COCCIDIANS

Coccidia Life Cycle

• Largest group of apicomplexan protozoa falling under Class
Conoidasida
• Subclass of microscopic, spore-forming, single-celled
obligate intracellular protozoan
• Members of Phylum Apicomplexa are provided with a
cluster of secretory organelles made up of
o rhoptries,
o micronemes
o polar rings
▪ microtubules
▪ conoid
• Secretory organelles allow the parasites to enter the host;
Coccidians enter the epithelial cells of the intestinal wall
• The secretion allows the parasite to enter the host cell.
• Almost all coccidian follow the same life cycle with slight
variations per genus; almost the same life cycle with malaria
• There is an alternation of sexual and asexual multiplication
• It is typically characterized by three sequential stages,
1. Sexual cycle or sporogony producing oocysts (oocysts
= cysts in protozoans)
2. Asexual cycle or schizogony (merogony) producing
merozoites (meronts),
3. Gametogony resulting in the development of male
(micro) and female (macro) gametocytes (gamonts)
• The complexity in the life cycles of coccidians is a challenge
in terms of taxonomy
Cryptosporidium
• Many reported species, however, only C. parvum and C.
hominis (more common) is seen in humans
• Recognized in mice in 1907
• Reported in humans in 1976
o Immunocompetent child
o Immunosuppressed adults
• Recognized globally in 1980s and 1990s
o AIDS patients
o Outbreak among veterinary students
• Cryptosporidium is a spore producing parasite found in the
intestine of infected people and animals.
• Intestine= disturbance of bowel movement and absorption
of nutrients; MOT= fecal oral route
• Cryptosporidium spp. is the most common cause of
Cryptosporidiosis.
• There are many species of Cryptosporidium that infect
animals, some of which also infect humans. The parasite is
protected by an outer shell
that allows it to survive
outside the body for long
periods of time and makes it
very tolerant to chlorine
disinfection
• There are several species of Cryptosporidium that are
currently recognized.
• It was initially reported that the only species that infect
mammals was C. parvum and was believed to be the
species infecting humans.
• However, molecular tools, especially DNA analysis,
described the existence of another species,
Cryptosporidium hominis found mainly in humans.

Coccidiosis
• Collective term for the disease caused by Coccidia
• Major problem in animal farming and zoo management
• Among humans, they are opportunistic in
immunocompromised and immunodeficient
• Self-limiting parasites; may be present in human but doesn’t
actually cause harm unless immunocompromised or
immunodeficient Oocysts which contains sporozoites

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 Coccidian

• Definitive host: Not specific but able to infect mammals Cryptosporidium Oocysts
e.g. humans • 4-5um wide
• Reservoir: Calves, sheep, fish, birds and turkeys • One oocyst contains 4 fusiform sporozoites
• All stages of development are completed in the • Does not stain with iodine and
gastrointestinal tract of the host; completed in the is acid-fast (since their wall is
definitive host, have no intermediate host but have very strong and resistant)
reservoir host, but development of stages happens mostly • Stains with Kinyoun and
in definitive host Safranin
• Very hard and resistant (60C)
Life Cycle of Coccidians (Cryptosporidium) • Infective for 2-6 months in the
environment
• Are released with fecal matter
during onset of the symptoms
• They are shed 5 days after
infection
• Takes only 5 days to
produce an oocysts from
ingestion until it is passed
out in the feces
• Incubation period between
1-14 days
• Sequential application of
ozone and chlorine –
eliminate the cysts

• Oocyst → Sporozoites → Trophozoites → Merozoites →

Gametes → Sexual cycle creating zygote → Oocysts Cryptosporidium Hominis
• Small cocci (2um-6um)
1. Oocysts • Opportunistic parasite
- ingestion of oocysts through fecal oral route • Zoonotic (cows, birds, reptiles,
(Cryptosporidium) fishes and humans)
- protective layer is strong; can withstand chlorine and
• Causes short term infection in
stomach acid; can invade the epithelium of GI tract
humans
- Once inside the epithelial cell it releases sporozoites
• Affects the host gastrointestinal and
2. Sporozoites
respiratory epithelial cells
- 1 oocysts=4 sporozoites
3. Trophozoites • Route of infection (fecal oral
- Still in the epithelial cell of the GI tract, sporozoites → contamination)
trophozoite • Causes watery diarrhea; expected
4. Type 1 meront since its inhabits the gastrointestinal
5. Type 2 meront
6. Microgamont Pathogenesis
7. Macrogamont } • Sporozoites adhere to the intestinal mucosa
- Purpose • Cells release cytokines (definitive host cells)
(gametogony • Increased intestinal secretion of sodium and chloride,
stage): to water absorption is inhibited
create oocysts; micro and macrogamete will mate to • Epithelial cells damaged by
produce zygote which will either become thin or thick- o Parasite invasion and multiplication
walled oocysts o T cell mediated villus atrophy; atrophy in
- Thin-walled oocysts can further reinfect the intestinal epithelial cells due to the presence of the parasite
wall crating an autoinfection • May produce up to 10-20 L of watery stools per day
- Thick-walled oocysts can pass through the feces: can
exist in the outside environment until it finds a Symptoms
definitive host again • Appear 2-10 days after infection and last to 30 days
• Diarrhea
• Stomach cramps

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 Coccidian

• Dehydration Wet Mount

• Nausea • Visualization of Oocysts
• Vomiting o 4 to 6 um
• Fever • Bright-Field Microscopy
• Weight loss • Differential Interference Contrast
• Sometimes no symptoms; asymptomatic especially if not (DIC)
immunocompromised • High Sensitivity and Specificity
Cryptosporidium
Innate Immune Response oocyst in wet mount

• White Blood Cells Phagocytize Parasites

o Segmented neutrophils Modified Acid-Fast Stain
o Macrophages • Visualization of Oocytes
o Lymphocytes o Light pink to dark red
o Eosinophils o Can also visualize
sporozoites
Cell Mediated Immune Response • Relatively High Sensitivity and
• CD4 + T cells Specificity
o Early infection • Irregular Staining
• CD8 + T cells o Cause “ghost” oocysts Modified Acid Fast Stain

o Elimination
• CD154 and CD40 Direct Fluorescent Antibody (DFA) Assay
o Stimulate nitric oxide • Use of differential or fluorescent stain
o IFN-γ, IL-12 and visualized under dark-field
o T cell response microscope
o Apoptosis • Fluorescence microscope
• Other Cytokines • “Gold Standard”
• TNF-α, IL-1β, IL-2, IL-4, IL-10, IL-15, etc. o High sensitivity and specificity
• Patients with AIDS • Does not provide archivable stained
o Decreased CD4+ count slide Fluorescent Stain
• These are produced by our body to fight against infection; • Requires special equipment
Auramine Rhodamine

cells that help fight the parasites

Safranin and Trichrome Stains
Humoral Immune Response
• Safranin stain
• IgM
o Oocysts stain a bright red orange
• IgG o Not widely used because oocysts may not stain
• IgA properly
• Antibodies that help fight the parasites • Trichrome stain
• X-linked immunodeficiency o Oocysts may appear unstained
o Mutations in CD154 gene • Lowest sensitivity and specificity among all tests
o Defected IgM cannot mount immune response • Can detect Oocysts, but Cryptosporidium should be
confirmed by diagnostic techniques
How to Diagnose C. hominis
• Cysts of amoeba does not stain in this
• Specimen Source
o Multiple Stool Specimens
o You cannot say that a person is negative of the
parasite with just one sample (3 samples)
• Diagnostic Techniques
o Wet mount
o Modified Acid Fast Stain
o Direct Fluorescent Antibody (DFA) Assay
Safranin Stain
• Detection Methods Trichrome Stain

o Safranin Stain Enzyme Immunoassay (EIA)

o Trichrome Stain
• Detects isolated antigens from a patients sample using
o Enzyme Immunoassay (EIA)
antibodies that are tagged with a color changing enzyme
o Polymerase Chain Reaction (PCR)
• Relatively high sensitivity and
o Rapid Immunochromatographic cartridge Assays
specificity
(RIA)
• Does not involve microscopy
• Post Mortem Lesions
o Gross lesions (not common) • Screens large numbers of
▪ Hyperemia of intestinal mucosa. The specimens
mucosal folds are
markedly thickened, and
there are numerous Polymerase Chain Reaction (PCR)
pinpoint foci of hyperemia • Separates DNA fragments based on size
o Microscopic lesions • 435 bp
▪ Mild to severe villous atrophy • High sensitivity and specificity
▪ Spherical organisms in the brush border
• Histopathology

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 Coccidian

Rapid Immunochromatographic Cartridge Assays
• Detects isolated antigens from sample using antibodies. A
positive test is indicated by a colored bar.
• Variable sensitivity and specificity
• Some assays have been recalled
• The microgametes fertilize the macrogametes to produce
oocysts, which are passed out with feces when the host
cells are sloughed off from the intestinal wall
• The oocysts undergo complete sporulation within 7-12 days
in a warm environment

Treatment
• Nitazoxanide
• Paromomycin
• Azithromycin
• Individuals with AIDS
o anti-retroviral therapy

Prevention
• Boiling and microfiltration of drinking water
• Micro filtration removes oocysts from the water supply
• Low levels of chlorine does not kill cysts
o C. parvum 240,000 times resistant to chlorination
than Giardia
o Chlorine dioxide – ineffective for oocysts

Waterborne Prevention
• Do not swallow recreational water
o Lakes, rivers, streams, untested wells
• Do not drink untreated water
o Travelers and hikers
o Boil water for 15 minutes or use filter rated for
“cyst removal”
o Don’t rely on chemical treatments
• Do not swim with GI infection

Foodborne Prevention
• Wash vegetables with detergent soap
• Proper human/animal waste disposal
• No bare hand contact of ready-to-eat foods
• No food workers with GI illness
o Until 2 weeks after end of diarrhea
• Handwashing-handwashing-handwashing
Cyclospora cayetanensis
• Was originally called a cyanobacterium-like body (CLB)
• Upon careful study, it was found to be a coccidian parasite
• Same life cycle with Cryptosporidium parvum
Pathogenesis and Clinical Manifestations of C.
cayetanensis
• Initial symptoms: malaise and low grade fever
• Chronic intermittent watery diarrhea
• Fatigue, anorexia, weight loss, nausea, vomitting,
abdominal pain, flatulence, bloating, dyspnea
• D-xylose malabsorption
• Infections are usually self-limiting and immunity may result
with repeated infections
Diagnosis of C. cayetanensis
• Direct microscopic examination of fecal smears under high
magnification (400x)
• Looks like fat or oil droplets in wet mount (NSS)
• Use phase contrast microscope to differentiate with fat
globules, RBC, or bubbles

Life Cycle of Cyclospora cayetanensis

• Ingestion of sporulated oocyst, which contains 2 sporocysts
with 2 sporozoites each • Various concentration techniques and acid-fast staining
• Sporozoites invade the epithelial cells of the small intestines (Kinyoun’s stain) are also useful
• Multiple fissions of these sporozoites take place inside the
cells to produce meronts, which contain 8-12 merozoites
during the first generation, and only 4 merozoites in the
second generation
• Some of the merozoites develop into male (micro) and
female (macro) gametes

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 Coccidian

• Oocysts are autofluorescent under fluorescent microscopy,
they appear as blue or green circles depending on the filter
(365-450DM); this technique is useful for screening of C.
cayetanensis
• UV microscopy is also used
• Sone of the merozoites undergo gametogeny to produce
macrogametes and microgametes (sexual stage), which
fuse to form a zygote that eventually matures to form an
unsporulated oocyst
• Sporulation occurs within 48 hours after passage with the
stool

Pathogenesis and Clinical Manifestations of

• Safranin staining and microwave heating are also helpful
Cytoisospora belli
• Self-limiting gastroenteritis (immunocompetent)
• Severe diarrhea and malabsorption (severe infections)
• Low grade fever, anorexia, vomiting, general body malaise,
weight loss and flatulence
• Stools usually contain undigested food, mucus, and
Charcot-Leyden crystals
• Typical watery diarrhea
• Immunocompromised
o May cause severe diarrheal illness
o Mucosal bowel biopsy may show flattened mucosa and
damaged villi
o Infiltration of the lamina propria with lymphocytes,
plasma cells, and eosinophils has been reported

Diagnosis of Cytoisospora belli

• PCR technique has been developed to differentiate • Oocyst may be detected in the feces by
Cyclospora from closely related Eimeria species o direct microscopy
o formalin ether/ethyl acetate concentration (FECT)
Cytoisospora belli o zinc sulfate
• Causative agent of a medical condition o sugar flotation
affecting the small bowel called
cytoisosporiasis Wet mount
• The other known species Isospora
hominis is now taxonomically grouped
under the genus Sarcocystis
• The sporulated oocyst contains 2 sporocysts each
containing 4 sporozoites (infective stage)
• When ingested via contaminated
water or food, the sporozoites
excyst in the small intestine
releasing sporozoites, which • Oocysts can be seen in a fecal smear stained by a modified
penetrate the epithelial cells, Ziehl-Neelsen method, where they stain granular red color
thus starting the asexual stage or against a green background
the schizogonic phase of the life
cycle
• Oocyst are thin walled,
transparent, and ovoid in shape
• Appear as translucent oval
structures; 20-33um by 10-19um
2 oocysts
Life Cycle of Cytoisospora belli
• The sporozoites develop in the epithelial cell to form a
• Phenol auramine (is a differential stain), as well as iodine
schizont, which ruptures the host epithelial cell liberating
staining of the specimen can
merozoites into the lumen
help visualize the organism;
• These merozoites will then infect new epithelial cells and uses dark field microscopy
the process of asexual reproduction in the intestine
• Acid fast stain such as Kinyoun’s
continues
stain or an auramine rhodamine
• This process may continue for weeks or months stain can also be useful

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 Coccidian

• String capsule (Enterotest) and duodenal aspirate • Multiplies by binary fission

examinations may be of value (endodyogeny) → can multiple
even without a complete
nucleus or can produce a
daughter cell without a nucleus
• Can infect phagocytic and
nonphagocytic cells

Toxoplasma gondii Bradyzoites

• Slow growing stage
inside tissue cyst
• Marks the chronic
stage of infection
Toxoplasma gondii • Resistant to low pH
• Different among coccidians since it has intermediate host and digestive
• Toxoplasma is an intracellular parasite, which infects enzymes during
different kinds of nucleated cells including macrophages stomach passage
• Definitive host: • Protective cyst wall is
o Members of Felidae (domestic cats and their relatives) finally dissolved and
• Life cycle: bradyzoites infect the
o Follows a typical coccidian life cycle consisting of tissue and transform into tachyzoites
schizogony, gametogony, and sporogony in the • Bradyzoites are released in the intestine and are highly
intestinal epithelium. infective when digested
o Extraintestinal stages are the asexual stages: • Attached to muscle tissues; when eating raw or improperly
tachyzoites and bradyzoites cooked meat, there is a high risk of ingesting it
• Toxoplasma gondii exist in four forms: • Resistant; not destroyed by stomach acid; in the intestine it
o Tachyzoites will transform into tachyzoites (infective stage)
o Tissue cysts • Chronic stage→ bradyzoites can stay in muscle tissues for
o Bradyzoit long periods of time without showing signs and symptoms
o Oocysts
Life Cycle of Toxoplasma gondii
Toxoplasma gondii oocyst
• Unsporulated Oocyst
o Non-infectious
o Subspherical to spherical
o Takes 1-5 days to sporulate
o Requires oxygen to sporulate
• Sporulated Oocyst
o 10u in diameter
o Subspherical to
ellipsoidal
o Each has ellipsoidal
sporocyst
o Each sporocyst
contains 4
sporozoites
o Infective (remain for months)

Toxoplasma gondii Tachyzoites

• Rapidly growing stage observed in the early stage of
infection (acute phase) habits in the body fluid
• Crescent shaped; length: 4-8um; width: 2-3um
• Organelles, such as rhoptries and micronemes, which are
associated with cell penetration, are found in a short conoid
on the anterior end
• Organelles causes penetration in cells
• A spherical nucleus is found in the posterior end
• Asexual form
• Extraintestinal stage: bradyzoites and tachyzoites
• Cats (Felidae) pass out feces; oocyst will be liberated
• Oocyst can be ingested by animals (sheep, pig) or humans
through fecally contaminated food and water
• When ingested, it can transform to tachyzoites and will go
to the muscle
• Once nasa muscle na, maiingest ng Felidae yung tissue
cyst (bradyzoites)
• When bradyzoites are ingested by Felidae, maliliberate
siya, magiging tachyzoites, then transforms into
trophozoites then merozoites then gametocytes, then
magpapass out ulit ng oocyst
• Oocysts may be ingested directly by the Felidae through
fecally contaminated food or water or ingestion of tissue
cysts

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 Coccidian

Transmission of T. gondii • Molecular diagnosis

• Contaminated food or water by oocysts o PCR
• Ingestion of tachyzoites and bradyzoites in the flesh of • Imaging
infected host o MRI and CT scan for CNS involvement
• Undercooked meat o USG for congenital toxoplasmosis
• Mother of fetus • Others
• Organ transplant (rare) o Animal inoculation
• Blood transfusion (rare) o Skin test of Frenkel
• To confirm diagnosis, we need to use more than one test
Pathogenesis and Clinical Manifestation of T. gondii
• Toxoplasmosis is commonly asymptomatic as long as the
immune system of the patient is functioning well
• Cysts can be found in the brain, skeletal, and heart muscles,
and retina
• Clinical manifestations become apparent when the immune
system is suppressed as in old age, drug-induced
immunosuppression after organ transplantation or in cases
of AIDS
• More often, symptoms appear when there is relapse of
Wet mount DIC
chronic infections as a result of a suppressed immune
system rather than as a response to an acute infection
• Among the immunocompromised patients, the most
common manifestation is encephalitis. Myocarditis and
focal pneumonia has also been reported.

Pathogenesis
• Acquired toxoplasmosis (mild lymphatic inflammation)
o Fever, fatigue, malaise and headache
o Myalgia
o Swollen lymph nodes
o Congenital impacts
• Congenital toxoplasmosis
o Intracerebral calcification
o Chorioretinitis
o Hydrocephaly
o Microcephaly
o Convulsions Sarcocystis spp.
o Mental retardation • Sarcocystis is a genus of intracellular protozoa reported to
o Cardiomegaly infect humans and animal worldwide
• Infection with this parasite is known as Sarcosporidiosis or
Sarcocystosis

S. hominis S. suihominis
Intermediate host Cattle Swine
Definitive host Human Human
Sporocyst Bigger Smaller
Effect Intestinal Intestinal
sarcocystosis sarcocytosis
Laboratory Diagnosis of T. gondii
• Microscopy History
o Tachyzoites and tissue cysts detected in blood, sputum • First reported in 1843 by Miescher as white threadlike cysts
and bone marrow aspirates in the striated muscle of a house mouse
o Stains used: Giemsa, PAS, GMS • Was simply referred to as Miescher’s tubules until 1899,
o Direct fecal smear (routine method) when the name Sarcocystis miescheriana was proposed to
• Serodiagnosis identify the said parasite
- Antibody detection: • There are about 130 recognized species under Sarcocystis
o For detecting IgG antibody: including S. hominis and S. suihominis (humans as
▪ ELISA definitive host)
▪ IFAT
▪ Latex agglutination test
▪ Sabin-Fieldman dye test
o For detecting IgM antibody:
▪ Double sandwich IgM ELISA
▪ IgM-ISAGA
o For detecting IgA antibody:
▪ Double sandwich IgA ELISA
o Antigen detection: ELISA

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 Coccidian

Sarcocystis Forms – Zoites Pathology and Clinical Manifestations

• Banana shaped • Rare, invasive form (lymph
• Pointed anterior end also known nodes, muscle, and the larynx)
as the apical complex which causing vasculitis and myositis
possesses micronemes, • Sarcocystosis has also been
micropores, and rhoptries, and associated with acute fever,
believed to be associated with myalgias, bronchospasm,
the host cell penetration and pruritic rashes,
creation of an intracellular lymphadenopathy,
environment suitable for subcutaneous nodules with
parasite growth and concurrent
development eosinophilia, elevated erythrocytes,
sedimentation rate (increase ESR =
Sarcocystis Forms – Oocyst inflammation) , and elevated creatinine
• Sporulated oocysts and individual sporocysts can be kinase levels
passed out in the feces of an infected definitive host • Intestinal form: nausea, abdominal
• The sporulated oocyst undergoes sporogony creating 2 pain, and diarrhea
sporocysts
• Once sporogony is complete, the oocyst itself undergoes Diagnosis
lysis, releasing the sporocysts into the environment • Stool examination
• Sporocysts of most species measure up to 15-19 um by o Fecal floatation (formalin ether/ethyl acetate and other
8-10 um and contain 4 sedimentation methods)
sporozoites and a discrete
refractile residual body
• Sporocysts are capable of
surviving on the ground and
infecting intermediate hosts

Life Cycle of Sarcocystis spp.

• Same with toxoplasma gondii; definitive host is human but
have an intermediate hosts which are pigs and cows/cattle

• Biopsy of infected muscles

o Sarcocystis of S. hominis are microscopic in muscles
of cattle, whereas those of S. suihominis are
macroscopic in cells of swine
o Sarcocysts are identifiable with hematoxylin and eosin
stain
o Confirmatory staining with the periodic acid-Schiff
(PAS) can be performed as the walls stain positively
• Polymerase Chain Reaction (PCR)
o PCR amplification of the 18S rRNA was demonstrated
to be useful in distinguishing S. hominis, S. fusiformis,
and S. cruzi sarcocysts and oocysts
o The technique makes possible amplification and
identification of species specific gene sequence based
on DNA extracted from as few as seven excreted
sporocysts (the equivalent of 3 ½ oocysts) from freshly
prepared material, or as few as 50 sporocysts from
fecal samples that had been stored in potassium
dichromate (K2Cr2O7) for as long as 6 years

Pader, Juliene Andrei B.