Protozoa Parasitology Labs.

Second Grade

University Of Baghdad College of science

Department of Biology

Protozoa Parasitology Lab Second Course – Feb. 2019 – May. 2019

Course Title: Parasitology

Lab Hours: 2 Hours

Instructor: Dr. Hayder Zuhair, Dr. Entsar Saheb, Dr. Harith Saeed, Dr. Amjed Qays, Dr. Rasha Hussein Israa Salim, Batool Kathim, Rula Raad

Grading:

Final grade for the Lab work will be based on the following:

Dates Total Points % of Final Grade

Lab Quizzes Each week 60 6%

First exam 40 7%

Second exam 40 7%

Total 20%

Student participation:

Attendance is expected in all classes. Students are expected to come prepared for class, and to contribute to and participate in all class discussion. Any absences must be justified and explained to the instructor in advance in order to make up any of the graded material.

Parasitic Protozoa

1-Sarcodina (Amoebae): Entameba histolytica, Entameba coli,

Endolimax nana, Iodameba butchlii, Entamoeba gingivalis

2- Ciliates: Balantidium coli

3-Mastigophora (Flagellates): Dientameba fragilis, Giardia lamblia,

Trichomonas vaginalis, Trypanosoma brucci, T. cruzi, Leishmania

donovani, L. tropica

4- Sporozoa: Plasmodium falciparum, Toxoplasma gondi,

Cryptosporidum parvum

Lab 1

Lab 1

General objectives of our Lab.

1- Scientific name and common name

• He is known as the father of modern taxonomy,

Many of his writings were in Latin

Carl Linnaeus

2- Identification: Shape – Nucleus – Cyst and/or Trophozoite – locomotion- Vacuoles.

3- Pathogenicity: - Location of parasite

- Name of Disease

- Symptoms

General objectives of our Lab.

Protozoa general characteristics:

1- Unicellular eukaryotes.

2- Have organelles that often function similarity to

organs and systems of multicellular organisms

3- locomotion: pseudopodia, flagella or cilia

4- Reproduction: Asexual (binary fission or multiple

fission) Sexual (conjugation or syngamy).

Phylum: Protozoa

• Super class: Sarcodina

• Super class: Flagellata

• Super class: Ciliata

• Super class: Sporozoa

Taxonomy

Protozoa Super Class: Sardcodina

Types of nuclei in Protozoa Karyosome or endosome

Chromatin granules

Class Sarcodina- Entamoeba histolytica • Trophozoite 10-60 µM Cyst 10-30 µM diameter.

• Found in the large intestine, cecum and terminal ileum.

• Endoplasm / ectoplasm.

• Endosome of nucleus is small and centric, peripheral

chromatin is identical.

• Pseudopodia are thin.

• Food vacuoles usually contain RBC.

• Cysts is the infective stage and has 4 nuclei, chromatoid

bodies are thick.

• Pathogenic, causes amebic ulcer/ amoebiasis/ amoebic

dysentery -blood in stool, complications- liver abscess.

Entamoeba histolytica

Trophozoite of E. histolytica Cyst of E. histolytica A:Nuclei B:Chromatoid bar

Entamoeba histolytica

Amoebic ulcer caused by E. histolytica

Cup or Flask shape

Class Sarcodina- Entamoeba coli

• Trophozoite (15-50 µM diameter) and Cyst.

• Found in the large intestine, colon.

• Endoplasm = ectoplasm.

• Endosome of nucleus is acentric, peripheral chromatin is non-

identical.

• Pseudopodia usually thick.

• Food vacuoles do not contain RBC.

• Cysts has 8 nuclei, chromatoid bodies are thin.

• Non-pathogenic, commensal.

Class Sarcodina- Entamoeba coli

Endosome of nucleus is acentric, peripheral

chromatin is non-identical

nuclei

Chromatoid bar

Lab 2

Lab 2

Class Sarcodina- Entamoeba gingivalis

• Only trophozoite, 10-20 µM diameter.

• Found in mouth, in teeth roots and gum, it is found

with 95% of gum diseases.

• Endosome is large, centric or acentric, peripheral

chromatin is non-identical.

• Highly vacuolated.

• Non-pathogenic.

Class Sarcodina- Entamoeba gingivalis Endosome is large, centric or acentric,

peripheral chromatin is non-identical

Entamoeba gingivalis

Class Sarcodina- Iodamoeba butschlii

• Trophozoite 8-20 µM, cyst 5-20 µM diameter.

• Found in large intestine.

• Endoplasm = ectoplasm.

• Large endosome surrounded by chromatic

globules.

• Non-pathogenic (commensal)

• Cyst has only one nucleus.

Class Sarcodina- Iodamoeba butschlii

Large endosome surrounded by chromatic globules, Cyst has only one nucleus

Class Sarcodina- Endolemax nana

• Very small – Trophozoite 6-12 µM

• Cyst 5-8 µM

• Live in large intestine, non-pathogenic

• Ectoplasm / Endoplasm

• Large endosome with no peripheral

chromatin.

Class Sarcodina- Endolemax nana

Large endosome with no peripheral chromatin

Trophozoite

Cyst – 4 nuclie غير الكروماتين )

(موجود

2- SUPERCLASS: Ciliata

(CILIOPHORA)

Balantidium coli

• Location: Ileum, colon and rectum

• Pathogenic: Balantidiasis

• Two forms: Trophozoit + Cyst

Cytostome

Cilia

macronecleus

Contractile vacuole

Trophozoite

• Oval shape

• Course cilia

• Contractile vacuoles

• Kidney shape macronucleous

Life cycle of Balantidium coli

Lab 3

Flagellata

INTESTINAL FLAGELATES

& Genital tract flagellates

HAEMOFLAGELATES

BLOOD& TISSUE FLAGELLATES

Lab 3

3- SUPERCLASS: (Flagellata)

MASTIGOPHORA

Include all protozoa that have one flagellum or more in

trophozoite stage

Dientamoeba fragilis

• Trophozoite 5-12 µM

• Found in large intestine

• Ectoplasm / endoplasm.

• Endosome consist of aggregated chromatin granules.

• 60% has 2 nuclei.

INTESTINAL FLAGELATES

Dientamoeba fragilis

Endosome consist of aggregated chromatin granules.

60% has 2 nuclei.

Giardia lamblia

• Location: Small intestine (Duodenum)

• Pathogenic: Giardiasis or lambiasis

• Two forms: Trophozoite + Cyst

INTESTINAL FLAGELATES

Trophozoit of Giardia lamblia Sucking disc Axostyle

Parapasal body

Trophozoite

• Bilaterally symmetrical

• Pear shape

• Broad round anterior and tapering

posterior. Convex dorsal surface and

concave sucking disc. Flat ventral

surface

• Two nuclei with large central

karyosomes

• Two axostyles, two parabasal bodies

• Four pairs of flagella

Cyst of Giardia lamblia

Cyst

• Oval in shape

• They have smooth well defined wall

• Contains four nuclei

• Contain parabasal bodies and flagellates

Trophozoit of Trichomonas

4 flagella 4 flagella

1 flagellum 1 flagellum

quiescent formفي دور السكون الدور المغتذي يتم انتقال العدوى بواسطة *

الذي يتكون بتكور الدور المغتذي الذي يصبح عديم الحزكة

Location: Vagina, Urethra

Pathogenic: Vaginitis, Urithritis

One form: Trophozoite

Genital tract flagellates

Lab 4

Lab 4

Blood and tissue flagellates or

Haemoflagellates

Haemoflagellates Forms

Leishmania tropica

Location:- Cutaneous

Pathogenic:- Baghdad boil or oriental sore

Form:- Amastigote + Promastigote

Vector:- Phlebotomus (Sand fly)

Leishmania donovani

Location:- visceral

Pathogenic:- Dum – Dum fever or kalazar

Form:- Amastigote + Promastigote

Vector:- Phlebotomus

Leishmania braziliensis

Location:-mucocutaneous

Pathogenic:- Uta, Chiclero ulcer, Espundia

Forms:- Amastigote + Promastigote

Vector:- Lutzomyia

In monocyte or phagocytic cells

macrophage

Nucleus of

macrophage

Amastigote of Leishmania

In the invertebrate vector or

the culture

Simple binary

fission

Leishmania promastigote

Life cycle of Leishmania spp.

Amastigotes phagocytized by macrophage

Macrophage

nucleus

Amastigotes

Nucleus

In vitro infection of macrophage with Leishmania amastigotes, DAPI

stain (Hayder Z. Ali et al. 2012)

Oriental sore or Baghdad boil

Case infected with kalazar

Chiclero ulcer or Uta

Lab 5

Lab 5

Trypanosoma gambiense

Location:- In plasma of blood

Pathogenic:- Mid and west African sleeping sickness

Forms:- Epimastigote and trypomastigote

Vector:- Tse-tse or Glossina palpalis

Anterior station development

Trypanosoma gambiense elongated spindle shape

trypomastigote

Trypanosoma cruzi

Location:- Muscles of heart and nervous system muscles

Pathogenic:-South America trypanosomiasis (Chaga’s

disease)

Forms:- Amastigote+ promastigote +

epimastigote + trypomastigote

Vector:- Triatomidae (Kissing bug)

Posterior station development

Trypanosoma cruzi

U or C shape Trypomastigote

Kinetoblast (big)

Lab 5

Lab 6

4- SUPERCLASS: Sporozoa

Lab 6

Sporozoa

o All of the species belong to this group are parasitic

o Produce spores, oocyst

o No clear organs for movement or locomotion

o Reproduction:

Asexual: Binary fission, Multiple fission, Endodyogeny

Sexual: Anisogametes, Isogametes

♀gametes ♂gametes

Zygote

Ookinate

Oocyst

sporocyst

spores

Sexual Reproduction

of humans caused by infectious diseaseborne -mosquitois a Malaria

Plasmodiumof the genus protists eukaryotic

and subtropical regions, including much of tropicalIt is widespread in

Americasand the Asia, Africa

red The disease results from multiplication of malaria parasites within

, headacheand fever, causing symptoms typically include blood cells

, and deathcomain severe cases progressing to

Four species of Plasmodium can infect humans:

Plasmodium falciparum

vivaxPlasmodium

ovalePlasmodium

malariaePlasmodium

Malaria

Plasmodium

• Include the sporozoa that have two replication cycle:

Schizogony (asexual)

• In the vertebrate host

Sporogony (sexual)

• In the invertebrate host

P.falciparum P.ovale P.malariae P.vivax

Malignant malaria Ovale tertian

malaria

Quatrain malaria Tertian malaria

36-48 h 48 h 72 h 48 h

Life cycle of Plasmodium in mammals (asexual)

P. falciparum P. ovale P. malariae P. vivax

Scanty cytoplasm and

small vacuoles,

multiple rings

common

Resembles that of P.

vivax, ring larger in

size and thicker.

smaller in size than

P. vivax, occupied

1/6 of the RBC,

heavy chromatin

dot, vacuole may

appeared filled in

pigment

Delicate cytoplasmic

ring measuring 1/3

RBC diameter, single

chromatin dot, ring

surround a vacuole

Ring form (early trophozoites)

Amoeboid stage (developing trophozoite)

P. falciparum P. ovale P. malariae P. vivax

Fine pigment

granules are

common, only

detected in sever

infection

Amoeboid

tendencies not as

evident as in P.

vivax

Solid cytoplasm,

band form

Irregular amoeboid

appearance. ring

remnant is common,

Mature Schizont P. falciparum P. ovale P. malariae P. vivax

Smaller merozoites

single pigment

mass , an average

of 8-26 merozoites

Meriozoite larger

than P.malariae

,irregular rosset ,

usually 8

Schizont smaller

but merozoites

larger an average

of 6-12

merozoites

Parasites occupy ¾

of RBCs, rossete of

an average of 12-24

merozoites

Microgametocyte P.falciparum P.ovale P.malariae P.vivax

Usually sausage

shape, chromatin

diffused

Similar to P.vivax

but smaller, never

abundant

Similar to P. vivax

but smaller, less

numerous

Spherical, compact,

no vacuole, large

nucleus, cytoplasm

stains light blue

Macrogametocyte P. falciparum P. ovale P. malariae P. vivax

Crescent often

longer and more

slender , chromatin

central , cytoplasm

stain darker blue

Similar to P. vivax

but smaller

Similar to P. vivax

but smaller and

less numerous

spherical , compact

,acentric chromatin

mass

Exflagellation: formation of flagelliform

microgametes from a microgametocyte

in some sporozoa

Zygote: the diploid cell that results from

the fusion of two gametes

Ookinete: The motile stage of the zygote

preceding the oocyst stage

Life cycle of Plasmodium in Mosquito (Sexual)

Oocyst : The resistant stage of the life

cycle of some sporozoal parasites. It

contains a zygote and under

appropriate conditions sporulates to

become a mature infective oocyst.

Sporozoite: Spore formed after

fertilization; a sickle shaped nucleated

germ formed by division of the

protoplasm of a spore of

an organism during sporogony.

Life cycle of Plasmodium in Mosquito

Lab 7

Lab 7

Toxoplasma gondii Final host:- members of family Felidae (domestic

cats and their relatives).

Intermediate host:- many warm-blooded animals, including humans.

Forms:- Oocyst (contains two sporocysts, each one contain four sporozoites

Tackyzoite, Bradizoite

Diagnosis:- is based on serology(IgM and IgG ElISA Test) and on

histologic examination of tissue.

Oocyst Tackyzoite

Toxoplasma gondii Life Cycle

T. gondii primarily exists in three forms: oocysts, tachyzoites, and bradyzoites.

Oocysts are only produced in the definitive host, members of the family Felidae.

When passed in feces and then ingested, the oocysts can infect humans and other

intermediate hosts. They develop into tachyzoites, which are the rapidly

multiplying trophozoite form of T. gondii. They divide rapidly in cells, causing

tissue destruction and spreading the infection. Tachyzoites in pregnant women are

capable of infecting the fetus. Eventually tachyzoites localize to muscle tissues and

the CNS where they convert to tissue cysts, or bradyzoites. This is thought to be a

response to the host immune reaction. Ingestion of cysts in contaminated meat is

also a source of infection, as bradyzoites transform back into tachyzoites upon

entering a new host

Toxoplasma life cycle

Eimeria

Host:- Small and large intestine of herbivores and carnivores

cattle, poultry, and smaller ruminants including sheep and goats.

Oocyst:- The sporulated oocyst is said to be tetrasporic meaning

it contains four sporocysts, each one contain two sporozoites

Disease:- Coccidiosis (diarrheal disease).

Diagnosis:- Oocyst in faeces.

Eimeria Oocyst

Eimeria life cycle

Eimeria species are

considered to be

monoxenous because

the life cycle is

completed within a

single host.

Cryptosporedium

Host:- Humans and a wide range of animals including

mammals, birds ,reptiles ,amphibians and fish.

Oocyst:- Sporulated oocysts, containing 4 sporozoites

Disease:- Cryptosporidiosis

Diagnosis:- Oocyst in faeces.

Cryptosporedium Oocyst

Cryptosporedium life cycle

Laboratory diagnosis and protozoan parasitic infection

The laboratory diagnosis is important in order to know

which parasite causes the disease, so it helps and provides

the clinical diagnosis. The responsibility for accurate

diagnosis requires special training, skill, and ability to

diagnose the parasite and to distinguish it from impurities,

and foreign matter, as well as the availability of essential

equipment and materials.

Lab 7 Laboratory diagnosis and protozoan parasitic infection

Specimen: it depends on the parasite habitat and its

distribution:

Stool

Blood

Urine

Sputum

Other body fluid

Fecal specimen: Immediately have to examine.

Liquid specimen should examining after 30 min. of passage

Soft (semi-formed) specimen after 1 hr.

Formed specimen after 24 hr. from passage.

Collection of specimen

Collecting Stool Samples for Testing

Preparation and Procedure

• Stool should be collected into a clean, dry container.

• Specimens must not be contaminated with urine or

water, so collection from toilet bowls is unacceptable.

• Print the patient name, date and time on the container

with the sample.

• Deliver the specimen to the lab immediately.

Direct examination:

This test is used to find parasites that cause diarrhea like Giardia

lamblia, loose or watery stools, cramping, flatulence (gas) and other

abdominal illness. CDC recommends that three or more stool samples,

collected on separate days, be examined. This test looks for ova (eggs)

or the parasite. You must put your stool specimens into special

containers. Specimens should be refrigerated, but not frozen, until

delivered to the lab. In the lab special stains or those special tests are

used to look for parasites. The stool examination consists of:

Macroscopy: Color, Consistency, Composition

Microscopic: Permanent, Temporary

Direct saline smear (1 drop of saline on slide + small amount of stool

take it by wooden stick).

Iodine smear (1 drop of Iodine solution on slide + small amount of

stool take it by wooden stick).

Concentration technique: separate parasites from fecal debris and

increase the chances of detecting parasitic organisms when these are in

small numbers. They are divided into flotation techniques and

sedimentation techniques.

1- Floatation: (most frequently used: zinc sulfate or Sheather’s sugar)

use solutions which have higher specific gravity than the organisms to

be floated so that the organisms rise to the top and the debris sinks to

the bottom. The main advantage of this technique is to produce a

cleaner material than the sedimentation technique. The disadvantages of

most flotation techniques are that the walls of eggs and cysts will often

collapse, thus hindering identification. Also, some parasite eggs do not

float.

• Liquefy small amount (1 gram) fecal with 5 ml of normal saline 9%.

• Apply the suspension with two layers of wet gauze.

• Collect the filtered suspension in centrifuge tubes

• Put the tubes in the centrifuge (1500 rpm for 1 min)

• Discard supernatant add 5 ml of N.S. and re-centrifuge suspension.

• Discard supernatant and add ZnSo4 to deposit (5 ml) and centrifuge.

2- Sedimentation: • Mix the specimen well.

• Strain 5ml of the fecal suspension (more or less depending on its consistency)

through wetted cheesecloth-type gauze placed over a disposable paper funnel into a

15 ml conical centrifuge tube. (Conical paper cups with the tips cut off are

sufficient).

• Add 0.85% saline or 10% formalin through the debris on the gauze to bring the

volume in the centrifuge tube to 15 ml. Distilled water may be used;

• Centrifuge at 500 × g for 10 minutes.

• Decant supernatant. Add 10 ml of 10% formalin to the sediment and mix

thoroughly with wooden applicator sticks.

• Add 4 ml of ethyl acetate, stopper the tube, and shake vigorously in an inverted

position for 30 seconds. Carefully remove the stopper.

• Centrifuge at 500 × g for 10 minutes.

• Free the plug of debris from the top of the tube by ringing the sides with an

applicator stick. Decant the top layers of supernatant.

• Use a cotton-tipped applicator to remove debris from sides of the centrifuge tube.

• Add several drops of 10% formalin to re-suspend the concentrated specimen.

Proceed with applicable testing.

Blood tests

Some, but not all, parasitic infections can be detected by

testing blood. Blood tests look for a specific parasite

infection; there is no blood test that will look for all parasitic

infections. There are two general kinds of blood tests that

the doctor may order:

1- Serology This test is used to look for antibodies or for

parasite antigens produced when the body is infected with a

parasite like: (Toxoplasma gondii) and the immune system

is trying to fight off the invader. This test is done in the lab

by different methods:

Latex test

ELISA test

Immunofloresent test

2- Blood smear This test is used to look for parasites that are found

in the blood. By looking at a blood smear under a microscope,

parasitic diseases can be diagnosed.

A- Thick and Thin Blood Smears for Malaria

Thick and thin blood smears are used to determine whether you have

malaria (Plasmodium spp.). If one test is negative and no parasites

are found, you will have repeated blood smears every 8 hours for a

couple of days to confirm that there is no malaria infection.

Blood smears are taken most often from a finger prick. Thick and thin

blood smears help to know the percentage of red blood cells that are

infected (parasite density) and what type of parasites are present.

A thick blood smear is a drop of blood on a glass slide.

Thick blood smears are most useful for detecting the

presence of parasites, because they examine a larger

sample of blood. (Often there are few parasites in the

blood at the time the test is done.)

Thick blood smear

A thin blood smear is a drop of blood that is spread

across a large area of the slide. Thin blood smears help

doctors discover what species of malaria is causing the

infection.

Thin blood smear

Results

Results of thick and thin blood smears may show:

Normal

No parasites are present in red blood cells. Your doctor will repeat the

test every 8 hours for 1 or 2 days if he or she still suspects that you

have malaria.

Abnormal

Parasites are present in red blood cells. The infecting species of

Plasmodium is identified. Also, the percentage of red blood cells

infected by the Plasmodium parasite (density) is determined.