Outbreak of Cryptosporidiosis Among Veterinary Students in a

Bovine Obstetrics Laboratory

Carrie Klumb1,2, Jeff Bender3,Kirk Smith1, Elizabeth Cebelinski1, Joni Scheftel1

1Minnesota Department of Health 2CSTE/CDC Applied Epidemiology Fellowship

3University of Minnesota Veterinary Population Medicine

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

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 1010 oocysts per day in

feces • C. parvum is present on 90% of dairy

farms in the United States

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

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

Outbreak Detection (cont.)

• Four, 2-day OB laboratory sessions were scheduled at the CVM on:

Lab 1: April 1 and 2Lab 2: April 8 and 9Lab 3: April 15 and 16Lab 4: April 29 and 30

• The first three sessions had already occurred

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

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)

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

Methods - Case Definition

A student who attended an OB laboratory session and subsequently had either:

a) A stool sample test positive for Cryptosporidium spp.

ORb) 3 or more days of diarrhea

(≥3 loose stools in 24 hours)

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

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

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

• 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

• 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

Mock Cow

Mock Cow

Mock Cow

Mock Cow

• Day 1: practiced re-positioning abnormal fetal presentations and manual assistance of difficult births

Results - OB Laboratory Description

• 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

• 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

OB Laboratory

OB Laboratory

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

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

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

Case Symptoms (n = 8)

Symptom No. (%)Diarrhea 8 (100)Cramps 8 (100)Gas 7 (88)Weight Loss 4 (50)Fever 3 (38)Vomiting 1 (13)Bloody Diarrhea 1 (13)

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

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

• 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

No.

of C

ases

16 17 18 19 20 21 2212 13 14 153129 30

March

1

4

23

1 1110987

April

62 43

First Obstetrics Lab

5

Second Obstetrics Lab

Third Obstetrics Lab

23 24 25 2628

Second Obstetrics Lab Attendee

Laboratory-confirmedVeterinary School Employee

Onset Date

Cryptosporidium parvum Cases Associated with a Veterinary School Bovine Obstetrics Laboratory by

Illness Onset Date, Minnesota, 2010

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

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

Self-Reported Personal Protective Equipment (PPE) Use Among Students

PPE No. (%) PPE No. (%)

Day 1 Day 2Sleeves 42 (100) Sleeves 42 (100)

Coveralls 28 ( 93) Coveralls 27 ( 96)

Boots 29 ( 97) Boots 29 (100)Rubber Apron 0 ( 0) Rubber

Apron 0 ( 0)

Mask/Face Shield 0 ( 0) Mask/Face

Shield 0 ( 0)

• 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

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

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

RecommendationsMDH 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

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

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

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

Follow Up (cont.)

Conclusion• Cryptosporidiosis is sometimes called a

“rite of passage” in veterinary school– Serious disease– Infections should be avoided to the

extent possible

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

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