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Home , Cystoisospora, Cystoisospora belli, Eugregarinorida, Gregarinasina, Neogregarinorida, Sarcocystis

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Learning objectives

Human Pathogens 2 • After class, students will be able to: infections in • Describe morphology, life cycle, signs and symptoms, immunocompromised host epidemiology, prevention and control, laboratory diagnosis and treatment of opportunistic protozoan Nimit Morakote, Ph.D. parasites of man. 23 July 2021

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General characteristics of Opportunistic parasites of humans apicomplixans • Sporozoa ( • Fungi • Obligate intracellular parasite ) • Pneumocystis jirovecii • Apical complex organelles for • • Microsporidia host cell invasion • Conoid • belli • Polar ring • spp. • Rhoptry • cayetanensis • Microneme • Subpellicular • Sexual and asexual reproduction

http://www.nature.com/scitable/topicpage/the-apicoplast-an-organelle-with-a-green-14231555

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Eukaryote classification, 2019 • Apicomplexa • • Haemospororida (, etc.) • • Conoidasia • • • (Cystoisospora, Toxoplasma, , Cyclospora, etc.) • • Archigregarinorida • • Drawing showing conoid structure • Cryptogregarinorida (Cryptosporidium) Anderson-White B, et al. Int Rev Cell Mol Biol 2012;298:1-31. Adl SM, et al. Revisions to the Classification, Nomenclature, and Diversity of . J Eukaryot Microbiol 2019, 66, 4–119.

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Host cell invasion- parasite active mechanism Asexual reproduction

Internal budding • Endodyogeny (1 mother cell → 2 daughter cells) • Endopolygeny (1 mother cell → many daughter cells)

Bang Shen, L David Sibley. The moving junction, a key portal to host cell invasion by apicomplexan parasites. Current Opinion in Microbiology, Volume 15, Issue 4, 2012, 449–455

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Multiple (merogony or schizogony)

1 cell/ 1 nucleus ↓ 1 cell/ multiple nuclei (schizont or meront) ↓ many cells (merozoites) Apicomplexan asexual cell division by endodyogeny. (A–E) Show progressive steps of cell division as observed for T. gondii tachyzoites. Two daughters bud internally, while the mother’s is maintained and only destabilized just before emergence of nearly mature daughters (E). Daughter buds emerge and the plasma membrane from the mother is incorporated onto the daughters. The mother falls away as a residual body. (Gubbels M-J, et al. (2020) Fussing About Fission: Defining Variety Among Mainstream and Exotic Apicomplexan Cell Division Modes. Front. Cell. Infect. Microbiol. 10:269.) 9 10

Sexual reproduction

Macrogametes Macrogamonts Zoite #1 (female sex cells) Asexual schizont Microgametes Microgamonts Zoite #2 multiplication (male sex cells) 2, Disassembly of the mother’s cytoskeleton shortly following completion of host cell invasion, resulting in an amoeboid or pleomorphic cell. Fertilization 3, Several cycles of DNA replication and nuclear division. Mitotic cycle not synchronous. Sexual Zygote 4, The last cycle of nuclear division is synchronous and coupled to the (Sporogony) Sporulated synchronous budding of the daughters at the end of schizogony. oocysts Oocysts (Sporozoites Gubbels M-J, et al. Fussing About Fission: Defining Variety Among Mainstream and Exotic Apicomplexan Cell Division Modes. sporulation inside) Front. Cell. Infect. Microbiol. 2020;10:269. 11 12

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Oocyst sporulation Opportunistic protozoan parasites

Meiosis and Life cycle Affected organs/ Oocyst wall mitosis Disease in man Zygote sporoblasts sporocysts sporozoite or 1N Toxoplasma gondii Heteroxenous -Toxoplasmic sporont (haploid) =DH Developing organs-Congenital 2N (diploid) homoxenous Small intestine- Cryptospridium homoxenous Small intestine-Diarrhea homoxenous Small intestine-Diarrhea Unsporulated Sporulated oocyst oocyst

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Toxoplasma gondii Intermediate hosts Asexual reproduction in cells and tissues • Disease: toxoplasmosis • Congenital toxoplasmosis Immature oocysts -> sporulated oocysts • Toxoplasmic encephalitis in AIDS • Stages in life cycle • Tachyzoites, 6 x 2 µm: during acute stage of infection carnivorism Definitive host • Bradyzoites, during chronic stage of infection Sexual reproduction in • with glycogen granules the small intestine • Pepsin-resistant Toxon = arc • Oocyst, 10 x 12 µm Gondii= gundi tissue cysts containing bradyzoites Image from https://www2.bc.edu/~gubbelsj/Toxoplasma.html

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Watch video Phase in (enteroepithelial)

• Toxoplasma gondii - Life • Ingestion of tissue cysts containing cycle and Invasion bradyzoites. https://www.youtube.com/ • Bradyzoites infect the small intestine. watch?v=25SjWK9smnw • Enteroepithelial stage: schizogony, gametogony, fertilization. • Immature oocysts released in feces. Prepatent period 3-10 days. Toxoplasma cyst. Attias M, et al. Parasites Vectors (2020) 13:588.

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Sporulation in environment Asexual phase in intermediate hosts

• Immature oocysts undergo sporulation to mature, infective oocysts in soil or environment. • Sporulation requires 1-5 days

• Ingestion of sporulated oocysts • Sporozoites enter epithelial cells, multiply by endodyogeny -> tachyzoites, hematogenous www2.bc.edu spread (acute phase)

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Chronic phase of infection  Bradyzoites in tissue cyst Human infection

• Tissue cysts • Consumption of improperly cooked (bradyzoites) • Sporulated oocysts • Drinking unfiltered water (oocysts) • Toxoplasma cyst in brain. Contaminated hand (oocysts) Attias M, et al. Parasites Vectors (2020) 13:588. • Tachyzoites • Organ transplant, blood transfusion (tachyzoites) From: Structures of Toxoplasma gondii Tachyzoites, Bradyzoites, and Sporozoites and Biology and Development of Tissue Cysts. Clin. Microbiol. Rev. 1998 11(2): 267-299; published 1 April 1998 21 22

Pathogenesis, Signs and Symptoms Diagnosis, prevention & treatment

• Acute phase- tachyzoites invade and destroy • Isolation of parasite impractical host cells • Primarily diagnosed by serology (antibody detection) • Lymphadenopathy, flu-like symptoms • Chronic phase • Serological survey shows worldwide distribution including • Immune pressure: Tachyzoites  bradyzoites Thailand (tissue cysts) in brain, muscle • Prevention by cook meat, proper handling of cat’s feces • Become asymptomatic • • Reactivate in AIDS  toxoplasmic encephalitis Treatment: and sulfadiazine, plus folinic acid. • Congenital toxoplasmosis: chorioretinitis, Austincc.edu hydrocephalus, intracerebral calcification

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Laboratory demonstrations

http://www.cdc.gov/parasites/toxoplasmosis/resources/printresources/catowners.pdf 25 26

Cystoisospora belli

• Former name= belli • Diagnosis: • Only in human and primate • Fecal exam: simple smear or acid fast stain • Unsporulated oocyst, 20-23 x 10-19 m • Merogony, gametogony, oocyst formation in epithelial cell • of the small intestine Contains 1 or 2 sporoblasts • Treatment: trimethoprim and • Oocyst sporulation outside host: 1-5 days sulfamethoxazole • Acute infection: diarrhea, self-limited • Immunodeficient person: severe diarrhea

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Unstained faecal smear Laboratory demonstrations Cryptosporidium (marvistavet.com)

• Facultative gregarine parasite (Order Cryptogregarinorida) • Parasite of , , , fish, amphibians: rather host-nonspecific • Human: mostly C. parvum, C. hominis • Oocyst 5 µm in diameter, smallest of all oocysts of human parasites • 4 naked sporozoites (oocyst without sporocyst)

Jstor.org

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• Life cycle similar to Cystoisospora belli (development occurs in GI epithelium) except: • Organism is intracellular, extracytoplasmic • Sporulation in host cells- • 80%=thick-wall oocysts • 20%=thin-wall oocysts (autoinfective) • Prepatent Period app. 2 days, Sateriale E, et al. PNAS 2021 Vol. 118 No. 2 Cunha FS, et al. New insights into the detection and molecular characterization of Cryptosporidium • Incubation Period app. 7-10 days e2007807118 with emphasis in Brazilian studies: a review. Rev Inst Med Trop Sao Paulo. 2019;61:e28.

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• Emerging infectious disease • Infected persons secrete large • Impaired intestinal absorption + amount of oocysts in feces enhanced secretion • Infectious dose 10-100 oocysts • Watery diarrhea, abdominal cramp, • The median maximum number of stool per day is 12 • Self-limited in 2 wk.

From: Davies AP, Chalmers RM. . BMJ | 24 october 2009 | Volume 339:963.

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• An important opportunist in • Outbreak in EU: settings or • Outbreaks associated AIDS patients vehicle with untreated • Fulminant infection, 2L • Nursery school recreational water- US watery stool daily (CD4 < • Playing on floodplain 2000-2014 50/microliter) • Open farm • Veterinary students • 4,958 cases of disease • Extraintestinal dissemination and two deaths can occur, most common= • Frisée salad biliary tract • contact • 12% by Cryptosporidium • Swimming pool • In population, most • Drinking water • Multiply in biofilms prevalent in children under Caccio SM and Chalmers RM. Human 5 years cryptosporidiosis in . Clinical Microbiology and Infection 2016: 22(6) : 471-480. MMWR Weekly / Vol. 67 / No. 25 June 29, 2018 35 36

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• Infection worldwide including Thailand • Account for 5% of patient diarrhea • Contaminated drinking water, food, direct contact • Clam • Outbreak in the past involving 403,000 persons in Milwaukee, USA, associated with drinking water • No effective, specific chemotherapy • nitazoxanide resolves diarrhoea in most immunocompetent individuals • Diagnosis by stool exam for oocysts Caccio SM and Chalmers RM. Human cryptosporidiosis in Europe. Clinical Microbiology and Infection 2016: • Acid fast stain fecal smear 22(6) : 471-480.

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Some data on occurrence in Thailand Laboratory demonstrations

• Trapped from Nan, Songkhla, and Tak provinces -overall prevalence 23.1 % : C. meleagridis C. parvum, C. suis, C. viatorum and C. wrairi (Amornrat M, et al 2014) • Water samples from Chao Phraya River, and sea water at Bang Pu- 11% and 6%: C. parvum, C. meleagridis, and C. serpentis (K. Koompapong and Y. Sukthana, 2012) • HIV-infected patients during 1999–2004 at the Siriraj Hospital C. hominis, C. meleagridis, C. canis, C. felis, C. suis and C. parvum, in order of frequency (Sannella et al., 2019)

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Cyclospora cayetanensis Unsporulated: cdc.gov

• Described in 1994 (Cayetano Heredia University, • Diarrhea (5-15 times/day) Lima, Peru) • Self-limited (19-57 days), can be • Man is the only host recurrent • Oocyst slightly bigger than Cryptosporidium • More severe in AIDS patients oocyst, i.e., 8.6 m • Traveler's diarrhea • Unsporulated when come out with feces • Implicated in Europeans returning • Sporulation requires 7-15 days in external from Asian/South American environment countries • 2 sporocysts, each with 2 sporozoites • Indonesia, Nepal, Thailand, • Incubation period app. 7 days Sporulated: asm.org Guatemala, Mexico

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• Waterborne outbreak reported Imported vegetables, salad & fruits = Also many foodborne outbreaks most frequent source of infection • Oocysts survive a week at 4 C • Treatment: Trimethoprim, • sulfamethoxazole Diagnosis • Stool exam for oocysts • Acid fast stain- must differentiate from Cryptoporidium • Staining not uniform: dark

red, pink or unstained Cyclospora oocyst

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Watch video Laboratory demonstrations

• New Food Poisoning Outbreak in at Least 15 States • https://www.youtube.com/watch?v=NASL43ZLjLM

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Microsporidia Some microsporidia infecting human

• A group of organisms: small single-celled, • Encephalitozoon cuniculi • Nosema ocularum obligate intracellular parasites belonging to • Encephalitozoon hellem • Anncaliia algerae Phylum Microspora • Encephalitozoon (Septata) intestinalis • Anncaliia connori • Early- eukaryotic protozoa • Enterocytozoon bieneusi • Anncaliia vesiculnarum • Recently- classified as fungi • Endoreticulatus spp. • Microsporidium africanum • >2,000 infecting vertebrates and • Trachipleistophora hominis • Microsporidium ceylonensis invertebrates • Pleistophora ronneafiei • Trachipleistophora anthropopthera • 14 species infecting human- truly opportunistic • Vittaforma corneae • Trachipleistophora hominis • Enterocytozoon bieneusi, Encephalitozoon intestinalis= most common

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Morphology Disease • Truly opportunistic, patient groups similar to P. jirovecii • Spores are relatively small, 1.0– 3.0 M × 1.5–4.0 M • Small intestine, eyes, muscle, respiratory tract, urinary tract, biliary • Special organelles- coiled polar tract, skin tube or filament- for infecting • E. bieneusi- the small intestine host cell  Persistent diarrhea, abdominal pain, weight loss  4-30 coils, used for species differentiation • E. intestinalis- • Merogony and sporogony in  Persistent diarrhea cytoplasm, mostly without  Extraintestinal dissemination: sinusitis, keratoconjunctivitis, parasitophorous vacuole encephalitis, tracheobronchitis, interstitial nephritis, hepatitis, or myositis

Epidemiology Encephalitozoon: 4-7 • Fecal-oral route (contaminated water and food) • Diagnosis coils in single rows  Majority of human-pathogenic microsporidia have been detected  Modified trichrome- red spore Enterocytozoon: 6 turns with two tiers in water with belt  Vegetable, fruit contamination found (100 cases outbreak of E.  EM- for species diagnosis bienneusi in Sweden was traced to cucumber) median incubation period=9 days • Treatment: • Reservoir hosts  fumagillin,  Immune restoration to  E. bienneusi: in , monkeys CD4>100 cells/µL  E. intestinalis: farm animals Modified trichrome stain

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Laboratory demonstrations Further readings

• Chapter 84. Toxoplasma gondii [http://www.ncbi.nlm.nih.gov/books/NBK7752/] • Chen et al. Cryptosporidiosis. N Engl J Med 2002; 346: 1723-31. • Ryan et al. Foodborne cryptosporidiosis. Int J Parasitol 2018;48:1-12. • Ortega YR, Sanchez R. Update on Cyclospora cayetanensis, a Food-Borne and Waterborne Parasite. Clin Mirobiol Rev 2010; 23(1): 218-234. • Han B, Takvorian PM, Weiss LM. Invasion of host cells by microsporidia. Front. Microbiol., 18 February 2020.

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