Lab 7 trypanosomiasis


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  • African trypanosomes are protozoan parasites responsible for human tropical diseases, such as sleeping sickness. This neglected disease is fatal in the absence of treatment. No vaccine is available and current therapy relies on the use of toxic drugs, including arsenic components that lead to patient death in 2-10% of cases. Trypanosomes proliferate in the blood stream of their host as extracellular parasites and escape the immune response by using a sophisticated process of antigenic variation. The surface of the trypanosome is covered with a dense coat made of a single type of protein (variant surface glycoprotein or VSG) that is periodically replaced. The genome contains ∼1000 different VSG genes and pseudogenes that can undergo recombination events to ensure rapid modification of this repertoire Absalon S. et al. (2008). Flagellum elongation is required for correct structure, orientation and function of the flagellar pocket in Trypanosoma brucei. J Cell Sci. 2008 Nov 15;121(Pt 22):3704-16. Epub 2008 Oct 21. They are also called kinetoplastida (contain kinetoplasts or modified mitochondria). The hemoflagellates include medically significant protozoan parasites of humans as well as other vertebrates. Two genera of the group that have a global impact on human health are Leishmania and Trypanosoma.   Hemoflagellates‑ Family Trypanosomidae : flagellated protozoans found in tissue or blood. These infections are arthropod borne and the organisms have evolved complex life cycles involving vertebrate and invertebrate hosts. There are two genera of medical importance Trypanosoma and Leishmania .
  • The kinetoplastids are a group of single-cell flagellate protozoa , including a number of parasites responsible for serious diseases in humans and other animals, as well as various forms found in soil and aquatic environments. They are members of the phylum Euglenozoa and their major distinguishing feature is the presence of a kinetoplast , a DNA -containing granule located within the single mitochondrion associated with the base of the cell's flagella (the basal body ). The kinetoplastids were first defined by Honigberg in 1961 as the flagellate order Kinetoplastida. [1] They are traditionally divided into the biflagellate Bodonidae and uniflagellate Trypanosomatidae ; the former appears to be paraphyletic to the latter. One family of kinetoplastids, the trypanosomatids , is notable as it includes several genera which are exclusively parasitic. Bodo is a typical genus within kinetoplastida and including various common free-living species which feed on bacteria . Others include Cryptobia and the parasitic Leishmania .
  • The name is derived from the Greek trypaô (boring) and soma (body) because of their corkscrew-like motion. A corkscrew is a kitchen tool for drawing stopping corks from wine bottles . Corkscrew Boring invagination
  • Pleomorphic (polymorphic) life cycles. Pleomorphism or polymorphism. ( There are also physiological, biochemical, and metabolic differences). Blepharoblast ( An organelle derived from the centriole (consists of a minute mass of chromatin embedded in the cytoplasm at the base of the flagellum ) and giving rise to the flagella. Found chiefly in Protozoa and Algae) = basal body= kinetosome.
  • T. Vivax infect Cattle, T evansi: horse and Camel, T. Lewisi: Rat, T. Melophagium: goat and sheep (Melon: Sheep, Phagien: to eat).
  • (Anterior station development) & (poterior station development). Salivarian transmission is via the insect proboscis. In stercorarian transmission, the infective stage is deposited in feces on the skin while the vector is feeding; the trypanosomes enter via a break in the skin or through mucous membranes, including the conjunctiva of the eyes. The conjunctiva (plural = conjunctivas or conjunctivae) is a clear mucous membrane consisting of cells and underlying basement membrane that covers the sclera (white part of the eye ) and lines the inside of the eyelids . It is composed of rare stratified columnar epithelium. It helps lubricate the eye by producing mucus and tears , although a smaller volume of tears than the lacrimal gland . It also contributes to immune surveillance and helps to prevent the entrance of microbes into the eye.
  • Greek axōn axis + nēma thread, from nēn to spin — the fibrillar bundle of a flagellum or cilium that usually consists of nine pairs of microtubules arranged in a ring around a single central pair . Numerous eukaryotic cells carry whip-like appendages ( cilia or eukaryotic flagella ) whose inner core consists of a cytoskeletal structure called the axoneme. The axoneme of primary cilia typically has a ring of nine outer microtubule doublets (called a 9+0 axoneme), and the axoneme of a motile cilium has two central microtubule doublets in addition to the nine outer doublets (called a 9+2 axoneme). A = without, mastigote (from subphylum Mastigophora)– mastix (Gr.) for whip. I ntracellular form, may see > 100 amastigotes/cell.
  • volutin granules : electron-dense cytoplasmic inclusions, are intracellular storages of complexed inorganic polyphosphate found as cytoplasmic granules in certain bacteria, yeasts, and protozoa (such as trypanosome flagellates) and is believed to serve as a phosphate store. - Pallerla SR, et al. (2005). Formation of volutin granules in Corynebacterium glutamicum. FEMS Microbiol Lett. 243(1):133-40. - PRO = forward Pocket on anterior end ( A single free flagellum extends anteriorly from the axoneme) , the nucleus is centrally located. Kinetoplastid parasites: such as trypanosomes contain a specialised plasma-membrane domain known as the flagellar pocket.The trypanosome flagellar pocket is a small invagination of the plasma membrane where the flagellum exits the cytoplasm and participates in many cellular processes. It is the only site of exocytosis and endocytosis and part of a multiorganelle complex that is involved in cell polarity and cell division (1 & 2) 1. 2. Mark C. Field 1 & Mark Carrington. (2009). The trypanosome flagellar pocket. Nature Reviews Microbiology 7, 775-786.
  • EPI = upon
  • Metacyclic Tryposmastigote is the same as the tryposmastigote but is the infectious stage in the vector.
  • are defined by the position of the kinetoplast in relation to the nucleus and the length of the undulating membrane
  • Endemic in 36 countries and affects from 20,000 to 50,000 annually increased awareness and programs initiated by the WHO have led to a decreasing incidence and in 2009 there were less than 10,000 cases (see Simarro et al, 2011 ). 􀂄 Untreated is universally fatal 􀂄 Animal infections may have more impact than human infections by decreasing the food supply ( eg cattle, sheep, goats, pigs, chickens) Dose: 300-500 and may inject over 40 000 during a blood meal
  • Systematic screening, treatment, and patient follow-up was established in western and central Africa for the gambiense form of the disease while, animal reservoir and vector control was mainly implemented in eastern and southern Africa for the rhodesiense form. By the 1960s, transmission was practically interrupted in all endemic areas, providing evidence that the elimination of the disease as a public health problem was feasible and could be achieved with basic tools. Thereafter, the rarity of cases led to a loss of interest in sustained surveillance, and the risk of re-emergence of the disease was overlooked. Thus in the 1980s the disease re-emerged. By the 1990s, flareups were observed throughout past endemic areas, leading to a worrisome increase in the number of reported cases. At this time, nongovernmental organizations (NGOs) played a crucial role in the control of HAT. However, their interventions were mainly focused on remote and insecure areas. As emergency operators, their policy understandably excluded support to National Sleeping Sickness Control Programmes (NSSCPs), which resulted in (i) the establishment of substitute HAT control systems (ii), the maintenance of a large part of the population at risk out of the umbrella of NGO projects, and (iii) the difficulty for national programmes to sustain control achievements after the NGOs’ withdrawal. Concurrently, bilateral cooperation continued to support NSSCPs in some historically linked countries.
  • Name stems from sound made in flight; means “fly” in Tswana
  • From Tswana: there are 31 species, 11 of them are important in the transmission of the disease to human. Flies can remain infected for life (2-3 months).
  • East African Trypanosomiasis. flies are primarily day feeders.
  • Cerebrospinal fluid is a fluid that circulates throughout the central nervous system . The cerebrospinal fluid is located between the brain and skull. Therefore, it has 2 important benefits to the central nervous system: 1. circulation the cerbrospinal fluid delivers nutrients to the structures of the nervous system the cerebrospinal fluid removes wastes from the brain and spinal cord, detoxifying the environment of the nervous system 2. shock absorption the cerbrospinal fluid protects the brain and spinal cord from trauma brought upon by movement, falls, blows, etc.
  • Live in holes, like dark, humid sites
  • A chancre will always look like a sore (lesion) and will always appear at the site of injection (the place where the germs got onto the skin).
  • Swelling of the posterior cervical lymph nodes, characteristic of early stages of African trypanosomiasis; useful for surveys or control of migrations from endemic areas of persons with preclinical infections. The term Winterbottom's sign derives from descriptions of the posterior cervical lymphadenopathy associated with African trypanosomiasis made by Dr. Thomas Masterman Winterbottom (1766-1859) in 1803.
  • A young woman watches over her husband who is comatose, suffering from sleeping sickness.
  • In the early stages of the disease the parasite can be demonstrated in lymph nodes aand blood: later, they appear in the cerebral fluid. (Thick or thin blood film). Card Agglutination Test for Trypanosomiasis. Immunoflourscent antibody (IFAT) and ELISA may be useful for screening. IFAT may be useful for assessing cure.
  • Trypanosoma cruzi is the causative agent of Chagas' disease. Chagas' disease exhibits a patchy distribution throughout south and central America. The infective stage of the organism was discovered by chance while Carlos Chagas was studying the vectors. He named the organism after his mentor Oswaldo Cruz. Named after the Brazilian physician Carlos Chagas who described the infection in 1909, Host – man and at least 100 other species and 8 orders of mammals ( eg dogs, cats, opossum, raccoon, armadillo, monkeys, rats, etc.) 􀂄 15 million infected with 32 million at risk. 􀂄 Zoonotic in US ( eg Washington DC, California, Texas) since the bug rarely colonized US homes. a very mobile, thin shape with an elongated nucleus, a subterminal kinetoplast and short flagellum; and a thick, slow-moving shape with an oval nucleus and a long, free flagellum21
  • 15 million infected in Central and South America, and chronic infection is the leading cause of heart failure in these regions. 􀂄 Important cause of death in Central and South America (added to the ppt). 􀂄 It is relatively common in immigrants from Central and South America. Its one of the main health problems in Latin America . However, its control is complex since the parasite can survive in the bloodstream of different vertebrate hosts (200) and hematophagous Triatominae (Hemiptera: Reduviidae) vectors (Brigada et. al., 2010).
  • Members of this subfamily are blood-feeding insects and are called many different names including: triatomine bugs, reduviid bugs, kissing bugs, cone nosed bugs and assassin قاتل bugs. Reduviid bugs live in mud طين filled walls of huts أكواخ in rural areas. T. cruzi infection is primarily a zoonosis and humans are only incidental hosts; thus, natural transmission occurs primarily in rural areas where insects are abundant.
  • The skin lesion in acute Chagas disease.
  • Romana's sign , also known as a chagoma, is a medical term for the unilateral painless palpebral swelling associated with the acute stage of Chagas' disease . It is named after Cecilio Romaña , an Argentinian researcher who first described the phenomenon in 1935.
  • a) Thick and thin blood films are made and stained with Fields stain and examined as for malaria parasites. Wet preparations of blood can also be examined for motile trypanosomes. b) Buffy coat examination - Trypanosomes are centrifuged in a microhaematoctit tube for 5 minutes. Parasites can be seen microscopically at the junction of the packed red cells and plasma. This technique is rapid and sensitive. Trypanosoma cruzi can often be seen in C, U or S shapes in stained films. Blood culture is as sensitive as xenodiagnosis but it requires sterile conditions. Other lab findings include: Raised ESR, marked lymphocytosis with atypical mononuclear lymphocytes   Chronic Phase Diagnosis: Latex Agglutination, Direct Agglutination Tests
  • a) Thick and thin blood films are made and stained with Fields stain and examined as for malaria parasites. Wet preparations of blood can also be examined for motile trypanosomes. b) Buffy coat examination - Trypanosomes are centrifuged in a microhaematoctit tube for 5 minutes. Parasites can be seen microscopically at the junction of the packed red cells and plasma. This technique is rapid and sensitive. Trypanosoma cruzi can often be seen in C, U or S shapes in stained films. Blood culture is as sensitive as xenodiagnosis but it requires sterile conditions. Other lab findings include: Raised ESR, marked lymphocytosis with atypical mononuclear lymphocytes   Chronic Phase Diagnosis: Latex Agglutination, Direct Agglutination Tests
  • Xenodiagnosis is a process to diagnose an infectious disease by exposing tissue to a vector and then examining the vector for the presence of a microorganism or pathogen . Xenodiagnosis is useful in chronic and sub acute (low parasitaemia) disease. Sterile bugs are fed on patients by attaching a black bag containing the bugs to the arm of the patient and allowing them to feed for 30 minutes. Twenty five to thirty days later the bugs are dissected and the contents of the hindgut and rectum are examined microscopically for the presence of trypanosomes.
  • Lab 7 trypanosomiasis

    1. 1. Hemoflagellates: Trypanosoma spp. University of Sulaimani School of Science Department of Biology Practical Parasitology 2 nd stage Lab 7 : Trichomoniasis
    2. 2. <ul><li>Objectives: Students should be able to: </li></ul><ul><ul><li>Identify developmental stages of the life cycle of Trypanosoma spp. </li></ul></ul><ul><ul><li>List methods of diagnosis of Trypanosoma spp. </li></ul></ul>Trypanosomiasis
    3. 3. <ul><li>Trypanosomiasis is a group of diseases caused Trypanosoma spp. in different location in the world. </li></ul>Trypanosomiasis <ul><li>African trypanosomiasis </li></ul><ul><ul><li>(African Sleeping sickness) </li></ul></ul><ul><li>American trypanosomiasis </li></ul><ul><ul><li>(Chaga’s disease) </li></ul></ul>
    4. 4. Trypanosomiasis <ul><li>West-Africa trypanosomiasis: </li></ul><ul><li>Trypanosoma gambiense </li></ul><ul><li>East-Africa trypanosomiasis : </li></ul><ul><li>Trypanosoma rhodesiense </li></ul><ul><li>American trypanosomiasis: </li></ul><ul><li>Trypanosoma cruzi </li></ul>
    5. 5. <ul><li>Malaria </li></ul><ul><li>Onchocerciasis </li></ul><ul><li>Lymphatic filariasis </li></ul><ul><li>African Trypanosomiasis </li></ul><ul><li>American Trypanosomiasis </li></ul><ul><li>Leishmaniasis </li></ul>Trypanosomiasis Major Vector-Borne Diseases
    6. 6. <ul><li>Salivaria: </li></ul><ul><ul><li>Anterior section – develops in anterior portions of digestive tract. </li></ul></ul><ul><ul><li>Trypanosoma gambiense,T. rodesiense T. vivax; T. evansi, </li></ul></ul><ul><li>Stercoraria ( sterc/o feces) </li></ul><ul><ul><li>Posterior section – develops in hindgut of insect. </li></ul></ul><ul><ul><li>T. cruzi; T. melophagium; T. lewisi </li></ul></ul>Trypanosomes: Classification
    7. 7. <ul><ul><li>Amastigote (Leishmania) stage: Intracellular stage, replicate within cells of reticuloendothelial system; 2-4mm in diameter, spheroid or slightly oval, without flagella, may see > 100 amastigotes/cell. </li></ul></ul>Trypanosomes: Morphology
    8. 8. <ul><ul><li>Promastigote (Leptomonas stage): elongated or fusiform (from short and fat to long and thin) with flagella extending forward, the kinetoplastid located distally at the anterior end. without undulating membrane. </li></ul></ul>Trypanosomes: Morphology
    9. 9. <ul><ul><li>Epimastigot (C rithidia stage ): elongated with anterior flagella and undulating membrane. The kinetoplastid located near the nucleus. </li></ul></ul>Trypanosomes: Morphology
    10. 10. <ul><ul><li>Trypomastigote – in the blood stream; flagellum runs entire length from posterior. </li></ul></ul><ul><ul><ul><li>The K is posterior to the N. </li></ul></ul></ul><ul><ul><ul><li>Includes metacyclic (infective) stage in tsetse fly. </li></ul></ul></ul>Trypanosomes: Morphology
    11. 11. Polymorphic spindle-shaped parasites
    12. 12. <ul><li>Trypomastigotes in the blood stream </li></ul>Trypanosomes: Morphology
    13. 13. African Trypanosomiasis <ul><li>A systemic protozoal disease </li></ul><ul><li>Organisms grow in blood, lymph, CSF, and intercellular spaces (in contrast to T. cruzi) </li></ul><ul><li>Endemic in 36 countries and affects from 20,000 to 50,000 annually. </li></ul><ul><li>Untreated is universally fatal. </li></ul>
    14. 14. African Trypanosomiasis
    15. 15. Trypanosoma gambiense <ul><li>G.D. : West and central Africa </li></ul><ul><li>Disease: mid and west African sleeping sickness </li></ul><ul><li>Habitat: extracellular parasite in blood and other body fluid of vertebrates. </li></ul><ul><li>Vector: tsetse fly ( Glossina palpalis ) </li></ul>
    16. 16. Trypanosoma gambiense <ul><li>Vector - Tse Tse fly </li></ul><ul><ul><li>Glossina palpalis </li></ul></ul>The Mouthpart of the Vector injected to the skin of the victim during a blood meal
    17. 17. Trypanosoma rhodesiense <ul><li>G.D. : East of Africa </li></ul><ul><li>Disease: Rhodesian or east African sleeping sickness </li></ul><ul><li>Habitat: extracellular parasite in blood and other body fluid of vertebrates. </li></ul><ul><li>Vector: tsetse fly ( Glossina morsitans ) </li></ul>
    18. 18. Transmission Transmitted by Tsetse fly A pregnant women pass it to her fetus (rare) Through a blood transfusion (rare)
    19. 20. Trypanosoma gambiense African trypanosomiasis Taking up & injecting the infectious form:
    20. 21. Where patients live
    21. 22. Tissue phase: chancre
    22. 23. Winterbottom , s sign Enlarged cervical lymph nodes <ul><ul><ul><li>Appear at the base of skull; sign of certain death according to slave traders. </li></ul></ul></ul>Hemolymphatic phase
    23. 24. CNS phase
    24. 25. <ul><li>Tissue phase </li></ul><ul><ul><ul><li>Fluid aspirated from a chancre </li></ul></ul></ul><ul><li>Hemolymphatic phase </li></ul><ul><ul><ul><li>Lymph-node aspirate </li></ul></ul></ul><ul><ul><ul><li>Concentrated of the blood buffy coat </li></ul></ul></ul><ul><li>CNS phase </li></ul><ul><ul><ul><li>Double centrifugation technique . </li></ul></ul></ul>Diagnosis – Stage related
    25. 26. Trypanosoma
    26. 27. American Trypanosomiasis Dr. Carlos Chagas Central and South America. Chagas Disease Trypanosoma cruzi typical C or S-shaped form.
    27. 28. <ul><li>G.D. : South America. </li></ul><ul><li>Disease: American Trypanisomiasis or Chagas disease </li></ul><ul><li>Habitat: Blood, tissue cell especially heart muscle, nerves, skeletal and smooth muscle of GIT by way of the blood and lymphatic system </li></ul><ul><li>Vector: triatomine bug (reduviid bug) </li></ul>American Trypanosomiasis Triatoma infestans
    28. 29. <ul><li>Kissing bugs ( Triatoma infestans ) transmit </li></ul><ul><li>T. cruzi while feeding, </li></ul><ul><li>not by inoculation but by faecal contamination . </li></ul>
    29. 31. Chagas' disease <ul><ul><ul><li>Romaña sign: bipalpebral edema (unilateral) </li></ul></ul></ul>
    30. 32. Laboratory diagnosis <ul><li>Acute phase </li></ul><ul><ul><ul><li>Giemsa stained buffy coat blood smear </li></ul></ul></ul><ul><ul><ul><li>Biopsy specimen – find Trypomastigotes and Amastigotes </li></ul></ul></ul><ul><li>Chronic phase </li></ul><ul><ul><ul><li>Xenodiagnosis </li></ul></ul></ul><ul><ul><ul><li>Culture on NNN media </li></ul></ul></ul><ul><ul><ul><li>Serology –, IHA, IFAT, ELISA, </li></ul></ul></ul>
    31. 33. Laboratory diagnosis
    32. 34. Xenodiagnosis