Biology of parasitic protozoa II. Euglenozoa (Excavata, Discoba) Andrea Bardůnek Valigurová andreav@sci.muni.cz Notice This presentation contains some material available on the web without the permission of the creator or the copyright owner. This presentation is to be used for educational purposes only. Educational purposes are defined as a communicating material for a particular course of instruction or for the administration of such course. Educational purposes do not cover the use of copyright material in PowerPoint for public lectures or other purposes. 5 supergroups = megagroups Excavata • have a conspicuous ventral feeding groove that is “excavated” from one side and through which pass one or more recurrent flagella; the ventral groove has characteristic ultrastructure and is supported by microtubules • originally 2 flagellated state - many changes: multiplication of flagella, reduction or disappearance of the ventral groove • variety of free-living and symbiotic forms • not a monophyletic group • paraphyletic group with the ancestors of other living eukaryotes • parasitic species in Discoba•: Euglenozoa••• Euglenozoa • monophyletic (non-monophyletic*) group consisting of flagellates with very different modes of nutrition, including predation, osmotrophy, parasitism and photoautotrophy • paddle-shaped, discoidal mitochondrial cristae • 2 flagella: an anteriorly directed dorsal flagellum and a posteriorly directed ventral flagellum • flagellar apparatus consisting of 3 microtubular roots: dorsal root, intermediate root and ventral root • microtubule-reinforced ventral or anterior feeding apparatus (MtR pocket) • mostly anaerobic • 4 groups in Discoba•: Euglenidida, Diplonemea, Symbiontida and Kinetoplastea Kinetoplastea (Kinetoplastida) • at least one stage in the life cycle equipped with one or two flagella, arising from a prominent flagellar pocket • presence of extensive mitochondrial DNA, termed kinetoplast DNA • kinetoplast - DNA-containing granule within the cell's single mitochondrion • kinetoplast - modified mitochondrion,10-100 x 20 μm • based on morphology: biflagellate bodonids and uniflagellate trypanosomatids • free-living or parasitic • Bodonida (Eubodonida, Parabodonida, Neobodonida), Prokinetoplastida, Trypanosomatida Kinetoplast • network of concatenated circular DNA molecules and their associated structural proteins along with DNA and RNA polymerases • found at the base of a cell's flagella and associated to the flagellum basal body by a cytoskeletal structure • about 40 % of a total DNA, maxicircles 20-28 kb (RNA editing), minicircles 0.5-10 kb (encoding the guide RNA genes) • maxicircles and minicircles catenated to form a planar network chainmail Variations of kinetoplast networks Catenated kinetoplasts • eukinetoplast • circles are catenated to form a planar network that has a topology resembling that of chain mail in medieval armour • genera Trypanosoma, Crithidia and Leishmania Variations of kinetoplast networks Noncatenated kinetoplasts • prokinetoplast (pro-kDNA kinetoplast) • a bundle-like structure in the mitochondrial matrix that superficially resembles a kDNA disk • contains very little catenation, maxicircles and minicircles are relaxed instead of supercoiled Dimastigella, Cruzella, Ichtyobodo Variations of kinetoplast networks Noncatenated kinetoplasts • polykinetoplast (poly-kDNA kinetoplast) • kDNA is distributed among various discrete foci throughout the mitochondrial lumen • little catenation and no supercoiling Variations of kinetoplast networks Noncatenated kinetoplasts • pankinetoplast (pan-kDNA kinetoplast) • fills most of the mitochondrial matrix, not limited to discrete foci like poly-kDNA • also a lesser degree of catenation, minicircles are not relaxed but are supercoiled relaxed, in supercoils concatenated megacircles kinetoplast Eubodonida genus Bodo • free-living and parasitic, 3-15 μm • two heterodynamic flagella: a short anterior projecting flagellum and a longer posteriorprojecting flagellum without hairs (acronematic) that extends beyond the length of the cell • free-living bacteriotrophs Bodo urinarius • contaminated water, cysts Bodo saltans • free-living, distributed throughout the world in both freshwater and marine environments • a key species to study the origin of the parasitic trypanosomatids; complete genome sequencing Cryptrobia Parabodonida genus Cryptobia • no cysts • ectocommensals or ectoparasites of fish and amphibians - gills and skin; endocommensals or endoparasites of invertebrates and poikilothermal vertebrates (blood, digestive tract) • two flagella: free anterior flagellum with folds and a recurrent posterior flagellum marking the outer margin of undulating membrane • attached by recurrent flagellum • ventral and dorsal stripes of microtubules Parabodonida genus Cryptobia Cryptobia branchialis • ectoparasites on fish skin or gills • fish are anorexic and swim close to a water surface Cryptobia salmositica • blood parasites causing anemia and lesions in the hematopoietic tissues of salmonids • transmission by blood-feeding leeches Cryptobia helicis • receptaculum seminis of snails Parabodonida genus Trypanoplasma • haematozoic, digenetic endoparasites transmitted by leeches (Piscicola sp.) • undulating membrane and short anterior flagellum • large kinetoplast, surface glycocalyx – thinner in stages from leeches • non-sterile immunity Trypanoplasma borreli • 18-30 x 3-5 μm, pathogenic in cyprinid fish  anaemia and splenomegaly • affecting wild fish and commercial fisheries Prokinetoplastida genus Ichtyobodo (syn. Costia) Ichthyobodo necator (syn. Costia necatrix) Ichthyobodo hippoglossi • complex of species • does not form cysts and lasts several hours without host • 10 x 5 μm, feeding via cytostome and cytopharyngeal canal protruding into the host cell • gills and skin of fish + occasionally amphibian tadpoles, attached by an attachment plate  prominent mucus production • heavy infected fish exhibit anorexia and petechial haemorrhagic lesions in the skin Ichtyobodo necator  cytopharyngeal canal protruding into the host cell Trypanosomatida (trypanosomatids) lower trypanosomatids higher trypanosomatids monoxenous dixenous intestine of insects invertebrate vector transmission to plants vertebrate host genera Leptomonas Trypanosoma Phytomonas Endotrypanum Crithidia Leishmania Blastocrithidia Herpetomonas Morphology of trypanosomatids Major morphological classes of trypanosomatids Promastigote (leptomonad)* – flagellum and kinetoplast anterior to the nucleus and flagellum not attached to the cell Opisthomastigote (herpetomonad)* - flagellum posterior to the nucleus, passing through a long groove in the cell Amastigote (leishmanial)* - very short flagellum, projecting only slightly beyond the flagellar pocket Epimastigote (crithidial)* - flagellum exits the cell anterior to the nucleus and is connected to the cell for a part of its length by undulating membrane, kinetoplast located between the nucleus and the anterior cell end Trypomastigote (trypanosomal)* - flagellum lies attached to the cell for most of its length by undulating membrane, kinetoplast located near the posterior cell end swimming direction * in Czech: leptomonádové, herpetomonádové, leishmaniové, crithidiové a trypanosomové stádium Choanomastigote – flagellum emerges through a collar-like extension surrounding the anterior cell end Spheromastigote - flagellum develops and begins to function Czech etymology of morphological forms o „a“ - řecky = bez o „pro“ - řecky = před o „epi“ - řecky = nad o „trypanon“ - řecky = vrták o „choane“ - řecky = nálevka o „opisthe“ - řecky = vzadu Morphological forms of higher trypanosomatids genus Leptomonas • promastigotes • cyst-like stages (amastigotes in the form of pseudocyst) Leptomonas pyrrhocoris • intestine of Pyrrhocoris apterus L. ctenocephali • intestine of Ctenocephalides canis Lower trypanosomatids genus Phytomonas • promastigotes • most identified species have not been associated with any plant pathology • only two species spread by different insects cause plant disease Phytomonas staheli • “hartrot“ (fatal wilt of palms) of coconut palm (Cocos nucifera) P. leptovasorum • coffee phloem necrosis Lower trypanosomatids genus Crithidia • epimastigotes • common parasites of insect gut Crithidia oncopeli • true bug Oncopeltus fasciatus genus Blastocrithidia • epimastigotes, cysts Blastocrithidia culicis • Aedes and Culex mosquitos B. triatomae • true bugs (Triatoma infestans) genus Herpetomonas • dominant promastigote stage in the life cycle but producing also opisthomastigotes • parasite of Diptera and possibly other insects Herpetomonas muscarum H. ztiplika Higher trypanosomatids genus Trypanosoma • more than 300 species in all vertebrates families Stercoraria (stercus = faeces) Stercorarian trypanosomes infect the insect when taking a blood meal, most often a triatomid kissing bug, develop in its posterior gut and infective stages are released in the faeces and deposited on the skin of host vertebrate. Parasites then penetrate and can disseminate throughout the host body. Salivaria (saliva) Salivarian trypanosomes develop in the anterior gut of insects, most importantly the Tsetse fly, and infective stages are inoculated into the vertebrate host via the insect bite prior to feeding. Salivaria Trypanosoma brucei Stercoraria Trypanosoma cruzi Morphology of Trypanosoma spp. Stercoraria • multiplication in vertebrate blood and tissues – epimastigotes or amastigotes • intracellular in visceral tissues • originally classified in subgenera Megatrypanum, Herpetosoma, Schizotrypanum subgenus Megatrypanum • largest mammalian blood trypanosomatids (40-100 µm) • ruminant hosts • epimastigotes multiplying in vertebrates • small kinetoplast situated very close to the nucleus Trypanosoma (Megatrypanum) theileri • host: cattle, other ruminants • vector: tabanid flies • distributed worldwide from the tropics to near the Arctic Circle, with higher prevalence in tropical and neotropical areas • considered non-pathogenic Trypanosoma (Megatrypanum) melophagium • sheep parasite transmitted by louse flies, the sheep restricted ectoparasite Melophagus ovinus (Diptera: Hippoboscidae) • non-pathogenic • in about 90 % of sheep louse flies subgenus Herpetosoma • medium sized trypanosomatids (20-40 µm) • mostly rodent hosts • epimastigotes multiplying in vertebrates • non-pathogenic Trypanosoma (Herpetosoma) lewisi • host: rats, but found also in primates including man • vector: fleas (Nosopsyllus fasciatus, Xenopsylla cheopis) Trypanosoma (Herpetosoma) musculi • host: house mouse (Mus musculus) • vector: fleas subgenus Trypanosoma (Schizotrypanum) • small trypanosomes (15-24 µm) • multiplication in vertebrates – amastigotes Trypanosoma (Schizotrypanum) cruzi • Chagas disease • host: > 100 mammal species (rodents, opossums, armadillos, dogs..) including man • vector: "barbieros“ or „kissing bugs„ triatomine of the family Reduviidae (Triatoma, Rhodnius, Panstrongylus + other 12 genera); aggregating in refuges during day and searching for blood during night when the host is asleep, and the air is cooler • transmission can occur through blood transfusions, organ transplantation, transplacentally, and in laboratory accidents History of Chagas disease • the oldest record of Chagas disease (T. cruzi DNA) has been found in almost 9,000 years old mummies from northern Chile and southern Peru • evidence of Chagas disease vectors (Triatoma infestans) in human dwellings in preColumbian Inca and Chinchorro cultures, suggesting progressive introduction of domestic transmission • over the past 200-300 years, with progressive deforestation for agriculture and livestock ranching and construction of transport routes (highways and railways), triatomine bugs increasingly lost their primary food source of wild-animal blood  more opportunities to spread • 1907: dr. Carlos Chagas first becomes aware of the barbiero • 1909: first publications on newly discovered trypanosome • 1930s: public health importance becomes known Carlos Justiniano Ribeiro Chagas (1879-1934) Chagas disease https://www.youtube.com/watch?v=1ais69H0li8 https://www.youtube.com/watch?v=di72_yCsUVY Life cycle of Trypanosoma cruzi Chagas disease Incubation period • 5-4 days after exposure to triatomine insect faeces • 20-40 days after blood transfusion Acute phase • restricted to the inoculation site • most adults asymptomatic • parasites found in blood • Romaña's sign, a chagoma (=localised painless induration) over the eyelid marker of acute Chagas disease infection • oedema of eyes, conjunctivitis • usually resolves in weeks to months Chagas disease Intermediate phase • asymptomatic phase of varying length • parasites disappear from blood  most patients enter chronic phase within 5 -15 years • indeterminate phase can last as long as 40 years Chronic phase • degenerative disease of hollow organs and organ failures • heart disease – most common chronic form • digestive organs abnormalities megaoesophagus, megacolon • up to 10% of deaths in endemic areas • successful treatment only in the acute phase (benznidazole, nifurtimox) Chagas disease Immunocompromised people can be severely affected Pregnant women • congenital infection • premature birth AIDS patients • brain abscesses • higher likelihood of reactivation https://www.sciencedirect.com/science/article/pii/S1471492215001439 Microscopy – acute stage ✓ blood ✓ CSF ✓ tissues Parasite isolation ✓ xenodiagnosis (exposition to and examination of kissing bug for parasites) ✓ cultivation ✓ Serology – chronic stage ✓ IFAT ✓ ELISA Molecular techniques ✓ PCR Diagnosis of Chagas disease Chagas disease Transmission of T. cruzi Three basic transmission cycles 1. sylvatic (wild) • wildlife-insect transmission • human infections rare 2. domestic • human-insect transmission 3. peridomestic Transmission: • blood, organs, ingestion, in utero, milk Increased global population mobility increased the possibility of establishing vector transmission to areas where Chagas disease was previously non-endemic (Asia, Australia). Red Endemic area of Chagas disease transmitted by local vectors. Yellow Endemic area of Chagas diseases transmitted by local vector occasionally. Blue Non-endemic areas of Chagas disease introduced by imported cases with nonvectorial transmission Global spreading patterns of Chagas disease no vaccine available vector control remains the most effective method of preventing transmission in Latin America blood screening has become increasingly more important to prevent infection through transfusion and organ transplantation Chagas disease control Drugs: nifurtimoxand, benznidazole Common side effects of benznidazole treatment: allergic dermatitis, peripheral neuropathy, anorexia and weight loss, insomnia Common side effects of nifurtimox treatment: anorexia and weight loss, polyneuropathy, nausea, vomiting, headache, dizziness or vertigo Chagas disease prevention prevent contact with triatomine bugs and their faeces improve substandard housing use screens/bed nets when sleeping spray homes with insecticides cook contaminated foods screen blood and organ donors travellers should wear thick clothing and avoid substandard housing Chagas disease prevention Chagas disease prevention Salivaria • originally parasites of tsetse flies • complex of species • parasites of vertebrates - trypomastigotes in blood, lymph and cerebrospinal fluid • taxonomy based on morphology and location of trypomastigotes subgenus Trypanozoon - Brucei group • small kinetoplast, undulating membrane subgenus Nannomonas - Congolense group • middle sized kinetoplast, without free flagellum subgenus Dutonella - Vivax group • large terminally located kinetoplast, free flagellum Salivaria African trypanosomes in domestic animals Glossina sp. Glossina sp. under scanning electron microscopy Glossina palpalis Glossina morsitans Glossina morsitans http://www.raywilsonbirdphotography.co.uk/Galleries/Invertebrates/vectors/Tsetse_Fly.html https://www.youtube.com/watch?v=odCtCote9U0 Distribution of tsetse fly Trypanosoma species T. b. rhodesiense T. b. gambiense tsetse vector G. morsitans G. palpalis ecology dry bush, woodland rainforest, riverine, lakes transmission cycle ungulate-fly-human human-fly-human non-human reservoir wild animals domestic animals epidemiology sporadic, safaris endemic, some epidemics disease progression rapid, often fatal slow (~1 yr.), chronic asymptomatic carriers rare common Sleeping sickness in humans Morphology of Trypanosoma brucei brucei slender forms stumpy form (without free flagellum) flagellar pocket with cytostomal activity subtending granular reticulum Golgi apparatus flagellum kinetoplast subpellicular microtubules glycosome basal body anterior granular reticulum food vacuole mitochondrion paraxial rod attached part of flagellum (undulating membrane) attaching zone of the flagellum Life cycle of Trypanosoma brucei • 20-30 days • metacyclic trypomastigotes  mammalian bloodstream • differentiation into proliferating long slender forms • differentiation into short stumpy forms (pre-adapted to survive in the tsetse fly) • differentiation into procyclic trypomastigotes in tsetse fly midgut • migration through the peritrophic matrix into the salivary gland, to generate one long epimastigote and one short epimastigote • attached short epimastigotes generate free metacyclic trypomastigotes in the salivary gland lumen • „T. brucei parasite has evolved an elegant mechanism to display a completely new coat of VSG antigen, rendering it once again invisible to the host’s immune system. The parasite’s genome has over 1,000 genes that code for different variants of the VSG protein.“ • around 107 Variant Surface Glycoprotein (~60kDa protein densely coating the cell surface) molecules expressed on the parasite’s cell surface, • 6-10% of the total genome is coding for VSGs (over 1,000 genes) • only one is expressed at a given time, others are „silent“ • this 12-15 nm thick coat is doffed periodically (internalised via the flagellar pocket) and replaced with an antigenically distinct version of VSG  this antigenic variation causes cyclical waves (5-8 days) of parasitemia Antigenic variation in African trypanosomes History Pathogenesis – general • trypanosomes live in blood, lymph nodes, spleen (= not intracellular) • they are particularly abundant in intercellular spaces in brain • clinical course depends on host susceptibility • T. b. brucei-vertebrate hosts (Equidae, dogs, some ruminants) exhibit acute disease with death in ~ 2 weeks • if the host survives, blindness develops (especially common in dogs) Clinical features of African trypanosomiasis https://youtu.be/EnsydwITLYk Humans • local reaction: painful sore at site of bite - disappears after a couple of weeks • trypanosomes reproduce rapidly after entering the blood and lymph system  lymphadenopathy, generalized invasion of all organs • Winterbottom’s sign – swollen lymph nodes at skull base (a sign of certain death according to slave traders) • binding of specific antibody to adsorbed trypanosome on host cell, coupled with complement, leads to lysis – cause of anaemia Pathogenesis of African trypanosomiasis Mechanisms under investigation • circadian rhythms - alterations in endogenous rhythms correlate with clinical symptoms • suprachiasmatic nucleus (SCN) – “biological clock” – regulating the hormonal, sleep, body thermostat activity • spontaneous rhythm of SCN is altered with trypanosome infection Pathogenesis of African trypanosomiasis • clinical features of infection are not sufficiently specific • based on finding the parasite in body fluid or tissue by microscopy • parasite load in T. b. rhodesiense infection is substantially higher than in T. b. gambiense • T. b. rhodesiense parasites are easily found in the blood, but it is difficult to detect T. b. gambiense there • parasites may also be found in lymph node fluid or in fluid or biopsy of a chancre • classic method of diagnosing T. b. gambiense infection is a microscopic examination of a lymph node aspirate, usually from a posterior cervical node Diagnosis of African trypanosomiasis Progression of African trypanosomiasis ✓ examination of cerebrospinal fluid obtained by lumbar puncture ✓ “buffy coat” (haematocrit centrifugation) ✓ serology ✓ CATT “card agglutination test for trypanosomes” ✓ PCR Diagnosis of African trypanosomiasis Distribution of human African trypanosomiasis in endemic countries HAT cases that were diagnosed and confirmed in non-endemic countries from 2011 to 2018 Risk factors ✓ geographic region ✓ occupation ✓ socioeconomic status ✓ host susceptibility – genetics? Epidemiology of African trypanosomiasis https://doi.org/10.1002/9780470688618.taw0183 Comparison of the biology of African trypanosomes subspecies: T. b. gambiense and T. b. rhodesiense Epidemiology of African trypanosomiasis The dynamic trend changes in HAT cases diagnosed in non-DECs and DECs DEC - disease-endemic countries Treatment of African trypanosomiasis First stage treatments • Pentamidine: discovered in 1941, used for the treatment of the first stage of T. b. gambiense sleeping sickness. Despite a few undesirable effects, it is well tolerated by patients. • Suramin: discovered in 1921, used for the treatment of the first stage of T. b. rhodesiense sickness. It provokes certain undesirable effects in the urinary tract and allergic reactions. Second stage treatments • Melarsoprol: discovered in 1949, used in both forms of infection. It derives from arsenic and has many undesired side effects - the most dramatic is reactive encephalopathy (encephalopathic syndrome) that may be fatal (3-10 %). An increase of resistance to the drug in several foci - particularly in central Africa. • Eflornithine: registered in 1990, a less toxic alternative to melarsoprol treatment. Effective only against T. b. gambiense. The regime is strict and difficult to apply. Trypanosoma species in domestic animals Salivaria African Trypanosoma spp. in domestic animals Economic impact on agriculture • >3 million deaths per year, major reduction in food production • 50% reduction in herd size • 25% reduction in milk production • 20% loss in calving zebu cattle N’Dama – trypanotolerant population Trypanosoma b. equiperdum Dourine = syphilis equorum • cosmopolitan distribution • in horses and other equids • in genital organs and secretions • genetic analyses  derivative of T. brucei • spreading primarily via sexual transmission = adaptation that has allowed the parasite to escape beyond the range of the tsetse fly • mother-foal transmission possible • donkeys are carriers but show no symptoms Other higher trypanosomatids Endotrypanum schaudinni • dixenous • unique among the Kinetoplastida in that they infect erythrocytes of their mammalian host (forest-dwelling two-toed sloths of the genus Choloepus) • probably transmitted by the bite of infected phlebotomine sandflies (Diptera: Psychodidae) • trypomastigotes in mammalian erythrocytes, promastigotes in vectors (sandflies) according to phylogenetic data, the genus Endotrypanum set inside the Leishmania group Trypanosomatids of fish and amphibians • transmission via blood sucking leeches – epimastigotes in gut, trypomastigotes in proboscis • transformation to metacyclic trypomastigotes Trypanosoma carassii • thought to be a non-pathogenic trypanosome offish in natural population • vascular system of many economically important fish such as carp (Cyprinus carpio), eel (Anguilla spp.) or tench (Tinca tinca) Trypanosoma rotatorium • trypomastigotes reaching up to 70 μm • host: frogs (Pelophylax esculentus, Rana temporaria) • vector: freshwater leeches Hemiclepsis marginata genus Leishmania • host: vertebrates (mostly hyraxes, canids, rodents) including human • vector: phlebotomine sand flies • obligate intracellular parasite – amastigotes in mononuclear phagocytes and circulatory systems of vertebrate host • motile and extracellular promastigotes in alimentary tract of sandflies • leishmaniasis classified as a neglected tropical disease (NTD), zoonosis, human infection is caused by more than 20 species • affecting 6 million people in 98 countries – cca 0.9-1.6 million new cases occur each year, and 21 species are known to cause disease in humans Life cycle of Leishmania https://www.youtube.com/watch?v=wzjSmxThgJ0 https://www.youtube.com/watch?v=d-ROuaznkSM Leishmania life cycle stages metacyclic promastigotes procyclic promastigotes amastigotes Morphology of Leishmania amastigote 2.5-5 x 1.5-2 μm promastigote 14-20 x 1.5-3.5 μm Morphology of Leishmania macrophage practically filled with amastigotes amastigotes are being freed from a bursting macrophage amastigotes with visible nuclei and kinetoplasts promastigotes from culture genus Phlebotomus • phlebotomine sand about 1.5–3.0 mm long with hairy bodies, wings, and and legs • yellowish in colour with conspicuous black eyes • found only in the Old World • number of Phlebotomus species occur in Europe, their range has increased in recent years • only females are blood-feeding P. duboscqi Phlebotomus papatasi Lutzomia longipalpis genus Lutzomia • phlebotomine sand flies with a hairy body, length of up to only 3 mm • nearly 400 species (at least 33 species are medically important vectors) • found only in the New World • only females are blood-feeding Suprapylaria - only in mammals transmitted by the bite of a sandfly, development occurs in the foreand midgut of the fly. Hypopylaria - in lizards that ingest the sandfly intermediate host. Development occurs in the hindgut of the fly. Peripylaria - in mammals and lizards, development occurs in the foregut and hindgut of the fly. Development of Leishmania in insect vector Leishmania does not infect salivary glands !!! Development of Leishmania in insect vector Development of Leishmania in insect vector https://doi.org/10.1016/j.ijpara.2004.07.010 • promastigote attach to the macrophage surface and after being phagocytosed they reside within phagolysosomal vacuoles and transform into amastigotes • survival and multiplication of amastigotes in phagolysosome ✓ inhibition of the production of oxygen radicals ✓ inhibition of hydrolases Interactions of Leishmania with host macrophages https://www.youtube.com/watch?v=0J6TMd-x6o0 Acute response to Leishmania infection by a sandfly bite: (1) Neutrophils, recruited from the blood to the infection site, undergo NETosis and phagocytose the promastigotes. (2) Infected neutrophils recruit dendritic cells by producing CCL3, which subsequently engulf the apoptotic bodies of infected neutrophils and (3) lose their ability to effectively activate Th1 response. (4) Macrophages become infected by the parasites released by the dying neutrophils. (5) CD11c+ monocytes are highly permissive to parasite replication and further promote infection. https://www.frontiersin.org/articles/10.3389/fimmu.2021.671582/full https://doi.org/10.3390/microorganisms9122434 „Silent“ invasion and host-parasite interactions in Leishmania https://doi.org/10.3390/microorganisms9122434 „Silent“ invasion and host-parasite interactions in Leishmania https://doi.org/10.3390/microorganisms9122434 „Silent“ invasion and host-parasite interactions in Leishmania • human infection caused by about 21 of 30 Leishmania species infecting mammals • different species are morphologically indistinguishable • antropozoonoses vs. antroponoses ▪ L. tropica, L. major, L. aethiopica; Old World cutaneous leishmaniasis ▪ L. mexicana complex with 3 main species: L. mexicana, L. amazonensis, and L. venezuelensis; New World cutaneous leishmaniasis ▪ subgenus Viannia with 4 main species: L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) panamensis, L. (V.) peruviana; New World leishmaniasis ▪ L. donovani complex with 2 species: L. donovani, L. infantum (also known as L. chagasi in the New World); Old World visceral leishmaniasis Human Leishmania species Human leishmaniosis • different forms of human leishmaniasis • cutaneous leishmaniosis - most common form ➢ skin sores ➢ sores usually are painless but can be painful • visceral leishmaniasis ➢ affects several internal organs (usually spleen, liver, and bone marrow) ➢ can be life threatening ➢ illness typically develops within months (sometimes years) after sand fly bite • mucosal leishmaniasis ➢ can be a sequela (consequence) of cutaneous leishmaniasis in parts of Latin America Species Disease Hosts Distribution L. major cutaneous leishmaniasis (wet form) humans, rodents Africa, Asia L. tropica cutaneous leishmaniasis (dry form) humans, hyrax Africa, Asia, Europe L. braziliensis espundia, mucocutaneous leishmaniasis humans, rodents South America L. mexicana localised & diffuse cutaneous leishmaniasis humans, rodents Central America L. infantum visceral leishmaniasis (mostly) humans, dogs Africa, Europe, South America L. donovani kala azar, visceral leishmaniasis humans, dogs Africa, Asia, Europe L. chagasi = synonymised recently with L. infantum Leishmania tropica • urban type • chronic dry sore, “oriental sore“ • vector: Phlebotomus sergenti • Mediterranean, Middle East, Central Asia, India • anthroponosis • possible visceralisation – Persian Gulf War syndrome??? Leishmania tropica • strong immune response and few parasites during relapse Leishmania major • rustic type, acute wet sore • incubation period lasts 1-4 weeks, healing in 3-6 months • vector: Phlebotomus duboscqi, P. papatasi • arid regions, semi-deserts - Africa, Middle East, Asia • reservoir: gerbils (Rhombomys opimus, Meriones spp. Arvicanthis niloticus, Psammomys obescus) • vaccination through leishmanisation with live L. major has been used successfully but is no longer practiced because it resulted in occasional skin lesions Histological section of ear pinna of BALB/c mouse with advanced leishmaniasis (left) and a healthy mouse ear pinna (right). Stained with Masson's green trichrome; ellipse indicates the lesion. Pathology of cutaneous leishmaniasis caused by Leishmania major Detail of the edge of a skin lesion in the ear pinna of a mouse infected with L. major. Haematoxylin-eosin (left) and Giemsa (right) stained histological sections. Circle - macrophage with Leishmania amastigotes (→), ➢ / ➢ - neutrophils, ➢ - lymphocytes, ➢ eosinophils, ➢ - mast cell, ➢ - cartilage. Pathology of cutaneous leishmaniasis caused by Leishmania major Leishmania aethiopica • cutaneous - dry sore, oedematous non-ulcerating lesions • often mucosal leishmaniasis or diffuse leishmaniasis • chronic slow disease course (3 years) • Ethiopia, Kenya • reservoir: hyrax Diagnosis of cutaneous leishmaniosis Leishmania mexicana complex • vector: Lutzomiya longipalpis, etc. • reservoir: forest rodents • Central America Leishmania m. amazonensis • diffuse cutaneous leishmaniasis Leishmania m. mexicana • “ulcera des chicleros” • on auricle, metastasis in the adjacent cartilages Achras zapota Leishmania (V.) braziliensis • “Espundia“ • forests in the Amazon basin • reservoir: small forest rodents • metastasizes to the nasopharyngeal mucosa Leishmania (V.) quyanensis • “pian bois” • vector: Lutzomyia umbratilis, etc. • reservoir: sloths, dogs • secondary lesions, spreading along the lymphatics - diffuse leishmaniasis • weak induction of immune responses (high parasite burden in the lesion, low antibody Leishmania donovani complex • visceral leishmaniosis - complex of two species: L. d. donovani, L. d. infantum William Leishman and Charles Donovan detected amastigotes within macrophages of people with Kala-azar Leishmania d. infantum • Old World infant visceral leishmaniasis • reservoir: canids; vector: Phlebotomus papatasi, P. perniciosus, etc. • viscerotropic and dermotropic variant Leishmania d. donovani • vector: Phlebotomus orientalis, P. martini • reservoir: canids • “Kala azar“ „Dum dum fever“ ➢ weight loss, weakness ➢ cough, fever that lasts for weeks or months ➢ enlarged spleen, enlarged liver ➢ decreased production of red blood cells (RBCs) ➢ bleeding, night sweats, thinning hair ➢ scaly skin, dark, ashen skin • estimated 200,000 to 400,000 infections each year worldwide • over 90% of new cases occur in 6 countries: Bangladesh, Brazil, Ethiopia, India, South Sudan and Sudan • fatality rate in developing countries can be as high as 100 % within 2 years Diagnosis of “Kala azar“ or „Dum dum fever“ Leishmania d. donovani ✓ biopsy of the spleen and culture ✓ bone marrow biopsy and culture ✓ direct agglutination assay ✓ indirect immunofluorescent antibody test ✓ Leishmania-specific PCR test ✓ liver biopsy and culture ✓ lymph node biopsy and culture ✓ Montenegro skin test (not approved in the USA) ✓ skin biopsy and culture Serological diagnosis of visceral leishmaniosis direct agglutination test rK39 immunochromatographic test The DAT and rK39 RDT are both based on the detection of antibodies in blood. Due to the persistence of antibodies over long periods, they cannot be used to differentiate between current and past infection. To overcome this limitation, tests are being developed that can detect VL antigen in urine and blood but their performance so far has been suboptimal. Post-kala-azar dermal leishmaniasis (PKDL) • developing usually 6 months to 1 or more years after kala-azar has apparently been cured, but can occur earlier • occurring mainly in East Africa and on the Indian subcontinent, where 5-10% of patients with kala-azar are reported to develop the condition • macular, papular or nodular rash on face, upper arms, trunks and other parts of the body • human with PKDL considered a potential source of Leishmania infection WHO's work on leishmaniasis control involves: ✓ supporting national leishmaniasis control programs technically and financially to produce updated guidelines and make disease control plans, including sustainable, effective surveillance systems, and epidemic preparedness and response systems ✓ monitoring disease trends and assessing the impact of control activities which will allow raising awareness and advocacy on the global burden of leishmaniasis and promoting equitable access to health services ✓ developing evidence-based policy strategies and standards for leishmaniasis prevention and control and monitoring their implementation ✓ strengthening collaboration and coordination among partners and stakeholders ✓ promoting research and use of effective leishmaniasis control including safe, effective and affordable medicines, as well as diagnostic tools and vaccines ✓ supporting national control programs to ensure access to quality assured medicines Thank you for your attention ☺ Lectures ✓ Introduction: BPP 2022 I ✓ Euglenozoa (Excavata): BPP 2022 II  Fornicata / Preaxostyla / Parabasala (Excavata): BPP 2022 III • Apicomplexa I (SAR): BPP 2022 IV • Apicomplexa II (SAR): BPP 2022 V • Amoebae (Excavata, Amoebozoa): BPP 2022 VI • Ciliophora, Opalinata (SAR): BPP 2022 VII • Pneumocystis (Opisthokonta, Fungi): BPP 2022 VIII • Microsporidia (Opisthokonta, Fungi): BPP 2022 IX • Myxozoa (Opisthokonta, Animalia): BPP 2022 X