Laboratory 4 Apicomplexans I

Lab 4
1
APICOMPLEXANS I
“SPORE FORMING PROTOZOA”
Phylum: Apicomplexa
Class: Coccidia
Order: Eimeriida
Family: Eimeriidae
Eimeria tenella
Isospora belli
Family: Sarcocystidae
Sarcocystis spp
Toxoplasma gondii (toxoplasmosis)
Cryptosporidium parvum (cryptosporidiosis)
APICOMPLEXANS:
GENERAL CHARACTERISTICS:
-All are parasitic
-No cilia or flagella (a few species have
flagellated microgametes)
-Organisms contain an “apical complex”
-polar ring
-subpellicular tubules
-conoid
-rhoptries
-micronemes
All of the components of the apical complex,
disappear after host cell penetration
This indicates that the apical complex plays a role in host cell penetration by the organism
-All have a life cycle that includes a “Spore” stage involved in transmission
-Most have sexual and asexual reproduction
-Direct and indirect life cycles are found in this group of parasites.
-Several pathogens of humans and domestic animals in this group
Lab 4
2
Terminology used in Life Cycles:
1. Schizogony (multiple fission) asexual reproduction in which multiple mitoses take
place followed by simultaneous cytokinesis resulting in many
daughter cells at once.
2. Merogony (schizogony) multiple fission produces many daughter cells called
Merozoites
3. Merozoites daughter cells produced from merogony
which will asexually reproduce more merozoites
4. Meront – the cell that undergoes merogony
it will contain many merozoites
5. Gametogony - merozoites or trophozoites maturing to form gametocysts
6. Macrogametocyte will produce female gamete(s) “macrogametes”
7. Microgametocyte will produce male gametes “microgametes” (only flagellated
stage in this group)
8. Sporogony – zygote undergoes multiple fission to produce sporozoites
9. Sporozoites – daughter cell produced during sporogony
-- infective stage
10. Zygote – produced by fusion of macrogamete and microgamete
11. Spore – Oocyst the cystic form resulting from sporogony
--contain sporozoites
- has resistant membrane secreted around it (coccidia, e. g.
Eimeria)
- no resistant membrane (malaria)
An oocyst is NOT THE SAME THING as a cyst!
Lab 4
3
APICOMPLEXANS OF Medical/Economic Significance:
Coccidia: Eimeria and Isospora
Toxoplasma
Sarcocystis
Cyclospora
Cryptosporidia
COCCIDIANS
Phylum: Apicomplexa
Class: Coccidea
General Characteristics of Class Coccidea:
-Parasitic niche in hosts includes GI tract and associated organs- liver, kidney, blood
-Alterations of sexual and asexual reproduction
- Life Cycle Phases:
-Merogony, Gametogony, Sporogony are the three phases in the life cycle
---merogony and gametogony occur within host cells
---sporogony occurs outside the host in soil or litter (There are several notable
exceptions. Examples: Toxoplasma, Sarcocystis, Cryptosporidia
-Infective stage: is the sporozoite within very resistant spores called “oocysts”
Family Eimeriidae:
Economically important genera: Eimeria and Isospora
Differ mainly in details of development within the oocysts:
Eimeria – oocysts have four sporocysts containing two sporozoites each
Isospora – oocysts have two sporocysts containing 4 sporozoites each
Note: cannot distinguish between Eimeria and Isospora oocysts until they are sporulated,
OOCYST MORPHOLOGY:
Lab 4
4
Eimeria spp - Economically important parasites in domestic animals
NOTE: NO Eimeria spp infect humans!
-Host Niche: digestive tract (some in liver and kidneys)
-Development within host is intracellular
-Merogony and Gametogony within host GI tract
-Sporogony outside host (requires oxygen for sporulation)
-Eimeria spp are host specific and within host, tissue specific
-Infections with Eimeria may be self-limiting but immunity which may develop is only
partial.
-taxonomic characteristics: size and shape of oocyst and the number of sporocysts within
Eimeria tenella
- Host: chickens
- Niche: GI tract –cecum
Mechanisms of Pathogenesis in Host:
“Bloody Cecum”
-necrosis of cecal epithelium
-bloody diarrhea
-high mortality rate especially in young chickens
-young more susceptible and shed more oocysts!
- No treatment once infection is established
-why all commercial feeds for chickens contain coccidiostats
-continuous administration of coccidiostats in food or water will prevent
transmission
- Infections are self limiting; asexual reproduction does not continue indefinitely
-if chicken survives primary infection, may have partial immunity
-Concurrent infections can occur with other Eimeria spp. (infection with one species
actually seems to enhance infection with a second species)
Lab 4
5
In chicken:
•
•
•
•
•
Transmission: ingestion of sporozoite within sporulated oocysts in soil/litter,
Sporozoites released in small intestines and penetrate epithelial cells of cecum
to become trophozoites
Trophozoite becomes a first generation schizont or “meront”
- undergoes schizogony ( multiple fission) producing many daughter cells called
“merozoites”
st
1 generation Merozoites released as cell ruptures
1st generation Merozoites :
-can penetrate other epithelial cells and repeat process of multiple fission
producing 2nd generation merozoites ----Æ3rd generation etc.
-some will penetrate epithelial cells and undergo gametogony transforming
into gamonts (gametocytes): Macrogametocytes and Microgametocytes
•
•
•
•
Most gametocytes will become Macrogametes
Some will become microgametocytes and undergo schizogony to produce many
bi-flagellated microgametes
Fusion of microgamete and macrogamete Æ zygote = unsporulated oocyst
Oocyst out with feces:
- zygote undergoes sporogony (multiple fission) outside host
-forms 4 sporocysts, each with 2 sporozoites = sporulated oocyst
Sporulated oocyst (excyst/release)
merozoites
gametogony
(merogony)
gamonts
sporozoite
2nd generation
merozoites
macrogametocyte
microgametocyte
fusion of microgamete and macrogamete
Oocyst
in oocyst, zygote (sporogony)
(merogony)
merozoites
(merogony) etc.
macrogamete
biflagellated microgametes
zygote
forms thick walled unsporulated
4 sporocysts, each with 2 sporozoites = sporulated
oocyst
Lab 4
6
Schizogony in cecal epithelium: (1-7) 1- Sporozoite enters epithelial cell 2-Growing trophozoite. 3Nucleus undergoes multiple divisions to form schizont. 4-Each nucleus together with a bit of cytoplasm
forms a merozoite. 5-Fully developed merozoites. 6-Schizont and epithelial cell rupture, releasing
merozoites into cecal lumen. 7-Merozoites enter other cecal cells to repeat schizogony cycle several
times.
Gametogony in cecal epithelium: (8-25) Microgametogony (8-13) 8-Some schizogonic merozoites are
destined to become microgametocytes. 9-Merozoite penetrates cecal epithelial cell to begin cycle. 10Growth of microgametocyte. 11-Multiple division of nucleus is beginning formation of microgametes. 12Formation and alignment of microgametes over surface of residual cellular cytoplasm. 13-Cecal cell
ruptures, liberating mature biflagellate microgametes into cecal lumen.
Macrogametogony (15-24) 14-Schizogonic merozoites predetermined to initiate macrogametogenous
cycle. 15-Merozoite penetrates cecal epithelial cell. 16-Formation of macrogametocyte. 17-Meiotic
division of nucleus with extrusion of half of chromosomes to form macrogamete. 18-Haploid
macrogametocyte. 19- Mature macrogametocyte. 20-Fertilization of macrogamete. 21-Fusion of
microgamete and macrogamete to form zygote. 22-Oocyst. 23-Mature oocyst ruptures cell epithelium.
24-Oocyst free in cecum. 25-Oocyst in large intestine will be voided with feces.
Sporogony in soil and litter: (26-30) 26-Mature oocyst has not begun development. 27-First nuclear
division. 28-Second and final nuclear division preparatory to forming sporoblasts. 29-Formation of four
sporoblasts. 30-Infective or ripe oocyst containing four sporocysts each with two sporozoites.
Infection of chicken host, excystation of sporozoites in crop, gizzard and intestine: (31-34)
31-Oocyst in crop where action of CO2 and enzymes cause its wall to weaken. 32-Oocyst in gizzard
under mechanical action and continued chemical action ruptures sporocysts, releasing
sporozoites. 33-Sporozoites freed from oocyst in small intestine. 34-Liberated sporozoites enter
cecal lumen preparatory to penetrating epithelial cells.
Lab 4
7
On Slides of Eimeria tenella:
Sporozoites are “banana” or “rod” shaped
This is the infective stage.
Trophozoite – the rounded up small single-cell uninuclear form that the
sporozoite becomes when it penetrates an epithelial cell.
-- small nucleus of parasite surrounded by a clear halo and is
easily distinguished from the large nucleus of the epithelial cell.
Schizont or Meront – the trophozoite which has undergone schizogony
--this cell is filled with many small nuclei scattered in the cytoplasm
Merozoites – within the schizont or meront, each small nucleus becomes visible as a
spindle-shaped organism, the merozoite.
-- may see these merozoites free in lumen, released by the rupture of cell
Remember, Merozoites can do one of two things:
1) each enters a new epithelial cell and undergoes merogony repeating the
asexual reproduction cycle to form another generation of merozoites
2) enter a new epithelial cell and transform into a gametocyte (sexual cell)
initiating the sexual reproduction cycle
Macrogametocyte – large cell located inside a cecal epithelial cell (will have crowded
the epithelial cell nucleus to one side) the “female” gametocyte
-have a large centrally located nucleus
-have large cytoplasmic granules (with H&E stain, will stain bright pink)
(may see granules lining periphery of macrogametocyte)
-Each macrogametocyte will become a mature macrogamete!
-Tend to be found in groups of cells
Microgametocyte– large cell located inside a cecal epithelial cell
- the “male” gametocyte
-will undergo multiple fission to form many microgametes within the
microgametocyte.
-cell ruptures releasing microgametes into lumen of intestine
Microgametes – minute, slender biflagellated microorganisms
Oocyst – formed when microgamete fertilizes macrogamete to form zygote
- ruptures from epithelial cell and is stage passed in feces
-has a thick cell wall
-contains a large spherical cytoplasmic body but no sporocysts
-is not infective
Sporulated Oocyst – infective for new host
-contains 4 sporocysts, each with two sporozoites
Lab 4
8
-sporulation occurs within 48 hours after oocyst passed from host
Eimeria slides 5.5 day PI: look for meronts with merozoites (host cell nucleus will be
displaced)
Eimeria slides 7 days PI: Oocyst takes up all of host cell.
Reproductive rate of Eimeria is phenomenal:
1 oocyst Æ 8 sporozoites
8 sporozoites can produce millions of merozoites
most of which will become macrogametes and
thus oocysts.
Good thing:
if organism enters epithelial cell about to be
sloughed, it’s “bye-bye” to the organism in that
cell. (chickens replace epithelial cells every 2-3
days)
Some get shipped on out with the feces before they can penetrate host cell
Host immune responses get some of the merozoites as they leave the cells
Macrophages can ingest and kill them.
E. tenella 5 DPI schizont with merozoites
7 DPI with macrogametocytes and
oocysts
Lab 4
9
Family: Sarcocystidae
-Toxoplasma gondii
-Sarcocystis spp
•
Heteroxenous cycle
-asexual development in vertebrate intermediate host
-sexual development in vertebrate definitive host (carnivorous mammal or bird)
•
Life cycle includes both intestinal and tissue (extra intestinal) stages
•
Morphology:
Oocyst contains two sporocysts, each with four sporozoites
•
Transmission: ingestion of sporozoites within sporulated oocysts or tissue
containing tissue cysts with bradyzoites.
Terms associated with Family Sarcocystidae:
tachyzoites – merozoites formed by multiple fission (endodyogeny) of sporozoites
bradyzoites – merozoites formed in tissue cysts in extra intestinal (outside the gut) sites
bradycyst – same thing as a tissue cyst; also called a “pseudocyst” or zoitocyst
•
•
Sexual reproduction occurs in intestine of definitive host and produces oocysts.
Asexual reproduction occurs in tissues and results in tachyzoites and
bradyzoites
Toxoplasma gondii
“toxoplasmosis”
-obligate intracellular parasite
-Intermediate host: humans, domestic animals, wild animals
-- Infects most mammalian species and most nucleated cell types
-Definitive host: Felidae only! (domestic and wild cats)
Intermediate host (asexual reproduction only and extra intestinal stages only)
Transmission:
-Ingestion of sporozoites in sporulated oocyst or bradyzoites in tissue cyst
-oocysts in cat litter box or garden soil
-tissue cysts from undercooked infected meat such as beef – not cat!!
- transplacentally. (tachyzoites can cross the placenta in pregnant women who
become infected.
Lab 4
10
Cycle in Intermediate Host: No intestinal stages!!
-Oocyst excysts and the released sporozoites penetrate through the intestinal wall
-Sporozoites are phagocytosed by macrophages
-Macrophages distribute the organisms throughout the host
-Virulence Factor: Macrophages do not kill because the parasite prevents the host
cell lysosomes from fusing with the vacuole containing the parasite
-Sporozoite undergoes schizogony producing tachyzoites in the macrophage
-Macrophages rupture releasing tachyzoites
-Tachyzoites invade other tissues, especially striated muscle, retina, brain, liver
and lymph nodes (any nucleated cell!)
This is the danger to the fetus because tachyzoites can cross the placenta.
-Tachyzoites undergo schizogony and repeat the asexual cycle rupturing out of cells.
(can be seen free in blood or peritoneal fluid for a short period when released
by ruptured cell)
-Host immune response eventually limits the parasite’s asexual reproduction.
-When this occurs, some tachyzoites differentiate into a second asexual stage, the
bradyzoite.
-Bradyzoites are found in cyst-like accumulations called “pseudocysts” or tissue
cysts.
-Bradyzoites are dormant within the tissue cysts (can persist for months to years)
Host immune response keeps the parasite infection latent (inactive). This is
why there is no inflammatory response around these cysts in tissues.
-Transmission requires that the intermediate host tissue containing the cysts be
eaten by another host.
Definitive host – the cat
(has both sexual and asexual reproduction)
-See both intestinal and extra intestinal stages in the cat
-Can acquire infection two ways:
1) ingesting sporozoites within infective oocysts (Remember, unsporulated oocyst does not
contain sporozoites yet!!!)
2) Eating tissue infected with pseudocysts (rodents commonly harbor Toxoplasma cysts)
-If oocyst ingested, sporozoites released and invade epithelial cells in small intestine
-If pseudocyst ingested, bradyzoites released and invade epithelial cells in small intestine
-Each sporozoite rounds up in cell forming trophozoite which undergoes schizogony to
form merozoites which are released when cell ruptures.
-Merozoites invade new epithelial cells and repeat the asexual reproduction
-Within 3-5 days post infection, some merozoites invade epithelial cells and undergo
gametogony forming macrogametocytes and microgametocytes.
-Within cell, macrogametocyte becomes a macrogamete
-Within cell, microgametocyte undergoes schizogony to form microgametocytes.
Lab 4
11
-Released microgametes fuse with macrogametes to form oocysts. This is syngamy.
-Zygote secretes cell wall to become unsporulated oocyst.
-Unsporulated oocysts are released and pass out with feces
-Sporulation occurs outside of definitive host
-Sporogony produces two sporocysts, each containing 4 sporozoites within
Only Cats produce Toxoplasma gondii oocysts!!
Cats can also be intermediate hosts and will also develop extra intestinal infections with
Toxoplasma gondii, resulting in production of tachyzoites and pseudocysts with bradyzoites
within their tissues.
Life cycle of Toxoplasma gondii in
definitive and intermediate hosts
Humans:
Acquired infection:
-Immunocompetent hosts are usually
asymptomatic.
-May see enlargement of lymph
nodes especially cervical lymph
nodes
-Possibly may have low grade fever
-Immunity to Toxoplasma is complete
Rupture of tissue cyst wall and release of bradyzoites can cause severe reaction in human
host tissues. Bradyzoites will be killed by the immune response. If this occurs in the brain,
however, the result can be encephalitis. If it occurs in the retina, blindness can result.
Lab 4
12
Severe disease in adults is rare. (80% of adults in France have antibody (IgG titers) to T.
gondii indicating prior exposure –BECAUSE: like to eat raw meat ground up and served as
“steak tartar.”
Seronegative organ-transplant recipients are at greatest risk – if get an organ from infected
donor, can result in disseminated toxoplasmosis, myocarditis, CNS lesions or
retinochoroiditis. Heart transplants are particularly at risk.
AIDS patients are another group at risk for severe disease caused by T. gondii, especially
for encephalitis.
Congenital Toxoplasmosis in humans:
-Mom will have no clinical signs but the organism crosses placental barrier during
pregnancy.
-Fetal infection can vary from asymptomatic to severe developmental damage
-Fetal damage is most acute if infection occurs between first through third months of
gestation (first trimester).
-Clinical effects can range from severe CNS damage to still births
-Cerebral and ocular lesions can be very severe
Mom does not need to get rid of the cat just because she is
pregnant, but she should not clean the litter box!!
Trophozoite stages: tachyzoites; bradyzoites
(banana-shaped crescents)
Oocysts: oval, surrounded by a smooth wall
Sporulated oocysts contain 2 oval sporocysts,
each with 4 elongate, curved sporozoites.
Sarcocystis spp.
-Heteroxenous
-Herbivorous intermediate host (hoofed mammals, birds, reptiles, small rodents etc.)
-Carnivorous definitive host
Cycle in definitive host:
-ingestion of bradyzoites
-transform into merozoites in intestine
-invade epithelial cells and undergo gametogony
-Oocyst are formed only in the definitive hosts
-Oocysts sporulated in intestine and rupture releasing sporocysts
-Sporocysts containing sporozoites pass out in feces.
Lab 4
13
Cycle in intermediate host:
-Ingest sporocysts (oocysts rupture in definitive host and only sporocysts pass
out with feces)
-Sporozoites released from ingested sporocysts in intestine and penetrate through
epithelial cells
-Disseminate to various tissues in host
-Invade tissue cells and undergo schizogony to produce tachyzoites
-Tachyzoites released from ruptured cells and repeat cycle
-Tachyzoites become bradyzoites and form tissue cysts called “sarcocysts” or
“zoitocysts” in striated muscle, cardiac muscle and occasionally in brain.
Shapes of these cysts in tissues may vary from round to spindle-shaped to
irregular in shape.
-Only bradyzoites are infective for definitive host!
Look for sarcocysts on slides of striated muscle.
Sarcocyst (zoitocyst) in muscle of experimentally infected
animal.
Note the bradyzoites within sarcocyst.
Lab 4
14
Family: Cryptosporiididae
Cryptosporidium sp
-Coccidian parasite of intestinal epithelium and respiratory epithelium of many
animals: mammals, including man; birds; reptiles and fish.
-Organism has direct life cycle with schizogony, gametogony and sporogony.
Cryptosporidium parvum
-opportunistic parasite of humans, especially young children
-common in AIDS patients
-The most likely culprit in cases of chronic diarrhea
-schizogony, gametogony and sporogony within microvilli of epithelial cells of GI
tract ( predominantly in small intestine but in heavy infections can be found from
esophagus to colon)
-Round oocysts are very small (4-5um) and are passed in feces
-very refractile
-no sporocysts – just 4 slender sporozoites
-Two types of oocysts:
-thick-walled which pass out with feces
-thin-walled which can rupture in lumen of gut and cause autoinfection of infected
host
Oral transmission route:
-Sporulated oocysts very infective, low number (30-100) is enough to infect host
-oocysts survive a long time in water (drying out, however, will kill the organism)
-contaminated water is the most common source (swimming pools, water parks,
day care centers)
Zoonosis – can spread from animals to man and vice versa
Organism attaches to surface of epithelial cells:
-the organism surrounds itself with a host cell derived membrane when it attaches to
the host cell membrane of the epithelial cells of digestive tract. The parasite causes
the microvilli of the host epithelial cells to fuse forming a membrane around the
organism. The parasite is NOT intracytoplasmic because it has not invaded the
cytoplasm of the epithelial cell.
-clinical signs: profuse watery diarrhea persisting for several days to several weeks
(1-10 days on average for healthy individuals)
Lab 4
15
-No treatment available
-- can evade drugs because it is surrounded by a membrane of host cell origin and
drugs ingested and present in the gut lumen cannot get to the parasite.
Slides: acid fast stain of oocysts (left)
Tissue section of intestinal epithelium with parasites at tips of villi (right)