The public health significance of zoonotic species of Cryptosporidium in drinking water

1. The public health significance of zoonotic species of Cryptosporidium in drinking water Norma J. Ruecker Department of Microbiology and Infectious Disease University of Calgary Alberta Provincial Laboratory for Public Health (Microbiology) Calgary

2. Cryptosporidiosis • Fecal-oral transmission • Infectious dose < 10 oocysts • Incubation 7-10 days • Symptoms: profuse watery diarrhea, abdominal cramping, nausea and vomiting, low-grade fever and headache. • Treatment – supportive therapy • Self-limiting in immunocompetent individuals lasting 3-12 days • ~ 600 cases reported yearly in Canada • ~ 3000 cases reported yearly in US

3. Cryptosporidium oocyst • Thick walled oocyst survives outside host • 4 – 8 µm (round or ovoid) • Oocyst wall has distinct inner and outer layer with suture at one end • Contains 4 sporozoites

4. C. molnari C. serpentis C. hominis C. parvum C. wrairi C. bovis C. muris C. suis C. baileyi C. canis C. felis C. saurophilum C. andersoni C. galli C. meleagridis Valid Cryptosporidium species

5. Pig genotype II Sheep genotype Horse genotype Rabbit genotype Marsupial genotypes I and II Opossum genotypes I and II Ferret genotype Fox genotype Coyote genotype Cervine genotype Deer genotype Muskrat genotypes I and II Monkey genotype Squirrel genotype Bear genotype Deer mice genotype Goose genotypes I and II Skunk genotype Snake genotype Lizard genotype Mouse genotype Tortoise genotype Woodcock genotype Duck genotype Mongoose genotype Numerous genotypes found in environment with no known host Additional host specific/adapted genotypes

6. Molecular phylogeny of the genus Cryptosporidium based on 18S rRNA Sequence Data(Xiao et al. 2004. Clin. Micro. Rev., 17:72)

7. Monkey genotype C. parvum C. andersoni C. felis Cervine genotype C. meleagridis C. suis C. hominis C. canis C. muris Epidemiology of human infections C. parvum ~ 29% of Human Infections ~ 69% of Human Infections

8. Water - a vehicle of transmission

9. Incidence of Cryptosporidium in water supplies • Surface waters • 4 – 100% of those tested • Finished water • up to 27% have been reported • 61 drinking water associated outbreaks • 53 recreational water associated outbreaks

10. Protect the public! • Regulatory changes in water treatment industry require utilities to monitor their source waters for Cryptosporidium • LT2-Rule – concentration of Cryptosporidium in source water will define the level of treatment required to minimize risk

11. Shortfalls of LT2 model for Cryptosporidium • Understanding transmission cycles • Concept of host specificity • Human-to-human transmission cycles are more important than zoonotic transmission • Monitoring water for parasites using existing methods proposed under the LT2-Rule (Method 1623) provide little meaningful public health data • Numbers do not equate to risk • high concentrations of C. andersoni are less of a risk than low concentrations of C. parvum or C. hominis

12. Studies examining diversity of Cryptosporidium species/genotypes in surface water • Chesapeake Bay area • C. hominis, C. parvum • Wachusett watershed • C. andersoni, C. parvum, C. baileyi • Storm water • Unique sources suspected to be wildlife • Germany and Switzerland • C. andersoni, C. parvum, C. muris

13. Detection of Cryptosporidium in Water:(Method 1623) Filter water (20 - 50 L) Elute parasites from filter Concentrate parasites # of Cryptosporidium oocysts in sample Purify by immunomagnetic separation Place on slide Stain with monoclonal antibodies Species? Genotypes? Enumerate by microscopy

14. Stained/counted microscope slide Remove material from slide (lysis buffer) Freeze/thaw (liquid N/65°C) DNA extraction (Qiagen kit) Nested PCR reactions (5 replicates) PCR positive – restriction digest Single genotype restriction patterns verified by sequence analysis Molecular forensic profiling byPCR-RFLP of 18S rRNA gene Sample # 1 (17 oocysts) Mw 1 2 3 4 5 Nested PCR product Ssp I digest Vsp I digest Dde I digest

15. Sampled weekly for 1 year Source tracking results: C. andersoni C. baileyi Skunk genotype Cervine genotype (2 occurences in 52 weeks) Muskrat genotype 2 unknown genotypes likely wildlife in nature Molecular profiling of Cryptosporidium from Lethbridge water utility 2003-2004

16. Lethbridge water utility 2003-2004 Occurrence data • 52 weeks at inflow into Lethbridge water utility • Poor correlation to indicators • Spike of Cryptosporidium in Aug 03 appears unlinked to an “ event” • Source tracking data indicated the appearance of Cryptosporidium skunk genotype

17. Eastern Ontario near Ottawa 14 sites weekly for 10 week period Mid October to mid December Agriculture based watershed South Nation Watershed 2004

18. Occurrence data Site 9 highest numbers of Cryptosporidium Week 8 overall highest values at all sites South Nation Watershed 2004

19. Nested PCR Ssp I Digest Vsp I Digest Dde I Digest South Nation Watershed 2004 Molecular characterization • 14 sequences isolated • Matches to published sequences • C. andersoni • C. baileyi • C. parvum (1 occurrence at 1 site) • Cervine genotype (1 occurrence at each of 2 sites) • Muskrat II genotype • 3 sequences 100% identity match to published environmental sequences • 5 sequences with no match to any published sequences

20. Phylogenetic relationships of isolates from SNW

21. Observations from initial watershed studies • C. andersoni is the dominant species in both these agriculturally based watersheds • In both watersheds the zoonotic species/genotypes of Cryptosporidium (C. andersoni and the cervine genotype) are infrequently observed

22. Estimating the public health risk of zoonotic species of Cryptosporidium • C. parvum is the only zoonotic species of Cryptosporidium know to have caused outbreaks of cryptosporidiosis • Does the infrequent occurrence of low numbers C. parvum in raw water pose a significant public health threat? • What is the risk to public health from other Cryptosporidium species? • Does the frequent occurrence of low numbers of zoonotic forms of Cryptosporidium pose a significant risk to public health? • Does the frequent occurrence of high numbers of Cryptosporidium not previously isolated from humans (specific to other hosts) pose a significant risk to public health?

23. The question of $$$ and ¢¢¢ At what level of risk to public health do we realistically spend multi-millions of dollars upgrading water treatment facilities? Current strategies are based solely on occurrence data

24. In summary • The current approach to protecting the public is treatment based • Need a scientific approach to develop a model incorporating both environmental risk assessment and public health risk assessment, based on the presence of human pathogenic species/genotypes • The key to protecting public health from the waterborne transmission of Cryptosporidium is an integrated and cost effective approach to both water treatment and watershed management

25. Acknowledgements • Dr. Norman Neumann (ProvLab, University of Calgary) • Support of staff and friends at Provlab Funding • NSERC Strategic Grant • Agriculture Policy Framework’s National Water Quality Surveillance Research Initiative and the National Agri-Environmental Standards Initiative • Canadian Water Research Network • FSWEP Research Affiliate work term