use direct transmission. T.
vaginalis is transmitted primarily through sexual intercourse. Many strains are of very
low pathogenicity, especially in men. Some strains, particularly when infecting women,
can cause an intense inflammation, with itching and copious white discharge that is
swarming with trichomonads. They feed on bacteria, leukocytes and cellular debris.
Giardia lamblia
Your slides contain the feeding form (trophozoite) and cysts of this, the most common
intestinal parasite of humans. It is the causative agent of giardiasis or ‘beaver-fever’.
This is the disease most associated with wilderness campers and is typically associated
with severe diarrhea. The parasite matures in many reservoir hosts (such as beavers and
cattle) which is an important factor in the epidemiology of this disease.
Giardia has four pairs of flagella arising from the centre of the cell; they are typically lost
during staining. Using the oil immersion lens you should be able to see two nuclei and a
central pair of median bodies. The trophozoites are cup-shaped and the surface of the
ventral side is concave and thickened to form a large adhesive disk, used for attachment
to the host’s intestinal villus (see diagram below). Locate a cyst using the high power
objective and advance to oil immersion power and note the thick cyst wall, enclosed
flagella, nuclei and median bodies.
Mammalian definitive hosts ingest infective cysts in drinking water. Giardia excysts in
the intestine, releasing trophozoites. These attach to villi and multiply by binary fission.
In victims of giardiasis massive infection is typical; the presence of several billion
trophozoites in a single stool is not unusual! Eventually cysts are produced and excreted.
Cysts are the infective, resistant stage. They can withstand freezing and stomach acidity.
Unlike the trypansomes, only one host is required to complete the life cycle.
Phylum Apicomplexa
All members of this phylum are parasitic (recall that this it is quite unusual for any phyla
to be exclusively parasitic) and infect members of all animal phyla. All are characterized
by an apical complex which is only revealed under the electron microscope. This
structure is important for recognition and penetration of host cells. Members of the
phylum include some of the most serious diseases of humans and domestic animals. The
life cycles of apicomplexans are complex; with direct or indirect pathways. Species with
direct life cycles have resistant spores or oocysts that bridge the gap in the external
environment between hosts. Those with an indirect life cycle involving vectors remain
within a host and thus have no need for a protective cyst.
a. Life-cycle of Monocystis in earthworms
Members of this Phylum Sporozoa is entirely parasitic and contains some of the most
pathogenic parasites of humans. It also contains species that have incredibly complex
life-cycles, often involving more than one host. The phylum is named after the presence
of an ‘apical complex’ that is typically used to penetrate host cells. The only other
generalized characteristic is that all of the approximately 5,000 species typically alternate
between sexual and asexual phases and often have complex life cycles. There are two
major groups within the Sporozoa: The gregarines (e.g. Monocystis), and the coccidians
(e.g. Plasmodium and Cryptosporidium).
Monocystis lumbrici is an Apicomplexan parasite that lives in the seminal vesicles of
terrestrial earthworms. The worm becomes infected when it ingests a spore containing
several sporozoites. These hatch in the gizzard, where the released sporozoites penetrate
the intestinal wall, enter the dorsal blood vessel, and then make their way to one of the
host’s 5 or so ‘hearts’. From there they penetrate the seminal vesicle, where they enter the
sperm-forming cells in the wall. At this point they ingest and destroy the developing
spermocytes. Then they move into the lumen of the vesicle where they become mature
trophozoites. After a period of feeding, two of these will come together, flatten against
each other, and secrete a common cyst around each other. This is the gametocyst, usually
containing 2 gamonts. Each now undergoes extensive division of their nuclei, pinches off
a small portion of cell cytoplasm, which together then bud off to become the gametes.
The fusion of a pair of gametes forms a zygote, each ultimately becoming a spore. Three
cell divisions later forms 8 sporozoites. Thus, each gametocyst now contains many
oocysts. New hosts become infected by ingesting gametocysts, or more commonly, by
ingesting individual oocysts. Thus, meiosis is zygotic. Only the zygote is diploid, and
reductional division in sporogony returns the sporozoites to the haploid condition.
Procedure:
Dissect the anterior end of a freshly anesthetized worm. Remove the seminal vesicles and
place in a drop of water. Take small pieces of seminal vesicle, squash under a cover slip
and look for the different stages of Monocystis (see figures). Make drawings of each
stage and construct an annotated life-cycle. Record as many different stages of infection
as you can.
b. Plasmodium falciparum
This intracellular parasite is the most dangerous of the four species that cause malaria in
humans. This species will be the focus of our discussions in lecture. It is the most
common and debilitating of human parasitic diseases. Over two million people each year
die from the disease. Moreover, the disease has played a major role in shaping our
history and civilizations. It is impossible for you to understand this disease without a
thorough understanding of its life cycle. Please spend time before lab going over the lifecycle diagrams.
Your slides only show a fraction of the various stages of the malaria life-cycle. One slide
shows gametocytes inside host red-blood cells as deeper-staining structures (often
crescent or bean-shaped). Using the oil immersion lens you should see that
microgametocytes have a large nucleus and irregularly distributed granules.
Macrogametocytes have a small compact nucleus with a dark red nucleolus. Further
devleopment of these forms only continues inside a mosquito’s stomach. Sometimes, the
gametocyte-infected cells have been distorted to such an extent that it ruptures during the
fixing process. Thus, you may see some gametocytes which are not enclosed within a red
blood cell membrane.
You also have slides which show trophozoites. They can be distinguished by the large
food vacuole surrounded by a thin layer of cytoplasm and including a peripheral nucleus.
This gives the characteristic ‘ring’ which forms the well-known ‘ring-stage’. Again, the
infected RBC may appear distended or abnormal in shape. Use the demonstration slides
of the various life-cycle stages and life-cycle diagrams to help you understand the
biology of this important parasite. We will also watch a David Suzuki video which
describes the recent worldwide increase in malaria. Take specific note of the video
footage of free-swimming sporozoites infecting liver cells.
c. Cryptosporidium parvum
This waterborn parasite, together with Giardia, represents one of the two major waterborn
parasites of humans. Although the genus was first discovered many years ago (as a
parasite of turkeys), it has only recently received attention from biologists and disease
specialists. In 1982 the American Centre for Disease Control, found 21 males from large
cities to be suffering from severe diarrhea; all had AIDS. Then, in 1993, there was a
severe outbreak in Milwaukee, affecting over 400,000 people. There has since been an
outbreak in Kelowna (1995) and again in Shaughnessy (near Picture Butte), Alberta in
1997. Not surprizingly, it is one of the main parasites under study by parasitologists,
agriculural agencies, water quality technicians and environmental groups. The Lethbridge
area is considered one of the most high-risk areas in Canada for an outbreak of
Cryptosporidiosis, due mostly to the numbers of cattle feed-lots and our climatic
conditions.
It is generally a non-lethal disease for humans but does cause severe diarrhea, vomiting,
weight loss and cramping. People with normal immune systems expel the parasites in
about 2 weeks, and are then immune to further infection. However, it is usually lethal in
patients with compromised immune systems (especially the elderly and HIV-infected
hosts). The main concern is that cysts are highly resistant to standard disinfectants such
as chlorine and chlorine dioxide. In the US, the main method of control is the conversion
of water-treatment plants to use ozone rather than chlorine as a disinfectant. Costs run
into the billions of dollars.
This parasite is a recent problem, so that biology supply houses do not have specimens.
However, there will be a demonstration in the lab, with the various life-cycle stages.
Concentrate on the various life-cycle stages. Note the similarities and differences in the
life cycle stages between this coccidian parasite and Plasmodium.
d. Toxoplasma gondi
This is a cosmopolitan parasite, found in humans and a wide variety of other carnivores.
It is intracellular within the cells of muscle tissue and the intestinal epithelium. The lifecycle always includes an intestinal and extra-intestinal stages in cats and other felines, but
extraintestinal stages only occur in other hosts. Sexual reproduction occurs in cats,
asexual reproduction occurs in the other hosts. Cats release oocysts in their feces and
then these hatch out in the guts of intermediate hosts, including humans. The normal lifecycle involves cats and either mice or rats. However, we ingest raw meat, exposure to
Toxoplasma can occur.
Most of us carry antibodies to Toxoplasma, so either we have come into contact with
oocyst-laden cat feces, or have eaten infected meat. Yet most of us don’t get sick from
this infection and never know we have it. Thus, context-dependent or a common feature
of this interaction. Factors such as stress, age, diet etc. can lead reduced
immunocompetence and increased pathogencity. Martina Navratilova lost the US Open
in 1982 after withdrawing due to complications with this parasite.
Certain stages of the parasite proliferate in almost any tissue, often killing the cells faster
than they are replaced. If they end up in the brain, and many do so, problems can result.
One tragic form of the disease is congenital toxoplasmosis. If a mother is infected, then
Toxoplasma often will infect the developing fetus. Most infections will be nonpathogenic but many will cause significant morbidity and death. Stillbirths and
spontaneous abortions are usually the cause. This is especially true in sheep, where
spontaneous abortions can reach epidemic propotions. Severe damage is usually caused
by CNS disorders due to the rapidly dividing parasites. It is for this reason that pregnant
mothers and cats are a bad mix.
Myxobolus cerebralis
This bizarre species is a member of an enigmatic group called the Myxozoans. Some
view these weirdos as highly adapted protozoans, others view them as previously
multicellular cnidarians (jellyfish?) that lost their cellular structure when they became
parasitic. All members of this diverse group are also parasitic. We will briefly consider
them for three reasons. First, it is another group which is locally common in most of our
fresh-water fish (although most are completely unknown and unidentified). Specimens
are usually easy to obtain, depending on the time of year. Second, one of the species,
Myxobolus cerebralis is a disease agent of fish, particularly important as agents of
mortality among salmonids. A third reason is that this obscure group provides an
excellent example of how controversial and acromonius the taxonomy of parasitic
organisms can become.