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Medical Protozoology

Experiment 5

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Objectives and Requirements

1.To study morphological structures

of these protozoa.

2. To study laboratory diagnostic met

hods of these protozoa.

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Person-to-person transfer

Trophozoite --active feeding stage,reproductive

stage, pathogenic stage

Cyst------- --- resting stage, infective stage

Entamoeba histolyticaEntamoeba histolytica

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1060μm

binary fission

endoplasm

Morphology 1.Trophozoite

ectoplasm pseudopodium

vesicular nucleus central karyosome

Ingested RBC

chromatin granules

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Entamoeba histolytica

trophozoite

Parasites stained with iron--hematoxylin

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8Micrograph of a trophozoite ingesting a red blood cell derived from its host.

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2. cyst (non-motile, resting stagenon-motile, resting stage))

spherical or round ， 1016μm

cyst wall, 125150 nm

chromatoid body

(rounded end)

glycogen vacuole 1 nucleus cyst

4 nuclei cyst

nuclei

using oil immersion lens

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iodine stained

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Diagnosis1.Stool examination

trophozoite cyst

specimenfeces feces

method direct smear with normal saline

direct smear with iodine stain

diseases amoebic dysenterychronic intestinal amoebiasis or carriers

remarks

1.container must clean2.examined soon after they have been passed.3.select bloody and mucous portion.

4.keep specimen warm.

5.drug using history. 　

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Diagnosis

2. Serologic studies: indirect hemagglutin

ation(IHA), skin tests, ELISA 3. Tissue exa

mination: sigmoidoscopic biopsy, aspiration

4. DNA probe

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Leishmania Donovani

Promastigote – in the digestive tract of sandfly

Amastigote – in the reticuloendothelial system of

man and other vertebrate animals –

visceral leishmaniasis.

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Morphology

Minute round or elliptical body, 2-5µm

No free flagellum Nucleus – deep red, lo

cated at one side Cytoplasm-blue, conta

in: kinetoplast; basal body, rhizoplast

1.Amastigote (1.Amastigote (Leishman-Donovan  body, L.Leishman-Donovan  body, L.D. bodyD. body ））

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Leishmania donovani

无鞭毛体

无鞭毛体

无鞭毛体

stained smear of amastigotes

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amastigote

Bone marrow smear -- Giemsa stain Blue cytoplasm, red nucleus

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Large numbers of amastigote in a M

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Spindle shaped

1420×1.51.8μm in size

Basal body

kinetoplast

flagellum

nucleus

Promastigote

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. Promastigote

in the digestive tract of sandfly

Giemsa stained specimen

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Usually gather together in the medium as a mum

• Spindle shaped with 1 free flagellum

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promastigote rosettepromastigote rosette

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(1)Parasitic diagnosis

1) Punctures:

①Direct smear of materials from

a) bone marrow aspiration with 80~90% of detectio

n rate , the safest procedure, first choice

b) lymph nodes aspiration: positive in 60% of cases

c) Splenic aspiration: It is the most sensitive (90% ~

99.3%) method, but with the risk of life threatening haemo

rrhage, rarely use

4. Diagnosis

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② Animal inoculation

Golden hamster used, for 1-2 months or more,

then impression smears of the spleen .

2) Biopsy: skin

(2) Serologic tests ：

ELISA, IHA, IFA, Skin test

(3) Other molecular methods: PCR ， DNA probe

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Giardia lamblia

Giardia lambilia Giardia lambilia lives in small intestinelives in small intestine

GiardiasisGiardiasis

Diarrhea Diarrhea

“traveler’s diarrhea”

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1. MorphologyNucleus核

Sucking disk

吸盘

Axostyle轴柱

Flagellum鞭毛

(two anterior, posterior, ventral and tail flagella)

Trophozoite

Median body中体

Inverse Pear-shaped

921×525μm

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Ventral surfaceVentral surface Lateral surfaceLateral surface

Tail flagellaTail flagellaventral flagellaventral flagella

Behind side Behind side flagellaflagella

Front side Front side flagellaflagella

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Cyst ：

ball-shaped or oval , thick wall , 2-4 nuclei,

10 14µm ×7.5 9µm

Nucleus

AxostylesFlagella

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4. Diagnosis

(1)Parasitic Diagnosis

1 ） Stool examination ： as same as in E.h

2 ） Duodenal fluid or bile examination:

①Duodenal aspiration( 十二指肠引流 )

② Duodenal capsule technique ( 肠检胶囊法 )

(2)Immunologic test ： mainly Ab detection

(3)Molecular method: DNA probe 、 PCR

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Trichomonas Vaginalis ( 阴道毛滴虫 )

Inhabit --- Female: vagina, urethra

--- Trichomonas vaginitis,

Trichomonas urethritis

--- Male: urethra, prostate gland

--- Trichomonas urethritis

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乒乓 (ping-pong) 感染

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1 nucleus

axostyle

4 anterior flagella

undulating membrane(1 posterior flagellum)

1. Morphology1. Morphology

Trophozoite

colorless

pyriform or oval

1030μm×1015μm

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Trophozoites – Gimsa stain

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4. Diagnosis (1)Parasitic diagnosis ：

1 ） Direct smear

① Saline smear( 生理盐水涂片 )

② Staining

specimen ：Discharge from posterior vaginal fornix, Urine, Secretion of prostate gland

2 ） Cultivation ： specimen above ， 37℃ ， 48h ， microscopic exam

(2)Immunologic test ： Ag detection

(3)DNA probe

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Plasmodium vivax （ P.v ）

P. falciparum （ P.f ）

Plasmodium

.Erythrocytic stage

.Exoerythrocytic stage

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Ring form

Trophozoite Immature schizont

Mature schizont

♀ gametocyte (macrogametocyte ）

♂ gametocyte （ microgametocyte ）

Erythrocytic stage

1 2 3 4

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The entire organism looks like a ruby-ring, which is about 1/3 that diameter of the infected RBC. Ring like plasma with one nucleus at one side.

The ring form ( 环状体）

P. vivax Thin blood film (Giemsa stained)

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mature trophozoites (amoeboid form)

The plasmodium grow with pseudopods, more cyto

plasm and malarial  pigment presented in the plasm

a

Red blood cell enlarged and became pale with Schü

ffner‘s  dots( 薛氏小点 )

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Immature schizonts

2-12 nuclei Schüffner‘s  dots Infected RBC enlarge,

getting pale

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Mature schizonts

12 ～ 24 merozoits ， average 16 The parasite occupied the whole enlarged RBC Malarial pigments gather together

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Mature schizonts

Merozoites lying free.

Malarial pigment is seen as a clump on one side.

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Schizonts

increasingly mature schizonts

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Female gametocyte of P.v

compact nucleus, usually at edge of the parasite scattered pigment granules blue staining cytoplasm The gametocyte is completely filling its host cell an

d the membrane of the erythrocyte is hardly visible

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♀gametocyte

cytoplasm

nucleus

(macrogametocyte)

P.vP.v

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♂gametocyte

Schuffner’s dots

hemozoin

Smaller parasites

large nucleus at the center of the cell

the pink staining cytoplasm (compared to the blue cytoplasm of female gametocytes)

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The P. falciparum ring forms

P.f: large trophozoite and schizont can’t find at the peripheral blood

They are in the internal organ blood capillary

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The ring form

•The ring form of P. f is about 1/5 that diameter of RBC

•some appear to have two nuclei

•appear to have two ring(multiple infection) in one RBC

The ring form P. v

The ring form P. f

•The ring form of P. vivax is about 1/3 that diameter of RBC, which looks like a ruby-ring

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Male gametocyte of P.f

Sausage shape

large compact nucleus at the center of the cell

blue staining cytoplasm malaria pigment diffused

distribution around the nucleus

It is bigger than the red cell ,sometimes the red cell may rupture

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Female gametocyte of P.f

crescent shape

smaller compact nucleus at the center of the cell

blue staining cytoplasm

malaria pigment compacted distribution around the nucleus

It is bigger than the red cell ,sometimes the red cell may rupture

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The procedure of microscope diagnosis

Collecting a finger-prick (or ear-prick) blood sample

Preparing a thick blood smear (in some settings a thin smear is also prepared)

Staining the smear (most frequently with Giemsa)

Examining the smear through a microscope (preferably with a 100X oil-immersion objective) for the presence of malaria parasites

5. Diagnosis(1) Parasitic diagnosis

1 ） Smear of peripheral blood with Giemsa or Wright Staining

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Thin smear--used for determining the species, but easy missing

Thick smear--used for concentrating the parasites, increasing the sensitivity, but hard to read, difficult to distinguish the species of P.

Thick sm

ear

Thin

sme

ar

Label

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•Blood examination should be made at the proper time to increase the positive rate, because the malarial parasites appear more abundantly in the peripheral blood at certain time.

Several to about 10 hours after paroxysm for P.vivax or P.malariae

Usually, blood examination is made at the beginning of the paroxysm for P. falciparum

•In vivaxinfections all asexual stages, as well as the gametocytes, occur in peripheral blood, but in falciparum infections only the rings and gametocytes may be present.

When does the blood be collected?

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Toxoplasma gondii

• It will probably infect almost any mammal.

• It parasitizes in all kinds of cells except RBC.

• Man is its intermediate host --- toxoplasmosis.

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(1)Trophozoite

crescent shaped 4~ 7 × 2 ~4 μm

1. Morphology

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Trophozoites from peritoneal fluid of an infected mouse;

Scanning electron microgragh of toxoplasma trophozoites

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Pseudocyst -- intracellul

ar tachyzoites of Toxopla

sma gondii

pseudocystTachyzoite

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Cyst (bradyzoite, 缓殖子 ), tissue cyst

The cyst have a elastic wall. They contain several to several hundreds trophozoites which called bradyzoite. Under certain conditions, the cyst may be break, and the bradyz

oites invade a new cell and form another cyst

Cyst wall

Bradyzoite

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3 ） Cultivation

4. Diagnosis

(1) Parasitic diagnosis

1 ） Smears ： Specimens

Blood, Sputum, bone marrow, cerebrospinal fluid

-Tissue Biopsy

2 ） Animal Inoculation ： inoculation to laborator

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rats or mice ,for 1w, collecting liquid from the abdome

n

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(2) Serological tests

　　 1 ） Dye test (DT) ---is sensitive and specific 2 ） IHA, IFA, ELISA

(3) PCR

DNA probe

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Exercise l. Draw ring form, trophozoite, schizont

and gametocytes of P. vivax. 2. Draw ring form and gametocytes of

P. falciparum. 3. Draw trophozoite of morphological st

ructures of Entamoeba histolytica, Trichomonas vaginalis, Giardia lamblia, Toxoplasma

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