Experiment 5 Medical Protozoology

Objectives and Requirements To study morphological structures of these protozoa. 2. To study laboratory diagnostic methods of these protozoa.

Entamoeba histolytica Person-to-person transfer Trophozoite --active feeding stage,reproductive stage, pathogenic stage Cyst------- --- resting stage, infective stage

Morphology 1.Trophozoite ectoplasm pseudopodium endoplasm binary fission ectoplasm pseudopodium endoplasm vesicular nucleus central karyosome chromatin granules Ingested RBC

Entamoeba histolytica trophozoite Parasites stained with iron--hematoxylin

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

using oil immersion lens 2. cyst (non-motile, resting stage) using oil immersion lens spherical or round，1016μm cyst wall, 125150 nm chromatoid body (rounded end) 1 nucleus cyst glycogen vacuole nuclei 4 nuclei cyst

iodine stained

Diagnosis 1.Stool examination trophozoite cyst specimen feces method direct smear with normal saline direct smear with iodine stain diseases amoebic dysentery chronic intestinal amoebiasis or carriers remarks 1.container must clean 2.examined soon after they have been passed. 3.select bloody and mucous portion. 4.keep specimen warm. 5.drug using history.　

Diagnosis 2. Serologic studies: indirect hemagglutination(IHA), skin tests, ELISA 3. Tissue examination: sigmoidoscopic biopsy, aspiration 4. DNA probe

Promastigote – in the digestive tract of sandfly Leishmania Donovani Promastigote – in the digestive tract of sandfly Amastigote – in the reticuloendothelial system of man and other vertebrate animals – visceral leishmaniasis.

Morphology 1.Amastigote (Leishman-Donovan body, L.D. body） Minute round or elliptical body, 2-5µm No free flagellum Nucleus – deep red, located at one side Cytoplasm-blue, contain: kinetoplast; basal body, rhizoplast

Leishmania donovani 无鞭毛体 无鞭毛体 stained smear of amastigotes 无鞭毛体

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

Large numbers of amastigote in a M

Promastigote Spindle shaped 1420×1.51.8μm in size Basal body kinetoplast 1420×1.51.8μm in size flagellum nucleus

. Promastigote Giemsa stained specimen in the digestive tract of sandfly Giemsa stained specimen

Spindle shaped with 1 free flagellum Usually gather together in the medium as a mum Spindle shaped with 1 free flagellum

promastigote rosette

4. Diagnosis (1)Parasitic diagnosis 1) Punctures: ①Direct smear of materials from a) bone marrow aspiration with 80~90% of detection 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 haemorrhage, rarely use

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

Giardia lamblia Giardia lambilia lives in small intestine Giardiasis Diarrhea “traveler’s diarrhea”

1. Morphology Trophozoite Inverse Pear-shaped 921×525μm Nucleus 核 Flagellum 鞭毛 (two anterior, posterior, ventral and tail flagella) Median body 中体 Sucking disk 吸盘 Axostyle 轴柱 Trophozoite

Front side flagella Behind side flagella ventral flagella Tail flagella Ventral surface Lateral surface

Cyst： 10  14µm ×7.5  9µm Nucleus ball-shaped or oval , thick wall , 2-4 nuclei, 10  14µm ×7.5  9µm Nucleus Axostyles Flagella

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

Trichomonas Vaginalis (阴道毛滴虫) Inhabit --- Female: vagina, urethra --- Trichomonas vaginitis, Trichomonas urethritis --- Male: urethra, prostate gland --- Trichomonas urethritis

乒乓(ping-pong)感染

undulating 1. Morphology Trophozoite colorless pyriform or oval 4 anterior flagella Trophozoite 1 nucleus colorless pyriform or oval 1030μm×1015μm undulating membrane (1 posterior flagellum) axostyle

Trophozoites – Gimsa stain

(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

Plasmodium Plasmodium vivax （P.v） P. falciparum （P.f） .Exoerythrocytic stage .Erythrocytic stage Malaria

Erythrocytic stage ♀ gametocyte ♂ gametocyte Ring form Trophozoite Immature schizont Mature schizont 1 2 3 4 ♀ gametocyte (macrogametocyte） 5 ♂ gametocyte （microgametocyte） 6

The ring form (环状体） 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. P. vivax Thin blood film (Giemsa stained)

mature trophozoites (amoeboid form) The plasmodium grow with pseudopods, more cytoplasm and malarial  pigment presented in the plasma Red blood cell enlarged and became pale with Schüffner‘s  dots(薛氏小点)

Immature schizonts 2-12 nuclei Schüffner‘s dots Infected RBC enlarge, getting pale

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

Mature schizonts Merozoites lying free. Malarial pigment is seen as a clump on one side.

Schizonts increasingly mature schizonts

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 and the membrane of the erythrocyte is hardly visible

cytoplasm nucleus P.v ♀gametocyte (macrogametocyte)

Schuffner’s dots hemozoin ♂gametocyte Smaller parasites large nucleus at the center of the cell the pink staining cytoplasm (compared to the blue cytoplasm of female gametocytes)

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

The ring form 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 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

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

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

(1) Parasitic diagnosis 1）Smear of peripheral blood with Giemsa or Wright Staining 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

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

When does the blood be collected? 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? 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. Usually, blood examination is made at the beginning of the paroxysm for P. falciparum Several to about 10 hours after paroxysm for P.vivax or P.malariae

Toxoplasma gondii It will probably infect almost any mammal. It parasitizes in all kinds of cells except RBC. Man is its intermediate host --- toxoplasmosis.

1. Morphology (1)Trophozoite crescent shaped 4~ 7 × 2 ~4 μm

Trophozoites from peritoneal fluid of an infected mouse; Scanning electron microgragh of toxoplasma trophozoites

Pseudocyst -- intracellular tachyzoites of Toxoplasma gondii

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 bradyzoites invade a new cell and form another cyst Cyst wall Bradyzoite

4. Diagnosis (1) Parasitic diagnosis 1）Smears： Specimens Blood, Sputum, bone marrow, cerebrospinal fluid -Tissue Biopsy 2）Animal Inoculation：inoculation to laboratory rats or mice ,for 1w, collecting liquid from the abdomen 3） Cultivation

(2) Serological tests 　　 1）Dye test (DT) ---is sensitive and specific 2）IHA, IFA, ELISA (3) PCR DNA probe

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