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Giardia & Cryptosporidium: Protozoan Parasites. FISH/MICROM 490 Spring 2007. Anne Mataia Vanessa Lowe Lisa Crosson Kelsey Davies. Giardia and Cryptosporidium. What disease do these microorganisms cause? What factors aid in transmission? How are these microorganisms related?

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giardia cryptosporidium protozoan parasites

Giardia & Cryptosporidium: Protozoan Parasites

FISH/MICROM 490

Spring 2007

Anne Mataia

Vanessa Lowe

Lisa Crosson

Kelsey Davies

giardia and cryptosporidium
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

giardia and cryptosporidium3
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.
review of infectious disease
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.

factors of disease in the u s

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
cryptosporidium outbreak
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.

cryptosporidium outbreak cont
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.
giardia outbreak
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.

giardia outbreak cont
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.
cryptosporidium and giardia on a global scale
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

water and health
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)

factors of global disease
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
slide13

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

www.microbialrosettastone.com

what are these organisms
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.
giardia lamblia

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

giardiasis
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
other potential effects
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

morphology

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

giardia life cycle
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

biochemistry and metabolism

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 

carbohydrate metabolism map
Carbohydrate Metabolism Map

Jarroll et al, 1989

visualization methods
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

cryptosporidium27

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

species
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)

cryptosporidiosis
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
pathogenesis
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.)

metabolic pathways
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
transmission
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

diagnosis
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

current research
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
slide37
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
giardia cryptosporidium in summary
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
treatment
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
methods of prevention
Methods of Prevention
  • Good Hygiene
  • Safe Traveling/Hiking
  • Vaccines
  • Halogenation
  • Filtration
prevention
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
prevention cont
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.

prevention cont43
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.

method 1623
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
prevalence worldwide
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
global warming implications
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
slide50

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

other human impacts
Other Human Impacts
  • Deforestation
    • Subsequent conversion to farmland
    • Different composition of bodies of water
    • Pushing wildlife, potential carriers, into closer proximity to humans
  • Human movement
    • Population expansion
    • Crowded areas with poor water quality/sanitation procedures
  • Water control projects: dams, reservoirs
    • Large-scale breeding areas
references
References

Centers for Disease Control and Prevention: Division of Parasitic Diseases. “Cryptosporidiosis Control and Prevention.” Available online at: http://www.cdc.gov/ncidod/dpd/parasites/cryptosporidiosis/crypto_control_prevent.htm.

Centers for Disease Control and Prevention: Division of Parasitic Diseases. Giardiasis. Available online at: http://www.cdc.gov/Ncidod/dpd/parasites/giardiasis/default.htm

Curriero, F.C., J.A. Patz, J.B. Rose, and S. Lele. 2001. The association between extreme precipitation events and waterborne disease outbreaks in the United States, 1948-1994. American Journal of Public Health 91: 1194-1199.

Danielle Moore, W. Ray Waters, Michael J. Wannemuehler, James A. Harp. Treatment with Agmatine Inhibits Cryptosporidium parvum Infection in Infant Mice.The Journal of Parasitology, Vol. 87, No. 1 (Feb., 2001), pp. 211-213

Ecker DJ, Sampath R, Willett P, Samant V, Massire C, Hall TA, Hari K, McNeil JA, Buchen-Osmond C, Budowle B. The Microbial Rosetta Stone Database: A common structure for microbial biosecurity threat agents. Journal of Forensic Science. 2005;in press

Fayer, R. Cryptosporidium: a water-borne zoonotic parasite. Veterinary Parasitology. (2004) 126:37-56.

Goldstein, S.T., D.D. Juranek, O. Ravenholt, A.W. Hightower, D.G. Martin, J.L. Mesnik, S.D. Griffiths, A.J. Bryant, R.R. Reich, and B.L. Herwaldt. 1996. Cryptosporidiosis: An Outbreak associated with drinking water despite state-of-the-are water treatment. Annals of Internal Medicine 124: 459-468.

Jarroll, E.L., P. Manning, A. Berrada, D. Hare, D. G. Lindmark. 1989. Biochemistry and Metabolism of Giardia. Journal of Protozoology, 36(2), pp. 190-197.

Jenkins, M.C. 2001. Advances and prospects for subunit vaccines against protozoa of veterinary importance. Veterinary Parasitology 101: 291-310.

Madigan, M. T., and J.M. Martinko. 2006. Brock biology of microorganisms. 11th ed. Prentice Hall, Upper Saddle River, NJ.

references53
References

 Murray, CJL.; Lopez, AD. The Global Burden of Disease: A Comprehensive Assessment of Mortality and Disability from Diseases, Injuries, and Risk Factors in 1990 and Projected to 2020 (Global Burden of Disease and Injury, Vol. 1). Murray CJL and Lopez PD. , editor. Vol. 1. Boston, WHO; 1996. p. 906.

Partovi, F., G. Khalili, A. Kariminia, H. Mahmaudzadeh-Niknam. 2007. Effect of Giardia lamblia infection on the cognitive function of school children. Iranian Journal of Public Health, 36 (1), pp.73-78.

Patz, J.A., T.K. Graczyk, N. Gellar, and A.Y. Vittor. 2000. Effects of environmental change on emerging parasitic diseases. International Journal of Parasitology 1-11.

Roubin, M.-R. de, J.-S. Pharamond, F. Zanelli, F. Poty, S. Houdart, F. Laurent, J.-L. Drocourt, S. Van Poucke. 2002. Application of laser scanning cytometry folled by epifluorescent and differential interference contrast microscopy for the detection and enumeration of Cryptosporidium and Giardia in raw and potable waters. Journal of Applied Microbiology, 93, pp. 599-607.

San Francisco Department of Public Health - Environmental Health Section. Cryptosporidium Surveillance Project. Available online at: www.dph.sf.ca.us/ ehs/phes/water/crypto.htm

San Francisco Public Utilities Commission."Cryptosporidium White Sheet" Available online at: http://sfwater.org/detail.cfm/MC_ID/10/MSC_ID/51/MTO_ID/71/C_ID/446

Smith, HD, and Corcoran, GD. New drugs and treatment for cryptosporidiosis. Cur Opin Infec Dis. (2004) 6:557-564.

Tzipori S et al. Cryptosporidiosis in hospital patients with gastro-enteritis, American journal of tropical medicine and hygiene, 1983, 32:931-4

U.S. Environmental Protection Agency. Microbiology. Giardia image sequence 1. Available online at: http://www.epa.gov/nerlcwww/gda_seq1.htm

Vaishnava, S. and Striepen, B., (2006) The cell biology of endosymbiosis - how parasites build, divide and segregate the apicoplast, Mol. Microbiol. 61: 1380–1387

Xiao, L.and Ryan, UM. Crytpsporidiosis: an update in molecular epidemiology. Curr Opin Infec Dis. (2004) 5:483-490.

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