Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis. It exists in three forms - trophozoite, tissue cyst, and oocyst. Humans can be infected by ingesting undercooked meat containing cysts or by ingesting oocysts from cat feces. Clinical symptoms vary from mild flu-like illness to severe disease in immunocompromised individuals or congenital infection. Treatment involves pyrimethamine and sulfadiazine. Naegleria fowleri is a free-living amoeba that causes PAM, a fatal brain infection. It is found in warm freshwater and soil and enters the body through the nose. A

1. ZL 604:
PROTOZOOLOGY AND HELMINTHOLOGY
PRESENTATION: TISSUE PROTOZOANS
PRESENTER
MR. EMMANUEL KAYUNI
MSc. Ed (Biol)

2. PRESENTATION OUTLINE
This presentation is going to cover the following
areas
1. Coccidia
Toxoplasma gondii causing toxoplasmosis
2. Amoebae
Naeglia fowleri that cause Primary Amebic
Meningoencephalitis (PAM)
Acanthamoeba spp that cause Glanulomatous Amebic
Encephalitis (GAE) and Amebic keratitis

3. COCCIDIA / COCCIDIAN

4. INTRODUCTION
The coccidia are unicellular protozoa and belong to the Phylum
Apicomplexa.
At some stage in their life cycle, they possess a structure called the apical
complex, hence included in Phylum Apicomplexa.
All coccidian show alteration of generation having both schizogony and
sporogony or gametogony.
Many of them also show an alteration of hosts- a definitive host and an
intermediate host.
Prominent example of Coccidian is Toxoplasma gondii
Other Coccidia includes………

5. TOXOPLASMA GONDII
Toxoplasma gondii is an obligate intracellular Coccidian parasite that cause a
disease called Toxoplasmosis in human being.
Etymologically, Toxoplasma is derived from a Greek word Toxon meaning arc
or brow referring to the curved shape of the Trophozoite.
HISTORY AND DISTRIBUTION
Toxoplasma gondii was first described in 1908 by Nicole and manceux in small
North American rodent called gundi.
It was first found in human in 1923 when scientist called Janku observed
T. gondii cyst in the retina of the small boy with Hydrocephalus and
micropthalmia.
Currently, T. gondii is recognized as global protozoan parasite with wider range
of hosts spreading over 200 species of birds, reptiles and mammals including
cat and human being

6. MORPHOLOGY AND ANATOMY
Toxoplasma gondii occurs in three form namely:
a) Trophozoites
b) Tissues cyst and
c) Oocyst.
All the three forms occur in the definitive host such as cats.
Trophozoites and tissue cyst appears in intermediate host which
are other animals including man and birds.
All the three forms are infectious to man.

7. Morphology of Trophozoites
Crescent shaped with one end pointed and other end rounded.
It measure 3-7um length
 Its nucleus is ovoid and situated at the blunt end of the parasite
The electron microscope reveals an apical complex at the pointed
end.
It can invade any nucleated cell and replicate within cytoplasmic
vacuole by the process called endogeny (internal budding).
When the host cell become distended with parasite, it disintegrate
releasing the trophozoites that affect the host cell.
a) TROPHOZOITE

8. Diagram of Trophozoite

9. b) TISSUE CYST (BRADYZOITES)
Tissue cysts are the resting form of the parasite, remain viable in tissue for
several years
Tissue cysts are round or oval, 10-20um in diameter containing numerous
parasites called Bradyzoites.
It is relatively resistant and when the raw or undercooked meat containing the
cysts is eaten, infection occurs.
The cyst wall is disrupted by peptic or tryptic digestion to release Bradyzoites
that initiate infection by invading intestinal epithelial cells.
They reach various tissues and organs through blood and lymphatic
dissemination.
During the chronic stage of the disease Bradyzoites can be found in the brain,
skeletal muscle and other organs.
 Cysts are susceptible to desiccation, freezing, and thawing, and heat above
60°C.

10. c) OOCYST
Oocyst develops only in definitive host - the intestine of the cats and other
felines but not human.
It is oval and measure 10-12um in size.
Each Oocyst is surrounded by a thick resistant wall.
A single cat shed approximately millions of oocyst per day in feces for
about two weeks during primary infection. The freshly passed oocyst is not
infectious.
They undergo sporulation in the soil with formation of two sporocysts,
each containing four sporozoites.
The sporulated oocyst is infective.
Oocyst is very resistant environmental conditions and can remain infective
in soil for about a year.
When the infective Oocyst is ingested, it releases sporozoites in the
intestine, which initiates infection.

11. LIFE CYCLE
T. gondii completes its life cycle in two hosts .
l. Definitive hosts: Cats and other felines, in which both sexual
and asexual cycles take place.
2. Intermediate hosts: Man and other mammals, in which only the
asexual cycle takes place.
T. gondii has involve two types of life cycles:
1. Enteric cycle (Feline cycle)
2. Exoenteric cycle (Human cycle)

12. ENTERIC CYCLE (FELINE CYCLE)
Enteric cycle occurs in cat and other definitive hosts.
Both (gametogony) and (schizogony) occur within the mucosa epithelial cells
of the small intestine of the cat.
Cat acquires infection by ingestion of tissue cysts in the meat of rats and other
animals or by ingestion of oocysts passed in its feces.
The bradyzoites are released in the small intestine and they undergo asexual
multiplication (schizogony) leading to formation of merozoites.
Some merozoites enter extrainrestinal tissues resulting in the formation of
tissue cysts in other organs of the body.
Other merozoites transform into male and female gametocytes and sexual cycle
(gametogony) begins, withthe formation of microgamete and macrogamete.

13. ENTERIC CYCLE CONT………
A macrogamete is fertilized by motile microgamete resulting
in the formation of an oocyst, which passes through maturation
stages (sporulation) in the soil after being excreted from host
through feces.
A mature oocyst containing eight sporozoites is the infective
form which may be ingested by rats or other mammals to repeat
the cycle.

14. EXOENTERIC CYCLE (HUMAN CYCLE)
Exoenteric cycle occurs in humans, mice, rats, sheep, cattle, pigs and birds,
which are the intermediate hosts.
Human acquire infections by different methods
Sporozoites from the oocysts and bradyzoites from the tissue cysts enter into
the intestinal mucosa and multiply asexually and tachyzoites are formed
(endodyogeny).
Tachyzoites continue to multiply and spread locally by lymphatic system and
blood.
Some tachyzoites also spread to distant extraintestinal organs like brain, eye,
liver, spleen, lung and skeletal muscles and form tissue cysts.
The slowly multiplying forms inside the tissue cysts a re known as
bradyzoiles,which remain viable for years.

15. The dormant bradyzoites inside the cyst may be reactivated in
immune suppression causing renewed infection in the host.
Human infection is a dead end for the parasite .
Human toxoplasmosis is a zoonosis.
The full natural cycle is maintained predominantly by cats and mice.
Mice eat materials contaminated with oocysts shed in cat's feces.
Tissue cysts develop in mice.
When such mice are eaten by cats, they get infected and again shed
oocysts in feces.
The Following diagram shows the life cycle of the T.gondii
EXOENTERIC CYCLE CONT…….

16. fig

17. TRANSMISSION AND EPIDEMIOLOGY
According to CDC, In the United States it is estimated that 11% of the
population of 6 years and older have been infected with Toxoplasma.
In various places throughout the world, it has been shown that more than
60% of some populations have been infected with Toxoplasma.
Infection is often highest in areas of the world that have hot, humid climates
and lower altitudes, because the oocysts survive better in these types of
environments.
Toxoplasmosis is not passed from person-to-person, except in instances of
mother-to-child (congenital) transmission and blood transfusion or organ
transplantation.
People typically become infected by three principal routes of transmission:
i. Foodborne
ii. Animal-to-human (zoonotic)
iii. Mother-to-child (congenital)

18. PATHOLOGY AND MANIFESTATION
 The outcome of Toxoplasma infection depends on the immune status of
the infected person.
 Most human infections are asymptomatic.
 Clinical toxoplasmosis may be congenital or acquired.
Congenital Toxoplasmosis
 Congenital toxoplasmosis results when T.gondii is transmitted
transplacentally from mother to fetus.
 Most infected newborns are asymptomatic at birth and may remain so
throughout.
 Some (0.3-1 %) develop clinical manifestations of toxoplasmosis within
weeks, months and even years after birth.

19. The manifestations of congenital toxoplasmosis include chorioretinitis,
cerebral calcifications, convulsions, strabismus, deafness, blindness, mental
retardation, microcephaly and hydrocephalus.
A few children are born with manifestations of acute toxoplasmosis, which
may include fever, jaundice, petechial rashes, microphthalmia, cataract,
glaucoma, lymphadenopathy, hepatosplenomegaly, myocarditis, cerebral
calcifications and chorioretinitis.
• Acquired Toxoplasmosis
Infection acquired postnatally is mostly asymptomatic.
The most common manifestation of acute acquired toxoplasmosis is
lymphadenopathy; the cervical lymph nodes being most frequently affected.
 Fever, headache, myalgia and splenomegaly are often present. the illness
may resemble mild flue and is selflimited, although the lymphadenopathy
may persist.

22. TREATMENT
Congenital Toxoplasmosis
Neonates with congenital infection are treated with the oral pyrimethamine
(1 mg/ kg) daily and sulfadiazine (100 mg/kg) with folinic acid for 1 year.
lmmunocompetent Patients
Immunologically competent adults and older children, who have only
lymphadenopathy, do not require specific therapy unless they have persistent
severe symptoms.
Patients with ocular toxoplasmosis are treated for 1 month with
pyrimethamine plus either sulfadiazine or clindamycin (600 mg QID).
 Folinic acid should be administered concomitantly to avoid marrow
suppressive effect of pyrimethamine.

23.  Obligate intracellular parasite.
Exists in three forms: (1) trophozoite, (2) tissue cyst, and (3) oocyst.
 Definitive host: Cat family (enteric cycle) and Intermediate host: Human
(exoenteric cycle).
•Human infection occurs by ingestion of food containing oocyst and tissue cyst.
 Congenital infection can also occur.
Clinical features: Acute encephalopathy, fever, chorioretinitis,
lymphadenopathy, myocarditis, hepatosplenomegaly.
• Diagnosis: By demonstration of parasite in tissue specimen, ELISA, IFAT,
Sabin-Feldman dye test, lgM-ISAGA.
 Treatment: Congenital infection is treated with pyrimethamine and
sulfadiazine.
SUMMARY ABOUT T.GONDII

24. AMOEBAE / AMEBA

25. INTRODCTION
Amoebae are structurally simple protozoans which have no
fixed shape.
Etymologically, the word ameba is derived from the Greek
word "amibe" meaning change.
They are classified under
Phylum: Sarcomastigophora,
Subphylum: Sarcodina,
Superclass: Rhizopoda and
Order: Amebida.

26. CHARACTERISTICS OF AMOEBAE
The cytoplasm of ameba is bounded by a membrane and can be differentiated
into an outer ectoplasm and inner endoplasm.
Pseudopodia are formed by the ameba by thrusting out ectoplasm, followed
by endoplasm.
Reproduction occurs by fission and budding.
Cyst is formed in unfavorable conditions and is usually the infective form for
vertebrate host (e.g. Entamoeba histolytica).
Amebae are classified as either free-living or intestinal amebae
The parasitic amebae inhabit the alimentary canal
Few of the free-living amebae occasionally act as human pathogens
producing meningoencephalitis and other infections, e.g. Naegleria and
Acanthamoeba

27. CLASSIFICATION OF AMOEBAE
Intestinal amebae includes
Entamoeba histolytica
Entamoeba dispar
Entamoeba coli
Entamoeba polecki
Entamoeba hartmanni
Entamoeba gingivalis
Endolimax nana
Free-living amebae included
Naegleria fowleri
Acanthamoeba spp.
 Among fore mentioned organism, only two will be discussed in details as
they have direct effect on body tissues

28. NAEGLERIA FOWLERI
Naegleria fowleri is a parasitic free living protozoa that causes the
disease Primary Amebic Meningoencephalitis (PAM), a brain infection that
leads to destruction of brain tissue.
It is also called brain eating Amoeba
HISTORY AND DISTRIBUTION
N. fowleri is named after Malcolm Fowler, who along with Carter described it
first from Australia in 1965.
N. fowleri is a heat-loving (thermophilic) ameba that thrives in warm water at
low oxygen tension and is commonly found in warm places, commonly found
in warm freshwater (e.g. lakes, rivers, and springs) and soil.
It is worldwide in distribution.
Though, it is frequently reported in US and India

29. N. fowleri occurs in three form each having specific shape and
characteristics as follows.
1. Cyst
2. Ameboid trophozoite form
3. Flagellate trophozoite form.
MORPHOLOGY AND ANATOMY

30. TROPHOZOITE FORM
The trophozoites occur in two forms, ameboid and flagellate.
Amoboid form: The ameboid form is about 10-20 µm, showing rounded
pseudopodia (lobopodia), a spherical nucleus with big endosome and pulsating
vacuoles.
When viewed wiith electron microscopy, vacuoles appear to be densely
granular in contrast to highly vacuolated body of ameba and are called as
amebostomes.
They are used for engulfing RBCs and white blood cells (WBCs) and vary in
number, depending on the species.
Ameboid form is the feeding, growing, and replicating form of the parasite,
seen on the surface of vegetation, mud and water.
It is the invasive stage of the parasite and the infective form of the parasite.

31. Flagellate form:
The biflagellate form occurs when trophozoites are transferred to
distilled water .
This transformation of trophozoites co biflagellate pear shaped
form occurs within a minute.
The flagellate can revert to the ameboid form,
Hence N. fowleri is classified as ameboflagellate.

32. Cyst stage:
Trophozoites encyst due co unfavorable conditions like food
deprivation, desiccation, cold temperature, etc.
The cyst is spherical 7-10 µm in diameter and has a smooth double
wall.
They are the resting or the dormant form and can resist unfavorable
conditions, such as drying,
The cyst can withstand moderate heat ( 45°C), but die at chlorine
levels of 2 ppm and salinity of 0.7%.
Cysts and flagellate forms of N. fowleri have never been found in
tissues of cerebrospinal fluid {CSF).

33. LIFE
CYCLE
Naegleria fowleri has 3 stages in its life cycle: cyst (1), trophozoite (2), and
flagellate (3).
The only infective stage of the ameba is the trophozoite.
Trophozoites are 10-35 µm long with a granular appearance and a single
nucleus.
The trophozoites replicate by binary division during which the nuclear
membrane remains intact (a process called promitosis) (4).
Trophozoites infect humans or animals by penetrating the nasal tissue (5) and
migrating to the brain (6) via the olfactory nerves causing primary amebic
meningoencephalitis (PAM).

34. Fig. life cycle of N.fowleri

35. The disease do not spread
from person to person
The parasite enter a man
either by swimming or
diving in warm stagnant
fresh water
Or by drinking unsafe
water (un boiled water)
TRANSMISSION

36. PATHOLOGY AND MANIFESTATION
Human infection comes from water containing the amebae and usually
follows swimming or diving in ponds.
The amebae invade the nasal mucosa and pass through the olfactory nerve
branches in the cribriform plate into the meninges, and brain to initiate an
acute purulent meningitis and encephalitis, called as primary amebic
meningoencephalitis (PAM).
The incubation period varies from 2 days to 2 weeks.
In the incubation period, the patient experiences anosmia.
The disease advances rapidly, causing fever, headache, vomiting, stiff neck,
ataxia, seizure and coma.
The disease almost always ends fatally within a week (average 5 days).

37. DIAGNOSIS
The diagnosis of PAM is based on the finding of motile Naegleria trophozoites
in wet mounts of freshly obtained in CSF examination
The CSF is cloudy to purulent, with prominent neutrophilic leukocytosis,
elevated protein and low glucose, resembling pyogenic meningitis.
Wet film examination of CSF may show trophozoites.
Cysts are not found in CSF or brain.
At autopsy, trophozoites can be demonstrated in brain histologically by
immunofluorescent staining.
Culture: N. fowleri can be grown in several kinds of liquid axenic media or
non nutrient agar plates coated with Escherichia coli. Both trophozoites and
cysts occur in culture.
Molecular diagnosis: Newer tests based on PCR technology are being
developed.

38. TREATMENT
PAM can be treated by drug called amphotericin B.
It can also be instilled directly into the brain.
 Treatment combining miconazole and sulfadiazine has shown
limited success, only when administered early.
More than 9.5% cases of PAM are fatal despite of treatment

39. ACANTHAMOEBA SPECIES
A. culbertsoni {formerly, Hartmannella culbertsoni) is the
species most often responsible for human infection but other
species like A. polyphagia, A. castellanii and A. astromyx have
also been reported.
DISTRIBUTION
This is an opportunistic protozoan pathogen found worldwide
in the environment esp in water and soil.
Approximately, 400 cases have been reported worldwide.

40. DISEASES CAUSED BY ACANTHAMOEBA
SP
Acanthamoeba keratitis: An infection of the eye that typically occurs in
healthy persons and develops from the entry of the amebic cyst through
abrasions on the cornea.
Granulomatous amebic encephalitis: It is a serious infection of the
brain and spinal cord that typically occurs in persons with a compromised
immune system.
NB, In immunocompromised states like acquired immunodeficiency
syndrome (AIDS), a widespread infection can affect skin lungs, sinuses,
and other organs independently or in combination.

41. MORPHOLOGY
Acanthamoeba exists as active trophozoite form and a resistant
cystic form.
 The trophozoite is large, 20-50 μm in size and characterized by spin
e-like pseudopodia (acanthopodia).
 It differs from Naegleria in not having a flagellate stage and in
forming cysts in tissues
The polygonal double-walled cysts are highly resistant.
The cysts are present in all types of environment, all over the world.

42. LIFE CYCLE OF A. CULBERTSONI
Unlike N. fowleri, Acanthamoeba has only two stages, cysts (1) and trophozoites
(2), in its life cycle. No flagellated stage exists as part of the life cycle.
The trophozoites replicate by mitosis (nuclear membrane does not remain intact)
(3). The trophozoites are the infective forms, although both cysts and trophozoites
gain entry into the body (4) through various means.
Entry can occur through the eye (5), the nasal passages to the lower respiratory
tract (6), or ulcerated or broken skin (7).
When Acanthamoeba spp. enters the eye it can cause severe keratitis in otherwise
healthy individuals, particularly contact lens users (8).
When it enters the respiratory system or through the skin, it can invade the central
nervous system by hematogenous dissemination causing granulomatous amebic
encephalitis (GAE) (9) or disseminated disease (10), or skin lesions (11) in
individuals with compromised immune systems.
Acanthamoeba spp. cysts and trophozoites are found in tissue.

44. Infection usually occurs in patients with immunodeficiency, diabetes,
malignancies, malnutrition, systemic lupus erythematosus (SLE), or
alcoholism.
The parasite spreads hematogenously into central nervous system.
Subsequent invasion of the connective tissue and induction of pro-
inflammatory responses lead to neuronal damage that can be fatal within days.
 A postmortem biopsy reveals severe edema and hemorrhagic necrosis.
A. keratitis is manifested through the eye pain, eye redness, blurred vision,
Sensitivity to light, ensation of something in the eye and excessive tearing.
GAE is manifested through Confusion, headache, and seizures are common.
People may have a low-grade fever, blurred vision, changes in personality,
and problems with speaking, coordination, or vision.
One side of the body or face may become paralyze
PATHOLOGY AND
MANIFESTATION

45. DIAGNOSIS
Diagnosis of amebic keratitis is made by demonstration of he cyst in corneal
scrapings by wet mount, histology and culture.
Growth can be obtained from corneal scrapings inoculated on nutrient agar,
overlaid with live or dead Escherichia coli and incubated at 30°C.
Rapid diagnosis can be made by identification of ameba or cyst in corneal
scraping by fluorescent microscopy using calcofluor white staining and IFA test
([FAT) procedure.
Diagnosis of GAE is made by demonstration of trophozoites and cysts in brain
biopsy, culture and immunofluorescence microscopy using monoclonal
antibodies.
Cerebrospinal fluid shows lymphocytic pleocytosis, slightly elevated protein
levels, and normal or slightly decreased glucose levels.
Computed tomography scan o f brain provides inconclusive findings.

46. TREATMENT
In Acanthamoeba keralitis, current therapy
involves topical administration of
biguanide or chlorhexidine with or without
diamidine agent.
In severe cases, where vision is threatened,
penetrating keratoplasty can be done.
No effective treatment is available for
"GAE
Multidrug combinations including
pentamidine, sulfadiazine, rifampicin and
fluconazole are being used with limited
success.

47. THANK YOU
THE END