Parasitology: Protozoa

Thurs 28th & Fri 29th Nov 2013 Parasitology: Protozoa

MVST BOD & NST PART 1B Pathology Practical Class 14 AIMS

These two classes will demonstrate the diversity of parasitic organisms. Three Phyla, Protozoa and two helminth Groups; the Platyhelminthes (Trematodes and Cestodes) and the Nematodes are represented which include some of the highly important human and veterinary pathogens. The life cycles of the selected parasites are given but please note that the diagrams are not drawn to scale. For the most part, the specimens provided demonstrate those stages of the life cycle that are used for diagnosis e.g. blood films for malaria. In addition to the slides and live specimens provided, two videos entitled "Malaria and mosquitoes" and "Schistosomiasis" will be shown at the beginning of the Protozoa and Helminth practicals respectively. It will be necessary to use the x100 objective oil-immersion lens for examination of all protozoan parasites.

MALARIA The life cycle of Plasmodium spp. is summarised in Figure 1 (over leaf). The blood forms of P. vivax and P. falciparum can be identified in human blood smears -slides 14.1A (99.31) & 14.1B (99.30) compare with (Figure 2). Schizonts are rarely seen in P. falciparum blood smears. Why?

FIGURE 2

THE ASEXUALLY REPLICATING BLOODSTREAM FORMS OF HUMAN MALARIA

Red cells infected with mature schizonts can be very fragile and rupture during preparation of the blood film. This sometimes gives the impression that mature schizonts are extracellular: this is an artifact. Gametocytes can be distinguished from trophozoites by the granular appearance of their cytoplasm and more diffuse nucleus. Use a x100 objective under oil for examination of this slide. teaching:DEPARTMENTAL_TEACHING:Pt1:Practicals:2013-2014:P14_13-14:Handouts:P14_13-14v01asc Page 1 of 9

Make drawings and notes as required. Use x10 objective to orientate yourself, then switch to a higher magnification to identify different stages of parasite in the erythrocytes. Catalogue Number

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P. falciparum

P. falciparum

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P. vivax

P. vivax

The blood forms of Plasmodium falciparum at various intervals in blood smears–slides are labeled: 1. 2. 3. 4. 5.

0-3 hrs. – rings ~24 hrs – early trophozoites ~36 hrs – late trophozoites ~48 hrs – Schizonts Gametocytes

(07-001) (07-002) (07-003) (07-004) (07-005)

Use a x100 objective under oil for examination of these slides. Make drawings and notes as required. Use x10 objective to orientate yourself, then switch to a higher magnification to identify different stages of parasite in the erythrocytes. Catalogue Number

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P. falciparum – Rings (0-3 hrs)

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P. falciparum hrs)

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P. falciparum – Late Trophozoites (~36 hrs)

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P. falciparum – Schizonts (~48 hrs)

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P. falciparum - Gametocytes

Early Trophozoites (~24

Demonstration 1: photograph of sporocysts on gut wall of mosquito Catalogue Number

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Demonstration 1

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Malaria Risk Table Catalogue Number

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Malaria Risk

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Diagnosis of Malaria

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Malaria Comparison 1

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Malaria Comparison 2

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P.vivax Schizont

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teaching:DEPARTMENTAL_TEACHING:Pt1:Practicals:2013-2014:P14_13-14:Handouts:P14_13-14v01asc Page 2 of 9

LIFE CYCLE OF PLASMODIUM SPP: MALARIA


Toxoplasma gondii. This parasite is related to malaria and the coccidian parasites (Eimeria spp.). It is responsible for abortions in sheep and is of human medical importance with respect to foetal damage following congenital transmission. Ocular lesions are also associated with congenital infection and immunosuppressed individuals, including AIDS patients. T. gondii is frequently implicated in the death of AIDS patients. The life cycle is summarised below.

Figure 3. Life cycle of Toxoplasma gondii

micro-gametocyte macro-gametocyte

Carnivorism sporozoites in oocyst

T. gondii life cycle

Ingestion

tachyzoite

Carnivorism bradyzoite tissue cyst

J.W. Ajioka

Congential

Toxoplasma gondii life cycle

a) Infection of intermediate host (herbivorous and omnivorous mammals) by ingestion of sporulated oocyst from cat faeces. The parasite penetrates the gut wall and enters numerous types of cells, particularly mononuclear cells. b) Initial growth in mononuclear cells is by asexual reproduction of fast growing tachyzoites in pseudocysts. c) As the hosts immune system begins to control the infection, slower growing tissue cysts form, (particularly in the muscle and nervous tissues, e.g. brain), containing bradyzoites. Here they may persist for many years, but if the host becomes immunocompromised may give rise to a rapidly fatal disseminating infection. d) Secondary infection of carnivorous hosts, on ingestion of tissues containing either tachyzoites or tissue cysts (e.g. undercooked meat). Therefore almost all mammals may be infected with this parasite. e) Free tachyzoites can cross the placenta, giving rise to congenital infections which may give rise to severe damage to the foetus. This only occurs in the initial stages of a primary infection. f)

Cats become infected after eating infected meat, (e.g. from rodents).

g)

Both sexual and asexual cycles occur in the intestinal epithelium of the cat.

h)

Sporogony of infective oocysts occurs in cat faeces.

14.2 (92.1454) Cysts (also described as pseudocysts) of Toxoplasma gondii in mouse brain. These cysts measure 15-100 microns in diameter and contain bradyzoites which are infective to both cats (the definitive host) and intermediate hosts teaching:DEPARTMENTAL_TEACHING:Pt1:Practicals:2013-2014:P14_13-14:Handouts:P14_13-14v01asc Page 4 of 9

such as man and sheep. Similar cysts can be found in meat. Ingestion of rare meat is the most common route of infection of humans in Western Europe. Locate cysts using a x40 objective and then examine using a x100 objective under oil.

14.3 (79-109) Tachyzoites in peritoneal exudate. This slide is of heavily infected mononuclear cells which have broken open as the slides are made. The tachyzoites appear much smaller than monocyte nuclei. Locate with x40 objective, then examine using a x100 objective under oil. Catalogue Number

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Toxoplasma gondii

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Demonstration 2, Photograph of oocysts of Toxoplasma gondii in cat faeces. These are the products of sexual reproduction. They measure 10-12 microns in diameter, possess a very resistant cell wall and are infective for intermediate hosts including man. Catalogue Number

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Demonstration 2

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Toxoplasma Table Catalogue Number

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Toxoplasmosis

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Toxoplasma Life Cycle

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Toxoplasma Brain Cysts

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Toxoplasma Tachyzoites

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Kinetoplastida. Members of this group include: the African trypanosomes which are the causative agents of human sleeping sickness (Trypanosoma brucei rhodesiense or T. b. gambiense) and the cattle disease nagana (T. b. brucei); the South American T. cruzi, causative agent of Chagas' disease; and Leishmania spp., the causative agents of cutaneous leishmaniasis (oriental sore, espundia or uta) and visceral leishmaniasis (kala azar). All are characterised by the presence of extrachromosomal DNA located in an organelle known as the kinetoplast. This organelle is easily recognised as the second blue/purple staining '1spot" in the cytoplasm by staining with any Romanowsky stain. The nucleus also stains blue/purple. Two morphological forms can be identified in the vertebrate host (Figure 4): the extracellular and flagellated trypomastigote and the intracellular amastigote. Not all forms are produced by all species. Thus, trypomastigotes are the only morphological form produced by the African trypanosomes, while amastigotes are the only form produced by Leishmania species. Both trypomastigotes and amastigotes are produced by T. cruzi.


Figure 4

NB. Only the trypomastigote (in blood, lymph or spinal fluid) and amastigote (intracellular) forms are found in the vertebrate host, epimastigote and promastigote forms are only seen in the insect vector hosts, as below and Figures 5 & 6:

Trypanosoma brucei spp

Vertebrate host

Insect Host

Trypomastigote

Epimastigote (in Tsetse fly) Trypomastigote

(sleeping sickness) Trypanosoma cruzi (Chagas disease)

Trypomastigote (Acute disease)

Epimastigote (in Reduvid bugs)

Amastigote (Chronic disease in Muscle + Nerve cells) Leishmania spp

Amastigote

Promastigote (in Sandfly)


Figure 5: Life Cycle of Trypanosoma brucei spp (Sleeping sickness)

a) Trypomastigote forms inoculated into vertebrate host in Tsetse fly blood meal. Here the extra cellular parasites undergo asexual reproduction by binary fission in the bloodstream, and, later in infection, lymph and spinal fluid. b) Trypomastigote forms transform to a form capable of infecting the Tsetse fly, the so-called "Stumpy" form. c) When taken up by the Tsetse fly in its blood meal into the fly's midgut, the parasites mitochondrion becomes active, and further cycles of binary fission take place. d) The parasites migrate to the fly's proventriculus and salivary glands, where they transform to the epimastigote forms and further cycles of binary fission take place. e) In the salivary glands, trypomastigote forms appear, capable of infecting the vertebrate host in the fly's blood meal ("Metacyclic" Trypomastigotes)

14.4 (92.1453) Trypomastigotes of Trypanosoma brucei in blood. Use a x100 objective under oil for examination of this slide. Catalogue Number

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Trypanosoma brucei

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Trypanosoma Table Catalogue Number

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African Trypanosomiasis

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Trypanosoma brucei

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14.5. (63.1086, 67.757 or 71.202) Amastigote of L. donovani (visceral leishmaniasis). Impression smears from spleens of infected mice. Splenic macrophages are often heavily infected, hence the usefulness of spleen biopsies in diagnosis. However, macrophages in any organ can become infected, depending on the parasite species. During preparation of spleen smears many macrophages rupture and the large blue/purple nuclei become smeared across the slide. The amastigotes are much smaller than the nuclei of the macrophages: look for 'double spots' surrounded by a faint membrane. Use a x100 objective under oil for examination of this slide.

14.6. (81.42) Section through the eyelid of a dog infected with L. tropica (cutaneous leishmaniasis). Amastigotes can be seen in the cytoplasm of macrophages, however the kinetoplasts are not easy to see in this specimen. First examine the whole cross section by holding it up to the light. The section of the eye and upper eyelid can easily be seen. Examine the inner portion of the eyelid under low power (x10) to identify the area of macrophage infiltration, seen as many densely packed blue staining nuclei. Examine the cytoplasm of these cells for the presence of the double spots of the much smaller amastigote nuclei. Catalogue Number

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Leishmania

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Leishmania Table Catalogue Number

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Leishmaniasis

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L.donovani amastigotes

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L.donovani amastigotes

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L.tropica dog eyelid

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L.tropica dog eyelid 10X

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Figure 6. Life cycle of Leishmania spp. 1)

Vertebrate Host

NB. With different species of leishmania, and particular individual hosts, the parasites infect macrophages in different parts of the body eg Leishmania donovani - normally infects mainly macrophages of internal organs, particularly the spleen and liver - Visceral leishmaniasis Leishmania major - normally infects mainly macrophages in the skin: Cutaneous leishmaniasis Leishmania braziliensis - infects macrophages mainly around nose and mouth: Mucocutaneous leishmaniasis

