Biology of parasitic protozoa III. Fornicata, Parabasala, Preaxostyla (Excavata) 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 • 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 Metamonada•: Fornicata•••, Parabasalia•••, Preaxostyla••• Fornicata • together with Preaxostyla (oxymonads) and Parabasalia named as metamonads = group of flagellate amitochondriate protozoa • unicellular heterotrophic flagellates • anaerobic / microaerophilic • mostly flagellated cells, usually with 4 kinetosomes per kinetid; • some are free-living, many endobiotic or parasitic • modified atypical, non-respiratory mitochondria, lacking the cristae and genome (DNA) (e.g. hydrogenosomes or mitosomes) Hydrogenosome • membrane-enclosed organelles of some anaerobic ciliates, trichomonads, fungi, and animals • approximately 1 μm in diameter, under stress conditions up to 2 μm • are so called because they produce molecular hydrogen • bound by distinct double membranes and one has an inner membrane with some cristae-like projections • genes for hydrogenosomal components in the nuclear genome Fornicata – atypical mitochondria Mitosomes • detected only in anaerobic or microaerophilic organisms that do not have mitochondria (microsporidia, Giardia intestinalis, Entamoeba spp.) • about 0,1 μm in diameter • just like mitochondria surrounded by a double membrane • genes for mitosomal components are contained in the nuclear genome Fornicata – atypical mitochondria Electron micrographs of different mitochondrial manifestations Fornicata • monophyletic taxon • unicellular heterotrophic flagellates with one or two karyomastigonts per cell • karyomastigont – first used by Janicki (1915) - conspicuous organellar system observed in certain protozoans • mastigont by definition is present in all ciliated or flagellated eukaryotes and bears the basal body, cilium or flagellum (undulipodium) and in some cases the parabasal body (Golgi complex supported by a parabasal fiber) • karyomastigont (= mastigont associated with nucleus) bears 1-4 flagella ▪ Carpediemonas-like organisms (carpediemonads) - free-living ▪ Retortamonadida (retortamonads) - almost exclusively parasitic ▪ Diplomonadida (diplomonads) - parasitic Carpediemonas-like organisms • marine, free-living flagellates • uninucleate (unikaryotic) • anaerobic / microaerophilic flagellated cells with a broad ventral suspension = the feeding groove • almost always biflagellated • Carpediemonas bialata and other „Carpediemonas-like“ organisms (= CLOs) Fornicata Carpediemonas-like organisms a-b) Carpediemonas membranifera c-d) Kipferlia bialata e) Dysnectes brevis f) Dysnectes sp. g) Hicanonectes teleskopos h) Ergobibamus cyprinoides i-j) Carpediemonas-like organism k-l) Aduncisulcus sp. m-n) Aduncisulcus paluster Fornicata Retortamonadida • uninucelate • phagotrophic • conspicuous apical cytostome associated with the posterior flagellum • system of microtubules underlying the plasmatic membrane-microtubular cytoskeleton/corset • genera Retortamonas, Chilomastix, both with trophozoites in host intestinal tract and piriform shaped cysts in environment genus Retortamonas • biflagellate, more than 20 described species genus Chilomastix • quadriflagellate, about 30 described species Fornicata genus Retortamonas • biflagellate - 1 anterior + 1 recurrent (trailing) flagellum extending through the cytostomal groove • 4 kinetosomes • intestinal commensals Retortamonas intestinalis • colon of man and monkeys Retortamonas caviae • guinea-pigs Retortamonas blattae • cockroaches Retortamonas testudae • tortoises Cysts and trophozoites of R. intestinalis are shed in faeces. Infection occurs after the ingestion of cysts in faecalcontaminated food/water, or on fomites. In the large (and possibly small) intestine it excysts and releases trophozoites and then resides in colon as a commensal (not known to cause disease). The presence of trophozoites and/or cysts in stool specimens can be an indicator of faecal contamination of a food or water source, and thus does not rule-out other parasitic infections. Life cycle of Retortamonas intestinalis ovoid or pyriform cysts measuring 4-7 x 3-5 μm genus Chilomastix • quadriflagellate - 3 anterior and 1 short recurrent (cytostomal) flagellum entirely located in the cytostomal pouch • spiral groove crossing over the middle half of the body • the beating of anterior flagella moves the cell, while the undulation of the recurrent flagellum propels food into the cytopharynx pear-shaped trophozoites measuring 6-24 x 3-10 μm piriform /lemon-shaped cysts measuring 6-10 μm Chilomastix mesnili • human non-pathogenic flagellate, found also in non-human primates and pigs Trophozoite of C. mesnili from a stool specimen stained with trichrome C. mesnili cysts - Iodine stain Trophozoites of Chilomastix mesnili Morphology and life cycle of Chilomastix mesnili Chilomastix caulleryi • amphibians and leeches C. equi • horses C. gallinarum • birds C. bursa • lizards C. motellae • marine fish Chilomastix bandicooti C. bettencourti C. rosenbuchi • rodents C. wenrichi • guinea pigs C. caprae • goats, cattle C. cuniculi • rabbits Diplomonadida • most diplomonads are diplozoic (= 2 karyomastigonts), i.e. all cellular structures are doubled and the cell is axially symmetric Hexamitidae • unizoic diplomonads: genus Enteromonas (enteromonads) • diplozoic diplomonads: genera Trepomonas, Hexamita, Spironucleus • tube-like cytostomes passing through the entire cell and opening posteriorly or pocketlike grooves harbouring 3 flagella • trophozoite x cyst Giardiidae • dorsoventrally flattened cell and the anterior part of the ventral surface is modified into an adhesive disc • lacking cytostomes (feeding by pinocytosis) • recurrent flagella passing through the cytoplasm as naked axonemes • genera Octomitus, Giardia • trophozoite x cyst Fornicata genus Enteromonas • oval trophozoites, 4-10 μm • quadriflagellate - 3 anterior flagella + 1 recurrent flagellum that adheres to the body ending in a tail and produces a rotational movement • no cytostome, only shallow oral gutter • cysts with 1, 2 or 4 nuclei, binucleate forms being the most common Diplomonadida - Hexamitidae Fornicata Enteromonas hominis • human non pathogenic flagellate, • indicator of faecal contamination of a food and water source Cyst of E. hominis in a stool specimen stained with trichrome Enteromonas intestinalis • appendix of rabbits E. ratti • rats E. caviae • guinea-pigs E. suis • pigs E. lagostomi • viscacha Lagostomus maximus E. wenyoni • big-eared opossum Didelphis aurita genus Trepomonas • all free-living, in ponds rich in decaying organic matter and infusions • oval to elliptical trophozoites, 7-15 μm • octaflagellate – 2 lateral flagella for rotational movements + 2 lateral pocket-like grooves each harbouring 3 flagella • 2 cytostomes Trepomonas agilis T. angulatus T. rotans T. simplex T. steinii Diplomonadida - Hexamitidae Fornicata https://doi.org/10.1186/s12915-016-0284-z Diplomonadida - Hexamitidae genus Hexamita • octaflagellate – 6 anterior + 2 posterior flagella • 2 cytostomes Fornicata Hexamita salmonis • significant anorexia in infected salmonids https://www.int-res.com/articles/dao/25/d025p229.pdf Hexamita axostyles H. giganti H. globulus H. guanqiaoensis • fish H. nelsoni • oysters H. cryptocerci H. periplanetae • cockroaches Hexamita gigas • hind-gut of horse-leech (Haemopis sanguisugae) https://doi.org/10.1017/S0031182000019351 Phases of cell division Diplomonadida - Hexamitidae genus Spironucleus • octaflagellate – 6 anterior flagella arising back to the anterior pole + 2 posterior flagella emerging posteriorly - recurrent flagella toward the posterior cytostomal apertures • 2 slender cytostomes • elongate kidney-shaped nuclei • elliptical cyst Fornicata Spironucleus elegans • amphibians and fish S. salpae • marine fish Box salpa Spironucleus anguillae S. barkhanus S. torosa S. vortens S. salmonicida S. mobilis • fish Spironucleus columbae • pigeons S. meleagridis • birds S. muris • rodents Spironucleus vortens https://www.int-res.com/abstracts/dao/v45/n3/p197-202/ Spironucleus meleagridis https://doi.org/10.1637/7250-080204R Spironucleus meleagridis ??? https://doi.org/10.1177/03009858103637 genus Octomitus • 12-17 μm, no adhesive disc • 2 anterior nuclei • octaflagellate – 6 antero-lateral flagella + 2 posterior flagella which traverse the cell axially and are lined by microtubular fibres • no cytostomal opening at the emergence of the trailing flagella Octomitus intestinalis • intestine of rodents O. neglectus • amphibians Diplomonadida - Giardiidae Fornicata axial columna striated lamella RER axonemes funis secondary MT genus Giardia Diplomonadida - Giardiidae https://www.youtube.com/watch?v=2R7fTTsXQzYGiardia intestinalis, trichrome stained stool specimen Fornicata https://www.youtube.com/watch?v=UbSUE0gYfxI Giardia intestinalis trophozoites 10-20 x 7-10 μm cysts 11-14 x 7-10 μm Giardia intestinalis https://www.youtube.com/watch?v=8X4bxITNPeg Giardia intestinalis Life cycle and infection of Giardia intestinalis Discovery of Giardia Correspondence between Anthoni van Leeuwenhoek and Robert Hooke, a curator of experiments of the Royal Society in London Letter to Robert Hook (1681) I weigh 160 pounds and have been of the same weight for some 30 years, and ordinarily in the morning I have a well-formed stool; however, now and then I have had a looseness at intervals of 2, 3, or 4 weeks when I went to stool 2, 3, or 4 times a day. My excrement being so thin I was at diverse times persuaded to examine it. I have generally seen in my excrement many irregular particles of sundry sizes.. All lay in a clear transparent medium, wherein I have sometimes seen animalcules moving very prettily; some of them a bit bigger, others a bit less than a blood globule…their bodies were somewhat longer than broad, and their belly, which was flat like, furnished with sundry little paws, wherewith they made such a stir in the clear medium and among the globules…they made a quick motion with their paws, yet for all that they made but slow progress. 1859 Vilém Lambl, Praha - Cercomonas intestinalis in humans 1875 Daviane - Hexamita duodenalis 1882 Kunstler - Giardia agilis 1888 Blanchard - Lamblia intestinalis 1914 Alexeieff - Lamblia / Giardia 1952 Filice - Giardia duodenalis Current synonyms: Giardia intestinalis, Giardia duodenalis, Giardia lamblia, Lamblia intestinalis, Lamblia lamblia History of Giardia intestinalis Karyomastigont of Giardia intestinalis Subcellular organisation of Giardia intestinalis TEM visualisation of funis: https://doi.org/10.1016/j.jsb.2004.01.017 ventrolateral flange peripheral vacuoles Subcellular organisation of Giardia intestinalis https://doi.org/10.1016/S0020-7519(02)00228-X Subcellular organisation of Giardia intestinalis Morphology of Giardia spp. • known as beaver fever • Giardia inhabits the digestive tract of a wide variety of domestic and wild animal, as well as humans • one of the most common pathogenic parasitic infections in humans worldwide • in 2013, there were about 280 million people worldwide with symptomatic giardiasis • incubation period 12-19 days – acute phase symptoms: diarrhoea, greasy stool without blood, weakness, convulsions, vomiting, malabsorption • most infected people are asymptomatic, only about a third of infected people exhibit symptoms • zoonotic potential Giardiosis https://www.youtube.com/watch?v=xjYgfjVZ-Vk https://doi.org/10.1016/j.ijpara.2013.06.001 https://doi.org/10.1016/j.pt.2008.11.006 Zoonotic potential of Giardia intestinalis https://doi.org/10.1016/j.pt.2008.11.006 Zoonotic potential of Giardia intestinalis Giardia assemblages and transmission to humans. Most human infections are anthroponotic, and involve subassemblages AI, AII, BIII and BIV. Sub-assemblage AI has zoonotic potential, and involve livestock, wild ruminants, nonhuman primates, cats and dogs. Occasional reports of transmission of assemblages C, E and F have been reported, likely originating from the animals predominantly infected by these assemblages. https://doi.org/10.1016/j.meegid.2017.12.001 Zoonotic potential of Giardia intestinalis Diagnosis of giardiosis ✓ microscopy ✓ detection of coproantigens ✓ examination of duodenal fluid ✓ ENTEROtest ✓ ELISA ✓ PCR SNAP Giardia Test ✓ detects soluble Giardia antigens, no more slide-scanning for evasive cysts ✓ no sample-prep time = easy to set up alongside faecal floats ✓ the first USDA-approved in-clinic rapid assay for the detection of Giardia-solution antigen https://www.youtube.com/watch?v=pscgOZerxhg Parabasalia • monophyletic group • parabasal apparatus = massively developed or even multiplied Golgi apparatus associated with 2 or more transversely striated microfibrils extending from the kinetosome = parabasal fibres • generally with 4 flagella / kinetosomes, but frequently with additional flagella (one to thousands) • 1 kinetosome bears sigmoid fibres that connect to a pelta–axostyle complex • reduction or loss of the flagellar apparatus in some taxa x multiplication of all or of parts of the flagellar apparatus in several taxa • closed mitosis with an external spindle, including a conspicuous microtubular bundle • hydrogenosomes instead of mitochondria • no cytostome • anaerobic organisms, commensals, mutualists and parasites of vertebrates and invertebrates karyomastigont / parabasal apparatus • basal bodies(1, 2 3, R) (kinetosomes) • parabasal fibres (PF1, PF2) • pelta-axostyle complex (PAX) • pelta (P) sheet microtubules (helmet) • axostyle (Ax) sheet microtubules that runs down the centre of the cell and in some cases projects past the end • undulating membrane (UM) • costa (C) microfibrillar structure to support undulating membrane • Golgi apparatus (G) • nucleus (N) Parabasalia Parabasalia Cell organisation in Parabasala (Trichomonas): 1) anterior flagella, 2) kinetosomes, 3) parabasal body, 4) costa, 5) parabasal fibers, 6) undulating membrane, 7) posterior flagellum, 8) hydrogenosomes, 9) axostyle, 10) nucleus, 11) pelta Karyomastigont – costa A-type costa B-type costa • mechanical support to the undulating membrane • different striations (A-type x B-type) • A-type (Tritrichomonas) - attached to kinetosome 2 • B-type (Trichomonas, Pentatrichomonas) - connected to kinetosome R (kinetosome of the recurrent flagellum) Parabasalia Parabasalia https://doi.org/10.1016/j.protis.2009.11.005 Parabasalia Trichomonadea Cristamonadea Hypotrichomonadea Spirotrichonymphea Tritrichomonadea Trichonymphea „trichomonads“ • 10-40 μm • up to 6 flagella • symbionts or parasites • urogenital tract, oral cavity, intestine of vertebrates „hypermastigids“ • up to 500 μm • thousands flagella • exclusively in the hindgut of termites and wood-eating Cryptocercus cockroaches Parabasalids were traditionally divided in orders Trichomonadida and Hypermastigida: Parabasalia https://doi.org/10.1016/j.protis.2009.11.005 Čepička et al. 2010: Critical taxonomic revision of Parabasalids with description of one new genus and three new species. Protist 161, 400–433 Parabasalia Trichomonadea • 4-6 flagella with 1 axoneme supporting lamelliform undulating membrane • B-type costa, sometimes absent • axostyle usually of “Trichomonas type” • Hexamastix, Pseudotrichomonas, Tricercomitus, Trichomonas, Tetratrichomonas, Pentatrichomonas, Cochlosoma https://doi.org/10.1016/j.protis.2009.11.005 Čepička et al. 2010: Protist 161 Trichomonas vaginalis • causative agent of human trichomoniasis • 10-20 ×2-14 μm, typically pyriform • pentaflagellate - 4 flagella from the anterior cell portion + 1 flagellum backwards to the middle of the organism, forming undulating membrane • undulating membrane without free flagellum • axostyle extending from the posterior aspect of the cell • V-shaped parabasal apparatus • large genome (strain G3, 176,441,227 bp) with ~ 60,000 protein coding genes organized into 6 chromosomes • obligate parasite phagocytosing bacteria, vaginal epithelial cells and erythrocytes and is itself ingested by macrophages Virulence factors in Trichomonas vaginalis • cytoadherence • level of iron intake • defence against complement and leukocytes Cytoadherence of Trichomonas vaginalis https://www.science.org/doi/10.1126/science.1132894https://doi.org/10.1016/j.pt.2012.10.004 Human urogenital trichomoniasis • humans are the only known host of T. vaginalis • infection of the urogenital tract • cosmopolitan, sexually transmitted diseases (STD) • possible contaminant transmission and transmission to newborns during childbirth • estimated 248 million new cases per year in the world • treatment with metronidazole • most infected people are asymptomatic • sexual partners, even if asymptomatic, should also be treated • without treatment, can persist for months to years in women, and is thought to improve without treatment in men microbial vaginal smear (MVS) Trichomoniasis https://www.youtube.com/watch?v=ze8jA2y1kIQ https://doi.org/10.1186/s12879-015-1055-0 Trichomonas tenax • 12-20 x 5-6 μm • long axostyle and tail • 4 anterior flagella + 1 recurrent flagellum raising an undulating membrane to 2/3 of the cell • oral cavity and bronchi of humans • likely involved in the degradation of periodontal tissue • affecting more than 50 % of the population • routine hygiene is generally not sufficient to eliminate the parasite Trichomonas gallinae • 6-15 x 4-8 μm • cosmopolitan parasite of pigeons and doves • 4 anterior flagella and undulating membrane • undulating membrane extending 2/3 of the cell length • lacking the free posterior flagellum • commonly found in the mouth, throat, gastrointestinal tract and upper respiratory tract of pigeons, doves, turkeys, chickens, canaries, raptors (predatory birds) and a variety of psittacine (parrot) birds including budgerigars, cockatiels and Amazon parrots Trichomonas gallinae canker in pigeon • causative agent of bird trichomonosis / trichomoniasis in young birds • yellow necrotic lesions in mouth and oesophagus • known as „canker“ (doves, pigeons) and as „frounce“ (raptors) • fatal for young pigeons aged 1-3 weeks • adult bird as carriers - infection via regurgitated crop content („pigeon milk“) • infection via contamination of food and water • transmission to the raptors • avirulent strains x virulent strains Trichomonas gallinae frounce in falcon • survives at least 5 days on some moist grains and several hours in water • extremely sensitive to desiccation (drying) • no cysts or resistant stage • caseous necrotic masses in the upper digestive tract and occasionally in the visceral organs • diagnosis based on the history, clinical signs, lesions and identification of the organism microscopically and by culturing Trichomonas gallinae • recent appearance of deadly infections in wild Passeriformes with T. gallinae obtained greater attention • trichomonosis in Europaen green finch (Carduelis chloris) https://doi.org/10.1051/parasite/2019022 The bird sat on the ground. It had ruffled feathers and some mucus dropped out of its beak. The bird was in poor nutritional condition and had a moderately developed pectoral muscle. Thick yellowish exudate covered part of the oral cavity, the crop and the upper oesophagus. Transmission of Trichomonas gallinae https://doi.org/10.1016/j.ijppaw.2013.12.002 Tetrarichomonas canistomae Tetratrichomonas felistomae • oral cavity of dogs • oral cavity of cats axostyle Tetratrichomonas brixi • oral cavity of cats and dogs • morphologically identical with T. tenax • new species description based on sequencing and phylogenetic analysis of ITS1-5.8S-ITS2 region • prevalence of T. brixi from the mouth of dogs and cats were 30,6 % (34/111) and 6,6% (8/122) Pentatrichomonas hominis • 4 anterior flagella + 1 trailing flagellum • long undulating membrane • no known cyst • infection via ingestion of trophozoites in faecal-contaminated food or water, or on fomites • non-pathogenic commensal in colon of humans and various mammals • human isolates are capable of infecting cats via oral inoculation with trophozoites Schematic view of (a) Trichomonas vaginalis, (b) T. tenax and (c) Pentatrichomonas hominis AF - free flagella, AX - axostyle, CO - costa, HY - hydrogenosome, N - nucleus, PP - parabasal body, PG - parabasal body + Golgi apparatus, RF - recurrent flagellum https://doi.org/10.1016/j.pt.2014.05.005 Cochlosoma anatis • associated with enteritis in ducklings, turkeys and finches Cochlosoma anatis https://doi.org/10.1099/ijs.0.63754-0 Parabasalia Hypotrichomonadea • 4 flagella with 1 axoneme supporting a lamelliform undulating membrane • comb-like structure present, but no infrakinetosomal body, biramous parabasal body • A-type costa • axostyle of “Trichomonas type” • symbionts in intestines of lizards, tortoises, kangaroos, pigs and cockroaches • Hypotrichomonas, Trichomitus https://doi.org/10.1016/j.protis.2009.11.005 Čepička et al. 2010: Protist 161 Parabasalia Hypotrichomonadea Trichomitus batrachorum • commensal in intestine of frogs Hypotrichomonas blattarum • commensal in hindgut of blaberid cockroaches http://dx.doi.org/10.1016/j.ejop.2015.02.003 https://doi.org/10.1016/j.protis.2009.11.005 Čepička et al. 2010: Protist 161 Parabasalia Tritrichomonadea • uninucleate or binucleate; 0–5 (4 ancestrally) flagella; ancestrally with comb-like structure • undulating membrane typically of a rail type (if present) • A-type costa • suprakinetosomal and infrakinetosomal body • axostyle of “Tritrichomonas type” or“ Trichomonas type” • Histomonas, Dientamoeba, Monocercomonas, Tritrichomonas Histomonas meleagridis • wide range of birds including chickens, turkeys, quail and pheasants • blackhead disease, infectious enterohepatitis, or histomoniasis • in two forms: amoeboid (8-15 μm) in tissues x flagellated (spherical, up to 30 μm) in lumen or free in the contents of caecum • both forms are shed in faeces and survive in the external environment for only a few hours • transmitted in the eggs of nematode Heterakis gallinarum (caecal parasite, eggs of which are transmitted by earthworms) Complex life cycle of Histomonas meleagridis https://doi.org/10.3389/fvets.2022.880738 Blackhead disease • symptoms within 7-12 days after infection - depression, reduced appetite, poor growth, increased thirst, sulphur-yellow diarrhoea, listlessness, drooping wings • symptoms highly fatal to turkey, less pathogenic in chickens; young birds most susceptible (particularly those 3-12 weeks old) - mortality up to 70 % • synergistic interaction with other enteropathogens (such as coccidia, Clostridium perfingens) https://doi.org/10.3389/fvets.2022.880738 Blackhead disease Pathology of blackhead disease Typical lesions resulting from Histomonas meleagridis infection (A,B) caseous cheese-like caecal core (C,D) focal necrosis resulting in target-like liver lesions Pathology of blackhead disease Diagnosis of blackhead disease ✓ clinical symptoms ✓ pathological findings ✓ trophozoites of H. meleadridis in tissues (edges of pathological lesions) ✓ cultivation ✓ histopathology – PAS (Periodic Acid Schiff) reaction ✓ PCR Histological section stained with H&E of liver showing H. meleagridis (black arrows) and necrotic liver cells (white arrows). Control of blackhead disease Control focused on Histomonas meleagridis ✓ good management of the farm and sanitation are the only effective strategies to control the spread of infection ✓ ban of effective therapeutic and prophylactic medication in the EU (5nitroimidazols: ronidazole, dimetronidazole, metronidazole, rodidazole) ✓ plant substances as an alternative treatment Control focused on Heterakis gallinarum ✓ anthelmintic treatment https://doi.org/10.1016/j.vetpar.2014.12.018 Dientamoeba fragilis • pleomorphic trophozoite (4–20 μm), typically binucleate • parabasal apparatus and hydrogenosomes • permanently lost flagella and kinetosomes • pseudopodia in fresh material https://doi.org/10.1128/JCM.00813-14 A-C) trophozoites, D-F) precysts, G-I) cysts stained with a modified iron haematoxyline Dientamoeba fragilis https://doi.org/10.1128/JCM.00813-14 Dientamoeba fragilis The complete life cycle of D. fragilis has not yet been elucidated. Assumptions have been made on the basis of clinical observations and the biology of related species (H. meleagridis in particular). Trophozoites that multiply via binary fission in the large intestine lumen are shed in the stool. Only the trophozoites of D. fragilis have been detected so far. Although rare putative forms of cysts and precysts have been described in human clinical specimens; it is not yet known whether and under what conditions transmission to humans occurs through their ingestion, in contrast to other faecal-oral transmission routes. Transmission via helminth eggs (e.g. Enterobius vermicularis) has been postulated. https://doi.org/10.1128/CMR.00076-15 Pleomorphic trophozoites of D. fragilis. (A) Binucleate trophozoite (modified iron hematoxylin staining); (B) live trophozoite displaying motility (phase-contrast microscopy). Putative life cycle of Dientamoeba fragilis https://doi.org/10.1016/j.pt.2014.01.005 Clinical presentation and diagnosis of dientamoebiasis • pathogenicity and clinical importance of D. fragilis continue to be investigated, including whether particular genotypes, subtypes, or strains are associated with symptomatic infection in humans • reported both the asymptomatic and symptomatic infection (e.g., with various nonspecific gastrointestinal symptoms such as intermittent diarrhoea, abdominal pain, nausea, anorexia, malaise, fatigue, poor weight gain and unexplained eosinophilia • symptoms often reported to be like colitis, appendicitis or irritable bowel syndrome Diagnosis • microscopic detection in faecal smears (Heidenhain‘s iron haematoxylin, trichrome, other permanent stains) • trophozoites not usually detectable by stool concentration methods, can easily be overlooked or misidentified due to their pale-staining nuclei that sometimes resemble those of Endolimax nana or Entamoeba hartmanni • cultivation on egg serum media (Dobell-Laidlaw) • PCR, antigen detection… More details in the review: https://doi.org/10.1128/JCM.00400-16 genus Monocercomonas • uninucleate, quadriflagellate • undulating membrane and costa absent • axostyle of Trichomonas type • non-pathogenic Monocercomonas colubrorum • widely distributed in cloaca of snakes and lizards M. ruminantium • rumen - beef cattle, sheep M. cuniculi • caecum of rabbits https://doi.org/10.1016/j.protis.2007.02.003 genus Tritrichomonas • quadriflagellate – 3 anterior flagella + well developed undulating membrane with a long free flagellum • rod-shaped parabasal apparatus • nutrition by pinocytosis Tritrichomonas muris • nonpathogen in cecum, colon, and small intestine of rodents Tritrichomonas foetus • spindle shaped (10-25 μm) cells resembling miniature tadpoles • traditionally identified as a cause of reproductive disease in cattle • currently important cause of diarrheal in cats Diagram and TEM of Tritrichomonas foetus. Hydrogenosomes (H) are preferentially located along the axostyle (Ax) and costa (C). AF- anterior flagella, BB - basal bodies, ER- endoplasmic reticulum, F - parabasal filament, G - Golgi, GL - glycogen granules, L - lysosomes, N - nucleus, Nu - nucleolus, P - pelta, R - recurrent flagellum, UM - undulating membrane, V - vacuoles Tritrichomonas foetus Bovine trichomoniasis • sexual transmission • bulls = asymptomatic carriers, parasite multiplication in foreskin • in cows causing endometritis, abortion and permanent infertility • pyometritis = death of the foetus in utero • artificial insemination in breeding cattle to eliminate organism from the cattle population Bovine trichomoniasis • reportable disease with no available treatment • bulls = primary carriers  most diagnostic sampling strategies focus on testing bulls and affected bulls and cows must be culled • sample collection methods: preputial scraping, flushing, and sponge sampling Preputial scraping for T. foetus ✓ microscopic examination of cultured preputial samples ✓ PCR and staining techniques to differentiate from other trichomonads ✓ … https://doi.org/10.1099/jmm.0.047365-0 https://doi.org/10.1111/j.1550-7408.2002.tb00360.x https://doi.org/10.1016/j.ijpara.2012.10.004 https://doi.org/10.1016/j.pt.2004.12.001 Cat trichomoniasis https://doi.org/10.1186/s13567-015-0169-0 https://doi.org/10.1016/j.protis.2009.11.005 Čepička et al. 2010: Protist 161 Parabasalia Cristamonadea • uninucleate to multinucleate • two to thousands of flagella/cilia per mastigont • kinetosomes, except for ‘privileged kinetosomes’, often discarded during cell division in highly flagellated/ciliated taxa • axostyle ancestrally of “Tritrichomonas type’’ • secondarily thin or reduced in some; multiple axostyles in multinuclear forms • parabasal body single or multiple, ellipsoid or rod-shaped, often spiralled or ramified • symbionts inhabiting the hindgut of the lower termites • Coronympha, Deltotrichonympha, Devescovina, Foaina, Joenia, Mixotricha, Calonympha Parabasalia Cristamonadea Calonympha grassii • termite Cryptotermes brevis https://doi.org/10.1016/j.protis.2009.11.005 Čepička et al. 2010: Protist 161 Parabasalia Trichonymphea • bilaterally or tetraradially symmetrical, with anterior rostrum divided into 2 hemirostra • each hemirostrum bears 1-2 areas with hundreds to thousands of flagella/cilia • flagella/cilia usually retained during cell division; one hemirostrum goes to each daughter cell; numerous parabasal fibres • numerous thin axostyles do not protrude outside the cell • symbionts in hindguts of lower termites and the woodfeeding cockroach • Barbulanympha, Hoplonympha, Staurojoenia, Trichonympha Parabasalia Trichonymphea Trichonympha spp. T. campanula Staurojoenina mulleri https://doi.org/10.1016/j.protis.2009.11.005 Čepička et al. 2010: Protist 161 Parabasalia Spirotrichonymphea • kinetosomes in counter clockwise spiral rows • flagella/cilia retained during cell division with the ciliary rows dividing between daughter cells • axostyle single of “Tritrichomonas type”, or multiple in thin bands, or reduced • symbionts in hindguts of lower termites • Holomastigotes, Holomastigotoides, Microjoenia, Spirotrichonympha Parabasalia Spirotrichonymphea A) protargol-stained juvenile Microjoenia sp. from Reticulitermes lucifugus B) protargol-stained adult Microjoenia sp. from R. lucifugus C) protargol-stained Spironympha sp. from Reticulitermes flaviceps D) living Spirotrichonympha flagellata from Reticulitermes hesperus, DIC E) protargol-stained juvenile Holomastigotes elongatum from Reticulitermes lucifugus F) protargol-stained adult H. elongatum from R. lucifugus G) living Holomastigotoides sp. from Coptotermes testaceus, DIC Scale bars = 10 μm for A-C, E and F; 20 μm for D, and 50 μm for G Preaxostyla Oxymonadida • single kinetid (occasionally multiple kinetids) consisting of 2 pairs of ciliated kinetosomes distantly • separated by preaxostyle (microtubular root R2, with paracrystalline lamina), from which arises a microtubular axostyle • axostyle is contractile or motile in some taxa, microtubular pelta usually present • many taxa attach to host using an anterior holdfast • closed mitosis with internal spindle • lacking Golgi and mitochondria • intestinal endosymbionts, mostly in lower termites and Cryptocercus cockroaches Preaxostyla Oxymonadida Preaxostyla Oxymonadida Polymastix melolonthae • 4 free anterior flagella + 1 recurrent • slender axostyle and a row of microtubules or pelta covering the anterior end • larvae of beetles Oxymonas spp. • 40-165 μm • 4 flagella, anterior rostellum for fixation • gut of termites Pyrsonympha spp. • spirally twisted and contractile body, 20-150 μm • 4 or 8 flagella wrapped around the cell body and having a posterior trailing portion • symbionts of gut of termites Preaxostyla Oxymonadida Monocercomonoides sp. • intestine of reptiles and intestine of beetles From marginated tortoise (Testudo marginata) (Vlasáková 2014, Master thesis) Preaxostyla Trimastigida • sister group to oxymonads genus Trimastix • heterotrophic free-living flagellates with 4 flagella • broad ventral feeding groove, in which beats the posteriorly directed flagellum (with 2 broad vanes) • small dense organelles bounded by two membranes in place of mitochondria 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