Classification, Characteristics, Life Cycle & Microscopy
Trypanosoma is a genus consisting of hemoflagellate protozoa that exist as obligatory parasites of plants, mammals and other animals (fish, birds, reptiles etc). Members of this genus, known as trypanosomes, are unicellular organisms whose life cycle is dependent on both vertebrate and invertebrate hosts.
While some of the most common species are found in Africa (responsible for African trypanosomiasis in various parts of sub-Saharan Africa) many other species can be found in various parts of the world (Parts of North America, Canada etc).
While some species in the genus are responsible for serious diseases, a majority of the species have been shown to be non-pathogenic. The different species vary in terms of size, general body shape as well as location of such organelles as the nucleus which has made it possible to distinguish between them.
Some of the species that belong to the genus Trypanosoma include:
- T. brucei
- T. equiperdum
- T. cruzi
- T. bancrofti
- T. anguillicola
- T. granulosum
- T. aulopi
Classification of the Genus Trypanosoma
· Domain: Eukaryota - As members of the domain Eukaryota, Trypanosoma species have membrane-bound organelles. As such, they have more complex structures compared to members of the domain prokaryota.
· Kingdom: Protista - Also known as Protoctista, the kingdom Protista is largely composed of simple (unicellular or colonies) eukaryotic organisms that either exist s parasites (e.g. members of the genus Trypanosoma that dependent on hosts) or as free-living organisms in water and terrestrial environments.
· Phylum: Protozoa - In some books, protozoa is ranked as a kingdom in itself or a subkingdom. It consists of single-celled eukaryotic organisms that either exist as parasites or as free-living organisms.
· Sub-phylum: Sarcomastigophora - The subphylum Sarcomastigophora falls under protozoa and includes a range of single-celled eukaryotic organisms that are characterized by locomotive structures; flagella or pseudopodia.
· Order: Kinetoplastida - Kinetoplastida is an order of flagellate protozoan characterized by a kinetoplast (a mass of mitochondrial DNA located close to the nucleus).
· Family: Trypanosomatidae - Members of this family, like many Trypanosoma species, are characterized by a single flagellum that allows them to swim in fluids and blood. The name Trypanosomatidae is derived from Greek words that describe corkscrew-like movement of the organisms in their respective environments.
Stercoraria and Salivaria
Apart from traditional taxonomy, different genera of Trypanosoma (mammalian Trypanosoma) have also been classified based on the mode of transmission. Whereas Salivaria consists of trypanosomes that are transmitted by African tsetse flies, Stercoraria includes genera that complete their development in the posterior station.
As such, their infective forms can be found in feces. As compared to organisms described as stercoraria, genera classified as salivaria complete their development in the salivary system (the anterior station).
Stercoraria species include:
- T. rangeli
- T. cruzi
- T. musculi
- T. theleri
Salivaria species include:
- T. suis
- T. congolense
- T. vivax
- T. godfreyi
- T. evansi
Characteristics (Life Cycle and Cell Biology)
The life cycle of various Trypanosoma species involves the transmission of the parasite from one host to another.
According to studies, there are three main modes of transmission, these include:
· Cyclical transmission - In the cyclical mode of transmission, the parasite is transmitted by infected tsetse flies. Here, the parasite undergoes development in the insect before being transmitted when the fly is feeding (through saliva). This allows the cycle to continue from infected hosts to infected tsetse flies.
· Mechanical transmission - In this mode of transmission, the parasite is transmitted by a number of biting flies including tsetse flies. According to studies, stomoxes and tabanids are the main biting flies involved in the transmission of the parasites. As compared to cyclical transmission, this type of transmission has also been shown to continue in new hosts within a group of animals. As a result, the parasite can still spread to other areas where a given vector may have been completely eliminated.
· Per-orale and vertical transmission - In per-orale transmission, transmission of the parasite is as a result of ingesting contaminated fluids. For instance, when a calf ingests contaminated fluids, an infection is likely to take place. Vertical transmission, on the other hand, may occur during pregnancy or shortly after birth (parent to offspring transmission).
As Trypanosoma parasites are transmitted from one host to another, they go through several stages of development which allows the parasite to successfully complete their life cycle and allow the cycle to continue. This section gives focus to the life cycle of Trypanosoma cruzi as a representative of the group.
The life cycle of this parasite can be said to start when the metacyclic trypomastigotes stage of the parasite is deposited on the skin of the host (mammal e.g. man). Rather than transmitting the parasite through a bite during feeding (as is the case with Salivaria species) the insect vector (reduviid bug in this case) excretes the parasite form along with its excreta onto the skin surface of the host.
Here, it is worth noting that the parasitic form develops in the posterior part of the vector's gut and ultimately deposited along with the fecal matter. Given that the parasite has no way of penetrating the skin to invade the underlying cells, they have to be mechanically introduced which may involve rubbing or scratching the irritated part (part of the skin where the insect bit the host).
* The the metacyclic trypomastigote is characterized by a blunt body shape as well as a short flagellum.
When the parasite is mechanically introduced into the skin and enters the bloodstream, it's phagocytosed by such cells as macrophages (being an invading organism in the body). However, to avoid being destroyed, it quickly transforms into the amastigote form under the influence of lysosomal contents that produce low pH.
This new form of the parasite is able to rapidly migrate from the vacuole (which contains lysosomal contents) to the cytoplasm. Here, the conditions of the lysosome influence dramatic transformation characterized by such activities as flagella involution forming a pore that is resistant to harsh conditions.
In the cell cytoplasm/cytosol, the amastigote form (the dividing form of the parasite in mammals) proliferates to produce many more forms that ultimately fill the infected cell. This division is then followed by the development of the amastigote to form trypomastigote forms that have elongated flagella. These new forms then migrate and infect the adjacent cells allowing the parasite to thrive.
For the trypomastigotes that do not invade adjacent cells, they enter the bloodstream (of lymph) which allows them to be transported to other tissues as they continue differentiating. This extracellular differentiation (as compared to differentiation within the cells) produces trypomastigotes and amastigote forms that are taken up by the vector (reduviid bug) during blood feeding.
Following ingestion (by the bug), trypomastigotes differentiate to form amastigotes which in turn transform to spheromastigotes (characterized by extending flagella). In turn, spheromastigotes lengthen to form epimastigotes that continue to develop as they consume nutrients available in the blood meal of the bug.
As the level of nutrients decreases, these forms undergo further transformation and ultimately form the metacyclic trypomastigotes which are the infectious form. Following the transmission of these forms to the appropriate host, the life cycle continues.
Some of the organs vulnerable to infection by the parasite include:
- Lymph glands
- Nervous system
* Within the cells of the host (mammal cells), amastigotes reproduce asexually through binary fission to form more forms. This form allows them to divide and create more parasites that ultimately rapture the cell. Following this rapture, however, the amastigotes have to transform into trypomastigotes that are capable of swimming in blood.
* While the parasites were initially thought to solely reproduce asexually, new findings suggest that sexual reproduction may also be part of their reproduction cycle.
How T. Cruzi causes Chagas Disease
* Cell destruction by dividing forms of the parasites is one of the negative impacts on the host.
While the entire mechanism is not yet fully understood, development of the disease in mammals has been associated with the metabolism of the parasite.
In the body of the host, the parasite is dependent on various nutrients (carbohydrates, lipids, proteins etc) for their survival. As the parasites divide and continue to increase in numbers in the body (both within the cells and blood) they continue utilizing the host's nutrients while releasing metabolites that negatively affect the host.
According to studies, the parasite has very small polysaccharide stores. For this reason, they have to continue utilizing the host's supply for their energy. On the other hand, forms of the parasite in blood are continually utilizing glucose for energy. Here, products of metabolism, including carbon dioxide and acetate, have negative impacts on the host.
Symptoms of Chagas diseases include:
- Swollen lymph nodes
- General fatigue
- High fever
- Skin rash
- Nausea and vomiting
- Skin redness
* The parasite has also been associated with heart conditions (chronic Chagas cardiomyopathy).
Some of the other diseases caused by Trypanosoma species include:
- Sleeping sickness - Caused by T. brucei
- Nagana in various animals - Caused by T. congolense
- Dourine in horses - Caused by T. equiperdum
- Surra in a number of animals - Cuased by T. suis and T. evansi
Morphological Characteristics of Tryanosome Cells
As members of the domain Eukaryota, Trypanosoma species are characterized by the genera features found in typical eukaryotic cells.
For instance, like normal eukaryotic cells, a trypanosome cell has a membrane-bound nucleus, Golgi apparatus, E.R, as well as a plasma membrane among other important organelles. On the other hand, as members of order Kinetoplastida, Trypanosoma have a number of unique features including a kinetoplast, glycosomes, as well as acidocalcisomes (site of mineral storage).
Trypanosome cells have also been shown to possess a unique cytoskeleton that is mostly composed of microtubules. It also lacks centrioles that play an important role in cell replication. As a result, poorly defined structures in the cell are responsible for the production of microtubule spindles that contribute to the closed mitosis in these parasites.
As for the distinctive general morphology of the parasite, it is defined by distinct microtubules that act in opposition to the plasma membrane.
Trypanosomes are also characterized by a single flagellum (ranging from 2 to 20um in length) that is supported by basal and probasal bodies within the cell. As is the case with motile cilia and flagella found in various eukaryotic cells, the flagella of trypanosome cells is characterized by a 9+2 configuration consisting of parallel microtubules.
A structure, known as a paraflagellar rod, also extends along the length of the flagella and has been shown to provide support (by enhancing rigidity) to the axoneme of the flagellum as well as promoting motility.
* At the point where the flagellum enters the cell, a gap known as a flagellar pocket exists. This gap is believed to be the point at which vesicular trafficking and nutrient uptake takes place.
A number of techniques may be used to prepare and observe trypanosome cells under the microscope, these include:
1. Mini Anion Exchange Centrifugation Technique (mAECT)
Using a micropipette, obtain about 400ul of the patient's blood and add it into the minicolumn containing diethylaminoethyl cellulose.
For about 15 minutes, centrifuge the sample at low speed.
Observe the sample under the microscope at low power (a special holder is used to hold the column when observing under the microscope).
When the tip of the column is observed under the microscope, trypanosome cells in the sample can be seen wiggling (moving) randomly.
2. Capillary Tube Centrifugation (CTC)
This technique involves the use of capillary tubes to collect and observe the sample under the microscope:
- Capillary tubes
- Blood sample
- Centrifuge (micro-hematocrit centrifuge)
- Compound microscope
- Using a disinfectant, wipe the fingertip (of the patient) and prick using a lancet
- Using a clean cotton swab, wipe off the first drop
- Press the finger (making sure not to touch the pricked part) and using a capillary rube, collect fresh blood - Fill the capillary (which contains EDTA) with blood
- Turn the capillary tube several times to ensure that the blood is properly mixed with the anticoagulant (EDTA)
- Using a sealer paste, seal the open end of the capillary tube
- Using a micro-Haematocrit centrifuge, centrifuge the tube at maximum speed for about 10 minutes
Mount the capillary tube and observe the cells under low power - This is achieved by using a holder to hold the capillary tubes on a microscope glass slide and filling the viewing chambers with distilled water and covering the chambers with a coverslip
When viewed under the microscope, the cells can be seen at the junction between the plasma and blood cells.
3. Blood Smear Staining with Wright's Stain
- Wright’s Stain
- Compound microscope
- Staining rack
- Buffered water
- Clean glass slides
- Compound microscope
- Place a drop of the blood sample on a clean slide and make a thin film using another slide/coverslip
- Allow the film to air-dry and dip the slide in methanol (fixation)
- Allow the slide to air-dry and cover with Wright’s Stain and allow the slide to stand for 2 minutes on a staining rack
- Add a few drops of buffered water on the slide and allow the slide to stand for about 5 minute
- Flood the slide with buffered water and then allow the slide to dry (wipe off excess water under the slide)
- Observe the slide under the microscope
See more on Cell Staining
Staining allows for better visualization of trypanosome cells that will appear as bluish/purple slender organisms with thin flagellum on one end.
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Fnu Nagajyothi et al. (2013). Mechanisms of Trypanosoma cruzi persistence in Chagas disease. ncbi.
J. D. Smyth and Derek Wakelin. (1994). Introduction to Animal Parasitology. Google Books.
John L. Capinera. (2008). Encyclopedia of Entomology.
Julien Bonnet, Clotilde Boudot, and Bertrand Courtioux. (2015). Overview of the Diagnostic Methods Used in the Field for Human African Trypanosomiasis: What Could Change in the Next Years? BioMed Research International
Volume 2015, Article ID 583262, 10 pages.
K. M. Tyler, C. L. Olson and D. M. Engman. (2001). The Life Cycle of Trypanosoma Cruzi.