Cyclosporiasis: An Emerging Zoonotic Disease?

1. Cyclosporiasis: An Emerging Zoonotic Disease?

2. Proposed Life Cycle • Ingested oocysts excyst in GI tract • Sporozoites invade small intestinal epithelial cells • Asexual forms • 2 different sized merozoites • type I meronts (8-12 merozoites) • type II meronts (4 merozoites) • Sexual forms • gametocytes • zygotes • oocysts shed in stools

3. Transmission • Fecal-oral route • Direct transmission from person-to-person UNLIKELY • Oocysts need to sporulate before becoming infectious • Food or waterborne USUALLY http://www.dpd.cdc.gov/dpdx/HTML/Cyclosporiasis.htm

4. Cyclosporiasis • GI illness • diarrhea - protracted ~ 6 weeks • cramps • dehydration • bloating • Constipation • nausea • Treatment • Trimethoprim-sulfamethozaxole (Bactrim) • Complications if untreated • Reiter’s • Guillain-Barré

5. Epidemiology Previously known as cyanobacterium-like body 1979: 1st probable report • undescribed coccidian in humans in PNG • (Ashford, L’pool Sch Trop Med, Ann Trop Med Parasitol) 1989: Expatriates in Nepal • (Shlim et al, 1991, Am J Trop Med Hyg) 1993: Study on Peruvian cases (Ortega et al, NEJM) 1994: New species designation (Ortega et al, J Parasitol) • Reports on travel-related cases • Australian traveller to Bali, Indonesia (Butcher et al, 1994) • US travellers to Mexico or Thailand (Berlin et al, 1994) • Bangladesh cases (Albert et al, 1994)

6. Cyclosporiasis OutbreaksWaterborne • 1990 Chicago hospital (Huang et al, 1995, Ann Intern Med) • cluster of 21 cases • associated with tap water in physicians’ dormitory • 1994 British soldiers in Nepal (Rabold et al, 1994, Lancet) • 12 cases • Cyclospora oocysts detected in water specimen from storage tank

7. Cyclosporiasis OutbreaksFoodborne • 1996 USA / Canada Herwaldt & Ackers 1997 NEJM • 1,465 non-travel cases • 20 US states + DC • 2 Canadian provinces • Ontario - 36 cases • Quebec - 3 cases • 55 event–related clusters • Associated with Guatemalan raspberries

8. More Foodborne Outbreaks Year & Location Cases Vehicle 1997 Florida 93 mesclun lettuce 1997 US/Ontario 1,012 raspberries 1997 Wash., DC 341 basil 1997 US bans Guatemalan raspberries 1998 Ontario 315 raspberries 1998 Canada bans Guatemalan raspberries 1999 Florida 94 berries 1999 Ontario 104 blackberries 1999 Missouri 64 basil Herwaldt 2000 Clin Inf Dis 31:1040-1057

9. Cyclosporiasis outbreaks in British Columbia (BC) Location Date Cases Association L. Mainland Jul 1999 15 Unknown Vancouver May 2001 17 Thai basil L. Mainland Jul 2003 11 Cilantro L. Mainland Jun 2004 18 Cilantro

10. Cyclosporiasis • North American outbreaks associated with the consumption of imported fruits and vegetables • A reportable communicable disease • 1999 - British Columbia • 2000 - Canada

11. Cyclosporiasis cases and rates in British Columbia and Canada Data: Epidemiology Services, BC Centre for Disease Control

12. refractile globules MICROSCOPY Wet mounts examined using: • DIC • UV fluorescence microscopy (excitation filter 330-365 nm) autofluorescence http://www.dpd.cdc.gov/dpdx/HTML/Cyclosporiasis.htm

13. Cyclospora sp. in Animals? Reported in: • Duck Peru Zerpa et al, 1995 • Chickens Mexico García-López et al, 1996 • Dogs Brazil Yai et al, 1997 Absent in: • Domesticated animals, Haiti Eberhard et al, 1999 • Livestock - pigs, cattle, horses, goats • Poultry - chicken, ducks, turkeys • pigeons • Companion animals - dogs, cats, guinea pigs • Dogs Brazil Carollo et al, 2001

14. Cyclospora sp. in Primates Reported in: • Baboons Tanzania Smith et al, 1996 Lopez et al, 1999 • Baboons / Monkeys Eberhard et al, 1999 Ethiopia • Primates Kenya Eberhard et al, 2001

15. CYCF1E1 CYCF3E1 18S rRNA ITS1 5.8S rRNA CYCR4B1 CYCR2B1 MOLECULAR METHODS • Nested PCR: conserved 18S rRNA gene • Diagnostic marker for Cyclospora cayetanensis • Cross-reacts with Eimeria tenella 1Relman et al. (1996) J.Inf.Dis. 173:440

16. Nested PCR Amplification of the 18S rRNA gene

17. 18S rRNA Gene Sequence

18. 18S rRNA Gene Sequence

19. Quartet puzzling maximum likelihood tree 18S rRNA gene sequences of Cyclospora and Eimeria species Outgroup: Toxoplasma gondii Eberhard et al (1999) Emerg. Inf. Dis. 5:651

20. Multiplex PCR for the differentiation of Cyclospora spp. and Eimeria spp. Specificity Primer Sequence Amplicon size (bp) C. cayetanensis5'-GTAGCCTTCCGCGCTTCG-3'298 C. cercopitheci, 5'-CTGTCGTGGTCATCTGTCCGC-3‘ 361 C. colobi Eimeria spp. 5'-GTTCTATTTTGTTGGTTTCTAGGACCA-3'174 Common 5'-CGTCTTCAAACCCCCTACTGTCG-3' reverse primer Orlandi et al, Appl Env Mic (2003), 69(8):4806

21. Multiplex PCR Specificity Sensitivity Orlandi et al, Appl Env Mic (2003), 69(8):4806

22. Cyclosporacayetanesis • Absence in Haitian domestic animals • Different Cyclospora species in primates NOT A ZOONOTIC PATHOGEN! REALLY?

23. C. cayetanensis in Nepal Monkeys 1/3 Dogs 2/14 Chickens 1/3 Chu et al., Am.J.Trop.Med.Hyg. (2004) 74(4):373-9

24. Animal Reservoirs? Can’t confirm • Infections? OR • Human fecal material passing through? • Coprophagic feral animals • Poor sanitary conditions • Contamination of feed

25. Future Work Required • Check animal scats for Cyclospora sp. oocysts • Confirm by molecular methods that species is C. cayetanensis • Perform tissue biopsies in animals shedding C. cayetanensis oocysts to determine if infections are really present

26. Questions? Photo: Jean Ryder / DHD Multimedia Gallery http://gallery.hd.org