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
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
Methods Surface Water Filtration Fecal Sample Collection Immunomagnetic Separation DNA Extraction Nested PCR Clone & Sequence
Surface Water Filtration: Gelman Envirochek Filter Capsules
Methods Surface Water Filtration Fecal Sample Collection Immunomagnetic Separation DNA Extraction Nested PCR Clone & Sequence
Immunomagnetic Separation MB MB MB
Immunomagnetic Separation MB MB MB
Immunomagnetic Separation MB MB MB
Methods Surface Water Filtration Fecal Sample Collection Immunomagnetic Separation DNA Extraction Nested PCR Clone & Sequence
1 10 10 2 5*10 2 10 3 10 4 Nested PCR: 18S rrna
Methods Surface Water Filtration Fecal Sample Collection Immunomagnetic Separation DNA Extraction Nested PCR Clone & Sequence
52 C. felis (cats) 54 74 C. andersoni (calves) C. muris (calves) 100 C. muris (mouse) C. serpentis (reptiles) C. baileyi (birds) 57 74 87 C. meleagridis (birds) C. parvum (human) C. parvum (calves) C. parvum (mouse) 80 C. parvum (pig) C. wrairi (guinea pigs) 0.005 substitutions/site C. parvum (dog)
MWRA Water Supply System Boston Harbor
Wachusett Reservoir Watershed: Surface Water Sampling Sites Brook JF Quinapoxet River Stillwater River Brook SF Gates Brook Wachusett Reservoir
58 51 78 61 67 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) 100 100 70 JF4 (8/01) Goose 3b (IL) C. baileyi Cormorant (8/00) 91 94 100 Cow (6/00) 0.005 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) 98 100 Goose 7 (N) JF1 (6/01) JF2 (6/01) C. serpentis 100 86 98 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
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
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
Methods Surface Water Filtration Fecal Sample Collection Immunomagnetic Separation DNA Extraction Immunofluorescent Staining Microscopic Density Counts Nested PCR Clone & Sequence Cell Culture Infectivity Assay
Kristen Jellison, Ph.D. Fritz Engineering Lab Lehigh University 13 E. Packer Avenue Bethlehem, PA 18015 kjellison@lehigh.edu (610) 758-3555
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)