1. Blood flagellates Mrs. Dalia Kamal Eldien MSC in Microbiology Lecture NO 7

2. Objectives  Introduction to family Trypanosomatidae family  Taxonomy  Common species of Trypanosomatidae family  Morphological characteristic of hemoflagellates  Vectors of hemo & tissue flagellates  African trypanosomiasis (life cycle, pathology &diagnosis)  American trypanosomiasis (life cycle, pathology &diagnosis)

3. Introduction The family Trypanosomatidae (include hemoflagellates), contain only two genera that parasitize humans.  Genus Trypanosoma contains members that may be found both in the circulating blood and intracellular  Genus Leishmania are always intracellular, mainly in the reticuloendothelial system.

4. Taxonomy  Kingdom: Protista  Subkingdom: Protozoa  Phylum: Euglenozoa  Subphylum: Kinetoplasta (unique mitochondrion with large disc of DNA called the Class Trypanosomatidea kinetoplast)  Order: Trypanosomatidea

5. Common species  Trypanosoma brucei brucei  Trypanosoma brucei rhodesiense  Trypanosoma brucei gambiense  Trypanosoma cruzi  Trypanosoma equiperidum  Trypanosoma lewisi  Leishmania tropica  Leishmania major  Leishmania donovani  Leishmania braziliensis  Leishmania chagasi  Leishmania mexicana Trypansoma Leishmania

6. Morphological characteristic of Kinetoplast family  Spindle-shaped body  Central nucleus and Kinetoplast (mitochondrial genome )  Motility by Flagellum or flagellum and undulating membrane  Most are heteroxenous, requiring development within two hosts in life cycle

7. Morphology of Kinetoplast family

8. Vectors  In all probability, the blood & tissue flagellates were originally parasites of insects  They are transmitted by insects, serves as intermediate host

9. Vectors InfectionVector American trypanosomiasisReduviid bugs African trypanosomiasisTsetse flies of the Glossina species "Old World” leishmaniasisSand flies of the Phlebotomus species South American leishmaniasisSand flies of the Lutzomyia species

10.  Genus Trypanosoma, cause 2 diseases in human 1- African trypanosomiasis (Sleeping sickness) (Tsetse flies are found only in Africa)  T. brucei rhodesiense, causing acute trypanosomiasis.  T. brucei gambiense, causing chronic trypanosomiasis. 2- American trypanosomiasis (Chagas’ disease)  Trypanosoma cruzi

11. Morphology  Trypomastigotes found in lymphatics and bloodstream, have a prominent subterminal kinetoplast, an elongated nucleus and an undulating membrane, appear with a typical C (T.cruzi) or S-shaped form.  Amastigote does not have visible external flagella, grows in a variety of tissue cells especially muscle. (T.cruzi)

12. Morphology  Epimastigotes are an extracellular and non infective form of the parasite found in the midgut of insect vectors, where they multiply by binary fission.  Metacyclic trypomastigotes, which are non dividing forms that have the capacity to infect mammalian cells.

13. Trypomastigotes

14. Amastigote

15. Epimastigotes

17. Life cycle  Both male and female tsetse flies suck blood and can therefore transmit the disease.  Once infected a tsetse fly remains a vector of trypanosomiasis for the remainder of its life.  Infected tsetse fly bites a host to take a blood meal & inoculates metacyclic trypomastigotes with saliva into the bite wound.  The organisms rapidly transform into trypomastigotes and which multiply at the site of inoculation by binary fission, and later in the blood, lymphatic system, and tissue fluid.

18.  They are carried to the heart and various organs of the body, and in the later stages of infection they invade the central nervous system (CNS).  The tsetse fly takes blood meal from an infected host containing trypanosomes.  In the midgut of the fly, the parasites develop and multiply. After 2–3 weeks, the trypomastigotes migrate to the salivary glands of the tsetse fly where they become epimastigotes, multiply, and develop into infective metacyclic trypomastigotes.  Blood transfusions are a rare cause of parasitic transmission

20. Tsetse fly

21. Epimastigote& trypomastigote

22. Pathology  African trypanosomiasis is a wasting disease which is usually fatal unless treated.  For T. b. gambiense human is the main reservoir  Rhodesiense trypanosomiasis is a zoonotic infection, many game animals and cattle are infected.  A painful swelling called a chancre develops and can be seen at the site of inoculation of the trypomastigotes.  It contains multiplying trypomastigotes. It disappears after about 10 days.

23. Chancre

24. Sings& symptoms  Early stage of infection:- o There is a high irregular fever, sweating, and an increased pulse rate. o There is a persistent headache, and usually pains in the neck and shoulders. o The lymph glands become swollen.

25. Sings& symptoms  Late stage of the disease:- o The trypanosomes invade the central nervous system, giving symptoms of meningoencephalitis including trembling, inability to speak properly, progressive mental dullness o Excessive sleeping, and incontinence. o There is usually rapid weight loss

26. Lymph node enlargement

27. Laboratory diagnosis Specimen:-  Blood  Bone marrow  Aspirate from chancre(skin sore develops at bite site )  Lymph node aspirate  In late stage collect CSF.

28. Note: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense are morphologically indistinguishable. A-Direct microscopical exam for trypomastigotes in the blood 1- Examination of a thick stained blood film. 2-Microhaematocrit concentration technique 3-Triple centrifugation tube technique to concentrate trypanosomes when a microhaematocrit centrifuge is not available.

29. Thick blood film  Collect a drop of capillary blood on a slide and spread it to cover evenly an area 15–20 mm in diameter.  Allow the smear to dry completely in a safe place  Stain the film using Field’s rapid technique for thick blood films or Giemsa staining technique  When dry, spread a drop of immersion oil on the film and examine it microscopically by X100

30. Thick blood film

31. Result:-  Trypomastigote stage (the only stage found in patients), posterior kinetoplast, a centrally located nucleus, an undulating membrane, and an anterior flagellum, length range is 14 to 33 µm.

32. Trypanosoma brucei sp. trypomastigotes

33.  Centrifuge the capillaries in a microhaematocrit centrifuge for 3–5 minutes  Examine immediately the plasma just above the buffy coat layer for motile trypanosomes  The preparation must be examined within a few minutes of the blood being centrifuged Microhaematocrit concentration

34. Microhaematocrit

35.  For CSF Examine sediment, wet or fixed & stained, examine the cells, it is increased with present of special cell name as Morula or Mott cells  Morula (Mott) cells are larger than small lymphocytes. The nucleus stains dark mauve, and the cytoplasm (may be scanty) stains blue. Characteristic vacuoles can be seen in the cytoplasm

36. Morula or Mott cells

37. B-Indirect methods [Serologic tests] o The main immune response in African trypanosomiasis is a humoral one with stimulated B lymphocytes producing large amounts of Ig M followed in the later stages of infection by Ig G. o Demonstrating anti- trypanosoma antibodies in serum can be done by different methodes: e.g. IFAT, IHAT, ELISA…..

38. C-Culture CSF, blood, bone marrow aspirate, or tissue specimens can be performed in liquid media. D-Other tests o Low hemoglobin with reticulocytosis o High ESR o Moderate leucocytosis with monocytosis& lymphocytosis

40. American trypanosomiasis  Trypanosoma cruzi causes Chagas disease, also referred to as American trypanosomiasis  The vector is Reduviid bugs - blood sucking bugs, Reduviidae has 22 subfamilies, including the Triatominae  T. cruzi is mainly transmitted through contact with the faeces of an infected blood-sucking bug  The faeces containing infective trypomastigotes are deposited on the skin or mucous membranes as the bug feeds from its host.

42. Trypomastigotes& Amastigote

43. Life cycle  Metacyclic trypomastigotes in bug faeces deposited on skin when a bug takes a blood meal.  Following penetration, invade muscle and other tissue cells near the point of entry and multiply intracellularly as amastigotes.  The amastigotes develop into trypomastigotes which are released into the blood.  No multiplication of the parasite occurs in its trypomastigote stage in the blood.  The trypomastigotes reach tissue cells, especially those of heart muscle, nerves, skeletal muscle.

44. Life cycle  The trypomastigotes become amastigotes, the amastigotes develop first into epimastigotes and then into trypomastigotes which are released into the blood when the host cell ruptures  Some of these trypomastigotes continue to circulate while the majority infect further tissue cells.  The life cycle is continued when a triatomine bug ingests trypomastigotes in a blood meal. In the vector, the trypomastigotes develop into epimastigotes which multiply by binary fission in the gut of the bug.

46. Morphology of T. cruzi

47. Pathogenicity  Following penetration, the metacyclic trypomastigotes invade muscle and other tissue cells near the point of entry and multiply intracellularly as amastigotes.  Multiplication of T. cruzi at the site of infection, together with a host cellular immune response, can produce an inflamed swelling known as a chagoma which usually persists for several weeks.  If the site of infection is the eye, usually the conjunctiva becomes inflamed and oedema forms. This is known as Romana’s sign.

48. Chagoma

49. Laboratory diagnosis – Microscopic examination a) of fresh anticoagulated blood, or its buffy coat, for motile parasites b) of thin and thick blood smears stained with Giemsa, for visualization of parasites. – Isolation a) culture in specialized media b) Xeno diagnosis, where uninfected reduviid bugs are fed on the patient's blood, and their gut contents examined for parasites 4 weeks later. – Serology

51. Trypomastigote

52. Amastigotes in heart muscle