Laboratory 1. Parasitic protozoaFor this lab, we have chosen to focus on a few parasites representing four protozoan orders that are areas of concern to humans around the world.

Organisms to study: Giardia lamblia Trichomonas vaginalis

Entamoeba histolytica Entamoeba coli Balantidium coli

The organisms you will study in this lab are unicellular protozoan parasites. Protozoans represent some of the oldest known eukaryotic groups that are highly adapted for each of their habitats. Unicellular protozoans are single celled organisms containing a nucleus (or at some stages nuclei), cytoplasm, plasma membrane, and highly specialized organelles. Protozoans are strictly aquatic organisms. They must have an aqueous environment for feeding, reproduction, and locomotion. Many protozoans form cysts that provide protection for stages of reproduction that do not occur entirely in a fluid. Members of this group are highly successful, having invaded every ecological niche imaginable. Nearly every species of metazoan has a complement of protozoans living in it. Many human and animal diseases are caused by unicellular organisms, making any knowledge of parasitic protozoans important to both doctors and parasitologists! This first lab will be one of your most challenging simply because these organisms are small and because the preparations are fecal smears and tissue smears. To make your job easier, we have included pictures within the lab manual to help you form a search image. The best way for you to get through this material is to become familiar with these pictures…..and to be patient. Have fun!

1. Order Diplomonadida

The order Diplomonadida contains parasites that are bilaterally symmetrical. Giardia lamblia is considered one of the deepest branching or most primitive eukaryotes in existence. Some scientists have called Giardia the “missing link” in the evolution of eukaryotic cells from prokaryotic cells. Giardia lamblia was the first eukaryotic cell to be seen using one of the first good quality microscopes developed by Antoine Van Leeuwenhoek back in the 1600’s.

Giardia lamblia is an intestinal parasite of humans and a variety of other mammalian hosts. Giardia is highly contagious and is the cause of a number symptoms that can include severe diarrhoea. A human or animal host becomes infected when it ingests food or water that is contaminated with the feces from another infected host. Transmission depends on the swallowing of mature cysts. Once swallowed, the cysts pass though the stomach, excyst in the duodenum and develop into trophozoites. Trophozoites develop into cysts as they pass through the intestine and into the colon. The feeding stage, or trophozoite, is 12-15 µm long, rounded at the anterior end and

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pointed at the posterior end. The ventral surface bears a concave, bi-lobed adhesive disc. The adhesive disc is a rigid structure that allows this organism to attach to the host intestinal cells. Trophozoites feed on the intestinal mucosal surface. In severe infections the free surface of nearly every cell is covered by a parasite and one person can pass millions of cysts each day.

Giardiosis is diagnosed by finding cysts or trophozoites in the feces. Both life cycle stages have a characteristic appearance making it the easiest human intestinal protozoan to diagnose.

Slide: Giardia lamblia trophozoite: this parasite is generally tear-drop shaped with two visible nuclei. If you use the fine focus knob to focus in and out on the parasite you will be able to see the bi-lobed adhesive disc. Note: Tear-drop shape may or may not be visible depending on your plane of vision.

Slide: Giardia lamblia cyst: Giardia cysts are slightly smaller than trophozoites and have 4 visibly distinct nuclei. A median rod, known as an axostyles, is often visible down the centre of the organism.

Trophozoite

Cyst

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2. Order Trichomonadida

Members of the Order Trichomonadida are nearly all parasitic. They are small flagellates that exist only as trophozoites. Members of this family are easy to recognize because they have an anterior tuft of flagella, a median rod (axostyle), and an undulating membrane that extends the length of the body. The characteristic number of flagella is five,

though there is some deviation from this number. In addition, Trichomonads have a single nucleus and a parabasal body (golgi apparatus). Trichomonads lack mitochondria but do have granules that serve roughly the same purpose (these granules contain many of the Kreb’s cycle enzymes).

Trichomonads are found widely as parasites of both vertebrates and invertebrates. Most are found in the intestinal tract but can be found in the reproductive system, urinary tract, and the respiratory tract. Transmission is direct. Trichomonads have no cyst stage. Reproduction is asexual (binary fission)

Trichomonads in humans

Slide: Trichomonas vaginalis: found in the genital and urinary tract of humans. T. vaginalis is transmitted from one person to the next through sexual intercourse. Although both men and women are equally infected with T. vaginalis, it causes disease almost exclusively in women. Diagnosis involves finding living organisms in material taken from an infected person. Swabs are most commonly examined under a microscope.

Pentatrichamonis hominis: There is no slide for P. hominis but know that it occurs in the large intestine of humans and is non-pathogenic.

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Trichomonads in other animals

Tritrichomonas foetus: causes trichomonad abortion, or bovine trichomonas. The principle host of T. foetus is the domestic ox however closely related organisms are also known to infect the horse and the pig. T. foetus infects the reproductive tract of both cows and bulls.

Trichomonas gallinae: causes avian trichomonas. T. gallinae infects the digestive tract, liver, and lungs. Infections to the liver and lungs will often result in death.

3. Order Amoebida: Family Entamoebidae

The term “amoeba” refers to an organism that moves and feeds with the aid of pseudopodia. The Entamoebidae lack flagella at all stages in their development. Species in the Entamoebidae are parasites or commensals of the digestive system. Several species occur in humans; most are harmless commensals but Entamoeba histolytica is a highly pathogenic species. The life cycles of all species in the Entamoebidae are similar: A host ingests food or water contaminated with Entamoeba cysts. After excysting in the small intestine, the cytoplasm and nuclei divide to form trophozoites. Trophozoites live in the intestinal tract where they feed on bacteria. The trophozoites are then passed with the faeces into the colon. As fecal matter becomes dehydrated the amoeba encysts. Transmission occurs when a host swallows these specialized multinucleate cysts.

Species 1: Entamoeba histolytica: This species is the third most common cause of parasitic death in the world and is estimated to be responsible for between 50,000 and 100,000 deaths each year. It is the causative agent of amoebic dysentery. Transmission is direct, involving ingestion of contaminated food and water. It normally feeds on bacteria in the intestine but will also erode the mucosa coating of the epithelium and enter the mucosa of the gut causing a typically flask-shaped ulcer. Trophozoites may even be carried by the lymphatics to other organs, most often the liver, where they cause further problems.

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Slide: Entamoeba histolytica trophozoite: Trophozoites are round with large lobular pseudopodia and a clock-face nucleus. The nuclear endosome is typically (but not always) centrally located. You may see erythrocytesin the

cytoplasm, indicating digestion of erythrocytes.

Slide : Entamoeba histolytica cyst: Cysts are smaller than trophozoites. They contain 4 nuclei and chromatoid bars (tightly packed messenger RNA with rounded ends).

Slide: Entamoeba histolytica in colon tissue. Trophozoites in tissue sections are often larger than those found in luminal samples. You will find the trophozoites within ulcers in the tissues. Search the slides for ulcerations and look for the inflammatory response.

Species 2: Entamoeba coli: This is another species that occurs in the intestine of humans. It is a harmless commensal of the human large intestine, but looks similar to Entamoeba histolytica. It feeds on bacteria, other protozoa, and yeasts. E. coli is more common than E. histolytica, however multiple infections are common therefore it is important to be able to distinguish between these two forms.

Slide: Entamoeba coli trophozoite: distinguished from E. histolytica by its coarser chromatin, eccentrically located endosome and the absence of erythrocytes in the cytoplasm.

Slide: Entamoeba coli cyst: E. coli cysts contain 8 nuclei (compared to 4 nuclei seen in E. histolytica) and smaller chromatid bodies that are often filamentous.

CystsTrophozoite

CystTrophozoite

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4:Phyllum Ciliophora: Family Balantidiidae

The family Balantidiidae contains one major genus, Balantidium, which can be found in the intestines of crustaceans, insects, fish, amphibians, and mammals. The species you will examine, Balantidium coli, is the largest protozoan parasite of humans. B. coli is the cause of balantidiosis or balantidial dysentery. Transmission is direct and usually occurs when the cyst stage is accidentally ingested via contaminated food or water. It is generally non-pathogenic but can cause ulceration of the intestinal epithelium followed by secondary bacterial infections.

Slide: Balantidium coli trophozoite: trophozoites are oblong with a prominent kidney shaped macronucleus and a smaller micronucleus. B. coli is uniformly ciliated over its body but this is rarely visible on these slides.

Slide: Balantidium coli cyst: cysts are round with a rounder and

thicker macronucleus

Slide: Balantidium coli intestinal tissue: search the tissue cross-section for ulcerations caused by the trophozoites. Trophozoites are easily distinguishable by their large size.The trophozoites attack intestinal epithelial tissue, causing flask-shaped ulcers (similar to amoebic ulcers). The liver, lungs and urogenital tract may also become infected.

Trophozoite

Cyst

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Learning Objectives

1. Know general features of protozoans

2. Order Diplomonadida- Visual ID Giardia lamblia – cyst + troph- Life cycles and roles of cyst + troph- Anatomy of troph + cyst- Transmission: from who, how, what stage, to who- Tissue it is found- Pathology = symptoms and disease-

3. Order Trichomonadida- What makes them special? (2 things)- Visual ID Trichomonas vaginalis- Life cycle, anatomy and transmission-

4. Order Amoebida, Family Entamoebidae- How do they move- Visual ID Entamoeba histolytica and Entamoeba coli (trophs + cyst)- Be able to visually differentiate between the two species- How do they differ in other ways- Life cycle, Transmission, Epidemiology, Pathology-

5. Phyllum Ciliophora, Family Balantidiidae- Visual ID Balantidium coli troph + cyst- What makes B. coli special- Life cycle, Pathology- Host, Tissue, Transmission

Vocabulary

Protozoan Trophozoite Cyst Giardiosis Flagella Axostyle Amoeba Pseudopodia Pathology

Transmission Epidemiology Erythrocyte Erythrocytosine Cilia Commensal Pathogenic Endosome Ulcer

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