Protozoa- Protista

1. Protozoa- Protista Over 45,000 species described- many parasitic Parasitic protozoa kill, mutilate, and debilitate more than any other group of disease organisms Protozoa used to be a phylum so many references will use protozoa and protista interchangeably

2. Protozoa Single cells Some have >1 nucleus during all or part of their life cycles Some stages (spores) may be constructed from >1 cell Largely recognized after the development of microscopes van Leeuwenhoek described all sorts of protozoa Electron microscopy indicated that all protozoa did not have a common body plan- used for grouping organisms into different phyla

3. Protozoa possess a great diversity of membranous organelles in the cytoplasm Mitochondria- organelles with enzymes of the oxidative phosphorylation system and tricarboxylic acid cycle-diversity in shape of mitochondria Golgi apparatus-elaborate and diverse in different protozoa-may occur as single or multiple large parabasal bodies often in association with kinetosomes. Golgi can play diverse roles in protozoa: protein assembly, source of skeletal plates and polar filaments

4. Membrane-bound bodies: Microbodies- spherical structures with a dense granular matrix- often contain oxidases and catalases Bodies often named due to role: Peroxisomes: Oxidase: reduces oxygen to H2O2 Catalase: converts H2O2 towater and oxygen Hydrogenosomes produce molecular hydrogen Microbodies may contain enzymes for glyoxylate cycle fat-carbohydrate conversion

5. Reproduction Asexual or sexual Asexual: binary fission: plane may be random (Sarcodina) longitudinal in flagellates (between the kinetosomes or flagellar rows) Nuclear division is mitiotic. Multiple fission (merogony, schizogony)-nucleus and other essential organelles divide repeatedly before cytokinesis- producing simultaneously a large number of daughter cells. In shizogony-the cell is called a shizont and daughter nuclei arrange themselves peripherally beneath the cell surface. Daughter cells are called merozoites. Merozoites are additional stages of the same life-cycle stage.

6. Sexual Reproduction Reductional division in meiosis: diploidy-haploidy gametes join to restore diploidy Amphimictic: union of gametes from 2 parents Automictic: one parent produces both gametes.

7. Many protozoa protected within cysts- resistant stage covered in chitin or chitin-cellulose mixes. Triggered by: temperature, adverse environment, dessication, lack of food, pH. In some situations we do not know what is the trigger. Entamoeba histolytica can be spread by persons who are asymptomatic, but are shedding cysts in their faeces. Under suitable conditions- excyst -triggered by change in environment: water, temperature, pH, host digestive enzymes, parasite produced enzymes

8. Feeding / Metabolism Some protozoa are holophytic – synthesize carbohydrates. Heterotrophic- require energy in form of complex carbon molecules, nitrogen as preformed amino acids Most are particle feeders-grazers and predators Mouth openings may be temporary (amoebas) or permanent cytostomes (ciliates) Food enters food vacuole, digestive organelle that forms around ingested food – undigestible material voided through temporary opening or permanent cytopyge (ciliates) Endocytosis: internalizing material: Pinocytosis-liquid, phagocytosis-particulate matter

10. Protozoa carry out glycolysis, Krebs cycle, ETS, transaminations, nucleic acid synthesis etc. ATP is principal form of available energy Glycogen is common energy storage Genes are transcribed in nucleus, peptides synthesized on ribosomes as in other systems Many parasites live in reduced oxygen environments. Often they derive energy from glycolysis, and partially oxidized products excreted

11. Non Pathogenic Protozoa: Each day all of us are infected with bacteria-viruses-protozoa- parasites Vast majority expelled due to their inability to take advantage of our essential and available niches. Many protozoa are commensals: Non pathogenic, flagellated commensals of humans: Trichomonas tenax, T. hominis, Enteromonas hominas, Retortamonas intestinalis, Chilomastix mesnili Commensal Amoebae Entamoeba dispar, E. hartmanii, E. coli, Iodamoeba butschlii

12. Giardia lamblia Amitochondriate flagellated protozooan Transmitted via fecal-oral route Aerotolerant anaerobes and require a reducing environment First known description by Van Leeuwenhoek in 1681 in stool samples

13. Two forms: Trophozoite and cyst Trophozoite is pear shaped, 10-20 µm long, 7-10 µm diameter 8 flagella Binucleate- both nuclei are transcriptionally active 2 rigid median bodies No mitochondria, peroxisomes, hydrogenosomes or other subcellular organelles for energy metabolism Anterior region contains structure for attachment to epithelial cells Structure is maintained with tubulin and giardins (calcium binding annexins) Surface is covered with cysteine-rich molecules

14. Infection: ingestion of cysts. Cysts excyst in response to physiological / environmental stimuli Following a series of stimuli: acid, pancreatic enzymes Stimuli activates the parasite within the cyst (quadrinucleate) Motile parasite divides into 2 binucleate parasites Trophozoites attach to epithelial cells- probably based on carbohydrate-binding lectins But will also attach to glass plates Parasites grow-divide by binary fission.

16. Giardia attaches to cells in intestine In the presence of trypsin trophozoites produce lectins Uses lectins to bind to cells This interaction may delineate where parasite occurs in the hosts

17. Nutrition G. Lamblia cannot synthesize nucleic acids de novo. No mitochondria, tricarboxylic acid cycle Uses glucose and arginine as major energy sources Obtains these from host- often through degradation of host mucous Lipids are absorbed directly or via endocytosis Food reserves are stored in the form of glycogen. Glucose catabolism via the glycolytic pathway results in production of the end products ethanol, acetate and carbon dioxide.

18. Some trophozoites encyst. Can be induced via exposure to bile and elevated pH Passed out in feces Cyst production continues for years- numbers produced vary depending on many factors Cysts are resistant to many treatments- mild chlorine Can survive in cold water Boiling can kill- but not at high altitudes

19. Pathogenesis Steatorrhea Malabsorption Associated weight loss Physiological changes experienced during symptomatic infection could relate to host-based responses Antigenic variation of trophozoite surface components occurs- changes every 12 G Helps parasite avoid elimination by humoral responses of the host (IgA antibodies) directed towards the trophozoite surface proteins. Switching of cysteine-rich variant surface proteins (VSP) occurs during excystment- allowing parasite to avoid immune response

20. G. Lamblia in immune deficient mice often do not show VSP switching- or at least not to the extent shown in normal hosts but may occur spontaneously or in response to physiological processes Constant interactions between host and parasite: mother’s milk is protective Lactoferrin/products produced by lipid hydrolyisis of milk are toxic Nitric oxide produced by epithelial cells is toxic Arginine required to produce nitric oxide- if Giardia can compete for arginine- less nitric oxide produced Duration and severity of infection depends on the immune responses of the host and the parasites’ response

21. Highly contagious Most infected people are asymptomatic but may become carriers Symptoms: protracted diarrhea- semi-solid stools to pure liquid There is no blood loss associated with the diarrhea, however, the stool characteristically has a fatty consistency (steatorrhea) as a result of fat malabsorption. This occurs in heavy infections where attached trophozoites can cover much of the intestinal epithelial surface. Can last months Chronic infections: malabsorption- with substantial weight loss, debility, constant fatigue Other symptoms include epigastric discomfort, pain, increased intolerance or allergic responses to specific foods but no cause/effect relationship has been established

22. How do we get Giardia??? By putting something in your mouth or accidentally swallowing something that has come in contact with the stool of a person or animal infected with Giardia.  By swallowing recreational water contaminated with Giardia. Recreational water is water in swimming pools, hot tubs, jacuzzis, fountains, lakes, rivers, springs, ponds, or streams that can be contaminated with sewage or feces from humans or animals. By eating uncooked food contaminated with Giardia. Thoroughly wash with uncontaminated water all vegetables and fruits you plan to eat raw. By accidentally swallowing Giardia picked up from surfaces (such as toys, bathroom fixtures, changing tables, diaper pails) contaminated with stool from an infected person.

23. Giardiasis is most frequently associated with the consumption of contaminated water. Outbreaks have been traced to food contamination by infected or infested food handlers, and the possibility of infections from contaminated vegetables that are eaten raw cannot be excluded. Cool moist conditions favor the survival of the organism. Giardiasis is more prevalent in children than in adults, possibly because many individuals seem to have a lasting immunity after infection. This organism is implicated in 25% of the cases of gastrointestinal disease and may be present asymptomatically. The overall prevalence of infection in the United States is estimated at 2% of the population. The disease is also common in child day care centers, especially those in which diapering is done. (EFFECT OF CROWDING) Who is susceptible? Everyone: Immunocompromised patients- HIV/AIDS, cancer patients, transplant rejection patients, cystic fibrosis, malnourished individuals

24. Diagnosis Microscopic examination of stool trophozoite cysts

26. Treatment. The drug of choice for the treatment of giardiasis remains Metronidazole (Flagyl), but quinacrin hydrochloride and furazolidone are also frequently used. However, drug resistance has been observed with each of these compounds. In addition, toxicity has restricted their use in women of child-bearing age, furazolidone has been used preferentially for children as it is can be administered as a suspension. Nevertheless, this compound has been recognised by the FDA in the U.S. as both a mutagen and carcinogen and can no longer be used.

27. Entamoeba histolytica Amitochondriate ameba Causes diarrhea and dysentery Rarely- spreads to extraintestinal sites Acquired by ingesting contaminated feces – in food / water Exists as a trophozoite in host and cysts outside host Some animals can become infected - none serve as important reservoirs Entamoeba dispar Morphologically identical- often misidentified Patients with diarrhea often tested- misdiagnosed and treated

29. Described in 1800’s Schaudinn 1903 depicted trophozoites and cysts and promptly died from overwhelming amebiasis Trophozoite is motile Facultative anaerobe Uses glucose as energy source Uses anaerobic metabolic pathways Trophozoites have single nucleus and lysozomes Cysts are smaller, no more than 4 characteristic nuclei

30. Direct Life Cycle Cysts ingested- one is enough to begin an infection (one of the most efficient protozoa) Excyst in small intestine- why? Receives environmental cues- acid/basic Stimuli required to excyst- release 4 trophozoites. Carried to large intestine Penetrate perimucosal space- attach to epithelial cells via lectin carbohydrate interactions- cytotoxic. They engulf and lyse living cells- causing ulcers- can result in hematogenous or lymphatic spread Some trophozoites encyst inside lumen- pass out in feces Cysts can survive weeks in warm moist conditions

31. E. Histolytica Adheres to colon cells Secretes enzymes-kills/damages cells Phagocytosis of cellular debris

32. Entamoeba histolytica must attach to host tissues Attachment based on interactions between epithelial cell membrane-bound N-acetyl-glucosamine and N-acetyl-galactosamine and two surface lectins The genes for parasite lectins have been cloned and characterized- In vitro- attachment to epithelia can be inhibited by adding free galactose to the medium

33. Attachment uses the secretion of a pore-forming peptide that is involved in lysing the host cell membrane Infections can cause increase in IL-8 in the surrounding epithelial cells- this brings more lymphocytes to the area, these can serve as target cells for the parasite

34. Immune mechanisms: Protective antibodies Antibodies in animal models directed to the carbohydrate-binding lectins of the parasite Cell mediated killing of parasites via reactive oxygen molecules

35. Most infections are asymptomatic- produces carriers Symptomatic- diarrhea, colic, flatulence, bloody stools, dysentery In chronic condition- form ameboma (large granuloma containing cells, parasites, necrotic colon tissue)- detectable / palpable masses- sometimes misdiagnosed as malignancies Colon becomes damaged- may stretch ulcerate, perforation, colon contents enter abdominal cavity- peritonitis Perforated, inflamed bowel may adhere to abdominal wall, amebas may attach to and infect skin tissue- cutaneous amebiasis

36. Extraintestinal Amoebas may damage mucosa down to circulation area of the submucosa- enter circulation- visit other tissues- especially liver This may occur after symptomatic or asymptomatic colon invasion. About 50% of patients with liver abscesses have not complained about amebic colitis Hepatic amebiasis is slow, progressive and insidious General fever, general pain in upper abdomen- may be diagnosed as tender liver of a swelling / mass on the liver Lungs can be involved-effusion, pleurisy, lung abscess, Can affect bronchia, pericardium, brain

37. Diagnosis Microscopy- hard to tell E. histolytica and E. dispar apart Detection of antigen (Elisa/Western) or PCR Treatment Several drugs- metronidazole Often given with another drug to kill cysts Prevent infection- heating or freezing kills cysts Normal chlorination levels do not kill cysts

38. A cyst of Entamoeba histolytica.  This is a mature cyst and, therefore, contains four nuclei.  However, only two nuclei are clearly visible in this plane of focus; approximate size = 18 µm. Entamoeba histolytica trophozoite.  The single nucleus with its central endosome and regularly distributed chromatin is visible; approximate size = 22 µm