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Home , Kinetoplast, Leishmania, Leishmania donovani, Leishmania tropica, Trypanosoma, Trypanosoma cruzi

Lab 2: Trypanosomes

Organisms to study: lewisi gambiense tropica Leishmani donovania

Order : Members in this order are principally parasitic, with the exception of one suborder that contains a few free-living . Kinetoplastids possess a , a DNA containing organelle which can be thought of as a mitochondrial compartment packed with and maxicircles of DNA. are self-replicating and contain genomic material in addition to that which is in the nucleus. In general, members have from one to four flagella, a self-replicating kinetoplast, a , and a nucleus.

Family Trypanosomatidae: The family Trypanosomatidae is defined as having a single nucleus, a single , and a leaf-like or rounded body. Members of this family are all parasitic, inhabiting the blood and (or) fixed tissues of all classes of . Most species spend one stage of their life cycle in a and another in a blood-sucking , which functions as a . During their life cycles most of the trypanosomatids change body form depending on the or the organ in which they are located. Stages: Trypanosomatids often undergo cyclic development as they pass from one host to the next. Although they shift back and fourth between forms during their life cycles, the basic structure remains much the same. There are six basic forms of trypanosomatid : (a) amastigote, (b) promastigote, (c) opisthomastigote, (d) epimastigote, (e) trypomastigote, and (f) choanomastigote. At least two of these stages occur in every member of the family. You can learn to identify the different developmental stages based on the position of the kinetosome () with respect to nucleus. The different stages you will examine are illustrated on the next page (K=kinetosome, N=nucleus, F=flagella):

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Amastigote Leishmania amastigote - spherical - very short flagellum

Promastigote - has elongated body form - kinetoplast and kinetosome at the anterior end, in front of nucleus

Trypomastigote - characteristic of blood stream forms and metacyclic forms in the invertebrate vector - has undulating membrane - kinetoplast and kinetosome at the posterior end, behind the nucleus

2 Trypanosoma: Trypanosomes represent some of the most economically important and animal parasites. Members of this genus are parasites of and all classes of vertebrates. The majority of trypanosomes are located within the blood or fluids, although some are intracellular parasites. Most are transmitted by a blood-sucking arthropod, some by leeches, and one is transmitted venereally. During their life cycle these parasites assume the amastigote, promastigote, epimastigote and (or) trypomastigote forms. Development: Members of this genus undergo one of two types of development in the insect vector: anterior station and posterior station. Anterior station development involves division of trypomastigotes in the midgut of the vector followed by migration of the parasite forward into the upper digestive tract (eg. salivary glands). Metacyclic trypomastigotes are then passed to the vertebrate host when the vector feeds. Anterior station development occurs with the salivarian trypanosomes (eg. Trypanosoma brucei gambiense) transmitted by vectors such as the tse-tse fly. Posterior station development describes parasite development in the hindgut of the invertebrate vector. In the hindgut the parasite transforms into epimastigotes and metacyclic trypomastigotes, which then move back through the digestive tract and are passed to the vertebrate host in the vector faeces. This form of development and transmission occurs in the trypanosomes (Trypanosoma cruzi) transmitted by insects such as the reduvid kissing bugs.

Slide: is a cosmopolitan parasite of rats. Rats become infected by ingesting or feces which contains trypomastigote forms of the parasite. Study this parasite with oil immersion optics making sure to note the nucleus, free flagellum, undulating membrane, and kinetoplast. Note the variety of shapes T. lewisi shows in this heavy . The "fat" forms are young trypomastigotes and the slender forms are older forms infective to fleas.

Slide: Trypanosoma equiperdum - This trypanosome causes a venereal disease in equines called dourine. It is transmitted venereally during coitus and is usually fatal, unless treated. Dourine has been eradicated from North America. Both T. evansi and T. equiperdum are morphologically similar to the T. brucei subspecies.

3 Trypanosoma brucei gambiense

Slide: Trypanosoma brucei gambiense: T. gambiense is morphologically indistinguishable from T. b. brucei and T. b. rhodesiense. These three subspecies are extremely important from a medical-veterinary standpoint, being the causative agents for nagana in wild antelope and livestock (T. b. brucei), and African sleeping sickness in (T. b. gambiense and T. b. rhodesiense). They are widely distributed throughout tropical , with their distribution being correlated with that of the , which serves as the vector.

These trypanosomes undergo anterior station development within their vector. Infection in the human or reservoir host begins when the infective stage, the metacyclic trypomastigotes, is injected by the tsetse fly. Once injected, the organisms transform into blood-stage trypomastigotes (long slender forms) and divide by binary fission. This flagellated stage then enters the blood stream through the lymphatics and divides further, producing patient parasitemia. At this stage, some parasites transform into a non-dividing short, stumpy form that is then ingested by the tsetse fly. Once inside the vector, the trypanosomes develop into procyclic trypomastigotes in the midgut, and continue to divide for approximately 10 days. When the division cycles are complete, the organisms migrate to the salivary glands, and transform into epimastigotes. These forms, in turn, divide and transform further into metacyclic trypomatigotes, the infective stages for humans and livestock.

4 Lifecycle of Trypanosoma brucei gambiense: Anterior Station

Trypanosoma cruzi Slide: Trypanosoma cruzi (=Schizotrypanum cruzi) trypomastigote. T. cruzi is a parasite and is the causative agent for in humans. Many wild and domestic animals serve as reservoir hosts and the arthropod vector is the reduviid bug or "kissing" bug. The trypomastigote form is passed from the insect vector to the definitive host. Tryps can be found in the blood of the definitive host during early because they do not reproduce by fission until they have entered host cells (primarily cardiac tissue and cells of the reticulo-endothelial system). Here they transform into the amastigote stage and multiply by fission to form pseudocysts. The 5 photograph below is a section of rat showing pseudocysts of amastigotes of T. cruzi. Both the epimastigote and trypomastigote stages occur in the vector. Trypomastigotes infect humans from the feces in what is termed posterior station transmission.

Slide: Trypansoma cruzi pseudocyst in rat cardia tissue. The pseudocyst is an enlarged cell containing amastigotes.

Lifecycle of Trypanosoma cruzi: Posterior Station

6 Genus Leishmania: Individuals in this genus are primarily parasites of , most commonly humans, dogs, and , but several species also occur in . Leishmania spp. are vectored principally by ( and ). Infection begins when a carrying the parasite takes a blood meal. The promastigotes resides in the anterior midgut and thorax of the sandfly and are injected into the host along with the fly’s salivary secretions. Once injected, the promastigotes are engulfed by . The promastigotes will either remain inside the macrophages (such as those species that cause cutaneous ) or are carried by macrophages to other locations. Once inside the macrophages, the parasites transform rapidly into the amastigote stage and proceeds to replicate intracellularly. The amastigote stage is the only stage found in the definitive host's tissues. Infection of the sandfly occurs when the fly takes a bloodmeal from an infected individual containing either infected macrophages or free amastigotes. Upon ingestion, amastigotes transform into procyclic promastigotes, which are not infectious but become attached to the sandfly midgut. Here, the procyclic promastigotes will differentiate to produce infectious metacyclic promastigotes, which are then transmitted back to their human hosts through the sandfly bite. See attached life cycle diagram. Lifecycle of Leishmania

7 The three most important species from a medical standpoint are: • , which causes Oriental sore or , • L. donovani, which causes Kala-azar or • L. braziliensis, the causative agent of espundia or mucocutaneous leishmaniasis. Again, these species cannot be differentiated morphologically. They are separated on the basis of the disease produced, the host and its immune response, and on the geographical distribution.

Slide: promastigote (culture smear)

Slides – Leismania donovani in tissue: study the tissue smears ( and ) containing amastigotes of L. donovani using oil immersion. The amastigotes are very small (2.5-5.0 μm)) and spheroid to ovoid in shape. Only the nucleus and a very large kinetoplast are visible with light microscopy. Some describe amastigotes as little snowmen.

Leishmania replicates as intracellular amastigotes in macrophages. In fact, amastigotes will eventually kill a and go on to infect others. Macrophages play a role in both adaptive and innate immunity. In the most basic terms, macrophages engulf foreign invaders and produce , such as superoxides to kill them. They can be very effective at ingesting and digesting exogenous antigens and endogenous matter. How then can Leishmania not only survive, but thrive in such an environment?

8 Their survival is believed to be a combination of any of the following strategies:

1. When macrophages first engulf a parasite such as Leishmania, macrophages undergo a . This respiratory burst involves enzymes such as superoxides which should effectively digest the parasite. Some Leishmania sp can produce an called superoxide dismutase which can neutralize the free radical damage.

2. Leishmania has evolved the ability to bind Compliment-like C3 , which can outright inhibits the respiratory burst.

3. Macrophages function by communication usually via lymphokines, with T-cells. Leishmania are able to “shut down” communication between these cells by interrupting the transcription of major histocompatibility genes (MHC) so that the macrophages they are infecting bare few if any MHC on the surface for T-cells to recognize.

4. Leishmania has been reported to stimulate the to produce more macrophages. However, the new macrophages being produced are immature; they can capture and engulf the parasite but are physiologically unable to kill the, even in the presence of stimulating cytokines.

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12 Learning Objectives

1. Order Kinetoplastida - Know general characteristics

2. Family Trypanosomatidae - General characteristics (leaf-like, flagellum, parasites of vertebrates) - undergo cyclic development as they pass from one host to the next - know life cycle stages (purpose, morphology, differentiation)

3. Genus Trypanosoma - general features - anterior vs. posterior station - visual ID T.lewisi, T.equiperdum, T.brucei gambiense, T.cruzi

4. Trypanosoma lewisi - host + vector transmission

5. Trypanosoma equiperdum - host + pathology + transmission

6. Trypanosoma brucei gambiense - distribution, pathology, vector - life cycle (hosts, stages, transmission, tissue) - vector + type of development in vector

7. Trypanosoma cruzi - Pathology - distribution, vector , life cycle, transmission - Visual ID pseudocyst in rat heart, what stage

8. Genus Leishmania - Hosts, vectors, life cycle, transmission - know 3 species of medical importance + disease associated with each - visual ID promastigotes to genus L.donovani - L.donovani amastigotes in liver tissue - What strategies does Leishmania use to overcome macrophage defenses

Vocabulary

• Kinetoplast • Nagana • Macrophage • Amastigote • Dourine • Superoxides • Promastigote • Vector • Respiratory burst • Trypomastigote • Reservoir host • Superoxide dismutase • Epimastigote • Definitve host • MHC • Anterior station • Pseudocyst • Posterior station

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