Cryptosporidium spp. Oocysts
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1 Sampling and Source Tracking of Cryptosporidium spp. Oocysts June 28, 2005 Kristen L. Jellison, Ph.D. Department of Civil & Environmental Engineering Lehigh University Bethlehem, Pennsylvania
2 Ultimate Goal of Source Tracking Identify important watershed sources of Cryptosporidium spp. oocysts Develop watershed management policies to prevent Cryptosporidium spp. contamination of surface waters
3 Methods Surface Water Filtration Fecal Sample Collection Immunomagnetic Separation DNA Extraction Nested PCR Clone & Sequence
4 Surface Water Filtration: Gelman Envirochek Filter Capsules
5 Methods Surface Water Filtration Fecal Sample Collection Immunomagnetic Separation DNA Extraction Nested PCR Clone & Sequence
6 Immunomagnetic Separation MB MB MB
7 Immunomagnetic Separation MB MB MB
8 Immunomagnetic Separation MB MB MB
9 Methods Surface Water Filtration Fecal Sample Collection Immunomagnetic Separation DNA Extraction Nested PCR Clone & Sequence
10 * Nested PCR: 18S rrna
11 Methods Surface Water Filtration Fecal Sample Collection Immunomagnetic Separation DNA Extraction Nested PCR Clone & Sequence
12 52 C. felis (cats) C. andersoni (calves) C. muris (calves) 100 C. muris (mouse) C. serpentis (reptiles) C. baileyi (birds) C. meleagridis (birds) C. parvum (human) C. parvum (calves) C. parvum (mouse) 80 C. parvum (pig) C. wrairi (guinea pigs) substitutions/site C. parvum (dog)
13 MWRA Water Supply System Boston Harbor
14 Wachusett Reservoir Watershed: Surface Water Sampling Sites Brook JF Quinapoxet River Stillwater River Brook SF Gates Brook Wachusett Reservoir
15 C. parvum (human) C. parvum (bovine) SR2 (2/00) C. parvum (ferret) C. parvum (mouse) C. wrairi C. meleagridis C. parvum (kangaroo) C. parvum (pig) 92 Deer (8/00) GB2 (11/01) GB1 (4/00) QR1 (11/00) C. parvum (dog) C. felis JF5 (11/01) 96 JF6 (11/01) JF7 (11/01) JF4 (8/01) Goose 3b (IL) C. baileyi Cormorant (8/00) Cow (6/00) substitutions/site Goose 9 (VA) Goose 5 (IL) SR3 (12/00) C. parvum C. meleagridis C. wrairi Goose 1, 2, 6, 8 (IL, MA, VA) Goose 3a (IL) Goose 7 (N) JF1 (6/01) JF2 (6/01) C. serpentis C. muris (mouse) SR1 (2/00) C. andersoni C. muris (bovine) 89 QR2 (7/99) SF1 (3/99) Manure (6/00) C. muris C. andersoni C. serpentis GenBank Sequences Surface Water Sequences Fecal Sample Sequences
16 Observations: Wachusett Reservoir Watershed The molecular detection method is sensitive and specific for Cryptosporidium spp. oocysts in water and fecal samples Nested PCR is required for detection of Cryptosporidium spp. in baseline environmental samples Multiple species of Cryptosporidium are detectable in environmental samples Suspected oocyst source is not always the observed source Evidence of wildlife, not human, impacts on GB Birds seem to be a bigger influence on JF than dairy farm activity
17 Method Comparison Quantitative estimate of oocyst density EPA Method 1622 es Molecular Detection Method No * Identification of oocyst infectivity No No * Detection of low oocyst densities (e.g., baseline environmental samples) Identification of oocyst genotype No No es es *But could be determined with method modifications
18 Methods Surface Water Filtration Fecal Sample Collection Immunomagnetic Separation DNA Extraction Immunofluorescent Staining Microscopic Density Counts Nested PCR Clone & Sequence Cell Culture Infectivity Assay
19 Kristen Jellison, Ph.D. Fritz Engineering Lab Lehigh University 13 E. Packer Avenue Bethlehem, PA (610)
20 Cryptosporidium Life Cycle Oocyst Sporozoite Type I Meront Ingested Exits Host Auto-infection Merozoite Cycle Asexual Thick-walled Oocysts Thin-walled Oocysts Host Enterocytes Type II Meront Sexual Cycle Microgamont Merozoites Zygote Macrogamont (Adapted from Current & Blagburn, 1990)
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