Toxoplasma and toxoplasmosis Medical Parasitology 2012 Silvia N.J. Moreno

1. Toxoplasma and toxoplasmosis Medical Parasitology 2012 Silvia N.J. Moreno

2. Alveolata Dinoflagellata: Many are photosynthetic. transverse flagellum that encircles the body (often in a groove known as the cingulum) and a longitudinal flagellum oriented perpendicular Ciliata: some or all of the surface is covered with short, densehairlike structures (cilia) whichbeat to propel the ciliate through the water and/or to draw infood particles. Apicomplexa: Diverse group. All parasitic. Elements of the Apical complex

3. Alveolata • Cortical alveoli or inner membrane complex (flattened membranous cisternae underlying the plasma membrane) • Subpellicular microtubuli • Mitochondria with tubular cristae • Molecular phylogeny on rRNA, tubulin and several other genes supports this grouping PM IMC MT

4. Balantidium coli • Balantidium coli is the only human pathogen from this phylum • Ciliates have 2 nuclei (one macronucleus and one micronucleus) and reproduce by transverse binary fission, conjugation, autogamy, and cytogamy. • Typical ciliate organization (cilia, digestive vacuole, pulsating vacuole etc.)

5. Balantidium coli • Cosmopolitan distribution. Relatively rare in humans (most reported cases are from Philippines). Association with pig reservoir • Balantidium coli has 2 developmental stages: a trophozoite stage and a cyst stage. • Causes diarrhea and dysentery in both swine and humans. • Trophozoites can invade the mucosa forming ulcers very much like E. histolytica. Disease can be fatal.

6. APICOMPLEXA • Apical complex: an assemblage of cytoskeletal elements and secretory organelles • No flagella or cilia except for the microgamete (sperm) • All members of the phylum are parasitic • Complex life cycle:asexual and sexual forms the development of these stages are tissue specific.

7. APICOMPLEXA • Gregarines:parasites of invertebrates, some quite big (used as early research models), Cryptosporidium might belong into this category • Coccidians:tissue parasites of vertebrates and invertebrates (can have single (e.g. Eimeria) or two host (e.g. Toxoplasma). Many parasites of medical and veterinary importance. Sex produces a sporelike oocyst • Haemosporidians (Plasmodium) and Piroplasms(Babesia & Theileria): small parasites of blood cells which are transmitted by arthropods Credit: James Marden Gregarine parasites that causes metabolic disorders in dragonflies, as it appears under an electron microscope at 100-micron scale. Eimeria Tenella labelled with antibodies against microneme proteins Plasmodium falciparum bursting from red blood cells

8. SPECIES OF MEDICAL IMPORTANCE Intestinal parasites:Eimeria, Cyclospora and cryptosporidium. Extraintestinalor tissue parasites: Toxoplasma, sarcocystis, neospora. Blood parasites: plasmodium, babesia and theileria

9. Toxoplasma gondii • Initially found in 1908 as a tissue parasite of gondi (an african rodent) • T. gondii is distributed worldwide. High prevalence in Europe. In the US approx. 20-30% of the population is infected • Broad host and tissue specificity: infects almost all nucleated cells. • Transmission by tissue cysts (containing bradyzoites) or the oocyst (containing sporozoites) • Infection through ingestion of undercooked or raw meat or contaminated water • Clinical significance: • Asymptomatic in healthy individuals • CNS involvement in immunocompromised patients • Congenital disease

10. PARASITE FORMS • TACHYZOITES: rapidly growing meront or zoite observed in the early stage of of infection • BRADYZOITES: Slow-growing zoite or meront inside the tissue cysts • TISSUE CYSTS: Found in the tissues contain bradyzoites. Infective form • OOCYSTS: Zygote form. Highly resistant form. Infective form • Sporocyst • Sporozoites This scanning electron micrograph shows a tissue cyst with its many bradyzoites within the brain of an infected mouse.Photo courtesy of David Ferguson, Oxford University.http://cmgm.stanford.edu/micro/boothroyd/boothroydlabdesc.html Oocyst from a fecal sample of a cat

11. Life cycle of Toxoplasma gondii From Chidoni, Moody & Manser, 2001 • Oocysts are only produced in the definitive host (DH), members of the family Felidae DEFINITIVE HOST • Ingestion of TC by the cat initiates the intestinal stage • Tissue cysts are also found in the cat • Sexual cycle produces the oocyst which is excreted in the feces • As the host • develops immunity replication rate slows down and parasite form tissue cysts (TC) containing bradyzoites • Sporulation results in the formation of sporozoites • Sporulated oocyst remain infective for months INTERMEDIATE HOST • IH gets infected by ingesting sporulated oocysts which release sporozoites which penetrate the intestinal epithelium, and invade macrophages and other types of cells. • Parasites multiply by endodiogeny • Tachyzoites are released which will invade new host cells

12. The intestinal phase in the cat Cat ingests tissue cystscontaining bradyzoites Number of merogonous cycles is variable Gametocytesdevelop in the small intestine but are more common in the ileum 2-4% of gametocytes are male, each produces around 12 microgametes Oocysts appear in the cat’s feces 3-5 days after infection by cysts with peak production around 5 and 8 Oocysts require oxygen and they sporulate in 1-5 days

13. TOXOPLASMA TRANSMISSION DEFINITIVE HOST Sources of infection: Contaminated water or food Undercooked meat Mother to fetus Organ transplant (rare) Blood transfusion (rare)

14. Latent bradyzoite cysts confer life-long infection • The acute infection is carried by tachyzoite stage • Tachyzoites divide rapidly and aggresively destroy tissues, all pathology is associated with tachyzoites • Tachys can cross the blood-brain barrier and the placenta • When the IR slows down tachy replication, they differentiate in bradys and form tissue cysts • Cysts form in brain and skeletal muscle • Bradyzoite cyst persist in the immune host for the rest of its life • Bradyzoites are resistant to all currently available drugs

15. Bradyzoite cysts are highly infective if ingested • Bradyzoites marks the beginning of the chronic phaseof infection • Tissue cysts show very little evidence of inflammation or immune cell infiltrates. • Bradyzoites (not tachyzoites) are resistant to low pH and digestive enzymesduring stomach passage • Protective cyst wall is finally dissolved and bradyzoites infect tissue and transform into tachys • Tachyzoites: pathogenesisBradyzoites: epidemiology

16. Toxoplasma is an opportunistic pathogen • 15-70% of the population is chronically infected (current seroprevalence in the US is 21.5%) • Most people show no or only benign symptoms (headache, sore throat, lymphadenitis, fever • Three situations can lead to severe disease: 1) congenital toxoplasmosis 2) occular toxoplasmosis in immunocompetent adults 3) loss of a functional immune system

17. PATHOGENESIS • Congenital disease: acute infection of the expectant mother. • Severity depends on the stage of pregnancy. • Spontaneous abortions or neurological disorders such as blindness and mental retardation can result. • Immunocompromised individuals: toxoplasmic encephalitis • Reactivation of a latent infection (>95% cases) • Recently acquired infection • Ocular toxoplasmosis

18. Congenital toxoplasmosis • If a women is infected for the first time during pregnancy the parasite can transverse the placenta and cause fetal disease • Both the probability and severity of the disease depend on when the infection takes place during pregnancy (early: low transmission, but severe disease, late: high transmission, more benign symptoms) • Several countries screen all pregnant women for acute infection but the efficacy of in utero treatment is hotly debated

19. Congenital toxoplasmosis Girl with hydrocephalus due to congenital toxoplasmosis. • Children who are asymptomatic at birth often develop disease later on, long term treatment of these kids is quite successful • This include antiparasitic treatment as well as symptomatic treatment against e.g. hydrocephalus • Despite calcification throughout the brain this 10 month old child developed completely normal

20. Toxoplasmic encephalitis (TE) • Only 25-30% of seropositive patients who develop AIDS develop TE • Involvement of the CNS. • Symptoms: lethargy, loss of memory to severe dementia, focal to major motor seizures MRI result of an homosexual male diagnosed with HIV admitted to a local hospital complaining of intermittent headache, lethargy, personality change, abdominal pain and vomiting.

21. T. gondii is a major pathogen in late stages of AIDS • In the majority of cases this is due to reactivation of the chronic infectionrather than new infection • Usually a multifocal process. Multiple brain abscesses: necrotic central zone surrounded by hyperemic region containing parasites and infiltrating mononuclear cells. Sometimes disseminated TE • TE can be treated with pyrimethamine and sulfa but not all patients tolerate side effects • As for all other opportunistic infections the incidence is much lower in patient on HAART (highly active antiretroviral therapy)

22. Ocular toxoplasmosis • Ocular toxoplasmosis can be a sequel of congenital infection (neonatal or late forms) • However recent epidemiological studies show that it also occurs after infection of seemingly fully immunocompetent adults • Severe retinochoroiditis (cleared yellowish zone), after treatment scaring occurs • The acute phase responds well to treatment, but • 80% probability of relapse and relatively poor prognosis of eyesight in the long term

23. DIAGNOSIS OF TOXOPLASMOSIS • Parasites can be detected in biopsied specimens or cerebral spinal fluid. Not very reliable. • Numerous serologic procedures available: Sabin-Feldman dye test, indirect haemagglutination assays, IFA, direct agglutination tests, etc. • Serologic results may be complicated because of the high prevalence of seropositive individuals. High titer is not evidence of an acute infection. • Best to collect two samples 2-4 weeks apart. If there is increase in the titer in the second sample may indicate acute infection. • Of particular interest is determining acute infection in pregnant women, due to the risk of congenital toxoplasmosis. This is complicated by the fact that many women have existing IgG and IgM antibodies to T. gondii. Fixed tachyzoites are recognized by the patient's antibody (IgG), and a fluorescent-labeled anti-human antibody (IgG) is added next to identify the antibody by fluorescent microscopy. If no antibody against the T gondii tachyzoite is seen, no fluorescence is present. (Photomicrographs and IFA preparation by Parkland Memorial Health and Hospital System Humoral Immunology Laboratory.)

24. CHEMOTHERAPY OF TOXOPLASMOSIS • Inhibitors of dihydrofolate reductasePyrimethamine in combination with sulfonamides • SulfonamidesSulfadiazine, sulfamethazine, sulfamerzaine. • Clindamycin • Macrolides and azalidesRoxithromycin, azythromycin, clarithromycin, spiramycin • TetracyclinesDoxycline, minocycline • Hydroxynaphthoquinones • Atovaquone. • OthersEimycine, aprinocid, dapsone, quinghasou, pentamidine • Combination therapy • pyrimethamine-dapsone, clarithromycin-minocycline, azithromycin-pyrimethamine, azithromycin-sulfadiazine, etc • Biological response modifiersCytokines (IFN-, IL-2, TNF, IL-1, IFN-) in combination with chemotherapy

25. Pyrimethamine and sulfadoxine Sulfadoxine: The sulfonamides are structural analogues of para-aminobenzoic acid (PABA), and competitive inhibitors of dihydropteroate synthetase. Pyrimethamine: An antifolate, a highly selective and powerful competitive inhibitor of dihydrofolate reductase.

26. Folate metabolism TOXOPLASMA HUMAN Pteridine + PABA Dihydropteroate synthetase sulfadoxine Dihydropteroic acid Dihydrofolate Tetrahydrofolate FAH4 cofactors GLUTAMATE Preformed dietary folates Tetrahydrofolate FAH4 cofactors NADPH NADP Dihydrofolate reductase pyrimethamine NADPH NADP Dihydrofolate reductase Thymidine PurinesMethionine Glycine f-met-tRNA DNA DNA RNA PROTEINS Thymidine PurinesMethionine Glycine f-met-tRNA DNA DNA RNA PROTEINS Parasites synthesize their own folic acid; unlike man, they cannot import preformed folic acid. Tetrahydrofolic acid is an essential cofactor for the synthesis of nucleic acid precursors, and some amino acids.

27. THE TACHYZOITE • Crescent shaped • 2 by 6 µm • Asexual form • Multiplies by endodyogeny • It can infect phagocytic and non-phagocytic, nucleated cells. • Organelles:pellicle, apical rings, polar rings, conoid, rhoptries, micronemes, dense granules, apicoplast , acidocalcisome, micropore, subpellicular microtubules, mitochondrion, endoplasmic reticulum, Golgi complex, ribosomes, rough and smooth endoplasmic reticulum, nucleus. • M. Black and J. C. Boothroyd (2000).The lytic cycle of Toxoplasma gondii. Microbiology and Molecular Biology Reviews, 64 (3) p. 607

28. THE TACHYZOITE Carey et al, (2004) PNAS 101: 7433 Am, amylopectin granule; Co, conoid; Dg, electron-dense granule; Go, Golgi complex; Mn, microneme; No, nucleolus, Nu, nucleus; Pv, parasitophorous vacuole; Rh, rhoptry. Dubey, et al (1998). Clinical Microbiology Reviews, 11: 267-299.

29. THE CYTOSKELETON OF TOXOPLASMA The anterior end of the parasite is enlarged in the box to the right so as to illustrate the preconoidal rings, the conoid, the two apical microtubules, and the polar ring from which 22 subpellicular microtubules emanate. The IMC is located just beneath the plasma membrane from the anterior to the posterior poles and is interrupted only by the micropore (MP) located in the middle of the parasite body. . Micrograph by J. Boothroyd and D. Ferguson

30. Diagram of endodyogeny As a parasite begins to divide, two IMCs begin to develop in the middle of the cell. As the IMC extends from these structures, the nucleus (N) and mitochondrion (Mitoch.) divide into these membranous outlines. Nascent apical organelles (NO) develop within the anterior poles as the daughter cells grow. Eventually, the entire cytoplasm is divided between the daughters and the IMC of the mother dissociates. A cleavage furrow divides the cells from the anterior pole. This division continues down the length of the cells until it reaches the posterior end, where it can leave a residual body connecting the two daughters. M. Black and J. C. Boothroyd (2000). The lytic cycle of Toxoplasma gondii. Microbiology and Molecular Biology Reviews, 64 (3) p. 607

31. Transmission electron micrograph of a tachyzoite undergoing endodyogeny within a mouse peritoneal macrophage Cd, conoid of developing tachyzoite;Co, conoid of mother tachyzoite;Dg, dense granule; Ga, Golgi adjunct; Hm, host cell mitochondrion;Hn, host cell nucleus; Id, inner membrane complex of developing tachyzoite;Im,inner membrane complex of mother tachyzoite; Mi, mitochondrion; Mn, microneme; Nu, nuclei of daughter tachyzoites;Pl,plasmalemma of mother tachyzoite; Pm,parasitophorous vacuolar membrane; Pv, parasitophorous vacuole;Rh, rhoptries of daughter tachyzoites. • Dubey, J.P., et al (1998). Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of Tissue Cysts. Clinical Microbiology Reviews, 11: 267-299.

32. BRADYZOITE TISSUE CYSTS • Crescent-shaped cell • 7 x 1.5 m • Morphologicaly similar to tachyzoites except that it has higher content of micronemes and amylopectin granules • Only stage that can initiate the sexual phase. When they invade the feline enterocytes they differentiate into micro- and macrogamete. • Intracellular and bradyzoites divide by endodyogeny. • Intact tissue cysts do not cause any harm and do not induce an inflammatory response. • The intracellular location protects them from the immune system. • More abundant in myocardium, striated muscle and CNS. • Some bradyzoites may escape from the cyst and form daughter cysts. • The cysts are resistant to HCl and digestive enzymes. • Vary in size; young cysts as small as 5 µm in diameter with only two bradyzoites, while older ones may contain hundreds

33. TISSUE CYST A highly stretched tissue cyst with more than 1,000 bradyzoites in an impression smear of brain homogenate from a rat 14 months after infection with the VEG strain of T. gondii. The cyst wall (arrow) is barely visible. • Dubey, J.P., Lindsay, D.S. and Speer, C.A. (1998). Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of Tissue Cysts. Clinical Microbiology Reviews, 11: 267-299.

34. THE OOCYST • The oocyst is noninfectious before sporulation. • Unsporulated oocysts are subspherical to spherical and are 10 by 12 m in diameter. • Sporulated oocysts are subspherical to ellipsoidal and are 11 by 13 m in diameter. • Each oocyst has two ellipsoidal sporocysts. • Sporocysts measure 6 by 8 m. • Each sporocyst contains four sporozoites. • Shedding occurs 3-5 days after ingestion of tissue cysts or 20-34 days after ingestion of oocysts.

35. Toxoplasmosis prevention • Avoid raw or undercooked meat. (Cooking kills Toxoplasma) • Wash raw fruits and vegetables well before eating. • Remove feces from the litter box every day, to eliminate any parasites before oocysts sporulate • Keep cats indoors to prevent hunting. Cats can become infected by eating infected rodents or birds. • Feed cats only commercial food or well-cooked meat. • Do not feed cats raw or undercooked meat. • Do not adopt cats who have lived outdoors or who have been fed raw meat. • Do not handle stray cats. • Wash hands well with soap and warm water after: • Gardening • Yard Work • Any Other Outdoor Activity Involving Contact With Soil that could be • contaminated with cat feces • There is no treatment available to prevent Toxoplasma in cats, or to • prevent an infected cat from shedding the parasite in its stool. Source: U.S. Centers for Disease Control and Prevention