Giardia & Cryptosporidium: Protozoan Parasites

1. Giardia & Cryptosporidium: Protozoan Parasites FISH/MICROM 490 Spring 2007 Anne Mataia Vanessa Lowe Lisa Crosson Kelsey Davies

2. Giardia and Cryptosporidium • What disease do these microorganisms cause? • What factors aid in transmission? • How are these microorganisms related? • Where do they live? • How are the diseases diagnosed? • How are the diseases treated? • What steps can be taken to reduce their impact in our society? http://216.54.19.111/~corp2002/epa/sb/crypto/gandcrypto/index.html Giardia cysts and Cryptosporidium oocysts

3. Giardia and Cryptosporidium • Causative agents of the diseases Giardiasis and Cryptosporidiosis respectively. • Lives in the intestine and passed in the stool. • Transmitted to humans via fecal contamination of water and food. • Characterized by mild to severe diarrhea and can cause death in individuals with impaired immunity such as AIDS.

4. Review of Infectious Disease • Infectious diseases cause approximately 26% of all deaths worldwide and 31% of all disability. • In both developing and developed nations, the leading cause of death by a wide margin is acute respiratory disease. • Water plays a role in the transmission of a significant number of these diseases. • 75% of emerging or re-emerging diseases are zoonotic. Zoonotic agents are a special class of pathogens that co-evolve with the reservoir host, not with humans. They have a tendency to cause severe disease in humans.

5. Pathogen Host Disease Environment Factors of Disease in the U.S. Pathogenic Factors: • Specificity to host • Ability to persist • Zoonotic agent • Environmental Factors: • - Water temperature • Watershed security • Reservoir population • Season Host Factors: • Food preparation • Sanitary bathroom habits • Safe sex practices • Immuno-compromised • Age

6. Cryptosporidium Outbreak • *More than 30,000 people become infected with Cryptosporidium each year from eating raw and undercooked meat. • Food worker handling is major cause. • *The undiagnosed prevalence of Cryptosporidium in the US is between 0.5 - 2 million cases/year. • Cryptosporidium parvum now one of the most commonly identified intestinal pathogens throughout the world. Among children aged 1-5 years with diarrhea, Cryptosporidium parvum may be the most frequently found pathogen.

7. Spring, 1993- Milwaukee. An outbreak sickened an estimated 400,000 people and contributed to the deaths of more than 50 AIDS and chemotherapy patients. Spring rains and runoff from surrounding farmland had drained into Lake Michigan and overburdened the water supply system. Dairy cattle were the most likely source of this outbreak. Cryptosporidium Outbreak cont.

8. Giardia Outbreak *Over 200,000 people in the US become infected with Giardia every year from contaminated food. Food borne outbreaks are the result of contamination of food by infected workers or household members *Giardia lamblia was implicated in 4 of the 15 recent drinking water infectious disease outbreaks in the US. • Giardia cysts have been found in 97% of surface water sources (lakes, ponds, and streams) Major route of transmission: fecal–oral transfer of Giardia cysts, and most occur in child day-care and nurseries.

9. Giardia Outbreak cont. 1998,An outbreak occurred on an USA Naval vessel, infecting hundreds of US sailors. Contaminated water was bunkered at a port in Indonesia. Chlorine residuals were reported as trace. No additional treatment of the ship’s tanks had been performed. Giardia cysts can survive in low amounts of chlorine • Ingestion of water from infected swimming pools, lakes can also be another route of transmission.

10. Cryptosporidium and Giardia on a Global Scale "Diarrhea, which is spread easily in an environment of poor hygiene and inadequate sanitation, kills about 2.2 million people each year, most of them children under five” UNICEF

11. Water and Health Water supply, distribution of unserved populations Sanitation, distribution of unserved population Asia shows the highest number of people unserved by either water supply or sanitation; yet it is important to note that proportionally, this group is bigger in Africa because of the difference of population size between the two continents. Source: WHO/UNICEF Joint Monitoring Programme, (2002)

12. Pathogenic Factors: Specificity to host Ability to persist Zoonotic agent Host Factors: Food preparation Access to water Sanitary bathroom habits Access to toilet paper Proximity to livestock Age Immuno-compromised Pathogen Host Disease Environment Factors of Global Disease • Environmental Factors: • - Water temperature • Watershed security • Reservoir populations • Season • Water Sanitation

13. Microbial Rosetta Stone Database: relates microorganism names, taxonomic classifications, diseases, specific detection and treatment protocols, and relevant literature. www.microbialrosettastone.com

14. Food and Waterborne Pathogens

16. What are these organisms? Unicellular flagellated Protists Diplomonads: • The Diplomonads are a group of mostly parasitic flagellates. • Most Diplomonads are double cells: they have two nuclei, each with four associated flagella. • They lack both mitochondria and a Golgi apparatus. • Mitosomes are involved in the maturation of iron-sulfur proteins • Apicomplexa: • Characterized by the presence of a unique organelle called an apical complex. • Cells contain a single plastid, called the apicoplast surrounded by either 3 or four membranes that synthesizes lipids among other compounds. • Apicoplast is thought to be derived from Green Algae • Cryptosporidium lacks apicoplast.

17. Giardia

18. www.med-chem.com Giardia lamblia • History • First observed 1681 by Anthony van Leeuwenhoek • Described ~200 years later by Vilem Lambl • First cultured in 1960’s • Confirmed pathogen 1970’s • One of most common intestinal parasites • Causes Giardiasis (beaver fever) • 2.5 million cases/year U.S. • > with poor water sanitation • Species details • Single-celled protist • 5 species of Giardia • G. intestinalis/lamblia • G. muris in rodents, birds, reptiles • G. agilis in amphibians • G. ardae in great blue heron • G. psittaci in budgerigar www.en.wikipedia.org

19. Host examples Humans Cats, dogs Cows, lamb Beavers, deer Manifest in 7-10 days Lasts 2- 6 weeks Symptoms Severe diarrhea Abdominal cramps Bloating Flatulence Nausea Vomiting May lead to Weight loss Dehydration Giardiasis Common Chronic vs. • More severe condition • Lasts 2 months - years • Symptoms • Increased gas • Dehydration • Burping • Loose stools • Slowed growth

20. Other Potential Effects? • Study of school children in Iran • Comparison of cognitive function in • Giardia infected children • uninfected children • Same nutritional status • 3 cognitive tests used • Results • Uninfected children improved significantly more than Giardia infected children in 2 tests • Test of Fluency • Digit-span forward/backwards test Partovi et al., 2007

21. www.tulane.edu www.oregon.gov www.med-chem.com Morphology • Cyst • Infective stage in the environment, hardy • Persist in cold water up to several months • egg-shaped, 8-14µm by 7-10µm • Organelle duplication w/out cytokinesis results in • four nuclei (Nu) • four median bodies (MB) • four axonemes (Ax) • Upon excystation, each cyst produces two trophozoites. • Trophozoite • Cannot survive in the environment • Motile 4 pairs of flagella • Pear shaped, bilaterally symmetrical • Relatively flattened, 10-12µm long • 5-7µm wide with a large sucking disk on the anterior ventral side • Two nuclei www.oregon.gov

22. Giardia Life Cycle 2 trophozoites released per cyst Multiply by binary fission every 12 h Sucking disc used for attachment Cyst forms in transit to colon

23. www.cdc.gov Biochemistry and Metabolism • Aerotolerant anaerobe • Respires in the presence of oxygen by a flavin, iron-sulfur protein-mediated electron transport system • Glucose is the only sugar catabolized • Energy produced by substrate level phosphorylation • Substrates incompletely oxidized to carbon dioxide, ethanol, and acetate • Trophozoites rely on preformed lipids and composition of lipids in cysts are similar • Trophozoites lack most carbohydrate splitting hydrolases 

24. Carbohydrate Metabolism Map Jarroll et al, 1989

25. Visualization Methods • Laser scanning cytometry • Immunofluorescent antibodies (Giardia –specific) for enumeration • Differential interference contrast (DIC) for confirmation • 4,6-diamidino 2-phenyl-indole dihydrochloride (DAPI) for viability • Propidium iodide (PI) for non-viability H.D.A. Lindquist, U.S. EPA

26. Cryptosporidium

27. Cryptosporidium Classification “hidden spore” Small (2-5µm) single- celled parasitic protist Invade and grow intracellularly in mucosal epithelial cells of the stomach and intestine History 1895 Clarke first to view Cryptosporidium 1910 Tyzzer named and described Cryptosporidium muris in mice Recognized as disease agent in 1976 1987 Carrollton,Georgia ~13,000 people ill 1993 Milwaukee, Wisconsin ~400,000 people ill

28. Species • ~ 23 species named • Human infections: C. Baileyi, C. canis, C. felis, C. hominis, C meleagridis, C. muris, and C. parvum • Humans are the primary hosts for C. hominis • C. Parvum widespread in non human hosts ~155 mammal species • Most frequently reported zoonotic species • Past ten years gained world wide recognition - caused more human illness and mortality than any other fecal-associated microorganism C. hominis invasion Thick-walled oocyst of C. parvum (Scanning Electron Micrograph)

29. Host Occurs naturally in a variety of animals fishes birds reptiles mammals humans Symptoms Most common-watery diarrhea Others include:Abdominal crampsNauseaLow-grade feverDehydrationWeight loss or no symptoms at all Cryptosporidiosis

30. Morphology

32. Pathogenesis • Osmotic (primary) • characterized by enterocyte malfunction • ↓ Na+ absorption • ↑ Cl- secretion • Inflammatory • generally associated with invasion of the mucosa • inflammation of the lamina propria • leukocytes in the stools • Secretory • generally associated with bacterial enterotoxins • characterized by watery diarrhea • Increased intercellular permeability and inflammation in the submucosal layer Schematic representation of Cryptosporidium pathogenesis. Many factors may be involved in the diarrhea associated with cryptosporidiosis. (Modified from Clark and Sears, Parasitology Today 12:221, 1996.)

33. Metabolic Pathways • Poorly understood • Apicoplast and its associated Type II fatty acid synthetic enzymes absent in C. parvum • Possesses: • Type I fatty acid synthase (CpFAS1) • Acetyl-CoA binding protein • a number of other enzymes involved in fatty acid metabolism • However, C. parvum lacks enzymes for the oxidation of fatty acids, indicating that fatty acids are not an energy source for this parasite • Genome analysis – reliance on host for nutrition

34. Transmission • Cryptosporidium lives in the intestines of infected individuals and is released with bowel movements • Minimum infective dose – 10 to 100 oocysts depending upon species • Thick cell walls of oocysts enable them to persist in the natural environment for long periods of time • C. parvum oocyst is able to survive for several days in mild chlorine Images courtesy of the San Francisco PUC

35. Diagnosis • Unable to diagnose Crypto from symptoms alone • Stool samples must be submitted for laboratory analysis • Common laboratory diagnostic methods of cryptosporidiosis include microscopy, enzyme immunoassays and molecular biology methods Image courtesy of CDC. Oocysts can be detected with fluorescent antibodies. Image courtesy of CDC. Diagnostics can involve PCR detection of C. parvum in stool specimen

36. Current Research Treatment with agmatine inhibits Cryptosporidium parvum infection in infant mice (Moore et al. 2001) • treated infant mice with oral doses of agmatine following experimental infection with C. parvum • Mice treated with agmatine were significantly less infected with C. parvum than were control mice • Suggest that exogenous agmatine alters the metabolism of C. parvum sufficient to interfere with its ability to colonize the mammalian intestine

37. The cell biology of endosymbiosis - how parasites build, divide and segregate the apicoplast (Vaishnava & Striepen 2006) • Chloroplast exciting new target for drug development • Role of specific plastid pathways for the development and pathogenesis of the parasite – genomic & genetic approach • Parasite depends entirely on salvage of purines and pyrimidines from its host cell • Enzymes obtained via horizontal gene transfer from bacterial source

38. Giardia & Cryptosporidium:In Summary • Aquatic, protozoan parasites • Infect multiple hosts: humans, pets, farm animals, wildlife • Similar symptoms: diarrhea, cramping, nausea, weight loss, dehydration • Similar methods of transmission • Hearty organisms • Different phases of life cycle • Can be hard to kill

39. Treatment • Giardia • Metronidazole (Flagyl) first-line treatment • Tinidazole (Tindamax) first-line treatment • Nitazoxanide (Alinia) inhibits trophozoite growth by disrupting their energy metabolism • Paromomycin (Humatin) may be used to treat severe infections in pregnant women, however worse side effects - long term use not recommended • Furazolidone (Furoxone) • Cryptosporidium • No reliable treatment • Nitazoxanide and paromomycin occasionally used, temporary effects only

40. Methods of Prevention • Good Hygiene • Safe Traveling/Hiking • Vaccines • Halogenation • Filtration

41. Prevention 1. Hygienic Practices • Wash hands thoroughly with soap and water, especially: • After using the bathroom • After handling animals • After touching dirt • Before eating/preparing food • Avoid consuming contaminated water • Don’t swallow recreational water or drink untreated water • Peel and rinse fruits and vegetables • Follow water advisories

42. Prevention (cont.) 2. Safe Traveling/Hiking • Be careful of drinking the water and consuming raw foods • If you must drink untreated water, use sand filtration or boil the water. Sand filtration filters are available in many outdoors-goods stores Vaccines • Not available for humans • In farm/herd and companion animals • Prevent cross-transmission • Available for both Giardia and Cryptosporidium • Problems: live-vaccine, must be refrigerated, short shelf-life 3.

43. Prevention (cont.) 4. Halogenation • Non-spore forming bacteria are very susceptible • Protozoan cysts are resistant, therefore, used but not as effective Filtration • Most effective way of removing Giardia & Cryptosporidium cysts • Many filtration systems implement other methods of disinfection • Halogenation (see above) • Ozone - produces small amt of carcinogen, no residual • UV radiation - no residual disinfectant • Method 1623 5.

44. Method 1623 • Assessment of the occurrence of Giardia and Cryptosporidium • Surface waters • 1996-influenced by Safe Drinking Water Act amendments • First developed Method 1622 • Detection of Cryptosporidium only • Combined procedure for detection of Cryptosporidium and Giardia • Steps: • Filtration • Immunomagnetic separation of oocysts and cysts • Immunofluorescence assay • Dye staining and contrast microscopy

47. Prevalence Worldwide • Cryptosporidium • 1-4% in Europe • 3-20% in Africa, Asia, South America • Giardia • 3-7% in developed countries • Average 20% (range 5-43%) in undeveloped countries • In immuno-suppressed individuals prevalence greater and more complications can occur

49. Global Warming Implications • Three potential areas of impact: • Rainfall events • Low rainfall can increase breeding sites • Increased vegetation can expand population of vertebrate host • Flooding • Increased surface water can can provide breeding sites for vectors • Force vertebrate hosts in closer proximity to humans • Increased temperatures • Changed rate of vector population growth • Changes in incubation period of vector

50. 51% of waterborne disease outbreaks were preceded by precipitation events above the 90th percentile. -of these 13% were attributed specifically to Giardia Curriero et al., 2001