Waterborne Organisms and Human Health

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July 18, 06 EnviroHealth Connections
Waterborne Organisms and Human Health
Thaddeus Graczyk
Department of Environmental
Health Sciences
Department of Molecular Microbiology and
Immunology
Bloomberg School of Public Health
Johns
Hopkins University, Baltimore, MD
Cryptosporidium, Giardia, Human-infective
microsporidia
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May 26, 2000 / 49(SS04)1-35
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• SIGNIFICANT CONTRIBUTION TO THE MORTALITY OF
  IMMUNOSUPRESSED PEOPLE

• LACK OF EFFECTIVE PROPHYLACTIC OR THERAPEUTIC
  MEDICATIONS

• ID50 FOR IMMUNOCOMPETENT PEOPLE 10-1000 OOCYSTS
  IOWA 100 oocysts UCP 1,000 oocysts TAMU
  10 oocysts MD 300 oocysts

• CALF DAILY, 109 OOCYSTS HUMAN DIARHOETIC FECES,
  106 OOCYSTS/ML

• LONG LASTING INFECTIVITY IN THE ENVIRONMENT

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1) Recovery of Oocysts from Water
Filtration, Continuous Flow Centrifugation,
Centrifugation
2) Detection of Oocysts in Recovered Substrate
Elution, Dissolution, Concentration,
Separation, Purification, Staining, Microscopic
examination, Flow cytometry

• Am Standards for Testing Materials Method (ASTM
  Standards)
• Standard Method (AWWA Standards)
• Alternate Method (Hansen Ongerth, 1991)
• Cellulose Acetate Membrane (CAM)-Filter
  Dissolution Method (Graczyk et al., 1997)
• Method 1622 (U.S. EPA) Method 1623 (U.S. EPA)
  (limitations 10L, drinking water (low turbidity)

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Cellulose Acetate Membrane (CAM)-Filter
Dissolution Method
2.5 - 39-cm-diameter 0.210.0 um pore
size
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Graczyk et al. 1977. Recovery of waterborne
oocysts of Cryptosporidium from water samples by
the membrane filter dissolution method. Parasitol
Res 83 121-125.
Recovery Efficiency 78.7, CV 5.6
34.7
oocysts were not drained with the water
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Host Specificity of Cryptosporidium
Cryptosporidium parvum (zoonotic humans)


C. hominis (humans)

C. muris (rodents)

C. andersoni (cattle)

C. felis (cats humans)

C. wrairi (guine
pigs)
C. canis (dogs
humans) C. meleagridis (birds-turkeys)

C. baileyi (birds) C. serpentis (snakes)

C. saurophilum (lizards) C.
nasorum (fish)
Molecular studies indicate that this genus is
complex and may contain many additional species
(Xiao, Ryan, Graczyk et al. 2004.
Appl Environ Microbiol 70 891-899)
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1995 - 1998
Graczyk et al. 1995 Graczyk et al.
1996 Graczyk et al. 1998
Graczyk Cranfield 1998
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Genotyping of Cryptosporidium PCR, RFLP
C. hominis
C. parvum
C. andersoni
SspI
C. muris
Sewage Wastewater discharges vs.
Agricultural runoff
VspI
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Fluorescent In Situ Hybridization (FISH)
Giardia duodenalis
Cryptosporidium parvum
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E. hellem
E. bieneusi
E. cuniculi
E. intestinalis
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• PHASE I - Method selection
• PHASE II Wastewater effluent sampling
• PHASE III Sedimentation (3 WWTP)
• PHASE IV Water Plant influent sampling
• Base flow
• Storm event
• PHASE V Data evaluation/report prep. Etc.
  Whats next?

• Sample Dataset
• Range 3 oocysts/liter - 570 oocysts/liter
• Average 171 oocysts/liter
• Median 20 oocysts/liter
• Highest concentrations found in samples collected
  during plant failure

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MCI-Antietam Sediment Sampling
No No No
Above
Stream Bank
98 132 67
Just Below
Farthest Downstream
10 8 14

• Samples from Hancock/Tolonoway showed similar
  trend

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Small watersheds
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Foodborne Illnesses Vectored by Molluscan
Shellfish

• Pathogens associated with (raw) sewage disposal,
  wastewater effluents, overboard disposal, and
  runoff
• Agents indigenous to coastal waters
• Post-harvest contamination

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Graczyk Schwab 2000, Gastroenterol Rpts 2
305-309
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Phagocytosis
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Giardia duodenalis
2 hr
Cyclospora cayetanensis
14 days
Cryptosporidium parvum
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1998-02 2002-06
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Cryptosporidium
Fecal coliform
Dissolved O2
Oyster meat
Temperature
Salinity
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• Monitoring of Water for Fecal Coliform Is not
  Sufficient to Indicate the Presence of Human
  Waterborne Pathogens
• Oysters can cause cryptoptosporidiosis if
  consumed raw
• Oyster contamination coincides with rainfall
  and stream flow into the Bay

Graczyk et al. 2002. Susceptibility of the
Chesapeake Bay to environmental contamination
with Cryptosporidium parvum. Environ Res 82
106-112.
Technological limitations mouse bioassay, PCR
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• 2002-06
• 54 commercial bars only (30 oysters/bar)
• Oysters processed by six ( 1 meal)

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Graczyk et al. Appl Environ Microbiol in press
Cumulative number of viable C. parvum oocysts
found in six oysters
Eastern Bay
Choptank River
Wye River
Upper Bay
Fluorescent In Situ Hybridization
Bars, 23 of 54 (42.3) Meals 130
of 324 (40.1)
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Graczyk et al. Appl Environ Microbiol in press
Cumulative number of viable C. parvum oocysts
found in six oysters vs. water quality
parameters
Eastern Bay
Wye River
MPN/100 ml
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Foodborne Outbreaks Vectored by Molluscan
Shellfish
Cases
Outbreaks
Graczyk Schwab 2000, Gastroenterol Rpts 2
305-309
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73
WHO 2003 2.3 million children under 5 yr of age
die from diarrhea-related causes
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