1. Outbreak of Cryptosporidiosis Among Veterinary Students in a Bovine Obstetrics Laboratory Carrie Klumb 1,2, Jeff Bender 3, Kirk Smith 1, Elizabeth Cebelinski 1, Joni Scheftel 1 1 Minnesota Department of Health 2 CSTE/CDC Applied Epidemiology Fellowship 3 University of Minnesota Veterinary Population Medicine

2. Background - Cryptosporidiosis Acute gastrointestinal illness caused by the protozoan parasite Cryptosporidium Estimated 748,000 cases and 46 deaths per year in the United States Incubation period 1-12 days, mean 7 days Profuse watery diarrhea, and cramping over days to weeks. Fever and vomiting also may occur

3. Background - Cryptosporidiosis (cont.) 19 species of Cryptosporidium –C. hominis – human primary reservoir –C. parvum – cattle primary reservoir Oocysts (infective stage) are shed in the feces of infected hosts In cattle, primarily disease of calves –Shed up to 10 10 oocysts per day in feces C. parvum is present on 90% of dairy farms in the United States

4. Background - Cryptosporidiosis (cont.) Transmission occurs through water and food, from person-to-person and through direct animal contact 1983: Published report of cryptosporidiosis outbreak due to direct cattle contact among veterinary researchers Numerous outbreaks of cryptosporidiosis have been reported among veterinary students

5. Outbreak Detection On April 22, 2010 gastrointestinal illness among second year veterinary students at a college of veterinary medicine (CVM) was reported to the Minnesota Department of Health (MDH) Students had participated in a 2-day bovine obstetrics (OB) laboratory prior to becoming ill

6. Outbreak Detection (cont.) Four, 2-day OB laboratory sessions were scheduled at the CVM on: Lab 1: April 1 and 2 Lab 2: April 8 and 9 Lab 3: April 15 and 16 Lab 4: April 29 and 30 The first three sessions had already occurred

7. Methods - Epidemiologic Investigation Obtained contact list of all students registered for any of four OB laboratory sessions Spoke to professor and employees about laboratory set-up and specific activities Developed a questionnaire regarding symptoms and risk factors both in and outside of the laboratory setting Interviewed students by phone or in person

8. Methods - Epidemiologic Investigation (cont.) Risk factors assessed –Tracking in small, mixed or large animal practice –Dates of laboratory attendance –Eating or drinking in the laboratory –Use of personal protective equipment (PPE)

9. Methods - Epidemiologic Investigation (cont.) Risk factors assessed –Hand hygiene –Being splashed in the face during the laboratory session –Events or meals in common with other students Analyses were performed using Epi-Info software, version 6.04d and SAS, version 9.2

10. Methods - Case Definition A student who attended an OB laboratory session and subsequently had either: a) A stool sample test positive for Cryptosporidium spp. OR b) 3 or more days of diarrhea (≥3 loose stools in 24 hours)

11. Methods - Laboratory Investigation Testing was performed on stool samples –Students and employees with symptoms –Calves from Dairy Farm A, where animals for class were obtained Fecal samples from the calves actually used in the laboratories were unavailable

12. Methods - Laboratory Investigation (cont.) The MDH Public Health Laboratory conducted routine testing for Salmonella, Shigella, Campylobacter, Yersinia and E. coli O157:H7 PCR for Shiga toxin-producing E. coli virulence factor genes (stx1, stx2, eae and hlyA) performed

13. Methods - Laboratory Investigation (cont.) Testing for Giardia and Cryptosporidium spp. performed using acid fast staining, direct fluorescent antibody tests Positive Cryptosporidium specimens identified to species by PCR fragment length polymorphism (PCR-RFLP) –Further identified to subtype by DNA sequence analysis of the 60-kDa glycoprotein (gp60) gene

14. Laboratory gives students hands-on experience assisting cows with difficult labors Course is highly physical and occurs in a small room Approximately 12 students working in pairs under the supervision of professor or lab technician Results - OB Laboratory Description

15. Mock cows consisting of a wooden box, plastic bag, and cow pelvic bones Dead calves are placed inside mock cow Calves used are generally healthy, 48-hour old bull calves euthanized by captive bolt Results - OB Laboratory Description

16. Mock Cow

20. Day 1: practiced re-positioning abnormal fetal presentations and manual assistance of difficult births Results - OB Laboratory Description

21. Day 2: Students practiced re-positioning abnormal presentations and performed a fetotomy –Used when calf has died in utero and too large to be delivered naturally –Calf is cut up in utero, removed in pieces Often results in laceration of the dead calf’s bowels Introduces fecal material into the classroom Results - OB Laboratory Description

22. Clean-up procedures –Students hosed down the inside of the boxes, the plastic linings, and the floor with a garden hose –No facial protection used Results - OB Laboratory Description

23. OB Laboratory

25. Results - OB Laboratory Description Calves from Dairy Farm A used during outbreak were: –Mix of male and female –Several weeks old –Some had died of diarrheal illness Calves stored together between day 1 and 2 and not labeled –Could not tell who was exposed to which calves

26. Results (cont.) 42 students had completed a lab at the time of the investigation 38 (91%) interviewed 8 (21%) met the case definition 22 (58%) reported no symptoms 8 (21%) reported gastrointestinal symptoms but did not meet case definition

27. Results (cont.) 8 of 8 (100%) and 4 of 22 (18%) controls attended Lab 2 on April 8 and 9 –Fisher’s exact p < 0.001 There were no cases among Lab 1 or Lab 3 attendees

28. Case Symptoms (n = 8) SymptomNo. (%) Diarrhea8 (100) Cramps8 (100) Gas7 (88) Weight Loss4 (50) Fever3 (38) Vomiting1 (13) Bloody Diarrhea1 (13)

29. Results (cont.) Median incubation period from Lab 2 was 7 days (range, 5 to 12 days) Median duration of illness for the 6 cases that had recovered at the time of the interview was 8.5 days (range, 7 to 14 days) 1 case was seen by their physician and 1 case was seen in an Emergency Department

30. Results (cont.) A CVM employee also became ill with bloody diarrhea, gas and cramps with illness onset on April 23 Employee still ill at time of interview Set up and cleaned up the OB laboratories on several different dates Incubation period calculated from the date of Lab 2 was 15 days

31. 4 of 8 human samples (3 student cases and CVM employee) positive for C. parvum of the same gp60 subtype (IIaA15G2R2) All human stool samples negative for Salmonella, Shigella, Campylobacter, Yersinia, E. coli O157:H7, Shiga toxin-producing E. coli virulence factors, and Giardia Results - Human Testing

32. No. of Cases 1617181920212212131415312930 March 1 4 2 3 11110987 April 6243 First Obstetrics Lab 5 Second Obstetrics Lab Third Obstetrics Lab 2324252628 Second Obstetrics Lab Attendee Laboratory-confirmed Veterinary School Employee Onset Date Cryptosporidium parvum Cases Associated with a Veterinary School Bovine Obstetrics Laboratory by Illness Onset Date, Minnesota, 2010

33. Results (cont.) Among the Lab 2 attendees, the only risk factor that approached significance was being splashed in the face at any point during day 2 of the lab –7 of 7 cases vs. 1 of 3 controls* –Fisher’s exact p = 0.07 *1 case and 1 control were not able to recall and could not answer the question

34. Results (cont.) Students in Lab 2 were significantly more likely to have been splashed in the face at any point during the lab session than students in Labs 1 and 3 –Odds ratio, 13.0 –95% confidence interval, 1.44 to 117.20 –p = 0.01

35. Self-Reported Personal Protective Equipment (PPE) Use Among Students PPENo. (%)PPENo. (%) Day 1Day 2 Sleeves42 (100)Sleeves42 (100) Coveralls28 ( 93)Coveralls27 ( 96) Boots29 ( 97)Boots29 (100) Rubber Apron 0 ( 0) Rubber Apron 0 ( 0) Mask/Face Shield 0 ( 0) Mask/Face Shield 0 ( 0)

36. 3 of 9 calf fecal samples collected at Dairy Farm A positive for C. parvum gp60 subtype (IIaA15G2R2) –Matched C. parvum in human samples –Subtype commonly found in Minnesota 3 fecal samples were positive for the E. coli virulence factor gene stx1 Calf fecal samples negative for Salmonella, Shigella, Campylobacter, Yersinia, E. coli O157:H7 and Giardia Results - Calf Testing

37. Conclusion This was an outbreak of cryptosporidiosis among veterinary students in an OB laboratory Calves that had died of a diarrheal illness and then used in the laboratories were the source of infection

38. Conclusion (cont.) Not surprising that stool samples from calves at Dairy Farm A tested positive for C. parvum –C. parvum is present on 90% of dairy farms in the U.S. –Higher prevalence in calves Highlights the zoonotic risk inherent in certain veterinary school activities

39. Recommendations MDH epidemiologists and infection control specialists from CVM made recommendations Have students read provided information about zoonoses and prevention measures Remind students about proper hand hygiene Restrict calves used to euthanized 48-hour old healthy bull calves

40. Recommendations (cont.) Provide face shields/masks and rubber aprons to students Offer laundry service at school Use an EPA-approved disinfectant to clean laboratory No illnesses were identified during the last laboratory on April 29 and 30

41. Follow Up Visited laboratory on May 12, 2011 to observe Warning was added to this year’s syllabus

42. Follow Up (cont.) Observations made –All students wore boots, coveralls, sleeves –No face shields/masks offered or worn by students –Laundry service not being provided by the school –Near-term fetuses were being obtained from slaughter facilities

43. Follow Up (cont.)

44. Conclusion Cryptosporidiosis is sometimes called a “rite of passage” in veterinary school –Serious disease –Infections should be avoided to the extent possible

45. Conclusion Prevention of future outbreaks –Educating students about the risks –Ensuring calves used in laboratories are healthy –Providing face shields or a mask and goggles Compendium of Veterinary Standard Precautions for Zoonotic Disease Prevention in Veterinary Personnel offers infection control guidance

46. Acknowledgments Minnesota Department of Health Team Diarrhea Foodborne, Vectorborne, and Zoonotic Disease Unit University of Minnesota Dr. Ricardo Chebel Amanda Doran Dr. Tom Molitor Leslie Hiber